U.S. patent application number 12/586342 was filed with the patent office on 2010-03-18 for methods of treating lysosomal storage related diseases by gene therapy.
Invention is credited to R. Scott Mclvor, Chester B. Whitley.
Application Number | 20100068183 12/586342 |
Document ID | / |
Family ID | 32599801 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100068183 |
Kind Code |
A1 |
Whitley; Chester B. ; et
al. |
March 18, 2010 |
Methods of treating lysosomal storage related diseases by gene
therapy
Abstract
Isolated nucleic acid-based vectors and lentivirus vectors, and
methods of using those vectors to inhibit or prevent metabolic
disorders in a mammal, are provided.
Inventors: |
Whitley; Chester B.;
(Brooklyn Park, MN) ; Mclvor; R. Scott; (St. Louis
Park, MN) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG & WOESSNER, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Family ID: |
32599801 |
Appl. No.: |
12/586342 |
Filed: |
September 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11057410 |
Feb 14, 2005 |
7592321 |
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12586342 |
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PCT/US03/25508 |
Aug 13, 2003 |
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11057410 |
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60403108 |
Aug 13, 2002 |
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60403586 |
Aug 14, 2002 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1 |
Current CPC
Class: |
C12N 9/16 20130101; C12N
2810/6081 20130101; C12N 2740/16043 20130101; C12N 2810/50
20130101; C12Y 302/01023 20130101; C12Y 302/01076 20130101; C12N
9/2471 20130101; C12Y 302/01045 20130101; C12N 2740/16045 20130101;
C12N 15/86 20130101; C12N 9/2465 20130101; A61K 48/00 20130101;
C12Y 302/01022 20130101; C12N 9/2402 20130101 |
Class at
Publication: |
424/93.2 ;
435/235.1 |
International
Class: |
A61K 45/00 20060101
A61K045/00; C12N 7/01 20060101 C12N007/01; A61P 43/00 20060101
A61P043/00 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] The invention was made at least in part with a grant from
the Government of the United States of America (grant P01-HD32652
from the National Institutes Health). The Government may have
certain rights to the invention.
Claims
1. A method to prevent, inhibit or treat a disorder characterized
by the absence or reduced levels of a lysosomal enzyme in a mammal,
comprising: administering to a vascular compartment of a mammal
having or at risk of the disorder, an effective amount of a
recombinant lentivirus comprising a nucleic acid segment encoding
the enzyme.
2. (canceled)
3. The method of claim 1 wherein the protein is
alpha-L-iduronidase, iduronate-2-sulfatase, heparan sulfate
sulfatase, N-acetyl-alpha-D-glucosaminidase, beta-hexosamine,
alpha-galactosidase, beta-galactosidase, beta-glucuronidase or
glucocerebrosidase.
4. The method of claim 1 wherein the recombinant lentivirus
comprises a heterologous promoter operably linked to the nucleic
acid segment.
5. The method of claim 1 wherein vascular compartment is a vein,
artery, bone marrow cavity, heart, spleen, umbilical cord vessel or
placenta.
6. The method of claim 1 wherein the mammal is a human.
7. (canceled)
8. The method of claim 1 wherein the recombinant lentivirus is a
pseudotyped virus.
9. The method of claim 1 wherein the lentivirus is a human
immunodeficiency virus-1 (HIV-1).
10. The method of claim 1 wherein the disorder is a
mucopolysaccharide disorder.
11. The method of claim 10 wherein the disorder is a
mucopolysaccharidosis type I disorder.
12. The method of claim 10 wherein the disorder is a
mucopolysaccharidosis type VII disorder.
13. (canceled)
14. The recombinant virus of claim 18 wherein the enzyme is
alpha-L-iduronidase, beta-hexosamine, alpha-galactosidase,
beta-galactosidase, beta-glucuronidase or glucocerebrosidase.
15. The recombinant virus of claim 18 wherein the lentivirus is
HIV-1.
16. The recombinant virus of claim 18 wherein the U3 of the 5'LTR
is modified by substantially replacing the U3 of the 5'LTR with a
heterologous promoter.
17. The recombinant virus of claim 18 which further comprises a
promoter operably linked to the nucleic acid segment.
18. Recombinant virus comprising a lentivirus vector comprising a
5N LTR and a 3N LTR and a nucleic acid segment encoding a lysosomal
enzyme, wherein the U3 region of at least one LTR is optionally
modified.
19. The recombinant virus of claim 18 which is a pseudotyped
virus.
20. A method for providing a biologically active lysosomal enzyme
to a cell of a mammal, comprising: contacting the cell with an
effective amount of a recombinant lentivirus comprising a nucleic
acid segment encoding the enzyme.
21. The method of claim 1 wherein the mammal is a newborn.
22. The method of claim 1 wherein the lentivirus is intravenously
administered.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/057,410, filed Feb. 14, 2005, which is a continuation under
35 U.S.C. 111(a) of PCT US/2003/025508, filed Aug. 13, 2003 and
published on Jul. 1, 2004 as WO 2004/055157 A2, which claims the
benefit under 35 U.S.C. 119(e) of U.S. Provisional Application Ser.
No. 60/403,108, filed on Aug. 13, 2002 and U.S. Provisional
Application Ser. No. 60/403,586, filed Aug. 14, 2002, which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Many human genetic diseases are due to the deficiency of an
enzyme or other protein. For example, the genetically determined
deficiency of the lysosomal enzyme alpha-L-iduronidase results in
the progressive accumulation of glycosaminoglycan substrates. In
vitro evidence that cells grown in culture can take up exogenously
supplied enzymes from the surrounding tissue culture medium led to
the concept of in vivo enzyme replacement. For instance, enzyme
replacement by intravenous infusion has been demonstrated to be
successful for adenosine deaminase deficiency and for Gaucher
disease, and some measures of efficacy have been found in human
patients receiving weekly infusions of recombinant human
alpha-L-iduronidase. However, these infusions must occur over a
period of hours every week, and it is unclear if weekly
administration of the enzyme would be efficacious, even if
treatments are started early in life. Moreover, it is likely that
enzyme infusions will not prevent progressive mental retardation
associated with particular protein deficiencies.
[0004] Enzyme replacement may also be accomplished by
transplantation of genetically normal cells and tissues, e.g., via
bone marrow transplantation for mucopolysaccharidosis (also known
as Hurler syndrome). For example, bone marrow transplantation was
found to reduce many of the consequences of mucopolysaccharidosis
type I and may prevent progression of mental retardation (Whitley
et al., 1986). However, transplantation procedures which include
the use of immunosuppressive medications are associated with an
increase in morbidity and mortality.
[0005] Enzyme replacement may also be accomplished via gene
therapy, e.g., with viral vectors such as HIV-based vectors, ex
vivo or in vivo. Lentiviral vectors are one type of viral vector
which has been proposed as useful for mammalian gene therapy.
HIV-based lentiviral vectors pseudotyped with the envelope of
another virus (most often the G protein of the vesicular stomatitis
virus, VSVG) have become promising tools for gene delivery into
nondividing cells. These vectors have been shown to be capable of
transferring genes into a range of nonproliferative cell types,
including neurons, retinal cells, muscle cells, and hematopoietic
pluripotent cells (Amado et al., 1999; Lever, 2000; Podsakoff,
2001) and, using local administration of those vectors, in vivo
gene delivery has been accomplished in rat brain (Naldini et al.,
1996; Blomer et al., 1997), liver and muscle (Kafri et al., 1997),
retina (Miyoshi et al., 1999), and airway epithelia (Johnson et
al., 2000).
[0006] However, the biosafety concerns surrounding HIV vectors have
received considerable attention because of the pathogenic nature of
HIV. Thus, efforts have been made to increase the safety of
lentivirus vectors by minimizing the potential formation of
replication-competent virus (RCR) via homologous recombination
events. One strategy to reduce RCR formation has been to use
nonoverlapping split-genome packaging constructs that require
multiple recombination events with the transfer vector for RCR
generation (Naldini et al., 1996; Reiser et al., 1996). Other
strategies have focused on eliminating all unnecessary HIV reading
frames from the system (Kim et al., 1998; Dull et al., 1998) or
truncating the 3N long terminal repeat (3N LTR) to generate
self-inactivating HIV vectors (Miyoshi et al., 1998).
[0007] Lentiviral vectors have been a preferred vector for ex vivo
modification of hematopoietic (i.e., blood-forming) stem cells as
lentiviral vectors are likely capable of transducing such
nondividing types of cells. Nevertheless, despite thousands of
experiments attempting ex vivo gene transfer into hematopoietic
stem cells, this vector-cell combination has not been successful in
animal models of disease. Moreover, there has never been a
successful clinical response in an animal using in vivo lentiviral
gene therapy, probably owing to insufficient delivery of vector, or
lack of expression of therapeutic protein, to the disease-causing
tissues or cells.
[0008] Thus, what is needed is an improved method to prevent,
inhibit or treat metabolic disorders characterized by a lack of, or
a reduction in the amount of, an enzyme in a mammal.
SUMMARY OF THE INVENTION
[0009] The invention provides a lentivirus vector comprising a
nucleic acid segment encoding a gene product such as a protein, the
absence or reduced levels of which are associated with a disorder
in a mammal, a disorder including, but not limited to, a metabolic
disorder, e.g., a lysosomal storage disease, hemophilia, or
adrenoleukodystrophy. A lentivirus includes ovine, caprine, equine,
bovine and primate, e.g., HIV-1, HIV-2 and SIV, lentiviruses. Also
provided is a method in which a recombinant lentivirus comprising a
nucleic acid segment encoding a gene product, the absence or
reduced levels of which in a mammal are associated with a disorder,
is administered to a mammal having or at risk of having such a
disorder, in an amount effective to prevent, inhibit or treat at
least one symptom associated with the disorder, e.g., a
neurological symptom. In one embodiment, the recombinant virus is
administered into a vascular compartment, e.g., intravenously, of
the mammal. Preferred amounts of virus include, but are not limited
to, 1.times.10.sup.3 to 1.times.10.sup.15 TU, e.g.,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10,
1.times.10.sup.11, 1.times.10.sup.12, 1.times.10.sup.13,
1.times.10.sup.14 or 1.times.10.sup.15 TU, although other amounts
maybe efficacious. Preferably, the mammal is a neonate or juvenile,
although it is envisioned that adult mammals, and the developing
embryo or fetus in utero, may also be treated.
[0010] In one embodiment, the recombinant lentivirus encodes a
lysosomal enzyme and is administered in an amount which is
effective to prevent, inhibit or treat a lysosomal storage disease
in a mammal. Lysosomal storage diseases include, but are not
limited to, mucopolysaccharidosis diseases, for instance,
mucopolysaccharidosis type I, e.g., Hurler syndrome and the
variants Scheie syndrome and Hurler-Scheie syndrome (a deficiency
in alpha-L-iduronidase); Hunter syndrome (a deficiency of
iduronate-2-sulfatase); mucopolysaccharidosis type III, e.g.,
Sanfilippo syndrome (A, B, C or D; a deficiency of heparan sulfate
sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl
CoA:alpha-glucosaminide N-acetyl transferase or
N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis
type IV e.g., mucopolysaccharidosis type IV, e.g., Morquio syndrome
(a deficiency of galactosamine-6-sulfate sulfatase or
beta-galactosidase); mucopolysaccharidosis type VI, e.g.,
Maroteaux-Lamy syndrome (a deficiency of arylsulfatase B);
mucopolysaccharidosis type II; mucopolysaccharidosis type III (A,
B, C or D; a deficiency of heparan sulfate sulfatase,
N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide
N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase);
mucopolysaccharidosis type IV (A or B; a deficiency of
galactosamine-6-sulfatase and beta-galatacosidase);
mucopolysaccharidosis type VI (a deficiency of arylsulfatase B);
mucopolysaccharidosis type VII (a deficiency in
beta-glucuronidase); mucopolysaccharidosis type VIII (a, deficiency
of glucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IX
(a deficiency of hyaluronidase); Tay-Sachs disease (a deficiency in
alpha subunit of beta-hexosaminidase); Sandhoff disease (a
deficiency in both alpha and beta subunit of beta-hexosaminidase);
GM1 gangliosidosis (type I or type II); Fabry disease (a deficiency
in alpha galactosidase); metachromatic leukodystrophy (a deficiency
of aryl sulfatase A); Pompe disease (a deficiency of acid maltase);
fucosidosis (a deficiency of fucosidase); alpha-mannosidosis (a
deficiency of alpha-mannosidase); beta-mannosidosis (a deficiency
of beta-mannosidase), ceroid lipofuscinosis, and Gaucher disease
(types I, II and III; a deficiency in glucocerebrosidase), as well
as disorders such as Hermansky-Pudlak syndrome; Amaurotic idiocy;
Tangier disease; aspartylglucosaminuria; congenital disorder of
glycosylation, type Ia; Chediak-Higashi syndrome; macular
dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickel
syndrome; Farber lipogranulomatosis; fibromatosis; geleophysic
dysplasia; glycogen storage disease I; glycogen storage disease Ib;
glycogen storage disease Ic; glycogen storage disease III; glycogen
storage disease IV; glycogen storage disease V; glycogen storage
disease VI; glycogen storage disease VII; glycogen storage disease
0; immunoosseous dysplasia, Schimke type; lipidosis; lipase b;
mucolipidosis II; mucolipidosis II, including the variant form;
mucolipidosis IV; neuraminidase deficiency with beta-galactosidase
deficiency; mucolipidosis I; Niemann-Pick disease (a deficiency of
sphingomyelinase); Niemann-Pick disease without sphingomyelinase
deficiency (a deficiency of a npc1 gene encoding a cholesterol
metabolizing enzyme); Refsum disease; Sea-blue histiocyte disease;
infantile sialic acid storage disorder; sialuria; multiple
sulfatase deficiency; triglyceride storage disease with impaired
long-chain fatty acid oxidation; Winchester disease; Wolman disease
(a deficiency of cholesterol ester hydrolase); Deoxyribonuclease
I-like 1 disorder; arylsulfatase E disorder; ATPase, H+
transporting, lysosomal, subunit 1 disorder; glycogen storage
disease IIb; Ras-associated protein rab9 disorder; chondrodysplasia
punctata 1, X-linked recessive disorder; glycogen storage disease
VIII; lysosome-associated membrane protein 2 disorder; Menkes
syndrome; congenital disorder of glycosylation, type Ic; and
sialuria. In particular, the invention is useful to prevent,
inhibit or treat lysosomal storage diseases wherein the lysosomal
enzyme is trafficked to the lysosome (within the cell and between
cells) by specific glycosylation. For most lysosomal enzymes and
their corresponding diseases, this would be by means of a terminal
mannose-6-phosphate, however, it also includes terminal mannose
glycosylation, e.g., in the case of beta-glucocerebrosidase
deficiency responsible for Gaucher disease. Thus, in one
embodiment, the lentivirus vector of the invention is useful to
prevent, inhibit or treat lysosomal storage diseases including but
are not limited to, mucopolysaccharidosis diseases, for instance,
mucopolysaccharidosis type I, e.g., Hurler syndrome and the
variants Scheie syndrome and Hurler-Scheie syndrome (a deficiency
in alpha-L-iduronidase); mucopolysaccharidosis type II, e.g.,
Hunter syndrome (a deficiency of iduronate-2-sulfatase);
mucopolysaccharidosis type III, e.g., Sanfilippo syndrome (A, B, C
or D; a deficiency of heparan sulfate sulfatase,
N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminide
N-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase);
mucopolysaccharidosis type IV, e.g., Morquio syndrome (a deficiency
of galactosamine-6-sulfate sulfatase or beta-galactosidase);
mucopolysaccharidosis type VI, e.g., Maroteaux-Lamy syndrome
(deficiency of arylsulfatase B); mucopolysaccharidosis type VII,
e.g., Sly syndrome (a deficiency in beta-glucuronidase); Tay-Sachs
disease (a deficiency in alpha subunit of beta-hexosaminidase);
Sandhoff disease (a deficiency in both alpha and beta subunit of
beta-hexosaminidase); GM1 gangliosidosis; Fabry disease (a
deficiency in alpha-galactosidase); metachromatic leukodystrophy (a
deficiency of aryl sulfatase A); Pompe disease (a deficiency of
acid maltase); fucosidosis (a deficiency of fucosidase);
alpha-mannosidosis (a deficiency of alpha-mannosidase);
beta-mannosidosis (a deficiency of beta-mannosidase), ceroid
lipofuscinosis, and Gaucher disease (types I, II and III; a
deficiency in glucocerebrosidase). As described herein, a single
administration of a lentivirus encoding alpha-L-iduronidase to
newborn Hurler syndrome mice resulted in normal patterns of
behavior for the treated mice relative to untreated mice.
Administration of the lentivirus to newborns likely resulted in an
increased efficiency of transduction which may in turn be due to
the presence of cells that are more susceptible to infection in the
newborn. Early therapy, e.g., prenatal or in newborns, for
metabolic disorders such as lysosomal storage diseases may thus be
particularly efficacious.
[0011] In one embodiment, a recombinant lentivirus encoding a
lysosomal enzyme is administered to a mammal in an amount which is
effective to increase the level and/or activity of one or more
lysosomal storage proteins, e.g., enzymes, and/or decrease skeletal
deformity including kyphoscoliosis, scoliosis, deformity or
arthritis of the hip joints, contractures of the digits or larger
joints at the knees, ankles, elbows and shoulders, disfigurement of
the face, recurrent and chronic ear infections, enlargement,
dysfunction of an organ such as the liver, spleen or heart,
obstruction of the coronary arteries causing myocardial infarction,
respiratory abnormality such as obstructive airway disease,
reactive airway disease or pneumonia, brain or other nervous system
damage, and/or dysfunction such as hydrocephalus, cranial nerve
compression, hearing loss, blindness, spinal cord compression.
[0012] In one embodiment, a recombinant lentivirus encoding a
lysosomal enzyme is administered to a mammal in an amount which is
effective to increase longevity, preserve intellect, e.g., measured
by intelligence quotient (IQ), reduce ear infections, reduce
skeletal deformity with improved ambulation, e.g., measured in a
6-minute walk test or other measurements of endurance, reduce organ
size (e.g., liver, spleen), improve respiratory function, e.g.,
measured by improved by spirometry, normalize of organ cellular
architecture, e.g., observed by decreased pathology (reduced
lyosomal vacuolization or other microscopic pathology), decrease
occlusion of the coronary arteries, reduce aberrant thickening of
the meninges of the central nervous system, prevent or reduce
hydrocephalus of the brain, decrease levels of pathologic
substrates such as decreased glycosaminoglycan in the liver and
other tissues, urine, or cerebrospinal fluid, and/or increase
levels of a deficient enzyme such as alpha-L-iduronidase in liver
tissue, white blood cells or plasma.
[0013] In one embodiment, a recombinant lentivirus encoding a
lysosomal enzyme is administered intravenously to a mammal, e.g., a
fetus (prenatal delivery), an infant (e.g., a human from birth to 2
years of age), a child (e.g., a human from over 2 years to 12 years
or age), a juvenile (e.g., a human from over 12 years to 18 years
of age), or an adult (e.g., a human older than 18 years of
age).
[0014] In another embodiment, the invention provides a lentivirus
vector comprising a nucleic acid sequence encoding a clotting
factor, e.g., Factor VIII or Factor IX, and a method to prevent,
inhibit or treat a mammal having or at risk of having the clotting
disorder which employs a recombinant lentivirus comprising the
vector. Preferably, the recombinant lentivirus is administered to a
vascular compartment of the mammal.
[0015] Further provided is a lentivirus vector comprising a nucleic
acid sequence encoding an ABC protein such as a peroxisomal
transport protein, e.g., the X-ALD protein (ALDP), the
adrenoleukodystrophy-related protein (ALDRP), the 70 kDa
peroxisomal membrane protein (PMP70), or a PMP70-related protein,
and a method to prevent, inhibit or treat a mammal having or at
risk of an adrenoleukodystrophy which employs a recombinant
lentivirus comprising the vector. Preferably, the lentivirus is
administered to a vascular compartment of the mammal.
[0016] The invention also provides a recombinant lentivirus of the
invention, a host cell transfected with a lentivirus vector of the
invention, e.g., eukaryotic host cells including mammalian host
cells such as human, non-human primate, canine, caprine, feline,
bovine, equine, swine, ovine, rabbit or rodent cells, a host cell
infected, e.g., ex vivo, with a recombinant lentivirus of the
invention, and a method of expressing a biologically active protein
in a cell which employs a lentivirus vector or lentivirus of the
invention which encodes the biologically active protein. A
"biologically active" protein is a protein which has substantially
the same activity, e.g., at least 80%, more preferably at least
90%, the activity of a corresponding wild-type (functional)
protein.
[0017] Also provided is a kit comprising a recombinant lentivirus
of the invention, e.g., a lyophilized or frozen preparation of
recombinant lentivirus.
[0018] Mucopolysaccharidosis (MPS) type VII is an autosomal
recessive lysosomal storage disease resulting from deficiency of
beta-glucuronidase due to mutations of the corresponding gene for
beta-glucuronidase, GUSB. As described herein, a plasmid was
constructed to express the human GUSB cDNA under the
transcriptional regulation of a hybrid promoter-enhancer (CAGGS)
containing the chicken beta-actin enhancer and CMV early promoter.
Sleeping Beauty transposon IR sequences were included to examine
the potential for integration into the cell chromosome. This
transposed plasmid pT-CAGGS-GUSB was administered by hydrodynamic
injection (i.e., intravenous infusion in a volume equal to 10% of
body weight, over about 8-10 seconds) into the tail vein of mice
ranging from 4 to 23 weeks of age. The pT-CAGGS-GUSB plasmid was
administered (25 mcg/animal) alone (Group 1), or with transposase
plasmid pSBI0, at a transposon:transposase molar ratio of 1:1
(Group 2), or 10:1 (Group 3). Forty-eight hours after injection,
plasma beta-glucuronidase enzymatic activity in treated MPS mice
was markedly elevated (1,552-7,711 nmol/ml/hr, n=14) in comparison
to that of wild-type, untreated or sham-treated mice (9-15
nmol/ml/hr, n=6). In liver, beta-glucuronidase activity in treated
MPS mice was also markedly elevated (1,860-6,185 nmol/mg/hr, n=4)
compared to normal levels (86-188 nmol/mg/hr). Notably, the liver
tissue of MPS mice receiving pTCAGGS-GUSB stained uniformly
positive for beta-glucuronidase activity, including both Kupffer
cells and hepatocytes. One week after injection, plasma
beta-glucuronidase activity was reduced relative to day 2 levels:
Group 1, 59-93% of the 2-day levels; Group 2, 21-36%; and Group 3,
33-63% (n=4 in each group). Beta-glucuronidase levels in the liver
and spleen were 184-185 nmol/mg/hr and 4,534-6,080, respectively,
while levels in other organs were lower (heart 94-98, lung 49-65,
kidney 59, and undetectable in the brain). Two months after
injection, beta-glucuronidase activity remained at therapeutic
levels in animals receiving pT-CAGGS-GUSB plasmid alone.
Histochemical studies showed staining for beta-glucuronidase
activity throughout the liver and spleen. Remarkably, mice
co-injected with pSBIO had much lower levels of beta-glucuronidase
activity. Morphometric analysis of inclusion morphology
demonstrated that clearing of hepatic lysosomal pathology was
related to the level of beta-glucuronidase, and that mice receiving
pT-CAGGS-GUSB plasmid alone were completely clear of pathology.
[0019] Thus, hydrodynamic infusion of the pT-CAGGS-GUSB transposon
delivered DNA to liver with marked increase in enzyme activity,
with the highest levels in blood ever achieved. GUSB enzymatic
activity was found throughout the liver transiently reaching levels
10- to 1,000-fold of normal levels, levels which are above those
that would be curative in newborns.
[0020] The results described herein with the lentivirus and plasmid
vectors of the invention were surprising as the intravenous
administration of other vectors did not show the extent of
correction observed with the lentivirus and plasmid vectors.
Moreover, PCR analysis of gonads, e.g., testes, showed virtually no
evidence of viral vector sequences, indicating a decreased risk for
germ line transmission. Further, viral vector sequences were
surprisingly detected in bone marrow stem cells after intravenous
administration of a lentivirus vector of the invention to a mammal
and so those vectors are particularly useful for systemic
expression of therapeutic genes.
[0021] The invention provides a method to prevent, inhibit or treat
a metabolic disorder in a mammal via the hydrodynamic infusion of a
plasmid encoding a gene product, the expression of which in the
mammal prevents, inhibits, or treats one or more symptoms of the
disorder. In one embodiment, a fetus or neonate is infused via the
umbilical cord with a vector of the invention.
[0022] The invention provides a method to prevent, inhibit or treat
a metabolic disorder such as one characterized by the absence or
reduced levels of a lysosomal protein in a mammal. The method
comprises administering to a mammal, e.g., to a vascular
compartment of a mammal having or at risk of the disorder an
effective amount of an isolated nucleic acid molecule comprising a
nucleic acid sequence encoding the protein, e.g., a biologically
active protein. In one embodiment, the nucleic acid molecule
comprises a promoter operably linked to the nucleic acid
sequence.
[0023] The invention also provides isolated nucleic acid-based
vectors to inhibit or treat metabolic disorders, e.g., lysosomal
storage disease such as mucopolysaccharidosis type I diseases,
e.g., Hurler syndrome, mucopolysaccharidosis type II diseases,
e.g., Hunter syndrome, mucopolysaccharidosis type II diseases,
e.g., Sanfilippo syndrome, mucopolysaccharidoses type VII diseases,
e.g., Sly disease, Fabry disease, Gaucher disease as well as
hemophilia, e.g., Factor VIII or factor IX deficiency. Further
provided is a method to prevent, inhibit or treat a metabolic
disorder in a mammal which employs an isolated nucleic acid vector
of the invention, e.g., in an amount effective to prevent, inhibit
or treat at least one symptom associated with the disorder, e.g., a
neurological symptom associated with the disorder. In one
embodiment, the mammal is an adult. In one embodiment, the isolated
nucleic acid vector of the invention is administered two or more
times to the mammal.
[0024] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising a nucleic acid sequence encoding a
clotting factor, e.g., Factor VIII or Factor IX, and a method to
prevent, inhibit or treat a mammal having or at risk of having the
clotting disorder which employs a vector comprising the nucleic
acid molecule. Preferably, the vector is administered to a vascular
compartment of the mammal.
[0025] Further provided is an isolated nucleic acid molecule
comprising a nucleic acid sequence encoding an ABC protein such as
a peroxisomal transport protein, e.g., the X-ALD protein (ALDP),
the adrenoleukodystrophy-related protein (ALDRP), the 70 kDa
peroxisomal membrane protein (PMP70), or a PMP70-related protein,
and a method to prevent, inhibit or treat a mammal having or at
risk of an adrenoleukodystrophy which employs a vector comprising
the nucleic acid molecule. Preferably, the lentivirus is
administered to a vascular compartment of the mammal.
[0026] The invention also provides an isolated nucleic acid
molecule of the invention, a host cell transfected with the
isolated nucleic acid molecule of the invention, e.g., eukaryotic
host cells including mammalian host cells such as human, non-human
primate, canine, caprine, feline, bovine, equine, swine, ovine,
rabbit or rodent cells, a host cell transfected, e.g., ex vivo,
with an isolated nucleic acid molecule of the invention, and a
method of expressing a biologically active protein in a cell which
employs a vector comprising a nucleic acid molecule of the
invention which encodes the biologically active protein.
BRIEF DESCRIPTION OF THE FIGURES
[0027] The patent or application file contains at least one drawing
executed in color. Copes of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0028] FIG. 1 shows the genetic map of a representative lentiviral
vector for intravascular administration. LTR is the "long terminal
repeat" of HIV-1 virus; .psi..sup.+ is the packing signal; CMV
refers to a promoter from cytomegalovirus that regulates gene
expression; PGK refers to a phosphoglycerate kinase promoter; the
inverted triangle indicates a modification which results in a "self
inactivating" vector; and IDUA refers to a cDNA sequence encoding
the therapeutic protein alpha-L-iduronidase.
[0029] FIG. 2 shows the increase in blood levels of therapeutic
IDUA in MPS I mice treated by intravenous administration of an IDUA
encoding recombinant lentivirus contrasting the increase in those
treated by intravenous administration of a control recombinant
lentivirus. The range of activity of IDUA in heterozygous IDUA
transgenic mice is shown as a hatched band marked HET/HIGH
HET/LOW.
[0030] FIG. 3 shows the therapeutic effect of intravenous
administration of an IDUA encoding recombinant lentivirus on the
facial pathology of a mouse with MPS I.
[0031] FIG. 4 shows preservation of normal behavior in MPS I mice
due to intravenous administration of an IDUA encoding recombinant
lentivirus.
[0032] FIG. 5 illustrates the pathology observed in untreated
Hurler syndrome mice (A), normal mice (B), and IDUA
lentivirus-treated Hurler syndrome mice (C). The sections were
stained with a horseradish peroxidase conjugated anti-alpha-GM2
ganglioside antibody.
[0033] FIG. 6 shows a schematic of pT-CAGGS-GUSB.
[0034] FIG. 7 shows histochemical visualization of
beta-glucuronidase catalytic activity in liver 48 hours after
hydrodynamic infusion of pT-CAGGS-GUSB plasmid. Transfected cells
stain intensively red. A) Untreated MPS VII mouse. B) Wild-type
mouse showing normal levels of beta-glucuronidase activity. C) MPS
VII mouse treated with pT-CAGGS-GUSB only. Dark red punctuate spots
are likely transfected cells expressing extremely high levels of
beta-glucuronidase enzyme while more diffusely red cells have taken
up enzyme by mannose-6-phosphate receptor-mediated endocytosis.
(Representative views of 6 micron sections at 10.times.
magnification.)
[0035] FIG. 8 shows data from two months after intravenous infusion
of pT-CAGSS-GUSB plasmid. Liver sections are stained for
beta-glucuronidase activity (left) and with toluidine blue to
visualize pathologic lysosomal vacuolization (right). A) and B) are
MPS VII mouse untreated. C) and D) are wild-type mouse, untreated.
E) and F) are MPS VII mouse treated with pT-CAGGS-GUSB alone. G)
and H) are MPS VII mouse treated by co-injection of pT-CAGGS-GUSB
and pSB plasmids in equal amounts, a 1:1 ratio. I) and J) represent
an MPS VII mouse treated by co-injection of pT-CAGGS-GUSB and pSB
plasmids in a ratio of 10:1.
[0036] FIG. 9 provides data from mice studied at 2 months after
treatment. There is a dose-related correspondence of hepatic
lysosomal pathology (area of vacuolization) to the level of hepatic
beta-glucuronidase enzyme activity. Mice receiving pT-CAGGS-GUSB
demonstrated high levels of enzyme activity and complete clearance
of lysosomal pathology, while those co-injected with pT-CAGGS-GUSB
and pSB showed intermediate levels of response.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0037] "Inducible expression system" includes a construct or
combination of constructs that includes a nucleotide sequence
encoding a transactivator, an inducible promoter that can be
transcriptionally activated by the transactivator, and a nucleotide
sequence of interest operably linked to the inducible promoter. For
example, an exemplary inducible expression system of the invention
includes a nucleotide sequence encoding a tetracycline operon
regulatable transactivator (tTA) and a nucleotide sequence of
interest operably linked to an inducible promoter composed of a
minimal promoter operably linked to at least one tetO sequence.
[0038] "Transactivator," "transactivating factor," or
"transcriptional activator" includes a polypeptide that facilitates
transcription from a promoter. Where the promoter is an inducible
promoter, the transactivator activates transcription in response to
a specific transcriptional signal or set of transcriptional
signals. For example, in the inducible expression system of the
invention, tTA is a transactivator that facilitates transcription
from the inducible tetO promoter when tTA is not bound to
tetracycline.
[0039] "Tetracycline repressor protein," "tetracycline repressor
polypeptide," "tetR polypeptide," and "tetR protein" are used
interchangeably herein to include a polypeptide that exhibits both
1) specific binding to tetracycline and/or tetracycline
derivatives; and 2) specific binding to tetO sequences when the
tetR polypeptide is not bound by tetracycline or a tetracycline
analog(s). "TetR polypeptide" is meant to include a naturally
occurring (i.e., native) tetR polypeptide sequence and functional
derivatives thereof.
[0040] "Transcriptional activation domain" includes a polypeptide
sequence that facilitates transcriptional activation from a
promoter. "Transcriptional activation domain" includes
transcriptional activation domains derived from the naturally
occurring amino acid sequence of a transcription factor as well as
functional derivatives thereof.
[0041] "Envelope protein" includes a polypeptide that 1) can be
incorporated into an envelope of a retrovirus; and 2) can bind
target cells and facilitate infection of the target cell by the RNA
virus that it envelops. "Envelope protein" is meant to include
naturally occurring (i.e., native) envelope proteins and functional
derivatives thereof that 1) can form pseudotyped retroviral virions
according to the invention, and 2) exhibit a desired functional
characteristic(s) (e.g., facilitate viral infection of a desired
target cell, and/or exhibit a different or additional biological
activity). In general, envelope proteins of interest in the
invention include any viral envelope protein that can, in
combination with a retroviral genome, retroviral Pol, retroviral
Gag, and other essential retroviral components, form a retroviral
particle. Such envelope proteins include retroviral envelope
proteins derived from any suitable retrovirus (e.g., an
amphotropic, xenotropic, ecotropic or polytropic retrovirus) as
well as non-retroviral envelope proteins that can form pseudotype
retroviral virions (e.g., VSV G). Envelope proteins of particular
interest include, but are not limited to, envelope protein of
vesicular stomatitis virus (VSV G), HTLV-1, gibbon ape leukemia
virus (GALV), Sindai virus, influenza virus, herpes virus,
rhabdovirus, and rabies virus.
[0042] "Functional derivative of a polypeptide" includes an amino
acid sequence derived from a naturally occurring polypeptide that
is altered relative to the naturally occurring polypeptide by
virtue of addition, deletion, substitution, or other modification
of the amino acid sequence. "Functional derivatives" contemplated
herein exhibit the characteristics of the naturally occurring
polypeptide essential to the operation of the invention. For
example, by "functional derivative of tetR" is meant a polypeptide
derived from tetR that retains both 1) tetracycline or tetracycline
analog binding and 2) the ability to inhibit transcriptional
activation by tTA when bound to tetracycline or an analog
thereof.
[0043] "Promoter" includes a minimal DNA sequence sufficient to
direct transcription of a DNA sequence to which it is operably
linked. The term "promoter" is also meant to encompass those
promoter elements sufficient for promoter-dependent gene expression
controllable for cell-type specific expression, tissue-specific
expression, or inducible by external signals or agents; such
elements may be located in the 5N or 3N regions of the naturally
occurring gene.
[0044] "Inducible promoter" includes a promoter that is
transcriptionally active when bound to a transcriptional activator,
which in turn is activated under a specific condition(s), e.g., in
the presence of a particular chemical signal or combination of
chemical signals that affect binding of the transcriptional
activator to the inducible promoter and/r affect function of the
transcriptional activator itself. For example, the transcriptional
activator of the present invention, tTA, induces transcription from
its corresponding inducible promoter when tetracycline is absent,
i.e., tetracycline is not bound to tTA.
[0045] "Construct" includes a recombinant nucleotide sequence,
generally a recombinant DNA molecule, that has been generated for
the purpose of the expression of a specific nucleotide sequence(s),
or is to be used in the construction of other recombinant
nucleotide sequences. In general, "construct" is used herein to
refer to a recombinant DNA molecule.
[0046] "Operably linked" includes a DNA sequence and a regulatory
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the regulatory sequence(s).
[0047] "Isolated" when used in relation to a nucleic acid, as in
"isolated oligonucleotide" or "isolated polynucleotide" includes a
nucleic acid sequence that is identified and separated from at
least one contaminant with which it is ordinarily associated in its
source. Thus, an isolated nucleic acid is present in a form or
setting that is different from that in which it is found in nature.
In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are
found in the state they exist in nature. For example, a given DNA
sequence (e.g., a gene) is found on the host cell chromosome in
proximity to neighboring genes; RNA sequences (e.g., a specific
mRNA sequence encoding a specific protein), are found in the cell
as a mixture with numerous other mRNAs that encode a multitude of
proteins. However, isolated nucleic acid includes, by way of
example, such nucleic acid in cells ordinarily expressing that
nucleic acid where the nucleic acid is in a chromosomal location
different from that of natural cells, or is otherwise flanked by a
different nucleic acid sequence than that found in nature. The
isolated nucleic acid or oligonucleotide may be present in
single-stranded or double-stranded form. When an isolated nucleic
acid or oligonucleotide is to be utilized to express a protein, the
oligonucleotide contains at a minimum, the sense or coding strand
(i.e., the oligonucleotide may single-stranded), but may contain
both the sense and anti-sense strands (i.e., the oligonucleotide
may be double-stranded). In one embodiment, the isolated nucleic
acid is one which is free of viral proteins, i.e., it is not a
viral particle, and/or does not encode one or more viral
proteins.
[0048] "Operatively inserted" refers to a nucleotide sequence of
interest is positioned adjacent a nucleotide sequence that directs
transcription and translation of the introduced nucleotide sequence
of interest (i.e., facilitates the production of e.g., a
polypeptide encoded by a DNA of interest).
[0049] By "packaging cell line" is meant a line of packaging cells
selected for their ability to package defective retroviral vectors
at a titer of generally greater than 10.sup.3 virions per
milliliter of tissue culture medium, having less than 10 helper
virus virions per milliliter of tissue culture medium, and capable
of being passaged in tissue culture without losing their ability to
package defective retroviral vectors.
[0050] "Transformation" includes a permanent or transient genetic
change, preferably a permanent genetic change, induced in a cell
following incorporation of new DNA (i.e., DNA exogenous to the
cell). Where the cell is a mammalian cell, a permanent genetic
change is generally achieved by introduction of the DNA into the
genome of the cell.
[0051] "Target cell" is a cell(s) that is to be transformed using
the methods and compositions of the invention. Transformation may
be designed to non-selectively or selectively transform the target
cell(s). In general, target cell as used herein means a eukaryotic
cell that can be infected by a VSV G pseudotyped retroviral vector
according to the invention.
[0052] "Transformed cell" is a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding a gene product (e.g., RNA
and/or protein) of interest (i.e., nucleic acid encoding a
therapeutic cellular product).
[0053] "Nucleotide sequence of interest", "gene of interest" or
"DNA of interest" includes any nucleotide or DNA sequence that
encodes a protein or other molecule that is desirable for
expression in a host cell (e.g., for production of the protein or
other biological molecule (e.g., a therapeutic cellular product) in
the target cell). The nucleotide sequence of interest is generally
operatively linked to other sequences which are needed for its
expression, .e.g., a promoter. In general, a nucleotide sequence of
interest present in the genome of a recombinant retroviral particle
of the invention encodes any gene product of interest, usually a
therapeutic gene product where the recombinant retroviral particle
is to be used to transform cells in vivo (e.g., in a gene therapy
application in humans).
[0054] A "therapeutic gene product" includes a polypeptide, RNA
molecule or other gene product that, when expressed in a target
cell, provides a desired therapeutic effect, e.g., repair of a
genetic defect in the target cell genome (e.g., by
complementation), expression of a polypeptide having a desired
biological activity, and/or expression of an RNA molecule for
antisense therapy (e.g., regulation of expression of a endogenous
or heterologous gene in the target cell genome).
[0055] By "subject" or "patient" is meant any subject for which
cell transformation or gene therapy is desired, including humans,
cattle, dogs, cats, guinea pigs, rabbits, mice, insects, horses,
chickens, and any other genus or species having cells that can be
infected with a viral vector having an envelope containing VSV G or
other envelope described herein.
[0056] A "transgenic organism" includes a non-human organism (e.g.,
single-cell organisms (e.g., yeast), mammal, non-mammal (e.g.,
nematode or Drosophila)) having a non-endogenous (i.e.,
heterologous) nucleic acid sequence present as an extrachromosomal
element in a portion f its cells or stably integrated into its germ
line DNA.
[0057] A "transgenic animal" includes a non-human animal, usually a
mammal, having a non-endogenous (i.e., heterologous) nucleic acid
sequence present as an extrachromosomal element in a portion of its
cells or stably integrated into its germ line DNA (i.e., in the
genomic sequence of most or all of its heterologous nucleic acid is
introduced into the germ line of such transgenic animals by genetic
manipulation of, for example, embryos or embryonic stem cells of
the host animal.
[0058] A "viral vector" includes a recombinant viral particle that
accomplishes transformation of a target cell with a nucleotide
sequence of interest.
[0059] A "virion," "viral particle," or "retroviral particle"
includes a single virus minimally composed of an RNA genome, a
viral polymerase, e.g., a Pol protein (for reverse transcription of
the RNA genome following infection), a viral glycoprotein Gag
protein (structural protein present in the nucleocapsid), and an
envelope protein. As used herein, the RNA genome of a retroviral or
lentiviral particle is usually a recombinant RNA genome, e.g.,
contains an RNA sequence exogenous to the native retroviral genome
and/or is defective in an endogenous retroviral lentiviral sequence
(e.g., is defective in pol, gag, and/or env, and, as used herein,
is normally defective in all three genes).
[0060] "Pseudotyped viral particle," or "pseudotyped retroviral
particle" includes a viral particle having an envelope protein that
is from a virus other than the virus from which the RNA genome is
derived. For instance, the envelope protein can be from a
retrovirus of a species different from a retrovirus from which the
RNA genome is derived or from a non-retroviral virus (e.g.,
vesicular stomatitis virus (VSV)). Preferably, the envelope protein
of the pseudotyped retroviral particle is VSV G.
[0061] By "VSV G" or "VSV G envelope protein" is meant the envelope
protein of vesicular stomatitis virus (VSV) or a polypeptide
derived therefrom or recombinant fusion polypeptide having a VSV G
polypeptide sequence fused to a heterologous polypeptide sequence,
where the VSV G-derived polypeptide of recombinant fusion
polypeptide can be contained in a viral envelope of a pseudotyped
retroviral particle and retains infectivity for a desired target
cell (e.g., a range of desired eukaryotic cells, or a specific
target cell of interest).
[0062] By "VSV G pseudotyped virus," "VSV G pseudotyped
retrovirus," "VSV G pseudotyped viral particle," or "VSV G
pseudotyped retroviral particle," is meant a retrovirus having the
envelope protein VSV G, e.g., either in combination with or
substantially substituted for the endogenous retroviral envelope.
Preferably, VSV G is present in the VSV G pseudotyped viral
envelope such that VSV G represents about 50% of the envelope
protein(s) present in the envelope, more preferably about 75%, even
more preferably about 90% to about 95%, still more preferably
greater than about 95%, most preferably about 100% or such that VSV
G is substantially the only envelope protein present in the
pseudotyped viral particle envelope.
Vectors for Recombinant Lentivirus Production
[0063] The lentiviral genome and the proviral DNA have the three
genes found in retroviruses: gag, pol and env, which are flanked by
two long terminal repeat (LTR) sequences. The gag gene encodes the
internal structural (matrix, capsid and nucleocapsid) proteins; the
pol gene encodes the RNA-directed DNA polymerase (reverse
transcriptase), a protease and an integrase; and the env gene
encodes viral envelope glycoproteins. The 5' and 3' LTR's serve to
promote transcription and polyadenylation of the virion RNA's. The
LTR contains all other cis-acting sequences necessary for viral
replication. Lentiviruses have additional genes including vif, vpr,
tat, rev, vpu, nef and vpx (in HIV-1, HIV-2 and/or SIV).
[0064] Adjacent to the 5' LTR are sequences necessary for reverse
transcription of the genome (the tRNA primer binding site) and for
efficient encapsidation of viral RNA into particles (the Psi site).
If the sequences necessary for encapsidation (or packaging of
retroviral RNA into infectious virions) are missing from the viral
genome, the cis defect prevents encapsidation of genomic RNA.
However, the resulting mutant remains capable of directing the
synthesis of all virion proteins.
[0065] The invention provides a method of producing a recombinant
lentivirus capable of infecting a cell, e.g., non-dividing cell,
comprising transfecting a suitable host cell with two or more
vectors carrying the packaging functions, namely gag, pol and env,
as well as rev and tat. As will be disclosed hereinbelow, vectors
lacking a functional tat gene are desirable for certain
applications. Thus, for example, a first vector can provide a
nucleic acid encoding a viral gag and a viral pol and another
vector can provide a nucleic acid encoding a viral env to produce a
packaging cell. Introducing a vector providing a heterologous gene,
herein identified as a transfer vector, into that packaging cell
yields a producer cell which releases infectious viral particles
carrying the heterologous gene of interest.
[0066] Generally the vectors are plasmid-based or virus-based, and
are configured to carry the essential sequences for incorporating
foreign nucleic acid, for selection and for transfer of the nucleic
acid into a host cell. The gag, pol and env genes of the vectors of
interest also are known in the art. Thus, the relevant genes are
cloned into the selected vector and then used to transform the
target cell of interest.
[0067] According to the above-indicated configuration of vectors
and heterologous genes, the second vector can provide a nucleic
acid encoding a viral envelope (env) gene. The env gene can be
derived from any virus, including retroviruses, e.g., lentiviruses,
and heterologous viruses such as VSV. The env preferably is an
envelope protein which allows transduction of cells of human and
other species.
[0068] It may be desirable to target the recombinant virus by
linkage of the envelope protein with an antibody or a particular
ligand for targeting to a receptor of a particular cell-type. By
inserting a sequence (including a regulatory region) of interest
into the viral vector, along with another gene which encodes the
ligand for a receptor on a specific target cell, for example, the
vector is now target-specific. Retroviral vectors can be made
target-specific by inserting, for example, a glycolipid or a
protein. Targeting often is accomplished by using an
antigen-binding portion of an antibody or a recombinant
antibody-type molecule, such as a single chain antibody, to target
the retroviral vector. Those of skill in the art will know of, or
can readily ascertain without undue experimentation, specific
methods to achieve delivery of a retroviral vector to a specific
target.
[0069] Examples of retroviral-derived env genes include, but are
not limited to: Moloney murine leukemia virus (MoMuLV or MMLV),
Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor
virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV),
human immunodeficiency virus (HIV), Rous sarcoma virus (RSV), and
env genes of amphotropic viruses. Other env genes such as Vesicular
stomatitis virus (VSV) protein G (VSV G), that of hepatitis viruses
and of influenza also can be used.
[0070] The vector providing the viral env nucleic acid sequence is
associated operably with regulatory sequences, e.g., a promoter or
enhancer. The regulatory sequence can be any eukaryotic promoter or
enhancer, including for example, the Moloney murine leukemia virus
promoter-enhancer element, the human cytomegalovirus (CMV) enhancer
or the vaccinia P7.5 promoter. In some cases, such as the Moloney
murine leukemia virus promoter-enhancer element, the
promoter-enhancer elements are located within or adjacent to the
LTR sequences.
[0071] Preferably, the regulatory sequence is one which is not
endogenous, i.e., it is heterologous, to the lentivirus from which
the vector is being constructed. Thus, if the vector is being made
from SIV, the SIV regulatory sequence found in the SIV LTR would be
replaced by a regulatory element which does not originate from
SIV.
[0072] While VSV G protein is a desirable env gene because VSV G
confers broad host range on the recombinant virus, VSV G can be
deleterious to the host cell. Thus, when a gene such as that for
VSV G is used, it is preferred to employ an inducible promoter
system so that VSV G expression can be regulated to minimize host
toxicity when VSV G is expression is not required. For example, the
tetracycline-regulatable gene expression system of Gossen et al.
(1992) can be employed to provide for inducible expression of VSV G
when tetracycline is withdrawn from the transfected cell. Thus, the
tet/VP16 transactivator is present on a first vector and the VSV G
coding sequence is cloned downstream from a promoter controlled by
tet operator sequences on another vector.
[0073] The heterologous nucleic acid sequence of interest, the
transgene, is linked operably to a regulatory nucleic acid
sequence. As used herein, the term "heterologous" nucleic acid
sequence refers to a sequence that originates from a foreign
species, or, if from the same species, it may be substantially
modified from the original form. Alternatively, an unchanged
nucleic acid sequence that is not expressed normally in a cell is a
heterologous nucleic acid sequence.
[0074] The term "operably linked" refers to functional linkage
between a regulatory sequence and a heterologous nucleic acid
sequence resulting in expression of the latter. Preferably, the
heterologous sequence is linked to a promoter, resulting in a
chimeric gene. The heterologous nucleic acid sequence is preferably
under control of either the viral LTR promoter-enhancer signals or
of an internal promoter, and retained signals within the retroviral
LTR can still bring about efficient expression of the
transgene.
[0075] The heterologous gene of interest can be any nucleic acid of
interest which can be transcribed. Generally the foreign gene
encodes a polypeptide. Preferably the polypeptide has some
therapeutic benefit. The polypeptide may supplement deficient or
nonexistent expression of an endogenous protein in a host cell. The
polypeptide can confer new properties on the host cell, such as a
chimeric signalling receptor, see U.S. Pat. No. 5,359,046. The
artisan can determine the appropriateness of a heterologous gene
practicing techniques taught herein and known in the art. For
example, the artisan would know whether a heterologous gene is of a
suitable size for encapsidation and whether the heterologous gene
product is expressed properly.
[0076] The method of the invention may also be useful for neuronal,
glial, fibroblast or mesenchymal cell transplantation, or
"grafting", which involves transplantation of cells infected with
the recombinant lentivirus of the invention ex vivo, or infection
in vivo into the central nervous system or into the ventricular
cavities or subdurally onto the surface of a host brain. Such
methods for grafting will be known to those skilled in the art and
are described in Neural Grafting in the Mammalian CNS, Bjorklund
& Stenevi, eds. (1985).
[0077] For diseases due to deficiency of a protein product, gene
transfer could introduce a normal gene into the affected tissues
for replacement therapy, as well as to create animal models for the
disease using antisense mutations. For example, it may be desirable
to insert a Factor VIII or IX encoding nucleic acid into a
lentivirus for infection of a muscle, spleen or liver cell.
[0078] The promoter sequence may be homologous or heterologous to
the desired gene sequence. A wide range of promoters may be
utilized, including a viral or a mammalian promoter. Cell or tissue
specific promoters can be utilized to target expression of gene
sequences in specific cell populations. Suitable mammalian and
viral promoters for the instant invention are available in the
art.
[0079] Optionally during the cloning stage, the nucleic acid
construct referred to as the transfer vector, having the packaging
signal and the heterologous cloning site, also contains a
selectable marker gene. Marker genes are utilized to assay for the
presence of the vector, and thus, to confirm infection and
integration. The presence of a marker gene ensures the selection
and growth of only those host cells which express the inserts.
Typical selection genes encode proteins that confer resistance to
antibiotics and other toxic substances, e.g., histidinol,
puromycin, hygromycin, neomycin, methotrexate etc. and cell surface
markers.
[0080] The recombinant virus of the invention is capable of
transferring a nucleic acid sequence into a mammalian cell. The
term, "nucleic acid sequence", refers to any nucleic acid molecule,
preferably DNA, as discussed in detail herein. The nucleic acid
molecule may be derived from a variety of sources, including DNA,
cDNA, synthetic DNA, RNA or combinations thereof. Such nucleic acid
sequences may comprise genomic DNA which may or may not include
naturally occurring introns. Moreover, such genomic DNA may be
obtained in association with promoter regions, poly A sequences or
other associated sequences. Genomic DNA may be extracted and
purified from suitable cells by means well known in the art.
Alternatively, messenger RNA (mRNA) can be isolated from cells and
used to produce cDNA by reverse transcription or other means.
[0081] Preferably, the recombinant lentivirus produced by the
method of the invention is a derivative of human immunodeficiency
virus (HIV). The env will be derived from a virus other than
HIV.
[0082] Thus, three or more vectors, e.g., in one or more plasmids,
which provide all of the functions required for packaging of
recombinant virions, such as, gag, pol, env, tat and rev, can be
employed to prepare recombinant lentivirus. As noted herein, tat
may be deleted. There is no limitation on the number of vectors
which are utilized so long as the vectors are used to transform and
to produce the packaging cell line to yield recombinant
lentivirus.
[0083] The vectors are introduced via transfection or infection
into the packaging cell line. The packaging cell line produces
viral particles that contain the vector genome. Methods for
transfection or infection are well known by those of skill in the
art. After cotransfection of the packaging vectors and the transfer
vector to the packaging cell line, the recombinant virus is
recovered from the culture media and titered by standard methods
used by those of skill in the art.
[0084] Thus, the packaging constructs can be introduced into human
cell lines by calcium phosphate transfection, lipofection or
electroporation, generally together with a dominant selectable
marker encoding, for example, neomycin resistance, DHFR, Gln
synthetase or ADA, followed by selection in the presence of the
appropriate drug and isolation of clones. The selectable marker
gene can be linked physically to the packaging genes in the
construct.
[0085] Stable cell lines wherein the packaging functions are
configured to be expressed by a suitable packaging cell are known.
For example, see U.S. Pat. No. 5,686,279; and Ory et al. (1996),
which describe packaging cells.
[0086] Zufferey et al. (1997) teach a lentiviral packaging plasmid
wherein sequences 3' of pol including the HIV-1 env gene are
deleted. The construct contains tat and rev sequences and the 3'
LTR is replaced with poly A sequences. The 5' LTR and psi sequences
are replaced by another promoter, such as one which is inducible.
For example, a CMV promoter or derivative thereof can be used.
[0087] The packaging vectors of interest may contain additional
changes to the packaging functions to enhance lentiviral protein
expression and to enhance safety. For example, all of the HIV
sequences upstream of gag can be removed. Also, sequences
downstream of env can be removed. Moreover, steps can be taken to
modify the vector to enhance the splicing and translation of the
RNA.
[0088] To provide a vector with an even more remote possibility of
generating replication competent lentivirus, lentivirus packaging
plasmids wherein tat sequences, a regulating protein which promotes
viral expression through a transcriptional mechanism, are deleted
functionally. Thus, the tat gene can be deleted, in part or in
whole, or various point mutations or other mutations can be made to
the tat sequence to render the gene non-functional. An artisan can
practice known techniques to render the tat gene
non-functional.
[0089] The techniques used to construct vectors, and to transfect
and to infect cells, are practiced widely in the art. Practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures.
[0090] A lentiviral packaging vector is made to contain a promoter
and other optional or requisite regulatory sequences as determined
by the artisan, gag, pol, rev, env or a combination thereof, and
with specific functional or actual excision of tat, and optionally
other lentiviral accessory genes.
[0091] Lentiviral transfer vectors (Naldini et al., 1996) have been
used to infect human cells growth-arrested in vitro and to
transduce neurons after direct injection into the brain of adult
rats. The vector was efficient at transferring marker genes in vivo
into the neurons and long term expression in the absence of
detectable pathology was achieved. Another version of the
lentiviral vector in which the HIV virulence genes env, vif, vpr,
vpu and nef were deleted without compromising the ability of the
vector to transduce non-dividing cells, represents a substantial
improvement in the biosafety of the vector (Zufferey et al.,
1997).
[0092] In transduced cells, the integrated lentiviral vector
generally has an LTR at each termini. The 5' LTR may cause
accumulation of "viral" transcripts that may be the substrate of
recombination, in particular in HIV-infected cells. The 3' LTR may
promote downstream transcription with the consequent risk of
activating a cellular protooncogene. The U3 sequences comprise the
majority of the HIV LTR. The U3 region contains the enhancer and
promoter elements that modulate basal and induced expression of the
HIV genome in infected cells and in response to cell activation.
Several of the promoter elements are essential for viral
replication. Some of the enhancer elements are highly conserved
among viral isolates and have been implicated as critical virulence
factors in viral pathogenesis. The enhancer elements may act to
influence replication rates in the different cellular target of the
virus (Marthas et al., 1993). Also, enhancers in either LTR can
activate transcription of neighboring genes.
[0093] As viral transcription starts at the 3' end of the U3 region
of the 5' LTR, this U3 region (including the promoter and enhancer)
is not included in the viral mRNA, and a copy thereof from the 3'
LTR acts as template for the generation of the U3 region of both
LTR's in the subsequently integrated provirus. If the U3 region of
the 3' LTR is altered in a retroviral vector construct so as to
eliminate the promoter and enhancer, the vector RNA still is
produced from the intact 5' LTR in producer cells, but cannot be
regenerated in target cells. Transduction of such a vector results
in the transcriptional inactivation of both LTR's in the progeny
virus. Thus, the retrovirus is self-inactivating (SIN) and those
vectors are known as Sin transfer vectors.
[0094] There are, however, limits to the extent of the deletion at
the 3' LTR. First, the 5' end of the U3 region serves another
essential function in vector transfer, being required for
integration (terminal dinucleotide+att sequence). Thus, the
terminal dinucleotide and the att sequence may represent the 5'
boundary of the U3 sequences which can be deleted. In addition,
some loosely defined regions may influence the activity of the
downstream polyadenylation site in the R region. Excessive deletion
of U3 sequence from the 3' LTR may decrease polyadenylation of
vector transcripts with adverse consequences both on the titer of
the vector in producer cells and the transgene expression in target
cells. On the other hand, limited deletions may not abrogate the
transcriptional activity of the LTR in transduced cells.
[0095] U3 deletions in a HIV LTR can span from nucleotide-418 of
the U3 LTR to the indicated position: SIN-78, SIN-45, SIN-36 and
SIN-18. Lentiviral vectors with almost complete deletion of the U3
sequences from the 3' LTR were developed without compromising
either the titer of vector in producer cells or transgene
expression in target cells. The most extensive deletion (-418 to
-18) extends as far as to the TATA box, therefore abrogating any
transcriptional activity of the LTR in transduced cells. Thus, the
lower limit of the 3' deletion may extend as far as including the
TATA box. The deletion may be of the remainder of the U3 region up
to the R region. Surprisingly, the average expression level of the
transgene is higher in cells transduced by the SIN vectors as
compared to more intact vectors.
[0096] The 5' LTR of a transfer vector construct can be modified by
substituting part or all of the transcriptional regulatory elements
of the U3 region with heterologous enhancer/promoters. The changes
were made to enhance the expression of transfer vector RNA in
producer cells; to allow vector production in the absence of the
HIV tat gene; and to remove the upstream wild-type copy of the HIV
LTR that can recombine with the 3' deleted version to "rescue" the
above described SIN vectors.
[0097] Thus, vectors containing the above-described alterations at
the 5' LTR, 5' vectors, can find use as transfer vectors because of
the sequences to enhance expression and in combination with
packaging cells that do not express tat. Such 5' vectors can also
carry modifications at the 3' LTR as discussed hereinabove to yield
improved transfer vectors which have not only enhanced expression
and can be used in packaging cells that do not express tat but can
be self-inactivating as well.
[0098] The transcription from the HIV LTR is highly dependent on
the transactivator function of the tat protein. In the presence of
tat, often expressed by the core packaging construct existing in
producer cells, vector transcription from the HIV LTR is stimulated
strongly. As that full-length "viral" RNA has a full complement of
packaging signals, the RNA is encapsidated efficiently into vector
particles and transferred to target cells. The amount of vector RNA
available for packaging in producer cells is a rate-limiting step
in the production of infectious vector.
[0099] The entire enhancer or the entire enhancer and promoter
regions of the 5' LTR can be substituted with the enhancer or the
enhancer and promoter of the human cytomegalovirus (CMV) or murine
Rous sarcoma virus (RSV).
[0100] The high level of expression of the 5' LTR modified transfer
vector RNA obtained in producer cells in the absence of a packaging
construct indicates the producing vector is functional in the
absence of a functional tat gene. Functional deletion of the tat
gene as indicated for the packaging plasmid disclosed hereinabove
would confer a higher level of biosafety to the lentiviral vector
system given the number of pathogenetic activities associated with
the tat protein.
Exemplary Packaging Cell Lines
[0101] Pseudotyped lentiviral or retroviral particles can be
produced by introducing a defective, recombinant lentiviral genome
into a packaging cell (e.g., by infection with defective retroviral
particle, or by other means for introducing DNA into a target cell,
such as conventional transformation techniques). The defective
retroviral genome minimally contains the long terminal repeats, the
exogenous nucleotide sequence of interest to be transferred, and a
packaging sequence (N). In general, the packaging cell provides the
missing retroviral components essential for retroviral replication,
integration, and encapsidation, and also expresses a nucleotide
sequence encoding the desired envelope protein. However, the
packaging cell does not have all of the components essential for
the production of retroviral particles. The nucleotide sequence(s)
encoding the missing viral component(s) in the packaging cell can
be either stably integrated into the packaging cell genome, and/or
can be provided by a co-infecting helper virus.
[0102] The nucleotide sequences encoding the retroviral components
and the lentiviral or retroviral RNA genome can be derived from any
desired lenti- or retrovirus (e.g., murine, simian, avian, or human
retroviruses). In general, the retroviral components can be derived
from any retrovirus that can form pseudotyped retroviral particles
with the desired envelope protein, e.g., VSV G. Where VSV G is the
desired envelope protein, the retroviral components can be derived
from MuLV, MoMLV, avian leukosis virus (ALV), human
immunodeficiency virus (HIV), or any other retrovirus that can form
pseudotyped virus with VSV G as the only envelope protein or with
VSV G and a relatively small amount of retroviral envelope
protein.
[0103] The present invention thus provides recombinant retroviral
particles, particularly pseudotyped retroviral particles. Exemplary
packaging cell lines are derived from 293, HeLa, Cf2Th, D17, MDCK,
or BHK cells. Retroviral particles are preferentially produced by
inducibly expressing an envelope protein of interest (e.g., a
retroviral envelope or the envelope protein of vesicular stomatitis
virus). Inducible expression of the envelope protein may be
accomplished by operably linking an envelope protein-encoding
nucleotide sequence to an inducible promoter (e.g., a promoter
composed of a minimal promoter linked to at least one copy of tetO,
the binding site for the tetracycline repressor (tetR) of the
Escherichia coli tetracycline resistance operon Tn10). Expression
from the inducible promoter is regulated by a transactivating
factor, composed of a first ligand-binding domain that negatively
regulates transcription from the inducible promoter (e.g., a
prokaryotic tetracycline repressor polypeptide (tetR)).
Transcription of the envelope-encoding nucleotide sequence under
control of the inducible promoter is activated by a transactivator
when tetracycline is absent.
[0104] The packaging cell line may comprise a first polynucleotide
having an HIV genome operably linked to a first inducible promoter
wherein the HIV genome is defective for cis-acting elements, for
self-replication and for expression of functional Env protein; a
second polynucleotide encoding a functional heterologous Env
protein operably linked to a second inducible promoter; and a third
polynucleotide encoding a regulatable transcriptional activator
controlling transcription from the first and second inducible
promoters.
[0105] In one embodiment, the first, second and third
polynucleotides are contained in vectors. These polynucleotides can
be contained in one or more vectors, preferably plasmid vectors. In
an exemplary packaging cell line, the first polynucleotide is
contained in a first plasmid vector and the second polynucleotide
is contained in a second plasmid vector. The third polynucleotide
encoding a regulatable transcriptional activator is exemplified
herein as containing a minimal CMV immediate-early gene promoter
linked to seven tandem copies of the tetR-binding site replacing
the CMV promoter (BglII/BamHI fragment). As discussed herein, other
viral envelopes and other indicator markers will be known to those
of skill in the art for use in the present invention.
[0106] In one aspect of the invention, one or more polynucleotides
encoding retroviral accessory proteins, are included as part of the
first or second polynucleotide constructs, for example. Accessory
proteins include vpr, vif, nef, vpx, tat, eve, and vpu.
[0107] Preferably, the transcriptional activator or transactivator
can be expressed at high levels in a eukaryotic cell without
significantly adversely affecting general cellular transcription in
the host cell transactivator expression that is sufficient to
facilitate transactivation of the inducible promoter, but that is
not detrimental to the cell (e.g., is not toxic to the cell). "High
Levels" can be a level of expression that allows detection of the
transactivator by Western blot The transactivator can preferably be
expressed in a wide variety of cell types, including mammalian and
non-mammalian cells such as, but not limited to, human, monkey,
mouse, hamster, cow, insect, fish, and frog cells.
[0108] The transactivator can be expressed either in vivo or in
vitro, and expression of the transactivator can be controlled
through selection of the promoter to which the nucleotide sequence
encoding the transactivator is operably linked. For example, the
promoter can be a constitutive promoter or an inducible promoter.
Examples of such promoters include the human cytomegalovirus
promoter IE (Boshart et al., 1985), ubiquitously expressing
promoters such as HSV-Tk (McKnight et al., 1984) and
.E-backward.-actin promoters (e.g., the human .E-backward.-actin
promoter as described by Ng et al., 1985).
[0109] For example, where the transactivator is a tetR polypeptide,
the inducible promoter is preferably a minimal promoter containing
at least one tetO sequence, preferably at least 2 or more tandemly
repeated tetO sequences, even more preferably at least 5 or more
tandemly repeated tetO sequences, more preferably at least 7
tandemly repeated tetO sequences or more. The minimal promoter
portion of the inducible promoter can be derived from any desired
promoter, and is selected according to tet cell line in which the
inducible expression system is to be used. Where the cell is a
mammalian cell, a preferred minimal promoter is derived from CMV,
preferably from the CMV immediate early gene 1A. In addition, other
inducible promoters could be employed, such as the
ecdysone-inducible promoters (Invitrogen Inc., San Diego, Calif.)
or the lacZ inducible promoters.
[0110] The promoter of the transactivator can be a cell
type-specific or tissue-specific promoter than preferentially
facilitates transcription of the transactivator in a desired cell
of tissue type. Exemplary cell type-specific and/or tissue-specific
promoters include promoters such as albumin (liver specific;
Pinkert et al., 1987), lymphoid-specific promoters (Calame et al.,
1988); in particular promoters of T-cell receptors (Winoto et al.,
1989) and immunoglobulins (Banerji et al., 1983; Queen and
Baltimore, 1983), neuron-specific promoters (e.g., the
neurofilament promoter (Byrne et al., 1989), pancreas-specific
promoters (Edlunch et al., 1985) or mammary gland-specific
promoters (milk whey promoter, U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Promoters for expression of
the transactivator can also be developmentally regulated promoters
as the murine homeobox promoters (Kessel et al., 1990) or the
.A-inverted.-fetoprotein promoter (Campes et al., 1989). The
promoter can be used in combination with control regions allowing
integration site independent expression of the transactivator
(Grosveld et al., 1987). Preferably, the promoter is constitutive
in the respective cell types. For instance, the promoter is a CMV
promoter, preferably a CMV immediate early gene promoter.
Isolated Nucleic Acid-Based Vectors of the Invention
[0111] The isolated nucleic acid-based vectors of the invention,
e.g., those which are not delivered in a viral particle and/or do
not encode one or more viral proteins but may comprise viral
transcriptional and/or translational regulatory elements, include a
heterologous nucleic acid sequence of interest optionally operably
linked to a regulatory nucleic acid sequence. The heterologous gene
of interest in the isolated nucleic acid-based vector of the
invention can be any nucleic acid of interest which can be
transcribed. Generally the foreign gene encodes a polypeptide.
Preferably the polypeptide has some therapeutic benefit. The
polypeptide may supplement deficient or nonexistent expression of
an endogenous protein in a host cell.
[0112] It may be desirable to modulate the expression of a gene
regulating molecule in a cell by the introduction of a molecule by
the method of the invention. The term "modulate" envisions the
suppression of expression of a gene when it is over-expressed or
augmentation of expression when it is under-expressed.
[0113] The method of the invention may also be useful for neuronal,
glial, fibroblast or mesenchymal cell transplantation, or
"grafting", which involves transplantation of transfected cells
into the central nervous system or into the ventricular cavities or
subdurally onto the surface of a host brain. Such methods for
grafting will be known to those skilled in the art and are
described in Neural Grafting in the Mammalian CNS, Bjorklund &
Stenevi, eds. (1985).
[0114] For diseases due to deficiency of a protein product, gene
transfer of an isolated nucleic acid-based vector of the invention
could introduce a normal gene into the affected tissues for
replacement therapy, as well as to create animal models for the
disease using antisense mutations.
[0115] The promoter sequence of an isolated nucleic acid-based
vector of the invention may be homologous or heterologous to the
desired gene sequence. A wide range of promoters may be utilized,
including a viral or a mammalian promoter. Cell or tissue specific
promoters can be utilized to target expression of gene sequences in
specific cell populations. Suitable mammalian and viral promoters
for the instant invention are available in the art.
[0116] Optionally during the cloning stage, the nucleic acid
construct referred to as the transfer vector also contains a
selectable marker gene. Marker genes are utilized to assay for the
presence of the vector, and thus, to confirm infection and
integration. The presence of a marker gene ensures the selection
and growth of only those host cells which express the inserts.
Typical selection genes encode proteins that confer resistance to
antibiotics and other toxic substances, e.g., histidinol,
puromycin, hygromycin, neomycin, methotrexate etc. and cell surface
markers.
Exemplary Disorders and Genes
[0117] The invention includes the use of a vector, e.g., a
lentiviral vector, comprising any open reading frame encoding a
gene product useful to prevent, inhibit or treat a disorder in a
mammal characterized by the lack of, or reduced levels of, that
gene product. For example, the disorder may be characterized by the
lack of, or reduced levels of one or more lysosomal enzymes (see,
e.g., enzymes described in FIG. 5 in U.S. Pat. No. 5,798,366, the
disclosure of which is specifically incorporated by reference
herein). Exemplary disorders include GM1 gangliosidosis, which is
caused by a deficiency in .beta.-galactosidase; Tay-Sachs disease,
a GM2 gangliosidosis which is caused by a deficiency of
.beta.-hexosaminidase A (acidic isozyme); Sandhoff disease, which
is caused by a deficiency of .beta.-hexosaminidase A & B
(acidic and basic isozymes); Fabry disease, which is caused by a
deficiency in .alpha.-galactosidase; Hurler syndrome, which is
caused by a deficiency of alpha-L-iduronidase,
mucopolysaccharidosis type VII, which is caused by a deficiency in
beta-glucuronidase, and Gaucher disease, which is a deficiency in
.beta.-glucocerebrosidase, as well as Hunter syndrome (a deficiency
of iduronate-2-sulfatase); Sanfilippo syndrome (a deficiency of
heparan sulfate sulfatase, N-acetylglucosaminidase); Morquio
syndrome (a deficiency of galactosamine-6-sulfate sulfatase or
beta-galactosidase); Maroteaux-Lamy syndrome (a deficiency of
arylsulfatase B); mucopolysaccharidosis type II;
mucopolysaccharidosis type III (A, B, C or D; a deficiency of
heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl
CoA:alpha-glucosaminide N-acetyl transferase or
N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis
type IV (A or B; a deficiency of galactosamine-6-sulfatase and
beta-galatacosidase); mucopolysaccharidosis type VI (a deficiency
of arylsulfatase B); mucopolysaccharidosis type VII (a deficiency
in beta-glucuronidase); mucopolysaccharidosis type VIII (a
deficiency of glucosamine-6-sulfate sulfatase);
mucopolysaccharidosis type IX (a deficiency of hyaluronidase);
Tay-Sachs disease (a deficiency in alpha subunit of
beta-hexosaminidase); Sandhoff disease (a deficiency in both alpha
and beta subunit of beta-hexosaminidase); GM1 gangliosidosis (type
I or type II); Fabry disease (a deficiency in alpha galactosidase);
metachromatic leukodystrophy (a deficiency of aryl sulfatase A);
Pompe disease (a deficiency of acid maltase); fucosidosis (a
deficiency of fucosidase); alpha-mannosidosis (a deficiency of
alpha-mannosidase); beta-mannosidosis (a deficiency of
beta-mannosidase), ceroid lipofuscinosis, and Gaucher disease
(types I, II and III; a deficiency in glucocerebrosidase), as well
as disorders such as Hermansky-Pudlak syndrome; Amaurotic idiocy;
Tangier disease; aspartylglucosaminuria; congenital disorder of
glycosylation, type Ia; Chediak-Higashi syndrome; macular
dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickel
syndrome; Farber lipogranulomatosis; fibromatosis; geleophysic
dysplasia; glycogen storage disease I; glycogen storage disease Ib;
glycogen storage disease Ic; glycogen storage disease III; glycogen
storage disease IV; glycogen storage disease V; glycogen storage
disease VI; glycogen storage disease VII; glycogen storage disease
0; immunoosseous dysplasia, Schimke type; lipidosis; lipase b;
mucolipidosis II; mucolipidosis II, including the variant form;
mucolipidosis IV; neuraminidase deficiency with beta-galactosidase
deficiency; mucolipidosis I; Niemann-Pick disease (a deficiency of
sphingomyelinase); Niemann-Pick disease without sphingomyelinase
deficiency (a deficiency of npc1, a cholesterol metabolizing
enzyme); Refsum disease; Sea-blue histiocyte disease; infantile
sialic acid storage disorder; sialuria; multiple sulfatase
deficiency; triglyceride storage disease with impaired long-chain
fatty acid oxidation; Winchester disease; Wolman disease (a
deficiency of cholesterol hydrolase); Deoxyribonuclease I-like 1
disorder; arylsulfatase E disorder; ATPase, H+ transporting,
lysosomal, subunit 1 disorder; glycogen storage disease IIb;
Ras-associated protein rab9 disorder; chondrodysplasia punctata 1,
X-linked recessive disorder; glycogen storage disease VIII;
lysosome-associated membrane protein 2 disorder; Menkes syndrome;
congenital disorder of glycosylation, type Ic; and sialuria. In
particular, the invention is useful to prevent, inhibit or treat
lysosomal storage diseases wherein the lysosomal enzyme is
trafficked to the lysosome (within the cell and between cells) by
specific glycosylation.
[0118] For instance, Tay-Sachs disease results from mutations in
the HexA gene, which encodes the alpha subunit of
.beta.-hexosaminidase, leading to a deficiency in the A isoenzyme.
The A isoenzyme is responsible for the degradation of GM2
ganglioside. When this enzyme is deficient in humans, GM2
ganglioside accumulates progressively and leads to severe
neurological degeneration. In the mouse model of Tay-Sachs disease
(generated by the targeted disruption of the HexA gene) (Sandhoff
et al., 1989), the mice store GM2 ganglioside in a progressive
fashion, but the levels never exceed the threshold required to
elicit neurodegeneration. In the mouse (but not in a human) a
sialidase is sufficiently abundant that it can convert GM2 to GA2
(asialo ganglioside 2), which can then be catabolized by the
hexosaminidase .beta. isoenzyme.
[0119] Gaucher disease is the name given to a group of lysosomal
storage disorders caused by mutations in the gene that codes for an
enzyme called glucocerebrosidase ("GC"). Gaucher disease is caused
by deficiency of GC. All of the mutations in the gene alter the
structure and function of the enzyme which lead to an accumulation
of the undegraded glycolipid substrate glucosylceramide, also
called glucocerebroside, in cells of the reticuloendothelial
system. Each particular mutation of the human GC gene leads to a
clinical disease collectively known as Gaucher disease. These
disorders are usually classified into three types; type 1
(non-neuronopathic), type 2 (acute neuronopathic) and type 3
(subacute neuronopathic), the type depending on the presence and
severity of neurologic involvement.
[0120] GC is a monomeric, membrane-associated, hydrophobic
glycoprotein with a molecular weight of 65,000 daltons. Human GC
contains 497 amino acids and is translated as a precursor protein
with a 19 amino acid hydrophobic signal peptide which directs its
co-translational insertion into the lumen of the endoplasmic
reticulum-golgi-lysosome complex as reported by Erickson et al.
(1985). GC acts at the acidic pH of the lysosome to hydrolyze
beta-glucosidic linkages in complex lipids ubiquitously found in
all membranes to form the byproducts of glucose and ceramide. The
catalytic activity of GC is increased in vitro by detergents,
lipids, and in vivo by a naturally occurring activator known as
sphingolipid activator protein-2 (SAP-2 or saposin C). See, Ho et
al. (1971); O'Brian et al. (1988). While more than twenty mutations
in the human GC gene are known, only two are common. See, Tsuji et
al. (1988). The two common mutations account for approximately 70%
of the mutant alleles, as reported by Firon et al. (1990). Mutant
GC genes code for aberrant proteins that are either catalytically
altered or unstable and rapidly disappear from the cell.
[0121] Although GC is deficient in all of a subject's cells, for
unknown reasons, the accumulation of the substrate glucosylceramide
occurs virtually only in macrophages. To correct the enzyme
deficiency in macrophages, two approaches have been used. The first
treatment is based allogeneic bone marrow transplantation, which
results in the repopulation of affected tissues with
enzyme-competent macrophages. See, Rappeport et al. (1986). The
second approach to treatment which has resulted in clinical
improvement in Gaucher disease patients is macrophage-targeted
enzyme replacement. This treatment takes advantage of naturally
occurring mannose receptors on macrophages and the exposition of
accessible mannose receptors in the oligosaccharides of
glucocerebrosidase to efficiently deliver the enzyme to
macrophages. See, Barranger (1989); Takasaki et al. (1984); and
Furbish et al., (1981). However, allogeneic bone marrow
transplantation has associated with it morbidity and mortality
risks that are unacceptable for many patients. Further, HLA matched
bone marrow donors do not exist for the majority of patients. As
for macrophage-targeted enzyme replacement, it is currently an
expensive and life-long therapy.
[0122] Hurler syndrome is an autosomal recessive disease resulting
from deficient alpha-iduronidase enzymatic activity and the
consequent systemic accumulation of glycosaminoglycan (GAG)
substrates. The disease is characterized by hepatosplenomegaly,
severe skeletal involvement, progressive mental retardation, and is
typically lethal in childhood.
[0123] To be an effective permanent treatment for any disease
capable of being treated by gene therapy, the transfer and
sustained expression of genes in cells important to the
pathogenesis of the particular disease is required. Sufficient and
long term expression of a transduced gene in the progeny of
transduced cells, e.g., transduced stem cells such as pluripotent
bone marrow stem cells, for example, using a lentivirus, could
correct the deficiency of the enzyme in many if not all relevant
cell types.
Dosages, Formulations and Routes of Administration of the Agents of
the Invention
[0124] The therapeutic agents of the invention are preferably
administered so as to achieve beneficial results. The amount
administered will vary depending on various factors including, but
not limited to, the agent chosen, the disease, whether prevention
or treatment is to be achieved, and if the agent is modified for
bioavailability and in vivo stability.
[0125] Administration of sense or antisense nucleic acid molecule
may be accomplished through the introduction of cells transformed
with an expression cassette comprising the nucleic acid molecule
(see, for example, WO 93/02556) or the administration of the
nucleic acid molecule (see, for example, Felgner et al., U.S. Pat.
No. 5,580,859, Pardoll et al., Immunity, 3, 165 (1995); Stevenson
et al., Immunol. Rev., 145, 211 (1995); Moiling, J. Mol. Med., 75,
242 (1997); Donnelly et al., Ann. N.Y. Acad. Sci., 772, 40 (1995);
Yang et al., Mol. Med. Today. 2, 476 (1996); Abdallah et al., Biol.
Cell, 85, 1 (1995)). Pharmaceutical formulations, dosages and
routes of administration for nucleic acids are generally disclosed,
for example, in Felgner et al., supra. Nucleic acid molecules may
be complexed with polyethyleneimine, polylysine or cationic lipids
such as DOTMA, DOTAP, DOGS, or DC-Chol
(N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride,
DOTAP; N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium
chloride, DOTMA;
3.beta.-[N--(N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol,
DC-Chol) In one embodiment, DNA is delivered under pressure into
the hepatic circulation.
[0126] The amount of therapeutic agent administered is selected to
treat a particular indication. The therapeutic agents of the
invention are also amenable to chronic use for prophylactic
purposes, preferably by systemic administration.
[0127] Administration of the therapeutic agents in accordance with
the present invention may be continuous or intermittent, depending,
for example, upon the recipient's physiological condition, whether
the purpose of the administration is therapeutic or prophylactic,
and other factors known to skilled practitioners. The
administration of the agents of the invention may be essentially
continuous over a preselected period of time or may be in a series
of spaced doses. Both local and systemic administration is
contemplated.
[0128] One or more suitable unit dosage forms comprising the
therapeutic agents of the invention, which, as discussed below, may
optionally be formulated for sustained release, can be administered
by a variety of routes including oral, or parenteral, including by
rectal, buccal, vaginal and sublingual, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, intrathoracic,
intrapulmonary and intranasal routes. The formulations may, where
appropriate, be conveniently presented in discrete unit dosage
forms and may be prepared by any of the methods well known to
pharmacy. Such methods may include the step of bringing into
association the therapeutic agent with liquid carriers, solid
matrices, semi-solid carriers, finely divided solid carriers or
combinations thereof, and then, if necessary, introducing or
shaping the product into the desired delivery system.
[0129] When the therapeutic agents of the invention are prepared
for oral administration, they are preferably combined with a
pharmaceutically acceptable carrier, diluent or excipient to form a
pharmaceutical formulation, or unit dosage form. The total active
ingredients in such formulations comprise from 0.1 to 99.9% by
weight of the formulation. By "pharmaceutically acceptable" it is
meant the carrier, diluent, excipient, and/or salt must be
compatible with the other ingredients of the formulation, and not
deleterious to the recipient thereof. The active ingredient for
oral administration may be present as a powder or as granules; as a
solution, a suspension or an emulsion; or in achievable base such
as a synthetic resin for ingestion of the active ingredients from a
chewing gum. The active ingredient may also be presented as a
bolus, electuary or paste.
[0130] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, douches,
lubricants, foams or sprays containing, in addition to the active
ingredient, such carriers as are known in the art to be
appropriate. Formulations suitable for rectal administration may be
presented as suppositories.
[0131] Pharmaceutical formulations containing the therapeutic
agents of the invention can be prepared by procedures known in the
art using well known and readily available ingredients. For
example, the agent can be formulated with common excipients,
diluents, or carriers, and formed into tablets, capsules,
suspensions, powders, and the like. Examples of excipients,
diluents, and carriers that are suitable for such formulations
include the following fillers and extenders such as starch, sugars,
mannitol, and silicic derivatives; binding agents such as
carboxymethyl cellulose, HPMC and other cellulose derivatives,
alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents
such as glycerol; disintegrating agents such as calcium carbonate
and sodium bicarbonate; agents for retarding dissolution such as
paraffin; resorption accelerators such as quaternary ammonium
compounds; surface active agents such as cetyl alcohol, glycerol
monostearate; adsorptive carriers such as kaolin and bentonite; and
lubricants such as talc, calcium and magnesium stearate, and solid
polyethyl glycols.
[0132] For example, tablets or caplets containing the agents of the
invention can include buffering agents such as calcium carbonate,
magnesium oxide and magnesium carbonate. Caplets and tablets can
also include inactive ingredients such as cellulose, pregelatinized
starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium
stearate, microcrystalline cellulose, starch, talc, titanium
dioxide, benzoic acid, citric acid, corn starch, mineral oil,
polypropylene glycol, sodium phosphate, and zinc stearate, and the
like. Hard or soft gelatin capsules containing an agent of the
invention can contain inactive ingredients such as gelatin,
microcrystal line cellulose, sodium lauryl sulfate, starch, talc,
and titanium dioxide, and the like, as well as liquid vehicles such
as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric
coated caplets or tablets of an agent of the invention are designed
to resist disintegration in the stomach and dissolve in the more
neutral to alkaline environment of the duodenum.
[0133] The therapeutic agents of the invention can also be
formulated as elixirs or solutions for convenient oral
administration or as solutions appropriate for parenteral
administration, for instance by intramuscular, subcutaneous or
intravenous routes.
[0134] The pharmaceutical formulations of the therapeutic agents of
the invention can also take the form of an aqueous or anhydrous
solution or dispersion, or alternatively the form of an emulsion or
suspension.
[0135] Thus, the therapeutic agent may be formulated for parenteral
administration (e.g., by injection, for example, bolus injection or
continuous infusion) and may be presented in unit dose form in
ampules, pre-filled syringes, small volume infusion containers or
in multi-dose containers with an added preservative. The active
ingredients 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 ingredients may be in powder form,
obtained by aseptic isolation of sterile solid or by lyophilization
from solution, for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
[0136] These formulations can contain pharmaceutically acceptable
vehicles and adjuvants which are well known in the prior art. It is
possible, for example, to prepare solutions using one or more
organic solvent(s) that is/are acceptable from the physiological
standpoint, chosen, in addition to water, from solvents such as
acetone, ethanol, isopropyl alcohol, glycol ethers such as the
products sold under the name "Dowanol", polyglycols and
polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain
acids, preferably ethyl or isopropyl lactate, fatty acid
triglycerides such as the products marketed under the name
"Miglyol", isopropyl myristate, animal, mineral and vegetable oils
and polysiloxanes.
[0137] The compositions according to the invention can also contain
thickening agents such as cellulose and/or cellulose derivatives.
They can also contain gums such as xanthan, guar or carbo gum or
gum arabic, or alternatively polyethylene glycols, bentones and
montmorillonites, and the like.
[0138] It is possible to add, if necessary, an adjuvant chosen from
antioxidants, surfactants, other preservatives, film-forming,
keratolytic or comedolytic agents, perfumes and colorings. Also,
other active ingredients may be added, whether for the conditions
described or some other condition.
[0139] For example, among antioxidants, t-butylhydroquinone,
butylated hydroxyanisole, butylated hydroxytoluene and
.alpha.-tocopherol and its derivatives may be mentioned. The
galenical forms chiefly conditioned for topical application take
the form of creams, milks, gels, dispersion or microemulsions,
lotions thickened to a greater or lesser extent, impregnated pads,
ointments or sticks, or alternatively the form of aerosol
formulations in spray or foam form or alternatively in the form of
a cake of soap.
[0140] Additionally, the agents are well suited to formulation as
sustained release dosage forms and the like. The formulations can
be so constituted that they release the active ingredient only or
preferably in a particular part of the intestinal or respiratory
tract, possibly over a period of time. The coatings, envelopes, and
protective matrices may be made, for example, from polymeric
substances, such as polylactide-glycolates, liposomes,
microemulsions, microparticles, nanoparticles, or waxes. These
coatings, envelopes, and protective matrices are useful to coat
indwelling devices, e.g., stents, catheters, peritoneal dialysis
tubing, and the like.
[0141] The therapeutic agents of the invention can be delivered via
patches for transdermal administration. See U.S. Pat. No. 5,560,922
for examples of patches suitable for transdermal delivery of a
therapeutic agent. Patches for transdermal delivery can comprise a
backing layer and a polymer matrix which has dispersed or dissolved
therein a therapeutic agent, along with one or more skin permeation
enhancers. The backing layer can be made of any suitable material
which is impermeable to the therapeutic agent. The backing layer
serves as a protective cover for the matrix layer and provides also
a support function. The backing can be formed so that it is
essentially the same size layer as the polymer matrix or it can be
of larger dimension so that it can extend beyond the side of the
polymer matrix or overlay the side or sides of the polymer matrix
and then can extend outwardly in a manner that the surface of the
extension of the backing layer can be the base for an adhesive
means. Alternatively, the polymer matrix can contain, or be
formulated of, an adhesive polymer, such as polyacrylate or
acrylate/vinyl acetate copolymer. For long-term applications it
might be desirable to use microporous and/or breathable backing
laminates, so hydration or maceration of the skin can be
minimized.
[0142] Examples of materials suitable for making the backing layer
are films of high and low density polyethylene, polypropylene,
polyurethane, polyvinylchloride, polyesters such as poly(ethylene
phthalate), metal foils, metal foil laminates of such suitable
polymer films, and the like. Preferably, the materials used for the
backing layer are laminates of such polymer films with a metal foil
such as aluminum foil. In such laminates, a polymer film of the
laminate will usually be in contact with the adhesive polymer
matrix.
[0143] The backing layer can be any appropriate thickness which
will provide the desired protective and support functions. A
suitable thickness will be from about 10 to about 200 microns.
[0144] Generally, those polymers used to form the biologically
acceptable adhesive polymer layer are those capable of forming
shaped bodies, thin walls or coatings through which therapeutic
agents can pass at a controlled rate. Suitable polymers are
biologically and pharmaceutically compatible, nonallergenic and
insoluble in and compatible with body fluids or tissues with which
the device is contacted. The use of soluble polymers is to be
avoided since dissolution or erosion of the matrix by skin moisture
would affect the release rate of the therapeutic agents as well as
the capability of the dosage unit to remain in place for
convenience of removal.
[0145] Exemplary materials for fabricating the adhesive polymer
layer include polyethylene, polypropylene, polyurethane,
ethylene/propylene copolymers, ethylene/ethylacrylate copolymers,
ethylene/vinyl acetate copolymers, silicone elastomers, especially
the medical-grade polydimethylsiloxanes, neoprene rubber,
polyisobutylene, polyacrylates, chlorinated polyethylene, polyvinyl
chloride, vinyl chloride-vinyl acetate copolymer, crosslinked
polymethacrylate polymers (hydrogel), polyvinylidene chloride,
poly(ethylene terephthalate), butyl rubber, epichlorohydrin
rubbers, ethylenevinyl alcohol copolymers, ethylene-vinyloxyethanol
copolymers; silicone copolymers, for example,
polysiloxane-polycarbonate copolymers, polysiloxane, polyethylene
oxide copolymers, polysiloxane-polymethacrylate copolymers,
polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylene
copolymers), polysiloxane-alkylenesilane copolymers (e.g.,
polysiloxane-ethylenesilane-copolymers), and the like; cellulose
polymers, for example methyl or ethyl cellulose, hydroxy propyl
methyl cellulose, and cellulose esters; polycarbonates;
polytetrafluoroethylene; and the like.
[0146] Preferably, a biologically acceptable adhesive polymer
matrix should be selected from polymers with glass transition
temperatures below room temperature. The polymer may, but need not
necessarily, have a degree of crystallinity at room
temperature. Cross-linking monomeric units or sites can be
incorporated into such polymers. For example, cross-linking
monomers can be incorporated into polyacrylate polymers, which
provide sites for cross-linking the matrix after dispersing the
therapeutic agent into the polymer. Known cross-linking monomers
for polyacrylate polymers include polymethacrylic esters of polyols
such as butylene diacrylate and dimethacrylate, trimethylol propane
trimethacrylate and the like. Other monomers which provide such
sites include allyl acrylate, allyl methacrylate, diallyl maleate
and the like.
[0147] Preferably, a plasticizer and/or humectant is dispersed
within the adhesive polymer matrix. Water-soluble polyols are
generally suitable for this purpose. Incorporation of a humectant
in the formulation allows the dosage unit to absorb moisture on the
surface of skin which in turn helps to reduce skin irritation and
to prevent the adhesive polymer layer of the delivery system from
failing.
[0148] Therapeutic agents released from a transdermal delivery
system must be capable of penetrating each layer of skin. In order
to increase the rate of permeation of a therapeutic agent, a
transdermal drug delivery system must be able in particular to
increase the permeability of the outermost layer of skin, the
stratum corneum, which provides the most resistance to the
penetration of molecules. The fabrication of patches for
transdermal delivery of therapeutic agents is well known to the
art.
[0149] For administration to the upper (nasal) or lower respiratory
tract by inhalation, the therapeutic agents of the invention are
conveniently delivered from an insufflator, nebulizer or a
pressurized pack or other convenient means of delivering an aerosol
spray. Pressurized packs may comprise a suitable propellant such as
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.
[0150] Alternatively, for administration by inhalation or
insufflation, the composition may take the form of a dry powder,
for example, a powder mix of the therapeutic agent and a suitable
powder base such as lactose or starch. The powder composition may
be presented in unit dosage form in, for example, capsules or
cartridges, or, e.g., gelatine or blister packs from which the
powder may be administered with the aid of an inhalator,
insufflator or a metered-dose inhaler.
[0151] For intra-nasal administration, the therapeutic agent may be
administered via nose drops, a liquid spray, such as via a plastic
bottle atomizer or metered-dose inhaler. Typical of atomizers are
the Mistometer (Wintrop) and the Medihaler (Riker).
[0152] The local delivery of the therapeutic agents of the
invention can also be by a variety of techniques which administer
the agent at or near the site of disease. Examples of site-specific
or targeted local delivery techniques are not intended to be
limiting but to be illustrative of the techniques available.
Examples include local delivery catheters, such as an infusion or
indwelling catheter, e.g., a needle infusion catheter, shunts and
stents or other implantable devices, site specific carriers, direct
injection, or direct applications.
[0153] For topical administration, the therapeutic agents may be
formulated as is known in the art for direct application to a
target area. Conventional forms for this purpose include wound
dressings, coated bandages or other polymer coverings,
ointments, creams, lotions, pastes, jellies, sprays, and aerosols,
as well as in toothpaste and mouthwash, or by other suitable forms,
e.g., via a coated condom. Ointments and creams may, for example,
be formulated with an aqueous or oily base with the addition of
suitable thickening and/or gelling agents. Lotions may be
formulated with an aqueous or oily base and will in general also
contain one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents. The active ingredients can also be delivered via
iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122;
4,383,529; or 4,051,842. The percent by weight of a therapeutic
agent of the invention present in a topical formulation will depend
on various factors, but generally will be from 0.01% to 95% of the
total weight of the formulation, and typically 0.1-25% by
weight.
[0154] When desired, the above-described formulations can be
adapted to give sustained release of the active ingredient
employed, e.g., by combination with certain hydrophilic polymer
matrices, e.g., comprising natural gels, synthetic polymer gels or
mixtures thereof.
[0155] Drops, such as eye drops or nose drops, may be formulated
with an aqueous or non-aqueous base also comprising one or more
dispersing agents, solubilizing agents or suspending agents. Liquid
sprays are conveniently delivered from pressurized packs. Drops can
be delivered via a simple eye dropper-capped bottle, or via a
plastic bottle adapted to deliver liquid contents dropwise, via a
specially shaped closure.
[0156] The therapeutic agent may further be formulated for topical
administration in the mouth or throat. For example, the active
ingredients may be formulated as a lozenge further comprising a
flavored base, usually sucrose and acacia or tragacanth; pastilles
comprising the composition in an inert base such as gelatin and
glycerin or sucrose and acacia; mouthwashes comprising the
composition of the present invention in a suitable liquid carrier;
and pastes and gels, e.g., toothpastes or gels, comprising the
composition of the invention.
[0157] The formulations and compositions described herein may also
contain other ingredients such as antimicrobial agents, or
preservatives. Furthermore, the active ingredients may also be used
in combination with other therapeutic agents.
[0158] The invention is further described by the following
non-limiting examples.
Example I
Materials and Methods
[0159] Production and concentration of lentiviral vector. The
lentiviral vector was generated from a first-generation
tetracycline-inducible VSVG-pseudotyped lentiviral packaging cell
line SODk1-CGFI (Kafri et al., 1999). The vector (HRNcmvGFP)
contained a humanized red-shift GFP gene driven by a human
cytomegalovirus immediate-early promoter. The vector producer cells
were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life
Technologies, Inc., Gaithersburg, Md.) containing 10%
tetracycline-free FCS (Clontech, Palo Alto, Calif.) and 0.7 :g/ml
doxycycline (Sigma, St. Louis, Mo.). Induction of cells was
initiated by splitting cells into polylysine (0.01% solution;
Sigma) precoated plates in the absence of doxycycline. The cells
were washed twice with PBS (Life Technologies Inc.) and fed daily
with doxycycline-free medium containing 5 mM sodium butyrate
(Sigma). Vector-containing medium was collected 3 and 4 days after
induction and filtered through a 0.2 :m pore filter. The vector
stocks were further concentrated by ultracentrifugation at 50,000 g
for 2 hours (Beckman SW-28 rotor), followed by resuspending and
incubating at 37 EC for 2 hours in 1/200 of starting volume of
Tris-buffered saline (TBS, pH 7.8) containing 10 mM MgCl.sub.2
dNTPs (0.1 mM each), 3 mM spermine, and 0.3 mM spermidine. After a
second ultracentrifugation at 50,000 g for 30 minutes (Beckman
TLA-100.3 rotor), the vector pellet was resuspended in 1/2000 of
the initial volume of TBS with 2 :g/ml Polybrene and was stored at
-80 EC.
[0160] Titration assay for vector potency. Human 293 embryonic
kidney cells or murine NIH 3T3 cells were subcultured into six-well
culture plates (Beckton Dickinson, Franklin Lakes, N.J.) at
10.sup.5 cells/well with DMEM containing 10% FBS (Life Technologies
Inc.). After 8 hours had passed, cells were exposed to serial
dilution of vector stocks in the presence of Polybrene (8 :g/ml).
Titers were scored 48 hours after transduction by FACS analysis to
quantitate GFP-expressing cells. All assays were done in
triplicate.
[0161] In vivo vector administration to mice. Thirty-one 8 week-old
normal BALB/c mice (15 male, 16 female) were obtained from Charles
River Laboratories (Wilmington, Mass.) and housed in a
pathogen-free facility on a 12-hour light/dark cycle. One week
later, the mice were randomly divided into control (7 male, 7
female) and treated groups (8 male, 9 female). The treated groups
were injected i.v. with 100 :l pooled concentrated vector stock
into the tail vein over 3-6 seconds; the control groups were
injected with 100 :l TBS containing 2 :g/ml Polybrene. All animal
procedures were done under aseptic conditions and in accordance
with protocols approved by the Institutional Animal Care and Use
Committee. Mice were periodically bled by the retro-orbital
technique.
[0162] Perfusion and organ collection. At either 4 or 40 days after
injection, the mice were euthanized by intraperitoneal
administration of an overdose of sodium nembutal (Abbott
Laboratories). Each mouse was perfused transcardially through the
aorta with PBS for 5-10 minutes until its liver turned pale,
followed by perfusion with 4% formaldehyde-PBS for 10-15 minutes.
Nine organs were removed in the following order: gonad, bladder,
gastrointestinal tract, lung, heart, kidney, liver, spleen, and
brain. Organs were post-fixed in 4% formaldehyde-PBS for 0.5 to 2
hours, and then transferred into a 30% sucrose-PBS solution for
storage at 4 EC until further processing. Bone marrow was harvested
in PBS from femur and tibia of each mouse, and stored in Cell Lysis
Solution (Puregene, Minneapolis, Minn.) for further DNA isolation.
The remains of mice were stored in 4% formaldehyde-PBS for
pathologic analysis. Aliquots of each organ were segregated for two
different assays. Genomic DNA was isolated from the first aliquot
using a DNA isolation kit (Puregene) for quantitation of GFP gene.
The second aliquot was immersed in 10% neutral-buffered formalin
for 1-3 days and embedded in paraffin by routine methods. Sections
of 4 to 6 :m, stained with H&E, were examined.
[0163] Real-time QPCR using concurrent reactions. Both GFP
transgene and endogenous mouse Apob sequence (as an internal
control) were quantitated simultaneously in the same reaction well
of a total 50 :l PCR volume by real-time PCR. The TaqMan probe for
detection of GFP transgene was labeled with fluorescent reporter
dye 6FAM at the 5N end and quencher dye TAMRA at the
TABLE-US-00001 3Nend (5N-CCGACAAGCAGAAGAACGGCATCA-3N;, SEQ ID NO:
1)
whereas the probe for Apob was labeled with fluorescent reporter
dye VIC at the 5N end and quencher dye TAMRA at the 3N end
(5N-CCTTGAGCAGTGCCCGACCATTC-3N; SEQ ID NO:2). Sequences of TaqMan
primer-probe sets for GFP (sense,
5N-ACTACAACAGCCACAACGTCTATATCA-3N; SEQ ID NO:3; antisense,
5N-GGCGGATCTTGAAGTTCACC-3N; SEQ ID NO:4) and Apob (sense,
5N-CGTGGGCTCCAGCATTCTA-3N; SEQ ID NO:5; antisense,
5N-TCACCAGTCATTTCTGCCTTTG-3N; SEQ ID NO:6) were designed using the
Primer Express program (PE Applied Biosystems, Foster City,
Calif.). The duplex reaction contained 0.01-1 :g genomic DNA, 200
nM of each GFP primer, 200 nM GFP probe, 40 nM of each Apob primer,
200 nM Apob probe, and 25 :l TaqMan 2 H Universal Master Mix (PE
Applied Biosystems) including 8% glycerol, 1 HTaqMan buffer A, 5 mM
MgCl.sub.2, 400:M dUTP, 200 :M dATP, dCTP, AND dGTP (each),
AmpliTaq Gold (0.025 U/:l), and AmpErase UNG (0.01 U/:l). All PCR
reactions were set up in a MicroAmp Optical 96 well Reaction Plate
(PE Applied Biosystems). Amplification conditions were 2 minutes at
50 EC and 10 minutes at 95 EC for the first cycle, followed by 50
cycles of 95 EC for 15 seconds and 60 EC for 1 minute. The TaqMan
probes were cleaved during amplification, generating specific
fluorescence emission for the FAM-labeled GFP probe or the
VIC-labeled Apob probe. The data were collected in real time from
the ABI PRIME 7700 Sequence Detector and transferred online to a
Macintosh 7100 for analysis using the Sequence Detector version 1.6
program (PE Applied Biosystems). Unknown samples were run in
triplicate, and standard samples were in duplicate. All threshold
cycle (Ct) values of GFP transgene were normalized by Ct of Apob
that measured the total DNA content in each individual reaction.
Transgene frequencies of unknown samples were interpolated from a
standard curve (ranging from 0.001% to 100%) that was established
by simultaneous amplification of a series of genomic DNA mixtures
derived from a mouse myeloid cell line (32Dp210) (Carlesso et al.,
1994) and a GFP-containing cell line (32Dp210-LNChRGFP) with 1 copy
per genome (as determined by Southern blot analysis).
[0164] Flow cytometry. Blood samples were collected using
heparinized hematocrit tubes and diluted 1:1 into heparin sodium
solution (ICN Biomedicals Inc., Aurora, Ohio). PBL were fixed in
Optilyse B solution (Immunotech, Marseille, France), while red
cells were further lysed by the addition of dH.sub.2O. Specific
subsets of mouse PBL were detected by staining with PE-conjugated
hamster monoclonal antibody to mouse CD3e for T lymphocytes
(Pharmingen, San Diego, Calif.), or PE-conjugated rat monoclonal
antibody to mouse CD45R/B220 for B lymphocytes (PharMingen).
PE-conjugated Armenian hamster immunoglobulin group 1 and rat
immunoglobulin G2a were used, respectively, as isotype controls.
Cultured cells were trypsinized, then fixed in 4% formaldehyde, and
diluted in PBS to a concentration of 5 H 10.sup.5 cells/ml.
Two-color FACS analysis for cellular GFP (FL-1) and PE staining
(FL-2) were carried out by FACSCalibur with the CellQuest program
(Becton Dickinson). Cells from mice injected with TBS were analyzed
as negative controls for GFP expression in blood samples. For
titration assays, human 293 cells or mouse 3T3 cells were used as
negative controls; whereas uninduced SODk1-CGFI packaging cells
were used as positive controls.
[0165] Immunohistochemical staining for GFP-expressing cells. After
several PBS rinses and an incubation in 3% hydrogen peroxide, the
fixed cryosections were blocked in 5% normal goat serum (Vector
Labs, Burlingame, Calif.). The sections were then incubated with
the primary anti-GFP antibody (1:50; Clontech Lab) in 5% goat
serum-0.1% Triton X-100 overnight at 4 EC. After rinsing, the
sections were incubated in the biotinylated rabbit anti-goat
secondary antibody (Vector Labs) for 1 hour, washed three times
with PBS, stained with biotinylated horseradish peroxidase-avidin
(ABC kit; Vector Labs), and then colorized by using a
diaminobenzidine (DAB) substrate kit (Vector Labs). After staining,
sections were washed in dH.sub.2O, air-dried, and mounted in
Permount (Fisher Scientific Co., Fairlawn, N.J.).
Results
Vector Concentration and Administration
[0166] To assess the potency of the first-generation lentiviral
vector HRNcmvGFP, vector stocks from several batches of inductive
production and concentration were thawed at 30 EC and pooled.
Transduction was evaluated with both human 293 cells and mouse NIH
3T3 cells by FACS analysis for GFP expression (Table 1). Up to
780-fold increase in titer was observed in concentrated vector
supernatants, resulting in 1.8.A-inverted.0.15 H 10.sup.8
transforming units (TU) per milliliter. Titers obtained from human
293 cells were always about 10-fold higher than those from mouse
NIH 3T3 cells. Thus, about 2H 10.sup.7 293 TU of recombinant
HIV-GFP (in 100 :l) was injected through the tail vein into each of
17 normal BALB/c mice.
TABLE-US-00002 TABLE 1 Potency of lentiviral vector generated from
packaging cell line Titer* determined by FACS analysis for GFP
expression (TU/ml) Before concentration After concentration Cells
Mean SD Mean SD Human 293 2.3 H 10.sup.5 .A-inverted. 0.87 H
10.sup.5 1.8 H 10.sup.8 .A-inverted. 0.15 H 10.sup.8 Murine NIH 2.6
H 10.sup.4 .A-inverted. 0.12 H 10.sup.4 1.2 H 10.sup.7 .A-inverted.
0.76 H 10.sup.7 3T3 *Vector-containing supernatants were stored at
-70EC and thawed in 30EC water-bath before titration assay.
Pathology
[0167] To evaluate toxicity due to the i.v. administration of
VSVG-pseudotyped lentiviral vector, hematoxylin and eosin
(H&E)-stained sections from 17 treated and 12 control mice were
examined by light microscopy (Table 2). Mucosal and submucosal
edema was found in the gastrointestinal tract of 14 treated and 6
control mice, and congestion was observed in the livers of both
control and treated mice. These changes appear to have resulted
from the perfusion and fixation procedure. One mouse (F11)
developed a brain abscess, presumed to be the result of injury from
the blood collection procedure. Lymphocyte infiltration or other
signs of inflammation were not observed in any of the examined
organs. Thus, no significant lesions attributable to the test
article were found in any of the tissues.
TABLE-US-00003 TABLE 2 Pathological findings of treated and control
animals Mouse Number Liver Spleen Lung Heart Kidney Brain GI tract
Gonad Skin* 4 Days post-administration Control ConM1 NSL NSL NSL
NSL NSL NSL -- NSL NSL ConM3 NSL -- NSL NSL NSL NSL edema NSL --
ConF4 Cong NSL -- -- -- -- edema -- NSL ConF5 -- -- NSL NSL NSL NSL
NSL NSL -- ConF6 NSL NSL NSL NSL NSL NSL edema NSL NSL Treated
TrM12 NSL NSL NSL NSL NSL NSL edema -- -- TrM13 NSL NSL NSL NSL NSL
NSL NSL NSL -- TrM14 NSL NSL NSL NSL NSL NSL edema NSL NSL TrF13
NSL NSL NSL NSL NSL NSL edema NSL -- TrF14 NSL NSL NSL NSL NSL NSL
edema NSL NSL TrF15 NSL NSL NSL NSL NSL NSL edema NSL NSL 40 Days
post-administration Control ConM5 Cong NSL Hemo NSL NSL NSL edema
-- NSL ConM6 NSL NSL NSL NSL NSL NSL NSL NSL -- ConM15 NSL NSL NSL
NSL NSL NSL edema NSL -- ConF1 NSL NSL NSL NSL NSL NSL edema -- NSL
ConF2 NSL NSL NSL NSL NSL NSL NSL NSL NSL ConF3 NSL NSL NSL NSL NSL
NSL NSL -- -- ConF16 NSL NSL NSL NSL NSL NSL NSL NSL -- Treated
TrM7 Cong NSL NSL NSL NSL NSL edema NSL -- TrM8 Cong NSL NSL NSL
NSL NSL edema NSL -- TrM9 NSL NSL NSL NSL NSL NSL edema NSL --
TrM10 Cong NSL NSL NSL NSL NSL edema -- NSL TrM11 NSL NSL NSL NSL
NSL NSL edema NSL -- TrF7 NSL NSL NSL NSL NSL NSL NSL -- NSL TrF8
NSL NSL NSL NSL NSL NSL edema NSL -- TrF9 Cong NSL NSL NSL -- NSL
edema -- NSL TrF10 NSL NSL NSL -- NSL NSL edema NSL -- TrF11 NSL
NSL NSL NSL NSL abscess NSL edema -- TrF12 Cong NSL -- -- -- --
edema -- -- NSL, no significant lesions were found. Cong, small
foci of sinusoidal congestion. Hemo, small foci of extravasation of
blood. Edema, mucosal and submucosal edema due to perfusion and
fixation procedures. --, Not examined. *Tail was examined at the
injection site.
Duplex Real-Time PCR for GFP and Apob
[0168] To achieve a high degree of sensitivity, reproducibility,
and accuracy in quantitating gene transfer efficiency, a real-time
QPCR assay was established for concurrent quantification of GFP and
endogenous mouse apolipoprotein B (Apob) in a single reaction
vessel. The amplification plots for GFP shift to the right as
initial transgene input is reduced (from 100% to 0.001%) while
same-well Apob amplifications remain constant because total DNA
content is similar in all samples (that is, 1 :g/well as determined
by spectrophotometry). A common threshold was selected in the
exponential phase of PCR reactions to determine a specific
threshold cycle number for each sample.
[0169] To determine whether the concurrent amplification of GFP and
Apob in the same well was occurring under optimized reaction
conditions, amplifications of GFP were compared with same well Apob
from a serial dilution (>5-log-fold) of a reference cell
preparation, that is, cells containing one copy of GFP per cell.
Theoretically, if the reaction efficiencies for both genes were the
same and constant across various concentrations of DNA template,
the difference in amplifications between the two targets should
remain constant. This proved to be true, as indicated by the two
parallel amplification curves for GFP and Apob, and further
confirmed by graphing the difference in threshold cycle numbers (
)Ct) against the log of the DNA dilution fold. Regression analysis
of these data demonstrated a horizontal line with a negligible
slope (0.04). This result indicated that the PCR efficiency for GFP
was comparable to that for Apob in this duplex reaction system,
thus confirming that concurrent amplification of Apob would serve
as a reliable internal reference against which to normalize GFP
measurements.
[0170] To evaluate further the sensitivity, accuracy, and
reproducibility of this real-time QPCR assay, standard curves were
established by plotting normalized threshold cycle number (nor CT)
against transgene frequency using a set of standard samples.
Although each of the standards contained a different percentage of
GFP transgene, the total concentration of DNA remained similar.
When Ct was normalized for DNA concentration on the basis of equal
optical density (OD) readings using a spectrophotometer, the linear
regression analysis of the curve indicated a slope of -4.02, with
r.sup.2 of 0.984. An even higher squared correlation coefficient
(0.999) was observed in standard curve using the same set of
samples when normalizing Ct-GFP with in-well Apob readings. These
results indicate an extremely efficient quantitation assay that
remains linear (5-log fold) from 100% to 0.001% transduced cells.
Considering that 1 :g DNA is equivalent to about 10.sup.5 cells,
this assay can detect as few as one copy of GFP per reaction.
Moreover, there was a high reproducibility of this real-time QPCR.
A "mean standard curve" was generated by analyzing nor Ct values
derived from 20 PCR reactions (amplified in 10 separate runs) of
the same set of samples. The standard deviation was <3% of nor
Ct for each of the standard samples, suggesting a stable high
reproducibility over the 5-log-fold quantitation.
Biodistribution of GFP Transgene 4 Days after Administration
[0171] To assess where, and to what degree, the HIV-based
GFP-containing lentiviral vector localized within the animals
shortly after vector administration, 10 different organs were
carefully collected from six treated and six control mice 4 days
after injection. Mice were perfused transcardially for 20 minutes
to minimize potential DNA contamination from blood. Cross sections
of each organ were examined for GFP transgene by real-time QPCR
assay. High levels of GFP were observed in bone marrow (ranging
from 5 to 37 GFP copies per 100 genome equivalents), liver (12-59),
and spleen (20-54) in treated mice. In contrast, transgene was
undetectable in the brain of one animal and the gastrointestinal
tract of all treated mice. Various amounts of transgene were
obtained in other organs from all treated animals, ranging from
0.01 to 1 GFP copy per 100 genome equivalents. These observations
demonstrated a variable distribution of in vivo-transduction
capability of lentiviral vectors.
Biodistribution of GFP Transgene 40 Days after Administration
[0172] To assess stably transduced transgene efficiency in treated
mice, organ distribution of GFP was determined in mice 40 days
after injection (Table 3). Relatively high levels of GFP were
observed in liver (ranging from 0.3 to 1.3%) and spleen (ranging
from 0.045 to 0.38%), which were substantially lower than those in
mice 4 days after injection. Gonads from all but one treated animal
(TrF9) contained undetectable to barely detectable levels of GFP
(<9 copies GFP per 10.sup.5 cells). From undetectable to 0.30%
transgene was found in other organs. Remarkably, very high levels
of transgene (4.7-22.7%) were observed in bone marrow from all but
one mouse. These levels are comparable to those observed in mice 4
days after treatment. These results suggested that bone marrow
might be the organ most accessible to VSVG-pseudotyped lentiviral
vector.
TABLE-US-00004 TABLE 3 Tissue distribution of GFP in mice 40 days
after vector injection (%) Mouse Number Gonad Bladder GI tract
Brain Kidney Heart Lung Spleen Liver BM Male Control ConM4 UD 0.027
UD na na UD UD UD UD UD ConM6 UD UD UD UD UD UD UD UD 0.02 UD
ConM15 UD UD UD 0.003 UD 0.002 UD UD UD na Treated TrM7 0.005 0.037
UD 0.061 UD na UD 0.267 0.639 17.94 TrM8 0.008 0.087 0.002 0.161
0.002 0.043 UD 0.137 0.550 na TrM9 0.003 0.060 0.004 UD 0.001 0.015
UD 0.235 0.468 13.56 TrM10 0.003 0.022 UD 0.007 0.002 0.023 0.003
0.202 0.762 na TrM11 UD 0.040 UD 0.023 0.003 0.077 0.304 0.045
1.263 14.54 Female Control ConF3 UD UD UD UD UD na 0.005 UD UD UD
ConF4 0.003 na UD 0.003 UD UD 0.001 UD UD na ConF16 UD UD UD UD UD
UD UD UD 0.01 UD Treated TrF7 0.006 na UD 0.005 UD 0.003 0.073
0.289 0.256 na TrF8 0.006 0.066 UD UD UD 0.001 0.002 0.183 0.713
4.743 TrF9 0.034 0.058 UD 0.053 0.003 0.007 0.008 0.166 0.878 7.895
TrF10 UD 0.202 UD 0.014 0.02 UD 0.060 0.072 1.292 15.12 TrF11 0.009
UD 0.001 0.111 0.001 0.022 0.026 0.132 0.969 0.208 TrF12 na 0.846
UD 0.102 0.003 UD 0.003 0.377 0.728 22.68 UD, un-detectable. na,
not available.
Transgene Frequency in Peripheral Blood Leukocytes
[0173] To monitor the transgene in blood cells, whole blood was
collected periodically on days 4, 11, 25, and 40 after injection
and analyzed by real-time QPCR. GFP transgene frequency in
peripheral blood leukocytes (PBL) decreased significantly
(P<0.0001) from a mean of 0.77% (.A-inverted.0.27) on day 4 to
0.07% (.A-inverted.0.04) on day 11. Interestingly, the GFP level
increased back to 0.73% (.A-inverted.0.47) on day 25, a comparable
result to that on day 4 (P=0.858). Remarkably, it continued to
increase to a level of 20.8% (.A-inverted.17.1) on day 40 following
injection (P<0.002), indicating the presence of an additional
source for GFP.sup.+ leukocytes. This latter result is consistent
with the earlier observation of high levels of GFP transgene in
bone marrow from mice assayed 40 days after injection.
Transgene Expression in PBL by FACS Analysis
[0174] To determine whether the genetically translated GFP is
biologically active, GFP expression and the murine B-cell or T-cell
subsets were analyzed by two-color flow cytometry with
phycoerythrin (PE) conjugates in whole blood collected
periodically. FACS analysis failed to identify GFP-expressing cells
in PBL on days 4, 11, and 25, although low levels of GFP transgene
were detected by real-time PCR (<1%). However, up to about 10%
of leukocytes were found to be GFP in mouse TrM7, whose blood
contained the highest level of transgene (62.7%) 40 days after
injection. Moreover, GFP-expressing cells included both
CD45R/B220.sup.+ B cells and CD3e.sup.+ T cells.
Transgene Expression in Liver Visualized by Immunochemical
Staining
[0175] To characterize transduced cell type(s) further, mouse
livers were studied that had relatively high levels of gene
transfer by QPCR by immunochemical staining for GFP cells. In
livers 4 days after injection (with 12-59% transgene frequency),
the GFP cells (reddish-brown staining in cytoplasm) were most
likely to be hepatocytes adjacent to blood vessels. However, no
identifiable GFP.sup.+ cells were detected in liver derived from
mice 40 days after injection; approximately 1% transgene frequency
was measured by real-time QPCR (Table 3).
Discussion
[0176] With >300 phase I/II clinical trials conducted worldwide
over the last decade, gene therapy represents one of the fastest
growing areas in experimental medicine (Romano et al., 2000).
Tissue biodistribution is an important aspect of characterizing new
vectors, one that has received great attention from the FDA and the
National Institute of Health's (NIH) Recombinant Advisory Committee
(FDA, 1991; FDA, 1998; Pilaro et al., 1999). However, only limited
data are available from preclinical effectiveness and toxicity
studies needed to evaluate these new products (Verdler et al.,
1999). HIV-based lentiviral vectors are promising tools for in vivo
gene therapy, but their safety issues are more critical because of
their origins. Although gene transfer and transgene expression of
VSVG-pseudotyped HIV-based vectors have been demonstrated by
several groups in various organs (Naldini et al., 1996; Kafri et
al., 1997; Johnson et al., 2000; Miyoshi et al., 1997; Woods et
al., 2001), the biodistribution and systemic effects have not been
assessed. In this study, the organ biodistribution and general
toxicity of a first-generation lentiviral vector after tail-vein
injection in mice was assessed. A real-time QPCR assay was
established with a broad range of quantitation (5-log fold) to
detect as few as one copy of GFP per 10.sup.5 cell genomes. Such
studies are crucial in understanding both the potential efficacies,
as well as the level of risk for germline transmission (Pilaro et
al., 1999). The unexpected observation of high bone marrow
transgene frequency (ranging from 0.21 to 22.7% of total
bone-marrow genome) has important implications for stem cell gene
therapy.
[0177] Accurate quantitation of gene transfer (or gene correction)
has been a universal challenge to the field of gene therapy. High
sensitivity and reproducibility of such assays are key requirements
for a successful biodistribution study, especially when extremely
low gene transfer is anticipated in some of the nontarget organs
such as gonad (Gordon, 1998). PCR-based DNA analysis has been
specified as an adequate method in biodistribution studies by the
FDA (Pilaro et al., 1999). A major impediment to the use of PCR as
a quantitative technique has been the inherent change in kinetics
of the chemical reaction over time as substrates and other
essential components are consumed (Orlando et al., 1998). With a
given PCR cycle number (for example, 25 cycles), PCR amplifications
from different amounts of initial target templates could be at
different stages (geometric, linear, and plateau phases) with
divergent amplification rates and efficiencies. Therefore,
conventional end-point PCR methods are limited by the sensitivity
of detection in both linear and plateau phases of the PCR. However,
a systemic biodistribution study of gene transfer frequency demands
the capability to quantitate target templates over several-log
fold.
[0178] A new technique for quantitating PCR products in real time
has been developed recently (Held et al., 1996; Becker et al.,
1999). It is able to identify and measure amplification signals in
"real time" (that is, every 7 seconds) during PCR reactions. The
real-time QPCR assay established here could measure as few as one
copy of target sequence in a background of about 10.sup.5 genomes
(1 :g DNA). Moreover, reproducibility and accuracy were
consistently high over a wide range of quantitation, with
5-log-fold difference in the amount of target templates (1 to
10.sup.5 copies). In addition, the assay was further optimized by
simultaneously quantitating both target GFP sequence and
internal-control Apob sequence in the same reaction. In each
reaction well, the amplification of Apob serves as an internal
reference to validate each reaction mixture (an extremely important
issue when the target sequence is very low or undetectable). Such a
modification also has the advantage of reducing sampling and other
systemic errors, and eliminates the need for a second set of
control reactions. Thus, it is more accurate and economical.
[0179] Wild-type vesicular stomatitis virus (VSV) has a broad host
range extending from insects to nearly all mammals (Schnitzlein et
al., 1985). In humans, VSV infections result in nonsevere
influenza-like symptoms (Fields et al., 1967). The VSV glycoprotein
G is the major antigenic determinant responsible for virus
attachment and membrane fusion (Coll, 1995). Unlike most viral
envelope proteins, which must bind to a specific cell-surface
protein receptor to mediate infection (Albritten et al., 1989;
Sattentau et al., 1986), VSVG interacts with an intrinsic
phospholipid component of the plasma membrane (Mastromanno et al.,
1987; Knoieczko et al., 1994). Moreover, the VSVG also has the
unique ability to withstand the shearing forces encountered during
ultracentrifugation. Therefore, to broaden the target cell range
and increase potency in vector preparations, VSVG has been utilized
to "pseudotype" gene therapy vectors such as retroviral (Burns et
al., 1993), HIV-1-based (Naldini et al., 1996), and FIV-based
(Poeschla et al., 1998) lentiviral vectors.
[0180] In vitro studies on VSVG-pseudotyped HIV-1-based vectors
have demonstrated efficient gene transfer into nondividing airway
epithelial cells (Goldman et al., 1997), unstimulated primary T
lymphocytes (Costello et al., 2000), non-prestimulated CD34.sup.+
cells (Douglas et al., 1999), terminally differentiated
macrophages, and peripheral blood monocyte-derived dendritic cells
(Schroers et al., 2000). By route of local administration, in vivo
gene delivery has been accomplished in rat brain (Naldini et al.,
1996; Blomer et al., 1997), in liver and muscle (Kafri et al.,
1997), in retina (Mixoshi et al., 1997), and in airway epithelia
(Johnson et al., 2000). In this study, overall in vivo organ
distribution was assessed by injecting a low dose of lentivirus
i.v. into mice (2 H 10.sup.7 IU/mouse). Relatively high gene
transfer was observed in bone marrow (ranging from 0.21 to 22.7%
transgene frequency), liver (0.26-1.3%), and spleen (0.045-0.38%)
from mice 40 days after injection. Variable low levels of transgene
were observed in bladder (from undetectable to 0.85%), lung (from
undetectable to 0.30%), heart (from undetectable to 0.021%), brain
(from undetectable to 0.16%), kidney (from undetectable to 0.003%),
and gastrointestinal tract (from undetectable to 0.004%). These
observations do not conflict with those observed by others (Park et
al., 2000), in which a relatively high dose of lacZ-containing
lentivirus (1 H 10.sup.8 TU) was injected into the portal vein of
mice. The expression of 3-galactosidase was detected by X-gal
staining in liver (0.16.A-inverted.0.08% of hepatocytes) and
spleen, but not in the brain, heart, lung, kidney, and
duodenum.
[0181] The GFP transgene frequency was surprisingly high in liver
(up to 59%) and spleen (up to 54%) from mice 4 days after
injection, and decreased dramatically to a maximum of only 1.3% in
liver and 0.38% in spleen from mice 40 days after injection. This
change may be due to the existence of abundant defective vector
particles that contain partial reverse transcripts, and the loss of
extrachromosomal proviral DNA. It has been reported that only
0.1-1% of the virus particles in VSVG-pseudotyped lentiviral vector
preparations were infectious, when using the minus strong-stop cDNA
fragment that was present in viral capsids as template for
real-time QPCR (Scherr et al., 2001). Therefore, in this study
about 2 H 10.sup.11 particles were injected into each mouse, with
about 2 H 10.sup.7 transduction units. Varied partial
reverse-transcription (RT) intermediates have been found to be
present in newly assembled HIV-1 particles (Trono, 1992). These
cDNA intermediates and their derivatives may have contributed to
the GFP transgene signal in 4-day animals. Moreover, it has been
found that unintegrated lentiviral proviral DNA may persist in
transduced TE671 (muscle), 293T (kidney), and HepG2 (liver) cells
for more than 4-5 passages, but disappear by 40 passages (Chang et
al., 1999). Transient GFP expression caused by integrase-defective
lentiviral vectors was observed for as much as 10 days in
CD34.sup.+ cells and 14 days in 293 cells (Haas et al., 2000). No
lymphocyte infiltration or other signs of inflammation were
observed in any liver or spleen samples from mice 4 days or 40 days
after injection in this study, although a transient elevation was
observed in serum alanine amino-transferase level by others (Park
et al., 2000). Thus, the loss of GFP transgene in the liver is
unlikely to be related to the loss of transduced cells resulting
from liver toxicity.
[0182] One of the most disturbing concerns for gene therapy in
humans is the possibility of germ line integration of transgene,
which might result in the introduction of heritable genetic changes
into the offspring of patients (Pilling, 1999). Germline
integration may lead to insertional mutations that might have
devastating consequences, as indicated in some of the transgenic
mice produced by pronuclear microinjection (Woychik et al., 1985;
McNeish et al., 1988). To assess the risk of germline integration
by a first-generation lentiviral vector, transgene frequency was
quantitated in whole gonads of mature mice (Table 3). From
undetectable to 9 copies/10.sup.5 genome levels of transgene were
found in all testes (n=5), and all but one ovary (n=5). A very low
level of transgene (3 copies/10.sup.5 genomes) was observed in one
of the six control animals, although special precautions were taken
during perfusion, necropsy, DNA isolation, and QPCR assay. Thus,
the possibility of cross-contamination cannot be ruled out. In
addition, very high levels of transgene (2.08.A-inverted.1.7 H
10.sup.4 copies/10.sup.5 genomes) detected in the PBL of treated
mice may contaminate the gonad if not eliminated completely by
perfusion. Even if the transgene found in the gonads is real, the
hematopoietic spread of lentiviral vector to spermatocytes or
developing oocytes is unlikely because they are relatively
inaccessible to large molecules or to infection by viruses (Gordon,
1998). Moreover, there are statistical considerations that mitigate
against the germline transfer of foreign DNA that reaches an
offspring. For example, integration of transgene into one
spermatogenic cell would lead to the genetic transformation of only
a few of the millions of cells that would ultimately reach the
ejaculate. Also, of the about 400,000 oocytes present in the human
ovary at the onset of menstruation, only a few hundred are ovulated
during the reproductive lifespan of a woman, and fewer than a dozen
of those ovulated oocytes contribute their genes to subsequent
offspring. Thus, together with the observations provided herein,
the risk of germline transmission of the first-generation
lentiviral vector by i.v. administration is very low.
[0183] The most surprising observation was that bone marrow
exhibited the highest transgene frequency (ranging from 0.21 to
22.7% of total bone-marrow genome) in all mice tested 40 days after
injection (Table 3). This was consistent with the observation that
high levels of transgene were detected in PBL from these animals
(ranging from 0.61 to 62.7%). It was also supported by the results
that up to 10% of PBL expressed GFP as determined by FACS analysis.
In addition, the transgene levels in PBL decreased significantly
from a mean of 0.77% on day 4 to 0.07% on day 11, and then
increased considerably back to 0.73% on day 25 and to 20.8% on day
40 following injection. This observation suggested the presence of
an additional resource for GFP.sup.+ leukocytes, implying the
transduction of hematopoietic progenitor cells. This study provides
the first indication that bone marrow may be a susceptible target
tissue for i.v. administration of VSVG-pseudotyped lentiviral
vectors. It is possible that intravenously delivered lentiviral
vector may reach stem cells; ex vivo transduction studies have
demonstrated the capability of lentiviral vectors to transduce more
primitive and quiescent stem cells (Woods, 2001; Case et al.,
1999). The i.v. approach may overcome some of the difficulties
encountered by ex vivo approaches, such as limited gene transfer
efficiency, maintenance of long-term engraftment of the transduced
cells, and in vitro manipulation steps (Richter et al., 2001).
Example II
[0184] Mucopolysaccharidosis type I (MPS I) is an inborn error of
lysosomal glycosaminoglycan (GAG) metabolism resulting from
deficiency of alpha-L-iduronidase (IDUA). While allogeneic
hematopoietic stem cell (HSC) transplantation results in systemic
metabolic correction, including prevention of neurologic damage,
attempts to exploit Moloney murine leukemia virus vectors have
failed to demonstrate the requisite qualities to merit substitution
of ex vivo HSC gene therapy for allogenic HSC transplantation.
Lentiviral vectors may integrate into non-dividing cells and may
have broad tropism when pseudotyped with VSV-G envelope, thus
potentially solving this problem.
[0185] A murine model of MPS I (kindly provided by Hong-Hua Li and
Elizabeth F. Neufeld; Zheng et al., 2001) was used to evaluate
transgene expression in fibroblasts from these mice. Mice were
genotyped with a SYBR Green assay from which primary skin
fibroblast cultures were established. Second- and third-generation
HIV-1 based vectors were generated to transduce these
IDUA-deficient murine fibroblasts with either GFP or IDUA. Second
generation (three-plasmid) and third generation (four-plasmid) SIN
lentiviral vectors were prepared with either GFP (pCS-CG) or IDUA
(pCS-P1) transgenes by co-transfection into 293T cells. Potency of
48 hour viral supernatants was assessed by two methods. In the
first assay, real-time quantitative PCR was exploited using a
primer-probe set for the minus strong-stop cDNA (U5/R region) of
encapsulated lentiviral genomes (Scherr et al., 2001) yielding
titers between 2.times.10.sup.6 genomes/mL and 5.times.10.sup.7
genomes/ml. In the second assay, 293T cells were exposed to GFP
vector supernatants, and then subjected to FACS analysis to select
for transduced cells yielding titers of 1.times.10.sup.5 TU/mL.
Third-generation vectors were found to be comparable to
second-generation preparations.
[0186] Murine IDUA-deficient fibroblasts (2.5.times.10.sup.5
cells/plate) were exposed to various concentrations of vector and
incubated for 24 hours before changing the media, and then cultured
for an additional 6 days prior to analysis. Cells transduced with
third generation IDUA (pCS-P1) were found to have markedly
increased IDUA enzymatic activity (60-120 nmol/mg/hr) comparable to
that of normal human fibroblasts or leukocytes. Quantitative PCR
assays for the IDUA and GFP transgenes quantified the level of
integration of the transgene within the genome (see Example I). The
real-time QPCR assays for the third generation IDUA and GFP vectors
found that the average percentage of transduced cells was 60%
whereas the negative control was 0%. Microscopy analysis of GFP
transduced cells showed similar results.
[0187] For in vivo transduction, a transgene plasmid (pCS-P1)
containing the human IDUA cDNA sequence under transcriptional
control of the human PGK promoter was prepared. Vector preparations
were generated by transient cotransfection of 293T cells using a
third-generation 4 plasmid packaging system with Rev function
(pEFRev) separated from other helper functions (p2NRF) (kindly
provided by T. Kafri et al. Real-time quantitative PCR methods were
exploited to determine lentiviral particle numbers in vector
preparations, and to assess transduction efficiency (Example
I).
[0188] Intravenous injection of an IDUA encoding
replication-defective (third generation) VSVG pseudotyped
lentiviral vector (e.g., 5.times.10.sup.7 TU; FIG. 1) into newborn
mice resulted in higher-than-normal levels of alpha-L-iduronidase
in blood (FIG. 2). The circulating levels were up to 1-log-fold
higher than normal circulating levels, and those levels were much
higher than those achieved in efficacious bone marrow
transplantation. Further, the circulating levels were up to at
least 1-log-fold higher than those achieved by any other means of
gene therapy, e.g., intravenous injection of a retrovirus vector in
a mouse model of mucopolysaccharidosis type VII (Xu et al., 2002).
Moreover, circulating levels of enzyme persisted throughout the
period of observation (i.e., 3 months after treatment). Because
lentiviral vectors integrate the therapeutic gene into the host
chromosome, therapeutic levels of protein (enzyme) are expected to
persist for long periods of time, probably for the lifetime of the
treated individual.
[0189] In addition, intravenous infusion of newborn MPS I mice with
an IDUA encoding recombinant lentivirus resulted in normal facial
appearance (FIG. 3) indicating the efficacious effect of transgene
expression on bone growth. Further, intravenous infusion of newborn
MPS I mice with the IDUA encoding vector also resulted in normal
parameters of behavior (FIG. 4) indicating the efficacy of this
treatment on the progressive brain degeneration of MPS I. Moreover,
FIG. 5 shows a micrograph of the pathology observed in mice with
Hurler syndrome as well a micrograph of IDUA lentivirus treated
mice which demonstrates that treated mice have a decrease or lack
of GM-2 ganglioside pathology.
Example III
Materials and Methods
[0190] Plasmids. The plasmid for 5B transposon expression,
pT-CAGGS-GUS (transposon), was constructed as follows. A 2.3 kb
fragment containing human GUSB cDNA was excised from pHUG13 (ATCC
95658) by EcoRI digestion and ligated to EcoRI sites in the poly
linker in pCAGGS. The resulting expression cassette for GUSB
expression included a cytomegalovirus enhancer, chicken
.beta.-actin promoter, the initial intron of the chicken
.beta.-actin gene, GUSB cDNA, and a rabbit .beta.-globin and SV40
polyadenylation signal. This expression cassette was inserted
between SspI and HindIII sites of the pT/BH transposon polylinker.
The SB transposase expression plasmid pCMV-SB10 has been previously
described (Ivics, 1997). Pyrogen-free plasmids were used in this
study and were isolated using QiaFree kit (Qiagen, City,
State).
[0191] Mice. MPS VII mutant mice (B6.C-H-2bml/ByBir-gus.sup.mps)
were obtained from Jackson Laboratories (Bar Harbor, Me.) and
maintained in the AAALAC-accredited Specific Pathogen-Free mouse
facility at the University of Minnesota. Homozygous mutant mice
were produced by breeding of heterozygotes. Genotyping was
performed by allelic discrimination assay using TaqMan
chemistry.
[0192] Injections. The plasmids were injected into the tail vein
using a 3-cc latex-free syringe with a 271/2 G needle. The
hydrodynamics-based procedure was performed as described in Wolff
et al. (2000). Each mouse received 25-37.5 .mu.g of plasmid DNA in
lactated Ringers solution, in a total volume equal to 10% of body
weight. One animal from each group died before completion of the
experiment: one mouse from Treatment Group 1 and one mouse from
Treatment Group 3 died on 1 week post-injection and one mouse from
Treatment Group 2 died 4 weeks post-injection. The organs from
these mice were resected within 8 hours of death and used for
enzyme quantification and GUSB histochemical staining.
[0193] Treatment regimens. In the short-term experiment, four
groups of MPS VII or wt mice (n=4 each) received 25 .mu.g of
pT/BH-CAGGS-GUS (MPSVII and treatment groups) or pBluescript (MPS
VII and wt control groups). The mice were euthanized by CO
inhalation 48 hours after injections. 400 .mu.l blood was drawn
from the heart for plasma isolation, and livers were extracted and
preserved for biochemical molecular, histochemical and pathological
analysis.
[0194] For the long-experiment, MPS VII mice age 4-26 weeks were
used. An aliquot (25 .mu.g) of a single preparation of transposon
pT-CAGG5-GUSB was injected either alone (Treatment Group 1), or
with pCMV-SB10 at 1:1 (Treatment Group 2) or 10:1 (Treatment Group
3) molar ratios. The amount of injected DNA was kept the same in
each group (37.5 .mu.g) with the filler plasmid, pBluescript. The
control group of MPS VII mice was injected with the filler plasmid
alone. All injections were performed only once.
[0195] The mice were bled by retroorbital phlebotomy 48 hours, 1
week, 2 weeks, and 4 weeks post-injection to obtain plasma for
enzyme assays. 8 weeks post-injection, mice were euthanized by
CO.sub.2 inhalation, and the organs (liver, spleen, heart kidney,
lung, brain, and gonads) were resected, cut into 2 mm.sup.3
sections and preserved in different ways for analysis. Prior to
organ resection, 400 ml of blood was drawn for plasma and white
blood cell (WBC) isolation.
[0196] For lysosomal enzyme quantification, plasma and tissues were
snap-frozen in liquid nitrogen and stored at -80.degree. C.
Activities of GUSB, alpha-galactosidase and total
.beta.-hexosaminidase were measured in tissue homogenates and
plasma using fluorometric assay. Protein concentrations were
determined with Bradford assay (Bradford, 1976) using Bio-Rad
reagent.
[0197] For histology and histopathology studies, tissues were fixed
in 10% neutral-buffered formalin, embedded in paraffin and
sectioned at 6 .mu.m for staining with Hematoxylin and Eosin.
[0198] Histochemical localization of GUSB was performed in 6-8
.mu.m frozen sections stored at -80.degree. C. using
AS-BI-naphthol-.beta.-D-glucuronic acid (Sigma) as described in
Wolfe and Sands (2000) and Ghodsi et al. (1998).
[0199] For detection of storage vacuoles, tissues were fixed in
2.5% glutaraldehyde in 0.1 N cacodylate buffer for at least 48
hours at 4.degree. C. Tissues were embedded in Epon 812 resin
(Electron Microscopy Sciences, Ft. Washington, Pa.). Sections (0.5
.mu.m) were prepared and stained with toluidene blue as described
in Wolfe and Sands (2000).
Results
[0200] The transposon plasmid carried human GUSB cDNA regulated by
a strong promoter (FIG. 6) which expressed very high levels of
.beta.-glucuronidase. Correct assembly of pT-CAGG5-GUSB was
validated by restriction enzyme analysis, and the structure of GUSB
was confirmed by sequencing (data not shown). GUSB expression was
confirmed by transfection of primary .beta.-glucuronidase-deficient
murine fibroblasts. To determine whether beta-glucuronidase can be
detected in plasma 48 hours after hydrodynamic infusion of adult
MPS VII mice and wild-type mice. Mice at 8-12 weeks of age were
injected with 25 .mu.g of either pT-CAGG5-GUS plasmid or
pBluescript. All mice tolerated the procedure well with only one
death of a wild-type mouse.
[0201] The plasmid pT-CAGGS-GUS mediated high levels
.beta.-glucuronidase expression in the liver after hydrodynamic
infusion. In GUS-deficient mice, as well as in wild-type mice, foci
of intensively bright red staining were evenly distributed in liver
tissue (FIG. 7). Counterstaining with methyl green permitted
localization GUSB activity to hepatocytes (predominantly) and
Kupffer cells. Varying degrees of staining were observed in all
cells, suggesting enzyme cross-correction. Beta-glucuronidase
activity (Table 4) in treated MPS VII animals exceeded that in
untreated wild-type mice by over 10-fold.
TABLE-US-00005 TABLE 4 Beta-glucuronidase Enzyme Activity in Plasma
and Liver 48-hours after Hydrodynamic Infusion of pT-CAGGS-GUSB
plasmid. Enzyme activities* MICE Injection GUS hexosaminidase
galactosidase .sctn.Gen Weight Vol. Time Quality** Liver Plasma
Liver Plasma Liver PT/CAGGS-GUS, 25 .mu.g MPSVII 1 M 25.0 g 2.5 ml
10 seconds Excellent 2,552 5,209 4,300 5890 124 MPSVII 2 M 24.7 g
2.5 ml 12 seconds Good 1,860 2,715 4,386 N/A 132 MPSVII 3 M 22.9 g
2.3 ml 19 seconds Poor 99 3 5,185 1,229 123 MPSVII 4 F 19.0 g 1.9
ml 12 seconds Fair 2 2 6,561 8,107 123 Unaffected 5 F 20.3 g 2.0 ml
10 seconds Good 75 119 936 953 49 Unaffected 6 M 30.6 g 3.0 ml 14
seconds Fair 16 123 675 611 43 Unaffected 7.infin.F 19.9 g 2.0 ml
10 seconds Excellent 314 297 911 453 49 Unaffected 8 M 26.8 g 2.7
ml 9 seconds Excellent Died within 1 hour post injection
pBluescript, 25 .mu.g MPSVII 9 F 20.4 g 2.0 ml Injection failed,
mouse survived but was not used further MPSVII 10 F 17.7 g 1.8 ml
10 seconds Excellent 1 2 6,786 1,365 130 MPSVII 11 F 21.4 g 2.1 ml
8 seconds Excellent 1 1 6,526 4,750 153 MPSVII 12 F 20.0 g 2.0 ml 8
seconds Excellent 1 2 7,634 1,520 179 Unaffected 13 M 28.6 g 2.9 ml
15 seconds Poor 118 7 696 491 42 Unaffected 14 F 28.0 g 2.8 ml 9
seconds Excellent 9 9 1,187 792 75 Unaffected 15 F 22.9 g 2.3 ml 12
seconds Poor 86 9 845 633 38 Unaffected 16 F 22.8 g 2.3 ml 9
seconds Excellent 94 12 1,031 649 37 Untreated MPSVII 17 M -- -- --
-- 1 1.7 5,210 1,365 125 MPSVII 18 F -- -- -- -- 1.3 1.4 7,231
2,679 148 MPSVII 19 F -- -- -- -- 1.1 1.9 6,718 1,868 130
Unaffected 20 F -- -- -- -- 87 14 985 675 44 Unaffected 21 F -- --
-- -- 84 9.6 1,004 ? ? Unaffected 22 F -- -- -- -- 188 15 685 589
42 *Enzyme activity is expressed as nmoles of 4 MU/mg protein/h for
liver and as nmoles of 4 MU/ml plasma/h for plasma .sctn.Gen stands
for gender and genotype **Time and Quality refer to the
microinjection time and apparent success at the time of injection
.infin.This mouse is a replacement for an unaffected mouse, in
which injection failed (the needle did not go into the vein)
[0202] .beta.-glucuronidase activity was easily detectable in
plasma (Table 4), and may be used as a presumptive indicator of the
presence/absence of pT-CAGGS-GUSB. This proved to be a convenient
method of monitoring the success of the procedure for administering
the test agent.
[0203] Eight-week .beta.-glucuronidase enzyme activity in plasma
after co-injection with SB plasmid. MPS VII mice were either
injected with pT/CAGGS-GUSB alone (Treatment Group 1) or
co-injected with pCMV-SB10 at two different molar ratios of the
transposon to transposase plasmid: 1:1 (Treatment Group 2) and 10:1
transposon to transposase plasmid (Treatment Group 3). All treated
mice received an equal amount of 25 .mu.g of pT/BH-CAGGS-GUS. The
injected DNA amount was kept equal in all mice by using pBluescript
as the filler plasmid. MPS VII mice from the negative control group
received pBluescript alone. Forty-eight hours after injection,
.beta.-glucuronidase activities in the plasma of treated animals
were equally high in all three surviving animals and in the same
range as those in the short-term experiment (Table 5). One week
post-injection, plasma .beta.-glucuronidase in mice from Treatment
Group 1 was reduced to 71.8% of initial 48 hour post injection
activity. In Treatment Groups 2 and 3, enzymatic activity had
decreased to 37.3% and 28.6% of initial activity, respectively. At
one month, .beta.-glucuronidase in plasma was virtually
undetectable in all three groups. Secondary elevations of other
lysosomal enzymes concomitant with the deficiency of the causative
enzyme have been observed in storage diseases and respond to
treatment. In this series of mice, hexosaminidase levels in
untreated mice were found to be pathologically elevated. At time
point 1 week, hexosaminidase levels in plasma decreased to 78.5% in
Treatment Group 1; 58.5% in Treatment Group 2; and 61.8% in
Treatment Group 3 as compared to time-point 2 days post-injection.
No reduction of hexosaminidase activity was observed in
sham-treated MPS VII mice. At a later time point (time point,
weeks/hours), levels remained/changed (Table 5).
TABLE-US-00006 TABLE 5 Beta-glucuronidase Activity in Plasma 2 and
7 days after co-injection by hydrodynamic infusion of pT-CAGGS-GUS
plasmid % Initial % initial 2 days 7 days Activity Mouse 2 Days 7
Days Activity HEX in plasma, nmole/ml/hr pT/CAGGS_GUS 1 5304 3855
72.7 3,300 2160 65.5 3 3602 2114 58.7 3492 1980 56.7 4 2002 1868
93.3 2211 113.4 Mean 3636 .+-. 1651 2612 .+-. 1083 74.9 .+-. 17.4%,
3001 .+-. 691 2216 .+-. 268 n = 3 pT/CAGGS-GUSB + pSB10 1:1 5 2692
566 21 3491 2132 61.1 6 3800 823 21.7 6600 2165 32.8 7 6405 2074
32.4 2376 2039 85.8 8 3119 1116 35.8 3432 1855 54.1 Mean 4004 1145
28.6%, n = 4 3975 2048 58.5 pTCAGGS-GUSB plus pSB10, 10:1 9 7711
2565 33.3 2343 1835 78.3 10 3372 1172 34.8 2970 1835 61.8 11 0 0
7946 3610 45.4 12 1552 982 63.3 3928 N/A Mean 4212 1573 37.3%, n =
3 4420 2427 61.8 pBluescript 13 Neg. control 0 2897 3274 113 14
Neg. control 0 3815 N/A Wild-type 6.9-14.9 (n = 6) Untreated
[0204] Eight-week enzyme levels in organs. Two months after
administration of pTCAGGS-GUSB transposon, beta-glucuronidase
persisted in liver and spleen in all three groups and the levels of
.beta.-glucurorudase in animals that did not receive transposase
were higher than in those that were co-injected with the
transposase plasmid. Histochemical staining revealed considerable
reduction of positively stained cells both in liver and in spleen
(FIG. 8). Fluorometric quantification of .beta.-glucuronidase
activity showed that in Treatment Group 1 it was approximately
8-fold higher than in either group 1 or 2 (Table 7).
TABLE-US-00007 TABLE 6 Beta-glucuronidase activity in MPS VII mouse
organs 1 week and 1 month after injection, nmol/mg protein/hr Mouse
Organ #2 (No SB) #14 (SB1:10) #8 (SB1:1) W.T. Liver 5184 6185 28
119-188 n = 6 Spleen 6080 4534 3.9 269-290 n = 3 Heart 98 94 2.1
10-13 n = 3 Kidney 59 59 1.9 60-74 n = 3 Lung 49 65 1.8 60-83 n = 3
Gonad N/A N/A 0 223 n = 1 (testis) (testis) (ovary) Brain N/A N/A 0
17-20 n = 3
TABLE-US-00008 TABLE 7 GUSB activity in liver and spleen 8 weeks
post-injection, nmol/mg/hr Transposon pT- CAGGS- Transposase Inert
DNA .beta.-glucuronidase Treatment GUSB pCMV-SB10 pBluescript Liver
Spleen Group (mcg) (mcg) (mcg) Mouse (nmol/mg/h) (nmol/mg/h)
Transposon 25 0 12.5 1 29.5 2.1 Alone 3 18.6 3.2 4 2.4 1.04
Transposon:Transposase 25 12.5 0 6 1.15 0.59 1:1 9 2.21 1.0 10 1.92
0.85 Transposon:Transposase 25 1.25 11.25 11 4.01 0.82 10:1 12 1.51
0.89 15 0.78 2.71 Control (-) 0 0 37.5 17 0.63 0.76 33 0.39 0.75
Control (+) Untreated 166.8; (119-188), 368; 269-290, n = 6 n =
3
[0205] Distribution of .beta.-glucuronidase expression in various
mouse organs was studied at one week (1 mouse from Treatment Group
1 and Treatment group 3), 4 weeks (1 mouse from Treatment Group 2)
(Table 6) and 8 weeks (n=3 from each group) (Table 4). This study
showed that during the first week following hydrodynamic-based
administration, .beta.-glucuronidase activities in liver and spleen
were comparably high; the heart had levels less than 2% that of
liver and spleen. Moreover, the lung had less than 1%, and the
ovary had undetectable, .beta.-glucuronidase activity.
[0206] Extent of correction of the pathology. No lesions were seen
in Hematoxylin and Eosin-stained 6 mm sections of liver, spleen,
lung, testis, ovary, gut, cerebellum, kidney and heart from
treated, sham-treated or untreated MPS mutant mice. Toluidene-blue
staining of 0.5 mm sections of liver and spleen revealed a dramatic
reduction in the number and size of storage vacuoles in all treated
groups (FIG. 8). In Treatment Group 1, where the sections were
indistinguishable from those of normal controls, loss of storage
vacuoles appeared complete. Partial reduction of storage was
observed in Treatment Groups 2 and 3. Remarkably, storage vacuoles
appeared to be not just prevented but actually eliminated in some
mice (e.g., mouse #3 was 215 days of age when killed but had no
vacuoles; the wt untreated control had many vacuoles at 50 days of
age. If treatment of #3 started after it was 50 days of age, the
data suggests that vacuoles were not just prevented, but actually
eliminated).
Discussion
[0207] Therapeutic gene transfer and expression is widely held to
require some extraneous mechanism(s) for molecular stabilization
and for transmission across the tissue and cellular membranes.
Thus, the most feasible forms of gene therapy have used extensively
modified replication-defective viral vectors, liposomes or even ex
vivo electroporation of DNA into cells prior to
transplantation.
[0208] While studying a potential means of intravenous injection of
unmodified DNA into mice, surprisingly it was found that
hydrodynamic administration of a particular plasmid structure was
capable of long-term expression with high levels of enzyme
expression, achieving a potentially curative response, in a murine
model of mucopolysaccharidosis type VII. In particular, expression
of the GUSB transgene was easily detected in plasma. Notably,
plasma levels of glucuronidase were exceedingly high 1 week
post-injection, but became barely detectable after 1 month. This
suggests that such transient expression was due to episomal
delivery, and that the majority of pT-CAGGS-GUSB plasmid did not
persist in an integrated form. By 8 weeks post-injection,
.beta.-glucuronidase activity was undetectable in plasma; however,
glucuronidase activity was detectable in the liver and spleen.
Beta-glucuronidase activity in two out of three mice in Treatment
Group 1 was over 10% that of wt, whereas in Treatment Groups 2 and
3 these levels were around 1% that of normal values. The levels
observed were sufficient to result in the first successful
treatment of a metabolic disease by intravenous injection of a
plasmid.
[0209] Further, the results show there was a "dose effect", with
levels of expression corresponding to the level of reversal of
pathology. These levels of expression were sufficient to reverse
accumulation of GAG.
[0210] As observed by the correlation of glucuronidase enzyme
activity to correction (e.g., FIGS. 7 and 8, and Table 7),
increases in enzyme activity in the liver corresponded to the
degree of metabolic correction. Animals that had the highest level
of glucuronidase activity 8 weeks after treatment were clear of
pathologic lysosomal accumulations. In these animals, there appears
to be a cure of metabolic disease. By extension to the work of
enzyme replacement in this same animal model (Sands et al., 1997),
this would correspond to a cure of the murine MPS VII phenotype in
other studies, especially using non-viral gene transfer
systems.
[0211] The remarkable aspect of the reported study is the apparent
ability to cure this storage disease with out the necessity of
using a viral vector. This is the first time a long-term effect has
resulted from naked DNA gene therapy (i.e., without the benefit of
a viral vector).
[0212] Interestingly, beta-glucuronidase levels were lower when
transposase was present. For example, eight-weeks after
pT-CAGGS-GU5B administration, glucuronidase expression in Treatment
Group 1 was higher than in any (p<0.01) or all (p<0.01) of
the others (Treatment Group 2 and Treatment Group 3). This
difference was not random, but was statistically significant.
Notably, mice Treatment Group 2 and Treatment Group 3 received the
same amount of pT-CAGGS-GUSB, but showed much lower levels of
glucuronidase expression (p<0.01). From this observation, SB
transposase may be responsible for these lower levels.
[0213] Based on these observations, it is likely that expression of
glucuronidase activity is predominantly from an episomal form.
Nevertheless, it appears that episomal gene expression by this
means has the potential for effecting a long-term treatment.
TABLE-US-00009 TABLE 8 Dose effect of .beta.-glucuronidase activity
on storage clearance 2-Day 8-Week 8-Week Liver Spleen Treatment
Glucuronidase Glucuronidase Glucuronidase Vacuole Vacuole Mb Group
Activity Activity Liver Activity Spleen Area Area 34 W.T. 14 166.8
368 0.18 0.09 1 pT- 5304 29.5 2.1 0.75 2.19 3 CAGGS- 3602 18.6 3.2
0.35 2.75 4 GUSB 2002 2.4 1.04 1.11 1.76 6 pT- 2692 1.15 0.59 2.02
2.83 9 CAGGS- 6405 2.21 1.0 3.13 3.35 10 GUSB + 3119 1.92 0.85 2.36
1.93 pCMV- SB10, 1:1 11 pT- 7711 4.01 0.82 1.03 1.83 12 CAGGS- 3372
1.51 0.89 1.55 6.15 15 GUS + 1552 0.78 2.71 1.32 4.69 pCMV- SB10,
10:1 13 Fail 0 0.26 0.52 9.13 11.85 Treatment 17 Sham- 0 0.63 0.76
13.65 N/A treated MPS 33 Untreated 0 .039 0.75 9.54 6.97 MPS
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[0308] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification, this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details herein may
be varied considerably without departing from the basic principles
of the invention.
Sequence CWU 1
1
6124DNAArtificial SequenceSynthetic oligonucleotide 1ccgacaagca
gaagaacggc atca 24223DNAArtificial SequenceSynthetic
oligonucleotide 2ccttgagcag tgcccgacca ttc 23327DNAArtificial
SequenceSynthetic oligonucleotide 3actacaacag ccacaacgtc tatatca
27420DNAArtificial SequenceSynthetic oligonucleotide 4ggcggatctt
gaagttcacc 20519DNAArtificial SequenceSynthetic oligonucleotide
5cgtgggctcc agcattcta 19622DNAArtificial SequenceSynthetic
oligonucleotide 6tcaccagtca tttctgcctt tg 22
* * * * *
References