U.S. patent application number 10/109799 was filed with the patent office on 2003-09-04 for methods and compositions for liver specific delivery of therapeutic molecules using recombinant aav vectors.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Dwarki, Varavani, Escobedo, Jaime, Ponnazhagan, Selvarangan, Schloemer, Robert H., Srivastava, Arun, Wang, Xu-Shan, Yoder, Mervin C., Zhou, Shang-Zhen.
Application Number | 20030166284 10/109799 |
Document ID | / |
Family ID | 26699974 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166284 |
Kind Code |
A1 |
Srivastava, Arun ; et
al. |
September 4, 2003 |
Methods and compositions for liver specific delivery of therapeutic
molecules using recombinant AAV vectors
Abstract
Provided are methods for selectively expressing therapeutic
molecules, such as secretory proteins, antisense molecules and
ribozymes, in the liver. The methods find use in treating hepatic
diseases or conditions. The methods also find use in treating any
disease or condition in which systemic administration of the
therapeutic substance, for example, a secretory protein, is
desired. The methods involve administering to a mammalian patient
having a need for liver expression of a therapeutic molecule an AAV
vector containing a therapeutically effective amount of the
therapeutic molecule. Also provided are novel vectors employable in
these methods.
Inventors: |
Srivastava, Arun;
(Indianapolis, IN) ; Ponnazhagan, Selvarangan;
(Cleveland, OH) ; Schloemer, Robert H.;
(Indianapolis, IN) ; Wang, Xu-Shan; (Carmel,
IN) ; Yoder, Mervin C.; (Indianapolis, IN) ;
Zhou, Shang-Zhen; (Alameda, CA) ; Escobedo,
Jaime; (Alamo, CA) ; Dwarki, Varavani;
(Alameda, CA) |
Correspondence
Address: |
ALISA HARBIN, ESQ.
CHIRON CORPORATION
INTELLECTUAL PROPERTY - R440
P.O. BOX 8097
EMERYVILLE
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
Emeryville
CA
|
Family ID: |
26699974 |
Appl. No.: |
10/109799 |
Filed: |
March 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10109799 |
Mar 28, 2002 |
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08921497 |
Sep 2, 1997 |
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6521225 |
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60025616 |
Sep 6, 1996 |
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60025649 |
Sep 11, 1996 |
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Current U.S.
Class: |
435/456 ;
424/93.2 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2750/14143 20130101; C07K 14/705 20130101; C12N 15/86
20130101; A61P 1/16 20180101 |
Class at
Publication: |
435/456 ;
424/93.2 |
International
Class: |
A61K 048/00; C12N
015/861 |
Claims
What is claimed is:
1. A method for realizing liver specific delivery of a therapeutic
molecule in a mammalian patient comprising administering to said
mammalian patient a therapeutically effective amount of an AAV
vector containing said therapeutic molecule.
2. A method according to claim 1 wherein said therapeutic molecule
is a nucleic acid sequence encoding a secretory protein, an
antisense molecule or a ribozyme.
3. A method according to claim 1 wherein the AAV vector is selected
from the group consisting of pD-5, pD-10, pD-15, pD-20.
4. A method according to claim 1 wherein said AAV vector is
administered by intravenous or intraportal injection.
5. A method according to claim 1 wherein said AAV vector is
administered ex vivo.
6. A method according to claim 1 wherein said AAV vector is
administered by direct injection.
7. A method according to claim 1 wherein said therapeutic molecule
is a nucleic acid encoding the LDL receptor.
8. A method according to claim 1 wherein said AAV vector
additionally contains a liver specific promoter.
9. A method according to claim 8 wherein said AAV vector
additionally contains a liver specific enhancer.
10. A method according claim 8 wherein said promoter is selected
from the group consisting of the hepatitis B virus X gene promoter,
the hepatitis B virus core protein promoter, the AFP gene promoter,
the albumin gene promoter, the .alpha.-1 antitrypsin gene promoter,
the fibrinogen gene promoter, the APO-A1 gene promoter and the
promoter genes for liver transference enzymes.
11. A method according to claim 10 wherein said AAV vector
additionally contains a liver specific enhancer.
12. A hybrid helper AAV vector comprising a DNA sequence encoding
hepatitis B virus surface antigen or a functional fragment thereof
linked to a DNA sequence encoding the AAV VP-1 protein to form a
chimeric DNA sequence.
13. A recombinant AAV vector comprising two AAV ITRs (inverted
terminal repeats) in which the native D-sequences of each of said
ITRs are modified by the substitution of nucleotides such that at
least 5 native nucleotides and up to 18 native nucleotides are
retained and the remaining nucleotides of the D-sequence are
deleted or replaced with non-native nucleotides.
14. A recombinant AAV vector according to claim 13 wherein said at
least 5 native nucleotides are 5' CTCCA 3'.
15. A recombinant AAV vector comprising two AAV ITRs (inverted
terminal repeats) in which the native D-sequences of each of said
ITRs are modified by the substitution of nucleotides such that at
least 10 native nucleotides up to 18 native nucleotides are
retained and the remaining nucleotides of the D-sequence are
deleted or replaced with non-native nucleotides.
16. A recombinant AAV vector comprising two AAV ITRs (inverted
terminal repeats) in which the native D-sequences of each of said
ITRs are modified by the substitution of nucleotides such that 10
native nucleotides are retained and the remaining nucleotides of
the D-sequence are deleted or replaced with non-native
nucleotides.
17. A recombinant AAV vector according to claim 16 wherein said 10
native nucleotides comprise nucleotides 5' CTCCA 3' and five other
native nucleotides of said D-sequence.
18. A recombinant AAV vector selected from the group consisting of
pD-5, pD-10, pD-15 and pD-20.
Description
BACKGROUND
[0001] The therapeutic treatment of diseases and disorders by gene
therapy involves the transfer and stable insertion of new genetic
information into cells. Although a variety of physical and chemical
methods have been developed for introducing exogenous DNA into
eukaryotic cells, viruses have generally been proven to be more
efficient for this purpose. Several DNA-containing viruses, such as
parvoviruses, adenoviruses and herpesviruses, and RNA-containing
viruses, such as retroviruses, have been used to construct
eukaryotic cloning and expression vectors and explored as gene
therapy vehicles.
[0002] Retrovirus- and adenovirus-based vectors are associated with
certain complications and disadvantages. For example, retroviruses
are intimately associated with neoplastic events. See Donahue,
Helper virus induced T cell lymphoma in non-human primates after
retroviral mediated gene transfer, J. Exp. Med. 176(1992)
1125-1135. Adenovirus induces a CTL response. See Yang, MHC class
1-restricted cytotoxic T lymphocytes to viral antigens destroy
hepatocytes in mice infected with E1-deleted recombinant
adenoviruses, Immunity 1 (1994) 433-442. It also requires a
relatively large (35 kb) viral genome, making its usefulness as a
vehicle to deliver large sequences limited.
[0003] Thus, an alternative vector which is neither pathogenic nor
immunogenic would be advantageous. In contrast to adenoviruses, the
parvovirus, adeno-associated virus (AAV), has a much smaller
genome, most of which can be replaced by foreign DNA. Parvoviruses
are small, icohedral viruses approximately 25 nm in diameter
containing a single strand DNA genome of approximately 5 kilobases
(kb). They consist of two major classes: the dependoviruses,
including AAV and its subtypes (AAV1, AAV2, AAV3, AAV4 and AAV5),
and the autonomous parvoviruses. The latter lytically infect
permissive, proliferating cells in nonintegrating manner without
helper virus assistance. On the other hand, AAV is a non-pathogenic
human parvovirus that requires co-infection with a helper virus,
usually adenovirus (or herpesvirus), for its optimal replication.
See for example, Berns, Parvovirus replication, Microbiol. Rev. 54
(1990) 316-329 and Berns and Bohenzky, Adeno-associated viruses: an
update, Adv. Virus Res. 32 (1987) 243-306.
[0004] In the absence of a helper virus, the wild-type (wt) AAV has
been shown to integrate into the human chromosome 19 in a
site-specific manner. See Kotin and Berns, Organization of
adeno-associated virus DNA in latently infected Detroit 6 cells,
Virol. 170 (1989) 460-467; Kotin, Mapping and direct visualization
of a region-specific viral DNA integration site on chromosome
19q13-qter, Genomics 10 (1991) 831-834; Kotin, Site-specific
integration by adeno-associated virus, Proc. Natl. Acad. Sci. 87
(1990) 2211-2215 and Samulski, Targeted integration of
adeno-associated virus (AAV) into human chromosome 19, EMBO J. 10
(1991) 3941-3950. Recombinant AAV vectors appear to lack this
site-specificity of integration. See Ponnazhagan, Adeno-associated
virus 2-mediated transduction of murine hematopoietic cells and
long-term expression of a human globin gene in vivo, 6th Parvovirus
Workshop, Montpellier, France. p29, (1995). Nevertheless, it has
been suggested that the AAV-based vector system may prove to be a
safer alternative to the more commonly used retrovirus- and
adenovirus-based vectors. See, for example, Muzyczka, Use of
adeno-associated virus as a general transduction vector for
mammalian cells, Curr. Top. Microbiol. Immunol. 158 (1992) 97-129.
Because approximately 90% of the human population is sero-positive
for AAV (see, for example, Blacklow, A sero-epidemiologic study of
adeno-associated virus infection in infants and children, Am. J.
Epidemiol. 94 (1971) 359-366), accidental infection by recombinant
AAV is not likely to be problematic. Furthermore, relatively higher
stability, higher titers, and higher transduction efficiency of AAV
have added to the desirable features of AAV vectors. See Carter,
Adeno-associated virus vectors, Curr. Opin. Biotechnol. 3 (1993)
533-538 and Srivastava, Parvovirus-based vectors for human gene
therapy, Blood Cells 20 (1994) 531-538.
[0005] A number of studies have reported AAV-mediated successful
transduction and expression of therapeutic genes in vitro. For
example, see Chatterjee, Dual target inhibition of HIV-1 in vitro
by means of an adeno-associated virus antisense vector, Science 258
(1992) 1485-1488; Walsh, Regulated high level expression of a human
.gamma.-globin gene introduced into erythroid cells by an
adeno-associated virus vector, Proc. Nat. Acad. Sci. 89 (1992)
7257-7261; Walsh, Phenotypic correction of Fanconi anemia in human
hematopoietic cells with a recombinant adeno-associated virus
vector, J. Clin. Invest. 94 (1994) 1440-1448; Flotte, Expression of
the cystic fibrosis transmembrane conductance regulator from a
novel adeno-associated virus promoter, J. Biol. Chem. 268 (1993)
3781-3790; Ponnazhagan, Suppression of human .alpha.-globin gene
expression mediated by the recombinant adeno-associated virus
2-based antisense vectors, J. Exp. Med. 179 (1994) 733-738; Miller,
Recombinant adeno-associated virus (rAAV)-mediated expression of
human .gamma.-globin gene in human progenitor-derived erythroid
cells, Proc. Natl. Acad. Sci. 91 (1994) 10183-10187; Einerhand,
Regulated high-level human beta-globin gene expression in erythroid
cells following recombinant adeno-associated virus-mediated gene
transfer, Gene Ther. 2 (1995) 336-343 Luo, Adeno-associated virus
2-mediated gene transfer and functional expression of the human
granulocyte-macrophage colony-stimulating factor, Exp. Hematol. 23
(1995) 1261-1267 and Zhou, Adeno-associated virus 2-mediated
transduction and erythroid cell-specific expression of a human
.beta.-globin gene, Gene Therapy 3 (1996) 223-229.
[0006] A few studies have examined the safety and efficacy of the
AAV vectors in vivo (see Flotte, Stable in vivo expression of the
cystic fibrosis transmembrane conductance regulator with an
adeno-associated virus vector, Proc. Natl. Acad. Sci. 90 (1993)
10613-10617 and Kaplitt, Long-term gene expression and phenotypic
correction using adeno-associated virus vectors in the mammalian
brain, Nature Genet. 8 (1994) 148-153).
[0007] A disadvantage of AAV vectors in some clinical indications
is the generalized nature of AAV infection. Previous studies have
indicated that AAV possesses a wide host range that transcends the
species barrier. See for example Muzyczka, Use of adeno-associated
virus as a general transduction vector for mammalian cells, Curr.
Top. Microbiol. Immunol. 158 (1992) 97-129. The autonomous
parvovirus, LuIII, appears to possess a similarly wide host range,
since liver specific expression has been obtained only via use of
recombinants containing a liver-specific enhancer and a regulated
promoter. See Maxwell, Autonomous parvovirus transduction of a gene
under control of tissue-specific or inducible promoters, Gene
Therapy 3 (1996) 28036. Surprisingly, we have discovered that AAV
exhibits organ tropism for the liver and is therefore uniquely
adapted for the treatment of diseases or conditions of the liver,
diseases or conditions characterized by involving a protein made in
the liver or diseases or conditions in which systemic
administration of a therapeutic via the liver is desirable or
advantageous.
INVENTION SUMMARY
[0008] In one aspect, the invention provides methods for
selectively expressing therapeutic molecules, such as secretory
proteins, antisense molecules and ribozymes, in the liver. The
methods find use in treating hepatic diseases or conditions. The
methods also find use in treating any disease or condition in which
systemic administration of the therapeutic substance, for example a
secretory protein, is desired. The methods also find use in
treating or diseases or conditions involving proteins that
originate or are normally made in the liver.
[0009] The methods involve administering to a mammalian patient
having a need for liver expression of a therapeutic molecule a
therapeutically effective amount of an AAV vector containing a the
therapeutic molecule. Therapeutic molecules useful in treating
hepatic diseases or conditions which can be administered employing
the methods described here include, for example, insulin and
thymidine kinase,. Therapeutic molecules comprising proteins
originating in the liver or protein normally made in the liver
include, for example, the LDL receptor, Factor VIII, Factor IX,
phenylalanine hydroxylase (PAH), ornithine transcarbamylase (OTC),
and .alpha.1-antitrypsin. Therapeutic molecules comprising
secretory proteins in which systemic administration is
advantageously attained via liver specific delivery include, for
example, cytokines, growth factors and the colony stimulating
factors, G-CSF and GM-CSF. Additional protein therapeutic molecules
contemplated for use in the methods and compositions of the
invention are described infra.
[0010] Also included are nucleic acid sequences that encode
antisense molecules that are useful in treating a hepatic disease.
The antisense molecule will be an RNA sequence that can prevent or
limit the expression of over-produced, defective, or otherwise
undesirable molecules by being sufficiently complementary in
sequence to the target sequence that it binds to the target
sequence. For example, the target sequence can be part of the mRNA
that encodes a protein, and the antisense RNA would bind to the
mRNA and prevent translation. The target sequence can be part of a
gene that is essential for transcription, and the antisense RNA
would bind to the gene segment and prevent or limit transcription.
For example, Group C adenoviruses Ad2 and Ad5 have a 19 kiloDalton
glycoprotein (gp 19) encoded in the E3 region of the virus that
binds to class I MHC molecules in the endoplasmic reticulum of
cells and prevents terminal glycosylation and translation of the
molecule to the cell surface. Prior to liver transplantation, the
liver cells may be infected with gp19-encoding AAV vectors or
virions which upon expression of the gp 19 inhibit the surface
expression of class I MHC transplantation antigens. These donor
cells may be transplanted with low risk of graft rejection and may
require a minimal immunosuppressive regimen for the patient. It may
also permit a donor-recipient state to exist with fewer
complications.
[0011] Similar treatments may be used to treat chronic hepatitis B
infections or non-A non-B hepatitis. The vector can be engineered
to include a structural hepatitis gene, polyadenylation signal or a
fragment thereof in reverse orientation such that the expression
product binds to hepatitis virus mRNA transcripts, preventing
translation of the structural protein and ultimately "inactivating"
the virus. See, for example, Wu, Specific inhibition of hepatitis B
viral gene expression in vitro by targeted antisense
oligonucleotides, J. Biol. Chem. 267 (1992) 12436-12439 and
Offensperger, In vivo inhibition of duck hepatitis B virus
replication and gene expression by phosphorothioate modified
antisense oligodeoxynyucleotides, EMBO J. 12 (1993) 1257-1262.
[0012] Also included are nucleic acid sequences that encode
ribozymes that are useful in treating various diseases and
conditions. Ribozymes are RNA polynucleotides capable of catalyzing
RNA cleavage at a specific sequence and hence useful for attacking
particular mRNA molecules. In chronic myelogenous leukemia for
example, the "Philadelphia chromosomal translocation" causes
expression of a bcr-abl fusion protein and abnormal function of the
abl oncoprotein. Because the fusion mRNA occurs only in cells that
have undergone the chromosome tanslocation and because the fusion
transcript contains only two possible sequences at the splice
junction, a ribozyme specific for either of the two bcr-abl fusion
mRNA splice junctions can inhibit expression of the oncoprotein.
Exemplary ribozymes include ribozymes to hepatitis A, hepatitis B
and hepatitis C. See Christoffersen and Marr, J. Med. Chem. 38
(1995) 2023-2037 and Barpolome, J. Hepatol. 22 (1995) 57-64.
[0013] Currently preferred therapeutic molecules are the LDL
receptor, Factor VIII, Factor IX, PAH, TPO (thrombopoietin) and EPO
(erythropoietin). Also preferred are growth factors and cytokines.
A therapeutically effective amount of the therapeutic molecule for
purposes of this invention is at least about 10.sup.9 to about
10.sup.11 particles/body. The patient may be any mammal, although
it is contemplated that primate patients, and especially human
patients, will benefit most from the methods of treatment. Other
patients may include murine, canine, feline, bovine and equine
species.
[0014] We contemplate that any AAV vector can be employed in the
methods of this invention. Leading and preferred examples of such
vectors for use in this invention are the AAV-2 basal vectors
disclosed in Srivastava, PCT Patent Publication WO 93/09239. Most
preferred are the vectors of the invention as disclosed herein.
Such vectors comprise the two AAV ITRs (inverted terminal repeats)
in which the authentic (i.e., native) D-sequences of the ITRs are
modified by the substitution of nucleotides such that at least 5
authentic nucleotides and up to 18 authentic nucleotides,
preferably at least 10 authentic nucleotides up to 18 authentic
nucleotides, most preferably 10 authentic (i.e., native)
nucleotides, are retained and the remaining nucleotides of the
D-sequence are deleted or replaced with non-native, i.e., exogenous
nucleotides. One preferred sequence of 5 native nucleotides that
are retained is 5' CTCCA 3'. The authentic (i.e., native)
D-sequences of the AAV ITRs are sequences of 20 consecutive
nucleotides in each AAV ITR (i.e., there is one sequence at each
end) which are not involved in HP formation. The exogenous or
non-native replacement nucleotide may be any nucleotide other than
the nucleotide found in the native D-sequence in the same position.
For example, appropriate replacement nucleotides for native
D-sequence nucleotide C are A, T and G and appropriate replacement
nucleotides for native D-sequence nucleotide A are T, G and C. The
construction of four such vectors is exemplified in Example 4, to
wit, preferred vectors pD-5, pD-15 and pD-20, and most preferred
vector pD-10, using the vector pXS-22 as starting material.
[0015] Other employable exemplary vectors are pWP-19, pWN-1 both of
which are disclosed in Nahreini, Gene 124 (1993) 257-262. Another
example of such an AAV vector is psub201. See Samulski, J. Virol.
61 (1987) 3096. Another example is the Double-D ITR vector. How to
make the Double-D ITR vector is disclosed in U.S. Pat. No.
5,478,745. Still other vectors are those disclosed in Carter, U.S.
Pat. No. 4,797,368 and Muzyczka, U.S. Pat. No. 5,139,941,
Chartejee, U.S. Pat. No. 5,474,935, and Kotin, PCT Patent
Publication WO 94/28157. Yet a further example of an AAV vector
employable in the methods of this invention is SSV9AFABTKneo, which
contains the AFP enhancer and albumin promoter and directs
expression predominantly in the liver. Its structure and how to
make it are disclosed in Su, Selective killing of AFP-positive
hepatocellular carcinoma cells by adeno-associated virus transfer
of the herpes simplex virus thymidine kinase gene, Human Gene
Therapy 7 (1996) 463-470. The disclosures of these scientific
articles, U.S. patents and patent publications are herein
incorporated by reference.
[0016] Although not an absolute requirement for the practice of the
invention, in a further embodiment, the AAV vectors of the
invention may contain a liver specific promoter to maximize the
potential for liver specific expression of the exogenous DNA
sequence contained in the vectors. The promoter is operably linked
to the nucleic acid encoding the therapeutic molecule upstream from
the latter and between the AAV vector sequences (for example
between the inverted terminal repeats in psub201 or downstream of
the Double D ITR sequence) Preferred liver specific promoters
include the hepatitis B X-gene promoter and the hepatitis B core
protein promoter. These liver specific promoters are preferably
employed with their respective enhancers. The enhancer element can
be linked at either the 5' or the 3' end of the nucleic acid
encoding the therapeutic molecule. The hepatitis B X gene promoter
and its enhancer can be obtained from the viral genome as a 332
base pair EcoRV-NcoI DNA fragment employing the methods described
in Twu, J. Virol. 61 (1987) 3448-3453. The hepatitis B core protein
promoter can be obtained from the viral genome as a 584 base pair
BamHI-BglII DNA fragment employing the methods described in
Gerlach, Virol 189 (1992) 59-66. It may be necessary to remove the
negative regulatory sequence in the BamHI-BglII fragment prior to
inserting it. Other liver specific promoters include the AFP (alpha
fetal protein) gene promoter and the albumin gene promoter, as
disclosed in EP Patent Publication 0 415 731, the .alpha.-1
antitrypsin gene promoter, as disclosed in Rettenger, Proc. Natl.
Acad. Sci. 91 (1994) 1460-1464, the fibrinogen gene promoter, the
APO-A1 (Apolipoprotein A1) gene promoter, and the promoter genes
for liver transference enzymes such as, for example, SGOT, SGPT and
.gamma.-glutamyle transferase. See also PCT Patent Publications WO
90/07936 and WO 91/02805.
[0017] We also contemplate that any hepatic disease or any defect
in hepatic function, whether inherited or acquired, is susceptible
to treatment with the methods of the invention. Exemplary hepatic
diseases or defects in hepatic function include hepatocellular
carcinoma, jaundice, infectious hepatitis, alcohol liver damage,
including alcohol induced cirrhosis, and non-alcohol induced liver
cirrhosis.
[0018] We also contemplate that any inherited or acquired disease
or defect, the treatment of which requires administration of a
therapeutic molecule that is normally made in the liver, is
susceptible to treatment with the methods of the invention.
Exemplary inherited diseases include familial hypercholesterolemia,
which is caused by an LDL receptor deficiency, phenylketonuria,
which is caused by a phenylalanine hydroxylase deficiency, urea
cycle disorders, organic acid disorders, Wilson's disease,
tyrosinemia, .alpha..sub.1-antitrypsin deficiency and
hyperammonemia which is caused by an inherited deficiency of
ornithine transcarbamylase function. Exemplary acquired diseases
include non-familial hypercholesterolemia and other
hyperlipoproteinemias.
[0019] We also contemplate that the methods described here find use
in treating any disease or condition in which the therapeutic
substance, for example a secretory protein, is advantageously
expressed in the liver in order to, for example, obtain systemic
administration via entry into the circulatory system through the
hepatic system. Genes encoding any of the cytokines and
immunomodulatory proteins described here can be expressed in an AAV
vector to achieve liver specific in vivo expression. Forms of these
cytokines other than the forms mentioned here that are known to the
skilled artisan can be used. For instance, nucleic acid sequences
encoding native IL-2 (interleukin 2) and .gamma.-interferon can be
obtained as described in U.S. Pat. Nos. 4,738,927 and 5,326,859
respectively, while useful mutants of these proteins can be
obtained as described in U.S. Pat. No. 4,853,332. As an additional
example, nucleic acid sequences encoding the short and long forms
of M-CSF (macrophage colony stimulating factor) can be obtained as
described in U.S. Pat. Nos. 4,847,201 and 4,879,227 respectively.
AAV vectors expressing cytokine or immunomodulatory genes can be
produced as described here.
[0020] AAV vectors producing a variety of known polypeptide
hormones and growth factors can be used in the methods of the
invention to produce therapeutic expression of these proteins. Some
such hormones, growth factors and other proteins are described in
EP patent 0 437 478 B1 for instance. Nucleic acid sequences
encoding a variety of hormones can be employed, including for
example, human growth hormone, insulin, calcitonin, prolactin,
follicle stimulating hormone, luteinizing hormone, human chorionic
gonadotropin, thyroid stimulating hormone. AAV vectors expressing
polypeptide hormones and growth factors can be prepared by methods
known to those of skill in the art. As an additional example,
nucleic acid sequences encoding different forms of human insulin
can be isolated as described in EP patent publication 026598 or
070632 and incorporated into AAV vectors as described here.
[0021] Any of the polypeptide growth factors can also be
administered therapeutically by liver specific expression in vivo
with an AAV vector. For instance, different forms of IGF-1 and
IGF-2 growth factor polypeptides are well known in the art and can
be incorporated into AAV vectors for liver specific expression. See
EP patent 0 123 228 B1. Liver specific expression of different
forms of fibroblast growth factor can also be effected by the
methods of the invention. See U.S. Pat. Nos. 5,464,774; 5,155,214
and 4,994,559.
[0022] There are a number of proteins useful for treating
hereditary disorders that can be expressed by the methods of the
invention. Many genetic diseases caused by inheritance of defective
genes result in the failure to produce normal gene products, for
example, severe combined immunodeficiency (SCID), hemophilia A,
hemophilia B, adenine deaminase deficiency, Gaucher's syndrome,
hereditary lactose intolerance and inherited emphysema. Also
contemplated are diseases that are caused by the inability of the
gene to produce adequate levels of the appropriate hormone, such as
diabetes and hypopituitarism.
[0023] Liver specific expression of Factor VIII or Factor IX,
useful for the treatment of blood clotting disorders such a
hemophilia, is obtainable using the methods of the invention. PCT
Patent Publication WO 96/21014 describes Factor VIII and HGH (human
growth hormone) constructs for retroviral expression which could
readily adapted by the skilled artisan for AAV expression. The
Factor VIII minigene (see EP Patent Publication 232 112 and PCT
Patent Publication WO 91/07490) could advantageously be employed
for AAV expression. Also contemplated is the expression of lactase
for the treatment of hereditary lactose intolerance, ADA for the
treatment of ADA deficiency and .alpha.-1 antitrypsin for the
treatment of .alpha.-1 antitrypsin deficiency. See Ledley, J.
Pediatrics 110: (1987) 157-174; Verma, Scientific American
(November 1987) pp. 68-84 and PCT Patent Publication
WO95/27512.
[0024] There are a variety of other proteins of therapeutic
interest that can be expressed in a liver specific manner using the
methods of the invention. For instance sustained expression of
tissue factor inhibitory protein (TFPI) is useful for the treatment
of conditions including sepsis and DIC and in preventing
reperfusion injury. See PCT Patent Publications WO 93/24143, WO
93/25230 and WO 96/06637. Nucleic acid sequences encoding various
forms of TFPI can be obtained, for example, as described in U.S.
Pat. Nos. 4,966,852; 5,106,833 and 5,466,783, and can be
incorporated into AAV vectors as described here.
[0025] Other proteins of therapeutic interest such as
erythropoietin (EPO) and leptin can be expressed in the liver by
AAV vectors according to the methods of the invention. EPO is
useful in gene therapy treatment of a variety of disorders
including anemia. See PCT Patent Publication WO 95/13376. Gene
therapy delivery of the leptin gene and its use in the treatment of
obesity is described in PCT Patent Publication WO 96/05309. AAV
vectors expressing EPO or leptin can readily be produced and liver
specific expression attained employing the described methods. Other
exemplary proteins and polypeptides include the cytokines such as
interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15, .alpha.-interferon,
.beta.-interferon, the .gamma.-interferons, GM-CSF, the tumor
necrosis factors (TNFs), CD3, ICAM-1, LFA-1, LFA-3, the chemokines
including RANTES 1.alpha., MIP-1.alpha., MIP-1.beta. (see Cocchi,
Science 720 (1996) 1811-1815) or analogs of such proteins. Because
soluble forms of receptors can often behave as antagonists, as can
mutated forms of the factors themselves, the nucleic acid sequences
of therapeutic interest may also be agonists, antagonists or
ligands for these proteins and polypeptides.
[0026] Even more proteins and polypeptides of therapeutic interest
that can be expressed in liver specific fashion employing the AAV
vectors and methods of the invention include Protein S and Gas6,
thrombin, Coagulation Factor Xa, CSF-1 or M-CSF, IGF-1, IGF-2,
acidic FGF, basic FGF, keratinocyte growth factor (KGF), TGF,
platelet derived growth factor (PDGF), epidermal growth factor
(EGF), hepatocyte growth factor (HGF) and HGF activators, PSA,
nerve cell growth factor (NCGF), glial cell derived nerve growth
factor (GDNF), VEGF, Arg-vasopressin, thyroid hormones,
azoxymethane, triiodothyronine, LIF, amphiregulin, soluble
thrombomodulin, stem cell factor, osteogenic protein 1, the bone
morphogenic proteins, MGF, MGSA, heregulins and melanotropin.
Growth factors can also be used in combination with mixtures
consisting of one or several of, for example, DGF, IGF, PDGF, FGF
or KGF. The full length growth factor can be employed or forms of
the growth factor, such as active fragments, truncated forms and
analogues can be employed. By "active fragment" we mean a
polypeptide containing less than a full-length sequence that
retains sufficient biological activity to be used in the methods of
the invention. By "analogue" we mean truncated forms, splice
variants, variants with amino acid substitutions, deletions or
additions, alleles and derivatives of the mature protein or
polypeptide which possess one or more of the native bioactivities
of the full length protein or polypeptide. Thus, polypeptides that
are identical or contain at least 60%, preferably, 70%, more
preferably 80% and most preferably 90% amino acid sequence homology
to the amino acid sequence of the mature protein wherever derived,
from human or non-human sources are included within this
definition. For example, a preferred truncated form of KGF is
described in PCT Patent Publication WO 95/10434. See also PCT
Patent Publication WO 90/08771 and U.S. Pat. No. 5,096,825 relating
to human EGF.
[0027] The growth factor polypeptides, fragments and analogues can
be produced by isolation from naturally occurring sources,
polypeptide chain synthesis by peptide synthesis methods and
production or recombinant proteins. These methods are well known to
those of skill in the art. For example, production of recombinant
PDGF is described in U.S. Pat. Nos. 5,045,633 and 4,769,328 and
production of recombinant FGF and analogues is described in U.S.
Pat. Nos. 5,229,501; 5,331,095 and 5,143,829.
[0028] A variety of other disorders can be treated by the methods
of the invention. For example, production of apolipoprotein E or
apolipoprotein A, useful in treating hyperlipidemia, can be
attained via administration of the liver specific AAV vectors of
the invention. See Breslow, Biotechnology 12 (1994) 365. Sustained
production of angiotensin receptor inhibitor (see Goodfriend, N.
Engl. J. Med. 334 (1996) 1469) or of angiostatin useful in the
treatment of tumors (see O'Reilly, Nature Med. 2 (1996) 689) can be
attained.
[0029] Nucleic acid sequences that encode the above-described
proteins and polypeptides are obtainable from a variety of sources.
For example, plasmids containing sequences the encode altered
cellular products may be obtained form a depository such as the
American Type Culture Collection (ATCC, Rockville, Md.) or from
commercial sources such as Advanced Biotechnologies (Columbia, Md.)
and British Bio-Technology Limited (Cowley, Oxford, Great Britain).
Exemplary plasmids include ATCC Nos. 41000 and 41049 containing
muteins of ras. Other nucleic acid sequences that encode the
above-described proteins and polypeptides, as well as other nucleic
acid molecules such as antisense sequences and ribozymes that are
advantageously used in the invention may be readily obtained from
such public sources. Exemplary are BBG12 containing the full length
GM-CSF coding sequence, BBG6 containing the .gamma.-interferon
coding sequence, ATCC No. 39656 containing sequences encoding TNF,
ATCC No. 20663 containing sequences encoding .alpha.-interferon,
ATCC Nos. 31902 and 39517 containing sequences encoding
.beta.-interferon, ATCC No. 67024 containing the interleukin-1b
coding sequence, ATCC Nos. 39405, 39452, 39516, 39626 and 39673
containing sequences encoding interleukin-2, ATCC No. 57592
containing sequences encoding interleukin-4, ATCC Nos. 59394 and
59395 containing sequences encoding interleukin-5 and ATCC 67153
containing sequences encoding interleukin-6. Molecularly cloned
genomes encoding the hepatitis B virus are obtainable from the
ATCC. ATCC No. 45020 contains the total genomic DNA of hepatitis B
(with correctable errors), extracted from purified Dane particles,
in the BamHI site of pBR322. See Blum TIG 5 (1989) 154-158 and
Moriarty, Proc. Natl. Acad. Sci. 78 (1981) 2606-2610.
Alternatively, cDNA sequences for use with the invention are
obtainable from cells that express or contain the sequences.
Briefly, within one embodiment, mRNA from a cell that expresses the
gene of interest is reverse transcribed with reverse transcriptase
using oligo dT or random primers. The single stranded cDNA may then
be amplified by PCR (see U.S. Pat. Nos. 4,683,202; 4,683,195 and
4,800,159, PCR Technology: Principles and Applications for DNA
Amplification, Erlich (ed.), Stockton Press, 1989) using
oligonucleotide primers complementary to sequences on either side
of desired sequences. In particular, a double stranded DNA is
denatured by heating in the presence of heat stable Taq polymerase,
sequence specific DNA primers, ATP, CTP, GTP and TTP.
Soluble-stranded DNA is produced when synthesis is complete. This
cycle may be repeated many times resulting in a factorial
amplification of the desired DNA. Nucleic acid sequences may also
be synthesized de novo, for example on an Applied Biosystems Inc.
DNA synthesizer.
[0030] In another embodiment, AAV hybrid (i.e., chimeric) vectors
are provided containing the DNA sequence, or functional fragment
thereof, encoding hepatitis B surface antigen and the DNA sequence
encoding the AAV capsid protein. An oligonucleotide sequence that
corresponds to this HBV surface antigen peptide is blunt-ended and
ligated at the 5' end of the AAV VP-1 gene. Specifically, the 27
amino acid sequence of HBV surface antigen corresponding to amino
acids 20-47 of the preS1 region (see, Ishikawa, Proc. Natl. Acad.
Sci. 92 (1995) 6259-6263; Klingmuller, J. Virol. 67 (1993)
7414-7422 and Neurath, Cell 46 (1986) 429-436 and Virol. 176 (1990)
448-457) are fused in-frame to the AAV viral capsid VP-1 gene (see
Srivastava, J. Virol. 45 (1983) 555-564). Nucleic acids encoding
therapeutic molecules cloned within recombinant AAV vectors may be
packaged into recombinant AAV virions using this AAV-HBV chimeric
helper vector. The AAV-2 capsid gene has been cloned and is
available. See Samulski, J. Virol. 63 (1989) 3822-3828. But capsid
genes from any AAV, specifically from AAV-1, AAV-3 or AAV-4, can be
employed. For a general review of the molecular biology, structure
and gene products of HBV see Yoffe, Progress and perspectives in
human hepatitis B virus research. Prog. Med. Virol. 40 pp.107-140
(Melnick, J. L. ed.) 1993.
[0031] To establish integration of the vector into the chromosome
of a host cell, host cells are transfected with the vector or
infected with mature virions containing the vector. Methods of
transfection are well-known in the art and include, for example,
naked DNA transfection, microinjection and cell fusion. Virions can
be produced by coinfection with helper virus such as adenovirus,
herpes virus or vaccinia virus. Following coinfection with the
vector and a helper virus, the host cells are isolated and the
helper virus is inactivated. The resulting helper free stocks of
virions are used to infect host cells. Alternatively, virions are
produced by cotransfecting helper virus-infected cells with the
vector and a helper plasmid. The plasmid will contain the
parvovirus rep gene and non-AAV ITRs, for example adenovirus ITRs.
Following cotransfection, mature virions are isolated using
standard methods and any contaminating adenovirus inactivated using
methods known to skilled artisans. The resulting mature virions can
be used to infect host cells in the absence of helper virus.
[0032] Methods of making recombinant AAV vectors and packaging cell
lines, purification methods, rescue methods and methods of
generating high-titer vector stocks are known in the art. See for
example, Samulski, A recombinant plasmid from which an infectious
adeno-associated virus genome can be excised in vitro and its use
to study viral replication, J. Virol. 61 (1987) 3096-3101 and
helper-free stocks of recombinant adeno-associated viruses: Normal
integration does not require viral gene expression, J. Virol. 63
(1989) 3822-3828, McLaughlin, Adeno-associated virus general
transduction vectors: Analysis of proviral structures, J. Virol. 62
(1988) 1963-1973, Flotte, An improved system for packaging
recombinant adeno-associated virus vectors capable of in vivo
transduction, Gene Therapy 2 (1995) 29-37, Holscher, Cell lines
inducibly expressing the adeno-associated virus (AAV) rep gene:
Requirements for productive replication of rep-negative AAV
mutants, J. Virol. 68 (1994) 7169-7177 and High-level expression of
adeno-associated virus (AAV) Rep78 protein is sufficient for
infectious-particle formation by a rep-negative AAV mutant, J.
Virol. 69 (1995) 6880-6885, Yang, Characterization of cell lines
that inducibly express the adeno-associated virus Rep proteins, J.
Virol. 68 (1994) 4847-4856, Ponnazhagan, Alternative strategies for
generating recombinant AAV vectors, VIth parvovirus Workshop,
Montpellier, France, p. 71(1995), Luhovy, Stable transduction of
recombinant adeno-associated virus into hematopoietic stem cells
from normal and sickle cell patients, Bio. Blood Marrow Transpl. 2
(1996) 24-30, Tamayose, A new strategy for large-scale preparation
of high-titer recombinant adeno-associated virus vectors by using
sulfonated cellulose column chromatography, Hum. Gene Therap. 7
(1996) 507-513, Maxwell, Improved method for production of
recombinant AAV and determination of infectious titer, VIth
Parvovirus Workshop, Montpellier, France, p72 (1995), Chiorini,
High-efficiency transfer of the T cell co-stimulatory molecule B7-2
to lymphoid cells using high-titer recombinant adeno-associated
virus vectors, Hum. Gene Therap. 6 (1995) 1531-1541 and Colosi, AAV
vectors can be efficiently produced without helper virus, Blood 10
(1995) 627a.
[0033] The vector or virions can be incorporated into
pharmaceutical compositions for administration to mammalian
patients, particularly humans. The vector or virions can be
formulated in nontoxic, inert, pharmaceutically acceptable aqueous
carriers, preferably at a pH ranging from 3 to 8, more preferably
ranging from 6 to 8. Such sterile compositions will comprise the
vector or virion containing the nucleic acid encoding the
therapeutic molecule dissolved in an aqueous buffer having an
acceptable pH upon reconstitution. Such formulations comprise a
therapeutically effective amount of a AAV vector or virion in
admixture with a pharmaceutically acceptable carrier and/or
excipient, for example saline, phosphate buffered saline, phosphate
and amino acids, polymers, polyols, sugar, buffers, preservatives
and other proteins. Exemplary amino acids, polymers and sugars and
the like are octylphenoxy polyethoxy ethanol compounds,
polyethylene glycol monostearate compounds, polyoxyethylene
sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose,
glucose, mannitol, dextran, sorbitol, inositol, galactitol,
xylitol, lactose, trehalose, bovine or human serum albumin,
citrate, acetate, Ringer's and Hank's solutions, cysteine,
arginine, carnitine, alanine, glycine, lysine, valine, leucine,
polyvinylpyrrolidone, polyethylene and glycol. Preferably, this
formulation is stable for at least six months at 4.degree. C.
[0034] The virions can be systemically administered by intravenous
injection. The dosage regimen will be determined by the attending
physician or veterinarian considering various factors known to
modify the action of drugs such as, for example, the physical
condition of the patient, the severity of the condition, body
weight, sex, diet, time of administration and other clinical
factors. Generally, the regimen should be in the range of about
10.sup.9 to about 10.sup.11 particles per body. A preferred dose is
about 10.sup.10 particles per body. The number of doses
administered may vary, depending on the above mentioned
factors.
[0035] The AAV vector or virions can also be administered ex vivo
employing art recognized methods, for example, by electroporation
following the procedures of Chakrabarti, J. Biol. Chem. 264 (1989)
15494-15500 or by protoplast delivery following the procedures of
Kaneda, Science 243 (1989) 375-78 and Ferguson, J. Biol. Chem 261
(1986) 14760-14763. Alternatively, hepatocyte precursor cells can
be transduced with a vector of the invention, grown in tissue
culture vessels, removed and introduced into the patient surgically
by grafting or transplantation. The precursor cells can be attached
to supports such as microcarrier beads that are injected into the
peritoneal space of the patient or directly into the liver, into
the portal venous system or into the spleen. The patient's liver
cells may be obtained through liver biopsy, partial hepatectomy or
from specimens harvested for orthotopic liver transplantation,
purified and grown in culture. AAV vectors may be introduced into
the liver cells by exposure to the virus and the liver cells
reintroduced into the patient by grafting or by placing the cells
in the abdominal cavity in contact with the unremoved portion of
the patient's liver. Such methods are known in the art. See, for
example, Chang, Gene Therapy: Applications to the Treatment of
Gastrointestinal and Liver Diseases, Gastroenterology 106 (1994)
1076-1084. For ex vivo administration, the dosage regimen should be
in the range of 1 to 100 m.o.i., preferably in the range of 5 to 20
m.o.i. The dosage regimen will be determined by the attending
physician considering various factors known to modify the action of
drugs such as for example, physical condition, body weight, sex,
diet, severity of the condition, time of administration and other
clinical factors. The number of doses administered may vary,
depending on the above mentioned factors.
[0036] The liver specific delivery methods of the invention may be
employed with or without pretreatment of the liver. Pretreatment
includes benign hyperplasia, which can be induced by treatment with
HGF and/or transforming growth factor.alpha.. See Lui, Hepatology
19 (1994) 1521. Different forms of HGF useful in inducing liver
cell proliferation are known in the art and can be employed. See
for example EP patent publication EP 0 461 560. HGF can also be
produced and administered to induce liver cell proliferation in
vivo as described in Joplin, J. Clin. Invest. 90 (1992) 1284. Liver
cells can also be stimulated by administration of agents that
mediate or potentiate the activation of endogenous HGF. HGF is
produced as a single chain protein that is inactive as a growth
factor. Single chain HGF is subsequently cleaved into a two-chain
form that is biologically active. Enzymes that are shown to convert
single-chain HGF to its bioactive form are useful for inducing
liver cell proliferation. Therefore, these enzymes can be
administered either alone or in combination with exogenous HGF to
enhance liver proliferation. Exemplary enzymes include coaglation
factor XI1a, HGF activator, HGF converting enzyme, urokinase and
tissue plasminogen activator. For example, HGF and urokinase can be
co-formulated and administered by IV injection or mixed
immediatedly prior to injection. If co-formulated, storage at low
pH would advantageously minimize the activity of urokinase. See PCT
Patent Publication WO 96/21014 entitled Production and
Administration of High Titer Recombinant Retroviruses.
[0037] In another embodiment of the invention the AAV vector is
co-administered with a cholesterol lowering drug to a primate
patient suffering from hypercholesterolemia. A preferred
cholesterol lowering drug is M-CSF. See U.S. Pat. Nos. 5,021,239
and 5,019,381. Other preferred cholesterol lowering drugs include
niacin, gemfibrozil, lovastatin and mevacor.
DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1: Illustration of the Southern blot analysis of the
PCR-amplified DNA fragments of the vCMVp-lacZ vector administered
to mice as described in Example 1, in various murine tissues.
[0039] FIG. 2: Illustration of the Southern blot analysis of the
PCR-amplified DNA fragments of the
vHS2-.beta.p-.sup.A.gamma.-globin vector administered to mice as
described in Example 2, in various murine tissues.
[0040] FIG. 3: Illustration of autoradiogram of semi-quantitative
PCR amplification results, as detailed in Example 2.
[0041] FIG. 4: Schematic structures of pSub201 and pD-10
recombinant AAV vectors, as detailed in Example 5. The D-sequence
is shown as a shaded box in plasmid pSub201. In plasmid pD-10, the
distal 10 nucleotides in the D-sequence have been replaced by a
substitute (s)-sequence.
DETAILED DESCRIPTION
[0042] In murine mammalian patients the fate of AAV vectors was
followed after direct intravenous injection and it was surprisingly
found that the AAV vectors possess organ-tropism for liver. Our AAV
vectors contained the lacZ reporter gene or the human globin gene.
In mice administered the lacZ reporter gene containing AAV vectors,
expression occured in hepatocytes but a cytotoxic T lymphocyte
response against .beta.Gal was not detected. The recombinant AAV
vectors, when directly injected intravenously in mice, accumulated
predominantly in liver cells.
[0043] The AAV recombinant virus stocks containing the CMV promoter
(CMV.sub.p) driven lacZ gene (vCMVp-lacZ) cloned in between AAV
inverted terminal repeats (ITR) and the AAV recombinant virus
stocks containing the genomic copy of the normal human
.sup.A.gamma.-globin gene driven by the human .beta.-globin
promoter (.beta.p) plus an upstream Hypersensitive site 2 enhancer
element cloned in between AAV ITR were generated from their
respective recombinant plasmids by the methods described in
Samulski, Helper-free Stocks of Recombinant Adeno-associated
Viruses: Normal Integration Does Not Require Viral Gene Expression,
J. Virol. 63 (1989) 3822-3828; Nahreini, Versatile Adeno-associated
Virus 2-based Vectors for Constructing Recombinant Virions, Gene
124 (1993) 257-262; Zhou, Adeno-associated Virus 2-mediated High
Efficiency Gene Transfer into Immature and Mature Subsets of
Hematopoietic Progenitor Cells in Human Umbilical Cord Blood, J.
Exp. Med. 179 (1994) 1867-1875; Ponnazhagan, Lack of Site-specific
Integration of the Recombinant Adeno-associated Virus Genomes in
Human Cells, 5th Parvovirus Workshop, Crystal River, Fla., USA
p.P1-29 (1993); Ponnazhagan, Adeno-associated Virus 2-mediated
Transduction of Murine Hematopoietic Cells and Long-term Expression
of a Human Globin Gene in Vivo, 6th Parvovirus Workshop,
Montpellier, France. p29 (1995); and Ponnazhagan, Differential
Expression in Human Cells from the P6 Promoter of Human Parvovirus
B19 Following Plasmid Transfection and Recombinant Adeno-associated
Virus 2 (AAV2) Infection: Human Megakaryocytic Leukaemia Cells Are
Non-Permissive for AAV Infection, J. Gen. Virol. 77 (1996)
1111-1122. The viral stocks were purified on CsCl density gradients
following the protocol described in Wang, Parvovirus B19 Promoter
at Map Unit 6 Confers Replication Competence and Erythroid
Specificity to Adeno-associated Virus 2 in Primary Human
Hematopoietic Progenitor Cells, Proc. Natl. Acad. Sci. 92 (1995)
12416-12420. Titers were determined on quantitative DNA slot blots
as described in Srivastava, Parvovirus B19-induced. Perturbation of
Human Megakaryocytopoiesis In Vitro, Blood 76 (1990) 1997-2004;
Srivastava, Construction of a Recombinant Human Parvovirus B19:
Adeno-associated Virus 2 (AAV) DNA Inverted Terminal Repeats Are
Functional in an AAV-B19 Hybrid Virus, Proc. Natl. Acad. Sci. 86
(1989) 8078-8082.; Nahreini and Srivastava, Rescue of the
Adeno-associated Virus 2 Genome Correlates with Alterations in
DNA-modifying Enzymes in Human Cells, Intervirol. 33 (1992)
109-115.; Zhou, Adeno-associated Virus 2-mediated Gene Transfer in
Murine Hematopoietic Progenitor Cells, Exp. Hematol. 21 (1993)
928-933; Zhou, Adeno-associated Virus 2-mediated High Efficiency
Gene Transfer into Immature and Mature Subsets of Hematopoietic
Progenitor Cells in Human Umbilical Cord Blood, J. Exp. Med. 179
(1994) 1867-1875 and Zhou, Adeno-associated Virus 2-mediated
Transduction and Erythroid Cell Specific Expression of a Human
.beta.-globin Gene, Gene Therapy 3 (1996) 223-229.
[0044] These highly purified recombinant AAV vectors were
administered to C57B1/6 mice by direct intravenous injection into
the tail vein.
EXAMPLE 1
[0045] Highly purified recombinant AAV vectors containing the
cytomegalovirus (CMV) promoter-driven lacZ gene (vCMVp-lacZ) were
directely injected into C57B1/6 mice. Approximately
1.times.10.sup.10 viral particles of vCMVp-lacZ were injected
intravenously into the tail-vein of 12 animals in four groups of
three animals each. These animals were sacrificed at various times
post-injection (p.i.), and equivalent amounts of tissues from
various organs were examined for the presence of the recombinant
AAV viral genome by polymerase-chain-reaction (PCR) amplification
using a lacZ-specific primer-pair followed by Southern blot
analysis.
[0046] Approximately 1.times.10.sup.10 particles of the vCMVp-lacZ
r-virus were injected in 0.2 ml Iscov's-modified Dulbecco's medium
into the tail-vein of 8-week old C57B1/6 mice. Three animals per
group were sacrificed at 1 hour, 24 hours, 72 hours, and 1 week
p.i. Individual tissues and organs were obtained, rinsed
extensively with phosphate-buffered-saline, and equivalent amounts
were used in a 35-cycle PCR-amplification reaction using the
lacZ-specific primer-pair (5'-GATGAGCGTGGTGGTTATG,
5'-TACAGCGCGTCGTGATTAG). Plasmids pCMVp-lacZ (Ponnazhagan et al.,
1996) and pUC19 (Sambrook, Fritsch, and Maniatis, Molecular
Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y.,
1989) were used as positive and negative controls, respectively.
The PCR products were electrophoresed on 1% agarose gels and
analyzed on Southern blots (Southern, Detection of specific
sequences among DNA fragments separated by gel electrophoresis. J.
Mol. Biol. 98 (1975) 503-517) using a lacZ-specific
.sup.32P-labeled DNA probe.
[0047] The results of the Southern blot analysis are shown in FIG.
1. The recombinant AAV genomes were detected predominantly in the
liver tissues up to 1-week p.i. in each group of animals. The
arrows indicate the 588-bp lacZ-specific DNA fragment.
EXAMPLE 2
[0048] The results in Example 1 were corroborated by injecting
recombinant vHS2-.beta.p-.sup.A.gamma.-globin virions under
conditions identical to those in Example 1 and examining tissues
from various organs seven weeks p.i. using the same techniques, but
employing a .beta.-globin promoter-.sup.A.gamma.-globin
gene-specific primer-pair.
[0049] Highly purified recombinant AAV vectors containing the human
.beta.-globin promoter-driven human .sup.A.gamma.-globin gene
containing the DNase hypersensitive-site 2 (HS-2) enhancer element
(see Tuan, An erythroid specific, development stage-independent
enhancer far upstream of the human ".beta.-like globin" genes,
Proc. Natl. Acad. Sci. 86 (1989) 2554-2559) from the locus control
region (LCR) from the human .beta.-globin gene cluster
(vHS2-.beta.p-.sup.A.gamma.-globin) were directly injected into
C57B1/6 mice.
[0050] Approximately 1.times.10.sup.10 particles of the
vHS2-.beta.p-.sup.A.gamma.-globin r-virus were injected i.v. as
described in Example 1. Seven weeks p.i., the various organs were
obtained and analyzed for the presence of the r-viral genome using
the human .beta.-globin promoter (5'-GATGGTATGGGGCCAAGAGA)- and
.sup.A.gamma.-globin gene (5'-GGGTTTCTCCTCCAGCATCT)-specific
oligodeoxynucleotide primer pair. Liver tissues obtained from a
mock-injected mouse was also included as a negative control. The
Southern blot results are shown in FIG. 2. The arrow indicates the
354-bp human .gamma.-globin-specific DNA fragment.
[0051] We then investigated copy number of the
vHS2-.beta.p-.sup.A.gamma.-- globin vector in liver cells.
Equivalent amounts of DNA isolated from the liver of mock-injected
and vHS2-.beta.p-.sup.A.gamma.-globin virus-injected mice were used
in a semi-quantitative PCR amplification assay using either the
human .beta.-globin promoter-.sup.A.gamma.-globin gene-specific
oligodeoxynucleotide primers or the mouse .beta.-actin-specific
oligodeoxynucleotide primers. Approximately equivalent amounts of
liver tissue from each animals were lysed in a buffer containing 10
mM Tris.HCl/50 mM KCl/2.5 mM MgCl.sub.2/0.5% Tween-20/100 .mu.g
proteinase K per ml at 55.degree. C. overnight. The lysates were
heated at 90.degree. C. for 10 min to inactivate proteinase K, and
5 .mu.l of each sample was subjected to a 30-cycle PCR
amplification with the two sets of primer-pairs under identical
conditions. The primers for amplifying the transduced human globin
gene sequences were the same as those described in Example 1 and
the primer sequences for the mouse .beta.-actin gene were as
follows: 5'-ACCTTCAACACCCCAGCCAT and 5'-TCAGGCAGCTCATAGCTCTT. The
primers were designed to yield a 354-bp DNA fragment from each
sequence. The PCR reactions were performed in the presence of 2
.mu.Ci [.alpha.-.sup.32P]dCTP (sp. act. 800 Ci/mmol) in each
reaction mix. Ten per cent of the DNA products from the human
globin gene and a 15-fold diluted samples from the .beta.-actin
gene amplification reactions were analyzed on 6% polyacrylamide
gels and autoradiographed. The relative intensities of the
corresponding bands were determined by scanning the autoradiograms
using the Photoshop 3.0 program. The transduced globin gene was
detected in approximately 4% of liver cells seven weeks p.i. See
FIG. 3.
EXAMPLE3
[0052] We next examined whether the lacZ gene delivered by direct
injection of the r-AAV was transcriptionally active. Livers from
mock-injected and vCMVp-lacZ-injected C57B1/6 mice were obtained
one week p.i., and cryopreserved. Tissue sections were fixed and
stained with 5-Bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside
(XGal) as described in Cheng, Separable Regulatory Elements
Governing Myogenin Transcription in Mouse Embryogenesis, Science
261 (1993) 215-218 and visualized under a light microscope.
[0053] Livers were obtained one week p.i. and frozen immediately in
iso-pentane at -40.degree. C. Sections of 15 .mu.m were prepared
using a cryostat and fixed in a solution containing 2%
formaldehyde/0.2% para-formaldehyde in phosphate-buffered saline
(PBS, 135 mM NaCl/2.5 mM KCl/8 mM Na.sub.2HPO.sub.4/0.6 mM
KH.sub.2PO.sub.4/0.55 mM dextrose/liter) for 5 min on ice, washed
twice with PBS and stained overnight at 37.degree. C. in a solution
containing 5 mM K.sub.3Fe(CN).sub.6/5 mM K.sub.4Fe(CN).sub.6/1 mM
MgCl.sub.2/1 mg XGal in 1 ml of PBS, as described previously in
Cheng, Separable regulatory elements governing myogenin
transcription in mouse embryogenesis. Science 261 (1993) 215-218.
Tissue sections were visualized under a light microscope
(magnification .times.40). No expression of the transgene occurred
in liver cells from mock-injected animal. Expression of the lacZ
gene was readily detected in liver hepatocytes.
EXAMPLE 4
[0054] Co-transfection of an rAAV vector containing the AAV ITRs
and the nucleic acid sequence encoding a therapeutic molecule and a
helper plasmid containing the necessary rep and cap functions into
adenovirus-2 (Ad2) infected 293 cells was expected to eliminate
homologous recombination events leading to the production of
contaminating wild-type (wt) AAV during the production of
recombinant vector stocks. However, contaminating "wild type-like
AAV" particles have been observed in such stocks ranging from 0.1%
to 10%.
[0055] To determine the mechanism of generation of contaminating wt
AAV, stocks were amplified through four successive round of
co-infection with Ad2 in 293 cells. Low molecular weight DNA
fragments were isolated, digested with Bal I restriction
endonuclease and molecularly cloned into a pBlueScript plasmid
vector. AAV sequence-positive clones were subjected to nucleotide
sequencing using T3 and T7 primers. Nucleotide sequence analysis of
12 independent clones revealed that most of the recombination
events leading to the contaminating wt AAV involved 10 nucleotides
in the AAV D-sequence distal to viral hairpin structures. In
addition, by analyzing 22 different clones generated with a helper
plasmid that lacks the Ad2 ITRs, we observed only a limited number
of recombination sites and concluded that Ad2 ITRs play a role in
illegitimate recombination with the AAV-ITRs that leads to
generation of biologically active wild type-like AAV. Consequently,
by removing the Ad2 ITRs from the helper plasmid, nearly 5-fold
reduction in the illegitimate recombination frequency can be
achieved.
[0056] The first 10 nucleotides in the D-sequence proximal to the
AAV hairpin structures are essential for successful replication and
encapsidation of the viral genome. See, Wang, J Virol 71: 3077-82
(1997). In each of the recombinant junctions sequenced, the same 10
nucleotides were retained. By deleting the distal 10 nucleotides in
the D-sequence in the next generation of AAV vectors, the
generation of the wt AAV-like particles in recombinant AAV vectors
stocks can be redueced or eliminated. See Example 5 below for
production of such vectors.
EXAMPLE 5
[0057] Four recombinant AAV vectors, pD-5, pD-10, pD-15 and pD-20,
were constructed as follows. Plasmid pXS-22 can be employed as
starting material. The plasmid pXS-22 can be obtained from a public
depository or constructed following the methods described in Wang,
J. Mol Biol. 250 (1995) 573-580 using pSub201 as starting material.
Plasmid pXS-22 contains only the right ITR (inverted terminal
repeat): one hairpin and one D sequence. The D-sequence is that
part of the AAV ITR which is not involved in HP formation. See
Wang, supra. The D-sequence can be replaced by a substitute (S)
sequence as described in Wang, J. Virol. 70 (1996) 1668-1677. The
nucleotide sequences are as follows:
1 D-sequence: 5' CTCCA TCACT AGGGG TTCCT 3' GAGGT AGTGA TCCCC AAGGA
5' S-sequence: 5' CCAA TATTA GATCT GATAT CA 3' 3' GGTT ATAAT CTAGA
CTATA GTGAT C 5'
[0058] Four additional oligonucleotide sequences were synthesized
which contained selected nucleotides identical to the authentic or
native D-sequence in place of nucleotides in the S-sequence. These
four oligonucleotides are:
2 D-5 oligonucleotide: 5' CCAA CTCCA GATCT GATAT CACTT 3' 3' GGTT
GAGGT CTAGA CTATA GTGAA GATC D-10 oligonucleotide: 5' CCAA CTCCA
TCACT GATAT CACTT 3' 3' GGTT GAGGT AGTGA CTATA GTGAA GATC 5' D-15
oligonucleotide: 5' CCAA CTCCA TCACT AGGGG CACTT 3' 3' GGTT GAGGT
AGTGA TCCCC GTGAA GATC 5' D-20 oligonucleotide: 5' CCAA CTCCA TCACT
AGGGG TTCCT 3' 3' GGTT GAGGT AGTGA TCCCC AAGGA GATC 5'
[0059] The selected nucleotides conforming to the authentic,
native, D-sequence in the AAV ITR are indicated above in bold.
[0060] The D-5, D-10, D-15 and D-20 oligonucleotide sequences were
each inserted between the Xba I and Bal I sites of plasmid pXS-22,
which is described in Wang, J. Mol. Biol. 250 (1995) 573-580 and J.
Virol. 70 (1996) 1668-1677. The resulting four plasmids were named
pXS-64D-5, pXS-64D-10, pXS-64D-15 and pXS-64D-20 respectively. The
blunted ClaI-PvuII fragments from pXS-64D-5, pXS-64D-10, pXS-64D-15
and pXS-64D-20 were then excised and ligated between the ClaI and
XbaI sites of these plasmids to generate plasmids pD-5, pD-10,
pD-15 and pD-20 respectively containing the D-5, D-10, D-15 and
D-20 oligonucleotide sequences in place of the S sequences in both
ITRs.
[0061] Each of the four foregoing recombinant AAV vectors, pD-5,
pD-10, pD-15 and pD-20 may be employed in the methods of the
invention. We have determined that to optimize packaging 10 of the
native D-nucleotides are sufficient. The most preferred native 10
D-nucleotides are those included in the pD-10 vector and indicated
in bold in the D-10 oligonucleotide sequence above. The pD-15 and
pD-20 vectors, or their respective indicated oligonucleotides (see
above), may be used but they contain extra, unnecessary nucleotides
that would advantageously be eliminated in order to allow for more
space in the AAV vector for nucleotides encoding the desired
therapeutic molecule. The pD-5 vector works, but with less
efficiency. Consequently, the absolute minimal necessary sequence
is the 5 nucleotide sequence enumerated in bold in the D-5
oligonucleotide sequence above and contained in the pD-5 vector.
The pD-10 vector allows for the insertion of an additional 106
nucleotides.
[0062] Nucleic acid sequences encoding therapeutic molecules can be
ligated between the ITRs of these vectors using known techniques.
The vectors or virions may be formulated into pharmaceutical
compositions for administration in human or other mammalian
patients.
[0063] Plasmid pXS-22 was deposited on Sep. 10, 1996 with the ATCC,
12301 Parklawn Drive, Rockville, Md., USA under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for Purposes of Patent Procedure. The Accession
Number is 97710. This deposit assures maintenance of a viable
culture for 30 years from the date of deposit. The organism(s)
deposited will be made available by the ATCC under the terms of the
Budapest Treaty, and subject to an agreement between applicant and
the ATCC that assures unrestricted availability upon issuance of
the pertinent U.S. patent. This deposit is provided as convenience
to those of skill in the art, and is not an admission that a
deposit is required under 35 U.S.C. 112. The nucleic acid sequence
of this deposit, as well as the amino acid sequence of the
polypeptide(s) encoded thereby, are incorporated herein by
reference and should be referred to in the event of an error in the
sequence described herein. A license may be required to make, use,
or sell the deposited materials, and no such license is granted
hereby.
[0064] All patents, patent publications, patent applications and
scientific articles mentioned in this specification are herein
incorporated by reference. The invention now being fully described,
it will be apparent to one of ordinary skill in the art that many
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims.
Sequence CWU 1
1
26 1 19 DNA Artificial Sequence The full sequence for lacZ from
plasmid PCMV p-lacZ is found in Ponnazhagan, et al., J. Gen Virol.,
771111-1122 (1996) 1 gatgagcgtg gtggttatg 19 2 19 DNA Artificial
Sequence The full sequence for lacZ from plasmid PCMV p-lacZ is
found in Ponnazhagan, et al., J. Gen Virol., 771111-1122 (1996) 2
tacagcgcgt cgtgattag 19 3 20 DNA homo sapiens misc_feature primer
for human beta-globin promoter 3 gatggtatgg ggccaagaga 20 4 20 DNA
homo sapiens misc_feature primer for human gamma-globin 4
gggtttctcc tccagcatct 20 5 20 DNA mouse misc_feature primer for the
mouse beta-actin gene 5 accttcaaca ccccagccat 20 6 20 DNA mouse
misc_feature primer for the mouse beta-actin gene 6 tcaggcagct
catagctctt 20 7 20 DNA adenoassociated virus misc_feature
D-sequence in ITRs 7 ctccatcact aggggttcct 20 8 20 DNA
adenoassociated virus misc_feature D-sequence in ITRs Antisense
strand 8 aggaacccct agtgatggag 20 9 21 DNA adenoassociated virus
misc_feature substitute D-sequence in ITRs 9 ccaatattag atctgatatc
a 21 10 25 DNA adenoassociated virus misc_feature substitute
D-sequence in ITRs Antisense strand 10 ctagtgatat cagatctaat attgg
25 11 24 DNA adenoassociated virus misc_feature D-sequence in ITRs
11 ccaactccag atctgatatc actt 24 12 5 DNA adenoassociated virus
misc_feature D-sequence in ITRs 12 ctcca 5 13 28 DNA
adenoassociated virus misc_feature D-sequence in ITRs Antisense
strand 13 ctagaagtga tatcagatct ggagttgg 28 14 5 DNA
adenoassociated virus misc_feature D-sequence in ITRs Antisense
strand 14 tggag 5 15 24 DNA adenoassociated virus misc_feature
D-sequence in ITRs 15 ccaactccat cactgatatc actt 24 16 10 DNA
adenoassociated virus misc_feature D-sequence in ITRs 16 ctccatcact
10 17 28 DNA adenoassociated virus misc_feature D-sequence in ITRs
Antisense strand 17 ctagaagtga tatcagtgat ggagttgg 28 18 10 DNA
adenoassociated virus misc_feature D-sequence in ITRs Antisense
strand 18 agtgatggag 10 19 24 DNA adenoassociated virus
misc_feature D-sequence in ITRs 19 ccaactccat cactaggggc actt 24 20
15 DNA adenoassociated virus misc_feature D-sequence in ITRs 20
ctccatcact agggg 15 21 28 DNA adenoassociated virus misc_feature
D-sequence in ITRs Antisense strand 21 ctagaagtgc ccctagtgat
ggagttgg 28 22 15 DNA adenoassociated virus misc_feature D-sequence
in ITRs Antisense strand 22 cccctagtga tggag 15 23 24 DNA
adenoassociated virus misc_feature D-sequence in ITRs 23 ccaactccat
cactaggggt tcct 24 24 20 DNA adenoassociated virus misc_feature
D-sequence in ITRs 24 ctccatcact aggggttcct 20 25 28 DNA
adenoassociated virus misc_feature D-sequence in ITRs Antisense
strand 25 ctagaggaac ccctagtgat ggagttgg 28 26 20 DNA
adenoassociated virus misc_feature D-sequence in ITRs Antisense
strand 26 aggaacccct agtgatggag 20
* * * * *