U.S. patent application number 09/219977 was filed with the patent office on 2002-06-20 for modified adipose tissue and related implants and methods.
Invention is credited to CRYSTAL, RONALD G., HOFFMAN, LLOYD, MAGOVERN, CHRISTOPHER J., ROSENGART, TODD, TALMOR, MIA.
Application Number | 20020076395 09/219977 |
Document ID | / |
Family ID | 24698636 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020076395 |
Kind Code |
A1 |
CRYSTAL, RONALD G. ; et
al. |
June 20, 2002 |
MODIFIED ADIPOSE TISSUE AND RELATED IMPLANTS AND METHODS
Abstract
The present invention provides adipose tissue modified with a
vector comprising or encoding, in which case it expresses, an
anti-angiogenic factor, an angiogenic substance, an apoptotic
factor, an adipsin protein or an Ob protein. Also provided is
adipose tissue modified with a vector comprising a promoter and,
operably linked thereto, a DNA sequence encoding a secreted
protein. Methods of therapeutically treating adipose tissue and
expressing a secreted protein in adipose tissue are also provided.
The adipose tissue is optionally in the form of an implant, which
can further comprise a lymphogenic protein or a vector that
comprises and expresses a lymphogenic gene.
Inventors: |
CRYSTAL, RONALD G.;
(POTOMAC, MD) ; MAGOVERN, CHRISTOPHER J.; (NEW
YORK, NY) ; ROSENGART, TODD; (TENAFLY, NJ) ;
HOFFMAN, LLOYD; (GREAT NECK, NY) ; TALMOR, MIA;
(NEW YORK, NY) |
Correspondence
Address: |
LEYDIG VOIT AND MAYER LTD
TWO PRUDENTIAL PLAZA SUITE 4900
180 NORTH STETSON
CHICAGO
IL
606016780
|
Family ID: |
24698636 |
Appl. No.: |
09/219977 |
Filed: |
December 23, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09219977 |
Dec 23, 1998 |
|
|
|
PCT/US97/11229 |
Jun 26, 1997 |
|
|
|
09219977 |
Dec 23, 1998 |
|
|
|
08672461 |
Jun 26, 1996 |
|
|
|
Current U.S.
Class: |
424/93.2 |
Current CPC
Class: |
A61P 3/04 20180101; C12N
2830/008 20130101; C12N 2710/10343 20130101; A61P 27/06 20180101;
A61P 3/06 20180101; C12N 5/0653 20130101; A61F 2/02 20130101; A61P
35/00 20180101; A61P 9/12 20180101; C12N 15/86 20130101; A61P 3/10
20180101; A61K 48/00 20130101; A61P 9/10 20180101; A61P 29/00
20180101; C07K 14/52 20130101; A61P 17/02 20180101; A61K 35/12
20130101 |
Class at
Publication: |
424/93.2 |
International
Class: |
A01N 063/00 |
Claims
What is claimed is:
1. A method of therapeutically treating adipose tissue, which
method comprises contacting said adipose tissue with a vector
comprising a nucleic acid sequence comprising or encoding, in which
case it expresses, a) an anti-angiogenic factor such that said
vector enters said adipose tissue and said anti-angiogenic factor
inhibits vascularity in said adipose tissue, b) an apoptotic factor
such that said vector enters said adipose tissue and said apoptotic
factor causes adipocyte cell death, c) an adipsin protein such that
said vector enters said adipose tissue and said adipsin protein
treats said adipose tissue therapeutically, d) an Ob protein such
that said vector enters said adipose tissue and said Ob protein
treats said adipose tissue therapeutically, or e) an angiogenic
substance such that said vector enters said adipose tissue and said
angiogenic substance increases the vascularity in said adipose
tissue, with the proviso that, when said vector comprises a nucleic
acid sequence encoding any one of (a)-(e), said nucleic acid
sequence is operably linked to a promoter.
2. The method of claim 1, wherein said vector is a viral
vector.
3. The method of claim 2, wherein said viral vector is an
adenoviral vector.
4. The method of claim 3, wherein said adenoviral vector is
replication-deficient.
5. The method of claim 1, wherein said promoter is
adipocyte-specific.
6. The method of claim 5, wherein said promoter is from the
regulatory region of either of the adipocyte P2 (aP2) gene or the
p154 polypeptide gene.
7. The method of claim 1, wherein said promoter is
constitutive.
8. The method of claim 1, wherein said anti-angiogenic factor is
selected from the group consisting of taxol, endostatin,
angiostatin, fumagillin and an analogue of fumagillin.
9. The method of claim 1, wherein said angiogenic substance is a
vascular endothelial growth factor (VEGF).
10. The method of claim 1, wherein said apoptotic factor is
selected from the group consisting of p53, a cell death-inducing
coding sequence of Bc1-2 which comprises an N-terminal deletion, a
cell death-inducing coding sequence of Bc1-x which comprises an
N-terminal deletion, Bax, Bak, Bid, Bad, Bik, Bif-2, IAP-1, IAP 2,
a caspase, TGF .beta.1, c myc, a protease, and a protein
kinase.
11. The method of claim 10, wherein said protein kinase is selected
from the group consisting of protein kinase C.theta., protein
kinase C.delta., Akt/PI(3)-kinase, DNA-PK, PITSLRE, DAP kinase,
RIP, JNK/SAPK, Daxx, Raf-1, Pim-1, NIK, MEKK1, ASK1, and PKR.
12. An isolated adipose tissue comprising a vector comprising a
nucleic acid sequence comprising or encoding, in which case it
expresses, a) an anti-angiogenic factor, b) an apoptotic factor, c)
an adipsin protein, d) an Ob protein, or e) an angiogenic
substance, wherein said isolated adipose tissue is optionally in
the form of an implant.
13. The isolated adipose tissue of claim 12, wherein said vector is
a viral vector.
14. The isolated adipose tissue of claim 13, wherein said viral
vector is an adenoviral vector.
15. The isolated adipose tissue of claim 14, wherein said
adenoviral vector is replication-deficient.
16. The isolated adipose tissue of claim 12, wherein said promoter
is adipocyte-specific.
17. The isolated adipose tissue of claim 12, wherein said promoter
is from the regulatory region of either of the adipocyte P2 (aP2)
gene or the p154 polypeptide gene.
18. The isolated adipose tissue of claim 12, wherein said promoter
is constitutive.
19. The isolated adipose tissue of claim 12, wherein said
anti-angiogenic factor is selected from the group consisting of
taxol, endostatin, angiostatin, fumagillin and an analogue of
fumagillin.
20. The isolated adipose tissue of claim 12, wherein said
angiogenic substance, anti-angiogenic factor, adipsin protein or Ob
protein is secreted.
21. The isolated adipose tissue of claim 12, wherein said apoptotic
gene is selected from the group consisting of p53, a cell
death-inducing coding sequence of Bc1-2 which comprises an
N-terminal deletion, a cell death-inducing coding sequence of Bc1-x
which comprises an N-terminal deletion, Bax, Bak, Bid, Bad, Bik,
Bif-2, IAP-1, IAP-2, a caspase, TGF-.beta.1, c-myc, a protease, and
a protein kinase.
22. The isolated adipose tissue of claim 21, wherein said protein
kinase is selected from the group consisting of protein kinase
C.theta., protein kinase C.delta., Akt/PI(3)-kinase, DNA-PK,
PITSLRE, DAP kinase, RIP, JNK/SAPK, Daxx, Raf-1, Pim-1, NIK, MEKK1,
ASK1, and PKR.
23. The isolated adipose tissue of claim 12, wherein said isolated
adipose tissue is in the form of an implant and further comprises a
lymphogenic protein or a vector that comprises and expresses a
lymphogenic gene.
24. The adipose tissue implant of claim 12, wherein said vector
comprises a gene encoding a vascular endothelial growth factor
(VEGF).
25. A method of expressing a secreted protein in adipose tissue,
which method comprises contacting said adipose tissue with a vector
comprising a promoter and, operably linked thereto, a DNA sequence
encoding a secreted protein such that said gene transfer vector
enters said adipose tissue and said protein is expressed and
secreted.
26. The method of claim 25, wherein said vector is a viral
vector.
27. The method of claim 26, wherein said viral vector is an
adenoviral vector.
28. The method of claim 27, wherein said adenoviral vector is
replication-deficient.
29. The method of claim 25, wherein said promoter is
adipocyte-specific.
30. The method of claim 29, wherein said promoter is from the
regulatory region of either of the adipocyte P2 (aP2) gene or the
p154 polypeptide gene.
31. The method of claim 25, wherein said promoter is
constitutive.
32. An isolated adipose tissue comprising a vector comprising a
promoter and, operably linked thereto, a DNA sequence encoding a
secreted protein, wherein said isolated adipose tissue is
optionally in the form of an implant.
33. The isolated adipose tissue of claim 32, wherein said vector is
a viral vector.
34. The isolated adipose tissue of claim 33, wherein said viral
vector is an adenoviral vector.
35. The isolated adipose tissue of claim 34, wherein said
adenoviral vector is replication-deficient.
36. The isolated adipose tissue of claim 32, wherein said promoter
is adipocyte-specific.
37. The isolated adipose tissue of claim 32, wherein said promoter
is from the regulatory region of either of the adipocyte P2 (aP2)
gene or the p154 polypeptide gene.
38. The isolated adipose tissue of claim 32, wherein said promoter
is constitutive.
39. The isolated adipose tissue of claim 32, wherein said isolated
adipose tissue is in the form of an implant and further comprises a
lymphogenic protein or a vector that comprises and expresses a
lymphogenic gene.
Description
[0001] This is a continuation-in-part application of International
patent application PCT/US97/11229, filed Jun. 26, 1997, which is a
continuation-in-part of U.S. patent application Ser. No.
08/672,461, filed Jun. 26, 1996.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to modified adipose tissue,
implants made from modified adipose tissue, a method of
therapeutically treating adipose tissue, and a method of expressing
a secreted protein in adipose tissue.
BACKGROUND OF THE INVENTION
[0003] Gene therapy entails the use of genetic information as the
pharmacologic agent. While originally conceived as a means of
treating hereditary disease, gene therapy is now recognized as a
powerful tool for delivering therapeutic mRNA or proteins for local
and/or systemic use (see, e.g., Friedmann, Science, 244, 1275-1281
(1989); Miller, Nature 357, 455-460 (1992)). Generally, there are
two approaches to gene therapy: ex vivo and in vivo. In the ex vivo
approach, cells removed from a host are genetically modified in
vitro before being returned to the host (see, e.g., U.S. Pat. No.
5,399,346 (Anderson et al.)). In the in vivo approach, the genetic
information itself is transferred directly to the host, without
employing any cells as a vehicle for transfer.
[0004] Both approaches have been employed to transfer a so-called
"therapeutic" gene to a host. Broadly considered, a therapeutic
gene is a gene that corrects or compensates for an underlying
protein deficit or, alternately, a gene that is capable of
regulating another gene, or counteracting the negative effects of
its encoded product, in a particular disease state, condition,
disorder or syndrome. For instance, the ex vivo approach has been
used for the modification of T lymphocytes in the treatment of
adenosine deaminase deficiency, modification of hepatocytes in the
treatment of familial hypercholesterolemia, and modification of
tumor infiltrating lymphocytes in the treatment of neoplastic
disease (reviewed in Setoguchi et al., J. Investig. Dermatol., 102,
415-421 (1994)). The in vivo approach has been used, among others,
for the treatment of cystic fibrosis and neoplastic disease
(Setoguchi et al., supra). For the majority of these applications,
the coding sequence of the therapeutic gene to be expressed has
been placed under the control of a heterologous promoter (in
particular, a constitutive or inducible promoter), generating a
recombinant therapeutic gene.
[0005] The predominant approach to gene therapy has employed a
retrovirus as a vehicle for gene transfer. However, retroviruses
have a number of drawbacks which severely limit their application,
particularly in vivo (Mastrangeli et al., J. Clin. Invest., 91,
225-34 (1993); Burns et al., Proc. Natl. Acad. Sci., 90, 8033-37
(1993)). Consequently, many researchers have turned to the
adenovirus as a vector for gene therapy (Horwitz, In: Virology 2nd
Ed., Fields et al., eds. (NY: Raven Press, 1990), 1679-1721;
Berkner, BioTechniques, 6, 606-629 (1988); Ginsberg (ed.), The
Adenoviruses (NY: Plenum Press, 1984); Horwitz, supra; Rosenfeld et
al., Science, 252, 431-434 (1991); Rosenfeld et al., Cell, 68,
143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89,
2581-2584 (1992); Crystal et al., Nucleic Acids Res., 21, 1607-12
(1993)). Replication-deficient, recombinant adenovirus vectors are
highly efficient at transferring genes in vitro and in vivo, and
currently are used in a wide variety of applications (see, e.g.,
Rosenfeld et al. (1991), supra; Rosenfeld et al. (1992), supra;
Crystal et al., Nat. Genet., 8, 42-51 (1994); Lemarchand et al.,
Circ. Res., 72, 1132-1138 (1993); Guzman et al., Circ. Res., 73,
1202-1207 (1993); Bajocchi et al., Nat. Genet., 3, 229-234 (1993);
Mastrangeli et al., supra).
[0006] Adenoviruses exist as non-enveloped double-stranded DNA
viruses (Horwitz, supra). The adenovirus provides an efficient
means for transferring biological materials to target cells (Otero
et al., Virology, 160, 75-80 (1987); FitzGerald et al., Cell, 32,
607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984);
Yoshimura, Cell Struct. Funct., 10, 391-404 (1985); Defer et al.,
J. Virol., 64, 3661-3673 (1990); Rosenfeld et al. (1991), supra;
Curiel et al., Proc. Natl. Acad. Sci., 88, 8850-8854 (1991);
Rosenfeld et al. (1992), supra; Quantin et al., supra; Curiel et
al., Hum. Gene Therapy, 3, 147-154 (1992)). The adenovirus enters
cells by a receptor mediated endocytosis pathway. In the initial
virus receptor interaction, the adenovirus binds specific receptors
present on the cell surface via fibers on its outer surface
(Ginsberg, supra; Horwitz, supra; Seth et al., In: Virus Attachment
and Entry into Cells, Colwell et al., eds. (DC: American Society
for Microbiology, 1986), 191-195). Following attachment, the
receptors with bound adenovirus cluster in coated pits, and the
virus is internalized within a clathrin-coated vesicle and,
subsequently, into an endosomal vesicle, termed an endosome, or
receptosome (FitzGerald et al., supra). The adenovirus ultimately
is translocated to the nucleus, where it directs the synthesis of
nascent nucleic acids (FitzGerald et al., supra; Seth et al.
(1984), supra; Seth et al. (1986), supra; Seth et al., J. Virol.,
51, 650-655 (1984a); Seth et al., J. Biol. Chem., 259, 14350-14353
(1984b); Seth et al., J. Biol. Chem., 260, 9598-9602 (1985); Seth
et al., J. Biol. Chem., 260, 14431-14434 (1985); Blumenthal et al.,
Biochemistry, 25, 2231-2237 (1986); Seth et al., J. Virol., 61,
883-888 (1987)).
[0007] The ability of the adenovirus to easily enter cells has been
seized upon as a means of transporting macromolecules into cells
(Otero et al., supra; FitzGerald et al., supra; Seth et al. (1984),
supra; Yoshimura, supra; Defer et al., supra; Rosenfeld et al.
(1991), supra; Curiel et al. (1991), supra; Rosenfeld et al.
(1992), supra; Quantin et al., supra; Curiel et al. (1992), supra).
There are two means by which such transfer has been effected.
First, the adenovirus has been employed to transfer non-viral
macromolecules packaged within the adenovirus either in place of,
or in addition to, normal adenoviral components (Rosenfeld et al.
(1991), supra; Rosenfeld et al. (1992), supra; Quantin et al.,
supra; Berkner, supra). Second, the adenovirus has been employed to
mediate the transfer of non-viral macromolecules either linked to
the surface of the adenovirus (e.g., by means of conjugation of the
nucleic acid through a polylysine residue to an antibody to
adenoviral capsid protein (Curiel et al. (1992), supra)) or in a
"bystander" process where the macromolecule is cointernalized and
taken along as cargo in the adenoviral receptor-endosome complex
(Otero et al., supra; FitzGerald et al., supra; Seth et al. (1984),
supra; Yoshimura, supra; Otero et al., supra; Defer et al., supra).
Such a bystander process has been employed to enhance the transfer
of a variety of non-viral macromolecules including plasmid DNA
linked to ligands (Curiel et al. (1991), supra; Curiel et al.
(1992), supra; Cotten et al., Proc. Natl. Acad. Sci., 89, 6094-098
(1992)); Rosenfeld et al. (1992), supra; Quantin et al., supra;
Cotten et al., J. Virology, 67, 3777-3785 (1993); Wagner et al.,
Proc. Natl. Acad. Sci., 78, 144-145 (1981)), and plasmid DNA
unmodified by nonspecific linkers or by linker-ligand complexes
(Yoshimura et al., J. Biolog. Chem., 268 2300-303 (1993); PCT
Application WO 95/21259 (Seth et al.)).
[0008] Recently, Setoguchi et al. (Setoguchi et al., supra)
disclosed adenoviral-mediated gene transfer to adipocytes in vivo
of a replication-deficient recombinant adenoviral vector carrying
the coding sequence of the .beta.-galactosidase reporter gene under
the control of the Rous sarcoma virus long terminal repeat as a
promoter. Similarly, Clayman et al. (Clayman et al., Cancer Gene
Therapy, 2, 105-111 (1995)) disclosed that submucosal injection in
mice of a recombinant adenoviral vector carrying a
.beta.-galactosidase reporter gene produces scattered staining of
adipocytes along the needle track.
[0009] Other investigators working with vectors and means of
delivery other than adenovirus have transferred genes other than
reporter genes to adipocytes in vivo. Specifically, Ross et al.
(Ross et al., Genes Devel., 7, 1318-1324 (1993)) disclosed the
reduction of adiposity via gene transfer to adipose tissue of an
attenuated diphtheria toxin A chain under the control of the
adipocyte-specific adipocyte P2 (aP2) promoter. Yamaizumi et al.
(Yamaizumi et al., Cell, 15, 245-50 (1978)) disclosed cell killing
through the introduction of diphtheria toxin fragment A, and
Gregory et al. (Gregory et al., PCT Application WO 95/11984)
disclosed means of inducing cell death, such as with use of the
conditional suicide gene thymidine kinase. Similarly, Graves et al.
(Graves et al., Genes & Development, 5, 428-37 (1991)) and Ross
et al. (Ross et al., Proc. Natl. Acad. Sci., 89, 7561-65 (1992);
Ross et al., Proc. Natl. Acad. Sci., 87, 9590-94 (1990)) each
disclosed an adipocyte-specific enhancer located in the
5'-regulatory region of the aP2 gene.
[0010] Other references similarly have disclosed methods for
deleting specific cell lineages by cell-specific expression of a
toxin gene (Palmiter et al., Cell 50, 435-43 (1987); Bernstein et
al., Mol. Biol. Med., 6, 523-30 (1989); Behringer et al., Genes
& Development, 2, 453-61 (1988); Hughes et al., PCT Application
WO 92/09616)). The method employed typically calls for
microinjecting into fertilized eggs a chimeric gene in which a
cell-specific enhancer/promoter is used to drive the expression of
a toxic gene product. In a modification of this approach, Hughes et
al. (Hughes et al., supra) disclosed reduction in the amount of
fatty tissues of a host due to introduction of a vector encoding
the chicken c-ski protein, which induces myogenic
differentiation.
[0011] References not involving adenovirus as a means of gene
transfer suggest further ways in which adipocytes can be modified
in vivo to achieve specific therapeutic aims. Specifically,
Spiegelman et al. (Spiegelman et al., J. Biol. Chem., 268(10),
6823-26 (1993)) reviewed the regulation of adipocyte gene
expression and disclosed "influencing metabolism by controlling
adipogenic gene expression" and "[interfering] with adipogenesis
and systemic metabolism by targeting these key regulators"
associated with cell differentiation or obesity. Graves et al.
(Graves et al., supra) disclosed that "the relationship between
obesity and diabetes in several obese/diabetic mouse models . . .
could be probed by directly suppressing adipose cell formation
and/or lipid accumulation through the delivery of toxins or various
receptors affecting lipid accumulation." Ross et al. (Ross et al.
(1990), supra) disclosed the production of transgenic mice
containing the adipocyte-specific aP2 gene regulatory region linked
to the coding sequence of a reporter gene as a means of monitoring
tissue-specific expression and suggest "adipose-directed expression
of exogenous genes may be an effective method to alter fat storage
and thus directly manipulate the fatness of transgenic animals."
Ross et al. (Ross et al. (1992), supra) further disclosed the
production of transgenic mice containing the adipocyte-specific aP2
gene regulatory region linked to the simian virus 40 (SV40)
transforming genes as a means of directing expression of linked
exogenous genes, such as oncogenes, to adipose tissue.
[0012] Other references also have relevance to adipocyte
modification. Specifically, U.S. Pat. No. 5,268,295 (Serrero)
relates to a mammalian adipocyte-specific polypeptide, termed p154,
which is expressed in high quantities in adipogenic cell lines
after differentiation. The '295 patent discloses the murine and
human p154 polypeptide, as well as the DNA and RNA molecules coding
therefor, methods for its preparation, and antibodies specific for
the polypeptide. Flier et al. (Flier et al., Science, 237, 405-8
(1987)) disclose that expression of an adipsin gene and,
correspondingly, circulating levels of the serine protease homolog
are decreased in obesity. More recently, researchers have
demonstrated that the protein product (Ob) of the mouse obese gene
causes weight loss and maintenance of the weight loss when injected
into animals (e.g., reviewed in Barinaga, Science, 269, 475-76
(1995)).
[0013] Accordingly, there is a need for an improved means of
modifying adipocytes and adipose tissue by transferring vectors
comprising nucleic acid sequences thereto. In addition, there is a
need for modified adipocytes, adipose tissue and adipose tissue
implants. It is an object of the present invention to provide such.
This and other objects and advantages of the present invention, as
well as additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention provides methods and vectors to modify
adipocytes and adipose tissue in vivo and ex vivo, including
adenoviral mediated gene transfer and other means. The present
invention also provides for the in vivo transfer of nucleic acids
to adipocytes and adipose tissue to provide a source of RNA, such
as antisense molecules and ribozymes, or genes encoding
polypeptides or proteins to be used in the local milieu of the
adipocyte tissue or to be secreted and used systemically. In
particular, the present invention provides for the transfer of
nucleic acids encoding apoptotic factors or toxic genes to
adipocytes and adipose tissue in vivo as a means of reducing
adiposity, the transfer of genes encoding an adipsin protein or an
Ob protein, the transfer of genes encoding angiogenic substances to
induce neovascularization, as well as the transfer of nucleic acids
comprising or encoding anti-angiogenic factors to inhibit the
vascularity of adipose tissue. Furthermore, the present invention
provides for the modification and transfer of adipocytes or adipose
tissue to other sites within a host, which provides improved
transplantation of the adipocytes or adipose tissue, a
transplantable source of therapeutic or diagnostic biological
molecules, and other advantages.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a graph of dose of AdCMVCAT (total pfu) delivered
to rat retroperitoneal adipose tissue versus chloramphenicol acetyl
transferase (CAT) conversion (% conversion).
[0016] FIG. 2 is a graph of time (days) following vector (AdCMVCAT)
administration to rat retroperitoneal adipose tissue versus
relative CAT activity (% conversion/mg protein). The arrow
indicates the time of vector administration.
[0017] FIG. 3 is a graph of time (days) versus level of VEGF (ng/mg
protein) following AdCMV.VEGF (.circle-solid.) or AdCMV.Null (o)
administration to rat retroperitoneal tissue. The arrow indicates
the time of vector administration.
[0018] FIG. 4 is a graph of time (days) versus gross vessel count
following AdCMV.VEGF (.circle-solid.), AdCMV.Null (o), sham
(.quadrature.) or AdCMV.VEGF (contralateral) (.DELTA.)
administration to rat retroperitoneal tissue. The arrow indicates
the time of vector administration.
[0019] FIG. 5 is a graph of time (days) versus capillary
number/mm.sup.2 following AdCMV.VEGF (.circle-solid.) or AdCMV.Null
(o) administration (10.sup.9 pfu) to rat retroperitoneal tissue.
The arrow indicates the time of vector administration.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides a method of modifying an
adipocyte or adipose tissue. The method comprises contacting the
adipocyte or adipose tissue with a vector, i.e., an RNA or DNA
vector, comprising a nucleic acid sequence comprising or encoding a
therapeutic polypeptide or protein or a therapeutic RNA. When the
vector comprises a nucleic acid sequence encoding a therapeutic
polypeptide or protein or therapeutic RNA, the nucleic acid
sequence is operably linked to a promoter. Desirably, the
contacting is done under conditions, particularly in vivo, such
that entry of the vector into the adipocyte or adipose tissue is
effected and the therapeutic polypeptide or protein or therapeutic
RNA exerts its effect.
[0021] While any suitable vector, in particular, any suitable viral
vector, can be utilized for transfer of a nucleic acid sequence to
adipocytes and adipose tissue, it is preferable to use an
adenoviral vector, which preferably is replication-deficient. A
constitutive promoter or an adipocyte-specific promoter, such as a
promoter from the regulatory region of either of the adipocyte P2
(a P2) gene or the p154 polypeptide gene, can be used. For
instance, an adenoviral vector comprising an adipocyte-specific
promoter and, operably linked thereto, a nucleic acid sequence
encoding a therapeutic polypeptide or protein or therapeutic RNA,
e.g., an anti-sense RNA or a ribozyme, can be used. Alternatively,
the nucleic acid transfer can be carried out using an adenoviral
vector comprising a constitutive promoter (e.g., a CMV promoter)
and, operably linked thereto, a nucleic acid sequence comprising or
encoding a therapeutic polypeptide or protein or a therapeutic RNA.
Preferably, the nucleic acid comprises or encodes as appropriate
(i) a secreted protein, such as a secreted protein that acts
systemically, (ii) a protein that acts upon or in the vicinity of
an adipocyte, (iii) a toxin (especially diphtheria toxin A), (iv)
an angiogenic substance, such as an angiogenic growth factor
(especially a vascular endothelial cell growth factor (VEGF,
including VEGF.sub.121, VEGF.sub.165, or VEGF.sub.189)), (v) an
adipsin (especially an adipsin that is a serine protease homolog),
(vi) a protein product of the obese gene, namely an Ob protein or
leptin (especially an Ob protein from mouse or human), (v) an
apoptotic factor, and (vi) an anti-angiogenic factor. The present
invention also provides host cells, particularly cells of adipose
tissue, comprising the vectors of the present invention.
[0022] To optimize the ability of the vector, e.g., adenovirus, to
enter the cell, preferably the nucleic acid transfer is carried out
in the absence of neutralizing antibodies directed against a
particular adenovirus being introduced in the host. In the absence
of such antibodies, there is no possibility of the antibody
impeding the adenovirus from binding to and/or entering the cell.
It is well within the ordinary skill of one in the art to test for
the presence of neutralizing antibodies. In the event the presence
of such neutralizing antibodies are an obstacle to the
intracellular delivery of an adenovirus, another adenoviral vector,
e.g., another serotype adenoviral vector (Crompton et al., J. Gen.
Virol., 75, 133-139 (1994)), or another adenovirus vector lacking
the epitope against which the antibody is directed, can be
employed.
[0023] The present invention also provides isolated adipose tissue
comprising a vector as described herein. The adipose tissue can be
in the form of an adipose tissue implant, in which case the implant
can comprise adipose tissue isolated from a donor for implantation
into a host and can further comprise a lymphogenic protein or a
vector that comprises and expresses a lymphogenic gene.
[0024] An adipose tissue implant can be produced by modifying
adipose tissue of a donor animal (including humans) either in vivo
or ex vivo with an angiogenic substance or composition to provide
increased vascularization of the adipose tissue used to form the
implant. When the implant (i.e., adipose tissue implant) is
transferred (implanted) to a second site in the same animal or into
an immunologically compatible host (or second animal), the action
of the angiogenic factor or composition causes an increase in
vascularization compared to implanted tissue that has not been
modified or processed with an angiogenic substance or composition.
The increased vascularization results in a better supply of
nutrients and oxygen to the implant and better removal of waste
products produced by the implant in the second site or host. This
improved exchange of nutrients and waste products results in a
lower level of adipocyte loss following implantation of the adipose
tissue implant. By immunologically compatible is meant a host whose
immune system will not or cannot attack and destroy the cells that
form the implant such that the implant is substantially
eliminated.
[0025] The present invention also provides for an adipose tissue
implant comprising isolated adipose tissue (e.g., isolated from a
donor for implantation into a host) and an anti-angiogenic factor,
an apoptotic factor, an adipsin or an Ob protein.
[0026] The present invention also provides a method of
therapeutically treating adipose tissue and a method of expressing
a secreted protein in an adipocyte or isolated adipose tissue. The
methods comprise contacting the adipose tissue with a vector as
described herein.
[0027] Definitions
[0028] A "therapeutic gene" comprises a promoter and a nucleic acid
sequence encoding a therapeutic polypeptide or protein or a
therapeutic RNA. Such a therapeutic gene can be subcloned into a
vector according to the present invention, such that, upon
introduction into a host cell, there will be a discernible change
in the intracellular environment (e.g., an increased level of
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide or
protein, or an altered rate of production or degradation thereof),
as further described herein, to provide a therapeutic benefit. A
"gene product" is either an as yet untranslated RNA molecule
transcribed from a given nucleic acid sequence (e.g., mRNA or
antisense RNA) or the polypeptide chain (i.e., protein or peptide)
translated from the mRNA molecule transcribed from the aforesaid
nucleic acid sequence. A nucleic acid sequence or gene is
"recombinant" if the sequence of bases along the molecule has been
altered from the sequence in which the nucleic acid sequence or
gene is typically found in nature, or if the sequence of bases is
not typically found in nature. According to this invention, a
therapeutic gene can be wholly or partially synthetically made, can
comprise genomic or complementary DNA (cDNA) sequences, and can be
provided in the form of either DNA or RNA.
[0029] Therapeutic genes include, but are not limited to
"angiogenic genes" such as those comprising promoters operably
linked to DNA encoding one of the VEGF proteins, particularly
VEGF.sub.121, VEGF.sub.165, or VEGF.sub.189. In addition to the
VEGF proteins, aFGF, bFGF, and epithelial growth factor are also
illustrative of other angiogenic proteins suitable for use in this
inventive method. Angiogenic genes encode "angiogenic mRNAs" and
"angiogenic polypeptides or proteins," by which is meant any
polypeptide or protein that is capable of mediating blood vessel
formation (angiogenesis). Therapeutic genes also include
"anti-angiogenic genes," i.e., any gene encoding an
"anti-angiogenic RNA," e.g., an anti-sense molecule or a ribozyme,
or an "anti-angiogenic protein or polypeptide" that inhibits the
vascularity of adipose tissue, such as genes comprising promoters
operably linked to DNA sequences encoding, for example, taxol,
angiostatin, endostatin, fumagillin, and an analogue of fumagillin,
as well as others known in the art. One of ordinary skill in the
art will appreciate that "inhibition of vascularity," in the
context of the present invention, refers to the inhibition of new
blood vessel formation, as well as the inhibition of the growth of
existing blood vessels.
[0030] Similarly, a lymphogenic DNA comprises a promoter operably
linked to a nucleic acid sequence encoding a "lymphogenic protein"
or "lymphogenic mRNA." VEGF-C is a suitable illustration of a
lymphogenic protein.
[0031] A "promoter" is a DNA sequence that directs the binding of
RNA polymerase and thereby promotes RNA synthesis. "Enhancers" are
cis-acting elements of DNA that stimulate or inhibit transcription
of adjacent genes. An enhancer that inhibits transcription also is
termed a "silencer." Enhancers differ from DNA-binding sites for
sequence-specific DNA binding proteins found only in the promoter
(which also are termed "promoter elements") in that enhancers can
function in either orientation, and over distances of up to several
kilobasepairs (kb), even from a position downstream of a
transcribed region. According to the invention, a nucleic acid
sequence encoding a therapeutic protein or therapeutic mRNA is
"operably linked" to a promoter (e.g., when both the nucleic acid
sequence and the promoter constitute a therapeutic gene) when the
promoter is capable of directing transcription of that nucleic acid
sequence.
[0032] Vector
[0033] A "vector" is a molecule (e.g., a virus such as adenovirus)
that serves to transfer coding information to a host cell. Any
suitable vector, such as a viral vector, in particular an
adenoviral vector, can be utilized in the present inventive method.
Thus, an adenoviral vector utilized in accordance with the present
invention can encompass any adenoviral vector that is appropriate
for the introduction of nucleic acids into eukaryotic cells and is
capable of functioning as a vector as that term is understood by
those of ordinary skill in the art. An adenoviral vector in the
context of the present invention contains one or more heterologous
and/or recombinant sequences, e.g., a therapeutic gene comprising a
promoter and a nucleic acid sequence encoding a therapeutic protein
or therapeutic mRNA, possibly one or more enhancers or silencers,
and the like. A sequence is "heterologous" if it is present in a
different genome from which it is typically found.
[0034] The adenovirus can be any serotype of adenovirus (see, e.g.,
Fields Virology, Fields et al. (eds.), 3rd Ed., NY: Raven Press,
1996, 2111-2171) and, preferably, is a serotype that can transduce
and/or infect a human cell. Desirably, the adenovirus comprises a
complete adenoviral virus particle (i.e., a virion) consisting of a
core of nucleic acid and a protein capsid, or comprises a protein
capsid to which DNA comprising a therapeutic gene is appended, or
comprises a naked adenoviral genome, or is any other manifestation
of adenovirus as described in the art and which can be used to
transfer a therapeutic gene. In the context of the present
invention, any suitable adenoviral genome can serve as, or be a
part of, the adenoviral vector. Preferred adenoviral genomes
include those derived from Ad5 and Ad2, which are easily isolated
from infected cells, are commercially available (e.g., from Sigma
Chemical Co., St. Louis, Mo.), or are generally available from
those skilled in the art who routinely maintain these viral
stocks.
[0035] For the purpose of this invention, the adenovirus employed
for transfer of a therapeutic gene can be wild type (i.e.,
replication-competent). However, it is not necessary that the
genome of the employed adenovirus be intact. In fact, to prevent
the virus from usurping host cell functions and ultimately
destroying the cell, the adenovirus can be inactivated prior to its
use, for instance, by UV irradiation. Alternately, the adenovirus
can comprise genetic material with at least one modification
therein, which can render the virus replication-deficient. Also,
the adenovirus can consist of a therapeutic gene linked to an
adenoviral capsid, and thus may not possess an adenoviral genome.
Moreover, the virus can be coupled to a DNA-polylysine complex
containing a ligand (e.g., transferrin) for mammalian cells such as
has been described in the art (see, e.g., Wagner et al.,
supra).
[0036] The modification to the adenoviral genome can include, but
is not limited to, addition of a DNA segment, rearrangement of a
DNA segment, deletion of a DNA segment, replacement of a DNA
segment, methylation of unmethylated DNA, demethylation of
methylated DNA, and introduction of a DNA lesion. For the purpose
of this invention, a DNA segment can be as small as one nucleotide
and as large as 36 kilobase pairs (kb) (i.e., the size of the
adenoviral genome) or, alternately, can equal the maximum amount
which can be packaged into an adenoviral virion (i.e., about 38
kb).
[0037] Such modifications to the adenoviral genome can render the
adenovirus replication-deficient. Preferably, however, the
modification does not alter the ability of the adenovirus to bind
to a suitable cell surface receptor. Preferred modifications to the
adenoviral genome include modifications in the E1, E2, E3, and/or
E4 regions.
[0038] The vector utilized in the context of the present invention
can comprise sequences so as to constitute any type of suitable
vector. One of ordinary skill in the art will appreciate that an
RNA vector can be used in the context of the present invention. RNA
vectors suitable for in vivo and ex vivo embodiments are well known
in the art. If used in the present inventive method, an RNA vector
can comprise, for example, an anti-sense molecule or a ribozyme to
contribute to the inhibition of adipose tissue vascularity.
[0039] In addition, the vector can comprise a mammalian expression
vector, a vector in which the subcloned coding sequence of the
therapeutic gene is under the control of its own cis-acting
regulatory elements, or a vector designed to facilitate gene
integration or gene replacement in host cells. Preferably the
vector comprises an expression vector appropriate for expression of
a therapeutic gene in a mammalian (optimally, human) cell.
[0040] The vector according to the invention also can comprise a
vector other than an adenoviral vector (e.g., a plasmid, phage,
liposomal or other viral vector), or a ligation of adenovirus
sequences with other vector sequences. However, while these other
vectors can be employed, for instance, in the construction of
adenoviral vectors, preferably an adenoviral vector (i.e., as
compared to a phage, plasmid or other vector) is employed to
transfer genes to adipocytes, particularly in vivo.
[0041] Vector identification and/or selection can be accomplished
using a variety of approaches known to those skilled in the art.
For instance, vectors containing particular nucleic acid sequences
can be identified by hybridization, the presence or absence of
so-called "marker" gene functions encoded by marker genes present
on the vectors, and/or the expression of particular sequences. In
the first approach, the presence of a particular sequence in a
vector can be detected by hybridization (e.g., by DNA-DNA
hybridization) using probes comprising sequences that are
homologous to the relevant sequence. In the second approach, the
recombinant vector/host system can be identified and selected based
upon the presence or absence of certain marker gene functions such
as resistance to antibiotics, thymidine kinase activity, and the
like, caused by particular genes encoding these functions present
on the vector. In the third approach, vectors can be identified by
assaying for a particular gene product encoded by the vector. Such
assays can be based on the physical, immunological, or functional
properties of the gene product.
[0042] Therapeutic Gene
[0043] The vector used in the context of the present invention can
comprise one or more therapeutic genes. Any suitable therapeutic
gene can be employed according to the present invention, so long as
the therapeutic gene is capable of being transcribed in a cell in
which the vector has been internalized.
[0044] The therapeutic gene being transferred can comprise DNA
which can be as small as one repeat unit (e.g., a nucleotide) and
as large as reasonably can be isolated, synthesized, or transferred
to a host cell using the methods of the present invention and
considering the packaging constraints of viral vectors. The
therapeutic gene comprises non-coding sequences (such as a
promoter) as well as a nucleic acid sequence encoding a therapeutic
protein or therapeutic mRNA. The "nucleic acid sequence" of the
therapeutic gene preferably comprises sense or antisense sequences,
including ribozymes, or catalytic RNA species such as described in
the art (Hampel et al., Nucleic Acids Research, 18, 299-304 (1990);
Cech et al., Annual Rev. Biochem., 55, 599-629 (1986)), as well as
engineered sequences, or sequences which are not normally present
in vivo.
[0045] The nucleic acid sequence of the therapeutic gene can be in
any orientation in the vector. The therapeutic gene nucleic acid
sequence can be placed under the control of (i.e., "operably linked
to") 5' and/or 3' regulatory sequences (e.g., promoters) which
typically either do or do not control the coding sequence (e.g.,
the sense or antisense mRNA sequence) in its native form. In
particular, any promoter can be substituted for the native promoter
of the nucleic acid sequence to generate a recombinant therapeutic
gene. Furthermore, the therapeutic gene can contain lesions
including, but not limited to, a missing base or altered base
(e.g., an alkylated base), a cyclobutyl dimer, strand breaks, and
cross-linking of nucleic acid strands.
[0046] The therapeutic gene typically will exert its effect at the
level of RNA or protein for the purpose of treating a disease or
condition. The therapeutic gene i.e., a gene encoding an
anti-angiogenic factor, can exert its effect at the level of RNA,
for instance, by comprising a nucleic acid sequence that encodes a
therapeutic RNA such as an antisense molecule or a ribozyme or a
protein that affects splicing or 3' processing (e.g.,
polyadenylation). Alternately, the nucleic acid sequence of the
therapeutic gene can encode a therapeutic protein that acts by
affecting the level of expression of another gene within the cell
(i.e., where gene expression is broadly considered to include all
steps from initiation of transcription through production of a
processed protein), including, among other things, by mediating an
altered rate of mRNA accumulation, an alteration of MRNA transport,
and/or a change in post-transcriptional regulation. In addition, a
therapeutic protein or a therapeutic peptide can act upon a signal
transduction cascade, for example, interacting with a receptor or a
signaling molecule resulting in altered cellular responses.
[0047] Also, a protein encoded by the nucleic acid sequence of a
transferred therapeutic gene can be employed in the treatment of an
inherited disease, such as, e.g., the cystic fibrosis transmembrane
conductance regulator cDNA for the treatment of cystic fibrosis.
The protein encoded by the therapeutic gene nucleic acid sequence
can exert its therapeutic effect by resulting in cell killing. For
instance, expression of the gene, itself, can lead to cell killing,
as with the expression of an apoptotic factor or the diphtheria
toxin A gene, or the expression of the gene can render cells
selectively sensitive to the killing action of certain drugs, e.g.,
expression of the HSV thymidine kinase gene renders cells sensitive
to antiviral compounds including acyclovir, gancyclovir, and FIAU
(1-(2-deoxy-2-fluoro-.beta.-D-arabinofur- anosil)-5-iodouracil).
This is of particular value in the reduction of adiposity according
to the invention, wherein adipocytes are killed by the transferred
gene. Similarly, expression of the gene can result in new blood
vessel growth, as where the therapeutic gene encodes an angiogenic
substance, or, alternatively, the therapeutic gene encodes an
anti-angiogenic factor, the expression of which inhibits blood
vessel formation or growth.
[0048] Accordingly, the therapeutic gene preferably encodes a
protein selected from the group consisting of a secreted protein,
such as a secreted protein that acts systemically, and a protein
that acts upon or in the vicinity of an adipocyte. More preferably,
the therapeutic gene encodes a protein selected from the group
consisting of an apoptotic factor, a toxin, especially diphtheria
toxin A or a similar gene encoding a toxin (Yamaizumi et al.,
supra; Ross et al. (1993), supra; Palmiter et al., supra; Bernstein
et al., supra; Behringer et al., supra; Hughes et al., supra), p154
polypeptide, especially the p154 polypeptide obtained from a human
or mouse gene (Serrero, supra), an adipsin, especially an adipsin
which is a serine protease homolog (Flier et al., supra), an Ob
protein such as a leptin, especially an Ob protein obtained from a
human or mouse obesity gene (Zhang et al., Nature, 372, 425 (1994);
Murakami et al., Biochem. Biophys. Res. Commun., 209, 944 (1995);
Considine et al., J. Clin. Invest., 95, 2986 (1995)) or Ob
polypeptides such as have been described in the art (see, e.g.,
Great Britain Patent Application 2,292,382), an anti-angiogenic
factor, and an angiogenic substance such as a growth factor,
especially VEGF, particularly VEGF.sub.121, VEGF.sub.165
(Muhlhauser et al., J. Cell Biochem., 18A, DZ315 (1994)), or
VEGF.sub.189, other angiogenic growth factors such as have been
described in the art (see, e.g., Cid et al., supra) and are further
described herein.
[0049] Promoter
[0050] Any promoter (i.e., whether isolated from nature or produced
by recombinant DNA or synthetic techniques) can be used in
connection with the present invention to provide for gene
transcription. The promoter preferably is capable of directing
transcription in a eukaryotic (desirably mammalian) cell. The
functioning of the promoter can be altered by the presence of one
or more enhancers and/or silencers present on the vector. The DNA
sequences appropriate for expression in eukaryotic cells (i.e.,
"eukaryotic promoters") differ from those appropriate for
expression in prokaryotic cells. Generally, eukaryotic promoters
and accompanying genetic signals are not recognized in or do not
function in prokaryotic systems, and prokaryotic promoters are not
recognized in or do not function in eukaryotic cells.
[0051] A comparison of promoter sequences that function in
eukaryotes has revealed conserved sequence elements. Generally,
eukaryotic promoters transcribed by RNA polymerase II are typified
by a "TATA box" centered around position -25 which appears to be
essential for accurately positioning the start of transcription.
The TATA box directs RNA polymerase to begin transcribing
approximately 30 base pairs (bp) downstream in mammalian systems.
The TATA box functions in conjunction with at least two other
upstream sequences located about 40 bp and 110 bp upstream of the
start of transcription. Typically, a so-called "CCAAT box" serves
as one of the two upstream sequences, and the other often is a
GC-rich segment (e.g., a "GC box" comprised, for instance, of the
sequence GGGCGG, or the sequences GCCACACCC and ATGCAAAT). The
CCAAT homology can reside on different strands of the DNA. The
upstream promoter element also can be a specialized signal such as
one of those which have been described in the art and which appear
to characterize a certain subset of genes.
[0052] To initiate transcription, the TATA box and the upstream
sequences are each recognized by regulatory proteins which bind to
these sites, and activate transcription by enabling RNA polymerase
II to bind the DNA segment and properly initiate transcription.
Whereas base changes outside the TATA box and the upstream
sequences have little effect on levels of transcription, base
changes in either of these elements substantially lower
transcription rates (see, e.g., Myers et al., Science, 229, 242-247
(1985); McKnight et al., Science, 217, 316-324 (1982)). The
position and orientation of these elements relative to one another,
and to the start site, are important for the efficient
transcription of some, but not all, coding sequences. For instance,
some promoters function well in the absence of any TATA box.
Similarly, the necessity of these and other sequences for promoters
recognized by RNA polymerase I or III, or other RNA polymerases,
can differ.
[0053] Accordingly, promoter regions can vary in length and
sequence and can further encompass one or more DNA binding sites
for sequence-specific DNA binding proteins and/or an enhancer or
silencer. Enhancers and/or silencers can similarly be present on a
vector outside of the promoter per se. The present invention
preferentially employs within a therapeutic gene a constitutive
promoter, in particular the cytomelagovirus (CMV) promoter, for
regulating a coding sequence of interest. Such promoters, as well
as mutations thereof, are known and have been described in the art
(see, e.g., Boshart et al., Cell, 41, 521-530 (1985)). Other
promoters, however, also can be employed, such as the Ad2 or Ad5
major late promoter and tripartite leader, the Rous sarcoma virus
(RSV) long terminal repeat, and other constitutive promoters such
as have been described in the literature. For instance, the herpes
thymidine kinase promoter (Wagner et al., supra), the regulatory
sequences of the metallothionine gene (Brinster et al., Nature,
296, 39-42 (1982)), promoter elements from yeast or other fungi
such as the Gal 4 promoter, the alcohol dehydrogenase promoter, the
phosphoglycerol kinase promoter, and the alkaline phosphatase
promoter, can be employed. Similarly, promoters isolated from the
genome of mammalian cells or from viruses that grow in these cells
(e.g., adenovirus, SV40, herpes simplex virus, and the like) can be
used.
[0054] Instead of being a constitutive promoter, the promoter can
be a promoter which is up- and/or downregulated in response to
appropriate signals. For instance, an inducible promoter, such as
the IL-8 promoter which is responsive to TNF or another cytokine,
can be employed. Other examples of suitable inducible promoter
systems include, but are not limited to, the metallothionine
inducible promoter system, the bacterial lacZYA expression system,
the tetracycline expression system, and the T7 polymerase system.
Further, promoters that are selectively activated at different
developmental stages (e.g., globin genes are differentially
transcribed in embryos and adults) can be employed. Such promoters
are particularly useful in the present inventive method regarding
transfer of an anti-angiogenic factor or an apoptotic factor, and
an adipose tissue implant comprising a vector that comprises or
encodes an anti-angiogenic factor or an apoptotic factor.
[0055] In addition, a tissue-specific promoter, i.e., a promoter
that is preferentially activated in a given tissue and results in
expression of a gene product in the tissue where activated,
particularly an adipocyte specific promoter, can be used. Preferred
adipocyte-specific promoters according to the invention include the
aP2 gene regulatory region (Ross et al. (1990, 1992 and 1993),
supra) and the p154 polypeptide gene regulatory region (Serrero,
supra).
[0056] Adenoviral Mediated Gene Transfer To Adipocytes
[0057] In adenoviral mediated gene transfer, one or more adenoviral
vectors are transferred to a host cell, which is preferably a
eukaryotic host cell, optimally an adipocyte. The eukaryotic host
cell can be present in vitro or in vivo. According to the present
invention, the "contacting" of cells with an adenoviral vector of
the present invention can be by any means by which the vectors will
be transduced into the cell. Such transduction can be by any
suitable method. Preferably the adenoviral vectors will be
transduced by means of infection or transduction, i.e., using the
natural capability of the virus to enter cells and mediate uptake
of bystander macromolecules (e.g., the capability of adenovirus to
undergo receptor-mediated endocytosis). However, the vectors also
can be introduced by any other suitable means, e.g., by
transfection, calcium phosphate-mediated transformation,
microinjection, electroporation, osmotic shock, and the like.
[0058] Genes can be effectively transduced into a wide variety of
different types of adipocytes. For example, genes can be
transferred to cells differing both in number of adenovirus
receptors as well as in the affinity of the cell surface receptors
for adenovirus. The types of cells to which gene delivery is
contemplated in vitro or in vivo in the context of the present
invention include avian cells, fish cells, and mammalian cells
including but not limited to rodent, ape, chimpanzee, feline,
canine, ungulate (such as ruminant or swine), and, preferably,
human cells.
[0059] Pharmaceutical Compositions
[0060] Suitable vectors for use in the present invention can be
made into compositions appropriate for contacting cells with
appropriate (e.g., pharmaceutically acceptable) excipients such as
carriers, adjuvants, vehicles, or diluents. The means of making
such a composition, and means of administration, have been
described in the art (see, for instance, Remington's Pharmaceutical
Science, 16th Ed., Mack, ed. (1980)). Where appropriate, the
vectors can be formulated into preparations in solid, semisolid,
liquid or gaseous forms such as tablets, capsules, powders,
granules, ointments, solutions, suppositories, injections,
inhalants, and aerosols, in the usual ways for their respective
routes of administration. Means known in the art can be utilized to
prevent release and absorption of the composition until it reaches
the target organ or to ensure timed-release of the composition. A
pharmaceutically acceptable form should be employed which does not
ineffectuate the compositions of the present invention. In
pharmaceutical dosage forms, the compositions can be used alone or
in appropriate association, as well as in combination, with other
pharmaceutically active compounds. For example, in applying the
methods of the present invention for delivery of a nucleic acid
encoding a VEGF polypeptide to cells in need of angiogenic
stimulation (e.g., in the enhancement of collateral circulation
where there has been vascular occlusion or stenosis or where there
is a need for vascularization or increased circulation), such
delivery can be employed in conjunction with other means of
stimulating angiogenesis, such as, for example, treatment with
other angiogenic growth factors, or use in combination with
matrigel (a complex mixture of tumor basement membrane components
and growth factors) (Muhlhauser et al., Circ. Res., 77, 1077-86
(1995)).
[0061] Accordingly, the pharmaceutical composition of the present
invention can be delivered via various routes and to various sites
in an animal body to achieve a particular effect (see, e.g.,
Rosenfeld et al. (1991), supra; Rosenfeld et al., Clin. Res.,
39(2), 311A (1991a); Jaffe et al., supra; Berkner, supra). One
skilled in the art will recognize that although more than one route
can be used for administration, a particular route can provide a
more immediate and more effective reaction than another route.
Local or systemic delivery can be accomplished by administration
comprising application or instillation of the formulation into body
cavities, inhalation or insufflation of an aerosol, or by
parenteral introduction, comprising intramuscular, intravenous,
peritoneal, subcutaneous, intradermal administration, as well as
topical administration.
[0062] The composition of the present invention can be provided in
unit dosage form wherein each dosage unit, e.g., a teaspoonful,
tablet, solution, or suppository, contains a predetermined amount
of the composition, alone or in appropriate combination with other
active agents. The term "unit dosage form" as used herein refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
the compositions of the present invention, alone or in combination
with other active agents, calculated in an amount sufficient to
produce the desired effect, in association with a pharmaceutically
acceptable diluent, carrier, or vehicle, where appropriate. The
specifications for the unit dosage forms of the present invention
depend on the particular effect to be achieved and the particular
pharmacodynamics associated with the pharmaceutical composition in
the particular host.
[0063] Accordingly, the present invention also provides a method of
transferring a therapeutic gene to a host, which comprises
administering the vector of the present invention, preferably as
part of a composition, using any of the aforementioned routes of
administration or alternative routes known to those skilled in the
art and appropriate for a particular application. The "effective
amount" of the composition is such as to produce the desired effect
in a host which can be monitored using several end-points known to
those skilled in the art. Effective gene transfer of a vector to a
host cell in accordance with the present invention can be monitored
in terms of a therapeutic effect (e.g., alleviation of some symptom
associated with the particular disease being treated). For
instance, in regard to adipsin or Ob protein expression, the
effective amount of the protein is determined by realization of
reduction in fat percentage in the subject. Effective gene transfer
of a vector to a host cell is also confirmed by evidence of the
transferred gene or expression of the gene within the host (e.g.,
using the polymerase chain reaction in conjunction with sequencing,
Northern or Southern hybridizations, or transcription assays to
detect the nucleic acid in host cells, or using immunoblot
analysis, antibody-mediated detection, mRNA or protein half-life
studies, or particularized assays to detect protein or polypeptide
encoded by the transferred nucleic acid, or impacted in level or
function due to such transfer). One such particularized assay
described in the examples herein includes the Western immunoassay
for the detection of the protein encoded by a VEGF gene.
[0064] These methods described herein are by no means
all-inclusive, and further methods to suit the specific application
will be apparent to the ordinary skilled artisan. Moreover, the
effective amount of the compositions can be further approximated
through analogy to compounds known to exert the desired effect
(e.g., compounds traditionally employed to stimulate angiogenesis
can provide guidance in terms of the amount of a VEGF nucleic acid
to be administered to a host).
[0065] Furthermore, the preferred amounts of each active agent
included in the compositions according to the invention (e.g., per
each cell to be contacted, preferably from about 1 to at least
about 1000 adenoviral plaque forming units (PFU), more preferably
from about 1 to at least about 100 adenoviral PFU, although any
suitable amount can be utilized either above, i.e., greater than
about 1000 adenoviral PFU, or below, i.e., less than about 1
adenoviral PFU) provide general guidance of the range of each
component to be utilized by the practitioner upon optimizing the
methods of the present invention for practice either in vitro or in
vivo. The adenoviral vector(s) can be carried in any suitable
volume of diluent or carrier. However, it is usually preferred that
the concentration of the adenoviral vectors be in the range of
2.times.10.sup.7 to 2.times.10.sup.14 pfu/ml, and more preferably
about 10.sup.10 pfu/ml. Moreover, such ranges by no means preclude
use of a higher or lower amount or concentration of a component, as
might be warranted in a particular application. For instance, the
actual dose and schedule can vary depending on whether the
compositions are administered in combination with other
pharmaceutical compositions, or depending on interindividual
differences in pharmacokinetics, drug disposition, and metabolism.
Similarly, amounts can vary in in vitro applications depending on
the particular cell line utilized (e.g., based on the number of
adenoviral receptors present on the cell surface, or the ability of
the particular vector employed for gene transfer to replicate in
that cell line). Furthermore, the amount of vector to be added per
cell will likely vary with the length and stability of the
therapeutic gene inserted in the vector, as well as also the nature
of the sequence. As such, the arrivant of vector to be added per
cell is particularly a parameter which needs to be determined
empirically, and can be altered due to factors not inherent to the
methods of the present invention (for instance, the cost associated
with synthesis). One skilled in the art can easily make any
necessary adjustments in accordance with the exigencies of the
particular situation.
[0066] Also, for these embodiments, when one or more different
vectors (i.e., each comprising or encoding one or more different
therapeutic genes) are employed in the methods described herein,
the contacting of cells with the various components of the present
invention can occur in any order or can occur simultaneously.
Preferably the contacting will occur simultaneously. In a preferred
embodiment, the component vectors of the present invention can be
mixed together and preincubated prior to contacting the cell. When
multiple vectors are to be administered, the cell is preferably
contacted with the first vector less than about 6 weeks after, or
less than about 6 weeks before, the cell is contacted with another
vector. Even more preferably the cell is contacted with the first
vector less than about 2 weeks after, or less than about 2 weeks
before, the cell is contacted with another vector.
[0067] Adipocytes infected in vivo in accordance with the present
invention can be transferred to another site within the host as a
vehicle for the delivery of the protein encoded by the transferred
gene to another anatomic locale. Such a transfer can be effected by
any suitable technique, such as those that are known in the art
(see, e.g., Zhang et al., Microsurgery, 15, 269-73 (1994); Boyce et
al. Otolaryngol. Clin. North Am., 27, 39-68 (1994); Moscona et al.,
Ann. Plast. Sur, 33, 500-6 (1994); Krabatsch et al., J. Card.
Surg., 10, 46-51 (1995)). The adipocytes so transferred need not be
from the same host, or even the same species of host. Preferably,
however, the adipocytes are transferred within the same host.
[0068] Adipose Tissue Implants
[0069] The present inventive adipose tissue implant is adipose
tissue removed from a donor that is treated to increase the
vascularity of the adipose tissue implant when it is implanted into
a second site in the body of the donor or into an immunologically
compatible host. In the alternative, an implant may be treated to
decrease the vascularity of the adipose tissue implant when
implanted. Similarly, the adipose tissue implant may be treated in
such a manner that adipocyte cell death results from apoptosis. The
implant can be made by contacting adipose tissue comprising
adipocytes in vivo with a vector comprising nucleic acid sequence
comprising or encoding an angiogenic or anti-angiogenic protein or
RNA as described herein (i.e., the inventive method described
above) as well as by other means. As such, the present invention
provides an isolated adipose tissue comprising a vector comprising
a nucleic acid sequence comprising or encoding an anti-angiogenic
factor, an apoptotic factor, an adipsin protein, an Ob protein, or
an angiogenic substance, wherein said isolated adipose tissue is
optionally in the form of an implant.
[0070] While adenoviral vectors are suitable for delivery of an
apoptotic gene or a gene encoding an adipsin, an Ob protein, an
angiogenic substance or an anti-angiogenic factor, such as an
anti-angiogenic protein, polypeptide or RNA, e.g., an antisense
molecule or a ribozyme, that inhibits blood vessel growth or
formation, to the adipocytes of the implant in vivo, they also can
be employed ex vivo. That is, adipose tissue can be removed from a
host and contacted with an adenoviral vector comprising, for
example, a gene encoding an angiogenic substance, an
anti-angiogenic factor, such as anti-angiogenic proteins,
polypeptides or RNAs, an apoptotic gene, an adipsin protein, an Ob
protein or a gene encoding a secreted protein to make the implant,
which then can be shaped (if appropriate or necessary) and
transplanted into a host from which the adipose tissue was
originally removed or a second, immunologically-compatible
host.
[0071] Any suitable means of transferring one or more nucleic acids
or genes to adipose tissue in vitro or in vivo to form an implant
also can be employed to form the present inventive adipose tissue
implant. Nucleic acids encoding an anti-angiogenic factor, for
example, can be complexed with polylysine conjugates and contacted
to the adipose tissue that is to be incorporated into the implant.
Alternatively, DNA can be co-precipitated in calcium phosphate and
contacted with the adipose tissue. Another alternative is to
incorporate the nucleic acid comprising or encoding as appropriate
the angiogenic substance or anti-angiogenic factor, an adipsin
protein, an Ob protein or an apoptotic factor into a viral vector
other than an adenoviral vector, e.g., into a retroviral,
adeno-associated viral, or herpes viral vector, having the ability
to transduce adipocytes or other cells in the adipose tissue and to
contact that vector with the adipose tissue. Additionally, the
nucleic acids comprising or encoding an anti-angiogenic angiogenic
factor, angiogenic substance, an adipsin or Ob protein, an
apoptotic factor or a protein to be secreted by an adipocyte, can
be transferred to the adipose tissue by way of liposomes, lipid
vesicles, or cationic or zwitterionic lipid transfer agents (e.g.,
Lipofectamine.TM., Gibco BRL, Bethesda, Md.). The in vivo and in
vitro utilities and limitations of these gene transfer methods are
well known to the skilled artisan.
[0072] Rather than, or in addition to, the transfer of a nucleic
acid encoding or comprising an angiogenic substance or an
anti-angiogenic factor into the cells of the adipose tissue, the
adipose tissue can be treated in any other suitable manner to
increase or inhibit the vascularization of the adipose tissue when
implanted into a host.
[0073] Thus, for example, while an angiogenic gene that expresses
an angiogenic protein, particularly VEGF.sub.121, VEGF.sub.165, or
VEGF.sub.189 can be usefully employed to form the inventive
implant, angiogenic proteins per se also can be usefully employed.
The VEGFs, acidic FGF, basic FGF, and epithelial growth factor are
illustrative of angiogenic proteins suitable for use in the context
of the present invention. Preferably, the adipose tissue is
perfused with a solution containing a quantity of angiogenic
protein sufficient to stimulate angiogenesis. About 10.sup.-18 to
about 10.sup.-6 grams of active protein/cell, preferably about
10.sup.-15 to about 10.sup.-12 grams of active protein/cell,
desirably is administered to the adipose tissue to form the
implant. Similarly, while a nucleic acid sequence encoding an
anti-angiogenic factor, particularly a protein or polypeptide that
inhibits angiogenic factors, or a protein selected from the group
consisting of taxol, endostatin, angiostatin, or fumagillin or an
analogue of fumagillin, can be usefully employed to form the
inventive implant, anti-angiogenic proteins, polypeptides and RNA,
e.g., mRNA, anti-sense RNA and ribozymes, also can be usefully
employed.
[0074] In addition to contacting the adipose tissue with, for
example, an angiogenic protein or a gene encoding an angiogenic
factor, the present inventive implant is optionally treated with a
lymphogenic gene or protein (e.g., VEGF-C). While not wishing to be
bound by any particular theory, it is believed that lymphogenesis
usefully prevents the pooling of extravasated blood components and
lymph from the site of implantation which helps prevent cell loss
from the implant (i.e., shrinkage).
[0075] Advantageously, transplanted adipose tissue that has been
contacted with an angiogenic protein or a gene encoding an
angiogenic factor, such as angiogenic proteins, polypeptides and
RNA, has improved vascularization when implanted into a host and/or
is less susceptible to cell loss, which is manifested by the
appearance of shrinkage after transplantation. Additionally, if the
adipocytes of the adipose tissue are modified such that they
comprise a second therapeutic protein in addition to an angiogenic
protein, the increased vascularization resulting from the
angiogenic protein provides for more efficient delivery of the
second therapeutic protein to sites outside the adipose tissue.
[0076] In the alternative, the transplanted adipose tissue that has
been contacted with an anti-angiogenic protein or a gene encoding
an anti-angiogenic factor, such as anti-angiogenic proteins,
polypeptides or RNA, demonstrates inhibition of vascularity when
implanted in a host. Similarly, implants treated with an apoptotic
gene, will, advantageously, exhibit adipocyte cell death when
implanted in a host. Any suitable apoptotic gene can be used in the
context of the present invention. For example, the apoptotic gene
is selected from the group consisting of p53, a cell death-inducing
coding sequence of Bcl-2 which comprises an N-terminal deletion, a
cell death-inducing coding sequence of Bcl-x which comprises an
N-terminal deletion, Bax, Bak, Bid, Bad, Bik, Bif-2, inhibitor of
apoptosis proteins 1 and 2 (IAP-1, IAP-2), a caspase, tumor growth
factor-beta (TGF-.beta.1), c-myc, a protease, a protein kinase and
others as known in the art. Preferably, the protein kinase is
selected from the group consisting of protein kinase C.theta.,
protein kinase C.delta., Akt/PI(3)-kinase, DNA-protein kinase
(DNA-PK), PITSLRE, death-associated protein kinase (DAP kinase),
receptor-interacting protein (RIP), Jun kinase/stress-activated
protein kinase (JNK/SAPK), Daxx, Raf-1, Pim-1, NFkB-inducing kinase
(NIK), mitogen-activated kinase kinase kinase (MEKK1), ASK1,
RNA-activated protein kinase (PKR) and others as known in the
art.
[0077] Other Considerations
[0078] With respect to the transfer and expression of therapeutic
genes according to the present invention, the ordinary skilled
artisan is aware that different genetic signals and processing
events control levels of nucleic acids and proteins/peptides in a
cell, such as, for instance, transcription, mRNA translation, and
post-transcriptional processing. Transcription of DNA into RNA
requires a functional promoter, as previously described. The amount
of transcription is regulated by the efficiency with which RNA
polymerase can recognize, initiate, and terminate transcription at
specific signals. These steps, as well as elongation of the nascent
mRNA and other steps, are all subject to being affected by various
other components also present in the cell, e.g., by other proteins
which can be part of the transcription process, by concentrations
of ribonucleotides present in the cell, and the like.
[0079] Protein expression also is dependent on the level of RNA
transcription which is regulated by DNA signals, and the levels of
DNA template. Similarly, translation of mRNA requires, at the very
least, an AUG initiation codon which is usually located within 10
to 100 nucleotides of the 5' end of the message. Sequences flanking
the AUG initiator codon have been shown to influence its
recognition by eukaryotic ribosomes, with conformity to a perfect
Kozak consensus sequence resulting in optimal translation (see,
e.g., Kozak, J. Molec. Biol. 196, 947-950 (1987)). Also, successful
expression of a therapeutic gene in a cell can require
post-translational modification of a resultant protein/peptide.
Thus, production of a recombinant protein or peptide can be
affected by the efficiency with which DNA (or RNA) is transcribed
into mRNA, the efficiency with which mRNA is translated into
protein, and the ability of the cell to carry out
post-translational modification. These are all factors of which the
ordinary skilled artisan is aware and is capable of manipulating
using standard means to achieve the desired end result.
[0080] Along these lines, to optimize protein production following
recombination, preferably the vector employed for transfer of a
therapeutic gene further comprises a polyadenylation site following
the coding region of the therapeutic gene. Also, preferably all the
proper transcription signals (and translation signals, where
appropriate) will be correctly arranged on the recombinant vector
such that the therapeutic gene will be properly expressed in the
cells into which it is introduced. If desired, the vector also can
incorporate splice sites (i.e., splice acceptor and splice donor
sites) to facilitate mRNA production. Moreover, if the therapeutic
gene being transferred encodes a protein, which is a processed or
secreted protein or, for instance, functions in an intracellular
organelle, such as a mitochondrion or the endoplasmic reticulum,
preferably the vector further comprises the appropriate sequences
for processing, secretion, intracellular localization, and the
like.
[0081] With respect to promoters, coding sequences, therapeutic
genes, marker genes, and the like, located on a vector according to
the present invention, such elements are as previously described
and can be present as part of a cassette, either independently or
coupled. In the context of the present invention, a "cassette" is a
particular base sequence that possesses functions which facilitate
subcloning and recovery of nucleic acid sequences (e.g., one or
more restriction sites) or expression (e.g., polyadenylation or
splice sites) of particular nucleic acid sequences.
[0082] Other Uses
[0083] The present invention provides methods and vectors, which
have particular utility with respect to diseases or conditions that
can be treated directly by transfer of nucleic acids to adipocytes.
Because of the widespread effects of adipocytes on host metabolism,
the vector mediated transfer of nucleic acids, such as the
adenoviral mediated gene transfer, is preferably employed for the
treatment of an energy storage disorder, such as a disorder
selected from the group consisting of obesity, diabetes, increased
body fat deposition, hyperglycemia, hyperinsulinemia, hypothermia,
hypertension, hypercholesterolemia, hyperlipidemia, and the
like.
[0084] The present inventive methods also have utility with respect
to the treatment of other diseases or conditions. Specifically,
according to the present invention, a vector, such as an adenovirus
can be employed to transfer genes to adipocytes, and, following
establishment of at least a limited infection in adipocyte tissue,
the infected adipocytes can be transferred to another site in the
host, at which site the protein encoded by the transferred gene can
exert its effect. Using adipocytes as a vehicle for the transfer of
the gene in this fashion is advantageous, since adipocytes
typically are non-immunogenic, unlike certain other tissue grafts
which might be employed as a vehicle to transfer genes.
[0085] In particular, this method of the present invention can be
employed to deliver proteins, such as angiogenic substances or
growth factors to areas of ischemia, such as the heart or muscle,
or, more generally, in the treatment of ischemic disease.
Angiogenesis is the process by which new blood vessels are formed
from extant capillaries. Thus, the angiogenic process and the
angiogenic factors which regulate the process are relevant to
embryonic development, inflammation, and wound healing, and also
contribute to pathologic conditions such as diabetic retinopathy,
rheumatoid arthritis, cancer, and chronic inflammatory diseases
(see, e.g., U.S. Pat. No. 5,318,957 (Cid et al.); Brooks et al.,
Science, 264, 569-571 (1994)). Accordingly, the present inventive
approach can further be employed to deliver angiogenic growth
factors to a host, or to particular regions of the host, to
stimulate angiogenesis as a means to facilitate wound healing, as
well as to treat cancer or inflammation (especially inflammation of
blood vessels or systemic vasculitis). In particular, the approach
can be employed in a method wherein, instead of performing a more
invasive procedure, such as a coronary bypass operation, a vector
comprising an angiogenic gene is injected, and new blood vessels
are induced to grow around the blocked region. Genes encoding the
following angiogenic growth factors, and which have been described
in the art, can be used according to the present invention along
with further angiogenic substances: vascular endothelial cell
growth factor (VEGF), particularly VEGF.sub.121, VEGF.sub.165, or
VEGF.sub.189 acidic fibroblast growth factor (aFGF), basic
fibroblast growth factor (bFGF), transforming growth factor, alpha
and beta tumor necrosis factor, platelet-derived growth factor, and
angiogenin.
[0086] In that angiogenesis contributes to several pathological
conditions, the present inventive method also can be used to
deliver anti-angiogenic factors, such as anti-angiogenic proteins,
polypeptides and RNAs, i.e., an anti-sense molecule or a ribozyme,
to a host to inhibit vascularization of adipose tissue. Any
suitable anti-angiogenic factor can be used in the context of the
present invention as long as the factor inhibits or contributes to
the inhibition of the formation or growth of blood vessels. An
anti-angiogenic factor can inhibit angiogenesis on any one of a
number levels. For example, anti-sense RNA can recognize and bind
to a nucleic acid encoding an angiogenic substance, thereby
inhibiting successful translation of the protein. Anti-angiogenic
peptides can bind to receptors, such as cell-surface receptors, or
interact with molecules involved in signal transduction pathways in
such a way as to disrupt the signaling required for angiogenesis.
An anti-angiogenic factor for use in the present invention can act
by any mechanism as long as formation of new blood vessels or
growth of existing blood vessels in adipose tissue is inhibited.
Preferably, the anti-angiogenic factor is an inhibitor of an
angiogenic factor. More preferably, the anti-angiogenic factor
blocks VEGF or platelet-derived growth factor (PDGF) signaling,
such as interferon-alpha. Preferably the anti-angiogenic factor is
taxol, angiostatin, endostatin, fumagillin, or an analogue of
fumagillin.
[0087] Furthermore, adipocytes in vivo can be employed as a site
for the transfer of a gene encoding a protein which exerts its
effects locally in the region of the adipocyte tissue or
systemically, as for genes encoding secreted proteins which,
following their production in adipocytes, diffuse into the
bloodstream. Thus, the present inventive methods and vectors also
can be used in the treatment of diseases or conditions not directly
associated with adipocytes and/or metabolic processes affected
thereby.
[0088] For instance, the method preferably can be employed to
transfer genes that encode VEGF (particularly VEGF.sub.121 or
VEGF.sub.165), aFGF and bFGF, as well as other angiogenic growth
factors which can act locally to stimulate angiogenesis in the
setting of tissue ischemia. Adenoviral vector transfer of genes
encoding angiogenic substances can be employed to provide high
concentrations of such substances in a regional fashion for a
sustained period, thus inducing angiogenesis in the local milieu,
yet minimizing the risk of chronic overinduction of angiogenesis in
the target tissue, and promiscuous induction of angiogenesis in
sensitive nondiseased organs, such as the retina or synovium, or in
occult tumors (Folkman et al., J. Biol. Chem., 267, 10931-34
(1992)). Similarly, the method preferably can be employed to
locally increase sensitivity to 5-fluorouracil (5-FU),
cis-platinum, and other chemotherapeutic agents that can act to
stimulate a cytopathic effect for the treatment of cancer
cells.
[0089] For secreted proteins that act systemically, the present
inventive methods can be employed using vectors which encode
various therapeutic genes. For instance, the therapeutic gene can
comprise, but is not limited to, the gene for a1-antitrypsin or
adenosine deaminase for the treatment of inherited deficiency,
factor VIII for hemophilia, other coagulation factors for bleeding
disorders, erythropoietin for chronic renal failure and marrow
suppressive disorders, proteins for enhancing the host defense
response, such as antiviral proteins or immunomodulators, and
antitumor agents, for example, tumor suppressor proteins and
interferons Moreover, the present inventive methods and vectors can
further be employed to deliver pharmacologics such as
antihypertensives and anticoagulants, or receptor agonists or
antagonists, using transfected or infected adipocytes as the means
of producing these agents.
[0090] The present inventive methods also have utility with respect
to decreasing adiposity. According to the present invention, a
vector can be employed to transfer an apoptotic factor to adipose
tissue, thereby resulting in adipocyte cell death. Any suitable
apoptotic factor can be used as is known in the art. Preferably,
the apoptotic gene is selected from the group consisting of p53, a
cell death-inducing coding sequence of Bcl-2 which comprises an
N-terminal deletion, a cell death-inducing coding sequence of Bcl-x
which comprises an N-terminal deletion, Bax, Bak, Bid, Bad, Bik,
Bif-2, IAP-1, IAP-2, a caspase, TGF-.beta.1, c-myc, a protease, and
a kinase, such as protein kinase C0, protein kinase C.delta.,
Akt/PI(3)-kinase, DNA-PK, PITSLRE, DAP kinase, RIP, JNK/SAPK, Daxx,
Raf-1, Pim-1, NIK, MEKK1, ASK1, PKR, and the like.
[0091] In regard to the use of a VEGF protein to induce therapeutic
angiogenesis, several studies have demonstrated that the
administration of VEGF protein in the setting of ischemia is
capable of inducing the development of networks of new blood
vessels in vivo. A single bolus or repeat administration of VEGF
induced increased vascularity and blood flow, and improved both
hemodynamic and clinical function in rabbit hind limb models of
ischemia (Ferrara et al., Ann. N.Y. Acad. Sci., 752, 246-256
(1995); Takeshita et al., Circulation, 90, II228-II234 (1994);
Takeshita et al., J. Clin. Invest., 93, 662-670 (1994a); Bauters et
al., Am. J. Physiol., 267, H1263-H1271 (1994)). A similar model has
been used to demonstrate a synergistic effect of VEGF and bFGF on
angiogenesis in vivo (Asahara et al., Circulation, 92,
II-365-II-371 (1995)). In a canine model of myocardial ischemia
using an ameroid constrictor on the left circumflex coronary artery
(LCx), daily administration of VEGF via an indwelling catheter in
the distal LCx for 28 days resulted in an increase in collateral
blood flow to ischemic myocardium and an increase in the density of
intramyocardial distribution vessels (Banai et al., Circulation,
89, 2183-2189 (1994)). Also, in a porcine model of chronic ischemia
using an ameroid constrictor, continuous administration of VEGF to
the myocardium over 6 weeks resulted in myocardial angiogenesis as
demonstrated by magnetic resonance imaging, showing a reduced
ischemic zone, less contrast arrival delay, and improved ejection
fraction and myocardial wall thickening (Pearlman et al., Nature
Med., 1, 1085-1089 (1995)).
[0092] Delivery of the VEGF gene (as well as other genes) using a
vector-mediated approach is advantageous since gene transfer
provides an equivalent of a "sustained-release capsule," providing
high concentrations of the therapeutic protein for a sustained
period. In comparison, the VEGF protein and certain other proteins
have a very short biologic half-life (e.g., 6 minutes for VEGF)
(Takeshita et al. (1994a), supra). While animal models of hind limb
ischemia do show induction of angiogenesis with a single
intraarterial bolus of the VEGF protein (Takeshita et al. (1994a),
supra,) intramuscular administration for limb ischemia requires
repetitive administration over several days (Takeshita et al.
(1994), supra); as does intracoronary administration for myocardial
ischemia (Banai et al., supra; Pearlman et al., supra). In
comparison, the AdCMV.VEGF vector can provide sustained expression
of the VEGF protein for at least 5 days. Also, gene transfer can be
strategized to provide regional delivery of a high concentration of
VEGF to the ischemic limb or ischemic myocardium. In comparison,
systemic administration of an angiogenic factor carries the
theoretical risk of inducing inappropriate angiogenesis at sites of
vascular derangement or at sites where angiogenesis might have
major adverse consequences, such as in the retina, the synovium,
and occult tumors (Folkman et al., J. Biol. Chem., 267 10931-10934
(1992)). Finally, systemic administration of VEGF has been reported
to cause hypotension in rats (Yang et al., Circulation, 92, I-713
(Abstract)(1995)). Clinical applications for which
adenoviral-mediated delivery of VEGF or other genes (particularly
genes encoding angiogenic substances) might be useful include
nonbypassable ischemic heart disease or peripheral vascular
disease, reinforcement of ischemic anastomoses, and acceleration of
wound healing.
[0093] In comparison to the use of adenovirus for gene delivery to
adipocytes, other gene transfer systems that presently are in
clinical trials (e.g., retrovirus, adeno-associated virus,
plasmid-liposome complexes, and the use of naked plasmid DNA))
(reviewed in Crystal et al., Science, 270, 404-410 (1995))
theoretically could be employed instead. While naked plasmids
delivered to a proximal artery appear to provide sufficient VEGF to
induce angiogenesis in the rabbit hind limb ischemia model
(Takeshita et al., "Therapeutic Angiogenesis Following Arterial
Gene Transfer of Vascular Endothelial Growth Factor in a Rabbit
Model of Hind Limb Ischemia," Proc. Natl. Acad. Sci.,0 (1995)) and
are being evaluated in a clinical trial (Isner, "Arterial Gene
Transfer for Restenosis," Recombinant DNA Advisory Committee (RAC)
Report No. 9508-118 (Office of Recombinant DNA Activities, NIH:
Bethesda, Md. (1995)), expression from naked plasmids delivered in
vivo is several orders of magnitude less than that observed using
an adenovirus vector system (Crystal et al. (1995), supra; Nabel et
al., Cardiovasc. Res., 28, 445-455 (1994)). For in vivo gene
transfer, retrovirus vectors are limited secondary to their
sensitivity to inactivation in vivo and their requirement for
target cell proliferation to transfer the new gene (Crystal et al.
(1995), supra). Plasmid-liposome complexes are relatively
inefficient for in vivo cardiovascular-related gene transfer
(Crystal et al. (1995), supra; Nabel et al., supra). In contrast,
adenoviral vectors have the aforementioned properties that make
them ideal for the delivery of genes to adipose tissue as described
herein and, particularly, for the delivery of VEGF-related genes
for therapeutic angiogenesis. For instance, adenoviral vectors are
effective at transferring genes to cardiovascular tissues, with
high levels of expression of the gene for at least one week. This
is particularly advantageous in view of the short half-life of VEGF
protein. Moreover, the self-limited nature of adenoviral-mediated
gene expression means a decreased (and decreasing over time) risk
of evoking too much angiogenesis in the target tissue. The new gene
transferred by an adenoviral vector functions in an epichromosomal
position, in contrast to adeno-associated virus and retrovirus
vectors that integrate the exogenous gene into the chromosome of
the target cell, and thus carry the risk of inappropriately
delivering the angiogenic stimulus long after it is needed, and the
risk of interference with the regulation/expression of a endogenous
gene. Furthermore, adenovirus vectors achieve gene transfer to both
dividing and non-dividing cells with high levels of efficiency, and
produce localized and sustained levels of protein expression in a
number of cardiovascular related sites, such as skeletal muscle,
myocardium, and vascular endothelium, as well as adipose
tissue.
[0094] In addition to the foregoing uses, the present inventive
adipose tissue implants have particular utility in plastic or
reconstructive surgery. For example, such implants can be used in
breast or penile augmentation.
[0095] Additional uses and benefits of the present invention will
be apparent to one of ordinary skill in the art.
EXAMPLES
[0096] The following examples further illustrate the present
invention and, of course, should not be construed as in any way
limiting its scope.
[0097] Example 1
[0098] This example describes the construction of the adenoviral
vectors employed in the experiments described further herein and
described in Magovem et al., "Regional Angiogenesis induced in
Non-Ischemic Tissue by an Adenovirus Vector expressing Vascular
Endothelial Growth Factor" (Human Gene Therapy, 8, 215-227
(1997)).
[0099] Several replication-deficient, recombinant adenoviral
vectors were employed to assess gene transfer to adipocytes in
vivo. These vectors include Ad.RSV.beta.gal, AdCMVCAT, and
AdCMV.VEGF. Ad.RSV.beta.gal is an E1-E3- Ad5-based vector which
contains the Escherichia coli .beta.-galactosidase coding sequence
under the control of the long terminal repeat of the Rous sarcoma
virus as a promoter, and which follows the SV40 nuclear
localization signal, as previously described (Setoguchi et al.,
supra). AdCMV.VEGF (i.e., AdCMV.VEGF.sub.165) is an E1a-, partial
E1b-, partial E3- adenoviral vector that contains an expression
cassette in the E1 position containing the cytomegalovirus (CMV)
immediate early promoter/enhancer driving the cDNA for the 165
amino acid form of human VEGF (i.e., VEGF.sub.165, Muhlhauser et
al., Circ. Res., 77, 1077-1086 (1995)). AdCMVCAT is similar to
AdCMV.VEGF, but contains the coding sequence for chloramphenicol
acetyltransferase (CAT) instead of the sequence for VEGF
(Kass-Eisler et al., Proc. Natl. Acad. Sci., 90, 11498-502 (1993)).
AdCMV.Null (which is similar to AdCMV.VEGF, but contains no gene in
the expression cassette) was used as a control vector (Williams et
al., J. Vasc. Surg., 19, 916-923 (1994)).
[0100] With respect to construction of AdCMV.VEGF, the cDNA for
VEGF.sub.165 including the signal sequence for secretion (Conn et
al., Proc. Natl. Acad. Sci., 87, 2628-32 (1990)) was inserted into
an expression plasmid (Muhlhauser et al. (1995), supra) such that
the cDNA was placed under the control of the constitutive CMV
immediate early promoter/enhancer. The expression plasmid also
contains the Ad5 sequence from nucleotide 3384 to nucleotide 5778
(i.e., 9.24 to 16.05 map units), which serves as the homologous
recombination sequence. The plasmid carrying the cDNA for
VEGF.sub.165 was cotransfected with the plasmid pJM17 (from F.
Graham) into 293 cells (ATCC CRL1573; a human embryonic kidney cell
line which has been transformed by Ad5 and expresses the E1 region
in trans). The plasmid pJM17 contains the full length Ad5 DNA (36
kb) and pBRX, a 4.3 kb insert placed in the E1 region, thus
exceeding by approximately 2 kb the maximum packaging limit of DNA
into the adenoviral capsid (McGrory et al., Virology, 163, 614-17
(1988)). Homologous recombination between the expression plasmid
and pJM17 in 293 cells resulted in replacement of the E1 region and
pBRX insert with the expression cassette from the expression
plasmid. Ad.RSV.beta.gal and AdCMVCAT were similarly prepared.
[0101] The growth of these E1-deleted adenoviruses is limited to
293 cells. For these experiments, 293 cells transduced with the
various vectors were propagated in Improved Minimal Essential
Medium (IMEM) with 10% heat inactivated fetal bovine serum (FBS), 2
mM glutamine, 50 U/ml penicillin, and 50 .mu.g/ml streptomycin (all
from Biofluids, Rockville, Md.). Following cotransfection,
individual viral plaques were isolated and amplified in 293 cells,
and were purified by CsCl density purification as previously
described (Rosenfeld et al., supra). Subsequently, the preparations
were dialyzed and stored in dialysis buffer (10 mM Tris-HCl, 1 mM
MgCl.sub.2, pH 7.4) with 10% glycerol at -70.degree. C. The titer
of each viral stock was determined by plaque assay in 293 cells as
previously described; titers consistently ranged between
5.times.10.sup.9 and 2.times.10.sup.11 pfu/ml.
Example 2
[0102] This example describes adenoviral-mediated gene transfer to
adipocyte tissue in vivo.
[0103] Male Sprague-Dawley rats (250 to 300 gm) were used for all
studies; all procedures and care of animals were in accordance with
institutional guidelines. Animals were anesthetized with
intramuscular ketamine (100 mg/kg) and xylazine (2 mg/kg), and a
midline laparotomy was performed under sterile conditions. The
intestines were displaced to the contralateral side of the abdomen,
and the retroperitoneal fat was identified. The side of vector
administration (right vs. left) was determined pre-operatively in a
randomized fashion. A single 6-0 non-absorbable monofilament suture
was placed in the center of the adipose tissue to mark the site of
injection. The adenoviral vector was administered in a volume of 50
.mu.l using a 0.5 ml syringe with a 30 gauge needle. The needle tip
was positioned at a depth of 5 mm from the surface of the fat to
achieve uniform delivery, evident by the appearance of a small
weal. Sham-treated animals had identifying sutures placed, but no
vector administration. The intestines were returned to their normal
position, and the abdomen was closed in a single layer with
non-absorbable suture.
[0104] Rats were infected with 2.2.times.10.sup.9 pfu of
Ad.RSV.beta.gal, and 48 hours later the animals were sacrificed.
Sections of rat retroperitoneal adipose tissue were removed and
fixed in 4% formalin for 3 hours at 4.degree. C. Gene transfer, in
particular, the presence of the lacZ gene product encoded by the
.beta.-galactosidase reporter gene, was determined by staining
cells with the X-gal reagent
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside, Boehringer
Mannheim Corp., Indianapolis, Ind.), as previously described
(Setoguchi et al., supra; Mastrangeli et al., supra). Expression of
the lacZ gene product was considered positive when the cells
stained blue, particularly in the region of the nucleus. Following
infection with Ad.RSV.beta.gal, adipocytes stained blue and were
visualized (100.times.) and photomicrographed as darkened regions
of the tissue sample. In comparison, non-infected cells did not
demonstrate blue staining, and .beta.-galactosidase was not evident
in AdCMV.Null treated and naive (untreated) animals. These results
confirm that the transfer of the .beta.-galactosidase reporter
gene, and the subsequent expression of this gene, occurred in
adipocytes in vivo.
[0105] In similar experiments, AdCMVCAT also was delivered to rat
retroperitoneal adipose tissue in vivo. Specifically, AdCMVCAT in a
total volume of 100 ml in doses of 0, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, and 10.sup.9 pfu was injected into rat adipose
tissue (n=3 animals/dose). The tissue was harvested after 48 hours,
and chloramphenicol acetyltransferase (CAT) levels were quantified
by thin layer chromatography and phosphorimager analysis
(Kass-Eisler et al., supra). The relative CAT activity was reported
as percent conversion of chloramphenicol to its acetylated
counterpart by chloramphenicol acetyl transferase.
[0106] These results (as depicted in FIG. 1) demonstrate a higher
conversion rate with injection of a higher dose of AdCMVCAT. This
presumably is due to increased gene transfer with higher
multiplicities of infection. Moreover, the results confirm the
transfer of the chloramphenicol acetyl transferase reporter gene to
adipocytes in vivo. The fact that the graph peaks at a dose of
10.sup.7 is a function of the assay becoming saturated at this
dose. It is likely that even higher levels of CAT production would
be detected at the higher doses but for this saturation.
[0107] Furthermore, a time course experiment demonstrated that
chloramphenicol acetyl transferase activity can be detected at high
levels for up to 7 days following gene transfer, and at lower
levels for up to 10 days following gene transfer (FIG. 2). For this
experiment, about 10.sup.9 pfu/50 .mu.l AdCMVCAT was delivered to
rat retroperitoneal fat. The animals were sacrificed at the
indicated times, and CAT assays were performed. The values were
determined as percent CAT conversion, which either was or was not
normalized to mg protein. Three animals were sacrificed per
condition at each time point.
[0108] The VEGF gene encoded by the AdCMV.VEGF vector also was
delivered in vivo to rats. Specifically, rat adipose tissue was
injected with either 10.sup.11 pfu of AdCMV.VEGF or with 50 ng of
recombinant human VEGF as a positive control. Rat adipose tissue
also was injected with 10.sup.11 pfu of Ad.RSV.beta.gal as a
negative control. Within 24 hours following gene transfer, rat
adipose tissue was excised, minced, and bathed in Dulbecco's
modified Eagle medium (2 ml/g tissue) for 6 hours at 37.degree. C.
Aliquots (25 .mu.l) of the medium in which the cells were grown
were separated on a 15% polyacrylamide gel under reducing
conditions, transferred to a nitrocellulose membrane, and assayed
by standard Western immunoassay procedures using polyclonal
antibodies to the first 20 amino acids of the mature human VEGF
N-terminus (Tischer et al., J. Biol. Chem., 266, 11947-54 (1991))
with the peptide being conjugated to a carrier protein, keyhole
limpet hemocyanin, using 0.2% glutaraldehyde at a 1:500 dilution
and secondary antibody biotinylated for use with a
streptavidinalkaline phosphatase conjugate (goat anti-rabbit IgG
Bio-Rad Laboratories, Inc., Hercules, Calif.) at a 1:10000
dilution. The results of the Western assay confirm the transfer of
the VEGF gene encoded by the AdCMV.VEGF vector to rat adipocytes in
vivo, and the production of VEGF.sub.165 protein by the host
adipocytes--i.e., a proper size VEGF protein band was observed with
use of the positive control recombinant VEGF, and upon introduction
of AdCMV.VEGF, but not upon introduction of Ad.RSV.beta.gal.
[0109] The amount of VEGF protein produced was quantified using an
enzyme-linked immunoassay (ELISA) for the detection of human VEGF
protein (Cytokit Red VEGF enzyme immunoassay, CytImmune Science,
College Park, Md.). Rat retroperitoneal fat was injected with
either the control negative vector AdNull (10.sup.11 particles/50
ml) or AdCMV.VEGF (10.sup.11 particles/50 ml) The animals were
sacrificed immediately, or 1, 2, 5, 10 or 20 days following vector
administration. The fat was excised, minced, and bathed in
Dulbecco's Modified Eagle Medium (2 ml/gm tissue) for 6 hours at
37.degree. C. to allow release of secreted proteins from the tissue
into the medium. Aliquots (25 .mu.l) of the tissue culture medium
were loaded into 96-well plates in preparation for the ELISA. The
assay was performed according to the manufacturer's instructions,
and VEGF concentration was normalized to mg protein. Two animals
were sacrificed per condition at each time point. CAT assays were
carried out in triplicate.
[0110] As can be seen in FIG. 3, quantification of VEGF expression
in adipose tissue over time confirmed that the administration of
AdCMV.Null did not result in significantly increased levels of VEGF
over baseline at any of the time points examined. In comparison,
the administration of AdCMV.VEGF resulted in a more than 6-fold
increase over baseline VEGF expression, with peak expression
occurring about 5 days following vector administration. By day 10,
VEGF levels had returned to baseline. The levels of VEGF in these
tissues at 1, 2, and 5 days following AdCMV.VEGF administration
were significantly greater than the VEGF levels in tissue following
administration of AdCMV.Null (p<0.05, each time point). The
levels of VEGF on day 0, obtained immediately following vector
administration, were similar to the levels in naive animals, which
confirms that the viral preparation was not contaminated with VEGF
protein (p>0.8). No increase over baseline levels of VEGF was
detected in the serum of treated animals, consistent with the
observation that adenoviral vector delivery provides a localized
gene transfer strategy.
[0111] Immunohistochemical staining of adipose tissue was carried
out to confirm the presence of VEGF protein 48 hours following
administration of AdCMV.VEGF. For these experiments, paraffin
sections on slides were blocked with 1.5% goat serum for 20 minutes
to prevent nonspecific binding, and then were exposed to primary
antibody (rabbit anti-human VEGF; Santa Cruz Biotechnology) at a
concentration of 1 .mu.g/ml for 1 hour. A negative control
antibody, rabbit polyclonal antichloramphenicol acetyl transferase
(5'.fwdarw.3', Boulder, Colo.), was applied to a replicate section
of each tissue at the same concentration. The test and control
antibodies were diluted with phosphate buffered saline (PBS). The
primary antibody was eliminated from a parallel slide as an assay
control. The slides were exposed sequentially (30 minutes each)
with biotinylated goat anti-rabbit IgG (affinity purified against
rat serum proteins), ABC reagents (Vector Laboratories, Burlingame,
Colo.), and diaminobenzidime (4 minutes) as a substrate for the
peroxidase reaction, and were then counterstained with
hematoxylin.
[0112] The immunohistochemical staining of adipose tissue confirmed
the presence of VEGF in the cytoplasm of adipocytes and endothelial
cells in AdCMV.VEGF treated tissue, and its absence in AdCMV.Null
treated tissue.
[0113] Tissue sections were examined 10 days following gene
transfer in vivo to determine whether the encoded VEGF gene product
exerted any effect on vascularity. Two strategies were used to
determine whether the administration of AdCMV.VEGF to
retroperitoneal adipose tissue evoked angiogenesis in the adipose
tissue: (1) quantification of blood vessels assessed at the
macroscopic level (30.times.magnification) in vivo in living
tissue, and (2) quantification of blood vessels <20 mm by
histology. All studies were carried out using vector doses of
10.sup.9 pfu. Control groups included: injected retroperitoneal
adipose tissue immediately after injection; retroperitoneal adipose
tissue on the contralateral, untreated side; sham-injected adipose
tissue; and retroperitoneal adipose tissue injected with the
AdCMV.Null control vector.
[0114] The quantification of the number of macroscopic blood
vessels in living tissue was accomplished by injecting
retroperitoneal adipose tissue with AdCMV.VEGF (minimum of 3
animals per time point) or AdCMV.Null (minimum of 3 animals per
time point) as described above. Immediately thereafter, and 5, 10,
20, and 30 days following vector administration, the animals were
anesthetized, the retroperitoneal adipose tissue was exposed, and
injected (ipsilateral) and uninjected (contralateral) tissues were
examined in situ under a dissecting microscope (Nikon SMZ-U,
Morrell Instrument Co., Inc., Melville, N.Y.) at a distance of 15
cm (.times.3O). Photographic slides were prepared (Ektachrome 64T;
Kodak, Rochester, N.Y.), and the slides projected onto a screen at
3 m. Using the identifying suture as the center, a circle was drawn
on the screen around the suture with a diameter that corresponded
to a distance of 1 cm in situ. The number of vessels that crossed
the circle were counted by 3 blinded observers, with a minimum of
three vessels counted per slide per observer. The mean of these 3
counts was reported as the number of macroscopic blood vessels in
the 1 cm diameter circle of adipose tissue for each animal at each
time point.
[0115] To quantify the number of vessels <20 .mu.m in the
adipose tissue, 1 cm.sup.3 samples of both the ipsilateral
(treated) retroperitoneal adipose tissue centered around the
identifying suture and the contralateral (untreated) adipose tissue
were harvested from the same groups of animals used for gross
vessel quantification. Tissue was rinsed in PBS and stored in 4%
formalin at 4.degree. C. Samples were embedded in paraffin, and
serial 5 mm cross-sections in a plane parallel to the surface of
the tissue were obtained at intervals of 50 .mu.m. Three sections
were counterstained with hematoxylin and eosin, and 3 sections were
counterstained with Masson's trichrome. Random fields were
generated by computer, and sections were examined at a
magnification of .times.400 in a blinded fashion, by a pathologist
not associated with the study. Five fields were counted per slide,
with a minimum of 4 vessels <20 mm counted per field; 6 slides
were evaluated per animal. The counts were averaged, and reported
as vessel number per mm.sup.2. Results were reported as
mean.+-.standard error of the mean. Statistical analysis was
performed by the unpaired two-tailed Student's t-test.
[0116] Enhanced vascularity was observed (100.times. and 600
.times. magnification) following delivery of 10.sup.11 pfu of
AdCMV.VEGF to rat retroperitoneal adipose tissue, but not following
delivery of 10.sup.11 pfu of Ad.RSV.beta.gal. Similarly,
photomicrographs taken in vivo of retroperitoneal adipose tissue
demonstrated a marked increase in vascularity at longer times
following administration of AdCMV.VEGF. In particular, evaluation
30 days after AdCMV.VEGF administration showed several-fold more
vessels in adipose tissue as compared to adipose tissue of naive
animals, animals receiving the control AdCMV.Null vector, and the
contralateral (untreated) adipose tissue of animals receiving the
AdCMV.VEGF vector to the opposite side. Quantitative assessment of
the vessel number showed an increase in the number of blood vessels
in the adipose tissue 10 days after the administration of
AdCMV.VEGF which was 667% that of the uninjected contralateral
control adipose tissue in the same animals, and 310% and 256% that
of the sham and AdCMV.Null control adipose tissue, respectively
(p<0.01, all comparisons, FIG. 4). Importantly, the increase in
the quantitative in vivo blood vessel counts were maintained in the
adipose tissue 20 and 30 days following vector administration
(p<0.004, all comparisons), despite the fact that VEGF could not
be detected in the adipose tissue at day 10 following
administration of the AdCMV.VEGF.
[0117] Histologic evaluation of capillary number was consistent
with the observations made of in vivo blood vessel quantification.
For these experiments, all samples were examined at a magnification
of 400.times., and were counterstained with .alpha.-actin. In the
AdCMV.VEGF-injected tissue, histologic evaluation showed more
capillaries 30 days after vector administration compared to the
naive and AdCMV.Null-injected controls, as well as the
contralateral (untreated) adipose tissue of an animal injected with
the AdCMV.VEGF vector. Quantitative assessment of the histologic
samples (FIG. 5) showed a 21 to 39% increase in capillary number in
the AdCMV.VEGF-injected adipose tissue compared to the AdCMV.Null
controls at days 5, 10, 20, and 30 (p<0.0002, all comparisons),
i.e., as with the in vivo blood vessel quantification of vessels
observed at 30.times. magnification, there was persistence in the
increased capillary number despite the fact that VEGF could not be
detected at day 10 and thereafter.
[0118] These results confirm that adenoviral-mediated gene transfer
to adipocytes in vivo can be employed to attain a therapeutic
effect. In particular, the results validate that an adenoviral
vector carrying the VEGF cDNA is capable of inducing the growth of
new blood vessels in a regional fashion in relatively avascular,
normal tissue. This indicates that in vivo adenoviral-mediated gene
transfer can be used, inter alia, for therapeutic angiogenesis with
respect to adipocytes.
[0119] All of the references cited herein, including patents,
patent applications, and publications, are hereby incorporated in
their entireties by reference. The section headings or captions
preceding various portions of the text are used to index or divide
the text but should not be construed to denote or imply any
limitations on the scope of the invention.
[0120] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations in the preferred embodiments can
be used, including variations due to improvements in the art, and
that the invention can be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications encompassed within the spirit and scope of the
invention as defined by the following claims.
* * * * *