U.S. patent application number 10/601610 was filed with the patent office on 2004-05-13 for pi 3-kinase fusion mutants and uses thereof.
Invention is credited to Harrison, Stephen D., Kavanaugh, W. Michael, Klippel, Anke, Williams, Lewis T..
Application Number | 20040091898 10/601610 |
Document ID | / |
Family ID | 27486480 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040091898 |
Kind Code |
A1 |
Klippel, Anke ; et
al. |
May 13, 2004 |
PI 3-kinase fusion mutants and uses thereof
Abstract
Polynucleotide constructs encoding growth factor independent
catalytically active membrane targeted PI 3-kinase mutants useful
for therapeutic and research purposes are described. In addition, a
method for using the polynucleotide constructs to screen for
inhibitors of PI 3-kinase, a method for making 3' phosphorylated
inositol phospholipids, methods of reducing cell death after
trauma, and methods of overcoming insulin resistance are
described.
Inventors: |
Klippel, Anke; (San
Francisco, CA) ; Kavanaugh, W. Michael; (Mill Valley,
CA) ; Harrison, Stephen D.; (Berkeley, CA) ;
Williams, Lewis T.; (Tiburon, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
27486480 |
Appl. No.: |
10/601610 |
Filed: |
June 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10601610 |
Jun 23, 2003 |
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08832571 |
Apr 2, 1997 |
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6613956 |
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60015387 |
Apr 4, 1996 |
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60017693 |
May 14, 1996 |
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60033470 |
Dec 19, 1996 |
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Current U.S.
Class: |
435/6.18 ;
435/128; 435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
A01K 67/0339 20130101; C12P 9/00 20130101; A01K 2217/05 20130101;
C12P 19/44 20130101; C07K 2319/00 20130101; A61K 48/00
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/128; 435/194; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/12; C12P 013/00 |
Claims
What is claimed is:
1. A polynucleotide sequence comprising: (a) a first nucleotide
sequence comprising a sequence selected from the group consisting
of: (i) a sequence encoding the p110 subunit of PI 3-kinase
protein, and (ii) a sequence encoding a derivative or mutant of (i)
having a single or multiple nucleotide substitution, deletion or
addition, said derivative or mutant having an activity of the p110
subunit of PI 3-kinase protein, and (b) a second nucleotide
sequence comprising a sequence encoding a cell membrane targeting
sequence, said second nucleotide sequence being attached to the 5'
or 3' end of said first nucleotide sequence.
2. A polynucleotide sequence of claim 1, wherein said first
nucleotide sequence further comprises an additional sequence
selected from the group consisting of: (i) a sequence encoding a
portion of the p85 subunit of PI 3-kinase protein that is capable
of binding the p110 subunit of PI 3-kinase protein, and (ii) a
sequence encoding a derivative or mutant. of (i) having a single or
multiple nucleotide substitution, deletion or addition, said
derivative or mutant being capable of binding the p110 subunit of
PI 3-kinase.
3. A polynucleotide sequence of claim 2, wherein said additional
sequence comprises the iSH2 domain of the p85 subunit of PI
3-kinase protein.
4. A polynucleotide sequence of claim 1 wherein said cell membrane
targeting sequence is selected from the group consisting of (a) a
myristoylation cell membrane targeting sequence, and (b)
farnesylation and palmitoylation cell membrane targeting
sequences.
5. A sequence of claim 3, wherein said first nucleotide sequence
comprises a sequence encoding p110* and said second nucleotide
sequence comprises a sequence encoding a cell membrane targeting
sequence selected from the group consisting of: (a) a
myristoylation sequence and (b) farnesylation and palmitoylation
sequences.
6. A polynucleotide sequence comprising: (a) a first nucleotide
sequence comprising a sequence selected from the group consisting
of: (i) a sequence encoding the p110 subunit of PI3 kinase protein,
and (ii) a sequence encoding a derivative or mutant of (i) having
single or multiple nucleotide substitutions, deletions or
additions, said derivative or mutant having an activity of the p110
subunit PI 3-kinase, (b) a second nucleotide sequence comprising a
sequence selected from the group consisting of: (i) a sequence
encoding the iSH2 domain of the p85 subunit of PI3 kinase protein
that is capable of binding the p110 subunit of PI 3-kinase protein,
and (ii) a sequence encoding a derivative or mutant of (i) having a
single or multiple nucleotide substitution, deletion or addition,
said derivative or mutant being capable of binding the p110 subunit
of PI 3-kinase protein, wherein said second nucleotide sequence is
attached to a linker nucleotide sequence encoding a linker, said
linker nucleotide sequence being attached to the 5' end of said
first nucleotide sequence and forming a first fusion sequence, and
(c) a third nucleotide sequence encoding a cell membrane targeting
sequence, attached to the 5' or 3' end of said first fusion
sequence.
7. A polynucleotide sequence of claim 6 wherein said cell membrane
targeting sequence comprises a sequence selected from the group
consisting of: (a) a myristoylation cell membrane targeting
sequence and (b) farnesylation and palmitoylation cell membrane
targeting sequences.
8. A cell transformed with said polynucleotide sequence of claim
1.
9. A cell transformed with said polynucleotide sequence of claim
6.
10. A transgenic fly comprising a transgene having a polynucleotide
sequence of claim 6 under regulatory control of an eye specific
promoter, wherein said fly exhibits a phenotypic change in eye
morphology from normal to rough eye morphology.
11. A method of screening for an inhibitor of PI 3-kinase
comprising: (a) administering a candidate inhibitor to a transgenic
fly of claim 10, (b) observing any reversion in phenotype to normal
eye morphology in said fly, said reversion being indicative of PI
3-kinase inhibitor activity.
12. A method of reducing cell death due to trauma, comprising
administering to a mammalian patient a viral or non-viral vector
comprising a polynucleotide sequence of claim 1.
13. A method of reducing cell death due to trauma, comprising
administering to a mammalian patient a viral or non-viral vector
comprising a polynucleotide sequence of claim 6.
14. A method of making a 3' phosphorylated inositol phospholipid
comprising: (a) contacting a purified p110 or p110* polypeptide
with a vesicle including a PI 3kinase substrate selected from the
group consisting of phosphatidylinositol (PI), phosphatidyl
4phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate
(PI4,5,P.sub.2), and (b) isolating a 3' phosphorylated inositol
phospholipid.
15. A method of making a 3' phosphorylated inositol phospholipid
comprising transforming a host cell with said polynucleotide of
claim 1 and expressing said polynucleotide.
16. A method of making a 3' phosphorylated inositol phospholipid
comprising transforming a host cell with said polynucleotide of
claim 6 and expressing said polynucleotide.
17. A 3' phosphorylated inositol phospholipid made by the method of
claim 14.
18. A 3' phosphorylated inositol phospholipid made by the method of
claim 16.
19. A method of activating an enzyme effector of PI 3-kinase having
a pleckstrin homology domain comprising: (a) incubating a
polynucleotide sequence of claim 1 with a 4' phosphorylated
phosphatidylinositol selected from the group consisting of
phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol
4,5 bisphosphate (PI4,5P.sub.2,) to generate a mixture of 3'
phosphorylated inositol phospholipids comprising
phosphatidylinositol 3,4 bisphosphate (PI3,4P.sub.2,), and
phosphatidylinositol 3,4,5 trisphosphate (PI3,4,5P.sub.3,), (b)
isolating a 3' phosphorylated inositol phospholipid of (a) and (c)
contacting an active polypeptide having a pleckstrin homology
domain with an effective amount of said isolated 3' phosphorylated
inositol phospholipid of (b).
20. A method of promoting activation in a mammalian patient of an
insulin signaling pathway comprising contacting a cell
characterized by insulin resistance with a vector comprising a
polynucleotide sequence of claim 6.
21. A method of reducing cell death associated with trauma in a
mammalian patient, comprising contacting a population of said
patient's cells with an effective amount of a pharmaceutical
composition comprising a 3' phosphorylated inositol phospholipid of
claim 18.
Description
FIELD OF THE INVENTION
[0001] This invention provides polynucleotide constructs encoding
constitutively active membrane-targeted PI 3-kinase mutants,
methods for making polynucleotide constructs, an in vivo method for
screening for inhibitors of PI 3-kinase using the constructs, use
of the polynucleotide constructs to prevent cell death, or to
restore insulin responsiveness in type II diabetes, use of the
polynucleotide constructs to express PI 3-kinase mutants that
generate 3' phosphorylated inositol phospholipids, and use of these
phospholipids to prevent cell death.
BACKGROUND OF THE INVENTION
[0002] Phosphotidylinositol (PI) 3-kinase, both a phospholipid
kinase, and a protein serine/threonine kinase, is implicated in
certain oncogenic or mitogenic responses. See Carpenter et al.,
Mol. Cell. Biol. 13:1657-1665 (1993), Cantley et al., Cell
64:281-302 (1991), Escobedo and Williams, Nature 335:85-87 (1988),
and Fantl et al, Cell 69: 413-423 (1992). It is an intracellular
heterodimer consisting of an 85-kDa regulatory subunit (p85), and a
110-kDa catalytic subunit (p110) that is stimulated by growth
factors. See Whitman et al., Nature 332:644-646 (1988). The p85
subunit contains several domains and links the catalytic subunit to
activated growth factor receptors. The cDNA for the p110 subunit
has recently been cloned and expressed in insect and mammalian
systems as described in Hiles et al., Cell 70:419-429 (1992). The
general structure and function of PI 3-kinase, including analysis
of the structure and function of its subunits p85 and p110, are
described in Klippel et al, Mol. Cell. Biol. 13:5560 5566 (1993),
and in Klippel et al., Mol. Cell. Biol. 14:2675-2685 (1994).
[0003] The p85 subunit of PI 3-kinase has several domains,
including a 200 amino acid region of p85 located between the two
SH2 domains. This domain, called the inter-SH2 or iSH2 domain, has
been found sufficient to promote interaction with p110 in vivo with
activity comparable to that of full-length p85. See Klippel et al.,
Mol. Cell. Biol. 13:5560-5566 (1993). Additionally, a complex
between a 102 amino acid segment of p85 and the p110 subunit has
been found to be catalytically active, as described in and Klippel
et al., Mol. Cell. Biol. 14: 2675-2685 (1994).
[0004] Previously, studies to elucidate the of PI 3-kinase
activation have been conducted by constructing receptor mutants to
alter the signal transduction of PI 3-kinase, or by constructing
mutant oncogenes to study a PI 3-kinase inducible oncogenic
response. It would be advantageous to study effects of PI 3-kinase
activation directly, without growth factor activation, so as to
identify the role of PI 3-kinase in oncogenesis, mitogenesis, and
other tyrosine kinase and PI related functions. Methods and
compositions derived from such knowledge and use of PI 3-kinase to
control oncogenesis or mitogenesis, would be advantageous in the
treatment of cancer. In addition, it would be advantageous to
develop methods and compositions for such applications as
preventing cell death or treating type II diabetes.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention provides polynucleotide
sequences comprising the p110 subunit of PI 3-kinase polynucleotide
attached to a cell membrane targeting sequence. More specifically,
the invention provides a polynucleotide sequence comprising a first
nucleotide sequence encoding the p110 subunit of PI 3-kinase
protein, or a derivative or mutant of this sequence having a single
or multiple nucleotide substitution, deletion or addition, this
derivative or mutant having p110 catalytic activity, and a second
nucleotide sequence encoding a cell membrane targeting sequence,
this second nucleotide sequence being attached to the first
nucleotide sequence at the latter's 5' or 3' end. Further, the
polynucleotide sequence of the invention can be structured so that
the first nucleotide sequence also includes a nucleotide sequence
encoding the p85 subunit of PI 3-kinase or a fragment of the p85
subunit, for example the iSH2 domain of the p85 subunit, capable of
binding the p110 subunit. The cell membrane targeting sequence is a
nucleotide sequence encoding a myristoylation, or a palmitoylation
and farnesylation amino acid sequence.
[0006] Other aspects of the invention include methods of screening
for inhibitors of PI 3-kinase, methods of making 3' phosphorylated
inositol phospholipids, and the 3' phosphorylated inositol
phospholipid produced thereby, and methods for activating enzyme
effectors of PI 3-kinase having a pleckstrin homology domain.
[0007] Therapeutic aspects of the invention include methods of
reducing cell death due to trauma, by administering to the cell a
viral or non-viral vector including a polynucleotide sequence of
the invention, or by administering a 3' phosphorylated inositol
phospholipid to the cell. Another aspect of the invention is a
method of promoting activation of an insulin signaling pathway by
contacting a cell characterized by insulin resistance with a vector
having a polynucleotide sequence of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a schematic representation of polynucleotide
sequences used in the Examples.
DETAILED DESCRIPTION
[0009] All patents, patent publications, and scientific articles
cited herein are hereby incorporated by reference.
[0010] The Polynucleotide Constructs
[0011] Provided here are polynucleotide constructs encoding
constitutively active forms of PI 3-kinase mutants. The
polynucleotide constructs encoding PI 3-kinase fusion mutants are
capable of inducing PI 3-kinase dependent signaling responses that
are higher than those previously achieved with an equal or greater
concentration of other PI 3-kinase mutants, due to the addition of
one or more membrane targeting sequences. The term "PI 3-kinase
mutant" or "PI 3-kinase fusion mutant" is a polypeptide sequence
that differs from the native, full length PI 3-kinase sequence.
Fusion mutant refers to a sequence encoding a PI 3-kinase sequence
or portion of a PI 3-kinase sequence (including, for example, fused
portions of PI 3-kinase subunits) fused to a cell membrane
targeting sequence. PI 3-kinase is described in Kapeller and
Cantley, Bioessays 16:565-576 (1994), and in Stephens et al.,
Biochim. Biophys. Acta. 1179:27-75 (1993). The p110 subunit of PI
3-kinase can be used to make the fusion mutants, including also all
or a portion of the p85 subunit. In addition, single or multiple
deletions, substitutions or additions of nucleic acids can be made
to the p110 or p85 sequences, and the construct can be tested for
retention of the desired function as described in Examples 2, 3,
and 6 below. PI 3-kinase mutants of p85 are described, for example,
in Klippel et al., Mol. and Cell. Bio. 13:5560-5566 (1993) and
mutants of p110, including p110* , are described, for example, in
Hu et al., Science 268:100-102 (1995). The native polynucleotide
sequence of the p110 subunit, and the native polypeptide sequence
it encodes are described in Klippel et al, Mol. Cell. Biol. 14:
2675-2685 (1994). It is on deposit with Genbank, accession number
U03279. The native polynucleotide sequence of the p85 subunit of PI
3-kinase and the native polypeptide sequence it encodes are
described in Escobedo et al., Cell 65:75-82 (1991). It is on
deposit with Genbank, accession number M60651.
[0012] The invention provides a polynucleotide that comprises a
first nucleotide sequence encoding the p110 subunit of PI 3-kinase
protein, or a sequence encoding a derivative or mutant of the p110
subunit including single or multiple nucleotide substitutions,
deletions or additions, this derivative or mutant having p110
subunit catalytic activity. The polynucleotide also comprises a
second nucleotide sequence encoding a cell membrane targeting
sequence that is attached to the 5' or 3' end of the first
nucleotide sequence. Thus, the invention provides a polynucleotide
that upon expression is targeted to the cell membrane.
[0013] In another aspect, the invention provides that the first
polynucleotide sequence can further comprise a nucleotide sequence
encoding the p85 subunit of PI 3-kinase protein or a sequence
encoding a derivative or mutant of the p85 subunit including a
single or multiple nucleotide substitution, deletion or addition,
this derivative or mutant capable of binding the p110 subunit. The
sequence encoding the p85-derived sequence can be a sequence
encoding the amino acid sequence between the two SH2 domains of the
p85 subunit, the inter-SH2 domain (iSH2) that is capable of binding
the p110 subunit. An example of such a fusion molecule is p110*
described below which incorporates the iSH2 domain of p85 into a
construct containing a catalytically active p110 subunit, described
in Hu et al., Science 268:100-102 (1995). To this fusion molecule,
the cell membrane targeting sequence or sequences are added at the
5' or 3' end or both of the polynucleotide construct. The p110*
fusion mutant also contains a linker region between the p85 and
p110 sequences. The linker nucleotide sequence encoding the linker
can comprise, for example, a sequence encoding a glycine rich
region.
[0014] Thus, the PI 3-kinase mutant that is targeted to the
membrane of a cell can be any p110-derived PI 3-kinase mutant that
retains p110 kinase activity, including, for example, the p110
subunit of PI 3-kinase, and biologically active variations thereof,
including but not limited those described in Klippel et al., Mol.
Cell Biol. 13:5560-66 (1993), Klippel et aL, Mol. Cell Biol.
14:2675-2685 (1994). The PI 3-kinase mutant can also be a
constitutively active PI 3-kinase mutant such as p110* which can be
constructed as described in Hu et al., Science 268:100-102 (1995).
P110* includes the additional p85 derived sequence iSH2. The
membrane targeting sequences for attachment at the polynucleotide
level (i.e. at the 5' or 3' end) can include sequences encoding
myristoylation or farnesylation and palmitoylation sequences.
Generally, the farnesylation and palmitoylation sequences are used
together at the same. end of the polynucleotide sequence that is to
be targeted to the membrane.
[0015] The PI 3-kinase mutants expressed from the polynucleotides
of the invention promote "constitutive activity" which refers to
the ability of the PI 3-kinase mutant to catalytically activate
downstream effectors in the absence of growth factor stimulation.
Thus, the fusion mutants demonstrate a growth factor independent
induction of the downstream effectors of PI 3-kinase activity, or a
catalytic activity, including but not limited to, for example,
induction of pp70S6 kinase and AKT kinase activities, and
generation of active phosphoinositol 3' phosphorylated
phospholipids. These improved membrane targeted PI 3-kinase mutants
have increase utility: with only a small amount of mutant, in the
absence of growth factor, greater amounts of PI 3-kinase catalytic
activity are demonstrated.
[0016] The cell membrane targeting sequence of the fusion mutant
can be any sequence that targets a protein to the membrane of a
cell. The polynucleotide constructs of the invention have a
nucleotide sequence encoding cell membrane targeting sequences
attached to the 5' or 3' end of the polynucleotide construct. In
the expressed proteins of the invention, the membrane targeting
sequence thus may be located at the N-terminal or the C-terminal
end. Also, membrane targeting sequence may be encoded in the
polynucleotide sequence that encodes the mutant or a polypeptide
cell membrane targeting sequence may be added to the expressed
mutant at the N or C terminus by post translational modification.
Exemplary membrane targeting sequences include myristoylation
sequences, such as those described in Buss et al., Science
243:1600-03 (1989), Kaplan et al., PNAS USA 83:3624-3628 (1990),
Schultz et al., Science 227:427-429 (1985), and Deichaite et al.,
Mol. Cell Biol 8:4295-301 (1988), and farnesylation sequences and a
palmitoylation sequences, such as those described in Cadwallader et
al., Mol. Cell Biol. 14:4722-4730 (1994). Preferably a
myristoylation sequence is added to the 5' end of a polynucleotide
construct or to the N-terminus of an expressed polypeptide.
Preferably, a farnesylation sequence is added to the 3' end of a
polypeptide construct or to the C-terminus of an expressed
polypeptide. Depending on whether the farnesylation sequence is
derived from H-Ras or K-Ras, the farnesylation sequence is most
preferably added in conjunction with either a palmitoylation
sequence or a polybasic region of several lysines, as described in
Example 1. The mutant polynucleotide may also contain both a
myristoylation and a farnesylation sequence with either a
palmitoylation or a polybasic sequence (chosing a palritoylation or
polybasic sequence depending on the protein from which the
farnesylation sequence is derived) also as described in Example 1.
Other mechanisms for creating a cell membrane targeted fusion
mutant exist, including, but not limited to, for example, the
addition of lipid moieties to the polypeptide once it has been
expressed that act as cell membrane targeting sequences.
[0017] Nucleotide sequences encoding derivatives or mutants of the
p110, p85, or cell membrane targeting sequences can have 50%, more
preferably 60%, more preferably 70%, more preferably 80%, more
preferably 90%, most preferably 95% nucleic acid sequence identity
to a native sequence from which the derivative or mutant sequence
is derived. For example, a polynucleotide sequence can include a
nucleotide sequence encoding a sequence derived from the p110
subunit having 95% nucleic acid sequence identity to the native
p110 nucleotide sequence, a nucleotide sequence encoding a sequence
derived from the p85 subunit having 80% nucleic acid sequence
identity to the native p85 nucleotide sequence, and a nucleotide
sequence encoding a myristoylation sequence having 95% nucleic acid
sequence identity to the native myristoylation sequence.
[0018] The intracellular polypeptides expressed upon expression of
the polynucleotides of the invention in a host cell can have 60%,
more preferably 70%, more preferably 80% more preferably 90% and
most preferably 95% sequence identity to the native amino acid
sequences of the particular PI 3-kinase subunit sequences and cell
membrane targeting sequences comprising the entire polynucleotide
construct. Thus, for example, a cell membrane targeted p110*
sequence can contain within its intracellular expression product an
iSH2 sequence of 90% amino acid sequence identity to the native
iSH2 sequence cited, 95% amino acid sequence identity to the native
p110 sequence, and 90% amino acid sequence identity to the native
myristoylation sequence.
[0019] The polynucleotide constructs are made by first constructing
the p110 polynucleotide sequence. To the p110 subunit-derived
polynucleotide sequence is added a cell membrane targeting
modification. Alternatively, a p85 derived polynucleotide sequence
can be attached to the p110 subunit sequence, for example using a
linker at the 5' end of the p110 sequence. To this fusion a
membrane targeting sequence can then be attached. In all cases,
attachment of nucleic acid sequences can mean fusion or ligation,
for example, using standard molecular biology techniques. The cell
membrane targeting sequence can be encoded in a polynucleotide
sequence that is attached to the polynucleotide sequence encoding
the sequence that it will modify. The membrane targeting sequence
may also be a sequence added to the mutant polypeptide after the
polypeptide has been expressed, such as, for example, a lipid
moiety or lipid modification of the expressed polypeptide, or an
expressed cell membrane targeting sequence. The membrane targeting
sequence, whether encoded in the polynucleotide, or whether added
after translation of the polypeptide, may be attached to the mutant
at any position in the mutant that will target the mutant to a cell
membrane.
[0020] These and all other polynucleotides of the invention can be
assayed for function as described in Example 3 below.
[0021] Polynucleotide constructs can be made for the expression of
fusion mutants with regulatable activity include the coding region
for the respective molecule downstream of a CMV of SR.alpha.
promoter and a viral translation initiation region. The p110 coding
region can be attached at its 5' terminus to a mutant form of the
regulatory domain of the mouse estrogen receptor, as described in
Example 5. The regulatory domain of the mouse estrogen receptor
comprises amino acids 281 to 599 and can be preceded by two or more
glycine residues at the junction to the p110 sequence to provide
for flexibility and for proper folding of the individual portions
of the chimeric molecule. The estrogen receptor portion carries a
mutation, GR525, which provides for the following characteristics:
the GR525 mutation renders the fusion protein tightly dependent on
4-OHT and totally unresponsive to estrogen. These characteristics
ensure a tightly regulated, nonleaky, induction of PI-3 kinase
activity in response to 4-OHT.
[0022] On the same polynucleotide construct, can be located a TATA
box domain and a virally derived translation initiation site. The
translational initiation domain can be, for example, derived from
the translational initiation domain from the SV40 large T antigen
regulatory regions or from the Herpes Simplex thymidine kinase
regulatory regions. These sequences can then be followed by the
polynucleotide sequence encoding polynucleotide mutant of the
invention. Expression ensues in an inducible fashion much as
described in Littlewood et al., Nucleic Acids Research 23:1686-1690
(1995) with the additional advantage that the mutant is efficiently
translated to result in a functional kinase mutant. As far as these
inventors are aware, efficient expression and translation of PI
3-kinase or of PI 3-kinase mutants has not been achieved prior to
this, and is advantageously achieved using the inducible expression
method of the invention.
[0023] A polynucleotide construct for inducible expression of a
mutant PI 3-kinase is also included in the invention. The construct
includes a polynucleotide sequence encoding the following domains
described 5' to 3': a binding site for a repressor protein, a TATA
box, a viral sequence sufficient for efficient initiation of
translation, and a polynucleotide sequence encoding a PI 3-kinase
mutant. This construct can be used to inducibly express the
polynucleotides of the invention, and generate useful products of
the resulting activated pathways. The polynucleotide construct of
the inducible expression system can have a binding site that can
bind a chimeric protein having a DNA binding domain of a repressor
protein and a transactivating domain of a different gene
activator.
[0024] The term "repressor protein" refers to that class of
proteins characterized by both an ability to bind DNA and alter
transcription, and by the repression of transcription that results
from this binding. Repressor proteins include, for example, the lac
repressor, the tet repressor, the lambda repressor, and others as
described in MOLECULAR BIOLOGY OF THE CELL, Alberts et al. ed.,
Garland Press, New York, pp. 407, 420-426 (1994). The term "viral
sequence sufficient for initiation of translation" as used herein
refers to a polynucleotide sequence identified in a viral genome
that functions to regulate translation, and which has been
identified as controlling and facilitating the initiation of
translation of viral proteins and which can be included for
expression of heterologous proteins to facilitate initiation of
translation of heterologous proteins. Such a region can, for
example, be derived from the herpes simplex thyrnidine kinase, tk,
gene for optimal initiation of translation region (hereafter "the
tk upstream region"). This region can be isolated from plasmid pCG,
described in Giese et al., Genes and Development 9:995-1008 (1995),
which is a pEVRF derivative, described in Matthias et al., Nucleic
Acids Res. (1989) 17:6418. Other such viral initiation of
translation regions exist, including for example, the SV40 virus
large T antigen initiation of translation region. The term
"transactivating domain" as used herein refers to the domain of a
gene regulatory protein such as, for example, those gene regulatory
proteins described in MOLECULAR BIOLOGY OF THE CELL, Alberts et al
ed., Garland Press, New York, pp. 407, 420-426 (1994), also
including, for example, the transactivating domain of VP16 as
described in Gossen et al, Science 268: 1766-1769 (1995). This
domain of the gene activator molecules have the ability to activate
transcription. In a preferred embodiment the transactivating domain
can be VP16, the binding site can be a multimer, the DNA binding
domain can have binding sites for Tet or Lac, and the repressor
protein can be Tet or Lac.
[0025] The polynucleotide constructs of the invention, once
designed, can be constructed by standard recombinant DNA technology
and manipulation. For example, polynucleotide constructs having
deletions, mutations, substitutions, fusions, and which otherwise
encode polypeptide variants, derivatives, mutants, analogues, or
chimeras can be constructed by conventional techniques of molecular
biology, microbiology, and recombinant DNA technology that are
within the skill of the art. The polynucleotide can be placed into
a vector construct that directs its expression. The vector
construct must include transcriptional promoter element(s), and
preferably includes a signal that directs polyadenylation. In
addition, the vector construct must include a sequence which, when
transcribed, is operably linked to the sequence(s) or gene(s) of
interest and acts as a translation initiation sequence.
[0026] Such techniques for polynucleotide and polypeptide
construction and expression are explained fully in the literature,
for example in Sambrook, et al. MOLECULAR CLONING; A LABORATORY
MANUAL, SECOND EDITION (1989); DNA CLONING, VOLUMES I AND II (D. N
Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984);
NUCLEIC ACID HYBRIDIZATION (B. D. Hames & S. J. Higgins eds.
1984); TRANSCRIPTION AND TRANSLATION (B. D. Hames & S. J.
Higgins eds. 1984); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR
CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press,
Inc.). Further, sequences that encode the above-described genes may
also be synthesized, for example, on an Applied Biosystems Inc. DNA
synthesizer (e.g., ABI DNA synthesizer model 392 (Foster City,
California)). Additionally, the polynucleotides can be constructed
and cloned as described in PCR PROTOCOLS, Cold Spring Harbor, N.Y.
1991. The desired gene can also be isolated from cells and tissues
containing the gene, using phenol extraction, PCR of cDNA, or
genornic DNA. The gene of interest can also be produced
synthetically, rather than cloned, as described in Edge, Nature
292: 756 (1981), Nambair et aL, Science 223:1299 (1984), and Jay et
al., J. Biol. Chem. 259:6311 (1984). Additionally, variations of
any polynucleotide or polypeptide can be made by conventional
techniques, including PCR or site-directed mutagenesis. The DNA
constructs so synthesized can be ligated to an expression plasmid
containing an appropriate promoter for expression in a desired host
expression system. The host system can be in vitro, in vivo or ex
vivo.
[0027] The polynucleotides of the invention can be used to
transform host cells and can thus be expressed in these cells. Host
cells appropriate for this transformation include bacterial, yeast,
insect, or mammalian host cells, for example, including those host
cells systems described in WO 96/35787. The polynucleotides can be
used to stably transform cells in order to construct stable cell
lines.
[0028] The Phospholipids and Methods for Making Them
[0029] The invention provides a method for making a 3'
phosphorylated inositol phospholipid in vitro. In step one of the
method vesicles containing PI-3 kinase phospholipid substrates,
including for example, phosphatidylinositol (PI),
phosphatidylinositol 4 phosphate (PI4P) and/or phosphatidylinositol
4,5 bisphosphate (PI4,5P.sub.2,) are incubated with a PI 3-kinase
mutant to generate 3' phosphorylated inositol phospholipid products
from each substrate. In step 2 the reaction product is
collected.
[0030] In the particular case where substrate comprises
phosphatidylinositol 4,5 bisphosphate (PI4,5P.sub.2,), the 3'
phosphorylated inositol phospholipid generated will be
phosphatidylinositol 3,4,5 trisphosphate (PI3,4,5P.sub.3,). In this
particular case, the phosphatidylinositol 3,4,5 trisphosphate
(PI3,4,5P.sub.3,) can be contacted prior to the collection step
with a signaling inositol polyphosphate 5' phosphatase (SIP)
polypeptide in order to generate phosphatidylinositol 3,4
bisphosphate (PI3,4P.sub.2,), which is then collected. SIP is
described in Egan et al, Nature 363:45 51 (1993), Zhang et al.,
Proc. Natl. Acad. Sci. USA 92:4853-4856 (1995), and York et al., J.
Mol. Biol. 236:584-589 (1994).
[0031] Thus, the invention includes a method of making a 3'
phosphorylated inositol phospholipid comprising contacting a
polynucleotide sequence of the invention with a PI3 kinase
substrate selected from the group consisting of
phosphatidylinositol (PI), phosphatidyl 4-phosphate (PI4P) and
phosphatidylinositol 4,5 bisphosphate (PI4,5,P.sub.2), and
isolating a 3' phosphorylated inositol phospholipid. Another aspect
of the invention is that the 3' phosphorylated inositol
phospholipid phosphatidylinositol 3,4,5 trisphosphate
(PI3,4,5P.sub.3,) is isolated. In some circumstances it may be to
advantage contact the phosphatidylinositol 3,4,5 trisphosphate
(PI3,4,5P.sub.3,) with a signaling inositol polyphosphate 5'
phosphatase (SIP) polypeptide to facilitate isolating
phosphatidylinositol 3,4 bisphosphate (PI3,4P.sub.2,).
[0032] Treatment of PI3,4,5P3 with the inositol polyphosphate 5'
phosphatase SIP leads to activation of Akt in vitro. This
activation can thus be used as an assay to indicate whether the 3,4
inositol polyphosphate has been synthesized, and is described in
more detail below.
[0033] The most effective way of making 3' phosphorylated inositol
phospholipid is an intracellular method. A cell is transformed with
a polynucleotide construct of the invention, and this host cell is
used to express the polynucleotide and generate 3' phosphorylated
inositol phospholipids from substrates naturally occurring in the
cell. Because there is an excess of these substrates in cells, a
constitutively active mutant such as embodied in any of
polynucleotides of the invention will generate a large amount of 3'
phosphorylated inositol phospholipids as compared to cell
expressing native PI 3-kinase. These phospholipids, so generated,
can be isolated or used within the cell for purposes such as those
described herein.
[0034] Use of the Polynucleotide Compositions
[0035] The cell membrane targeted polynucleotide fusion mutants of
the invention are useful for many applications. The polynucleotide
constructs are useful in a research context for identifying and
studying cellular processes by PI 3-kinase, without the need for
prior growth factor activation. The use of polynucleotide
constructs of the invention facilitates testing whether PI 3-kinase
activation alone is sufficient for the induction of a signaling
event.
[0036] The signaling events that can be tested for dependence on PI
3-kinase activation include, for example, many of the cellular
responses that appear to be regulated by PI 3-kinase, including
mitogenesis and oncogenesis; the reorganization of actin
cytoskeleton as described in Kapeller et al., Mol. Cell Biol.
13:6052-6063 (1993); receptor internalization; histamine secretion;
neutrophil activation; platelet activation as described in Zhang et
aL, J. Biol. Chem. 267:4686 4692 (1992); cell rnigration; glucose
transport and antilipolysis; and vesicle sorting as described in
Stack et al., EMBO J. 12:2195-2204 (1993). Thus, the
polynucleotides can be used for functional studies of PI 3-kinase
activation in a variety of applications, including but not limited
to study of the PI 3-kinase dependent effects of reorganization of
actin cytoskeleton, receptor internalization, histamine secretion,
neutrophil activation, platelet activation, cell migration, glucose
transport and antilipolysis, vesicle sorting, apoptotic rescue,
mitogenesis, and oncogenesis.
[0037] Further, studies of PI 3-kinase activation can be
facilitated by using the membrane targeted polynucleotides for
elucidating whether a particular PI 3-kinase dependent effect is
due to a lipid phosphorylation event or a protein phosphorylation
event, or both, by, for example, demonstrating the accumulation of
a PI 3-kinase downstream effector in the presence of a
polynucleotide of the invention.
[0038] The invention also includes an expression system for
inducibly expressing the polynucleotides of the invention, as
described above. The expression system is useful for conducting in
vivo studies by, for example, overexpressing the polynucleotides of
the invention, accumulating PI 3-kinase activation products for
studies relating to the PI 3-kinase pathway, studying PI 3 kinase
involvement in a particular cellular response in which PI 3-kinase
is implicated, and screening for inhibitors of PI 3-kinase
activity. The expression system is useful for study of PI 3-kinase
function by stable expression in mammalian cells rather than by
transient overexpression surmounting a previous problem in the art,
that was the difficulty of expressing p110-derived subunits in a
stable manner. This inducible system combines the system of
post-translational activation by 4-hydroxy tamoxifen in mammalian
cells, described, for example, in Littlewood et aL, NAR
23:1686-1690 (1995), and the 5' upstream translation initiation
site of a virus to accomplish an inducible expression of any of the
polynucleotides of the invention. The viral translation initiation
site can be derived from, for example, the Herpes Simplex virus
thymidine kinase (TK) gene or the SV40 virus large T antigen gene.
Finally, the invention provides a method for screening for
inhibitors of PI 3-kinase using a transgenic fly expressing a
polynucleotide of the invention under the control of an eye
specific promoter. The eye tissue specific expression of the PI
3-kinase mutant results in a morphological change in the eye of the
fly. This variant morphological change can revert to a wild type
morphology upon administration to the fly of an inhibitor of PI
3-kinase. The transgenic fly screen for inhibitors of PI 3-kinase
can be used as a primary screen for inhibitors of PI 3-kinase, or
as a secondary screen for inhibitors that appear to inhibit PI
3-kinase in an in vitro or cell-based assay.
[0039] The cell membrane targeted polynucleotides of the invention
may be used in a transgenic assay to screen for inhibitors of PI
3-kinase activity. The screening assay is conducted by feeding the
flies food containing a candidate inhibitor. If the inhibitor is
functional, the eye morphology reverts from mutant to wild type.
The candidate inhibitor can be a small molecule, including a small
organic molecule, a peptide, a peptoid, a ribozyme or an antisense
polynucleotide, for example. This screening assay can be applied to
screening for inhibitors of any kinase capable of generating a
mutant phenotype when expressed in the eye tissue under control of
an eye-specific promoter.
[0040] Another embodiment of the invention is a method of screening
for an inhibitor of PI 3-kinase activity by providing a transgenic
insect expressing a polynucleotide of the invention under the
control of an eye-specific promoter, resulting in a mutant eye
morphology, administering to the transgenic insect a candidate
inhibitor, and identifying a functional inhibitor by a reversion of
the eye morphology to normal upon administration of the inhibitor.
This method can include the condition where the insect is a fly,
and where the fly is Drosophila melanogaster. The mutant eye
morphology in the fly is rough eye. The candidate inhibitor can be
a polynucleotide (for example a ribozyme or an antisense molecule),
a polypeptide (for example, an intra-body or intracellular
antibody), a small molecule, a peptide, or a peptoid.
[0041] Another embodiment of the invention is a transgenic fly
containing a transgene comprising a polynucleotide of the invention
under the regulatory control of an eye specific promoter, for
example a sevenless or a GMR promoter, as described in Hay et al.,
Development 120:2121-9 (1994). The inhibitor is fed to the fly
throughout the third instar larval development. Such a transgenic
fly can be made from Drosophila melanogaster. The Drosophila is
transformed with the transgene using standard techniques, and the
transgenic fly is fed the inhibitor throughout the third instar
larval development. Transgenic control flies and flies for which
the inhibitors are ineffective exhibit a rough eye morphology as
compared to a normal phenotype of the wild type fly. An effective
inhibitor reverts the rough eye phenotype to normal upon
administration. The rough eye and other such aberrant morphology
can be detected under a dissecting microscope as described in
Kaufman et al., Proc. Natl. Acad. Sci. USA 92:10919-23 (1995). This
assay has the advantage over in vitro assays in that inhibitors
that revert the eye phenotype must also possess additional
important properties required of a PI 3-kinase inhibiting
pharmaceutical including that the inhibitor must be able to enter
cells, that the inhibitor must be specific to the kinase target
expressed as the transgene. The Drosophila eye screen can be used
as a secondary or tertiary assay to test inhibitors that have been
previously identified by other means.
[0042] The polynucleotides of the invention are useful as
therapeutic agents in the context of trauma or potential cell
death, for administration in a gene therapy vehicle for preventing
the cell death that would result due to the trauma. The trauma can
be, for example, a stroke or heart attack. Administration of such
therapeutic agents is described below. The polynucleotides of the
invention are also useful for treating type II diabetes in humans
by administration of a gene therapy vehicle to human cells or
tissue normally expected to produced an insulin-induced response
but for the defectiveness of the cells or tissue to do so.
[0043] The inventors have observed that the expression of the
polynucleotides of the invention markedly increases intracellular
levels of PI3,4P.sub.2 and PI3,4,5P.sub.3. The level of
phospholipid products induced correlates with the relative
efficiencies of the activated p110-derived polynucleotides used.
This observation supports the view that the 3' phosphorylated
inositol phospholipid products mediate PI 3-kinase-induced
signaling responses. However, as PI 3-kinase is a dual specificity
kinase, that can phosphorylate phospholipids and proteins, a system
to observe the phosphorylation of phospholipids in isolation was
therefore devised.
[0044] Activation of the serine-threonine kinase Akt (also known as
RAC-PK or PKB) has been shown to be dependent on PI 3-kinase,
cotransfection of increasing amounts of Akt expression vectors with
p110* results in increased levels of Akt activation, Akt contains a
pleckstrin homology (PH) domain, and PH domains have been
implicated in the binding of phospholipids and in the regulation of
Akt activity, as described in Burgering et al., Nature 376: 599-602
(1995) and Harlan et al., Nature 371:168-70 (1994). By stimulation
of intracellular protein kinase activity of Akt using purified
p110* , it can be shown that this response is selectively mediated
by the phosphatidylinositol product PI3,4P.sub.2 and not by p110*
protein kinase.
[0045] The assay comprises the steps of incubating a polynucleotide
sequence of the invention with phosphatidylinositol 4 phosphate
(PI4P) or phosphatidylinositol 4,5 bisphosphate (PI4,5P.sub.2,) to
generate a 3' phosphorylated inositol phospholipid comprising
phosphatidylinositol 3,4 bisphosphate (PI3,4P,), or
phosphatidylinositol 3,4,5 trisphosphate (PI3,4,5P.sub.3,),
incubating the phosphatidylinositol 3,4,5 trisphosphate
(PI3,4,5P.sub.3,), with a signaling inositol polyphosphate 5'
phosphatase (SIP) polypeptide, collecting phosphatidylinositol 3,4
bisphosphate (PI3,4P.sub.2,), and contacting an active polypeptide
having a pleckstrin homology domain with an effective amount of the
phosphatidylinositol 3,4 bisphosphate (PI3,4P.sub.2,). The enzyme
effector of PI 3-kinase having a pleckstrin homology domain can be,
for example, Akt kinase, one of several guanine nucleotide exchange
factors, one of several GTPase activating proteins, and any other
PH domain containing enzymes.
[0046] The use of inositol polyphosphate 5' phosphatase SIP
(signaling inositol polyphosphate 5' phosphatase) converts the
"inactive" (with respect to Akt activation) phospholipid product of
PI 3-kinase, PI3,4,5P.sub.3, into PI3,4P.sub.2 that can stimulate
Akt. Thus, the assay is useful to make PI3,4P.sub.2, and to show
that PI3,4P.sub.2 is a specific membrane-bound product of PI
3-kinase (and perhaps also a product of SIP) that can directly
activate PH domain-containing cytoplasmic signaling molecules. The
protein SIP and nucleic acid encoding it is described in patent
application Serial No. 08/624,190 filed Mar. 28, 1996.
[0047] By developing this assay, the inventors have developed a
method of activating an enzyme effector of PI 3-kinase that has a
pleckstrin homology domain, such as, Akt kinase, a guanine
nucleotide exchange factor, GTPase activating proteins, or
phospolipases. Also the method is a method of making the activating
phospholipids. Once the phosphatidylinositol 3,4 bisphosphate
(PI3,4P.sub.2,). is made, a sufficient amount of it is placed in
contact with an active kinase polypeptide having a pleckstrin
homology domain, for example, Akt kinase, to test whether the
synthesis is successful. Activation of Akt kinase is measured as
described in the Example 6.
[0048] Using this method we determined that PI3,4P.sub.2, but not
PI3P or PI3,4,5P.sub.3, increases Akt activity. We observed Akt
activation by PI3,4P.sub.2 using synthetic dipalmitoylated
PI3,4P.sub.2 for in vitro stimulation of Akt, by generating
PI3,4P2-containing vesicles in vitro using p110* polynucleotide
constructs and subsequently treating immobilized Akt, and by
generating PI3,4P.sub.2 by treating p110*-produced PI3,4,5P.sub.3
with the inositol polyphosphate 5' phosphatase SIP in vitro. p110*
did not appear to stimulate Akt by its protein kinase activity
either in the presence or absence of phospholipid vesicles, because
no Akt phosphorylation was detected in the presence of p110*
despite the fact that p110* was able to autophosphorylate under the
same reaction conditions. The possibility that the p110* protein
kinase activity requires PI3,4P.sub.2 to stimulate Akt does not
seem likely, since Akt activation in vitro was also achieved using
synthetic PI3,4P.sub.2 in the absence of p110*. Thus, Akt is an
immediate downstream effector of PI 3-kinase and the
phosphatidylinositol products of PI 3-kinase can function as second
messengers by directly activating Akt. This assay can be used to
make and measure the production of intracellular 3' phosphorylated
inositol phospholipids.
[0049] The assay allowed determination that the stimulatory effect
of PI3,4P.sub.2 on the kinase activity of Akt was dependent on the
presence of a functional PH domain with the generation of a point
mutation in the Akt-PH domain that abrogated growth factor- or PI
3-kinase-mediated activation of Akt in vivo and that no longer
allowed Akt stimulation by PI3,4P.sub.2 in vitro. The discovery
demonstrates that the PH domain is directly involved in the
regulation of the enzymatic activity of Akt by PI3,4P.sub.2. The
experiments suggest that PI 3-kinase can activate signaling
pathways through its 3' phosphorylated inositol phospholipid
products that act on PH domains of effector molecules.
[0050] The phospholipids of the invention are useful as therapeutic
agents also in the context of trauma or potential cell death, for
reducing any potential cell death occurring from the trauma.
[0051] Administration
[0052] 1. Gene Delivery Vehicles
[0053] Gene delivery vehicles (GDVs) are available for delivery of
polynucleotides to cells, tissue, or to a the mammal for
expression. For example, a polynucleotide sequence of the invention
can be administered either locally or systemically in a GDV. These
constructs can utilize viral or non-viral vector approaches in in
vivo or ex vivo modality. Expression of such coding sequence can be
induced using endogenous mammalian or heterologous promoters.
Expression of the coding sequence in vivo can be either
constitutive or regulated.
[0054] The invention includes gene delivery vehicles capable of
expressing the contemplated polynucleotides. The gene delivery
vehicle is preferably a viral vector and, more preferably, a
retroviral, adenoviral, adeno-associated viral (AAV), herpes viral,
or alphavirus vectors. The viral vector can also be an astrovirus,
coronavirus, orthomyxovirus, papovavirus, paramyxovirus,
parvovirus, picornavirus, poxvirus, togavirus viral vector. See
generally, Jolly, Cancer Gene Therapy 1:51-64 (1994); Kimura, Human
Gene Therapy 5:845-852 (1994), Connelly, Human Gene Therapy
6:185-193 (1995), and Kaplitt, Nature Genetics 6:148-153
(1994).
[0055] Retroviral vectors are well known in the art and we
contemplate that any retroviral gene therapy vector is employable
in the invention, including B, C and D type retroviruses,
xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1
(see O'Neill, J. Vir. 53:160, 1985) polytropic retroviruses (for
example, MCF and MCF-MLV (see Kelly, J. Vir. 45:291, 1983),
spumaviruses and lentiviruses. See RNA Tumor Viruses, Second
Edition, Cold Spring Harbor Laboratory, 1985.
[0056] Portions of the retroviral gene therapy vector may be
derived from different retroviruses. For example, retrovector LTRs
may be derived from a Murine Sarcoma Virus, a tRNA binding site
from a Rous Sarcoma Virus, a packaging signal from a Murine
Leukemia Virus, and an origin of second strand synthesis from an
Avian Leukosis Virus.
[0057] These recombinant retroviral vectors may be used to generate
transduction competent retroviral vector particles by introducing
them into appropriate packaging cell lines (see U.S. Ser. No.
07/800,921, filed Nov. 29, 1991). Retrovirus vectors can be
constructed for site-specific integration into host cell DNA by
incorporation of a chimeric integrase enzyme into the retroviral
particle. See, U.S. Ser. No. 08/445,466 filed May 22, 1995. It is
preferable that the recombinant viral vector is a replication
defective recombinant virus.
[0058] Packaging cell lines suitable for use with the
above-described retrovirus vectors are well known in the art, are
readily prepared (see U.S. Ser. No. 08/240,030, filed May 9, 1994;
see also WO 92/05266), and can be used to create producer cell
lines (also termed vector cell lines or "VCLs") for the production
of recombinant vector particles. Preferably, the packaging cell
lines are made from human parent cells (e.g., HT1080 cells) or mink
parent cell lines, which eliminates inactivation in human
serum.
[0059] Preferred retroviruses for the construction of retroviral
gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia,
Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus,
Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma
Virus. Particularly preferred Murine Leukemia Viruses include 4070A
and 1504A (Hartley and Rowe, J. Virol. 19:19-25, 1976), Abelson
(ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC
No. VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.
VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such
retroviruses may be obtained from depositories or collections such
as the American Type Culture Collection ("ATCC") in Rockville,
Maryland or isolated from known sources using commonly available
techniques.
[0060] Exemplary known retroviral gene therapy vectors employable
in this invention include those described in GB 2200651; EP No.
415,731; EP No. 345,242; PCT Publication Nos. WO 89/02468, WO
89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 90/07936, WO
94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, and
WO 91/02805, in U.S. Pat. Nos. 5,219,740, 4,405,712, 4,861,719,
4,980,289 and 4,777,127, in U.S. Ser. No. 07/800,921 and in Vile,
Cancer Res. 53:3860-3864 (1993); Vile, Cancer Res 53:962-967
(1993); Ram, Cancer Res 53:83-88 (1993); Takamiya, J. Neurosci.
Res. 33:493-503 (1992); Baba, J Neurosurg 79:729-735 (1993); Mann,
Cell 33:153 (1983); Cane, Proc Natl Acad Sci 81:6349 (1984) and
Miller, Human Gene Therapy 1 (1990).
[0061] Human adenoviral gene therapy vectors are also known in the
art and employable in this invention. See, for example, Berkner,
Biotechniques 6:616 (1988), and Rosenfeld, Science 252:431 (1991),
and PCT Patent Publication Nos. WO 93/07283, WO 93/06223, and WO
93/07282. Exemplary known adenoviral gene therapy vectors
employable in this invention include those described in the
above-referenced documents and in PCT Patent Publication Nos. WO
94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO
95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO
94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO
95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO
95/25807, WO 95/05835, WO 94/18922 and WO 95/09654. Alternatively,
administration of DNA linked to killed adenovirus as described in
Curiel, Hum. Gene Ther. 3:147-154 (1992) may be employed.
[0062] The gene delivery vehicles of the invention also include
adenovirus associated virus (AAV) vectors. Leading and preferred
examples of such vectors for use in this invention are the AAV-2
basal vectors disclosed in Srivastava, PCT Patent Publication No.
WO 93/09239. Most preferred AAV vectors comprise the two AAV
inverted terminal repeats in which the native Dsequences are
modified by substitution of nucleotides, such that at least 5
native nucleotides and up to 18 native nucleotides, preferably at
least 10 native nucleotides up to 18 native nucleotides, most
preferably 10 native nucleotides are retained and the remaining
nucleotides of the Dsequence are deleted or replaced with
non-native nucleotides. The native D-sequences of the AAV inverted
terminal repeats are sequences of 20 consecutive nucleotides in
each AAV inverted terminal repeat (i.e., there is one sequence at
each end) which are not involved in HP formation. The non-native
replacement nucleotide may be any nucleotide other than the
nucleotide found in the native D-sequence in the same position.
Other employable exemplary AAV vectors are pWP-19, pWN-1, both of
which are disclosed in Nahreini, Gene 124:257-262 (1993). Another
example of such an AAV vector is psub201. See Samulski, J. Virol.
61:3096 (1987). Another exemplary AAV vector is the Double-D ITR
vector. How to make the Double D ITR vector is disclosed in U.S.
Pat. No. 5,478,745. Still other vectors are those disclosed in
Carter, U.S. Pat. No. 4,797,368 and Muzyczka, U.S. Pat. No.
5,139,941, Chartejee, U.S. Pat. No. 5,474,935, and Kotin, PCT
Patent Publication No. WO 94/288157. Yet a further example of an
AAV vector employable in this invention is SSV9AFABTKneo, which
contains the AFP enhance and albumin promoter and directs
expression predominantly in the liver. Its structure and how to
make it are disclosed in Su, Human Gene Therapy 7:463-470 (1996).
Additional AAV gene therapy vectors are described in U.S. Pat. Nos.
5,354,678; 5,173,414; 5,139,941; and 5,252,479.
[0063] The gene therapy vectors of the invention also include
herpes vectors. Leading and preferred examples are herpes simplex
virus vectors containing a sequence encoding a thymidine kinase
polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and
EP No. 176,170 (Roizman). Additional exemplary herpes simplex virus
vectors include HFEM/ICP6LacZ disclosed in PCT Patent No. WO
95/04139 (Wistar Institute), pHSVlac described in Geller, Science
241:1667-1669 (1988) and in PCT Patent Publication Nos. WO 90/09441
and WO 92/07945, HSV Us3:pgC-lacZ described in Fink, Human Gene
Therapy 3:11-19 (1992) and HSV 7134, 2 RH 105 and GAL4 described in
EP No. 453,242 (Breakefield), and those deposited with the ATCC as
accession numbers ATCC VR-977 and ATCC VR-260.
[0064] Alpha virus gene therapy vectors may be employed in this
invention. Preferred alpha virus vectors are Sindbis viruses
vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC
VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC
VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC
VR923; ATCC VR-1250; ATCC VR1249; ATCC VR-532), and those described
U.S. Pat. Nos. 5,091,309 and 5,217,879, and PCT Patent Publication
No. WO 92/10578. More particularly, those alpha virus vectors
described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, and
U.S. Ser. No. 08/198,450 and in PCT Patent Publication Nos. WO
94/21792, WO 92/10578, and WO 95/07994, and U.S. Pat. Nos.
5,091,309 and 5,217,879 are employable. Such alpha viruses may be
obtained from depositories or collections such as the ATCC in
Rockville, Maryland or isolated from known sources using commonly
available techniques. Preferably, alphavirus vectors with reduced
cytotoxicity are used (see co-owned U.S. Ser. No. 08/679640).
[0065] DNA vector systems such as eukaryotic layered expression
systems are also useful for expressing the nucleic acids of the
invention. See PCT Patent Publication No. WO 95/07994 for a
detailed description of eukaryotic layered expression systems.
Preferably, the eukaryotic layered expression systems of the
invention are derived from alphavirus vectors and most preferably
from Sindbis viral vectors.
[0066] Other viral vectors suitable for use in the present
invention include those derived from poliovirus, for example ATCC
VR-58 and those described in Evans, Nature 339:385 (1989), and
Sabin, J. Biol. Standardization 1:115 (1973); rhinovirus, for
example ATCC VR-11 0 and those described in Arnold, J Cell Biochem
(1990) L401; pox viruses such as canary pox virus or vaccinia
virus, for example ATCC VR-111 and ATCC VR-2010 and those described
in FisherHoch, Proc Natl Acad Sci 86(1989) 317, Flexner, Ann NY
Acad Sci 569:86 (1989), Flexner, Vaccine 8:17(1990); in U.S. Pat.
Nos. 4,603,112 and 4,769,330 and in WO 89/01973; SV40 virus, for
example ATCC VR-305 and those described in Mulligan, Nature 277:108
(1979) and Madzak, J Gen Vir 73:1533 (1992); influenza virus, for
example ATCC VR-797 and recombinant influenza viruses made
employing reverse genetics techniques as described in U.S. Pat. No.
5,166,057 and in Enami, Proc. Natl. Acad. Sci. 87:3802-3805 (1990);
Enami and Palese, J. Virol. 65:2711-2713 (1991); and Luytjes, Cell
59:110(1989), (see also McMicheal., New England J. Med. 309:13
(1983), and Yap, Nature 273:238 (1978) and Nature 277:108, 1979);
human immunodeficiency virus as described in EP No. 386,882 and in
Buchschacher, J. Vir. 66:2731(1992); measles virus, for example,
ATCC VR-67 and VR-1247 and those described in EP No. 440,219; Aura
virus, for example, ATCC VR-368; Bebaru virus, for example, ATCC
VR-600 and ATCC VR-1240; Cabassou virus, for example, ATCC VR-922;
Chikungunya virus, for example, ATCC VR-64 and ATCC VR-1241; Fort
Morgan Virus, for example, ATCC VR-924; Getah virus, for example,
ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example, ATCC
VR-927; Mayaro virus, for example, ATCC VR-66; Mucambo virus, for
example, ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example,
ATCC VR-371; Pixuna virus, for example, ATCC VR-372 and ATCC
VR-1245; Tonate virus, for example, ATCC VR-925; Triniti virus, for
example ATCC VR-469; Una virus, for example, ATCC VR-374; Whataroa
virus, for example ATCC VR-926; Y-62-33 virus, for example, ATCC
VR-375; O'Nyong virus, Eastern encephalitis virus, for example,
ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for
example, ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252;
and coronavirus, for example, ATCC VR-740 and those described in
Hamre, Proc. Soc. Exp. Biol. Med. 121:190 (1966).
[0067] Delivery of the compositions of this invention into cells is
not limited to the above mentioned viral vectors. Other delivery
methods and media may be employed such as, for example, nucleic
acid expression vectors, polycationic condensed DNA linked or
unlinked to killed adenovirus alone, for example see U.S. Ser. No.
08/366,787, filed Dec. 30, 1994, and Curiel, Hum Gene Ther
3:147-154 (1992) ligand linked DNA, for example, see Wu, J. Biol.
Chem. 264:16985-16987 (1989), eucaryotic cell delivery vehicles
cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994,
and U.S. Ser. No. 08/404,796, deposition of photopolymerized
hydrogel materials, hand-held gene transfer particle gun, as
described in U.S. Pat. No. 5,149,655, ionizing radiation as
described in U.S. Pat. No. 5,206,152 and in PCT Patent Publication
No. WO 92/11033, nucleic charge neutralization or fusion with cell
membranes. Additional approaches are described in Philip, Mol.
Cell. Biol. 14:2411-2418 (1994) and in Woffendin, Proc. Natl. Acad.
Sci. 91:1581-585 (1994).
[0068] Particle mediated gene transfer may be employed, for example
see U.S. provisional application No. 60/023,867. Briefly, the
sequence can be inserted into conventional vectors that contain
conventional control sequences for high level expression, and then
be incubated with synthetic gene transfer molecules such as
polymeric DNA-binding cations like polylysine, protamine, and
albumin, linked to cell targeting ligands such as
asialoorosomucoid, as described in Wu and Wu, J. Biol. Chem.
262:4429-4432 (1987), insulin as described in Hucked, Biochem.
Pharmacol. 40:253-263 (1990), galactose as described in Plank,
Bioconjugate Chem 3:533-539 (1992), lactose or transferrin.
[0069] Naked DNA may also be employed. Exemplary naked DNA
introduction methods are described in PCT Patent Publication No. WO
90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be
improved using biodegradable latex beads. DNA coated latex beads
are efficiently transported into cells after endocytosis initiation
by the beads. The method may be improved further by treatment of
the beads to increase hydrophobicity and thereby facilitate
disruption of the endosome and release of the DNA into the
cytoplasm.
[0070] Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120, PCT Patent Publication Nos.
WO 95/13796, WO 94/23697, and WO 91/144445, and EP No. 524,968. As
described in co-owned U.S. provisional application No. 60/023,867,
on non-viral delivery, the nucleic acid sequences can be inserted
into conventional vectors that contain conventional control
sequences for high level expression, and then be incubated with
synthetic gene transfer molecules such as polymeric DNA-binding
cations like polylysine, protamine, and albumin, linked to cell
targeting ligands such as asialoorosomucoid, insulin, galactose,
lactose, or transferrin. Other delivery systems include the use of
liposomes to encapsulate DNA comprising the gene under the control
of a variety of tissue-specific or ubiquitously-active promoters.
Further non-viral delivery suitable for use includes mechanical
delivery systems such as the approach described in Woffendin et
al., Proc. Natl. Acad. Sci. USA 91(24): 11581-11585 (1994).
Moreover, the coding sequence and the product of expression of such
can be delivered through deposition of photopolymerized hydrogel
materials. Other conventional methods for gene delivery that can be
used for delivery of the coding sequence include, for example, use
of hand-held gene transfer particle gun, as described in U.S. Pat.
No. 5,149,655; use of ionizing radiation for activating transferred
gene, as described in U.S. Pat. No. 5,206,152 and PCT Patent
Publication No. WO 92/11033.
[0071] Exemplary liposome and polycationic gene delivery vehicles
are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915, in
PCT Patent Publication Nos. WO 95/13796, WO 94/23697, and WO
91/14445, in EP No. 524,968 and in Stryer, Biochemistry, pages
236-240 (1975) W.H. Freeman, San Francisco, Szoka, Biochem.
Biophys. Acta. 600:1 (1980); Bayer, Biochem. Biophys. Acta. 550:464
(1979); Rivnay, Meth. Enzymol. 149:119 (1987); Wang, Proc. Natl.
Acad. Sci. 84:7851 (1987); and Plant, Anal. Biochem. 176:420
(1989).
[0072] Administration of the Phosipholipids
[0073] The phospholipids of the invention can be used to treat cell
death in humans or other mammalian patients by contacting the a
pharmaceutical composition containing the phospholipids with a cell
that has experienced trauma, for example a trauma from a heart
attack or a stroke.
[0074] In a therapeutic context, the phospholipids of the invention
can be administered as described in Franke et al,
[0075] Science 275:665-668 (1997), for example by placing the
phospholipid containing vesicles in contact with cells in which
they can be internalized. Therefore administration of the
phospholipids of the invention include all the local and systemic
modes of administration possible.
[0076] Pharmaceutical Compositions and Therapeutic Methods
[0077] The gene delivery vehicles containing the polynucleotides or
phospholipids of the invention can be administered, locally or
systemically to mammals, especially humans or primates, or placed
in direct contact with a cell or population of cells. The
phospholipids and gene therapy vectors can be formulated into
pharmaceutical compositions as described below. The pharmaceutical
compositions comprise gene therapy vectors containing a
polynucleotide of the invention or a phospholipid made by the
method of the invention in a pharmaceutically acceptable carrier or
diluent.
[0078] Suitable carriers may be large, slowly metabolized
macromolecules such as proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers,
and inactive viruses in particles. Such carriers are well known to
those of ordinary skill in the art. Pharmaceutically acceptable
salts can be used therein, for example, mineral acid salts such as
hydrochlorides, hydrobromides, phosphates, sulfates, and the like;
and the salts of organic acids such as acetates, propionates,
malonates, benzoates, and the like. A thorough discussion of
pharmaceutically acceptable excipients is available in REMINGTON's
PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).
Pharmaceutically acceptable carriers in therapeutic compositions
may contain liquids such as water, saline, glycerol and ethanol.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles. Typically, the therapeutic compositions are prepared
as injectables, either as liquid solutions or suspensions; solid
forms suitable for solution in, or suspension in, liquid vehicles
prior to injection may also be prepared. Liposomes are included
within the definition of a pharmaceutically acceptable carrier.
[0079] A "therapeutically effective amount" is that amount of any
of the pharmaceutical compositions that are administered will be
that amount sufficient to generate the desired therapeutic
outcome.
[0080] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent that does not
itself induce the production of antibodies harmful to the
individual receiving the composition, and which may be administered
without undue toxicity. Pharmaceutical compositions can include a
recombinant viral vector as described above, in combination with a
pharmaceutically acceptable carrier or diluent. Such pharmaceutical
compositions may be prepared either as a liquid solution, or as a
solid form (e.g., lyophilized) which is suspended in a solution
prior to administration. In addition, the composition may be
prepared with suitable carriers or diluents for either surface
administration, injection, oral, or rectal administration.
Pharmaceutically acceptable carriers or diluents are nontoxic to
recipients at the dosages and concentrations employed.
Representative examples of carriers or diluents for injectable
solutions include water, isotonic saline solutions which are
preferably buffered at a physiological pH (such as
phosphate-buffered saline or Tris-buffered saline), mannitol,
dextrose, glycerol, and ethanol, as well as polypeptides or
proteins such as human serum albumin. A particularly preferred
composition comprises a vector or recombinant virus in 10 mg/ml
mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl. In this
case, since the recombinant vector represents approximately 1 mg of
material, it may be less than 1% of high molecular weight material,
and less than 1/100,000 of the total material (including water).
This composition is stable at -70.vertline.C. for at least six
months.
[0081] Pharmaceutical compositions of the present invention may
also additionally include factors that stimulate cell division, and
hence, uptake and incorporation of a recombinant retroviral vector.
Preserving recombinant viruses is described in US applications
entitled "Methods for Preserving Recombinant Viruses" (U.S. Ser.
No. 08/135,938, filed Oct. 12, 1993) which is incorporated herein
by reference in full.
[0082] The pharmaceutically acceptable carrier or diluent may be
combined with the gene delivery vehicles of with the phospholipids
to provide a composition either as a liquid solution, or as a solid
form (e.g., lyophilized) which can be resuspended in a solution
prior to administration. The two or more gene delivery vehicles are
typically administered via traditional direct routes, such as
buccal/sublingual, rectal, oral, nasal, topical, (such as
transdermal and ophthalmic), vaginal, pulmonary, intraarterial,
intramuscular, intraperitoneal, subcutaneous, intraocular,
intranasal or intravenous, or indirectly.
[0083] Any therapeutic of the invention, including, for example,
polynucleotides for expression in the mammal or phospholipids, can
be formulated into an enteric coated tablet or gel capsule
according to known methods in the art. These are described in the
following patents: U.S. Pat. No. 4,853,230, EP No. 225,189, AU
9,224,296, AU 9,230,801, and WO 92144,52. Such a capsule is
administered orally to be targeted to the jejunum. At 1 to 4 days
following oral administration expression of the polypeptide, or
inhibition of expression by, for example a ribozyme or an antisense
oligonucleotide, is measured in the plasma and blood, for example
by antibodies to the expressed or non-expressed proteins.
[0084] Administration can be accomplished by any means appropriate
for the therapeutic agent, for example, by parenteral or oral
delivery. The parenteral delivery can be subcutaneous, intravenous,
intramuscular, intra-arterial, injection into the tissue of an
organ, mucosal, pulmonary, topical, or catheter-based. Oral means
is by mouth, including pills or other gastroenteric delivery means,
including a drinkable liquid. Mucosal delivery can include, for
example, intranasal delivery. Pulmonary delivery can include
inhalation of the agent. Catheter-based delivery can include
delivery by iontophoretic catheter-based delivery. Administration
will generally also include delivery with a pharmaceutically
acceptable carrier, such as a buffer, a polypeptide, a peptide, a
polysaccharide conjugate, a liposome, and a lipid. A gene therapy
protocol is considered an administration in which the therapeutic
agent is a polynucleotide capable of accomplishing a therapeutic
goal when expressed as a transcript or a polypeptide in the mammal,
and can be applied to both parenteral and oral delivery means. Such
administration means will be selected as appropriate for the
disease being treated. For example, where the disease is
organ-based, delivery may be local, and for example, where the
disease is systemic, the delivery may be systemic.
[0085] The term "in vivo administration" refers to administration
to a patient, for example a mammal, of a polynucleotide encoding a
polypeptide for expression in the mammal. In particular, direct in
vivo administration involves transfecting a mammal's cell with a
coding sequence without removing the cell from the mammal. Thus,
direct in vivo administration may include direct injection of the
DNA encoding the polypeptide of interest in the region afflicted by
trauma, or to the region where glucose uptake is regulated.
[0086] The term "ex vivo administration" refers to transfecting a
cell, for example, a cell from a population of cells that are
deficient in their normal function of glucose-uptake, after the
cell is removed from the patient. After transfection the cell is
then replaced in the patient. Ex vivo administration can be
accomplished by removing cells, transforming them with a
polynucleotide of the invention, including also a regulatory region
for facilitating the expression, and placing the transformed cells
back into the patient for expression.
[0087] The gene delivery vehicle or phospholipid can be introduced
into a population of cells or a mammal, for example, by injection,
particle gun, topical administration, parental administration,
inhalation, or iontophoretic delivery, as described in U.S. Pat.
Nos. 4,411,648; 5,222,936; and 5,286,254; and PCT Patent
Publication No. WO 94/05369.
[0088] The gene delivery vehicle may be administered at single or
multiple sites to a mammal directly, for example by direct
injection, or alternatively, through the use of target cells
transduced ex vivo. The present invention also provides
pharmaceutical compositions (including, for example, various
exipients) suitable for administering the gene delivery vehicles.
Within the context of the present invention, it should be
understood that the removed cells may be returned to the same
animal, or to another allogenic animal or mammal. In such a case it
is generally preferable to have histocompatibility matched animals
(although not always, see, e.g., Yamamoto et al., "Efficacy of
Experimental FIV Vaccines," 1st International Conference of FIV
Researchers, University of California at Davis, September,
1991.
[0089] The multiple gene delivery vehicles or phospholipids may be
administered to animals, plants, or to a population of cells. In
preferred embodiments, the animal is a warm-blooded animal, further
preferably selected from the group consisting of mice, chickens,
cattle, pigs, pets such as cats and dogs, horses, and humans.
[0090] For polynucleotide therapeutics, depending on the expression
of the polynucleotide in the target cell, vectors containing
expressable constructs of coding sequences, or non-coding sequences
can be administered in a range of about 100 ng to about 200 mg of
DNA for local administration in a gene therapy protocol, also about
500 ng to about 50 mg, also about 1 ug to about 2 mg of DNA, about
5 ug of DNA to about 500 ug of DNA, and about 20 ug to about 100 ug
during a local administration in a gene therapy protocol, and for
example, a dosage of about 500 ug, per injection or administration.
Where greater expression is desired, over a larger area of tissue,
larger amounts of DNA or the same amounts readministered in a
successive protocol of administrations, or several administrations
to different adjacent or close tissue portions of for example, a
tumor site, may be required to effect a positive therapeutic
outcome.
[0091] Phospholipid therapeutic agents can be administered in
dosage effective for the amount of cells targeted, in such
quantities as analogously appropriate to the amounts of
phospholipids effective as described in Franke et al., Science
275:665-668 (1997).
[0092] Administration of a gene delivery vehicle having a
polynucleotide of the invention for preventing or reducing cell
death, or administration of a 3' phosphorylated phospholipid, or a
vesicle containing such, for the purpose of preventing or reducing
cell death, can be made directly to a putative site of trauma, or
can be made systemically, but the vehicle or vesicle can be
targeted specifically to the putative site of trauma.
Administration of a polynucleotide of the invention can also be
made directly to cells exhibiting insulin resistance, such as for
example liver cells, or other cells expected under normal
conditions to be responsive to insulin.
[0093] In all cases, routine experimentation in clinical trials
will determine specific ranges for optimal therapeutic effect, for
each therapeutic, each administrative protocol, and administration
to specific mammals will also be adjusted to within effective and
safe ranges depending on the mammal condition and responsiveness to
initial administrations.
[0094] Further objects, features, and advantages of the present
invention will become apparent from the detailed description. It
should be understood, however, that the detailed description, while
indicating preferred embodiments of the invention, is given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description. Also,
the invention is not limited by any theories of mechanism of the
method of the invention.
EXAMPLE 1
Construction of Growth Factor Independent Membrane Targeted PI
3-Kinase Mutants
[0095] Growth factor independent membrane targeted p110 derived PI
3-kinase mutants were constructed by the addition of heterologous
membrane targeting signals to the polynucleotide sequences encoding
either a p110 subunit, p110* or any other p110 derived variant of
PI 3-kinase. p110* can be constructed, for example, as described in
Hu et al, Science 268: 100-102 (1995), and consists of the basic
functional elements of the PI 3-kinase subunits, providing for the
iSH2 domain of p85 attached to a p110 subunit.
[0096] As depicted schematically in FIG. 1, p110 constructions were
tagged either at the N- or C-terminal end with the Myc epitope
depicted by the oval. The iSH2 fragment of p85 contained a
C-terminal influenza virus hemagglutinin (HA) epitope tag depicted
by the diamond. The p110 region with homology to the catalytic
domain of protein kinases is depicted by a box labeled "kinase".
The domain responsible for the interaction with the iSH2 domain of
the p85 subunit is shown as a small box at the p110 N-terminus.
p110 .DELTA.kin is a kinase-deficient p 110, in which the arginine
at position 802 was mutated to a lysine residue as indicated by an
asterik within the catalytic domain. The iSH2 domain of p85 that is
required for catalytic activity is represented by a hatched bar.
The first and last amino acids of fragments are numbered with
respect to their position in the wt p85 or p110 sequence. p110* is
a constitutively active chimera that contains the iSH2 domain of
p85 attached to the N-terminus of p110 via a flexible "glycine
linker" as described in Hu et al., Science 268:100-102 (1995). The
p110*.DELTA.kin is the kinase-deficient version of p110*. Myr-P110
and Myr-P110* as well as their kinase deficient versions were
modified at their respective N-terminal ends with the
myristoylation sequence of pp60 c-Src as described in Kaplan et
al., PNAS USA 83: 3624-8 (1990), Schultz et al., Science 227:427-9
(1985), and Deichaite et al, Mol. Cell Biol 8:4295-301 (1988).. The
C-terminal ends of p110-H and p110*-H and their kinase-deficient
versions were extended by the farnesylation and palmitoylation
sequences of H-Ras, as described by Cadwallader et al., Mol. Cell
Biol. 14:4722-30 (1994). Similarly, polynucleotide sequences
containing the farnesylation sequence and polybasic stretch of
K-Ras were generated as described in Cadwallader et al., Mol. Cell
Biol. 14:4722-30 (1994).
[0097] The p110 and p110* variants that were constructed for this
example were, M-p110 and M-p110* which contains the N-terminal
myristoylation sequence of phosphoprotein 60 (pp60) c-Src and a
C-terminal Myc epitope tag, p110-H and p110-K, and p110*-H and
p110*-K that carry an N-terminal Myc epitope-tag. pp110-H and
p110*-H have a C-terminal farnesylation and palmitoylation signal
which sequences are derived from H-Ras, and p110-K and p110*-K have
a C terminal farnesylation signal and a polybasic sequence, which
sequences are derived from K-Ras. These variants were constructed
by N-terminus and C-terminus modifications of polynucleotides
encoding polypeptides of p110* and p110. The p110* or p110 mutants
were modified at the N-terminus by the pp60 c-Src myristoylation
sequence as described in Kaplan et al., Mol. Cell. Biol. 10: 1000-9
(1990), Schultz, et al., Science 227:427-9 (1985), and Deichaite et
al., Mol. Cell. Biol. 8:4295-301 (1988), using primers Src-M-sense
(5' C ATG GGG AGC AGC AAG AGC AAG CCC AAG GAC CCC AGC CAG CGC GGG
GGA CA 3) SEQ ID NO. 12, and SrcM antisense (5' TAT GTC CCC CGC GCT
GGC TGG GGT CCT TGG TCG TCT TGC TGC TCC C 3') SEQ ID NO. 11 flanked
by NcoI and NdeI restriction sites where A at position 2 is the
cSrc start codon. The annealed DNA fragment was attached in frame
via the respective restriction sites to the N-terminus of a
Myc-tagged p110 cDNA constructed as described in Klippel et al.,
Mol. Cell. Biol. 14:2675-2685 (1994) into a mammalian expression
vector that directs expression from the SR.alpha. promoter as
described in Takabe et al., Mol. Cell Biol. 8:466-72 (1988).
[0098] To modify the C-terminal end of any p110 subunit derived
mutant with the H-Ras farnesylation and palmitoylation signals or
the K-Ras famesylation signals and polybasic sequences, as
described in Cadwallader et al., Mol. Cell. Biol. 14:4722-30
(1994), a C-terminal fragment of the p110 cDNA was amplified using
primer p110- 3' HindIII (5' CTG AGC AAG AAG CTT TGG 3'), SEQ ID NO.
10, consisting of nucleotides 3092 to 3109 of the coding strand
overlapping a HindIII site, and a primer p110-H (5' GGA TCC TCA GCT
CAG CAC GCA CTT GCA GCT CAT GCA GCC GGG GCC GCT GCT GGC GCC CCC GAG
CTCGTT CAA AGC ATG CTG 3') SEQ ID NO. 9 where the underlined
portion indicates nucleotides that are changed with respect to the
wild-type sequence, overlapping nucleotides 3109 to 3204 of the
noncoding strand, where A of the start codon is designated
nucleotide 1. This extended the p110 C-terminal end by a sequence
encoding amino-acids DLGGA (SEQ ID No. 3) as a hinge region
containing overlapping restriction site for SacI and Ecl136II, and
KasI and NarI, which precedes the coding region for the H-Ras CAAX
box, a stop-codon, and a BamHI restriction site. The C-terminal end
of p110 was modified with K-Ras farnesylation sequence plus a
polybasic region as described in Cadwallader et al, Mol. Cell.
Biol. 14: 4722-30 (1994), by PCR using primer p110 3' HindIII and
primer p110-K (5' GCA TTC TCA CAT GAT CAC GCA CTT GGT CTT GGA CTT
CTT CTT CTT CTT TTT GCC ATC TTT GGA GGC GCC GAG CTCGTT CAA AGC ATC
CTG 3'), SEQ ID NO. 8. This extended the p110 C-terminal end by a
sequence encoding amino-acids DLGGA (SEQ ID No. 3) as a hinge
region containing overlapping restriction sites for SacI and
Ecl136II, and KasI and NarI, which precedes the coding region for
the K-Ras farnesylation and polylysine sequence, a stop-codon, and
a BamHI restriction site. The Myc-tagged C-terminal end of p 110,
constructed as described in Klippel et al., Mol. Cell. Biol.
14:2675-2685 (1994) was exchanged against the H-Ras or K-Ras
CAAX-box modified sequences using HindIII and BamHI. For the
C-terminal farnesylated p110 constructs, the N-terminal end of the
p110 coding region was modified with the 10-amino acid Myc epitope
consisting of EQKLISEEDL, SEQ ID NO. 7, as described in Evan et
al., Mol. Cell Biol. 5:3610-6 (1985), using primer p110 5' Myc
sense (5' CT AGA ATG GAT GAG CAG AAG CTG ATT TCC GAG GAG GAC CTG
AAC GGG GGA CA 3') SEQ ID NO. 6, and primer p110 5'Myc-antisense
(5' T ATG TCC CCC GTT CAG GTC CTC CTC GGA AAT CAG CTT CTG CTC ATC
CAT T 3'), SEQ ID NO. 5, flanked by restriction sites for XbaI and
NdeI. The Myc-coding region was attached in frame to the wild-type
p110 N-terminus by ligating the annealed oligonucleotide via
XbaI-NdeI ends into pCG-P110 as described in Klippel et al., Mol.
Cell. Biol. 14:2675-2685 (1994).
[0099] Kinase deficient control mutants, called generically
p110.DELTA.kin, were constructed by changing a lysine at position
802 to an arginine residue, which alteration was accomplished by
site-directed mutagenesis using the gapped duplex DNA method as
described in Stanssens et al, Nuc. Acids Res. 17:4441-54 (1989)
with primer p110-KR802 (5' C GTC GCC ATT TCT AAA GAT GAT CTC 3'),
SEQ ID NO. 4, where the underlined C indicates the point of
mutation, and annealing to nucleotides 2392 to 3016 of the p110
coding region. The correct sequence of the p110 fragments modified
by PCR or oligonucleotides was confirmed by DNA sequence
analysis.
[0100] The N-terminal myristoylation or C-terminal H-Ras
farnesylation or K-Ras palmitoylation sequences were furthermore
used to modify the coding regions for p110.DELTA.kin, p110*, and
p110*.DELTA.kin by using restriction described above for p110. For
expression of p110 molecules in COS-7 cells, the respective DNA
fragments were cloned into mammalian expression vector pCG via
XbaI-BamHI ends as described in Klippel et al., Mol. Cell. Biol.
14: 2675-2685 (1994). Plasmid pCG is a derivative of vectors
described by Matthias et al, Nuc. Acids Res. 17:6418 (1989), and
directs expression in mammalian cells from the human
cytomegalovirus promoter/enhancer region. COS-7 cells were obtained
from the American Type Culture Collection and cultured at
37.degree. C. in Dulbecco's modified Eagle medium containing 10%
bovine calf serum, and penicillin at a concentration of 50 .mu.g/ml
and streptomycin at a concentration of 50 .mu.g/ml.
EXAMPLE 2
Transient Expression of Recombinant p110 Derivatives in COS Cells
and the Intracellular Distribution of p110 Molecules After
Expression
[0101] COS-7 cells that were 60% to 70% confluent on a 10 cm plate
were transfected with mammalian expression vectors using the
DEAE-dextran method as described in Gorman, Glover ed. DNA Cloning:
A Practical Approach, v.II, IRL Press, Oxford, p.143-190 (1985).
The cells were starved for at least 30 hours in medium containing
0.5% dialyzed fetal bovine calf serum and then treated with or
without platelet-derived growth factor hormone (PDGF) at a
concentration of 2 nM for 10 minutes at 37.degree. C. The COS07
cells were washed twice with cold phosphate-buffered saline and
lysed at 4.degree. C. in a mammalian cell lysis buffer containing
the following: 20 mM Tris at pH 7.5, 137 mM NaCl, 15% v/v glycerol,
1% v/v Triton X-100, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride,
10 mg aprotinin per ml, 20 mM leupeptin, 2 mM benzamide, 1 mM
sodium vanadate, 25 mM .beta.-glycerolphosphate, 50 mM NaF and 10
mM NaPpi. The lysates were cleared by centrifugation at
14,000.times.g for 5 minutes and aliquots of the lysates were
analyzed for protein expression and enzyme activity.
[0102] To investigate the intracellular distribution of p 110
molecules, hypotonic lysates were prepared as described in
Cadwallader et al, Mol. Cell Biol. 14:4722-30 (1994), including
that COS-7 cells were scraped in ice-cold PBS into microfuge tubes
and collected at 400.times.g for 2 minutes. The cells were lysed by
Dounce homogenization on ice in 500 .mu.l of 10 mM
N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) pH 7.5,
10 mM Kcl, 1.5 MgCl.sub.2, 0.3 mM ethylene glycol-bis
(.beta.-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), 2
mM phenylmethylsulfonyl fluoride, 10 mg aprotinin per ml, 20 mM
leupeptin, 2 mM benzamidine, 1 mM sodium vanadate, 25 mM
.beta.-glycerolphosphate, 50 mM NaF and 10 mM NaPPi for 10 minutes.
After removal of the nuclei and unbroken cells at centrifugation at
1,500.times.g for 5 minutes, the membranes were pelleted for 30
minutes at 120,000.times.g in a TLA 120.2 rotor made by Beckman
Instruments, Palo Alto, Calif. The supernatant called S 100 and the
pellet called P100 fractions were collected and equal proportions
were analyzed for protein distribution by immunoblotting with
antibodies specific for the tagged proteins.
[0103] Immunoblotting for purposes of determining the protein
distribution was performed by boiling immunoprecipitates in
Lammli-sample buffer, separating the immunoprecipitates from the
unprecipitated proteins by SDS-PAGE and by transferring the
immunoprecipitates to nitrocellulose filters. The filters were
blocked in TBST buffer composed of 10 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 0.5% v/v Tween 20, and 0.5% w/v sodium azide containing 5%
w/v dried milk. The respective antibodies were added in TBST at
appropriate dilutions. Bound antibody was detected with anti-mouse
or anti-rabbit conjugated to alkaline phosphatase made by Promega
Corporation, Madison, Wis., in TBST, washed, and developed with
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate also
by Promega. Alternatively, horseradish peroxidase conjugated
anti-mouse antibody was used and developed by enhanced
chemiluminescence made by Amersham Life Sciences, located at
Arlington Heights, Ill.
EXAMPLE 3
In Vitro Protein Kinase Assays and Determination of PI 3-kinase
Activity in p110 Precipitates
[0104] Cell lysates containing HA-tagged pp70S6 kinase, Akt-kinase,
MAP-kinase or cJun terminal kinase (JNK) were incubated with
monoclonal anti-HA antibody 12CA5 for 1 hour at 4.degree. C.
Protein A-Sepharose beads (Sigma, St. Louis, Mo.) were used to
precipitate the immune complexes. The beads were washed with 50 mM
Tris-HCl (pH 7.5), 0.5 M LiCl, 0.5% v/v Triton X-100, twice with
PBS and once with 10 mM Tris-HCI (pH 7.5), 10 mM MgCi.sub.2, 1 mM
dithiothreitol, all containing 0.1 mM sodium vanadate and 20 mM
.beta.-glycerolphosphate. For analyzing the immune complexes in an
S6 kinase activity assay the beads were divided in three aliquots:
two aliquots were subjected to a S6 kinase activity assay using
[.gamma.- .sup.32.sub.32P] ATP (5,000 Ci/mmol) based on a peptide
substrate described in Terada et al, J. Biol. Chem. 268: 12062-8
(1993), in 30 .mu.l, one aliquot was analyzed for the amount of
recombinant pp70S6 kinase in the precipitate. After 25 minutes at
22.degree. C. the reaction was stopped by the addition of 10 .mu.l
of 500 mM EDTA. Twenty two .mu.l of the supernatant was applied to
phosphocellulose paper P81 made by Whatman Products, Fisher
Scientific, Pittsburg, Pa., and washed four times in 75 mM
H.sub.3PO.sub.4. The relative amounts of incorporated radioactivity
was determined in a liquid scintillation counter. Specific
phosphorylation of the S6-derived peptide was obtained after
subtracting counts with protein A-Sepharose beads in the absence of
anti-HA-antibody from counts of label incorporated in the presence
of anti-HA-antibody.
[0105] For all the other kinase assays, one-third of the
immunobeads were subjected to an in vitro kinase reaction, and
two-thirds were analyzed for the amount of the respective
recombinant kinase protein. For analyzing Akt kinase activity
histone H2B was used as substrate as described in Franke et aL,
Cell 81:727-36 (1995), according to the reaction conditions
described by Jones et al., PNAS USA 88:4171-5 (1991). JNK-activity
was determined using GST-Jun (amino acids of Jun 1 through 89,
which a slight variation from a standard version that contains
amino acids 1 through 79) as a substrate as described in Derijard
et al., Cell 76:1025-37 (1994). For MAP kinase activation, the
phosphorylation of myelin basic protein (MBP) was analyzed as
described by Ray et al., PNAS USA 85:3753-7 (1988). The in vitro
protein kinase reactions were carried out in 30 .mu.l in the
presence of [.gamma.-.sup.32P] ATP (5,000 Ci/mmol) and incubated at
22.degree. C. for 25 minutes. The reactions were stopped by the
addition of 8 .mu.l Lammli-sample buffer and 22 .mu.l of the
reaction mixtures were analyzed by SDS-PAGE. The relative amounts
of incorporated radioactivity was determined by autoradiography and
quantitated using a Molecular Imager System produced by BioRad,
Richmond, Calif. The complexes were analyzed by immunoblotting with
the indicated antibodies.
[0106] The presence of PI 3-kinase activity in immune complexes was
determined by incubating the beads with 30 mM HEPES, 30 mM
MgCl.sub.2, 50 .mu.M ATP, 200 .mu.M adenosine, 0.2 mg sonicated PI
per ml and 10 .mu.Ci [.gamma.-.sup.32P] ATP (5,000 Ci/mmol) for 20
minutes at 25.degree. C. Adenosine was added to inhibit any
contaminating PI 4-kinase activity as described by Whitman et aL,
Nature 332:644-6 (1988). Reactions were stopped by adding 100 .mu.l
of 1M Hcl and the phospholipids were extracted with 200 .mu.l of a
1:1 mixture of chloroform/methanol. The reaction products were
separated by thin layer chromatography as previously described in
Kaplan et al., PNAS USA 83:3624-8 (1986). The conversion of PI to
PI 3-phosphate was determined by autoradiography.
[0107] In addition, phosphoprotein 70 (pp70) S6 kinase activation
by coexpression of the polynucleotide sequences with pp70 S6 kinase
was demonstrated. The indicated Myc-tagged polynucleotide sequences
were coexpressed with HA-tagged pp70 S6 kinase. p110 kinase or
p110* kinase with either a myristoylation (M) cell membrane
targeting sequence added to the N-terminus, or a famesylation and
palmitoylation sequence (H) cell membrane targeting sequence added
to the C-terminus resulted in increased levels of pp70 S6 kinase
activity. pp70 S6 kinase is a known downstream effector of PI
3-kinase.
EXAMPLE 4
Fly Eye Screen for Inhibitors of PI 3-Kinase Activity
[0108] In this example, p110* expressing Drosophila melanogaster is
created for use as a screen for PI 3-kinase inhibitors. p110*, a
growth factor independent PI 3-kinase mutant is expressed in the
developing eye tissue of the fruit fly Drosophila melanogaster,
using the GMR promoter described in Hay et al., Development
120:2121-9 (1994). The expression of p110* under the control of the
eye specific promoter leads to developmental defects which result
in obvious aberrations in the external morphology of the external
eye tissue. The mutant morphology that results in the transgenic
flies is called "rough" eye. "Rough eye" morphology is a fly eye
with aberrant morphology that is detectable under a dissecting
microscope. These defects may depend on PI 3-kinase activity, as
indicated by a control experiment transforming developing flies
with a PI 3kinase mutant that contains a mutated kinase domain. The
fly eye cells transformed with a catalytically inactive PI 3-kinase
mutant are incapable of eliciting the rough eye morphological
effects of the catalytically active counterpart.
[0109] The results of the transformation of the developing fly
tissue result in ectopic production of R7 photoreceptor cells is
observed in p110* expressing eyes of the Drosophila, which is a
phenotype characteristic of the activation of the Ras signaling
molecule. Thus, as in mammalian cells, p110* appears to activate
Ras in Drosophila tissue and indeed mutations that reduce Ras
activity in Drosophila, reduce the phenotypic effects of p110*.
Even with this information, the invention is not limited to any
theories of mechanism of how the invention works.
[0110] Drosophila embryos are transformed by the method described
in Karess and Rubin, Cell 38:135-146 (1984) with a polynucleotide
construct made up of a p110* coding sequence under the regulatory
control of a GMR promoter. The flies are allowed to develop
normally and are selected by eye morphology for successful
transformants. Successful transformants will have a rough eye
morphology. The transgenic flies are then fed food spiked with an
appropriate dose of a candidate inhibitor. The amount of the
inhibitor will depend on the deduced possible potency of the
molecule as an inhibitor. In this case, the flies are fed different
small molecule inhibitors; a different inhibitor is selected for
each population of transformants. The flies are fed a candidate
inhibitor throughout third instar development during which time
they are observed for reversions of their eye morphology to wild
type or normal. Positives are identified and the inhibitors are
then retested by this assay, or by a kinase or binding assay. This
screening method may also be applied as a secondary or tertiary
screen using candidate inhibitors that have already been found
positive in prior screens such as the kinase or binding assay
screening protocols.
[0111] Alternative screens are conducted by injecting a candidate
inhibitor into the third instar larvae of the transformants that
are then observed for a reversion of the rough eye morphology to
normal.
EXAMPLE 5
Inducible Expression System for PI 3-kinase Mutant p110*
[0112] Expression of inducible p110* was achieved fusing the coding
region for p110* with the GR525 mutant of the regulatory domain of
the mouse estrogen receptor (ER) as described in Littlewood et aL,
Nucleic Acids Research 23:1686-1690 (1995). Activation is regulated
by the addition of 4-hydroxy tamoxifen (4-OHT), a natural breakdown
product of estrogen: in the presence of 4-OHT the p110*-ER chimera
is activated. The respective expression vector was further modified
by addition of the 5' untranslated leader sequence from the Herpes
simplex virus tk-gene to provide for efficient translation
initiation of p110*.
[0113] With the ability to regulate p110* activity it is possible
to generate stable cell lines, since in the uninduced state of the
system background activity of p110* is kept low. Pathways induced
by PI 3-kinase can be studied simply by 4-OHT to the culture
medium. Time-course experiments after induction give information
about the successive order (early/late) and the duration of the
respective responses. An inducible expression system for p110* will
aid in the determination of whether activation of PI 3-kinase is
sufficient for a mitogenic response and/or anchorage-independent
cell growth. Using this system allows us to address several key
questions about the importance of PI 3-kinase activation for the
regulation of cell growth and mitogenesis. After inducing PI
3-kinase activity by 4-OHT we found that activation of PI 3-kinase
is sufficient for a mitogenic response as measured by incorporation
of radiolabeled thymidine (DNA synthesis) and for
anchorage-independent cell growth as analyzed by colony formation
in soft agar.
[0114] Additionally, the 4-OHT-regulatable expression system is
reversible, so that p110* expression can be switched on and off.
Regulatable expression of constitutively active forms of PI
3-kinase is an ideal system to identify genes that are induced in
response to PI 3-kinase activation. This can be achieved either by
subtractive hybridization or by differential display after mRNA
isolation from cells grown in the absence or presence of 4-OHT.
EXAMPLE 6
Production of PI 3-kinase Induced Phospholipids and Their Direct
Activation of Akt
[0115] PI3,4P.sub.2 activates Akt in a defined phospholipid vesicle
system in vitro as described in Klippel et al., Mol. Cell. BioL
16(8):4117-4127 (1996). To optimize reaction conditions
commercially available synthetic PI3P and PI3,4P.sub.2 dipalmitoyl
derivatives were tested for in vitro Akt stimulation. Phospholipid
vesicles were prepared containing dipalmitoylated PI3P or
dipalmitoylated PI3,4P.sub.2, PI4P and phosphatidylserine (PS),
phosphatidylcholine (PC), phosphatidylethanolamine (PE), or
combinations of these as described previously to mimic their
relative concentrations found in cells. The phospholipid vesicles
were preincubated with immobilized Akt protein. The kinase activity
of Akt was analyzed by an in vitro kinase assay using histone H2B
as substrate. In this system PI3,4P.sub.2 in PC vesicles induced an
approximately 2.5 fold increase in kinase activity of Akt. No
activation was observed with PI3,4P.sub.2 in either PE or PS
vesicles. Conditions using PE/PS vesicles that were shown to
promote in vitro activation of Ca.sup.2+-independent PKC isoforms
by PI3,4P.sub.2 and PI3,4,5P.sub.3 did not allow activation of Akt.
The presence of PS in the PI3,4P.sub.2/PC vesicles interfered with
Akt activation, whereas certain concentrations of PE were
tolerated. No stimulation of Akt kinase activity was observed with
vesicles containing PI3P under any conditions. An aliquot of each
immunocomplex was analyzed in parallel for protein levels. No
further increase in Akt activation using synthetic dipalmitoyl
derivatives of PI3,4P.sub.2 at concentrations ranging from 260 nM
to 1.3 .mu.M was observed. Next we established a.system in which
all 3' phosphorylated inositol phospholipids generated by PI
3-kinase could be analyzed in vitro.
[0116] We observed that Akt activation in vitro by a constitutively
active PI 3-kinase is mediated by PI3,4P.sub.2. To generate the
known cellular products of PI 3-kinase, PI3P, PI3,4P.sub.2 and
PI3,4,5P.sub.3, and to compare these products with other
phospholipids for the activation of Akt in vitro, we used the
constitutively active PI 3-kinase, p110*. We have also showed that
p110* and its derivatives exhibit high specific enzymatic
activities in vitro and can efficiently induce signaling events
when expressed in mammalian cells, as described earlier. p110* was
extended at the C-terminus with six histidine residues
(p110*.multidot.6His), expressed in Sf9 insect cells, and purified
on a Ni-chelating column. To generate 3' phosphorylated inositol
phospholipids in vitro purified p110* was incubated in the presence
of ATP with the PI 3-kinase substrates PI, PI4P or PI4,5P.sub.2,
each in vesicles containing phosphatidylcholine (PC). A fraction of
each sample was subjected to phospholipid extraction and analyzed
for production of PI3P, PI3,4P.sub.2 and PI3,4,5P.sub.3.
Approximately 5% to 10% of the substrates were converted into 3'
phosphorylated inositol phospholipids under these conditions. In
order to assess the ability of these lipids to stimulate Akt,
phospholipid vesicles were preincubated with Akt. The kinase
activity of Akt was analyzed in an in vitro kinase assay using
histone H2B as substrate. Although comparable amounts of all three
3' phosphorylated inositol phospholipid products had been
generated, only Akt molecules that were preincubated with vesicles
containing PI3,4P.sub.2 exhibited a substantial increase in kinase
activity. Control samples containing either untreated phospholipid
vesicles or p110* only failed to activate Akt. The addition of 1
.mu.M PI3,4P.sub.2 resulted in an average of 3-fold stimulation of
Akt. A greater degree of stimulation (9 fold) was observed at
higher concentration (4 .mu.M) of PI3,4P.sub.2. In parallel half of
the Akt immunocomplexes were analyzed by Western-blotting to insure
equal protein concentration in all samples. Akt did not appear to
be activated through direct phosphorylation by the protein kinase
activity of p110*, since the presence of p110* protein per se did
not result in increased Akt kinase activity. p110* was an active
protein kinase under the reaction conditions employed, since its
autophosphorylation could be detected. Under the same conditions no
Akt phosphorylation was detected. Additional control samples in
which p110* was added to phospholipid vesicle substrates
immediately before incubation with Akt, did not show activation of
Akt. This suggests that PI3,4P.sub.2 has to accumulate at
sufficiently high concentration in the PI 3-kinase reaction before
Akt activation can be observed. These results demonstrate that it
is possible to reconstitute PI 3-kinase-mediated activation of Akt
in vitro with defined components and that Akt is an immediate
downstream effector of PI 3-kinase. Furthermore, they suggest that
the PI 3-kinase produced phosphatidylinositides can act as second
messengers.
[0117] Akt-kinase activation was demonstrated by coexpression of
p110 derivatives. Myc-tagged p110 molecules were coexpressed with
HA-tagged Akt-kinase. p110 kinase or p110* kinase with either a
myristoylation (M) cell membrane targeting sequence added to the
N-terminus or a farnesylation and palmitoylation sequence (H) cell
membrane targeting sequence added to the C-terminus in the case of
a farnesylation and palmitoylation resulted in increased levels of
AKT kinase activity. AKT kinase is a known downstream effector of
PI 3-kinase.
[0118] In addition, in vitro stimulation of Akt by p110*-generated
PI3,4P.sub.2 was demonstrated. The PI 3-kinase substrates PI, PI4P
and PI4,5P.sub.2 in PC vesicles were phosphorylated by purified
p110* protein in the presence of ATP (with 2500 cpm/pmol [g-
.sup.32P]ATP) to obtain PI3P, PI3,4P.sub.2 and PI3,4,5P.sub.3. In
order to monitor the production of phosphatidylinositides by p110*
a fraction of each reaction was subjected to phospholipid
extraction. The lipids were resolved by thin-layer chromatography
(TLC) and visualized by autoradiography. The amount of labeled
phospholipid products was quantitated by scraping the respective
areas of the TLC plate and counting in a scintillation counter. The
in vitro assay for activation of Akt by PI3,4P.sub.2. was conducted
using the phospholipid vesicles prepared as just described, mixed
with Akt for 10 min. Subsequently, the kinase activity of Akt was
assayed as described above. The following reaction conditions were
tested: Akt was incubated with reaction buffer alone, with reaction
buffer containing PI, PI4P or PI4,5P.sub.2 phospholipid vesicles,
and with PI, PI4P or PI4,5P.sub.2 phospholipid vesicles that had
been treated with p110*; 1 and 4 .mu.M of each PI 3-kinase product
were tested, respectively. As additional controls, Akt was
incubated in mixed vesicles, then p110* was added and Akt was
incubated with p110* protein in the absence of phospholipid
vesicles. In samples containing phospholipid vesicles the total
lipid concentration was maintained at approximately 1000 .mu.M. The
3' phosphorylated inositol phospholipids were presented in an
excess of PI, PI4P or PI3,4P.sub.2 (80 to 100 .mu.M) and PC (800 to
900 .mu.M). Relative amounts of Akt were analyzed by
immunoblotting. An aliquot of each immunocomplex was analyzed in
parallel for protein levels by Western-blotting with anti-Akt
antibody. Akt kinase activities were quantitated using a Molecular
Imager (BioRad). The increase in Akt kinase activity is expressed
relative to samples containing unstimulated Akt.
[0119] We determined that the PH domain at the Akt N-terminus is
essential for PI3,4P.sub.2 mediated stimulation. It has previously
been reported that the stimulation of the kinase activity of Akt by
PI 3-kinase in vivo is dependent on the PH domain at its immediate
N-terminus. We determined that PI3,4P.sub.2 mediates its
stimulatory effect on Akt through its PH domain by introducing a
point mutation in the PH domain of Akt (Akt RC25) which abrogates
PI 3-kinasemediated Akt activation in vivo. A kinase-deficient Akt,
Akt KA179, was tested as an additional control. We incubated Akt,
Akt RC25 and Akt KA179 proteins with PI3,4P.sub.2 in phospholipid
vesicles using p110* as described above. Akt was efficiently
activated by PI3,4P.sub.2 containing vesicles, while Akt RC25 was
not activated. The basal kinase activity of Akt RC25 remained
unaffected under the same conditions suggesting that the RC25
mutation in the PH domain does not interfere with basic kinase
function, but rather affects the ability of the mutant Akt
molecules to become activated. The kinase-deficient Akt KA179 had
no detectable enzymatic activity.
[0120] COS-7 cells were obtained from the American Type Culture
Collection and cultured at 37.degree. C. in Dulbecco's modified
Eagle medium containing 10% bovine calf serum, penicillin (50
.mu.g/ml) and streptomycin (50 .mu.g/ml). Spodoptera frugiperda
(Sf9) cells (from M. Summers, Texas A&M University, College
station) were grown in ISFM-7 medium. Recombinant baculovirus
expressing p110*.multidot.6His was prepared from the supernatant of
Sf9 cells as described previously.
[0121] Ascites fluid with the murine anti-influenza virus
hemagglutinin 1 (HA1) monoclonal antibody 12CA5 and hybridoma 9E10
are available commercially and using these mouse ascites fluid
containing murine monoclonal anti-Myc antibody was prepared. Rabbit
polyclonal anti-Akt/RAC-PK antibody is also commercially available.
Rabbit polyclonal anti-SHC antiserum has been described.
[0122] The mammalian expression vectors for the HA-tagged kinase
Akt/RAC-PK and Akt RC25 were described previously The cDNA for
Akt/Rac KA179 was cloned into the same expression vector as wt
Akt.
[0123] To generate p110*.multidot.6His the C-terminal end of p110
was modified using primer 6-His sense-(5' GC GCC CAC CAT CAT CAC
CAC CAT TGA GTC GAC G) SEQ ID NO.1 and primer 6His -antisense-(5'
GA TCC GTC GAC TCA ATG GTG GTG ATG ATG GTG G) SEQ ID NO. 2 flanked
by restriction sites for Kas I and Bam HI. The 6His coding region
was attached in frame to the p110 C-terminus by ligating the
annealed oligonucleotide via Kas IBam HI ends into
pCG-p110.multidot.H. This extended the p110 C-terminal end by a
sequence encoding amino-acids DLGGA (SEQ ID NO. 3) as a hinge
region (overlapping restriction sites SacI/Ecl136II and KasI/NarI),
which precedes the coding region for the six histidine residues, a
stop-codon and a BamHI restriction site. For expression in insect
cells the coding region for p110*.multidot.6His was reconstituted
using p110.multidot.6His and DNA fragments from previously
described p110* constructs and cloned into baculovirus expression
vector pVL1392 (available from Pharmingen) via XbaI-BamHI ends.
[0124] p110*.multidot.6His was transiently expressed in insect
cells Sf9 cells were infected with recombinant baculovirus
directing the expression of p110*.multidot.6His protein. The cells
were harvested after 50 h by centrifugation at 1000.times.g, washed
with ice-cold PBS and lysed at 4.degree. C. in lysis buffer
containing 20 mM Tris (pH 7.5), 137 mM NaCl, 15% (vol/vol)
glycerol, 1% (vol/vol) Triton X-100, 2 mM EDTA, 2 mM
phenylmethylsulfonyl fluoride, 10 mg aprotinin per ml, 20 mM
leupeptin, 2 mM benzamidine. Lysates were cleared by centrifugation
at 14,000.times.g for 5 minutes. The Sf9 cell extract was loaded on
a 1.times.7 cm Ni-chelating chelating Se pharose FF column
equilibrated in buffer A (20 mM HEPES
[N-2-hydroxyethyl-piperazine-N'-2ethanesulfonic acid, pH 7.5 ], 150
mM NaCl, 20 mM imidazole [pH 7.5 ], 5 mM bmercaptoethanol, 10%
[vol/vol] glycerol) containing 0.5% Triton X-100. The column was
washed in buffer A and developed with a 20 to 200 mM gradient of
imidazole in buffer A. Fractions containing purified
p110*.multidot.6His protein (100 to 200 .mu.g/ml) were pooled.
[0125] For preparing GST-SIP-110 protein the coding region of
SIP-110 was expressed in insect cells as GST-fusion using the
baculovirus expression vector pVIKS. The cells were lysed as
described above and SIP- 110 was immobilized by binding to
GST-agarose according to manufacturer's instructions (Pharmacia,
located in New Jersey). Alternatively SIP was immunoprecipitated
from stimulated B-cell lysates using anti-SHC antibodies as
described earlier. Akt/RAC-PK/PKB was transiently expressed in
COS-7 cells. COS cells (60 to 70% confluent on a 10 cm plate) were
transfected with mammalian expression vectors encoding HA-tagged
Akt, Akt RC25 or Akt KA179 using the DEAE-dextran method. Cells
were starved for 36 hours. COS cells were washed twice with cold
phosphate-buffered saline and lysed at 4.degree. C. in 20 mM Tris
(pH 7.5), 137 mM NaCl, 15% (vol/vol) glycerol, 1% (vol/vol) Triton
X-100, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10 mg
aprotinin per ml, 20 mM leupeptin and 2 mM benzamidine. Lysates
were cleared by centrifugation at 14,000.times.g for 5 minutes.
Cell-llysates containing HA-tagged Akt were incubated with
monoclonal anti-HA antibody 12CA5 for 1 h at 4.degree. C. Protein
A-Sepharose beads (Sigma) were used to precipitate the
immunocomplexes. The beads were washed with 50 mM Tris-HCl (pH
7.5), 0.5M LiCl, 0.5% (vol/vol) Triton X-100, twice with PBS and
once with 10 mM Tris-HCI (pH 7.5), 5 mM bglycerolphosphate, 2 mM
dithiothreitol. Half of the immunobeads was subjected to an in
vitro kinase reaction, the second half was analyzed for the amount
of protein by immunoblotting.
[0126] Phospholipid mixes containing phosphatidyl-serine (PS),
phosphatidylcholine (PC) or phosphatidylethanolamine (PE) (Avanti
Polar Lipids, Sigma) as carriers were dried under a stream of
nitrogen and sonicated (at 2 mg/ml) in 50 mM HEPES (pH 7.2) using a
bath sonicator. To generate vesicles containing synthetic 3'
phosphorylated phosphatidylinositides the sonication was carried
out in the presence of dipalmitoylated PI3P or PI3,4P.sub.2
(Matreya). Alternatively, vesicles containing the PI 3-kinase
substrates PI, PI4P or PI4,5P.sub.2 (Avanti Polar Lipids,
Boehringer Mannheim) were treated with purified p110*. The lipids
were incubated with p110*.multidot.6His protein (10 .mu.g/ml) in 50
.mu.M HEPES (pH 7.2), 5 mM MgCl.sub.2, 50 .mu.M [g-.sup.32P]ATP
(2500 cpm/pmol) and 2 mM dithiothreitol. A typical reaction mix
contained 80 to 100 .mu.M PI, PI4P or PI4,5P.sub.2 and 880 .mu.M PC
Under these conditions approximately 5% to 10% of the substrates
were converted into 3' phosphorylated phosphatidylinositides. The
reaction conditions employed were not optimal for the PI 3-kinase
reaction, but allowed for maximal Akt stimulation in the subsequent
protein kinase assay (see below). The phospholipid reactions were
either used directly in the Akt kinase assay or were stopped by the
addition of an equal volume of 1 M HCl and extracted using twice
the volume of methanol/chloroform (1:1). Extracted lipids were
dried and stored at -75.degree. C. or sonicated in reaction buffer
and subjected to treatment with immobilized preparations of SIP
proteins on glutathione- or immunobeads. The generation/conversion
of 3' phosphorylated phosphatidylinositides was monitored using a
fraction of the respective reactions. Reaction products were
extracted and separated by thin layer chromatography (TLC) using
H20, acetic acid, methanol, acetone and chloroform(14:24:26:30:80
[vol/vol]) The production of PI3P, PI3,4P.sub.2 and PI3,4,5P.sub.3
was visualized by autoradiography. PI, PI4P and PI4,5P.sub.2 in the
reaction mixture served as internal standards and were visualized
after staining in iodine-vapor. Labeled phospholipid products were
quantitated by scraping the respective areas of the TLC plate and
counting in a scintillation counter. The amounts of PI3P,
PI3,4P.sub.2 and PI3,4,5P.sub.3 produced were calculated based on
the specific activity of the [g-.sup.32P]ATP used.
[0127] The assay for the in vitro protein kinase activity of Akt
was conducted with immobilized Akt that was preincubated with or
without mixed phospholipid vesicles (20 .mu.l) for 10 min and
subjected to an in vitro protein kinase assay using histone H2B
(Boehringer Mannheim) as a substrate. The reactions were carried
out in 30 .mu.l at 22.degree. C. for 20 min in the presence of 5
.mu.Ci [g-.sup.32P]ATP. The reactions were stopped by the addition
of 8 .mu.l Lmmili-sample buffer and 22 .mu.l of each reaction
mixture were analyzed by 16% SDS-PAGE. The relative amounts of
incorporated radioactivity were visualized by autoradiography and
quantitated using a Molecular Imager System (BioRad).
[0128] Immunoprecipitates were boiled in Lammli-sample buffer,
separated by SDS-PAGE and transferred to nitrocellulose-filters
(Schleicher & Schuell). Filters were blocked in TBST buffer (10
mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% (vol/vol) Tween 20, 0.5%
(wt/vol) sodium azide) containing 5% (wt/vol) dried milk.
Antibodies were added in TBST at appropriate dilutions. Bound
antibody was detected with anti-mouse or anti-rabbit conjugated to
alkaline phosphatase (Promega, located in Madison, Wis.) in TBST,
washed, and developed with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate (Promega).
EXAMPLE 7
Method of Treating Cell Death From Trauma
[0129] A patient is diagnosed with having had a stroke. The site of
the, affected tissue in the brain is determined. A gene therapy
vehicle is prepared with a nonviral vector and a polynucleotide
sequence having a p110 subunit sequence, an iSH2 sequence, a linker
sequence and a myristoylation encoding sequence for membrane
attachment. This sequence is delivered in the vehicle to the
patient at the site of brain tissue damage. Cell death from the
trauma is thereby reduced.
EXAMPLE 8
Method of Treating Cell Death From Heart Attack Trauma
[0130] A patient is diagnosed with having had a heart attack, and
an ischemic lesion in the heart is identified. Vesicles containing
3' phosphorylated inositol phospholipids made in cells expressing
membrane targeted p110* polynucleotides are administered by
catheter to the region of the heart having the ischemic lesion,
thereby restoring some of the cells from loss due to the
trauma.
EXAMPLE 9
Method of Promoting Activation of Insulin Signaling Pathway
[0131] A patient having reduced responsiveness to insulin in cells
that would normally be expected to be responsive to insulin where
glucose has been released, for example after a meal, is
administered a viral-based gene therapy vehicle having a
polynucleotide of the invention systemically, in the portal vein,
targeting the liver organ. The gene therapy vehicle provides
expression in cells of a membrane targeted PI 3-kinase mutant of
the invention, providing activation of insulin signaling in the
non-responsive cells, or cell exhibiting a reduced responsiveness
to insulin.
Sequence CWU 1
1
* * * * *