Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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INPROVED P~ARNACEUTICAL COMPO~ITION8
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical
composition for delivery of DNA to cells or tissues bearing
the insulin receptor.
BACKGROUND ~F THE INVENTION
Gene therapy relies on efficient delivery of DNA to
target cells, and expression of the delivered DNA in the
nucleus of such cells. Different modes of DNA delivery have
been proposed, and these involve both viral and non-viral
delivery of gene sequences.
Early experiments on introducing DNA into mammalian
cells ln vitro utilized DNA in precipitated form with low
efficiency of transfection and required selectable mar~er
genes (Wigler et al. (1977) Cell 16, 777-85; Graham and Van
der Erb (lg79) Proc. Natl. Acad. Sci. USA 77, 1373-76 and
(1973) Virology 52, 456)). Since this time molecular
biologists have developed many other more efficient
techniques for introducing DNA into cells, such as
electroporation, complexation with asbestos, polybrene,
DEAE-Dextran, liposomes, lipopolyamines, polyornithine,
particle bombardment and direct microinjection (reviewed by
Kucherlapati and Skoultchi (1984) Crit. Rev. Biochem. 16,
349-79; Keown et al. (1990) Methods Enzymol. 185, 527). Many
of these methods are unsuitable for use clinically since
they give highly variable and relatively poor levels of
transfection. Another obstacle to the wider use of existing
gene delivery vehicles resides in their instability in vivo.
It has been shown that particles of a similar size to the
gene delivery vehicles of the prior art are rapidly and
3S efficiently removed from the blood by the
reticuloendothelial system (Posse and Kirsch, Bio/Technology
1, 869 (1984)).
Loyter and Volsky ~Cell Sur. Rev. 8, 215-266 (1982))
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and Kaneda et al. (Exp. Cell Res. 173, 56-69 (1987))
describe the reconstitution of viral envelopes as biological
carriers including carriers of DNA. In this approach,
naturally occurring viruses are isolated, dissolved in
detergent containing solvents, the viral nucleic acid
removed and the remaining viral components reconstituted in
the presence of plasmid DNA. However, this technology has
proven to be extremely expensive and difficult to scale up.
Moreover, serious safety concerns are connected with the
pharmaceutical use of extracted viruses.
Other non-viral gene delivery systems described in
the literature merely extend observations on transfection
using DNA condensed by synthetic polymers, for example,
soluble DNA/polylysine complexes can be generated (Li et
al., Biochem. J. 12, 1763 (1973)). Polylysine complexes
tagged with asialoglycoprotein have been used to target DNA
to hepatocytes in vitro (Wu and Wu, J. Biol. Chem. 262, 4429
(1987); U.S. Patent 5,166,320). Lactosylated polylysine
(Midoux et al. (1993) Nuc. Acids Res. 21, 871-878) and
galactosylated histones (Chen et al. (1994) Human Gene
Therapy 5, 429-435) have been used to target plasmid DNA to
cells bearing lectin receptors, and insulin conjugated to
polylysine (Rosenkrantz et al. (1992) Exp. Cell Res. 199,
323-329) to cells bearing insulin receptors. However, Wagner
et al. ( ibid) have shown that the latter approach is even
less efficient than standard methods of transfection, and
may therefore be considered unsuitable for pharmaceutical
development. Monoclonal antibodies have been used to target
DNA to particular cell types (Machy et al.- (1988) Proc.
Natl. Acad. Sci. USA 85, 8027-8031; Trubetskoy et al. (1992)
Bioconjugate Chem. 3, 323-27 and WO 91/17773 and Wo
92/19287).
The insulin receptor has a}so been used to target
gene delivery to cells derived from liver. Huchet et al.
(Biochem. Pharmacol. 40, 253 (1990)) obtained low level
transfection by using serum albumin derivatized with
dimethylaminopropyl groups as a DNA carrier and crosslinked
this complex to insulin. Higher transfection activity was
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obtained by Rosenkrantz et al. (Exp. Cell. Res. 199, 323
(1992)) in which the epsilon-amino group of the C-terminal
lysine residue of the insulin beta chain was derivatized
with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and
coupled to derivatized polylysine.
SUMMARY OF THE INVEN~ION
The invention is based on a gene delivery vehicle
that is capable of targeting cells or tissue types which
bear the insulin receptor on t~eir surface, and delivering
a gene to that cell or tissue.
The invention encompasses a gene delivery vehicle
which includes a nucleic acid binding peptide, HzN-Thr-
Lys18-(S-Acetimidomethyl-Cys)-COOH, linked to insulin or an
insulin derivative, and associated with condensed nucleic
acid (NA) coding for sequences of therapeutic benefit. The
insulin or insulin derivative allows for targeting of the
nucleic acid delivery vehicle to mammalian, preferably
human, cells or tissue that bear the insulin receptor. The
nucleic acid binding peptide thus allows the vehicle to form
a complex with condensed nucleic acid and thus to deliver a
selected nucleic acid to the insulin receptor- bearing
target cell.
The invention also encompasses a pharmaceutical
preparation for use in gene therapy, comprising a gene
delivery vehicle comprising a therapeutic nucleic acid
associated with Insulin-N~-Co-CH2-O-N=CH4-CO-Lysl8-Cys~S-
Pyridyl)-OH in combination with a pharmaceutically
acceptable carrier.
The gene delivery vehicle is useful for treating
diseases associated with the liver, such as cirrhosis of
the liver, hypercholesterolemia, cancer, and infection by
hepatitis A, B, C, D or E. Sequences of therapeutic benefit
for treatment of such diseases include, for example,
ribozymes directed against RNA of infectious organisms or
sequences encoding such ribozymes, genes encoding growth
factors and growth factor receptors, genes whose products
influence proqression of the cycle of cell division (e.g.,
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CDK genes and the p53 gene), and the LDL receptor gene.
As used herein, "associated with" or 'l~ound to" refer
to noncovalent forms of molecular association, such as
charge interactions, hydrogen bonding, and hydrophobic
interactions; e.g., positively charged amino groups of the
nucleic acid binding component are attracted to negatively
charged phosphate groups on the nucleic acid phosphodiester
backbone. Alternatively, 'associated with" or "bound to" may
refer to base pairing; e.g., the hydrophobic and hydrogen
bonding interactions found between two strands of DNA.
The nucleic acid binding component of the invention
includes an amino acid sequence that is capable of binding
to nucleic acid.
The protein hormone insulin or a derivative of insulin
acts as the targeting ligand to direct the nucleic acid
delivery vehicle to cells expressing the insulin receptor,
where the insulin or insulin derivative retains receptor
binding properties when conjugated to a nucleic acid binding
component. As used herein, "insulin derivative" includes any
form of insulin that is capable of specific binding to the
insulin receptor and being internalized into the target cell
when linked to the nucleic acid delivery vehicle; such as
natural or synthetic fragments of the insulin molecule,
chemically modified insulin molecules, or chemically
modified synthetic or naturally occurring fragments of
insulin.
Examples of cells which bear the insulin receptor
include but are not limited to hepatocytes, brain cells,
adipocytes, lymphoid cells, muscle, epithelial cells, and
cancerous tissue, all of which are known to bear a high
density of the insulin receptor.
The invention also encompasses methods of ex vivo,
in vitro, and in vivo cell-type specific targeting. As used
herein, ex vivo targeting refers to targeting of a nucleic
acid to an insulin receptor-bearing cell that has been
removed from a patient; in vitro targeting refers to
targeting of a nucleic acid to an insulin receptor-bearing
cell from a cultured cell line; and in vivo cell targeting
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refers to targeting of an ins~lin receptor-bearing cell in
a mammal such as in a human being.
The invention thus also includes methods of treating
an infectious disease, such as is caused by infection by
hepatitis virus, particularly hepatitis C, which method
includes targeting and incorporating nucleic acids coding
for anti-hepatitis C ribozyme genes, into insulin
receptor-bearing cells using the a~ove-described delivery
vehicle. -
The invention thus also encompasses a method of
treating a disease of a patient, comprising the steps of:
(a) providing a pharmaceutical preparation comprising a gene
delivery vehicle comprising a therapeutic nucleic acid
associated with Insulin-NH-CO-CH2-O-N=CH-CO-Lys1a-Cys(S-
Pyridyl)-OH in combination with a pharmaceutically
acceptable carrier; and (b) administering said
pharmaceutical preparation to a patient suffering from a
genetic disease
Ex vivo and in vltro methods will, include the step
of contacting the vehicle with an insulin receptor-bearing
target cell, whether that cell be in a substantially
homogenous population of target cells or in a heterogenous
cell population, for a time and under conditions sufficient
to allow cell targeting and nucleic acid uptake to occur.
One in vivo method includes administering a therapeutically
effective amount of the gene delivery vehicle to a mammal,
preferably a human. Another in vivo method includes
administering a therapeutically effective amount of a
homogenous or heterogenous insulin receptor-bearing cell
population that has been prepared by contacting the gene
delivery vehicle with a target insulin receptor-bearing cell
for a time and under conditions sufficient to allow cell
targeting and nucleic acid uptake to occur. As used herein,
a "therapeutically effective amount" is an amount which
confers a therapeutic benefit on a patient.
The invention also encompasses a method of making a
delivery vehicle for delivery of a gene to an insulin
receptor-bearing cell, comprising the steps of a) oxidizing
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Thr-~ysl8-Cys(S-Pyridyl) peptide, and b) conjugating the
oxidized peptide to amino-oxy-acetyl-insulin.
The invention also encompasses a method of making
kits for carrying out therapeutic delivery of a gene to a
target cell that expresses the insulin receptor, a kit
comprising the gene delivery vehicle described herein and
packaging materials therefore.
Further features and advantages of the invention will
become more fully apparent in the following description of
the embodiments and drawings thereof, and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based on the discovery of a vehicle
for delivery of a gene to insulin receptor-bearing cells,
which vehicle includes the ligand insulin or an insulin
derivative to target those cells bearing the insulin
receptor.
Z0 The Structure of Insulin or an Insulin Derivative
The protein hormone insulin, or an insulin
derivative, is used to target the nucleic acid delivery
vehicle to cells expressing the insulin receptor.
Derivatives of insulin include any form of insulin that is
capable of binding specifically to the insulin receptor and
being internalized into the target cell when linked to the
nucleic acid delivery vehicle. The insulin or insulin
derivative must recognize and bind with high and specific
affinity to the insulin receptor on the target cell type,
In practice, the most useful forms of insulin are wild type
insulin, or fraqments of insulin that are capable of binding
to the insulin receptor and being internalized. There are
over 500 fragments and derivatives of insulin having
biological activity which are known in the art. The
invention encompasses those fragments and derivatives of
insulin and proinsulin which have at least 10% of the
receptor binding affinity of native insulin, The receptor
binding affinity of native insulin is determined as taught
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by Martin et al., 1984, Diabetologia pages 118-120.
Described below is the insulin derivative
amino-oxy-acetyl-Insulin.
5 Structure of the r)NA Bindinq Component
The DNA binding component of the gene delivery vehicle
is H2N-Thr-Lysl8-(S-Acetimidomethyl-cys)-COOH.
EXAMPLE I
8ynthe~is of Nucleic Acid ~inding Peptide
1. Preparation of H2N-Thr-Lysl8-(S-Acetimidomethyl-Cys)-COOH
The peptide was synthesized using a Millipore 9050
plus peptide synthesizer in extended synthesis cycle mode
(30 mins - 1.25 hour couplings increasing during the
synthesis). Fmoc-Cys(Acm)-O-PEG-PS-Resin was used. After
20 deprotection of the of the Fmoc group using 20% piperidine
in DMF, the subsequent amino acids were coupled in four-
fold excess using o- (lH-benzotriazol-1-yl) -
tetramethyluronium tetraf luoroborate
( T B T U ) / 1 - h y d r o x y b e n z o t r i a z o l e a n d
25 N,N'-diispropylCarbOdiimide as activating agents. When
necessary, a four-fold excess of the amino acid,
0-(lH-7-aza- benzotriazol-l-yl) -tetramethyluronium
hexafluorophosphate and diisopropy1ethylamine was used to
ensure complete coupling to the growing peptide. After the
30 synthesis of the peptide was complete, the N-terminal Fmoc
group was removed as described above to give the free amino
side chain protected peptide bound to the resin. This was
c l e a v e d f r o m t h e r e s i n u s i n g a
TFA/water/phenol/thioanisole/1, 2-ethanedithiol
35 (82.5:5:5:5:2.5) mixture. Following precipitation with ether
and centrifugation, the peptide was purified using gel
filtration to give the desired product. When necessary, the
Acetimidomethyl (Acm) thiol protecting group may be removed
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using mercury (II) acetate in 30~ acetic acid in water
followed by precipitation of the mercury with
2-mercaptoethanol. The resulting free thiol peptide can be
purified using gel filtration to give the desired product.
The peptide was synthesized using Millipore 9050 plus
peptide synthesizer in extended synthesis cycle mode (30
mins - 1.25 hour couplings increasing during the synthesis).
Fmoc-Cys(Acm)-O-PEGPS-Resin was used. After deprotection of
the Fmoc group, using 20~ piperidine in DMF, the subsequent
amino acids were coupled in four fold excess using
O-(lH-benzotriazOl-l-yl)-tetramethyluronium
tetrafluoroborate (TI3TU)/l-hydroxybenzotriazole and
N,N'diisopropylcarbodiimide as activating agents. When
necessary, a four-fold excess of the amino acid
O-(lH-7-aza-benzotriazol-l-yl)tetramethyluronium
hexafluorophosphate and diisopropylethylamine was used to
ensure complete coupling to the growing peptide. After the
synthesis of the peptide was complete, the N-terminal Fmoc
group was removed as described above to give the free amino
side chain protected peptide bound to the resin. This
peptide was cleaved from the resin using a
TFA/water/phenollthioanisole/l,2- ethanedithiol
t82.5:5:5:5:2.5) mixture. Following precipitation with ether
and centrifugaticn, the peptide was purified using gel
2S filtration to give the desired product. When necessary, the
acetimidomethyl (Acm) thiol protecting group may be removed
using mercury (II) -acetate in 30t acetic acid in water
followed by precipitation of the mercury with
2-mercaptoethanol. The resulting free thiol peptide can be
purified using gel filtration to give the desired product.
The S-pyridyl derivative was obtained by reaction with a 5
fold molar excess of dithiopyridine in O.lN sodium acetate
buffer containing 50% acetonitrile. A~ter 2h incubation at
room temperature the product was purified by reverse phase
hplc.
2. Synthesis of F_ller Component
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A filler component may be added to the assembly
reaction during preparation of the gene delivery vehicle.
The filler component may be synthesized according to the
above-noted procedure for synthesis of the DNA ~inding
component. The filler NBC-II (H2N-NBC-II-
(Acetimidomethyl-Cys)-COOH) has the following sequence: NH2
PRKxRxvEKKspKKAE~RpARspAKA}tA~vKpKAAKpKK~tKKK
RKVEKKSPKKAKKPAAC-COOH.
EXAMPLE II
Functionalization of In~ulin
Insulin may be chemically modified according to
previously described methods (Offord, ~.E. "Semisynthetic
Proteins" pp. 235, Wiley, Chichester and New York (1980)l,
with slight modifications. Briefly, lOO mg Zn-free insulin
is dissolved in l mL of lM NaHCO3, diluted with 4 mL
dimethylformamide (DMF) and reacted with an equimolar amount
of MSC-ONSu (~-~ydroxy succinimide derivative of
Methylsulfonyloxycarbonate, Tesser, 1975, in ~Peptides",
John Wiley, NY, pp 53-56) relative to protein amino groups.
After lh incubation at room temperature, the mixture is
acidified and subjected to prepara~ive HP~C on a Waters Prep
Nova-Pak Hz C~ column ~flow rate 20ml min using a 25-50% B
gradient (B ~radient is a mixture of O.l tw/v) aqueous TFA
and acetonitrile:TFA water 900:l:l00 tv/w/v) over 50 min.
The peak corresponding to all-substituted insulin (as judged
by subsequent ESI-MS) is collected and desalted in a double
Chromabond (C18 solid phase extraction cartridge (2xlg of
resin in a polypropylene column) Macherey-Nagel, Dormstadt,
Germany) equilibrated in 0.1% TFA. The derivative obtained
in such reactions is known to be preponderantly the desired
N-~, A1-MSC, N- -B30MSC substituted molecule.
Analysis of the modified protein after overnight
incubation in 50 mM DTT allows identification of the B-chain
with only a single MSC group (calcd. m/z 3547.8; found m/z,
3549.6+0.4), which is in agreement with the desired
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structure.
mg MSC2-insulin are dissolved in 1 mL
N-methyl-pyrrolidone and reacted with a lO-fold molar excess
of Boc-AoA-OSu (Vilaseca et al., Bioconjugate Chem. 4
515-520 tl993)), in the presence of equimolar amounts of
HOBt and of sufficient N-ethylmorpholine to bring the pH to
approximately 8. After lh incubation at room temperature,
the reaction medium is acidified and diluted with 0.1% TFA,
and the derivatized insulin isolated by semipreparative HPLC
on a C8 reverse phase column equilibrated in 0.1% TFA in
conjunction with a 35-45B% gradient (described above) over
20 min.
The ~SC groups are then cleaved by treatment with
sodium hydroxide as described by Offord (loc cit) and the
material repurified on the C18 column using 35-45% gradient
over 20 mins. The final compound, BOC-AoA-insulin, may be
characterized by ESI-MS (calcd. m/z 5950.6; found m/z 5948.1
+ O.l) and is deprotected by TFA treatment (30 minutes at
room temperature~ just before conjugation to a polylysine
peptide.
EXAMPLE III
Oxidation of the Thr-Lysl8-Cy~(S-Pyridyl) Peptide
and Conjugation to Amino-oxy-acetyl ~AOA)-Insulin
The cys-protected peptide (10 mg/ml) is dissolved in
50 mM imidazole, pH 6.9, and O.2 M methionine in water is
added as a anti-oxidant scavenger to a lO-fold molar excess
over peptide. 50 mM sodium periodate is added to a five-fold
molar excess over peptide, and the solution allowed to stand
in the dark for 5 minutes at 220~C. The mixture is purified
by semipreparative HPLC on a C8 reverse phase column using
0.1% aqueous TFA and a 10% to 60% gradient of 0.1% aqueous
TFA in 90~ acetonitrile over 25 min.
The isolated oxidized peptide is dissolved into a
solution of 5 mg of the AoA-insulin derivative
(approximately 2-fold molar excess of peptide over insulin)
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made up in 0.5 mL 0.1 M sodium acetate buffer to which had
been added 50 ~L acetonitrile, followed by adjustment to pH
3.8 with glacial acetic acid. After 15h incubation at room
temperature, the conjugate is isolated and characterized by
ESI-MS (calcd. mtz 8426.1, found m/z 8429.3+ 0.5). 4 mg of
material were isolated by semipreparative HPLC with a 30-45%
gradient from the bulk of the reaction mixture. The pea~
fraction was dried in a speedvac (yield is approximately 4
mg of conjugate).
EXAMPLE IV
During gene transfer a fusogenic peptide may be
included in the transfection mix in order to enhance
efficiency of transfer of the therapeutic gene. A fusogenic
peptide FP useful according to the invention includes the
follow sequence: NH2- GLFEAIAGFIENGWEGMIDGGGC(Acm)-COOH, and
is synthesized as follows.
1. Synthesis of H2N-FP-(S-acetimidomethyl-Cys)-COOH:
The FP peptide is synthesized using a Millipore 9050
plus peptide synthesizer in extended synthesis cycle mode (1
hour couplings). Fmoc-Cys(Acm)-o-PEG-PS-was used. After
deprotection of the Fmoc group using 20% piperidine in DMF,
the subsequent amino acids were coupled in four-fold excess
using o-(lH-benzotriazol-l-yl)-tetramethyluronium
tetrafluoroborate (TBTU)/1- hydroxybenzotriazole and
N,N'-diisopropylcarbodiimide as activating agents. When
necessary, a four fold excess of the amino
acid,O(lH-7-aza-benzotriazol-1-yl)
-tetramethlyroniumhexafluorophosphate and
diisopropyleth~lamine was used to ensure coupling to the
growing peptide. After the synthesis of the peptide was
complete, the N-terminal Fmoc group was removed as described
above to give the free amino side chain-protected peptide
bound to the resin. This was cleaved from the resin using a
TFA/water/phenol/thioanisole/1,2- ethanedithiol
(82.5:5:5:5:2.5) mixture. Following precipitation with ether
and centrifugation, the peptide can be purified using gel
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filtration to give the desired product.
The acetamidomethyl (Acm) thiol protecting group on
the peptide may be removed using mercury (II) acetate with
water/acetonitrile tl:l, O.lt TFA) as solvent followed by
precipitation of the mercury with 2-mercaptoethanol. The
resulting free thiol peptide can be purified using gel
filtration to give the desired product.
EXAMPLE V
8ynthe~is of Transfection ComplexeQ
DNA is made up to 20 mg/ml in a transfection buffer
(0.15 M to 1.0 NaCl; 25 mM HEPES, pH 7.4) The conjugate and
peptide is made to an equal volume to the DNA in safe
buffer. The DNA is shaken or vortexed while the condensing
agent is added at the rate of O.l volume per minute. The
complex is left at room temperature for at least 30 minutes
prior to adding to cells, and can be stored at 4~ C if
necessary. Transfection complexes consist of plasmid DNA
containing the therapeutic gene or reporter gene, insulin
conjugated to NH2-Thr-Lys~8-Cys-COoH and unconjugated NBC-II.
The transfection complex is synthesised by incubating the
three components together, for 30 minutes to 24 hours at
room temperature. The prepared complex is centrifuged to
remove any aggregated material and then assayed for gene
transfer.
Gel Retardation A~say for DNA Condensation
Conjugates or peptides are assayed for their ability
to condense DNA using the following method:
The DNA is made up to 20 mg/ml in 150 mM NaCl; 25 mM
HEPES, pH 7.4, or in 0.6 M NaCl; mM HEPES, pH 7.4 and
aliquoted between wells on a multiwell plate. The amount of
conjugate or peptide required to give (positive
charge:phosphate) ratios of between O.l and 5.0 is
calculated. This amount is made up to an equal volume to
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13
the DNA aliquots (0.05-0.5 ml) in either 150 mM sodium
chloride; 25 mM HEPES, pH 7.4 or 0.6 M sodium chloride; 25
mM HEPES, pH 7.4. The plate containing the DNA is placed on
a plate shaker and shaken while the conjugate or peptide is
added at a rate of O.l volume per minute. After addition of
the condensing agent is complete, the solution is incubated
at room temperature for at least 30 minutes. A sample for
each (positive charge: phosphate) ratio is analyzed by
electrophoresis on an agarose gel. The gel is stained with
ethidium bromide and visualized under W light. Condensed
DNA remains in the well of the gel and does not migrate in
the electric Field.
EXAMPLE VI
A~say for Gen~ Transfer
The transfection complexes may be assayed for their
ability to transfer genes into hepatocytes (HepG2 cells)
expressing the insulin receptor. For studies aimed at
determining transfection efficiency, the plasmid DNA
contains a marker gene for firefly luciferase. For
pharmaceutical applications, the plasmid contains a gene
whose expression will have a beneficial effect. The
transfection complex is incubated with blood cells and the
mixture is subjected to electroporation. After incubation,
the cells are lysed and assayed for gene expression. In the
case of the luciferase reporter gene, luciferin and ATP are
added to lysed cells and the light emitted is measured with
a luminometer.
Cells are harvested on the day of assay by
centrifugation at 1200 rpm for 5 min at room temperature.
The cell pellet is resuspended in phosphate buffered saline
(PBS) and re-centrifuged. This operation is performed twice.
The cell pellet is then suspended in RPMI 1640 (Gibco Ltd.)
to make up a suspension of approximately 2.7 x lO6 cells per
ml. The cells are then aliquoted into tubes and .75 ml of
RPMI medium added, followed by 0.04 -0.08ml of lOO ~m
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Chloro~uine (CQ) or FP peptide and finally 0.25 ml of
DNA-complex solution. The transfection is then allowed to
proceed by incubating the cells at 37~C for 4 h. After this
time, the cells are harvested by centrifugation at 2000 rpm,
suspended in lml of RPMI and re-centrifuged. Finally, the
cells are in 0.5 ~l RPMI containing 10% fetal bovine serum.
At this stage~ if necessary, the cells are electroporated at
300 V and 250 ~F using conventional electroporation.
Each 0.5 ml of transfected cell suspension is
~0 transferred to a well of a 12 well plastic culture plate
containing l.~ml of RPMI 10% FBS. The original transfection
tube is rinsed with a further lml of medium and the wash
transferred to the culture dish making a final volume of
3ml. The culture plate is then incubated at 37~C for 24-72h
in an atmosphere of 5% C02. The contents of each well in the
culture dish are transferred to centrifuge tubes and the
cells collected by centrifugation at 13,000 rpm. The pellet
is resuspended in 0.12 ml of Lysis Buffer (100 mM sodium
phosphate, pH 7.8, 8 mM MgCl2, lmM E~TA, 1% Triton X-lO0 and
15% glycerol) and agitated with a pipette. The lysate is
centrifuged at 13,000 rpm for 1 minute and the supernatant
collected. 80 pl of the supernatant are transferred to a
luminometer tube. The luciferase activity is then assayed
using a Berthold Lumat L9501 luminometer. The assay buffer
used is Lysis buffer containing lOmM Luciferin and 100 mM
ATP. Light produced by the luciferase is integrated over 4
sec and is described as relative light units (RLU). The data
are converted to RLU/ml of lysate, RLU/cell or RLU/mg
protein (protein concentration of the lys~te having been
determined in this case by the BioRAD Lowry assay).
The transfe~tion efficiency resulted in the delivery
such that 200,000-500,000 relative luciferase units per mg
of protein were expressed in the transfected cells.
EXAMP~E VII
Dosage and Pharmaceutical Formulation
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The delivery vehicle and plasmid DNA may be formulated
separately for parenteral administration or formulated
together as the transfection complex. In the latter case the
transfection complex may be assembled just prior to use. In
the case of a pharmaceutical composition, the plasmid DNA
includes a gene whose expression would have some beneficial
therapeutlc effect on the cells of the recipient.
The delivery vehicle and DNA are exchanged into
isotonic phosphate free buffer and sterile filtered through
a 0.45 or 0.22 ~ filter. The formulated solution or
transfection complex (a mixture of the delivery vehicle, DNA
and free DNA condensing component) may be sterile filled and
aliquotted into suitable vials. The vials may be stored at
4~C, 20~C or 80~C or alternatively the DNA, delivery vehicle
or transfection complex may be freeze dried from a buffer
containing an appropriated carrier and bulking agent. In
these cases, the dosage form is reconstituted with a sterile
solution before administration.
Use of this type of pharmaceutical composition in
vivo or ex vivo with nucleic acid containing a gene of
physiological importance, such as replacement of a defective
gene or an additional potentially beneficial gene function,
is expected to confer long term genetic modification of the
cells and be effective in the treatment of disease. A
delivery vehicle of the invention can be administered to the
patient, preferably in a biologically compatible solution or
a pharmaceuticallY acceptable delivery vehicle, by
ingestion, injection, inhalation or any number of other
methods. The dosaqes administered will vary from patient to
patient; a "therapeutically effective dose" will be
determined by the level of enhancement of function of the
transferred genetic material balanced against any risk of
deleterious side effects. Monitorinq levels of gene
introduction, gene expression will assist in selecting and
adjusting the dosages administered. Generally, a composition
including a delivery vehicle will be administered in a
single dose in the range of lo ng - lOo ug/kg body weight,
preferably in the range of lon ng - 10 ug/kg body weight,
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W097/22363 PCT/GB96/03137
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such that at least one copy of the therapeutic gene is
delivered to each target cell.
Ex ~ivo treatment is also contemplated within the
present invention. A cell population comprising insulin
receptor-bearing cells can be removed from the patient or
otherwise provided, transduced with a therapeutic gene in
accordance with the invention, then reintroduced into the
patient.
OTHER EMBODIMENTS
Other embodiments will be evident to those of skill
in the art. It should be understood that the foregoing
detailed description is provided for clarity only and is
merely exemplary. The spirit and scope of the present
invention are not limited to the above examples, but are
encompassed by the following claims.
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