U.S. patent application number 10/954858 was filed with the patent office on 2005-06-02 for systemic delivery of serum stable plasmid lipid particles for cancer therapy.
This patent application is currently assigned to Protiva Biotherapeutics, Inc.. Invention is credited to Graham, Roger W., MacLachlan, Ian.
Application Number | 20050118253 10/954858 |
Document ID | / |
Family ID | 56289884 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118253 |
Kind Code |
A1 |
MacLachlan, Ian ; et
al. |
June 2, 2005 |
Systemic delivery of serum stable plasmid lipid particles for
cancer therapy
Abstract
The present invention relates to methods and compositions for
treating a neoplasia in a mammal.
Inventors: |
MacLachlan, Ian; (Vancouver,
CA) ; Graham, Roger W.; (Vancouver, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Protiva Biotherapeutics,
Inc.
Burnaby
CA
|
Family ID: |
56289884 |
Appl. No.: |
10/954858 |
Filed: |
September 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10954858 |
Sep 29, 2004 |
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09243102 |
Feb 2, 1999 |
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60112384 |
Dec 14, 1998 |
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60101429 |
Sep 22, 1998 |
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60086917 |
May 27, 1998 |
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60073598 |
Feb 3, 1998 |
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Current U.S.
Class: |
424/450 ;
435/458; 514/44R |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 38/45 20130101; A61K 38/50 20130101; A61K 31/382 20130101;
A61K 48/00 20130101; A61K 38/51 20130101; A61K 45/06 20130101; A61P
43/00 20180101; A61K 31/522 20130101; Y10S 977/804 20130101; A61K
31/70 20130101; A61K 2300/00 20130101; A61K 31/70 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 31/382 20130101; A61K 2300/00 20130101;
A61K 38/50 20130101; A61K 31/675 20130101; A61K 38/47 20130101;
A61K 38/208 20130101; A61P 35/00 20180101; A61K 31/675 20130101;
A61K 38/47 20130101; C12N 15/88 20130101; A61K 31/522 20130101 |
Class at
Publication: |
424/450 ;
435/458; 514/044 |
International
Class: |
A61K 048/00; A61K
009/127; C12N 015/88 |
Claims
1. A method of treating a tumor in a mammal, said method comprising
delivering to said tumor a serum-stable nucleic acid-lipid particle
comprising a nucleic acid portion that is fully encapsulated within
the lipid portion, wherein said delivering is by injection at an
injection site that is distal to said tumor in said mammal; wherein
said lipid portion of said nucleic-acid lipid particle comprises a
cationic lipid, a neutral lipid, and a lipid conjugate that
prevents aggregation during formulation: wherein cells of said
tumor are responsive to said nucleic acid; and wherein cells of
said tumor are transfectable by said nucleic acid.
2. A method of treating a tumor in a mammal in accordance with
claim 1, wherein said nucleic acid comprises an expressible
gene.
3. A method of treating a tumor in a mammal in accordance with
claim 2, wherein said expressible gene encodes a member selected
from the group consisting of therapeutic polypeptides and
therapeutic polynucleotides.
4. A method of treating a tumor in a mammal in accordance with
claim 2, wherein said gene is exogenous.
5. A method of treating a tumor in a mammal in accordance with
claim 3, wherein said gene is a member selected from the group
consisting of genes encoding suicide enzymes, toxins and
ribozymes.
6. A method of treating a tumor in a mammal in accordance with
claim 2, wherein said gene encodes a member selected from the group
consisting of herpes simplex virus thymidine kinase (HSV-TK),
cytosine deaminase, xanthine-guaninephosphoribosyl transferase,
purine nucleoside phosphorylase, cytochrome P450 2B1 and analogs
thereof.
7. A method of treating a tumor in a mammal in accordance with
claim 2, wherein said gene is homologous to a gene in the
mammal.
8. A method of treating a tumor in a mammal in accordance with
claim 2, wherein said gene encodes a member selected from the group
consisting of proto-oncogenes, cytokines, immune stimulatory
proteins and anti-angiogenic proteins.
9. A method of treating a tumor in a mammal in accordance with
claim 2, wherein said gene is a member selected from the group
consisting of IL-2, IL-12, IL-15 and GM-CSF.
10. A method of treating a tumor in a mammal in accordance with
claim 2, wherein a therapeutically effective amount of said gene is
generated at said tumor.
11. A method of treating a tumor in a mammal in accordance with
claim 1, wherein said nucleic acid-lipid particle comprises a
protonatable lipid having a pKa in the range of about 4 to about
11.
12. A method of treating a tumor in a mammal in accordance with
claim 11, wherein said protonatable lipid is a member selected from
the group consisting of DODAC, DODAP, DODMA, DOTAP, DOTMA, DC-Chol,
DMRIE, DSDAC and mixtures thereof.
13. (canceled)
14. A method of treating a tumor in a mammal in accordance with
claim 13, wherein said lipid conjugate is a member selected from
the group consisting of PEG-lipids and PAO-lipids.
15. A method of treating a tumor in a mammal in accordance with
claim 13, wherein said lipid conjugate is reversibly associated
with an outer lipid monolayer, and wherein said lipid conjugate
exchanges out of said outer lipid monolayer at a rate faster than
PEG-CerC20.
16. A method of treating a neoplasia tumor in a mammal in
accordance with claim 1, wherein said nucleic acid-lipid particle
is substantially devoid of detergents and organic solvents.
17. A method of treating a tumor in a mammal in accordance with
claim 1, wherein a therapeutically effective amount of said nucleic
acid-lipid particle accumulates at said neoplasia tumor.
18. A method of treating a tumor in a mammal in accordance with
claim 1, wherein a therapeutic effect is detected at the site of
said tumor.
19. A method of treating a tumor in a mammal in accordance with
claim 17, wherein said therapeutically effective amount comprises
greater than about 0.5% of an administered dose.
20. A method of treating a tumor in a mammal in accordance with
claim 1, wherein said nucleic acid-lipid particle has a diameter of
about 50 nm to about 200 nm.
21. A method of treating a tumor in a mammal in accordance with
claim 20, wherein said nucleic acid-lipid particle has a diameter
of about 60 ni to about 130 nm.
22. A method of treating a tumor in a mammal in accordance with
claim 20, wherein said nucleic acid-lipid particles are of a
uniform size.
23. A method of treating a tumor in a mammal in accordance with
claim 1, wherein said nucleic acid-lipid particle has a nucleic
acid to lipid ratio of greater than about 3 mg nucleic acid to
mmole of lipid.
24. A method of treating a tumor in a mammal in accordance with
claim 23, wherein said particle has a nucleic acid to lipid ratio
of greater than about 14 mg nucleic acid to mmole of lipid.
25. A method of treating a tumor in a mammal in accordance with
claim 23, wherein said particle has a nucleic acid to lipid ratio
of greater than about 25 mg nucleic acid to mmole of lipid.
26. A method of treating a tumor in a mammal in accordance with
claim 1, wherein said nucleic acid remains at least 90% intact when
said particle containing about 1 .mu.g DNA is treated with about
100 U DNAse 1 in digestion buffer at 37.degree. C. for 30 min.
27. A method of treating a tumor in a mammal in accordance with
claim 1, further comprising administering a chemotherapeutic
agent.
28. A method of treating a tumor in a mammal in accordance with
claim 1, wherein said administering is performed at least once per
eight weeks.
29. A method of sensitizing a neoplastic cell to a compound, said
method comprising: a) transfecting said neoplastic cell with a
serum-stable nucleic acid-lipid particle encoding a gene-product
comprising a nucleic acid that is fully encapsulated within a
lipid, wherein administration of said nucleic acid-lipid particle
is by injection at an injection site that is distal to said
neoplastic cell; and b) delivering to said cell a first compound
which is processed by said gene-product into a second compound,
wherein said cell is more sensitive to said second compound than
said first compound.
30. A method of sensitizing a neoplastic cell in accordance with
claim 29 wherein said first compound is formulated in a lipid.
31. A method of sensitizing a neoplastic cell in accordance with
claim 29 wherein said gene product is a member selected from the
group consisting of therapeutic polypeptides and therapeutic
polynucleotides.
32. A method of sensitizing a neoplastic cell in accordance with
claim 29 wherein said gene product is a member selected from the
group consisting of suicide enzymes, toxins and ribozymes.
33. A method of sensitizing a neoplastic cell in accordance with
claim 29 wherein said gene product is a member selected from the
group consisting of herpes simplex virus thymidine kinase (HSV-TK),
cytosine deaminase, xanthine-guaninephosphoribosyl transferase,
purine nucleoside phosphorylase, cytochrome P450 2B1 and analogs
thereof.
34. A method of sensitizing a neoplastic cell in accordance with
claim 29 wherein a therapeutic effect is detected at the site of
said neoplasia cell.
35. A method of treating a tumor in a mammal, in accordance with
claim 5, wherein said gene encodes a suicide enzyme and said method
further comprises administering a prodrug.
36. A method of treating a tumor in a mammal in accordance with
claim 35 , wherein said prodrug is administered after the
serum-stable nucleic acid-lipid particle.
37. A method of treating a tumor in a mammal in accordance with
claim 35, wherein said prodrug is administered before the
serum-stable nucleic acid-lipid particle.
38. A method of treating a tumor in a mammal in accordance with
claim 9, further comprising administering a chemotherapeutic
agent.
39. A method of treating a tumor in a mammal in accordance with
claim 38, wherein the chemotherapeutic agent is administered after
the serum-stable nucleic acid-lipid particle.
40. A method of treating a tumor in a mammal in accordance with
claim 38, wherein the chemotherapeutic agent is administered before
the serum-stable nucleic acid-lipid particle.
41. A method of treating a tumor in a mammal in accordance with
claim 1, wherein the cationic lipid is DODAC.
42. A method of treating a tumor in a mammal in accordance with
claim 1, wherein the neutral lipid is DOPE.
43. A method of treating a tumor in a mammal in accordance with
claim 1, wherein the lipid portion further comprises a
PEG-lipid.
44. A method of treating a tumor in a mammal in accordance with
claim 1, wherein the lipid portion further comprises
cholesterol.
45. A method of treating a tumor in a mammal, said method
comprising delivering to said tumor a serum-stable nucleic
acid-lipid particle comprising a nucleic acid portion that is fully
encapsulated within the lipid portion, wherein said delivering is
by injection at an injection site that is distal to said tumor in
said mammal; and wherein said tumor is responsive to the gene
product- of the nucleic acid; and wherein said nucleic-acid lipid
particle comprises a cationic lipid, a neutral lipid, and a lipid
conjugate that prevents aggregation during formulation.
46. A method of treating a tumor in a mammal, said method
comprising delivering to said tumor a serum-stable nucleic
acid-lipid particle comprising a nucleic acid portion that is fully
encapsulated within the lipid portion, wherein said delivering is
by injection at an injection site that is distal to said tumor in
said mammal; wherein cells of said tumor are transfectable by said
nucleic acid-lipid particle; and and wherein said lipid portion of
said nucleic-acid lipid particle comprises a cationic lipid, a
neutral lipid, and a lipid conjugate that prevents aggregation
during formulation.
47. The method of claim 45, wherein said nucleic acid encodes a
member selected from the group consisting of: suicide enzymes,
toxins, tumor suppressor genes, and cytokines.
48. The method of claim 45, wherein said nucleic acid encodes a
suicide enzyme; and said method further comprises administering a
prodrug.
49. The method of claim 45, wherein said nucleic acid encodes a
toxin.
50. The method of claim 45, wherein said nucleic acid encodes a
tumor suppressor protein.
51. The method of claim 45, wherein said nucleic acid encodes a
cytbkine.
52. The method of claim 48, wherein the suicide enzyme is a member
selected from the group consisting of: HSV-TK, purine nucleoside
phosphorylase, and cytosine deaminase.
53. The method of claim 48, wherein the tumor is a melanoma.
54. The method of claim 48, wherein the tumor is a colorectal
tumor.
55. The method of claim 48, wherein the tumor is a sarcoma.
56. The method of claim 49, wherein the toxin is Pseudomonas
exotoxin.
57. The method of claim 50, wherein the tumor suppressor protein is
apoptin.
58. The method of claim 51, wherein the cytokine is IL-12.
59. The method of claim 39, wherein delivery of the serum-stable
nucleic acid-lipid particle is intravenous.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. Nos. 60/101,429, filed Sep. 22, 1998 and
60/086,917, filed May 27, 1998, which are incorporated herein by
reference in their entirety for all purposes. This application is
also related to U.S. Patent Application Ser. No. ______, filed Feb.
2, 1999 (Attorney Docket No. 016303-006220), and claims the benefit
of U.S. Provisional Patent Application Ser. Nos. 60/112,384, filed
Dec. 14, 1998, and 60/073,598, filed Feb. 3, 1998, which are
incorporated herein by reference in their entirety for all
purposes.
FIELD OF THE INVENTION
[0002] This invention relates to methods and compositions for
treating a neoplasia and, in particular, a tumor in a mammal.
BACKGROUND OF THE INVENTION
[0003] Systemic delivery of therapeutic nucleic acids for gene
therapy applications is a highly desirable goal. Systemic delivery
using blood or lymph circulatory systems will permit therapeutic
nucleic acids to seek out multiple distal disease sites, and to
approach a disease site from multiple points. Successful systems
will be useful against cancers (i.e., solid, nonsolid or metastatic
tumors), infectious diseases, inflammation and other disease
targets that are distal to the site of administration.
[0004] Currently, the most popular working systems for in vivo
human gene therapy are not systemic delivery systems. Current
systems employ direct, i.e., local, injection or inhalation of
modified adenoviruses (see, Englehardt, "Methods for
Adenovirus-Mediated Gene Transfer to Airway Epithelium," Chapter 11
in Methods in Molecular Medicine, Gene Therapy Protocols. Ed. P.
Robbins, 1997. Humana Press Inc., Totowa, N.J.), retroviruses
(Olsen, et al., "Methods for the Use of Retroviral Vectors for
Transfer of the CFTR Gene to Airway Epithelium," Chapter 10,
Methods in Molecular Medicine, supra), cationic lipid-plasmid
aggregates (Nabel, et al., "Methods for Liposome-Mediated Gene
Transfer to Tumor Cells in vivo," Chapter 21, Methods in Molecular
Medicine, supra; Son, et al., "Cationic Liposome-Mediated Gene
Transfer to Tumor Cells in Vitro and In vivo," Chapter 23, Methods
in Molecular Medicine, supra), or simply delivery of naked DNA
(see, U.S. Pat. No. 5,589,466 to Felgner, et al.).
[0005] Unfortunately, there are well-known drawbacks to these
popular methods that prevent their use as systemic delivery
systems. For instance, cationic lipid-plasmid aggregates are
rapidly cleared by the liver, lung or spleen after intravenous
delivery and, therefore, do not satisfy the criteria for systemic
delivery since they demonstrate little useful transfection
elsewhere. This may be a result of their large diameter, i.e.,
>200 nm, and powerful cationic surface charge. Moreover,
adenoviral and retroviral systems are immunogenic, are rapidly
cleared from circulation and do not permit repeat dosing. In
addition, naked DNA is rapidly degraded in the blood and, thus, is
not suitable for systemic delivery.
[0006] Systemic delivery for in vivo gene therapy, i.e., delivery
of a therapeutic nucleic acid to a distal target cell via body
systems such as the circulation, a less well explored avenue, has
been achieved using lipid-plasmid particles such as those disclosed
in published PCT Patent Application WO 96/40964, U.S. Pat. No.
5,705,385, and U.S. Patent Applications Ser. Nos. 08/485,458,
08/484,282, 08/660,025, 08/856,374, 60/073,598 and 60/063,473, all
of which are assigned to the assignee of the instant invention and
incorporated herein by reference. This latter format provides a
fully encapsulated lipid-plasmid particle that protects the
therapeutic nucleic acid from nuclease degradation in serum, is
nonimmunogenic, is small in size and is suitable for repeat
dosing.
[0007] Once the systemic delivery system has been established, the
next question is to determine which disease condition to treat and
which therapeutic nucleic acid to deliver. To date, no publication
has reported therapeutic data employing, in a systemic delivery
system, the variation of the gene therapy technique known as
gene-delivered enzyme prodrug therapy ("GDEPT") or, alternatively,
the "suicide gene/prodrug" system, which was first developed by
Moolten, F. L., Cancer Res., 46:5276-5281 (1986). For a review of
the GDEPT system, see Moolten, F. L., The Internet Book of Gene
Therapy, Cancer Therapeutics, Chapter 11 (Sobol, R. E., Scanlon, N
J (Eds) Appelton & Lange (1995)). In this method, a
heterologous gene is delivered to a cell, the heterologous gene
encoding an enzyme that promotes the metabolism of a first compound
to which the cell is less sensitive (i e., the "prodrug") into a
second compound to which is cell is more sensitive. The prodrug is
delivered to the cell either with the gene or after delivery of the
gene. The enzyme will process the prodrug into the second compound
and respond accordingly. A suitable system proposed by Moolten is
the herpes simplex virus-thymidine kinase (HSV-TK) gene and the
prodrug ganciclovir. This method has recently been employed using
cationic lipid-nucleic aggregates for local delivery (i.e., direct
intra-tumoral injection), or regional delivery (i.e.,
intra-peritoneal) of the TK gene to mouse tumors by Zerrouqui, et
al., Can. Gen. Therapy, 3(6):385-392 (1996); Sugaya, et al., Hum.
Gen. Ther., 7:223-230(1996) and Aoki, et al., Hum. Gen. Ther.,
8:1105-1113 (1997). Human clinical trials using a GDEPT system
employing viral vectors have been proposed (see, Hum. Gene Ther.,
8:597-613 (1997), and Hum. Gene Ther., 7:255-267 (1996)) and are
underway.
[0008] Patent applications relating to the GDEPT method have been
published under the following numbers: WO 97/19180; WO 97/07118; WO
96/22277; WO 97/19183; WO 96/16179; WO 96/03515; WO 96/03515; WO
96/03151; EP 690129; EP 657541; EP 657539; WO 95/05835 and EP
415731.
[0009] From the foregoing, it is readily apparent that systemic
delivery of therapeutic nucleic acids to distal disease sites would
clearly provide significant advantages over existing gene therapy
modalities. It is an object of this invention to provide methods
and compositions to achieve this goal.
SUMMARY OF THE INVENTION
[0010] The present invention provides, inter alia, methods and
compositions for treating a neoplasia, e.g, a tumor, in a mammal.
In one embodiment, the present invention provides a method of
treating a neoplasia in a mammal, the method comprising
administering to the mammal a serum-stable nucleic acid-lipid
particle comprising a nucleic acid that is fully encapsulated
within a lipid, wherein the administration is by injection at an
injection site that is distal to the neoplasia in the mammal.
[0011] In another embodiment, the present invention provides a
method for sensitizing a neoplastic cell, the method comprising:
(a) transfecting the neoplastic cell with a serum-stable nucleic
acid-lipid particle comprising a nucleic acid that is fully
encapsulated within a lipid and that encodes a gene-product that
promotes the processing, i.e., conversion, of a first compound
(e.g., a prodrug) into a second compound, wherein the
administration is by injection at an injection site that is distal
to the neoplasia in the mammal; and (b) delivering to the
neoplastic cell the first compound, wherein the cell is more
sensitive to the second compound than to the first compound.
[0012] In the above methods, the nucleic acid and the first
compound can be delivered in lipid formulations that can be the
same or different. The lipid formulations, whether used to deliver
the nucleic acid or first compound (e.g., prodrug), can be prepared
from a variety of lipids, lipid conjugates and additional
compatible components known in the art. The lipid formulations can
be prepared, for example, from sphingomyelin and cholesterol.
Moreover, the lipid formulations can contain additional components
that improve the properties or characteristics of the formulations,
such as leakiness, longevity in circulation, reduced toxicity,
encapsulation efficiency, etc. Such components include, for
example, cationic lipids, ATTA-lipid conjugates, PEG-lipid
conjugates, targeting agents, etc. Once prepared, the lipid-nucleic
acid formulations and/or lipid-prodrug formulations can be
administered or delivered to the mammal using a variety of
techniques known to those of skill in the art. In a preferred
embodiment, the lipid-nucleic acid formulation is delivered
systemically (by, for example, intravenous injection), whereas the
lipid-prodrug formulations can be delivered systemically or
regionally or locally.
[0013] Any nucleic acid useful in treating neoplasia in a mammal
can be administered using the compositions and methods of the
present invention. For instance, when the GDEPT system is employed,
the nucleic acid can be any nucleic acid that encodes a gene
product that promotes the processing, i.e., conversion, of a first
compound (e.g., a prodrug) into a second compound to which the
mammal or cell of interest is more sensitive or receptive. Examples
of suitable gene-products include, but are not limited to, herpes
simplex virus thymidine kinase, cytosine deaminase,
xanthine-guaninephosphoribosyl transferase, purine nucleoside
phosphorylase, cytochrome P450 2B1 and their analogs. Other
gene-products suitable for use in the methods of the present
invention will be readily apparent to those of skill in the
art.
[0014] In a preferred embodiment, the first compound is a prodrug,
i.e., a compound to which the cell of interest in not initially
sensitive to, but which the gene-product converts into a compound
to which the cell of interest is more sensitive.
[0015] Examples of suitable prodrugs include, but are not limited
to, ganciclovir, acyclovir, bromovinyldeoxyuridine,
5-fluorocytosine, 6-thioxanthine, MeP-dr and cyclophosphamide.
Other prodrugs suitable for use in the methods of the present
invention will be readily apparent to those of skill in the
art.
[0016] In another embodiment, the present invention provides a
method of sensitizing a cell to a compound, the method comprising:
a) delivering to a cell an enzyme which promotes the processing of
a first compound into a second compound; and b) delivering to the
cell the first compound in a lipid formulation; wherein the cell is
more sensitive to the second compound than the first compound. In a
presently preferred embodiment, both the enzyme and the first
compound are delivered in lipid formulations.
[0017] In yet another embodiment, the present invention provides a
composition for treating a neoplasia in a mammal, the composition
comprising a nucleic acid in a lipid formulation and a
pharmaceutically acceptable carrier. In a presently preferred
embodiment, the composition further comprises a prodrug in a lipid
formulation.
[0018] In still another embodiment, the present invention provides
a kit for the treatment of a neoplasia in a mammal, the kit
comprising: a) a nucleic acid in a lipid formulation; and b) a
prodrug in a lipid formulation.
[0019] In addition, the present invention provides methods for
preparing lipid formulated nucleic acids (e.g., vectors and
enzymes) and prodrugs that can be used in carrying out the methods
of the present invention.
[0020] Other features, objects and advantages of the invention and
its preferred embodiments will become apparent from the detailed
description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 illustrates the relationship of citrate concentration
in the dialysis buffer and the DODAC mol % in the lipid for the
preparation of lipid-plasmid particles. The solid dots represent
good quality formulations having high association efficiencies
(>40%), small size (<100 nm) and low values of size
polydispersity (chi-square less than 10, preferably less than 3) on
a NICOMP particle sizer. The stars represent formulations
containing aggregates having large polydispersity values; and the
open circles represent formulations having low association
efficiencies (<40%). Proper tuning of the citrate buffer
concentration to the cationic lipid charge appears to improve the
formulation. Alternative anionic buffers can also be used if the
counterions can prevent the cationic lipid from aggregating during
the detergent removal step.
[0022] FIG. 2 illustrates the biodistribution of 303i in
various-organs (i.e., blood, spleen and liver) in C57-Lewis Lung
mice.
[0023] FIG. 3 illustrates the accumulation of 303i at the tumor
site in C57-Lewis Lung mice.
[0024] FIG. 4 illustrates a time course of gene product activity at
distal (metastatic) tumor sites.
[0025] FIG. 5 illustrates gene expression in LS180 tumors (dose
response of 303i after 48 hours).
[0026] FIGS. 6(A) and 6(B) illustrate the pattern of HSV-TK gene
expression within peritoneal tumors.
[0027] FIG. 7 illustrates the pINEX-TK10 construct which consists
of a pBR322 derived plasmid containing a CMV promoter linked to a
"hyper" HSV-TK gene, bovine growth hormone polyadenylation sequence
and kanamycin resistance gene.
[0028] FIG. 8(A) illustrates in vivo efficacy studies using a tumor
model.
[0029] FIG. 8(B) illustrates a 16-day treatment regimen on test
mice after tumor inoculation.
[0030] FIG. 9(A) illustrates the in vivo gene expression
protocol.
[0031] FIG. 9(B) illustrates an assessment of the tumor growth,
with the empty formulation showing the largest tumor volume.
[0032] FIG. 9(C) illustrates the efficacy of the suicide gene SPLP
of this invention.
[0033] FIG. 10 illustrates the long-term survival following
treatment with the suicide gene SPLP (i.e., Formulation 1.1 or,
alternatively, INEX 303 or 303i) of this invention.
[0034] FIG. 11 illustrates the efficacy of systemic delivery of
TK303 in the BALB/c CT26 tumor model, i.e., a colorectal tumor
model.
[0035] FIG. 12 illustrates the effect of the TK FS construct and TK
on tumor volume.
DEFINITIONS
[0036] "Sensitizing," as used herein, refers to the ability to
increase the sensitivity of a designated system, such as a cell.
This term includes changing a cell to make it responsive or more
responsive to a compound to which it previously was not responsive,
sensitive or was less sensitive. Sensitizing and "more sensitive"
also includes changes to a cell such that exposure to a previously
nonkilling substance results in cell death.
[0037] "Nucleic acid vector" or "vector," as used herein, refers to
a composition comprising a nucleic acid sequence encoding a gene
product. This is usually a plasmid or viral genome, but can also
include other compositions, such as linear nucleic acids,
protein/nucleic acid conjugates, etc. Depending on the use, vector
can also refer to a nucleic acid delivered in a virus encapsulated
or protein coated format, wherein the entire composition is known
as a vector.
[0038] "Neoplasia," as used herein, refers to any aberrant growth
of cells, tumors, malignant effusions, warts, polyps, nonsolid
tumors, cysts and other growths. A site of neoplasia can contain a
variety of cell types, including neoplastic cells, which harbor
deleterious genetic mutations, normal cells which are induced by
neoplasia, such as vascular endothelia, or immune system cells,
such as macrophages and leukocytes, etc.
[0039] "Therapeutically effective amount," as used herein, refers
to an amount that is sufficient or necessary to give rise to a
desired therapeutic effect. The therapeutic effect can be obtained
directly or indirectly. For instance, the therapeutic agent can
lead to activation of other therapeutic agents or can act in
combination with additional therapeutic agents. For neoplasia, a
therapeutic effect can be, for example, a reduction in growth,
inhibition or reduction in size of the neoplasia or inhibition or
reduction of metastasis and other malignant attributes, or other
beneficial effects, such as subjective or objective observations of
physicians and patients.
[0040] "Gene product," as used herein, refers to a product of a
gene such as an RNA transcript. The RNA transcript can be
therapeutic of its own accord as in the case of antisense or
ribozyme transcription plasmids, or the RNA transcript can be
translated into a polypeptide that is also a gene product.
[0041] "Distal site," as used herein, refers to a physically
separated site, which is not limited to an adjacent capillary bed,
but includes sites broadly distributed throughout an organism.
[0042] "Serum-stable" in relation to lipid/therapeutic nucleic acid
particles means that the particle is not significantly degraded
after exposure to a serum or nuclease assay that would
significantly degrade free DNA. Suitable assays include, for
example, a standard serum assay or a DNase assay such as those
described in the Example section.
[0043] "Systemic delivery," as used herein, refers to delivery that
leads to a broad biodistribution of a compound within an organism.
Some techniques of administration can lead to the systemic delivery
of certain compounds, but not others. Systemic delivery means that
a useful, preferably therapeutic, amount of a compound is exposed
to most parts of the body. To obtain broad biodistribution
generally requires a blood lifetime such that the compound is not
rapidly degraded or cleared (such as by first pass organs (liver,
lung, etc.) or by rapid, nonspecific cell binding) before reaching
a disease site distal to the site of administration. Systemic
delivery of lipid/therapeutic nucleic acid particles is preferably
obtained by intravenous delivery.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0044] The present invention provides methods and compositions
suitable for treating a neoplasia with a therapeutic nucleic acid,
preferably a plasmid encoding an expressible gene, wherein the
therapeutic nucleic acid is encapsulated in a lipid particle and
administered at a site distal to the site of neoplasia, preferably
intravenously, and delivered to the site of neoplasia by systemic
delivery.
[0045] The invention takes advantage of lipid-nucleic acid
particles, wherein the nucleic acid is fully encapsulated and
protected from nuclease degradation, and wherein the particles have
a small diameter (50-200 nm) and have other attributes suitable for
systemic delivery.
[0046] In general, patient therapy using the invention is achieved
as follows. First, the particles are prepared using lipids and a
therapeutic nucleic acid. Thereafter, the particles are
administered to a mammal having a neoplasia, preferably by
intravenous injection at a site that is distal to the site of
neoplasia. The particles are delivered to the site of neoplasia by
the blood, lymph or other internal body fluid. A therapeutically
effective amount of particles accumulates at the site of neoplasia,
and are taken up by cells at the site. Therapeutic effects are
obtained at the site of neoplasia generally by transcription of the
nucleic acid that leads either to a therapeutic transcription
product (i.e., a ribozyme or antisense oligonucleotide or other
RNA), or to an mRNA that is translated into a therapeutic
polypeptide.
[0047] The benefits of systemic gene therapy against tumors using
lipid/therapeutic nucleic acid particles include, but are not
limited to, the following benefits.
[0048] First, nucleic acids are typically nontoxic in comparison to
other chemotherapeutic agents employed. Second, the lipid carrier
systems of the present invention demonstrate preferential
accumulation at many tumor sites. This phenomena depends, in part,
on the "leakiness" of the neo-vasculature of the tumor. This
preferential "passive" targeting can be enhanced by targeting
agents attached to the outer lipid monolayer, such as FGF which
enhances localization of lipid particles at the tumor site (see,
e.g., Forum, et al., "Liposome Targeting in Animal Models," L.
Huang (Ed.), Journal of Liposome Research, 7(4):315-534 (1997)).
Third, therapeutic nucleic acids potentially provide more efficient
therapeutics than convention drugs because they are capable of
replication at the site of disease. The many copies of transcripts
or gene products generated reduce the total number of active agents
that must be delivered to the cells. Further, therapeutic nucleic
acids can be used to deliver a substrate to a cell that can be used
to generate polypeptides, which are too expensive to manufacture or
too difficult to deliver. Fourth, therapeutic nucleic acids, by
leading to synthesis of polypeptides within the disease cell, can
be used to deliver polypeptides to sites inside the disease cell to
which the polypeptide could not be delivered from exogenous routes.
Finally, because of their low toxicity, therapeutic nucleic acids
can be suitable candidates for combination therapy with other
chemotherapeutic agents. A suitable combination therapy approach is
described in U.S. Provisional Patent Application Ser. No.
60/082,665 (TTC Attorney Docket No. 16303-007100), which is
assigned to the assignee of the instant invention and incorporated
herein by reference.
[0049] To date, several approaches for introducing nucleic acids
into cells in vivo have been used. These include liposome based
gene delivery, intratracheal instillation, aerosolized gene
delivery or direct injection. For example, Debs and Zhu WO
93/12240, Debs WO 92/1108 and Debs U.S. Pat. No. 5,641,662 all
describe aerosolized gene delivery of lipid DNA complexes to
mammals. Similarly, Stribling, et al., PNAS, 89:11277-11281 (1992),
describe lipid delivery to mice. McLachlan, et al., Gene Therapy,
2:614-622 (1995), describe DOTAP-mediated lipid delivery of hCFTR
to mice. Canonico, et al., Am. J. Respir. Cell Mol. Biol., 10:24-29
(1994), and Canonico, et al., The American Physiological Society,
415-419 (1994), describe lipofectin-mediated gene delivery of hlAT
to rabbits by aerosolized gene delivery. Alton, et al., Nature
Genetics, 5:135-142 (1993), describe DC-chol:DOPE/DOTAP-mediated
delivery of hCFTR and -gal by aerosol or tracheal instillation to
mice. Capelen, et al., Nature Medicine, 1(1):39 (1995), describe
delivery of CFTR to the nasal epithelia of Humans using a
DC-Chol/DOPE mediated procedure, as does McLachlan, et al., Gene
Ther., 3(12):1113-23 (1996). A variety of reports of administration
of lipid-DNA complexes by parenteral administration have also been
made, including Brigham WO 91/06309, U.S. Pat. No. 5,676,954, and
Debs and Zhu WO 93/24640. Accordingly, a variety of procedures for
transducing cells in vivo using lipid-mediated techniques are
known. However, such procedures have not been shown to be useful
for systemic delivery. Details of preferred formulations of the
present invention are given below.
[0050] A. Therapeutic Nucleic Acids
[0051] The compositions and methods of the present invention are
useful for delivering a wide variety of therapeutic nucleic acids.
These nucleic acids can encode therapeutic polypeptides (ie., any
therapeutic gene product), or therapeutic polynucleotides (i.e.,
antisense or ribozyme transcription plasmids). The therapeutic
nucleic acids of the invention can be expressible genes, such as
those just described, or they can be nucleic acids, which by
themselves, induce some form of response, perhaps by immune system
stimulation.
[0052] For use with the instant invention, the most preferred
therapeutic nucleic acids are those which are useful in
gene-delivered enzyme prodrug therapy ("GDEPT"). Any suicide
gene/prodrug combination can be used in accordance with the present
invention. Several suicide gerie/prodrug combinations suitable for
use in the present invention are cited in Sikora, K. in OECD
Documents, Gene Delivery Systems at pp.59-71 (1996), incorporated
herein by reference, include, but are not limited to, the
following:
1 Suicide Gene Product Less Active ProDrug Activated Drug Herpes
simplex virus ganciclovir(GCV), phosphorylated dGTP type 1
thymidine kinase acyclovir, bromovinyl- analogs (HSV-TK)
deoxyuridine, or other substrates Cytosine Deaminase
5-fluorocytosine 5-fluorouracil (CD) Xanthine-guanine-
6-thioxanthine (6TX) 6-thioguano- phosphoribosyl sinemonophosphate
transferase (XGPRT) Purine nucleoside MeP-dr 6-methylpurine
phosphorylase Cytochrome P450 2B1 cyclophosphamide [cytotoxic
metabolites] Linamarase amygdalin cyanide Nitroreductase CB 1954
nitrobenzamidine Beta-lactamase PD PD mustard Beta-glucuronidase
adria-glu adriamycin Carboxypeptidase MTX-alanine MTX Glucose
oxidase glucose peroxide Penicillin amidase adria-PA adriamycin
Superoxide dismutase XRT DNA damaging agent Ribonuclease RNA
cleavage products
[0053] Any prodrug can be used if it is metabolized by the
heterologous gene product into a compound to which the cell is more
sensitive. Preferably, cells are at least 10-fold more sensitive to
the metabolite than the prodrug.
[0054] Modifications of the GDEPT system that may be useful with
the invention include, for example, the use of a modified TK enzyme
construct, wherein the TK gene has been mutated to cause more rapid
conversion of prodrug to drug (see, for example, Black, et al.,
PNAS (USA), 93:3525-3529 (1996)). Altematively, the TK gene can be
delivered in a bicistronic construct with another gene that
enhances its effect. For example, to enhance the "bystander effect"
also known as the "neighbor effect" (wherein cells in the vicinity
of the transfected cell are also killed), the TK gene can be
delivered with a gene for a gap junction protein, such as connexin
43. The connexin protein allows difflusion of toxic products of the
TK enzyme from one cell into another. The TK/Connexin 43 construct
has been assembled as follows: CMV promoter; TK gene; internal
ribosome entry sequence; and Connexin 43 (prepared by Dr. Clay
Flowers, Inex Pharmaceuticals Corp.).
[0055] In the second step, the prodrug is delivered to the cells.
The prodrug can be the free drug or, alternatively, it can be in a
lipid formulation. The use of lipid formulations has many
surprising and previously undiscovered advantages over the delivery
of free drug in the GDEPT system including, but not limited to,
improved targeting to the disease site transfected by the vector,
prolonged circulation half-life, increased drug loading, reduced
toxicity towards nontarget tissues, improved treatment modalities,
such as a single bolus injection as opposed to IV drip, and the
like. These advantages overcome the limitations of the prior art
GDEPT systems. Further, the liposomal formulation of the prodrug
will preferably provide similar biodistribution to a lipid vector
formulation, thereby concentrating both the vector and the prodrug
at the disease site.
[0056] Usually, the vector will be delivered to the target cell in
advance of the prodrug in order to allow synthesis of the suicide
gene product prior to the arrival of the prodrug. Temporal
separation can be obtained either by separate administration of
vector and prodrug or, alternatively, by providing the formulations
simultaneously, wherein the vector formulation rapidly accumulates
at the target site and delivers the vector, and the prodrug
formulation accumulates or delivers its payload more slowly. As
such, using the compositions and methods of the invention, the
vector is delivered to the cell to direct synthesis of the suicide
gene product, the cell is thereby sensitized, the prodrug is
delivered to the cell, and patient therapy, e.g., reduction of
tumor size, inflammation or infectious load and the like, is
achieved.
[0057] In addition to the GDEPT systems, there exists a very wide
variety of therapeutic nucleic acids that can be employed in the
instant invention. The nucleic acids can be human, nonhuman (i.e.,
from any other plant, animal or microorganism) or entirely
synthetic (i.e., non-naturally occurring). The nucleic acids can be
heterologous or homologous with patient DNA. Since treatment of
neoplasia does not necessarily require long term or stable
expression of the delivered nucleic acid, genes effective in
transient expression systems, such as toxins or immune stimulatory
proteins, are also useful in the methods of the present
invention.
[0058] When the gene is endogenous, a modified sequence, copy
number or transcriptional activity of the gene can be delivered.
The gene product can be directly toxic, indirectly toxic or it can
induce apoptosis or cell differentiation. In the most preferred
system, the gene product of the therapeutic gene will demonstrate
low toxicity to nontarget tissues, and high toxicity to the disease
site. For example, when delivered in the preferred lipid-nucleic
acid particles of the invention, the gene product preferably has
greater toxicity to tumor cells than liver or spleen cells, where a
large portion of particles are normally cleared. Disease site
specificity can also be enhanced by employing tissue/disease
specific promoters for gene transcription or translation. Tissue
specific promoters, and methods of associating them with
therapeutic nucleic acids are known to those skilled in the
art.
[0059] Heterologous genes can be advantageous because their gene
products can also serve to induce an immune response. For example,
genes used in a suicide gene/pro-drug system can have this effect.
Preferred homologous (endogenous) genes suitable for use in the
methods of this invention include, but are not limited to,
pro-apoptotic genes; poreifirin; tumor suppressor genes (p53 and
the like); cytokines (IL-2, IL-12, IL-15, GM-CSF, etc.); Heat Shock
Proteins; Immunodominant Ag (or tumor-specific protein genes);
genes activated in embryos only; TIMP-2 (tissue inhibitor of
metallo proteinase-2) and other metastasis inhibiting proteins;
replacement genes, such as CFTR, DMD; LDL-R and the like; and
anti-angiogenic genes, such as endostatin or angiostatin (see, WO
97/15666; WO 95/29242; Boehm, et al., Nature, 309:404-407 (1997);
and Kerbel, et al., Nature, 309:335 (1997)).
[0060] IL-12 is a preferred homologous gene that can be employed as
a therapeutic nucleic acid in the instant invention (see, Tahara,
H. and Lotze, M. T., Gene Ther., 2:96-106 (1995)). A suitable IL-12
plasmid construct for delivery is pNGVL3-mIL12 provided by the
National Gene Therapy Vector Laboratory at the University of
Michigan (Ann Arbor, Mich.).
[0061] Preferred heterologous genes include, but are not limited
to, genes used in GDEPT combinations (treatment in conjunction with
pro-drugs); ribozymes or transcription plasmids encoding ribozymes
or antisense transcripts; toxin genes, such as saporin, ricin,
diphtheria toxin and cholera toxin (or any other plant, bacterial
or fungal gene); viral protein genes, such as EIA; mutated E6; SV40
Tag, etc. Other heterologous genes suitable for use in the methods
of the present invention will be readily apparent to those of skill
in the art.
[0062] Methods of constructing plasmids or other vectors that carry
the therapeutic nucleic acids disclosed herein are well known to
those skilled in the art (see, e.g., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (Ausubel, et al. (eds.) 1995, the teachings of which are
incorporated herein by reference). Therapeutic activity can be
enhanced by the addition of transcription or translation promoters
and other nucleic acid elements, again, all of which are known to
those skilled in the art.
[0063] Lipid-nucleic acid and the lipid-prodrug formulations can be
achieved by any prior art method. The preferred methods result in a
high efficiency encapsulation, thereby reducing wastage and expense
of the formulation. Lipid-nucleic acid and the lipid-prodrug
formulations can be synthesized using standard freeze-thaw and
extrusion techniques disclosed by Hope, et al., Biochim. Biophys.
Acta, 812:55-65 (1985). In addition, other drug loading and
encapsulation techniques that can be used are disclosed in U.S.
patent application Ser. Nos. 08/399,692, 08/607,614, 08/588,542,
08/741,622, the teachings of which are incorporated herein by
reference. Sizing of the lipid formulations can be achieved using
extruders, pressure cells, and other tools known to those of skill
in the art.
[0064] The possible lipid components of the lipid-nucleic acid and
lipid-prodrug formulations of the invention include those
components typically used in the art. For instance, sphingosomes,
which are disclosed in U.S. Pat. No. 5,543,152, and U.S. patent
applications Ser. Nos. 08/536,584, 08/316,399, 08/485,608,
08/442,267, can be used in the formulations.
[0065] B. Preparing the Lipid/Therapeutic Nucleic Acid Particle
[0066] The lipid-nucleic acid formulations can be achieved using
any prior art method. The preferred methods for systemic (i.e.,
intravenous or other parenteral) delivery result in a
high-efficiency encapsulation, wherein little of the nucleic acid
is exposed to free solution or adsorbed to the outer surface of the
lipid particle. Such methods are disclosed in published PCT Patent
Application WO 96/40964, U.S. Pat. No. 5,705,385, and U.S. patent
applications Ser. Nos. 08/485,458, 08/484,282, 08/660,025,
08/856,374, 60/073,598 and 60/063,473, all of which are assigned to
the assignee of the instant invention and incorporated herein by
reference. Generally, high efficiency encapsulation provides low
immunogenicity and improved tolerance when injected for systemic
delivery. Further, these lipid-nucleic acid particles are
relatively easy to characterize and define compared to cationic
lipid-plasmid aggregates used in local delivery methods.
[0067] Preferred encapsulation methods are set out in the Example
section. The lipid-therapeutic nucleic acid particles obtained by
these methods have identifiable characteristics which make them
suitable for use in the invention. For instance, they are small
particles typically having a mean particle size of about 50 to
about 200 nm and, preferably, of about 60 to about 130 nm. Most
preferably, particles are of a relatively uniform size and have a
.chi..sup.2 (chi-squared) value of less than about 3, more
preferably of less than about 1 and, more preferably, of less than
about 0.5.
[0068] Moreover, the lipid-therapeutic nucleic acid particles of
the present invention are serum-stable and, thus, not significantly
degraded after exposure to a serum or nuclease assay that would
significantly degrade free DNA. Suitable assays for measuring serum
stability include a standard serum assay or a DNase assay (which
are described in the Example section). Nuclease resistance/serum
stability is a measure of the ability of the formulation to protect
the therapeutic nucleic acid from nuclease digestion either in an
in vitro assay or in circulation. The encapsulated particles of the
present invention have greater nuclease resistance and serum
stability than lipid-plasmid aggregates (also known as cationic
complexes), such as DOTMA/DOPE (LIPOFECTIN.TM.) formulations.
[0069] In addition, the lipid-therapeutic nucleic acid particles of
the present invention have a nucleic acid to lipid ratio that can
be formulated at various levels. For use in the methods of this
invention, the particles have a drug to lipid ratio of at least
about 3 mg of nucleic acid per mmol of lipid, more preferably, at
least about 14 mg of nucleic acid per mmol of lipid and, even more
preferably, greater than about 25 mg of nucleic acid per mmol of
lipid. The preferred particles, when prepared to an administration
ready formulation, are about 60-80 mg nucleic acid per mmol lipid
(i.e., they are "high ratio" formulations). The method used for
making high ratio formulations can also be employed using reduced
amounts of DNA to obtain lower ratio formulations. As used herein,
"drug to lipid ratio" refers to the amount of therapeutic nucleic
acid (i.e., the amount of nucleic acid that is encapsulated and
that will not be rapidly degraded upon exposure to the blood) in a
defined volume of preparation divided by the amount of lipid in the
same volume. This may be determined on a mole per mole basis, on a
weight per weight basis, or on a weight per mole basis. For final
administration ready formulations, the drug to lipid ratio is
calculated after dialysis, chromatography and/or nuclease digestion
have been employed to remove as much of the externally associated
therapeutic agent as possible. Drug to lipid ratio is a measure of
potency of the formulation, although the highest possible drug to
lipid ratio is not always the most potent formulation.
[0070] An alternative description of the lipid-nucleic acid
particles of the present invention is "high efficiency"
formulations that emphasizes the active loading process involved
and contrasts with low efficiency or passive encapsulation. Passive
encapsulation of nucleic acid in lipid particles, which is known in
the art, achieves less than 15% encapsulation of therapeutic agent,
and results in low ratio particles having less than 3 mg of nucleic
acid per mmol of lipid. The preferred lipid/therapeutic nucleic
acid particles of the present invention have an encapsulation
efficiency of greater than about 30%. As used herein,
"encapsulation efficiency" refers to absolute efficiency, i.e., the
total amount of DNA added to the starting mixture that ends up in
the administration competent formulation. Sometimes the relative
efficiency is calculated, wherein the drug to lipid ratio of the
starting mixture is divided by the drug to lipid ratio of the
final, administration competent formulation. The amount of lipid
lost during the formulation process may be calculated. Efficiency
is a measure of the wastage and expense of the formulation.
[0071] Other beneficial features that flow from the use of the
preferred particles of the present invention, such as low
nonspecific toxicity, improved biodistribution, therapeutic
efficacy and ease of manufacturing, will be apparent to those of
skill in the art. It is possible to develop particles as described
above by alternative methods of encapsulation. These methods may
employ standard techniques for loading of liposomes that are well
known for use with conventional drugs. These methods include
freeze-thaw extrusion, dehydration/rehydration, reverse phase
evaporation, and the like, some of which are disclosed in Monnard,
et al., "Entrapment of nucleic acids in liposomes," Biochem.
Biophys. Acta., 1329:39-50 (1997). These methods are not high
encapsulation efficiency formulations, nor high ratio formulations,
but the instant disclosure suggests the utility of such particles
in the use of gene therapy against distal tumor sites.
[0072] In addition to the lipids employed in the methods used
above, there are a tremendous number of additional lipid and
nonlipid components which can be used to enhance delivery or
targeting of particles. Additional lipid components include, but
are not limited to, lipids with neutral, anionic, cationic or
zwitterionic headgroups, and the like. These standard components
are set out in the art and in the patent applications referred to
above which are incorporated herein by reference. Charged lipids
that are particularly preferred with the invention are
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), the subject of
recently issued U.S. Pat. No. 5,753,613, incorporated herein by
reference and assigned to the assignee of the instant invention;
and 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP), the subject of
U.S. patent application Ser. No. 08/856,374, the teachings of which
are incorporated herein by reference.
[0073] Both the nucleic acid and prodrug formulations can include
additional components selected from a wide variety of lipids, lipid
conjugates and compatible additional components known in the art.
For instance, cholesterol and its derivatives can be used in the
nucleic acid and prodrug formulations of the present invention.
Still other formulations can employ polycationic compounds that can
condense DNA into small sizes before lipid encapsulation.
Polylysine and polyethyleneimine, among other compounds, have been
used by those of skill in the art in this capacity. Condensed
particles can also be used in the methods of this invention.
[0074] In addition, cloaking agents can be used to reduce
elimination by the host immune system. Such cloaking agents
include, for example, polyamide oligomer-lipid conjugates, such as
ATTA-lipids, disclosed in U.S. patent application Ser. No.
08/996,783, filed Feb. 2, 1998 (TTC Attorney Docket No.
16303-005800) and PEG-lipid conjugates disclosed in U.S. patent
application Ser. Nos. 08/486,214, 08/316,407 and 08/485,608, the
teachings of which are incorporated herein by reference. These
components can also be targeting agents that encourage the lipid
formulations to accumulate at the area of the disease or target
site. In addition, these components can be compounds that improve
features of the formulation, such as leakiness, longevity in
circulation, reduction in toxicity, encapsulation efficiency, etc.
Examples of these components and others that can usefully be
included in the formulations of the invention are known to and used
by those skilled in the art.
[0075] With respect to both the nucleic acid and prodrug lipid
formulations, it is sometimes preferable to employ a programmable
fusogenic lipid formulation. This refers to a formulation that has
little tendency to fuse with cell membranes and deliver its payload
until a given signal event occurs. This allows the lipid
formulation to distribute more evenly after injection into an
organism or disease site before it starts fusing with cells. The
signal event can be, for example, a change in pH, temperature,
ionic environment, or simply time. In this last event, a fusion
delaying or "cloaking" component, such as an ATTA-lipid conjugate
or a PEG-lipid conjugate, can simply exchange out of the liposome
membrane over time. By the time the formulation is suitably
distributed in the body, it is calculated to have lost sufficient
cloaking agent so as to be fusogenic. With other signal events, it
may be desirable to choose a signal event that is associated with
the disease site or target cell, such as increased temperature at a
site of inflammation.
[0076] One of the great advantages of the invention is its
versatility in targeting a broad range of disease sites. In
particular, lipid encapsulated formulations are usefully employed
in targeting and killing tumor cells and other neoplasia, or other
cell types at disease sites that can usefully be modified or
sensitized to perform some other function. Other cell types
include, but are not limited to, cells at sites of inflammation,
sites where genes are aberrantly expressed in sites of infection
and the like.
[0077] In a preferred embodiment, the nucleic acid, e.g., vector,
is delivered in a lipid encapsulated formulation by intravenous
administration. This mode of administration takes advantage of the
known tendency of lipid encapsulated formulations to accumulate at
tumors and neoplasia even without specific targeting aspects. This
ability is thought to be the result of "leaky" vasculature at sites
of neoplasia which is easily invaded by small sized lipid particles
(see, Jain, Sci. Am., 271:58-65 (1994)).
[0078] Where specific cell type targeting is preferred, the lipid
formulation can contain, on the outer surface, antigens or markers
that are recognized by moieties or that recognize receptors on the
target cell. Examples of such targeting can be found in, for
example, Forum, et al., "Liposome Targeting in Animal Models," L.
Huang (Ed.), Journal of Liposome Research, 7(4):315-534 (1997), the
teachings of which are incorporated herein by reference.
[0079] C. Administration-Ready Pharmaceutical Preparations
[0080] Generally, when administered intravenously, the nucleic acid
and/or the prodrug formulations are formulated with a suitable
pharmaceutical carrier. Many pharmaceutically acceptable carriers
may be employed in the compositions and methods of the present
invention. Suitable formulations for use in the present invention
are found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES,
Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). A
variety of aqueous carriers may be used, for example, water,
buffered water, 0.4% saline, 0.3% glycine, and the like, and may
include glycoproteins for enhanced stability, such as albumin,
lipoprotein, globulin, etc. Generally, normal buffered saline
(135-150 mM NaCl) will be employed as the pharmaceutically
acceptable carrier, but other suitable carriers will suffice. These
compositions can be sterilized by conventional liposomal
sterilization techniques, such as filtration. The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc. These compositions can be
sterilized using the techniques referred to above or,
alternatively, they can be produced under sterile conditions. The
resulting aqueous solutions may be packaged for use or filtered
under aseptic conditions and lyophilized, the lyophilized
preparation being combined with a sterile aqueous solution prior to
administration. Carriers may also be employed when delivering the
vector or prodrug formulations by other parenteral methods known in
the art, such as subcutaneous, intratumoral or intramuscular
injection, inhalation, and the like.
[0081] When preparing pharmaceutical preparations of the
lipid/therapeutic nucleic acid particles of the invention, it is
preferable to use quantities of the particles which have been
purified to reduce or eliminate empty particles or particles with
nucleic acid associated with the external surface.
[0082] D. Disease Indications Suitable for Treatment by the
Invention
[0083] The instant invention is particularly useful for treatment
of neoplasia in mammals. Treatment means obtaining a therapeutic
effect at the site of neoplasia. Treatment of a diverse range of
tumors can be obtained using the compositions of this invention. In
addition, the compositions and methods of this invention can be
tested against standard NIH-recommended models. See, for example,
Driscoll, "The preclinical new drug research program of the
National Cancer Institute," Cancer Treatment Reports, 68:63-76
(1984). Further, in vivo and/or in vitro models that are routinely
employed in National Cancer Institute sponsored drug screening
evaluations for identifying utility against human neoplasia can be
employed to confirm the utility of the instant invention (See,
Boyd, M. R., "The NCI In Vitro Anticancer Drug Discovery Screen. In
Anticancer Drug Development Guide" (B. Teicher (ed.), 1995, Humana
Press, Totawa, N.J.).
[0084] Characteristics of neoplasia against which this invention is
useful include, but are not limited to, tumors or neoplasia that
are 1) reasonably transfectable by lipid/therapeutic nucleic acid
particles; 2) are responsive to the gene product of the nucleic
acid; and 3) are not readily accessible to surgical approaches.
Such tumors include metastatic or nonsolid tumors, particularly
micro-metastases, metastasis found outside of the lung, liver or
spleen ("first pass organs"), and the like. Thus, the invention is
applicable to a variety of tumor types. A key characteristic of a
suitable disease indication will be its accessibility to the
lipid/therapeutic nucleic acid particles of the invention.
[0085] E. Combination Therapy
[0086] Combination chemotherapy, a well-known technique employing
conventional drugs for treating cancer, can also be employed using
the lipid-nucleic acid particles of the invention. Combination
chemotherapy treatment has advantages in that: 1) it avoids single
agent resistance; 2) in a heterogenous tumor population, it can
kill cells by different mechanisms; and 3) by selecting drugs with
nonoverlapping toxicities, each agent can be used at full dose.
Combination regimens, which are curative when single agent
treatment is not, include, but are not limited to, the following:
acute lymphocytic leukemia--vincristine, prednisone, doxorubicin
and L-asparaginase; Hodgkin's disease--mechoroethamine,
vincristine, procarbazine and prednisone (MOPP); histiocytic
lymphoma--cyclophosphamide, vincristine, procarbazine and
prednisone (C-MOPP); and testicular carcinoma--bleomycin,
vinblastine, cisplatin.
[0087] Some considerations that are well known in selecting drug
combinations include the following:
[0088] Kinetic considerations: A heterogenous tumor will be treated
first with noncell cycle specific agents (e.g., cyclophosphamide
and doxorubicin) to debulk the tumor and recruit slowly dividing
cells into active DNA synthesis; followed by a cell cycle specific
drug, such as methotrexate and 5-FU. The drug cycle is repeated at
regular intervals.
[0089] Drug resistance considerations: Two or more
noncross-resistant drugs are used simultaneously to avoid selection
of resistant tumor cells. Double resistant mutants may arise by
sequential chemotherapy.
[0090] Drug interactions: Some combinations (i.e., methotrexate and
5-FU) are synergistic when given in the proper sequence,
antagonistic when the order is reversed. Some combinations (i.e.,
L-Asparaginase and methotrexate) are antagonistic initially, but
after an extended period (about 10 days), tumor cells are found to
be more sensitive to methotrexate.
[0091] All these considerations may play a role in the proper
selection of therapeutic genes and anti-neoplastic agents employed.
Some possible combination therapies employing gene therapy and
chemotherapeutic agents are set out in U.S. patent application Ser.
No. 60/082,665 (TTC Attorney Docket No. 16303-007100), assigned to
the assignee of the instant invention and incorporated herein by
reference. It is particularly useful to deliver therapeutic nucleic
acids to neoplasia which have been identified as multi-drug
resistant, in order to resensitize the tumor to the
chemotherapeutic agent.
[0092] F. Dosages of Drugs
[0093] The precise dosage to be administered to a patient, whether
as part of the GDEPT system or as part of combination chemotherapy,
will ultimately be dependant upon the discretion and professional
judgement of the attendant physician and will be in part dependent
on such factors as the age, weight and the particular neoplasia of
the patient. The amounts and precise regime will of course depend
on other factors including the severity of the condition to be
treated.
[0094] In other systems, the exact dosage regime will need to be
detennined by individual clinicians which will be controlled by the
exact nature of the nucleic acid to be delivered and the condition
to be treated, but some general guidance can be given. In general,
dosage can easily range from about 0.1 .mu.g to 1 g or more of
nucleic acid. More preferably, the dose of nucleic acid will range
from about 0.1 .mu.g to about 5 mg per kilogram for a typical 70
kilogram patient, and doses of vectors, which include a viral
particle, are calculated to yield an equivalent amount of
therapeutic nucleic acid.
[0095] In GDEPT systems, a suitable dose of the nucleic-acid lipid
particle will be the amount of nucleic acid which will produce
about 500 to about 200,000 enzyme units/m.sup.2 (e.g., 20,000
enzyme units/m.sup.2). The dose of the prodrug will advantageously
be in the range of about 0.1 to 250 mg per kilogram of body weight
of recipient per day, preferably about 0.1 to 100 mg per kilogram
bodyweight.
[0096] In combination chemotherapy, a suitable dose of the nucleic
acid will typically range from about 0.1 .mu.g to about 5 mg per
kilogram for a typical 70 kilogram patient. In addition, a suitable
dose of the other drug, e.g., the other anti-cancer agent, will
advantageously be in the range of about 0.1 to 250 mg per kilogram
of body weight of recipient per day and, more preferably, in the
range of about 0.1 to 100 mg per kilogram body weight of recipient
per day.
[0097] Typically, the particle will be administered to the patient
and then the uptake and transfection into cells will be monitored,
for example by recovery and analysis of a biopsy sample of the
targeted neoplastic tissue. This can be determined by clinical
trials which involve administering a range of trial dosages to a
patient and measuring the degree of transfection in a target cell
or tumor. In the methods of the current invention, the prodrug will
usually be administered following administration of the nucleic
acid encoding a gene product.
[0098] The invention will be described in greater detail by way of
specific examples carried out in accordance with Canadian Council
on Animal Care, 2nd Ed., "Guide to the care and use of experimental
animals," Eds. Olfert, E., Cross, B. and McWilliam, A. (1993). The
following examples are offered for illustrative purposes, and are
not intended to limit the invention in any manner. Those of skill
in the art will readily recognize a variety of noncritical
parameters which can be changed or modified to yield essentially
the same results.
EXAMPLES
Example 1
[0099] This example illustrates the synthesis of 5 lipid-plasmid
particle formulations for systemic delivery.
[0100] Materials: Plasmids are preferably supercoiled, 4000 to
15000 bp in length, encoding genes and enhancer elements, etc. as
desired. Cationic lipid, N,N-dioleyl-N,N-dimethyl ammonium chloride
("DODAC") and monomethoxy polyethylene2000 glycol
succinate-(C8:0-ceramide) ("PEG-Cer-C8") were synthesized at Inex
Pharmaceuticals Corp. Dioleyl-phosphatidylethanolamine (DOPE) was
supplied by Northern Lipids, Vancouver. Standard dialysis
membranes: Spectro/Por 5 regenerated Cellulose (12-14,000 MWCO) was
purchased from VWR (Manufactured by Spectrum Medical Industries
Inc.). Sodium Citrate was purchased from BDH. Sodium Chloride,
Triton X-100 and Octyl-beta-D-glucopyranoside ("OGP") were obtained
from VWR Scientific, Fisher Scientific or Sigma Chemical
Company.
[0101] Formulation 1.1 (Or, Alternatively, INEX 303 or INEX
303i)
[0102] Plasmid (50-400 .mu.g) is incubated with DODAC in 500 .mu.L
of the prep solution containing 0.2 M OGP in 150 mM NaCl; 5 mM
HEPES pH 7.4, for 30 min at room temperature. This-mixture is added
to a mixture of DOPE and PEG-Cer-C 14 or PEG-Cer-C20 or PEG-Cer-C8
in 500 .mu.L of the same prep solution. The total lipid
concentration was either 5 or 10 mg/mL, with the molar ratio of
DOPE:DODAC:PEG-Cer being 84:6:10. The mixture was dialyzed against
150 mM NaCl; 5 mM HEPES (pH 7.4) for 36-48 h with two buffer
changes.
[0103] Nonencapsulated DNA was removed by anion exchange
chromatography on DEAE-Sepharose column (1.times.4 cm). Empty
liposomes were removed by pooling lipid/DNA samples that co-eluted
on the DEAE column on top of a sucrose density gradient in 12.5 mL
ultracentrifnge tubes. The gradient was formed with 3 mL each of
10% sucrose, 2.5% sucrose and 1% sucrose in HBS layered
consecutively from bottom to top. The gradients were centrifuged at
36,000 rpm (160,000.times.g) for 2 h at 20.degree. C. in a Beckman
Optima XL-100K ultracentriflge using an SW-28 rotor. Separated
bands were removed from top to bottom. Fractions were assayed for
3H-plasmid and 14C-CHE by dual-label scintillation counting using a
Beckman LS6500 scintillation counter. The lipid encapsulated
plasmid DNA banded tightly at the interface between 2.5% and 10%
sucrose, while the unassociated lipid was present as a smear from
the top of the gradient to the interface between 1% and 2.5%
sucrose. The formulation can be concentrated in 12-14,000 MWCO
dialysis tubing against 500,000 MW PEG (Aquacide II). When the
desired volume is reached, the formulation was transferred into a
new dialysis bag and dialyzed overnight against HBS to adjust the
NaCl concentration to 150 mM.
[0104] Formulation 1.2 (Or, Alternatively, INEX351)
[0105] In formulation 1.2 the following concentrations were used.
Lipid concentration: 5.0 mg/mL (or 5.3 mM); plasmid concentration,
200 ig; initial volume, 1.0 mL; lipid stock solutions (in 95:5
benzene:methanol, 2:1 chloroform:methanol or ethanol). Calculated
by molarity (dissolved in 95:5 benzene:methanol or 2:1
chloroform:methanol). DOPE (744 g/mol), 40 mM; DODAC (582 g/mol),
40 mM; and PEG-C8 (2515 g/mol), 20 mM.
[0106] Formulation for 351: 42.5:42.5:15 (mole %)
DOPE:DODAC:PEG-C8
2 DOPE DODAC PEG-C8 mg 1.68 1.315 2.005 mole % 42.5 42.5 15 .mu.mol
2.25 2.25 0.8 .mu.l 56.2 56.2 40
[0107] Formulation Procedure (1 ml scale):
[0108] Aliquot lipid stock solutions into a clean, dry test tube
and dry to a lipid film using a stream of N.sub.2 gas and then dry
under vacuum for at least 2 hrs. Add 50 .mu.L 2M OGP and add 500
.mu.L of 2.times. strength dialysis buffer, add 200 .mu.g of
plasmid and mix by vortexing to dissolve the lipid film. Make up to
1.0 mL with sterile deionized H.sub.2O, mix and allow to incubate
approximately 30 min at room temperature. Place the solution into a
dialysis bag and dialyze for 40-48 hrs against 2 L of dialysis
buffer with 1-2 changes of buffer after approximately 24 hrs, and
determine the volume of the sample by weighing in a tarred tube
(assume density of 1.0). These steps may be followed by DEAE
cleaning and/or sucrose density gradient centrifugation, as
described above.
[0109] After DEAE cleaning and sucrose density centrifugation, as
described above, the final INEX 351 formulation has a concentration
of about 200 .mu.g/ml plasmid and 5 mg/ml total lipid.
[0110] NOTES for INEX351:
[0111] Note 1: Appropriate dialysis buffer concentrations:
[0112] p53: 150 mM NaPO.sub.4+150 mM NaCl (try 140-160 mM NaCl), pH
7.4
[0113] pLuc: +175 mM NaCl (about 150-170 mM NaCl), pH 7.4
[0114] Note 2: 150 mM NaPO.sub.4 buffer, pH 7.4:
[0115] 35.77 g dibasic sodium phosphate (Na.sub.2HPO.sub.4)
[0116] 6.62 g monobasic sodium phosphate (NaH.sub.2PO.sub.4) add
appropriate quantity of NaCl dissolve in 2 L (final volume) of
deionized water with stirring. The final pH may vary between a pH
of about 7.3 and about 7.4; this has not normally been adjusted and
has not affected the performance of the formulation.
[0117] Note 3: Use 0.2 .mu.m filtered buffer with the
lipid/plasmid/detergent solution
[0118] Note 4: As an alternative to adding 2.times. dialysis
buffer, the plasmid may be pre-dialyzed against dialysis buffer and
the formulation may be diluted to its final volume normal strength
dialysis buffer. While this means that there will be a slight
difference in the buffer concentration, this does not affect the
encapsulation efficiency or resulting particle size.
[0119] Note 5: If the volume of the formulation is increased (i.e.,
above 5 mL), add another dialysis change.
[0120] Note 6: DEAE-Sepharose columns are often pre-treated by
eluting 50 .mu.L of a 10 mg/ml extruded or sonicated 1:1
phosphatidylcholine:cholest- erol vesicle formulation (diluted in 2
mL) to block any nonspecific lipid binding to the column.
[0121] To reduce the cationic surface charge of INEX 351
formulations, it may be desirable to reduce the amount of cationic
lipid (i.e., DODAC) employed. If the amount of DODAC is changed,
the amount of DOPE is changed to maintain the same total amount of
lipid. Formulations below 30% DODAC are preferably made in 10 mg
total lipid. Dialysis buffer may be changed as in Table 1,
below:
3TABLE 1 Characterization of representative large scale
formulations. Starting Encapsulation Nicomp particle Conc. volume
Buffer efficiency size (nm).sup.a 42.5% 30 ml 150 mM NaPO.sub.4,
49% 131 130 mM NaCl 30% 12 ml 150 mM NaPO.sub.4 56.8% 109 24% 30 ml
130 mM NaPO.sub.4 50.7% 250 20% 15 ml 105 mM NaPO.sub.4 63% 178
.sup.aNicomp analysis of mean particle size, gaussian dist., volume
weighting, before DEAE cleaning and isolation.
[0122] Formulation 1.3 (Or, Alternatively, INEX321)
[0123] Lipid-plasmid particles with 10-30% DODAC are also useful in
the present invention. These can be formulated, as described above,
or as follows.
[0124] Lipid stock solutions: Individual stock solutions of each
lipid were dissolved in chloroform/methanol (2:1 v/v) to a final
concentration of 2 or 20 mg/mL.
[0125] OGP solution: 1.0 M OGP solution was prepared in MilliQ
grade water.
[0126] Citrate buffer: Sodium citrate buffer was used for dialysis
to remove detergent from the formulation. The citrate
concentrations were varied according to the amount of DODAC. Buffer
also contains 150 mM NaCl and 5 mM HEPES at pH 7.4, unless
indicated otherwise. In general, a 10.times. solution was prepared
and diluted 1:10 in MilliQ Plus water for dialysis using a
graduated cylinder.
[0127] Preparation of lipid/DNA/OGP mixture: A typical formulation
contained 10 mg of lipid of DODAC/DOPE/PEG-Cer-C8 and 200 .mu.g
DNA. Appropriate amounts of stock solutions containing DODAC, DOPE
and PEG-Cer-C8 (normally 15 mol % in this study) were mixed in a
glass test tube. If the amount of DODAC is changed, the amount of
DOPE is changed to maintain a total of 10 mg lipid. The solvent was
first removed under a stream of N.sub.2 gas followed by incubation
under vacuum for 3-5 h. To the lipid, 0.2 mL of 1 M OGP was added.
The suspension was vortexed until the lipid was totally dissolved
and the solution-became clear. Then a 0.2 mL DNA (1 mg/mL) solution
containing 200 .mu.g DNA and 0.6 mL HBS (HEPES buffered saline) or
citrate buffer (concentrations designated in FIG. 1) were added to
a final total volume of 1 mL. If the solution did not become clear,
a small amount of OGP (50 .mu.L) was added. The solution was
incubated at room temperature for 1 h to allow the components to
equilibrate.
[0128] Dialysis: Dialysis tubes were soaked in 60% ethanol (or in
distilled water if sterilization was not required) for 30 min. The
mixture of DNA/lipid/OGP solution was then transferred to the
dialysis tube. The sample was dialyzed for 2 days in 2-4 L citrate
buffer (concentration as described in FIG. 1) with two changes of
buffer daily.
[0129] After preparation, empty liposomes can be removed by DEAE
cleaning and sucrose density centrinugation, as described above.
Having been taught the various lipid-plasmid particle formulations
suitable for systemic delivery in this example, it would be obvious
to one skilled in the art to modify them, for example, for improved
plasmid delivery and/or intracellular expression using one or more
possible variations. Variations of the following type are
suggested: percentage of PEG-lipid; size of PEG; length of
hydrophobic (anchor) chain; pH sensitive PEG-lipids; replacement of
PEG by ATTA (disclosed in U.S. patent application Ser. No.
08/996,783, filed Dec. 22, 1997 (bearing TTC Attorney Docket No.
16303-005800 and which is assigned to the assignee of the instant
invention); addition of membrane modifying lipids, such as
cholesterol or DOPE; use of alternative cationic lipids, such as
DMRIE, DOTAP, DOTMA, DODMA, AL-1, etc.; use of fusogenic
components, such as pH sensitive lipids, peptides (EALA) or
polymers (PEAA); use of targeting agents; use of DNA condensing
peptides (i.e., polylysine or spermine) or polymers (i.e., PEI);
use of negatively charged lipids, such as phosphatidylserine; or
use of alternative PEG-lipid linkers, such as SPDP or PDPH
(disclosed in U.S. patent application Ser. No. 08/536,584, which is
assigned to assignee of the instant invention).
[0130] Formulation 1.4
[0131] Formulation 1.4 contains DOPE:DODAC:PEG-Cer-C20
(83:7:10)-mol %. The synthesis protocol is as follows: Aliquot the
lipid stock solutions (in ethanol) into an autoclaved, clean, dry
round bottom flask. The solution is dried to a lipid film using a
rotavap in a 65.degree. C. water bath and vacuumed overnight. Add
HBS with octylglucopyranoside (OGP) to a final OGP concentration of
200 mM. Swirl the mixture to dissolve the lipid film and, if
necessary, heat to 37.degree. C. to ensure the lipid is fully
dissolved. Plasmid DNA is then added (400 .mu.g/10 mg lipid) to the
dissolved lipid films. After incubation at room temperature for 30
min, place the resulting solution in a dialysis bag that has been
pre-soaked in filter sterilized distilled H2O and autoclaved.
Dialyze overnight against 20 L of dialysis buffer (5 mM HEPES, 150
mM NaCl, pH 7.4, filter sterilized through a 0.2 micron sterile
filter) with two buffer changes.
[0132] Nonencapsulated DNA was removed by anion exchange
chromatography on a DEAE-Sepharose CL-6B column. Collect the
particle suspension as it appears in the eluate, and concentrate
using the Amicon difiltration system (YM 30 membrane). Next, empty
liposomes were removed using a sucrose density gradient. The
gradient was formed by layering 10% sucrose, 5.0% sucrose, and 2.5%
sucrose in HBS, pH 7.4. The sample is loaded by floating it on top
of the 2.5% sucrose layer and centrifuged at 28,000 rpm for 18 h at
20.degree. C. using a Beckman Optima XL-100K ultracentrifuge and an
SW-28 rotor. After centrifugation, remove the lower band with a
syringe and needle and pool the samples. The sucrose is removed and
the sample is concentrated simultaneously using the Amicon system.
Filter sterilize the final volume through a 0.2 micron filter. DNA
concentration is analyzed by Picogreen assays, lipid concentration
by HPLC and particle size by Nicomp analysis.
[0133] Formulation 1.5
[0134] This method, set out in PCT patent, publication WO 96/40964,
which is incorporated herein by reference, is an alternative
high-efficiency formulation of the lipid/nucleic acid particle. It
is, in essence, a preparation of lipid therapeutic nucleic acid
particles in organic solvent. The following stock solutions of
lipid are prepared in 100% ethanol: DSPC--20 mg/mL (20 mol %)=128.4
.mu.L; Chol--20 mg/mL (25 mol %)=113.1 .mu.L; DODAP--40 mg/mL (45
mol %)=44.5 .mu.L; PEG-Cer-C20 (or C14)--50 mg/mL (10 mol %)=67.6
.mu.L.
[0135] The lipids are mixed together and the volume is increased to
a total volume of 0.400 mL with 100% ethanol. An appropriate volume
of 300 mM citrate buffer (pH 3.3) is added to the DNA to a final
volume of 600 .mu.L and pH 3.8. Warm the two solutions to
65.degree. C. for 2 min. While vortexing the DNA tube, use a
Pasteur pipette to add lipid (in ethanol) in a dropwise manner to
the DNA solution. The resulting solution will get cloudy and can
bubble, but no aggregates should be present. Place the solution in
presoaked dialysis tubing (12-14,000 MWCO) and dialyze for 1 h
against 300 mM citrate buffer (pH 3.7-4.0). Transfer the dialysis
tubing to HBS (pH 7.5) and dialyze for 12 h. Nonencapsulated DNA
was removed by anion exchange chromatography using a DEAE-sepharose
column equilibrated in HBS. If necessary, the fmal preparation can
be concentrated using the Amicon system (YM 30 membrane). DNA
concentration is analyzed by Picogreen assays and the lipid
concentration by HPLC.
[0136] All of the above lipid-therapeutic nucleic acid formulations
have beneficial characteristics that make them suitable for use in
the methods of the present invention. Such characteristics include,
but are not limited to, the following: First, they are small
particles with mean sizes of about 50 about 200 nm and, more
preferably, of about 60 to about 130 nm. Most preferably, particles
are of a relatively uniform size and have a 2 value of less than 3,
more preferably, of less than 1 and, even more preferably, of less
than 0.5. Second, they are serum-stable and, thus, are not
significantly degraded after exposure to a serum or nuclease assay
that would significantly degrade free DNA. Third, they have a
nucleic acid to lipid ratio that can be formulated at various
levels. For use in the methods of this invention, particles are
preferably at least about 3 mg nucleic acid per mmol lipid, more
preferably at least about 14 mg per mmol lipid and, most
preferably, greater than about 25 mg per mmol. The lipid-nucleic
acid particles of the present invention have other advantageous
features, such as low nonspecific toxicity, improved
biodistribution, therapeutic efficacy and ease of
manufacturing.
[0137] Assays for Serum Stability
[0138] Lipid/therapeutic nucleic acid particles formulated
according to the above noted techniques can be assayed for serum
stability by a variety of methods.
[0139] For instance, in a typical DNase 1 digestion, 1 .mu.g of DNA
encapsulated in the particle of interest is incubated in a total
volume of 100 .mu.L of 5 mM HEPES, 150 mM NaCl, 10.0 mM MgCl2 pH
7.4. DNase treated samples are treated with either 100 or 10 U of
DNase I (Gibco--BRL). 1.0% Triton X-100 can be added incontrol
experiments to ensure that lipid formulations are not directly
inactivating the enzyme. Samples are incubated at 37.degree. C. for
30 min after which time the DNA is isolated by addition of 500
.mu.L of DNAZOL followed by 1.0 mL of ethanol. The samples are
centrifuged for 30 min at 15,000 rpm in a tabletop microfuge. The
supernatant is decanted and the resulting DNA pellet is washed
twice with 80% ethanol and dried. This DNA is resuspended in 30
.mu.L of TE buffer. 20 .mu.L of this sample is loaded on a 1.0%
agarose gel and subjected to electrophoresis in TAE buffer.
[0140] In a typical serum assay, 50 .mu.g of DNA in free,
encapsulated, or encapsulated +0.5% Triton X100 was aliquoted into
1.5 mL Eppendorf tubes. To the tubes were added 45 .mu.l normal
murine or human serum, dH.sub.2O (to make fmal volume 50 .mu.L).
The tubes were sealed with parafilm and incubated at 37.degree. C.
A sample of the free, encapsulated, or encapsulated +0.5% Triton
X100 not digested by nuclease (standard) was frozen in liquid
nitrogen in an Eppendorf tube and stored at -20.degree. C. Aliquots
were taken at various time points, added to GDP buffer containing
proteinase K (133 .mu.g/mL) and immediately frozen in liquid
nitrogen to stop the reaction. Once all of the time points were
collected, the samples were incubated at 55.degree. C. in a
waterbath to activate proteinase K enabling it to denature any
remaining exonuclease. Proteinase K digested samples were applied
to polyacrylamide gels to assess levels of exonuclease
degradation.
[0141] Particles disclosed above demonstrate serum stability by
showing less than 5% and preferably undetectable amounts of DNA
degradation (partial or total) as a result of such treatment, even
in the presence of 100 U DNase 1. This compares favorably to free
DNA, which is completely degraded, and plasmid/lipid complexes
(such as DOTMA or DODAC:DOPE complexes), wherein DNA is
substantially (i.e., greater than 20%, often 80%) degraded after
such treatment.
Example 2
[0142] This example illustrates the measurement of the therapeutic
effect of lipid formulated ganciclovir on subcutaneous tumors
transfected with lipid encapsulated HSV-TK.
4 Mice per Group Tumor Plasmid Prodrug Route Assay Group A B16 L018
PBS IV Tumor Volume 6 C57 B B16 L018 GCV IV Tumor Volume 6 C57 C
B16 pTK010 PBS IV Tumor Volume 6 C57 D B16 pTK010 GCV IV Tumor
Volume 6 C57
[0143] On day zero, 24 female C57 mice (Harlan Sprague Dawley,
Inc., Indianapolis, Ind.) are seeded sub-cutaneously with 100,000
B16 mice melanoma cells (NCI catalog B 16BL-6) in a total volume of
50 .mu.L (groups A, B, C, D). Tumor volume is determined daily by
measuring the length, width and height of the tumor with skin
calipers as soon as possible and every day thereafter. Groups A to
D are treated with 100 .mu.g plasmid of the appropriate
lipid-formulated plasmid, formulated according to Example 1, once
daily beginning at 9:00 a.m. on day five and on every day
following. The plasmid formulation is injected IV in the tail vein
in a total volume of 200 .mu.L PBS. Groups B and D are treated with
lipid formulated ganciclovir, prepared according to Example 4, once
daily beginning at 3:00 p.m. on day five and on every day
following. 0.5 mg ganciclovir (.about.25 mg/kg) are injected IV in
the tailvein in a total volume of 200 .mu.L PBS. On day 21, mice
are sacrificed. Tumors are collected and weighed.
[0144] The results obtained demonstrate that the mice of group D
either did not develop tumors, or else developed tumors
significantly more slowly than mice of control groups A, B and
C.
[0145] For a 1 mL preparation use 100 mg (180 .mu.mole) of lipid,
of which 55 mole % is sphingomyelin (99 .mu.moles) and 45 mole %
(81 .mu.moles) is cholesterol (Northern Lipids, Vancouver, BC).
Dissolve each lipid in 1 mL of chloroform. Aliquot the required
amounts of each lipid into one tube to obtain a 55/45 SM/Chol
mixture. Next, add 4500 dpm/.mu.mole of lipid of .sup.14C-CHE
(.sup.14C-cholesteryl hexadecyl ether) and dry the lipid to near
dryness under nitrogen. Apply to the lyophilizer overnight and make
up a 70/30% solution of HBS/ethanol (HBS is 20 mM Hepes, 145 mM
NaCl, pH 7.45). Next, dissolve 100 mg ganciclovir (109 mg
ganciclovir-Na, Hoffman LaRoche Ltd.) in 1 mL of 70/30% HBS/ethanol
and vortex well. Add 42000 dpm/.mu.mole 3H-GCV (7.5 .mu.L of a 1
Ci/mL stock) and add ganciclovir solution to the lipid film and
vortex well. Vortex until the solution appears homogeneous.
Freeze-thaw for 5 cycles (liquid nitrogen and 65.degree. C.). Warm
the cryovial up slightly before putting in the water bath. Next,
take 2-10 .mu.L pre-extrusion samples and assay for radioactivity
using the dual label program. Take note of the final volume and use
this to determine specific activity for both the lipid and GCV.
Extrude the sample 2.times. through 3.times.100 nm filters at
65.degree. C. at 350 psi. At this point, the sample becomes quite
viscous. Add 1 mL HBS to the samples and mix by pipetting up and
down. Continue extrusion for a total of 10 passes. Take 2-10 .mu.L
post-extrusion samples and assay for radioactivity. Hydrate some
dialysis tubing (MW cutoff 12,000-14,000) in dH.sub.20 for 15 min.
Next, put the extruded sample in the tubing and dialyze for 1 h
against 2 L HBS. Change to fresh buffer and dialyze overnight. Take
2-10 .mu.L samples and assay for radioactivity and determine the
percentage loading by comparing the pre-extrusion and post-dialysis
ratios of .sup.3H/.sup.14C. For example: .sup.3H/.sup.14C
pre-extrusion=12.0; .sup.3H/.sup.14C post-dialysis=1.2;
1.2/12.0.times.100% =10% encapsulation.
Example 5
[0146] This example sets forth the protocol for stable transfection
of B16 tumor cells with HSV-TK, for use in Examples 6 and 7, as
described in Short Protocols in Molecular Biology, Third Edition,
page 9-13 to 9-15, with the following modifications.
[0147] According to the method, the following materials were used.
Plasmids: pCMVTKIRESneo is based on plasmid pBR322 and includes a
CMV promoter, HSV-TK gene, internal ribosome entry site and
neomycin resistance gene. L018 is also based on pBR322, but
carrying a CMV promoter and a luciferase gene. First, plate B 16
murine melanoma cells in a tissue culture flask (T-75) at
5.times.10.sup.5 cells/flask in 10 mL MEM media with addition of
10% FBS and Glutamine and grow overnight in CO.sub.2 incubator at
37.degree. C. to 70% confluency. Next, aspirate media and feed
cells with 3.8 mL fresh media per flask 2 h prior to transfection
and prepare plasmid/lipid Lipofectin (GIBCO BRL) aggregate in a
polystyrene tube according to manufacturer's instructions.
Alternatively, dilute plasmid to 20 .mu.g/mL in sterile distilled
water. Use Luciferase L018 plasmid as a control for negative
selection in Geneticin (G418), use Thymidine Kinase (neomycin) 20 A
for TKneo stable cells; dilute lipid to 1 mM in sterile distilled
water; dilute lipid to charge ratio 1 in sterile distilled water
(1.2 mL lipid/8 mL water); add volume of DNA (20 mg/mL) to
equivalent volume of lipid (CR1) dropwise while vortexing; and
incubate DNA/lipid complex for 30 min at room temperature. Next,
slowly add 1.2 mL DNA/lipid complex/T75 flask, mix gently and
incubate 24 h in CO.sub.2 incubator at 37.degree. C. (complex is
diluted 1:4 in media). Aspirate media, wash with PBS buffer and
split each T75 flask into 2-100.times.20 mm tissue culture dishes.
Next, 24 h after plating into dishes, add the selective agent,
Geneticin(G418), at the appropriate concentration to kill
nontransfected cells, yet allow cells with transfected TKneo to
stay alive. The Luciferase control cells should die. Every 2-3 days
change the media to remove dead cell debris. Within 10 days clones
are visible on bottom of 100 mm dish which are neomycin resistant
and TK positive. Scrape clones into 1 mL media in 24-well plate and
expand up into T-75 flask. Cells that stably express TK can then be
used for local, regional or systemic injection into mice. To screen
identified clones for TK expression, plate 2000 cells/well in 96
well plate in 150 .mu.L volume and grow 48 h in CO.sub.2 incubator
at 37.degree. C.; add the specific prodrug for TK, ganciclovir, in
a dilution series across the plate made up at 2.5.times.
concentrated and add 100 .mu.L/well (add to 150 .mu.L volume);
incubate 3 days in CO.sub.2 incubator at 37.degree. C.; aspirate
media from wells and add Alamar Blue as per manufacturers
instructions (Biosource International) (1:10 dilution in media);
and 100 .mu.L/well and incubate for 1, 2, 4 h and read plate at
time intervals on fluorescent plate reader (550, 595 nm; 750V; 70
offset; 100 ms integration time).
Example 6
[0148] This example illustrates the effects of systemically
delivered lipid-formulated ganciclovir on tumor growth in mice
having B16 intradermal tumors stably transfected with HSV-TK.
5 Timepoint Mice per Group Tumor Prodrug Route Assay (assay) Group
A B16 PBS IV Tumor DAILY 8 C57 Volume B B16 TK PBS IV Tumor DAILY 8
C57 Volume C B16 LIPO-GCV IV Tumor DAILY 8 C57 Volume D B16 TK
LIPO-GCV IV Tumor DAILY 8 C57 Volume * It is noted that the "Route"
refers to the delivery of the prodrug, i.e., gangciclovir
(GCV).
[0149] 32 female C57 mice (Harlan Sprague Dawley, Inc.,
Indianapolis) were seeded intradermally in groups A and C with B16
parental control cells, and in groups B and D with B16 tumor cells
stably transfected and expressing HSV-TK (prepared as in previous
described) at a dose of 150,000 cells in a total volume of 50 .mu.L
phosphate buffered saline on day zero. Intradermal tumor volume was
determined daily by measuring the length, width and height of the
tumor with skin calipers as soon as possible and every day
thereafter.
[0150] The mice were treated with the ganciclovir prodrug, lipid
formulated as in Example 4, once every two days beginning on day
four and on every second day following. The ganciclovir dosage of
0.5 mg (.about.25 mg/kg) was injected IV in a total volume of 200
.mu.L PBS (phosphate buffered saline). Mice received a total of
nine treatments. On day 21, mice were sacrificed. Tumors were
collected and weighed prior to fixation for sectioning.
[0151] Intradermal tumors stably transfected with HSV-TK showed no
measurable growth when treated systemically with lipid formulated
ganciclovir. Untreated B16 tumors, and treated B16 tumors without
TK, were not effected by the drug.
Example 7
[0152] This example was carried out to determine the effect of
lipid formulated ganciclovir on TK gene expression in B16 tumor
cells stably transfected with HSV-TK and implanted
intravenously.
6 Timepoint Mice per Group Tumor Prodrug Route Assay (assay) Group
A B16 TK PBS IV Tumor DAILY 8 C57 Volume B B16 TK LIPO-GCV IV Tumor
DAILY 8 C57 Volume * It is noted that the "Route" refers to the
delivery of the prodrug, i.e., gangciclovir (GCV).
[0153] 16 female C57 mice were seeded with B16 tumor cells stably
expressing HSV-TK by tail vein injection at a dose of 150,000 cells
in a total volume of 200 .mu.L phosphate buffered saline on day
zero. The mice were treated with the ganciclovir prodrug, lipid
formulated as in Example 4, once every day beginning on day two and
on the two days following. The ganciclovir dosage of 0.5 mg
(.about.25 mg/kg) was injected IV in a total volume of 200 .mu.L
PBS (phosphate buffered saline). Mice received a total of three
treatments. On day 21, mice were sacrificed and tumors were scored.
Livers, lungs, spleen and pancreas were photographed. There was a
significant reduction in both size and number of metastatic tumor
nodules.
Example 8
[0154] This example illustrates gene expression in distal
metastatic tumors using Formulation 1.1 lipid plasmid
particles.
[0155] On day zero, C57BL/6 mice (Harlan Sprague Dawley, Inc.,
Indianapolis, Ind.) are seeded with 300,000 LL/2 (Mouse Lewis Lung
Carcinoma) cells (ATCC CRL-1642) by intravenous/tail vein injection
with total volume 200 .mu.L. On day 10, the mouse is intravenously
injected with formulation 1.1 plasmid-lipid particles. The
particles carry plasmid L018, which is a standard construct
containing the luciferase gene linked to the CMV promoter. At
various time points after plasmid injection, mice are sacrificed,
and organs and tumors are quickly frozen in liquid nitrogen, then
stored at -70.degree. C. Organs/tumors are assayed for the
luciferase gene to demonstrate delivery to the organ/tumor site.
Biodistribution results for organs are shown in FIG. 2.
Accumulation at the tumor site is illustrated in FIG. 3. Southern
blot data shows presence of intact plasmid at the tumor site
increasing to at least 96 h. Cell protein from organs/tumors is
also prepared and assayed for luciferase according to standard
techniques. A time course of gene product activity at distal
(metastatic) tumor sites is demonstrated in FIG. 4.
Example 9
[0156] This example illustrates the systemic vector delivery and
gene expression in an in vivo human tumor.
[0157] SCID mice (Harlan Sprague Dawley, Inc., Indianapolis, Ind.)
are seeded with 1.times.10.sup.6 LS180 human colon adenocarcinoma
cells (ATCC CL-187) by subcutaneous injection on day zero. On day
11, mice in groups A, B and C are injected intravenously with
indicated doses of L018 plasmid in lipid formulation 1.1, in 200
.mu.L total volume. On day 17, mice in group D and E are injected
intravenously with L018 plasmid in lipid Formulation INEX 320,
using C8 or C20 PEG-Cer) according to Example 1, in 200 .mu.L total
volume. At the times indicated after plasmid injection, mice are
sacrificed and organs (liver, spleen and lungs) and tumors are
harvested. Expression of the enzyme Luciferase is assayed according
to standard techniques on all samples.
[0158] The data obtained demonstrates the excellent transfection
and expression of the reporter gene Luciferase achieved in an in
vivo human tumor using the lipid-nucleic acid particles of the
present invention (see, FIG. 5).
7 Group Formulation Assay Time Point Mice per group A 1.1 (75
.mu.g) Luciferase 48 h 5 B 1.1 (100 .mu.g) Luciferase 48 h 5 C 1.1
(125 .mu.g) Luciferase 48 h 5 D 320 (100 .mu.g) Luciferase 24 h 4 E
320 (100 .mu.g) Luciferase 48 h 4 F PBS Luciferase 48 h 1
Example 10
[0159] This example demonstrates systemic delivery and expression
at an in vivo tumor site of a vector containing the HSV-TK gene,
using a lipid-nucleic acid particle prepared according to Example
1.
[0160] C57 mice are intraperitoneally seeded with 100,000 B16 tumor
cells in a total volume of 200 .mu.L PBS on day zero. On day 14,
test mice are injected with Formulation 1.2 (100 .mu.g DNA in 500
.mu.L PBS) prepared according to Example 1. The plasmid vector used
is pINEX-TK10 as described earlier. 24 h later, mice are
sacrificed, and tumors are isolated, fixed within 5 min, and
prepared in paraffm sections using standard techniques. The
expression of the HSV-TK gene at the distal tumor site is assayed
by in situ RNA/RNA hybridization using techniques standard in the
art. One such technique is summarized below.
[0161] The pattern of HSV-TK gene expression within peritoneal
tumors is demonstrated in FIGS. 6(A) and (B). In all cases of gene
expression, positive signal is observed as a cellular content of
B16 cells or endothelial cells. Positive stained cells are
localized in proliferative zone associated with blood vessels or
peripheral area.
Example 11
[0162] This example describes the treatment of tumors using the
method of the invention. In particular, this example demonstrates
the effect of pINEX-TK10 in Formulation 1.1, in inhibiting the
growth of MCA-207 tumors following treatment with ganciclovir. The
general method is set out in FIG. 8.
[0163] The pINEX-TK10 construct consists of a pBR322 derived
plasmid containing a CMV promoter linked to a "hyper" HSV-TK-gene,
bovine growth hormone polyadenylation sequence and kanamycin
resistance gene. "hyper" HSV-TK is a more active form of the HSV-TK
gene as disclosed by Black, et al., PNAS (USA), 93:3525-3529
(1996). The plasmid construct employed is set forth in FIG. 7.
8 # of Group Formulation GCV Route* Assay Timepoint Mice A PBS PBS
IP Volume/ -- 6 C57 CTL B Empty 1.1 PBS IP Volume/ -- 6 C57 CTL C
1.1 TK PBS IP Volume/ -- 6 C57 CTL D 1.1 TK GCV IP Volume/ -- 6 C57
CTL *It is noted that the "Route" refers to the delivery of the
prodrug, i.e., gangciclovir.
[0164] 24 female C57 mice were seeded with 100,000 MCA-207
fibrosarcoma tumor cells (provided by S. Rosenberg, National Cancer
Institute, Frederick/Bethesda, MD) by intra-dermal injection on day
zero. The tumor cells had been cultivated and prepared according to
standard techniques using RPMI media with 10% Fetal Bovine Serum
(see for example Current Protocols in Molecular Biology). Beginning
on day 5, all animals were treated with the lipid/therapeutic
nucleic acid formulation listed in the chart, supra. The
formulation was delivered intravenously through the tail vein. 80
.mu.g of pINEX-TK10 DNA were injected in a total volume of 200
.mu.L. Treatments were administered on days 5, 7, 9, 11 and 13.
[0165] Beginning on day 5, all animals were treated with
ganciclovir twice daily. 1 mg (.about.50 mg/kg) were injected
intra-peritoneally in a total volume of 200 .mu.L PBS. Treatments
continued twice daily for 12 days (see, FIG. 9(A)). Mice were
monitored for tumor growth.
[0166] FIG. 9(B) sets out in more quantitative terms the effect of
the treatments. Mice treated with HSV-TK in formulation 1.1 have
greatly reduced tumors compared to control treated mice. Not shown
is data of control mice which demonstrates that treatment with
empty liposomes and ganciclovir has no effect on tumor reduction.
FIG. 9(C) demonstrates the effect of the treatment on test mice in
comparison with control mice at day 16 after tumor inoculation.
Example 12
[0167] This example illustrates the protocol for RNA/RNA in situ
hybridization of in vivo tumors transfected by systemically
delivered plasmid.
[0168] Tumors are prepared for in situ investigation by paraffin
embedding and staining. Specifically, peritoneal tumors are
collected into 4% paraformaldehyde/PBS fixative (Sigma Chemical
Co.) and fixed overnight at 4.degree. C. Serial 5-.mu.m sections
are prepared after the samples have been dehydrated in graded
ethanol solutions, cleared in chloroform and embedded in paraffin
wax (Paraplast Plus, Fisher).
[0169] When ready to be used, prepared samples are treated with two
changes of xylene for 10 min, each followed by rehydration in
graded ethanol solutions to 50% ethanol. Samples are prehybridized
by standard rinsing, incubation with 0.1 M triethanolamine (TEA)
buffer, pH 8.0, containing 0.25% (v/v) acetic anhydride, followed
by treatment at 56.degree. C. for at least 60 min in hybridization
buffer containing: 40% deionized formamide, 10% dextran sulfate,
1.times. Denhardt's solution (0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 10 mg/mL RNase-free bovine serum albumin),
4.times.SSC, 10 mM DTT, 1 mg/mL yeast t-RNA, and 1 mg/mL denatured
and sheared salmon sperm DNA.
[0170] Labelling of RNA probe by in vitro transcription of DNA was
as follows. The fragment of 599 bp (532-1131) from HSV-TK (PTK10)
was cloned into KpnI and BamHI sites of the vector pGEM-7Zf(+)
(PTK11). The plasmid is cloned by standard techniques and prepared
using Qiagene 500 (Qiagen, Inc.). For the anti-sense probe, this
plasmid is linearized by cutting it with KpnI at the original 5'
end of the cDNA HSV-TK and purified. The same logic is used for
sense (control) probe (i.e., cut at the side of the 3-end of insert
by BssHII or BamH or SacI). The plasmid is purified by ethanol
precipitation. The following are then mixed in a 1.5 mL sterile
RNase free microcentrifuge tube on ice: 4 .mu.L (4 .mu.g) purified,
linearized plasmid DNA, 5 .mu.L of 10.times. concentrated DIG RNA
Labeling Mix (supplied by manufacturer), 10 .mu.L 5.times.
concentrated Transcription Buffer (400 mM Tris-HCl (pH 8.0,
20.degree. C.), 60 mM MgC12, 100 mM Dithiothreitol (DTT), 20 mM
spermidine], 2 .mu.L RNasin, 3 .mu.L RNA polymerase (SP6 for
antisense or T7 for sense), and sterile, redistilled water to make
a total reaction volume of 50 .mu.L.
[0171] The components are mixed and centrifuged briefly, and then
incubated for 2 h at 37.degree. C. (for T7 RNA polymerase) or at
40.degree. C. (for SP6 polymerase). After incubation, add 3 .mu.L
DNase I, RNase free and 1 .mu.L RNasin to the tube and incubate for
15 min at 37.degree. C. Then add 2.5 .mu.L 0.5 M EDTA (pH 8.0) to
the tube to stop the polymerization reaction.
[0172] The labeled RNA transcript is precipitated by performing the
following steps. Add to the reaction tube 6.25 .mu.L 4 M LiCi and
180 .mu.L prechilled (-20.degree. C.) 100% ethanol incubate
overnight at -70.degree. C. Centrifuged the tube (at
13,000.times.g) for 15 min at 4.degree. C. Discard the supernatant.
Wash the pellet with 50 .mu.L ice-cold 70% (v/v) ethanol.
Centrifuge the tube (at 13,000.times.g) for 5 min at 4.degree. C.
Discard the supernatant and dry the pellet at room temperature.
Dissolve the RNA pellet for 30 min at 37.degree. C. or (R.T.) in 20
.mu.L DEPC (diethylpyro-carbonate)-treated, sterile, redistilled
water, added 20 .mu.L deionized formamide and 1 .mu.L RNasin. Keep
transcript at -20.degree. C. or -70.degree. C.
[0173] An accurate quantification of DIG-labeled RNA obtained in
the labeling reaction is most important for optimal and
reproducible results in various membrane or in situ hybridization
techniques. Too high of a probe concentration in the hybridization
mix usually causes background, while too low of a concentration
leads to weak signals. The estimation of yield can be performed in
a side by side comparison of the DIG-labeled sample nucleic acid
with a DIG-labeled control, that is provided in the labeling kits.
Dilution series of both are prepared and spotted on a piece of
membrane. Subsequently, the membrane is colorimetrically detected.
Direct comparison of the intensities of sample and control allows
the estimation of labeling yield.
[0174] The hybridization reaction is then performed. Drain
pre-hybridization buffer from the pre-hybridized slides and overlay
each section with 200 .mu.L of hybridization buffer containing
0.2-1 ng of digoxigenin-labeled RNA probe (0.2 ng/.mu.L). Cover
samples with a 24.times.30 mm hydrophobic plastic coverslip.
Incubate sections at 56.degree. C. overnight in a humid chamber.
Washes can include an RNAse step which reduces the background, but
decreases the signal as well. It is important to keep the tissue
sections moist at all times during washing. Wash the slides in
2.times.SSC at 55.degree. C. for 30 min; wash in 50% formamide,
2.times.SSC at 65.degree. C. for 30 min; wash in 2.times.SSC 3
times at 37.degree. C. for 5 min each; wash in RNase 10 .mu.g/mL,
lxwashing solution at 37.degree. C. for 30 min; wash in 50%
formamide, 2.times.SSC at 65.degree. C. for 30 min; wash in
2.times.SSC at 37.degree. C. for 15 min; wash in 02.times.SSC 5
times at 37.degree. C. for 5 min each.
[0175] After hybridization, cells are incubated DIG-specific
antibody. Wash the slides in TBS at RT for 30 min. Incubate
sections with blocking solution (TBS and 2% goat serum) at RT for 1
h. Decant blocking solution and incubate sections with goat
anti-DIG-alkaline phosphatase (Fab fragment) dilution 1:500 at RT
for 1 h. Wash the slides in TBS at RT for 30 min. Wash the slides
in substrate buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM
MgCl2) at RT for 30 min. Prepare a color solution containing: 10 mL
substrate buffer, 50 .mu.L NBT (nitroblue tetrazolium) and 37 .mu.L
BCIP. Slides are immersed in color solution at room temperature for
1-2 h or at 4.degree. C. for 2-3 days. Slides are washed with water
and observed by light microscopy. Results are shown in FIGS. 6(A)
and (B).
Example 13
[0176] This example illustrates the treatment of tumors using the
methods and compositions of the invention. In particular, this
example demonstrates the long-term survival of mice bearing MCA-207
tumors that were treated with a course of intravenous pINEX-TK10 in
Formulation 1.1 combined with intra-peritoneal injections of
ganciclovir (GCV).
[0177] The pINEX-TK10 construct, as described above, was formulated
in Formulation 1.1, as described above. C57 mice were seeded with
100,000 MCA-207 fibrosarcoma tumor cells (provided by S. Rosenberg,
National Cancer Institute, Frederick/Bethesda, Md.) by intra-dermal
injection on day zero. The tumor cells had been cultivated and
prepared according to standard techniques using RPMI media with 10%
Fetal Bovine Serum (see for example Current Protocols in Molecular
Biology).
[0178] There were a total of 20 mice in each of the treatment
groups and the groups were treated as follows: Group I, ganciclovir
alone (GCV); Group II, empty SPLPs and ganciclovir (Empty
SPLP+GCV); Group III, formulated plasmid alone (INXC-gTK); and
Group IV, formulated plasmid together with ganciclovir
(INXC-gTK+GCV). The lipid/therapeutic nucleic acid formulation was
delivered intravenously through the tail vein. 80 .mu.g of
pINEX-TK10 DNA were injected in a total volume of 200 .mu.L PBS.
Treatments with the lipid/therapeutic nucleic acid formulation were
administered on days 5, 7, 9, 11 and 13. Animals were treated with
ganciclovir twice daily. 1 mg (.about.50 mg/kg) were injected
intra-peritoneally in a total volume of 200 .mu.L PBS. Treatments
continued twice daily for 12 days. Mice were monitored for
long-term survival.
[0179] FIG. 10 sets out in quantitative terms the effect of the
treatments. Mice treated with HSV-TK in Formulation 1.1 together
with ganciclovir have greatly increased long-term survival. Only
modest effects were observed following treatment with HSV-TK in
formulation 1.1 alone compared to untreated controls. Such results
demonstrate the successful use of a nonviral gene transfer system
that can produce regression in distal tumors following intravenous
delivery.
Example 14
[0180] This example illustrates the efficacy of systemic delivery
of TK303 in the BALB/c CT26 tumor model, i.e., a colorectal tumor
model.
9 Group Formulation GCV Route Assay # of Mice A HBS PBS IP
Volume/CTL 8 BALB/c B Empty 303 PBS IP Volume/CTL 8 BALB/c C Empty
303 GCV IP Volume/CTL 8 BALB/c D 303 TK PBS IP Volume/CTL 8 BALB/c
E 303 TK GCV IP Volume/CTL 8 BALB/c
[0181] On day zero, 48 female BALB/c mice were seeded with 100,000
CT26 tumor cells (ID) using the protocol described in the above
examples. Beginning on day 5, all animals were treated with
formulation delivered IV through the tail vein. 50 .mu.g of DNA was
injected IV in a total volume of 200 .mu.L PBS. Treatment was
continued every other day for the next eight days.
[0182] Groups C and E were treated with lipid formulated
ganciclovir once daily on day five and on every day following. 1.5
mg (.about.75 mg/kg) was injected IV in the tail vein in a total
volume of 300 .mu.L PBS. Mice were monitored for survival. If
tumors developed, mice were sacrificed and the tumors collected and
weighed. The results obtained demonstrate that the mice of Group E
either did not develop tumors or else developed tumors
significantly more slowly than the other mice (see, FIG. 11).
Example 15
[0183] This example illustrates the efficacy of systemic delivery
of TK303 in the BALB/c CT26 tumor model, i.e., a colorectal tumor
model.
10 Group Formulation GCV Route Assay # of Mice A HBS PBS IP Volume
8 BALB/c B Empty 303 PBS IP Volume 8 BALB/c C Empty 303 GCV IP
Volume 8 BALB/c D 303 TK FS* PBS IP Volume 8 BALB/c E 303 TK FS*
GCV IP Volume 8 BALB/c F 303 TK PBS IP Volume 8 BALB/c G 303 TK GCV
IP Volume 8 BALB/c *The vectors used in this experiment were as
follows: "TK" has the thymidine kinase coding sequence in its
proper reading frame, such that thymidine kinase protein is
expressed. "TK FS" has the thymidine kinase coding sequence out of
frame relative to the translation initiation codon so that no
thymidine kinase protein is produced.
[0184] On day zero, 65 female BALB/c mice were seeded with 100,000
CT26 tumor cells (ID) using the protocol described in the above
examples. Beginning on day 5, all animals were treated with
formulation delivered IV through the tail vein. 50 .mu.g of DNA-was
injected IV in a total volume of 200 .mu.L PBS. Treatment was
continued every other day for the next eight days.
[0185] Groups C, E and G were treated with lipid formulated
ganciclovir once daily on day five and on every day following. 1.0
mg (.about.50 mg/kg) was injected IV in the tail vein in a total
volume of 200 .mu.L PBS. Mice were monitored for survival. If
tumors developed, mice were sacrificed and the tumors collected and
weighted. Mice in Group G, which received GCV and the lipid
formulated TK construct, and thus expressed the TK protein,
exhibited a marked reduction in tumor growth rate (see, FIG.
12).
[0186] It is to be understood that the above description is
intended to be illustrative and not restrictive. Many embodiments
will be apparent to those of skill in the art upon reading the
above description. The scope of the invention should, therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the appended claims, along
with the full scope of equivalents to which such claims are
entitled. The disclosures of all articles and references, including
patent applications and publications, are incorporated herein by
reference for all purposes.
* * * * *