U.S. patent application number 10/003494 was filed with the patent office on 2002-06-27 for nucleic acid delivery.
Invention is credited to Conroy, Susan E., Engler, Heidrun, Maneval, Daniel C..
Application Number | 20020081736 10/003494 |
Document ID | / |
Family ID | 27357417 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081736 |
Kind Code |
A1 |
Conroy, Susan E. ; et
al. |
June 27, 2002 |
Nucleic acid delivery
Abstract
The present invention provides formulations and methods to
enhance the delivery of nucleic acids to cells. Formulations
comprising dextrin polymers in combination with sugars provide
enhanced delivery of nucleic acids, particularly eucaryotic
expression vectors, demonstrate enhanced delivery of nucleic acids
to cells in vivo. The present invention also provides methods of
treatment in combination with such formulations.
Inventors: |
Conroy, Susan E.; (London,
GB) ; Engler, Heidrun; (San Diego, CA) ;
Maneval, Daniel C.; (San Diego, CA) |
Correspondence
Address: |
Richard B. Murphy
Chief Counsel
Canji, Inc.
3525 John Hopkins Court
San Diego
CA
92121
US
|
Family ID: |
27357417 |
Appl. No.: |
10/003494 |
Filed: |
November 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60245539 |
Nov 3, 2000 |
|
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60287871 |
Apr 30, 2001 |
|
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Current U.S.
Class: |
435/455 ;
514/44R; 514/53; 514/58 |
Current CPC
Class: |
A61K 31/715 20130101;
C12N 15/86 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 38/1758 20130101; A61K 2300/00
20130101; C12N 2710/10343 20130101; A61K 31/724 20130101; A61K
48/00 20130101; A61K 38/1758 20130101; A61K 48/0008 20130101; Y10S
435/932 20130101; C12N 15/87 20130101; C12N 2710/10332 20130101;
A61K 35/761 20130101; A61K 31/724 20130101; A61K 35/761 20130101;
A61K 31/715 20130101 |
Class at
Publication: |
435/455 ; 514/44;
514/53; 514/58 |
International
Class: |
A61K 048/00; C12N
015/87; A61K 031/715 |
Claims
1. A method to deliver nucleic acid to a cell wherein the nucleic
acid is carried in a solution comprising dextrin and at least one
sugar, the osmolarity of which corresponds substantially to the
physiological osmolarity of the milieu surrounding the cell.
2. The method according to claim 1 wherein the molecular weight of
the dextrin is in the range from about 1,000-200,000.
3. The method according to claim 2 wherein the molecular weight of
the dextrin is between about 2,000-55,000.
4. The method according to claim 3 wherein the dextrin contains
more than about 15% w/v of polymers of a degree of polymerisation
greater than 12.
5. The method according to claim 4 wherein the dextrin contains
more than about 50% w/v of polymers of a degree of polymerisation
greater than 12.
6. The method according to claim 5 wherein the dextrin is present
in the solution in an amount of less than about 20% w/v.
7. The method according to claim 6 wherein the dextrin is present
in the solution in an amount selected from about: 1% w/v; 2% w/v;
3% w/v; 4% w/v; 5% w/v; 6% w/v; 7% w/v; 8% w/v; 9% w/v; 10% w/v;
11% w/v; 12% w/v; 13% w/v; 14% w/v; 15% w/v; 16% w/v; 17% w/v; 18%
w/v; 19% w/v; 20% w/v.
8. The method according to claim 7 wherein the dextrin is present
from 1-5% w/v.
9. The method according to claim 8 wherein the dextrin is
preferably about 4% w/v.
10. The method according to any of claim 1 wherein the sugar is a
disaccharide.
11. The method according to claim 10 wherein the amount of
dissacharide is between about 1 and 10% w/v.
12. The method according to claim 11 wherein the dissacharide is an
amount of between about 2 and 5% w/v.
13. The method according to claim 12 wherein the dissacharide is
sucrose and the amount of sucrose is about 3% w/v.
14. The method according to claim 1 wherein the amount of dextrin
is about between 2%-20% w/v and the amount of sucrose is between
about 1-10% w/v.
15. The method according to claim 14 wherein the amount of dextrin
is about 15% w/v and the amount of sucrose is about 3% w/v.
16. The method according to claim 15 wherein the amount of dextrin
is about 4% w/v and the amount of sucrose is about 3% w/v.
17. The method according to claim 1 wherein the solution further
comprises a divalent cation.
18. The method according to claim 17 wherein the divalent cation is
in a concentration of at least 0.2 mM.
19. The method according to claim 19 wherein the divalent cation
concentration is between 0.2-3.0 mM.
20. The method according to claim 19 wherein the divalent cation is
provided by MgCl.sub.2 and the concentration is about 2.0 mM.
21. The method according to claim 20 wherein the solution comprises
about 4% w/v dextrin, about 3% w/v sucrose and about 2.0 mM
MgCl.sub.2.
22. The method according to claim 1 wherein the nucleic acid
molecule is a vector.
23. The method according to claim 22 wherein the vector is adapted
for eukaryotic expression.
24. The method according to claim 22 wherein the vector is a
recombinant virus derived from adenovirus, retrovirus,
adeno-associated virus, herpesvirus, lentivirus, vaccinia virus, or
baculovirus.
25. The method of claim 24 wherein said vector is an
adenovirus.
26. The method of claim 25 wherein said adenovirus further encodes
an exogenous transgene.
27. The method of claim 26 wherein said transgene is a tumor
suppressor gene.
28. The method of claim 27 wherein said tumor suppressor gene is
p53.
29. The method of claim claims 24 to 26 wherein said vector is
replication competent.
30. The method of claim 29 wherein said vector is a conditionally
replicating replication competent vector.
31. A composition for delivery of a recombinant viral vector to a
cell comprising a recombinant viral vector in a solution comprising
dextrin and at least one sugar, the osmolarity of which corresponds
substantially to the physiological osmolarity of the milieu
surrounding the cell.
32. The composition of claim 31 wherein the recombinant viral
vector is derived from adenovirus, retrovirus, adeno-associated
virus, herpesvirus, lentivirus, vaccinia virus, or baculovirus.
33. The composition of claim 32 wherein the viral vector is derived
from adenovirus.
34. The composition of claim 33 wherein the viral vector is a
selectively-replicating replication competent adenoviral
vector.
35. A pharmaceutical formulation comprising a recombinant
adenoviral vector, a dextrin, a sugar and a divalent cation.
36. A method to deliver nucleic acid to a cell comprising:
providing a solution of dextrin, a sugar, a divalent cation and
nucleic acid and contacting the solution with a cell to be
transfected or transformed.
Description
RELATION TO OTHER APPLICATIONS
[0001] The present application claims the benefit of U.S. Patent
Provisional Application Serial No. 60/245,539 filed Nov. 3, 2000
and U.S. Provisional Application Serial No. 60/287,871 filed Apr.
30, 2001 pursuant to 35 U.S.C. .sctn. 119(e).
FIELD OF THE INVENTION
[0002] The invention relates to a method for the delivery of
nucleic acid to cells, particularly but not exclusively, in gene
therapy, including compositions the osmolarity of which corresponds
substantially to the physiological osmolarity which surrounds the
cell or tissue
BACKGROUND OF THE INVENTION
[0003] Many methods have been developed over the last 30 years to
facilitate the introduction of nucleic acid into cells which have
greatly assisted, inter alia, our understanding of the control of
gene expression.
[0004] Conventional methods to introduce DNA into cells are well
known in the art and typically involve the use of chemical
reagents, cationic lipids or physical methods. Chemical methods
which facilitate the uptake of DNA by cells include the use of
DEAE-Dextran (Vaheri and Pagano, Science 175:434). DEAE-dextran
associates with and introduces the DNA into cells. However this can
result in loss of cell viability. Calcium phosphate is also a
commonly used chemical agent which, when co-precipitated with DNA,
introduces the DNA into cells (Graham et al Virology (1973) 52:
456).
[0005] The use of cationic lipids (e.g. liposomes (Felgner (1987)
Proc.Natl.Acad.Sci USA, 84:7413) has become a common method since
it does not have the degree of toxicity shown by the above
described chemical methods. The cationic head of the lipid
associates with the negatively charged nucleic acid backbone of the
DNA to be introduced. The lipid/DNA complex associates with the
cell membrane and fuses with the cell to introduce the associated
DNA into the cell. Liposome mediated DNA transfer has several
advantages over existing methods. For example, cells which are
recalcitrant to traditional chemical methods are more easily
transfected using liposome mediated transfer.
[0006] More recently still, physical methods to introduce DNA have
become effective means to reproducibly transfect cells. Direct
microinjection is one such method which can deliver DNA directly to
the nucleus of a cell (Capecchi (1980) Cell, 22:p479). This allows
the analysis of single cell transfectants. So called "biolistic"
methods physically insert DNA into cells and/or organelles using a
high velocity particles coated with DNA (Neumann (1982) EMBO J, 1:
841).
[0007] Electroporation is arguably the most popular method to
transfect DNA. The method involves the use of a high voltage
electrical charge to momentarily permeabilise cell membranes making
them permeable to macromolecular complexes. However physical
methods to introduce DNA do result in considerable loss of cell
viability due to intracellular damage. These methods therefore
require extensive optimisation and also require expensive
equipment.
[0008] More recently still a method termed immunoporation has
become a recognised techinque for the introduction of nucleic acid
into cells, see Bildirici et al Nature (2000) 405, 298. The
technique involves the use of beads coated with an antibody to a
specific receptor. The transfection mixture includes nucleic acid,
typically vector DNA, antibody coated beads and cells expressing a
specific cell surface receptor. The coated beads bind the cell
surface receptor and when a shear force is applied to the cells the
beads are stripped from the cell surface. During bead removal a
transient hole is created through which nucleic acid and/or other
biological molecules can enter. Transfection efficiency of between
40-50% is achievable depending on the nucleic acid used.
[0009] Typically, gene therapy involves the transfer, and
optionally the stable insertion, of new genetic information into
cells for the therapeutic treatment of disease. Genes that have
been successfully expressed in mice after transfer by retrovirus
vectors include human hypoxanthine phosphoribosyl transferase
(Miller A et al, 1984, Science 255:630). Bacterial genes have also
been transferred into mammalian cells, in the form of bacterial
drug resistance genes. Transformation of hematopoietic progenitor
cells to drug resistance by eukaryotic virus vectors has also
accomplished with recombinant retrovirus based vector systems (Hock
R A and Miller A D 1986, Nature 320:275-277; Joyner, et al. (1983)
Nature 305:556-558; Williams D A et al 1984, Nature 310:476-480;
Dick J E et al, 1985, Cell 42:71-79); Keller G et al 1985, Nature
318: 149-154; Eglitis M et al, 1985, Science 230: 1395-1398).
Adeno-associated virus vectors have been used successfully to
transduce mammalian cell lines to neomycin resistance (Hermonat P L
and Muzyczka N, 1984, supra; Tratschin J D et al, 1985, Mol. Cell.
Biol. 5:3251). Other viral vector systems that have been
investigated for use in gene transfer include papovaviruses and
vaccinia viruses (See Cline, M L (1985) Pharmac. Ther.
29:69-92).
[0010] The main issues with respect to gene therapy relate to the
efficient targeting of nucleic acid to cells and the establishment
of high level transgene expression in selected tissues. A number of
methodologies have been developed which purport to facilitate
either or both of these requirements. For example, U.S. Pat. No.
6,043,339 discloses the use of signal peptides which when fused to
nucleic acid, can facilitate the translocation of the linked
nucleic acid across cell membranes. U.S. Pat. No. 6,083,714
discloses a combined nucleic acid and targetting means which uses
the polycation poly-lysine coupled to an integrin receptor thereby
targetting cells expressing the integrin. EP1013770 discloses the
use of nuclear localisation signals (NLS) coupled to
oligonucleotides. The conjugate may be covalently linked to vector
DNA and the complex used to transfect cells. The NLS sequence
serves to facilitate the passage of the vector DNA across the
nuclear membrane thereby targetting gene delivery to the
nucleus.
[0011] Nucleic acid, for example vector DNA, may be introduced into
an animal via a variety of routes including enterally (orally,
rectally or sublingually) or parenterally (intravenously,
subcutaneously, or by inhalation).
[0012] It is known that introduction of certain aqueous solutions
into the peritoneal cavity can be useful in the treatment of
patients suffering from renal failure. Such treatment is known as
peritoneal dialysis. The solutions contain electrolytes similar to
those present in plasma; they also contain an osmotic agent,
normally dextrose, which is present in a concentration sufficient
to create a desired degree of osmotic pressure across the
peritoneal membrane. Under the influence of this osmotic pressure,
an exchange takes place across the peritoneal membrane and results
in withdrawal from the bloodstream of waste products, such as urea
and creatinine, which have accumulated in the blood due to the lack
of normal kidney function. While this exchange is taking place,
there is also a net transfer of dextrose from the solution to the
blood across the peritoneal membrane, which causes the osmolality
of the solution to fall. Because of this, the initial osmolality of
the solution must be made fairly high (by using a sufficiently high
concentration of dextrose) in order that the solution continues to
effect dialysis for a reasonable length of time before it has to be
withdrawn and replaced by fresh solution.
[0013] Other osmotic agents have been proposed for use in
peritoneal dialysis and in recent years dextrin (a starch
hydrolysate polymer of glucose) has been used. When instilled in
the peritoneal cavity, dextrin is slowly absorbed via the lymphatic
system, eventually reaching the peripheral circulation. The
structure of dextrin is such that amylases break the molecule down
into oligosaccharides in the circulation. These are cleared by
further metabolism into glucose.
[0014] Typically, a medium chosen to introduce gene therapy
materials to a patient via a body cavity might be a buffered saline
solution, for instance, phosphate buffered saline (PBS).
[0015] Dextrin solutions have been proposed as the medium for
delivery of drugs to the body via the peritoneum. In GB-A-2207050,
such a solution is proposed for the intraperitoneal administration
of drugs for which enteral administration is unsatisfactory. Such
an approach is stated to be particularly useful for the delivery of
peptide drugs such as erythropoetin and growth hormones. Reference
is also made to cephalosporin antibiotics. Dextrin solutions have
also been described for the administration of chemotherapeutic
agents in the treatment of ovarian cancers. The use of icodextrin
formulations to increase the efficacy of chemotherapeutics
(especially 5FU) by increasing their dwell time in the peritoneal
space is well described in Dobbie J W. (1997) Adv Perit
Dial.13:162-7 and McArdle C S, et al. (1994) Br J Cancer
70(4):762-6.
[0016] The present invention is directed to a dextrin containing
solution which shows enhanced ability to deliver nucleic acid to
cells resulting in high level expression of transfected genes.
SUMMARY OF THE INVENTION
[0017] The present invention provides a pharmaceutical formulation
comprising a nucleic acid in a solution comprising dextrin and at
least one sugar, the osmolarity of said solution corresponding
substantially to physiological osmolarity.
[0018] The present invention further provides a method to deliver a
nucleic acid to a cell wherein the nucleic acid is carried in a
solution comprising dextrin and at least one sugar, the osmolarity
of which corresponds substantially to the physiological osmolarity
of the milieu surrounding the cell.
[0019] The invention further provides a method of treating an
mammal with a nucleic acid wherein the nucleic acid is administered
to said mammal in a pharmaceutical formulation comprising dextrin
and at least one sugar.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a histogram providing measuring the expression of
the beta-galactosidase gene in epithelial tissues of the peritoneum
in rabbits following the intraperitoneal instillation of a 100 ul
of a solution containing a recombinant adenoviral vector encoding
beta-galactosidase in various formulations as more fully described
in Example 1 herein. The vertical axis represents the levels of
viral RNA in tissues as determined by RT-PCR.
[0021] FIG. 2 is a graphical representation of the results of the
murine xenograft prostate cancer model described in Example 2
herein. The vertical axis represents cumulative survival as a
fraction of the starting number of animals remaining alive. The
horizontal graph represents time in Days.
[0022] FIG. 3 is a graphical representation of the data generated
from the murine model of ovarian cancer more fully described in
Example 4 herein. drawings. Formulation H/Treatment Group 2 is
represented by X, Formulation I/Treatment Group 1 is represented by
squares, Formulation J/Treatment Group 3 is represented by circles;
and Formulation L/Treatment Group 4 is represented by
triangles.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides a pharmaceutical formulation
comprising a nucleic acid in a solution comprising dextrin and at
least one sugar, the osmolarity of said solution corresponding
substantially to physiological osmolarity.
[0024] Dextrin:
[0025] The term "dextrin" means a glucose polymer which is produced
by the hydrolysis of starch and which consists of glucose units
linked together by means mainly of .alpha.-1,4 linkages. Typically
dextrins are produced by the hydrolysis of starch obtained from
various natural products such as wheat, rice, maize and tapioca. In
addition to .alpha.-1,4 linkages there may be a proportion of
.alpha.-1,6 linkages in a particular dextrin, the amount depending
on the starch starting material. Since the rate of biodegradability
of .alpha.-1,6 linkages is typically less than that for .alpha.-1,4
linkages, for many applications it is preferred that the percentage
of .alpha.-1,6 linkages is less than 10% and preferably less than
about 5%.
[0026] Any dextrin is a mixture of polyglucose molecules of
different chain lengths. As a result, no single number can
adequately characterise the molecular weight of such a polymer.
Accordingly various averages are used, the most common being the
weight average molecular weight (Mw) and the number average
molecular weight (Mn). Mw is particularly sensitive to changes in
the high molecular weights content of the polymer whilst Mn is
largely influenced by changes in the low molecular weight of the
polymer. It the preferred practice of the invention, the Mw of the
dextrin is in the range from about 1,000 to 200,000, more
preferably from about 2,000 to 55,000.
[0027] The term "degree of polymerisation" (DP) can also be used in
connection with polymer mixtures. For a single polymer molecule, DP
means the number of polymer units. For a mixture of molecules of
different DP's, weight average DP and number average DP correspond
to Mw and Mn. In addition DP can also be used to characterise a
polymer by referring to the polymer mixture having a certain
percentage of polymers of DP greater than a particular number or
less than a particular number. It is preferred that, in the present
invention, the dextrin contains more than about 15% of polymers of
DP greater than 12 and, more preferably, more than about 50% of
polymers of DP greater than 12.
[0028] Preferably the dextrin is present in the solution in an
amount of less than about 20%. Preferably the dextrin is present in
the solution in an amount selected from about: 1% (w/v); 2% (w/v);
3% (w/v); 4% (w/v); 5% (w/v); 6% (w/v); 7% (w/v); 8% (w/v); 9%
(w/v); 10% (w/v); 11% w/v; 12% w/v; 13% w/v; 14% w/v; 15% w/v; 16%
w/v; 17% w/v; 18% w/v; 19% w/v; 20% w/v. More preferably the
dextrin is present from about 2 to 5% by weight, most preferably
about 4% by weight.
[0029] Physiological Osmolarity:
[0030] As indicated, the solution possesses an osmolarity
essentially isotonic with the osmolarity of the physiological
milieu of the cell to be treated. Generally, the physiological
osmolarity maintained in mammalian body cavities is approximately
330 milliosmolar and will vary somewhat with the particular body
cavity. For example, an isotonic solution for instillation in the
human peritoneal cavity would have an osmolarity of approximately
290-300 milliosmolar. In the preferred practice of the invention
for intraperitoneal instillation, the solution will possess an
osmolarity from about 250 to 350 milliosmolar, more preferably from
about 275 to 330 milliosmolar. The adjustments to maintain
approximate isotonic osmolarity depending on the particular
physiological milieu will be readily apparent to those of skill in
the art.
[0031] Sugar:
[0032] The term "sugar" refers to a monosaccharide, disaccharide or
oligosaccharide. Monosaccharides have the empirical formula
(CH.sub.2O).sub.n wherein n=3 or greater. Examples of
monosaccharides, which are merely meant to be illustrative and not
restrictive, are glucose, galactose, mannose, allose, altrose,
gulose, idose, talose, fructose. Disaccharides consist of two
monosaccharides linked by a glycoside bond. Non-restrictive
examples of dissacharides are sucrose, maltose, cellobiose,
gentiobiose, lactose. Typically oligosaccharides are sugars with
more than two monosaccharide units. Non-restrictive examples of
oligosaccharides are raffinose and melezitose. Sugars can be
naturally occuring or industrially manufactured.
[0033] In a preferred method of the invention the sugar is a
disaccharide. More preferably still the amount of disaccharide is
between about 1-10% w/v. Preferably the amount of disaccharide is
between about 2-5% w/v. In a preferred embodiment of the invention,
the disaccharide is sucrose in is about 3% w/v.
[0034] In a further method of the invention the amount of dextrin
is about between 2%-20% w/v and the amount of sucrose is about
1-10% w/v.
[0035] More ideally still the amount of dextrin is about 4% w/v and
the amount of sucrose is about 3% w/v.
[0036] It will be apparent that the exact combination will depend
on the osmolarity of the fluid surrounding the cell or tissue.
Typically, it is desirable to maintain an isosmotic equilibrium
between the solution including the nucleic acid and the fluid
surrounding the cells/tissues.
[0037] Divalent Cation:
[0038] More preferably still said solution further comprises a
divalent cation. Preferably the divalent cation is at least 0.2 mM.
More preferably still the divalent cation concentration is between
about 0.2- 3.0 mM. Ideally the divalent cation is MgCl.sub.2 and
the concentration is about 2.0 mM. Alternatively the divalent
cation is provided by CaCl.sub.2.
[0039] Preferably the solution comprises about 4% w/v dextrin,
about 3% w/v sucrose and about 2.0 mM MgCl.sub.2. Alternatively,
the dextrin concentration is about 15% w/v.
[0040] It will be apparent to one skilled in the art that the
solution has utility with respect to the in vitro delivery of
nucleic acid to cells for the recombinant production of
polypeptides encoded by the nucleic acid. The invention also
encompasses gene therapy, both the in vivo introduction of nucleic
acid into cells and ex vivo introduction of nucleic acid into cells
followed by introduction of transfected cells into an animal in
need of gene therapy.
[0041] Nucleic Acid:
[0042] Preferably said nucleic acid molecule is adapted for
eukaryotic expression. Typically said adaptation includes, by
example and not by way of limitation, the provision of
transcription control sequences (promoter sequences) which mediate
cell/tissue specific expression. These promoter sequences may be
cell/tissue specific, inducible or constitutive.
[0043] "Promoter" is an art recognised term and, for the sake of
clarity, includes the following features which are provided by
example only, and not by way of limitation. Enhancer elements are
cis acting nucleic acid sequences often found 5' to the
transcription initiation site of a gene (enhancers can also be
found 3' to a gene sequence or even located in intronic sequences).
Enhancers function to increase the rate of transcription of the
gene to which the enhancer is linked. Enhancer activity is
responsive to trans acting transcription factors (polypeptides)
which have been shown to bind specifically to enhancer elements.
The binding/activity of transcription factors (please see
Eukaryotic Transcription Factors, by David S Latchman, Academic
Press Ltd, San Diego) is responsive to a number of
physiological/environmental cues which include, by example and not
by way of limitation, intermediary metabolites (eg glucose,
lipids), environmental effectors (eg light, heat,).
[0044] Promoter elements also include so called TATA box and RNA
polymerase initiation selection (RIS) sequences which function to
select a site of transcription initiation. These sequences also
bind polypeptides which function, inter alia, to facilitate
transcription initiation selection by RNA polymerase.
[0045] Adaptations also include the provision of selectable markers
and autonomous replication sequences which facilitate the
maintenance of said vector in either the eukaryotic cell or
prokaryotic host. Vectors which are maintained autonomously are
referred to as episomal vectors. Episomal vectors are desirable
since these molecules can incorporate large DNA fragments (30-50 kb
DNA). Episomal vectors of this type are described in
WO98/07876.
[0046] Adaptations which facilitate the expression of vector
encoded genes include the provision of transcription
termination/polyadenylation sequences. This also includes the
provision of internal ribosome entry sites (IRES) which function to
maximise expression of vector encoded genes arranged in
bi-cistronic or multi-cistronic expression cassettes. Expression
control sequences also include so-called Locus Control Regions
(LCRs). These are regulatory elements which confer
position-independent, copy number-dependent expression to linked
genes when assayed as transgenic constructs. LCRs include
regulatory elements that insulate transgenes from the silencing
effects of adjacent heterochromatin, Grosveld et al., Cell (1987),
51: 975-985.
[0047] Expression control sequences also encompass, ubiquitous
chromatin opening elements (UCOE's), see WO/GB00/05393. UCOE's are
nucleic acid elements that are responsible for establishing an open
chromatin structure across a locus that consists exclusively of
ubiquitously expressed, housekeeping genes. These elements are not
derived from an LCR. A UCOE is a polynucleotide which opens
chromatin or maintains chromatin in an open state and facilitates
reproducible expression of an operably-linked gene in cells of at
least two different tissue types.
[0048] These adaptations are well known in the art. There is a
significant amount of published literature with respect to
expression vector construction and recombinant DNA techniques in
general. Please see, Sambrook et al (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring
Harbour, N.Y. and references therein; Marston, F (1987) DNA Cloning
Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA
Cloning: F M Ausubel et al, Current Protocols in Molecular Biology,
John Wiley & Sons, Inc. (1994).
[0049] Vectors are typically viral based and may be derived from
viruses including adenovirus; retrovirus; adeno-associated virus;
herpesvirus; lentivirus; vaccinia virus; and baculovirus.
[0050] The terms "therapeutic virus" and "therapeutic viral vector"
are used interchangeably herein to refer to viruses used as
therapeutic agents (e.g. wild-type viruses, attenuated viruses),
vaccine vectors or recombinant viruses containing modifications to
the genome to enhance therapeutic effects. The use of viruses or
"viral vectors" as therapeutic agents are well known in the art as
previously discussed. Additionally, a number of viruses are
commonly used as vectors for the delivery of exogenous genes.
Commonly employed vectors include recombinantly modified enveloped
or non-enveloped DNA and RNA viruses, preferably selected from
baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae,
poxviridae, adenoviridiae, or picornnaviridiae. Chimeric vectors
may also be employed which exploit advantageous elements of each of
the parent vector properties (See e.g., Feng, et al.(1997) Nature
Biotechnology 15:866-870). Such viral vectors may be wild-type or
may be modified by recombinant DNA techniques to be replication
deficient, conditionally replicating or replication competent.
[0051] Preferred vectors are derived from the adenoviral,
adeno-associated viral and retroviral genomes. In the most
preferred practice of the invention, the vectors are derived from
the human adenovirus genome. Particularly preferred vectors are
derived from the human adenovirus serotypes 2 or 5. The replicative
capacity of such vectors may be attenuated (to the point of being
considered "replication deficient") by modifications or deletions
in the E1a and/or E1b coding regions. Other modifications to the
viral genome to achieve particular expression characteristics or
permit repeat administration or lower immune response are
preferred. Most preferred are human adenoviral type 5 vectors
containing a DNA sequence encoding the p53 tumor suppressor gene.
In the most preferred practice of the invention as exemplified
herein, the vector is replication deficient vector adenoviral
vector encoding the p53 tumor suppressor gene A/C/N/53 as described
in Gregory, et al., U.S. Pat. No. 5,932,210 issued Aug. 3, 1999
(the entire teaching of which is herein incorporated by
reference).
[0052] Alternatively, the viral vectors may be conditionally
replicating or replication competent. Conditionally replicating
viral vectors are used to achieve selective expression in
particular cell types while avoiding untoward broad spectrum
infection. Examples of conditionally replicating vectors are
described in Pennisi, E. (1996) Science 274:342-343; Russell, and
S. J. (1994) Eur. J. of Cancer 30A(8):1165-1171. Additional
examples of selectively replicating vectors include those vectors
wherein an gene essential for replication of the virus is under
control of a promoter which is active only in a particular cell
type or cell state such that in the absence of expression of such
gene, the virus will not replicate. Examples of such vectors are
described in Henderson, et al., U.S. Pat. No. 5,698,443 issued Dec.
16, 1997 and Henderson, et al., U.S. Pat. No. 5,871,726 issued Feb.
16, 1999 the entire teachings of which are herein incorporated by
reference.
[0053] Additionally, the viral genome may be modified to include
inducible promoters which achieve replication or expression only
under certain conditions. Examples of inducible promoters are known
in the scientific literature (See, e.g. Yoshida and Hamada (1997)
Biochem. Biophys. Res. Comm. 230:426-430; Iida, et al. (1996) J.
Virol. 70(9):6054-6059; Hwang, et al.(1997) J. Virol
71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol.
17(9):5097-5105; and Dreher, et al.(1997) J. Biol. Chem 272(46);
29364-29371.
[0054] The viruses may also be designed to be selectively
replicating viruses. Particularly preferred selectively replicating
viruses are described in Ramachandra, et al. PCT International
Publication No. WO 00/22137, International Application No.
PCT/US99/21452 published Apr. 20, 2000 and Howe, J., PCT
International Publication No. WO WO0022136, International
Application No. PCT/US99/21451 published Apr. 20, 2000. A
particularly preferred selectively replicating recombinant
adenovirus is the virus d101/07/309 as more fully described in
Howe, J.
[0055] It has been demonstrated that viruses which are attenuated
for replication are also useful in the therapeutic arena. For
example the adenovirus dl1520 containing a specific deletion in the
E1b55K gene (Barker and Berk (1987) Virology 156: 107) has been
used with therapeutic effect in human beings. Such vectors are also
described in McCormick (U.S. Pat. No. 5,677,178 issued Oct. 14,
1997) and McCormick, U.S. Pat. No. 5,846,945 issued Dec. 8, 1998.
The method of the present invention may also be used in combination
with the administration of such vectors to minimize the
pre-existing or induced humoral immune response to such
vectors.
[0056] Additionally, the therapeutic virus may incorporate a
therapeutic transgene for expression in an infected cell. The term
"therapeutic transgene" refers to a nucleotide sequence the
expression of which in the target cell produces a therapeutic
effect. The term therapeutic transgene includes but is not limited
to tumor suppressor genes, antigenic genes, cytotoxic genes,
cytostatic genes, pro-drug activating genes, apoptotic genes,
pharmaceutical genes or anti-angiogenic genes. The vectors of the
present invention may be used to produce one or more therapeutic
transgenes, either in tandem through the use of IRES elements or
through independently regulated promoters.
[0057] The term "tumor suppressor gene" refers to a nucleotide
sequence, the expression of which in the target cell is capable of
suppressing the neoplastic phenotype and/or inducing apoptosis.
Examples of tumor suppressor genes useful in the practice of the
present invention include the p53 gene, the APC gene, the DPC-4
gene, the BRCA-1 gene, the BRCA-2 gene, the WT-1 gene, the
retinoblastoma gene (Lee, et al. (1987) Nature 329:642), the MMAC-1
gene, the adenomatous polyposis coli protein (Albertsen, et al.,
U.S. Pat. No. 5,783,666 issued Jul. 21, 1998), the deleted in colon
carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene,
nasopharyngeal carcinoma tumor suppressor gene that maps at
chromosome 3p21.3. (Cheng, et al. 1998. Proc. Nat. Acad. Sci.
95:3042-3047), the MTS1 gene, the CDK4 gene, the NF-1 gene, the NF2
gene, and the VHL gene. A particularly preferred adenovirus for
therapeutic use is the A/C/N/53 vector encoding the p53 tumor
suppressor gene as more fully described in Gregory, et al., U.S.
Pat. No. 5,932,210 issued Aug. 3, 1999, the entire teaching of
which is herein incorporated by reference.
[0058] The term "antigenic genes" refers to a nucleotide sequence,
the expression of which in the target cells results in the
production of a cell surface antigenic protein capable of
recognition by the immune system. Examples of antigenic genes
include carcinoembryonic antigen (CEA), p53 (as described in
Levine, A. PCT International Publication No. WO94/02167 published
Feb. 3, 1994). In order to facilitate immune recognition, the
antigenic gene may be fused to the MHC class I antigen. Preferably
the antigenic gene is derived from a tumour cell specific antigen.
Ideally a tumour rejection antigen. Tumour rejection antigens are
well known in the art and include, by example and not by way of
limitation, the MAGE, BAGE, GAGE and DAGE families of tumour
rejection antigens, see Schulz et al Proc Natl Acad Sci USA, 1991,
88, pp991-993.
[0059] It has been known for many years that tumour cells produce a
number of tumour cell specific antigens, some of which are
presented at the tumour cell surface. These are generally referred
to as tumour rejection antigens and are derived from larger
polypeptides referred to as tumour rejection antigen precursors.
Tumour rejection antigens are presented via HLA's to the immune
system. The immune system recognises these molecules as foreign and
naturally selects and destroys cells expressing these antigens. If
a transformed cell escapes detection and becomes established a
tumour develops. Vaccines have been developed based on dominant
tumour rejection antigen's to provide individuals with a preformed
defence to the establishment of a tumour.
[0060] The term "cytotoxic gene" refers to nucleotide sequence, the
expression of which in a cell produces a toxic effect. Examples of
such cytotoxic genes include nucleotide sequences encoding
pseudomonas exotoxin, ricin toxin, diptheria toxin, and the
like.
[0061] The term "cytostatic gene" refers to nucleotide sequence,
the expression of which in a cell produces an arrest in the cell
cycle. Examples of such cytostatic genes include p21, the
retinoblastoma gene, the E2F-Rb gene, genes encoding cyclin
dependent kinase inhibitors such as p16, p15, p18 and p19, the
growth arrest specific homeobox (GAX) gene as described in
Branellec, et al. (PCT Publication WO97/16459 published May 9, 1997
and PCT Publication WO96/30385 published Oct. 3, 1996).
[0062] The term "cytokine gene" refers to a nucleotide sequence,
the expression of which in a cell produces a cytokine. Examples of
such cytokines include GM-CSF, the interleukins, especially IL-1,
IL-2, IL-4, IL-12, IL-10, IL-19, IL-20, interferons of the .alpha.,
.beta. and .gamma. subtypes, consensus interferons and especially
interferon .alpha.-2b and fusions such as interferon
.alpha.-2.alpha.-1.
[0063] The term "chemokine gene" refers to a nucleotide sequence,
the expression of which in a cell produces a cytokine. The term
chemokine refers to a group of structurally related low-molecular
cytokines weight factors secreted by cells are structurally related
having mitogenic, chemotactic or inflammatory activities. They are
primarily cationic proteins of 70 to 100 amino acid residues that
share four conserved cysteine. These proteins can be sorted into
two groups based on the spacing of the two amino-terminal
cysteines. In the first group, the two cysteines are separated by a
single residue (C--x--C), while in the second group, they are
adjacent (C--C). Examples of member of the `C--x--C` chemokines
include but are not limited to platelet factor 4 (PF4), platelet
basic protein (PBP), interleukin-8 (IL-8), melanoma growth
stimulatory activity protein (MGSA), macrophage inflammatory
protein 2 (MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig
alveolar macrophage chemotactic factors I and II (AMCF-I and -II),
pre-B cell growth stimulating factor (PBSF), and IP10. Examples of
members of the `C--C` group include but are not limited to monocyte
chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2
(MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte
chemotactic protein 4 (MCP-4), macrophage inflammatory protein
1.alpha. (MIP-1-.alpha.), macrophage inflammatory protein 1.beta.
(MIP-1-.beta.), macrophage inflammatory protein 1-.gamma.
(MIP-1-.gamma.), macrophage inflammatory protein 3.alpha.
(MIP-3-.alpha., macrophage inflammatory protein 3.beta.
(MIP-3-.beta.), chemokine (ELC), macrophage inflammatory protein-4
(MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78.beta.,
RANTES, SIS-epsilon (p500), thymus and activation-regulated
chemokine (TARC), eotaxin, I-309, human protein HCC-1/NCC-2, human
protein HCC-3, mouse protein C10.
[0064] The term "pharmaceutical protein gene" refers to nucleotide
sequence, the expression of which results in the production of
protein have pharmaceutically effect in the target cell. Examples
of such pharmaceutical genes include the proinsulin gene and
analogs (as described in PCT International Patent Application No.
WO98/31397, growth hormone gene, dopamine, serotonin, epidermal
growth factor, GABA, ACTH, NGF, VEGF (to increase blood perfusion
to target tissue, induce angiogenesis, PCT publication WO98/32859
published Jul. 30, 1998), thrombospondin etc. Also, the
pharmaceutical protein gene may encompass immunoreactive proteins
such as antibodies, Fab fragments, Fv fragments, humanized
antibodies, chimeric antibodies, single chain antibodies, and human
antibodies derived from non-human sources.
[0065] The term "pro-apoptotic gene" refers to a nucleotide
sequence, the expression thereof results in the induction of the
programmed cell death pathway of the cell. Examples of
pro-apoptotic genes include p53, adenovirus E3-11.6K(10.5K), the
adenovirus E4orf4 gene, p53 pathway genes, and genes encoding the
caspases.
[0066] The term "pro-drug activating genes" refers to nucleotide
sequences, the expression of which, results in the production of
protein capable of converting a non-therapeutic compound into a
therapeutic compound, which renders the cell susceptible to killing
by external factors or causes a toxic condition in the cell. An
example of a prodrug activating gene is the cytosine deaminase
gene. Cytosine deaminase converts 5-fluorocytosine to 5
fluorouracil, a potent antitumor agent). The lysis of the tumor
cell provides a localized burst of cytosine deaminase capable of
converting 5FC to 5FU at the localized point of the tumor resulting
in the killing of many surrounding tumor cells. This results in the
killing of a large number of tumor cells without the necessity of
infecting these cells with an adenovirus (the so-called bystander
effect"). Additionally, the thymidine kinase (TK) gene (see e.g.
Woo, et al. U.S. Pat. No. 5,631,236 issued May 20, 1997 and
Freeman, et al. U.S. Pat. No. 5,601,818 issued Feb. 11, 1997) in
which the cells expressing the TK gene product are susceptible to
selective killing by the administration of gancyclovir may be
employed.
[0067] The term "anti-angiogenic" genes refers to a nucleotide
sequence, the expression of which results in the extracellular
secretion of anti-angiogenic factors. Anti-angiogenesis factors
include angiostatin, inhibitors of vascular endothelial growth
factor (VEGF) such as Tie 2 (as described in PNAS(USA)(1998)
95:8795-8800), endostatin.
[0068] It will be readily apparent to those of skill in the art
that modifications and or deletions to the above referenced genes
so as to encode functional subfragments of the wild type protein
may be readily adapted for use in the practice of the present
invention. For example, the reference to the p53 gene includes not
only the wild type protein but also modified p53 proteins. Examples
of such modified p53 proteins include modifications to p53 to
increase nuclear retention, deletions such as the .DELTA.13-19
amino acids to eliminate the calpain consensus cleavage site
(Kubbutat and Vousden (1997) Mol. Cell. Biol. 17:460-468,
modifications to the oligomerization domains (as described in
Bracco, et al. PCT published application WO97/0492 or U.S. Pat. No.
5,573,925, etc.).
[0069] It will be readily apparent to those of skill in the art
that the above therapeutic genes may be secreted into the media or
localized to particular intracellular locations by inclusion of a
targeting moiety such as a signal peptide or nuclear localization
signal (NLS). Also included in the definition of therapeutic
transgene are fusion proteins of the therapeutic transgene with the
herpes simplex virus type 1 (HSV-1) structural protein, VP22.
Fusion proteins containing the VP22 signal, when synthesized in an
infected cell, are exported out of the infected cell and
efficiently enter surrounding non-infected cells to a diameter of
approximately 16 cells wide. This system is particularly useful in
conjunction with transcriptionally active proteins (e.g. p53) as
the fusion proteins are efficiently transported to the nuclei of
the surrounding cells. See, e.g. Elliott, G. & O'Hare, P. Cell.
88:223-233:1997; Marshall, A. & Castellino, A. Research News
Briefs. Nature Biotechnology. 15:205:1997; O'Hare, et al. PCT
publication WO97/05265 published Feb. 13, 1997. A similar targeting
moiety derived from the HIV Tat protein is also described in Vives,
et al. (1997) J. Biol. Chem. 272:16010-16017.
[0070] It may be valuable in some instances to utilize or design
vectors to achieve introduction of the exogenous transgene in a
particular cell type. Certain vectors exhibit a natural tropism for
certain tissue types. For example, vectors derived from the genus
herpesviridiae have been shown to have preferential infection of
neuronal cells. Examples of recombinantly modified herpesviridiae
vectors are disclosed in U.S. Pat. No. 5,328,688 issued Jul. 12,
1994. Cell type specificity or cell type targeting may also be
achieved in vectors derived from viruses having characteristically
broad infectivities by the modification of the viral envelope
proteins. For example, cell targeting has been achieved with
adenovirus vectors by selective modification of the viral genome
knob and fiber coding sequences to achieve expression of modified
knob and fiber domains having specific interaction with unique cell
surface receptors. Examples of such modifications are described in
Wickham, et al.(1997) J. Virol 71(11):8221-8229 (incorporation of
RGD peptides into adenoviral fiber proteins); Arnberg, et al.(1997)
Virology 227:239-244 (modification of adenoviral fiber genes to
achieve tropism to the eye and genital tract); Harris and Lemoine
(1996) TIG 12(10):400-405; Stevenson, et al.(1997) J. Virol.
71(6):4782-4790; Michael, et al.(1995) Gene Therapy 2:660-668
(incorporation of gastrin releasing peptide fragment into
adenovirus fiber protein); and Ohno, et al.(1997) Nature
Biotechnology 15:763-767 (incorporation of Protein A-IgG binding
domain into Sindbis virus). Other methods of cell specific
targeting have been achieved by the conjugation of antibodies or
antibody fragments to the envelope proteins (see, e.g. Michael, et
al. (1993) J. Biol. Chem 268:6866-6869, Watkins, et al. (1997) Gene
Therapy 4:1004-1012; Douglas, et al. (1996) Nature Biotechnology
14: 1574-1578. Alternatively, particularly moieties may be
conjugated to the viral surface to achieve targeting (See, e.g.
Nilson, et al. (1996) Gene Therapy 3:280-286 (conjugation of EGF to
retroviral proteins)). Additionally, the virally encoded
therapeutic transgene also be under control of a tissue specific
promoter region allowing expression of the transgene preferentially
in particular cell types.
[0071] Vectors may also be non-viral and are available from a
number of commercial sources readily available to the man-skilled
in the art. For example the vectors may be plasmids which can be
episomal or integrating plasmids.
[0072] In a further preferred embodiment of the invention said
nucleic acid is an antisense nucleic acid, preferably an antisense
oligonucleotide.
[0073] As used herein, the term "antisense oligonucleotide" or
"antisense" describes an that is an oligoribonucleotide,
oligodeoxyribonucleotide, modified oligoribonucleotide, or modified
oligodeoxyribonucleotide which hybridizes under physiological
conditions to DNA comprising a particular gene or to an mRNA
transcript of that gene and thereby, inhibits the transcription of
that gene and/or the translation of that mRNA. Antisense molecules
are designed so as to interfere with transcription or translation
of a target gene upon hybridization with the target gene. Those
skilled in the art will recognize that the exact length of the
antisense oligonucleotide and its degree of complementarity with
its target will depend upon the specific target selected, including
the sequence of the target and the particular bases which comprise
that sequence.
[0074] It is preferred that the antisense oligonucleotide be
constructed and arranged so as to bind selectively with the target
under physiological conditions, i.e., to hybridize substantially
more to the target sequence than to any other sequence in the
target cell under physiological conditions.
[0075] In order to be sufficiently selective and potent for
inhibition, such antisense oligonucleotides should comprise at
least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and
more preferably, at least 15 consecutive bases which are
complementary to the target. Most preferably, the antisense
oligonucleotides comprise a complementary sequence of 20-30
bases.
[0076] Although oligonucleotides may be chosen which are antisense
to any region of the gene or mRNA transcripts, in preferred
embodiments the antisense oligonucleotides correspond to N-terminal
or 5' upstream sites such as translation initiation, transcription
initiation or promoter sites. In addition, 3'-untranslated regions
may be targeted. The 3'-untranslated regions are known to contain
cis acting sequences which act as binding sites for proteins
involved in stabilising mRNA molecules. These cis acting sites
often form hair-loop structures which function to bind said
stabilising proteins. A well known example of this form of
stability regulation is shown by histone mRNA's, the abundance of
which is controlled, at least partially,
post-transcriptionally.
[0077] The term "antisense oligonucleotides" is to be construed as
materials manufactured either in vitro using conventional
oligonucleotide synthesising methods which are well known in the
art or oligonucleotides synthesised recombinantly using expression
vector constructs. Modified oligonucleotide is construed in the
following manner. The term "modified oligonucleotide" as used
herein describes an oligonucleotide in which;
[0078] i) at least two of its nucleotides are covalently linked via
a synthetic internucleoside linkage (i.e., a linkage other than a
phosphodiester linkage between the 5' end of one nucleotide and the
3' end of another nucleotide). Alternatively or preferrably said
linkage may be the 5' end of one nucleotide linked to the 5' end of
another nucleotide or the 3' end of one nucleotide with the 3' end
of another nucleotide; and/or
[0079] ii) a chemical group not normally associated with nucleic
acids has been covalently attached to the oligonucleotide or
oligoribonucleotide. Preferred synthetic internucleoside linkages
are phosphorothioates, alkylphosphonates, phosphorodithioates,
phosphate esters, alkylphosphonothioates, phosphoramidates,
carbamates, phosphate triesters, acetamidates, peptides, and
carboxymethyl esters.
[0080] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. Modified oligonucleotides also
can include base analogs such as C-5 propyne modified bases (Wagner
et al., Nature Biotechnology 14:840-844, 1996).
[0081] The present invention, thus, contemplates pharmaceutical
preparations containing natural and/or modified antisense molecules
that are complementary to and, under physiological conditions,
hybridizable with nucleic acids encoding proteins the regulation of
which results in beneficial therapeutic effects, together with
pharmaceutically acceptable carriers (eg polymers,
liposomes/cationic lipids).
[0082] Antisense oligonucleotides may be administered as part of a
pharmaceutical composition. Such a pharmaceutical composition may
include the antisense oligonucleotides in combination with any
standard physiologically and/or pharmaceutically acceptable
carriers which are known in the art (eg liposomes). The
compositions should be sterile and contain a therapeutically
effective amount of the antisense oligonucleotides for
administration to a patient. The term "pharmaceutically acceptable"
means a non-toxic material that does not interfere with the
effectiveness of the biological activity of the active ingredients.
The term "physiologically acceptable" refers to a non-toxic
material that is compatible with a biological system such as a
cell, cell culture, tissue, or organism.
[0083] In yet a still further preferred method of the invention
said nucleic acid is a double stranded RNA molecule (RNA). A
technique to specifically ablate gene function is through the
introduction of double stranded RNA, also referred to as inhibitory
RNA (RNAi), into a cell which results in the destruction of mRNA
complementary to the sequence included in the RNAi molecule. The
RNAi molecule comprises two complementary strands of RNA (a sense
strand and an antisense strand) annealed to each other to form a
double stranded RNA molecule. The RNAi molecule is typically
derived from exonic or coding sequence of the gene which is to be
ablated. In a preferred method of the invention the length of the
RNAi molecule is between 100 bp-1000 bp. More preferably still the
length of RNAi is selected from about 100 bp; 200 bp; 300 bp; 400
bp; 500 bp; 600 bp; 700 bp; 800 bp; 900 bp; or 1000 bp. More
preferably still said RNAi is at least 1000 bp.
[0084] In a further preferred method of the invention said RNAi is
derived from an exon.
[0085] Alternatively said RNAi molecule is derived from intronic
sequences or the 5' and/or 3' non-coding sequences which flank
coding/exon sequences of genes. Recent studies suggest that RNAi
molecules ranging from 100-1000 bp derived from coding sequence are
effective inhibitors of gene expression. Suprisingly, only a few
molecules of RNAi are required to block gene expression which
implies the mechanism is catalytic. The site of action appears to
be nuclear as little if any RNAi is detectable in the cytoplasm of
cells indicating that RNAi exerts its effect during mRNA synthesis
or processing.
[0086] In yet a further preferred method of the invention said RNAi
molecules comprise modified ribonucleotide bases. It will be
apparent to one skilled in the art that the inclusion of modified
bases, as well as the naturally occuring bases cytosine, uracil,
adenosine and guanosine, may confer advantageous properties on RNAi
molecules containing said modified bases. For example, modified
bases may increase the stability of the RNAi molecule thereby
reducing the amount required to produce a desired effect.
[0087] The exact mechanism of RNAi action is unknown although there
are theories to explain this phenomenon. For example, all organisms
have evolved protective mechanisms to limit the effects of
exogenous gene expression. For example, a virus often causes
deleterious effects on the organism it infects. Viral gene
expression and/or replication therefore needs to be repressed. In
addition, the rapid development of genetic transformation and the
provision of transgenic plants and animals has led to the
realisation that transgenes are also recognised as foreign nucleic
acid and subjected to phenomena variously called quelling (Singer
and Selker, Curr Top Microbiol Immunol. 1995;197:165-77), gene
silencing (Matzke and Matzke, Novartis Found Symp. 1998;214:168-80;
discussion 181-6. Review) and co-suppression (Stain et. al., .
Plant J. 2000;21(1):27-42.
[0088] Initial studies using RNAi used the nematode Caenorhabditis
elegans. RNAi injected into the worm resulted in the disappearance
of polypeptides corresponding to the gene sequences comprising the
RNAi molecule(Montgomery et. al., 1998; Fire et. al., 1998). More
recently the phenomenon of RNAi inhibition has been shown in a
number of eukaryotes including, by example and not by way of
limitation, plants, trypanosomes (Shi et. al., 2000) Drosophila
spp. (Kennerdell and Carthew, 2000). Recent experiments have shown
that RNAi may also function in higher eukaryotes. For example, it
has been shown that RNAi can ablate c-mos in a mouse ooctye and
also E-cadherin in a mouse preimplanation embryo (Wianny and
Zernicka-Goetz, 2000).
[0089] In a yet further preferred method of the invention said
nucleic acid is a ribozyme. A ribozyme is a catalytic RNA which is
well known in the art. A ribozyme comprises a catalytic core having
flanking sequences adjacent to the sequence which hybridises to the
substrate RNA. The simplest catalytic core is an RNA motif known as
a hammerhead. Since the discovery of catalytic RNA there has been a
desire to design ribozymes which have a targetted gene function
such that viral mRNA and disease gene mRNA's can be selectively
ablated. For example, U.S. Pat. No. 6,069,007 discloses ribozymes
active against HIV1 mRNA and their use in AIDS therapy. U.S. Pat.
No. 6,087,172 discloses ribozymes designed to ablate mRNA encoding
IL-15, an interleukin invloved in rheumatoid arthritis. U.S. Pat.
No. 6,077,705 discloses a method of gene therapy to inhibit the
expression of mutated genes combined with the replacement of the
mutated gene, in this example .alpha.-1-antitrypsin, with a
wild-type copy.
[0090] Modes of Administration/Treatment:
[0091] The formulations of the present invention are useful for
enhancing the transfer of nucleic acids into tissues. It will be
apparent to one skilled in the art that solutions according to the
invention may be introduced into an animal subject in a variety of
ways including enterally (orally, rectally or sublingually) or
parenterally (intravenously, subcutaneously, or by inhalation). The
solutions may be provided to the mammal by implanted catheters, In
the preferred practice of the invention, the solutions are
instilled into a body cavity to facilitate transduction of the
surrounding tissues. Examples of such body cavities into which the
solutions may be provided for the delivery of nucleic acids include
the peritoneal cavity, pleural cavity, and the abdominal cavity.
Additionally the solutions may be provided in other fluid
containing spaces such as cerebral spinal fluid, joints, the colon,
the bladder, the eye and gall bladder. It will also be apparent to
one skilled in the art that the solutions can be administered
simultaneously, (as an admixture), separately or sequentially to an
animal.
[0092] According to a further aspect of the invention there is
provided a composition comprising dextrin, a sugar, divalent cation
and a nucleic acid molecule. Preferably said composition is for use
in the delivery of nucleic acid for gene therapy. In the preferred
practice of the invention, this procedure is employed in
conjunction with recombinant adenoviral therapy for the treatment
of human cancers. In accordance with conventional oncology
practice, patients are dosed at the maximum tolerated dose of the
therapeutic agent. In the course of clinical investigation, a dose
of 7.5.times.10.sup.13 recombinant adenoviral particles was well
tolerated in human subjects. Clinical experience with replication
deficient recombinant adenoviruses expressing p53 has indicated
that a course of therapy of injection of approximately
7.5.times.10.sup.13 recombinant viral particles for a period of 5
day course of therapy repeated monthly up to five months is
effective in the treatment of ovarian cancer in human beings.
[0093] In a particularly preferred embodiment of the present
invention, a formulation of the present invention comprising a
replication deficient recombinant adenovirus encoding p53 is
instilled into the peritoneum for the treatment of ovarian cancer.
A typical course of therapy with this agent involves administration
of 7.5.times.10.sup.13 viral particles each day for a period of 5
days. A typical clinical protocol for the treatment of ovarian
cancer using the A/C/N/53 virus calls involves a typical 5 day
course of therapy described above in conjunction with the
administration of the chemotherapeutic agents carboplatin and
paclitaxel. In the preferred practice of the invention the mammal
is a human being which receives three or more courses of therapy,
preferably 5-6 courses of therapy, with intervening rest periods.
Modifications to this procedure for therapeutic viruses other than
adenovirus will be readily apparent to the skilled artisan
[0094] The formulations of the present invention may further
comprise additional carriers, excipients or diluants. 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. The
concentration of the compositions of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
EXAMPLES
[0095] The following examples are merely illustrative of the
practice of the present invention and are not intended to limit the
scope thereof.
Example 1
Enhancement of rAd-mediated Transgene Expression in Rabbits
(Intraperitoneal Administration) Using an Icodextrin
Formulation
[0096] In order to evaluate the ability to enhance transgene
expression from a recombinant adenoviral vectors, an experiment was
conducted to compare the relative levels of transgene expression. A
recombinant adenoviral vector encoding the beta-galactosidase gene
(rAd-bgal) was prepared in substantial accordance with the teach of
Gregory, et al., U.S. Pat. No. 5,932,210. The following solutions
were preparedin a volume of 100 ml.
1 Solution Components A 1 .times. 10.sup.9 particles/ml of rAd-bgal
in 15% w/v icodextrin contain- ing 3% sucrose, 2 mM magnesium, 95
mM sodium, 1.75 mM calcium, 59 mM chloride, 40 mM lactate B 1
.times. 10.sup.9 particles/ml of rAd-bgal, 3% sucrose, 2 mM
magnesium chloride in phosphate buffered saline
[0097] Ten female New Zealand white rabbits were anaesthetized with
ketamine/xylazine and the foregoing solutions instilled
intraperitoneally. The solution was allowed to incubate for 1 hour
(at dorsal side) and one hour (at ventral side). The animals were
sacrified and biopsies of the peritoneal wall were harvested.
Levels of viral RNA in harvested tissues was assayed using RT-PCR.
The results of transgene specific RNA concentrations isolated from
the peritoneal wall are presented in FIG. 1 of the accompanying
drawings. As can be seen from the data presented addition of 15%
icodextrin to the virus buffer solution (solution A) resulted in a
marked increase in transgene expression relative to the buffer
control solution alone (solution B).
Example 2
Efficacy of Icodextrin rAd-p53 Formulation in Murine Xenograft
Prostate Cancer Model
[0098] In order to demonstrate that the icodextrin containing
formulations of recombinant adenoviruses provide an enhanced
therapeutic effect, a experiment was conducted to compare the
anti-tumor efficacy of replication deficient recombinant adenoviral
vectors encoding the p53 tumor suppressor gene ("rAd-p53"). The
efficacy of the vectors was compared in a murine xenograft prostate
cancer model as described in Paine-Murrieta G D et al. Cancer
Chemother Pharmacol 1997, 40: 209. Increased survival was used as
the measure of efficacy.
[0099] The rAd-p53 vector designated ACN53 was prepared in
substantial accordance with the teaching of Gregory, et al., U.S.
Pat. No. 5,932,210. PC-3 prostate cancer cells were obtained from
the American Type Culture Collection, Bethesda, Md. under accession
number CRL-1435. Fifty-one female nude mice, approximately 5 weeks
old were obtained from Harlan Laboratories. Approximately
5.times.10.sup.6 PC3 cells in a volume of 0.2 ml of HBSS-FBS (Hanks
Balanced Salt Solution w/10% fetal bovine serum; HBSS was obtained
from Fisher Scientific, FBS was obtained from BioWhittaker) were
injected intraperitoneally into each animal. The cells were allowed
to establish tumors for a period of nine days prior to the
initiation of treatment.
[0100] Seven different formulations were prepared in accordance
with Table 2 below:
2TABLE 2 Formulations vPBS (1) vICO (2) virus (3) Name Description
(microliters) (milliliters) (microliters) A 15% vICO control 0 5 0
B 1 .times. 10.sup.10 PN ACN53; 549 7.2 251 15% vICO C 1 .times.
10.sup.10 PN ACN53; 7749 0 251 vPBS D 1 .times. 10.sup.9 PN ACN53;
775 7.2 25.1 15% vICO E 1 .times. 10.sup.9 PN ACN53; 7975 0 25.1
vPBS F 1 .times. 10.sup.8 PN ACN53; 775 7.2 2.51* 15% vICO G 1
.times. 10.sup.8 PN ACN53; 7975 0 2.51* vPBS Notes: (1) "vPBS"
solution is a sterile solution 3% sucrose, 2mM MgCl.sub.2 in
phosphate buffered saline, pH 7.4. (2) "vICO" is a sterile 15%
icodextrin solution 95 millimolar Na.sup.+, 1.75 millimolar
Ca.sup.++, 2.0 millimolar Mg.sup.++, 59 millimolar Cl.sup.-, 40
millimolar lactate, 88 millimolar sucrose, 160 g/liter icodextrin,
having a final osmolarity of 285.75. (3) "virus" refers to a stock
virus suspension containing 3.19 .times. 10.sup.11 ACN53 viral
particles per milliliter. As it is difficult to measure 2.51
microliters with conventional equipment, formulations F and G were
prepared by the addition of 25.1 microliters of a 1:10 dilution of
the stock viral suspension in vPBS to achieve the equivalent of
2.51 microliters of virus suspension.
[0101] The animals were divided up into eight treatment groups as
more fully described in Table 3 below:
3TABLE 3 Treatment Groups Total ACN53 Group n Formulation Dose
(particles) 1 5 untreated -- 2 4 A -- 3 7 B .sup. 5 .times.
10.sup.10 4 7 C .sup. 5 .times. 10.sup.10 5 7 D 5 .times. 10.sup.9
6 7 E 5 .times. 10.sup.9 7 7 F 5 .times. 10.sup.8 8 7 G 5 .times.
10.sup.8
[0102] Treatment was initiated on Day 9. (Note: All treatment days
denoted by "Day" mentioned herein are referenced from the date of
injection of the PC-3 cells) All animals appeared healthy upon
initiation of the treatment regimen. Other than the untreated
control group 1, each group was provided a treatment regimen
consisting of treatments each consisting of a single
intraperitoneal injection of 1.0 ml of the appropriate formulation
on Days 9, 11, 14, 15 and 18 following injection of tumor cells.
Upon completion of the treatment regimen, the animals were randomly
caged and monitored daily (blinded) for sick animals as charactized
by visible loss of body weight hunched back, and sedation. Sick
animals were sacrificed and examined gross pathologically. Animal
number, date of sacrifice (or found dead) and gross pathological
findings were recorded results are summarized in Table 4 below:
4TABLE 4 Results of PC-3 Tumor Model Survival Time (Days) Group
Formulation median minimum maximum 1 untreated 30 23 33 2 A 30 21
33 3 B 53 30 100* 4 C 33 21 42 5 D 36 21 55 6 E 21 21 36 7 F 36 21
42 8 G 36 21 39 *the experiment was concluded on Day 100
[0103] Days of survival after injection were plotted using a Kaplan
Meier survival and the results of treatment groups 1-4 are
presented graphically in FIG. 2 of the attached drawings.
Comparison among groups was performed using the Logrank Test
(Statview Software) Differences were considered significant if
p<0.05.
[0104] As can be seen from the data presented, treatment with ACN53
formulations containing icodextrin resulted in a statistically
significant prolongation of survival as compared to ACN53
formulated in vPBS or controls at a viral dose of 5.times.10.sup.10
viral particles. One animal receiving the maximal viral dose in
icodextrin was free of clinical signs of tumor growth upon
completion of the study (Day 100). This animal had minimal tumor
growth in the peritoneal cavity indicated that the animal was
injected with tumor cells and that tumor formed but that tumor
growth was inhibited.
Example 3
Efficacy of Icodextrin rAd-p53 Formulation in Murine Xenograft
Ovarian Cancer Model
[0105] The effect of icodextrin containing adenoviral formulations
containing (Formulations B and C in Table 2 above) was evaluated in
a murine intraperitoneal xenograft model of human ovarian cancer.
The experiment was conducted in substantial accordance with the
teaching of Example 2 above with the following variations. Animals
were inoculated intraperitoneally with 1.times.10.sup.7 human
MDL-H2774 ovarian cancer cells (ATCC CRL-10303) cells in 0.2 ml of
HBSS. The animals were dosed intraperitoneally with 0.5 ml
formulations B and C above on Days 2, 5, 8 and 12. Control groups
were treated as in Example 2. Similar prolongation of survival was
observed in this model system with adenovirus formulations
comprising icodextrin.
Example 4
vIco Enhanced Efficacy of an Oncolytic Adenovirus in an Orthotopic
Model of Human Ovarian Cancer
[0106] A murine model of human ovarian cancer was employed to
evaluate the effect of icodextrin containing formulations of a
conditionally replicating adenoviral vector. The model was
established in nude mice (20-25 g commercially available from
Harlan, Indianapolis, Ind.) by the administration of a single
intraperitoneal injection of a suspension of 1.times.10.sup.7 MDA
H2774 human ovarian cancer cells (available from the American Type
Culture Collection under Accession Number CRL-10303) in 0.5 ml in
Hank's Balanced Salt Solution (HBSS). Tumors were permitted to grow
for 7 days and the mice were evaluated for the presence of palpable
tumors. Those mice evidencing tumors were separated into groups for
treatment.
[0107] A conditionally replicating recombinant adenoviral vector
designated K9TB was prepared in substantial accordance with the
teaching of Ramachandra, et al. (PCT International Publication
Number WO 00/22137 published Apr. 20, 2000). The K9TB virus is a
conditionally replicating virus containing a deletion of a amino
acids 4-25 of the E1a region, a p53 response element driving
expression of the E2F-Rb fusion protein (Antelman, et al., U.S.
Pat. No. 6,074,850 issued Jun. 13, 2000) inserted into the E3
region.
[0108] The following formulations provided in Table 5 below were
prepared for evaluation in the tumor model described above.
5TABLE 5 Formulations vPBS (1) vICO (2) virus (3) Name Description
(microliters) (milliliters) (microliters) H 15% vICO control -- 3
-- I vPBS control 3 -- -- J 4.5 .times. 10.sup.8 PN K9TB; 3.757 --
743 vPBS K 4.5 .times. 10.sup.8 PN K9TB; -- 3.757 743 15% vICO
Notes: (1) "vPBS" solution is a sterile solution 3% sucrose, 2mM
MgCl.sub.2 in phosphate buffered saline, pH 7.4. (2) "vICO" is a
sterile 15% icodextrin solution 95 millimolar Na.sup.+, 1.75
millimolar Ca.sup.++, 2.0 millimolar Mg.sup.++, 59 millimolar
Cl.sup.-, 40 millimolar lactate, 88 millimolar sucrose, 160 g/liter
icodextrin. (3) "virus" refers to a 1:1000 dilution of a stock
virus suspension containing 6.06 .times. 10.sup.11 K9TB viral
particles per milliliter.
[0109] The tumor bearing animals were segregated into the following
groups for treatment:
6TABLE 6 Treatment Groups Total K9TB Group n Formulation Dose
(particles) 1 4 H -- 2 4 I -- 3 7 J 2 .times. 10.sup.8 4 7 K 2
.times. 10.sup.8
[0110] Each group was provided a treatment regimen consisting of a
single intraperitoneal injection of 0.5 ml of the appropriate
formulation on Days days 7, 9, 12, and 14 following injection of
tumor cells. Upon completion of the treatment regimen, the animals
were randomly caged and monitored daily (blinded) for moribund
animals as characterized by visible loss of body weight hunched
back, and sedation. Moribund animals were sacrificed and examined
gross pathologically. Animal number, date of sacrifice (or found
dead) and gross pathological findings were recorded and the results
summarized in Table 7 below.
7TABLE 7 Results of H2774 Ovarian Tumor Model Survival Time (Days)
Group Formulation mean minimum maximum 1 H 28.5 28 29 2 I 28.3 28
29 3 J 41.0 36 47 4 K 55.7 41 80* *the experiment was concluded on
Day 80
[0111] Days of survival after injection were summarized using a
Kaplan-Meier plot and the results of treatment groups 1-4 are
presented graphically in FIG. 3 of the attached drawings.
Comparison among groups was performed using the Logrank Test
(Statview Software). Differences were considered significant if
p<0.05.
[0112] A graphical representation of the data is presented in FIG.
3 of the attached drawings. Formulation H/Treatment Group 2 is
represented by X, Formulation I/Treatment Group 1 is represented by
squares, Formulation J/Treatment Group 3 is represented by circles;
and Formulation L/Treatment Group 4 is represented by
triangles.
[0113] As can be seen from the data presented, the treatment of
tumor bearing mice with the recombinant adenoviral vector K9TB in
both the vIco and vPBS formulations prolonged survival compared to
the vehicle controls. However, K9TB formulated in vIco demonstrated
a prolongation of survival compared to K9TB formulated in vPBS
(p<0.01, n=7 animals/treatment group).
* * * * *