Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR DELIVERING NUCLEIC ACID MOLECULES INTO CELLS
AND ASSESSMENT THEREOF
REl'ATED APPLICATIONS
Benefit of priority is claimed to U.S. application Serial No.
09/815,979 entitled "METHODS FOR DELIVERING NUCLEIC ACID
MOLECULES INTO CELLS AND ASSESSMENT THEREOF", filed on
March 22, 2001 by De Jong et al. (published as U.S. 20030059940 Al on
March 27, 2003), U.S. application Serial No. 09/815, 981 entitled "METHODS FOR
DELIVERING NUCLEIC ACID MOLECULES INTO CELLS AND ASSESSMENT
THEREOF", filed on March 22, 2001 by De Jong et al. (published as US
20030003435 Al on January 2, 2003) and U.S. application Serial No. 10/086,745
entitled "METHOD FOR DELIVERING NUCLEIC ACID MOLECULES INTO CELLS
AND ASSESSMENT THEREOF", filed on February 28, 2002 by De Jong et al.
(published as U.S. 20030186390 Al on October 2, 2003).
FIELD OF THE INVENTION
The present invention relates to methods of delivering nucleic acid
molecules into cells and methods for measuring nucleic acid delivery into
cells and the expression of the nucleic acids therein.
BACKGROUND OF THE INVENTION
A number of methods of delivering nucleic acid molecules,
particularly plasmid DNA and other small fragments of nucleic acid, into
cells have been developed. These methods are not ideal for delivery of
larger nucleic acid molecules. Thus, there is a need for methods of
delivering nucleic acid molecules of increasing size and complexity, such
as artificial chromosomes, into cells. Methods are required for use with in
vitro and in vivo procedures such as gene therapy and for production of
transgenic_ animals and plants. Furthermore, there is a need for the ability
to rapidly and simply determine and assess the efficiency of delivery of
DNA into cells.
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Therefore it is an object herein to provide methods for delivering
nucleic acid molecules, particularly larger molecules, including artificial
chromosomes, into cells. Methods for assessing delivery are also
provided.
SUMMARY OF THE INVENTION
Methods for delivery of large nucleic acid molecules into cells are
provided. The methods, which can be used to deliver nucleic acid
molecules of any size, are suitable for delivery of larger nucleic acid
molecules, such as natural and artificial chromosomes and fragments
thereof, into cells. The methods are designed for in vitro, ex vivo and in
vivo delivery of nucleic acid molecules for applications, including, but not
limited to, delivery of nucleic acid molecules to cells for cell-based protein
production, transgenic protein production and gene therapy. Methods of
protein production in cells and in transgenic animals and plants, methods
of introducing nucleic acid into cells to produce transgenic animals and
plants, and methods for ex vivo and in vivo gene therapy are also
provided.
Methods provided herein are designed for delivering a large nucleic
acid molecule into a cell, but may also be used to deliver smaller
molecules. Some of the methods include the steps of exposing the
nucleic acid molecule to a first delivery agent, typically an agent that
increases contact between the nucleic acid molecule and the cell; and
exposing the cell to a second delivery agent, which is generally different
from the first agent, and is particularly an agent, such as energy, that
enhances permeability of the cell. Selected delivery agents and
combinations thereof are those that result in delivery of the nucleic acid
into the cell to a greater extent than in absence of the agent or in the
presence of one of the agents alone. Generally, in all of these methods, if
the permeability enhancing agent is energy, such as electroporation or
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sonoporation, the cell is contacted therewith in the absence of the nucleic
acid molecule.
Also provided are methods in which the cells are contacted with a
lipid agent, particularly a dendrimer, such as SAINT-2' (1-methyl-4-(1-
octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride, also designated
1-methyl-4-(19-cis,cis-hepatritiaconta-9,28-dienyl) pyridinium chloride),
simultaneously with or sequentially with application of energy. The
nucleic acid, which is optionally treated with a delivery agent, is
contacted with the so-treated cell.
The selected delivery methods vary depending on the target cells
(cells into which nucleic acid is delivered), the nucleic acid molecules, and
the type(s) of delivery agent(s) selected. Exemplary methods for delivery
of large nucleic acid molecules into cells provided herein include, but are
not limited to, methods involving any of the following:
mixing the nucleic acid molecule with a delivery agent, such
as a cationic lipid that neutralizes the charge of the nucleic acid, and
contacting the cell with the mixture of nucleic acid and delivery agent;
contacting a cell with the nucleic acid molecule, and then
contacting the cell with a delivery agent or contacting a cell with a
delivery agent then contacting the ce!l with the nucleic acid molecule;
contacting a cell in the absence of the nucleic acid molecule
with a delivery agent, applying ultrasound or electrical energy to the cell
contacted with the delivery agent, and contacting the cell with the nucleic
acid molecule upon the conclusion of the application of the energy;
applying ultrasound or electrical energy to a cell, and
contacting the cell, upon conclusion of the application of the energy, with
a mixture of the nucleic acid molecule and a delivery agent;
applying ultrasound or electrical energy to a cell, contacting
the cell with a delivery agent upon conclusion of the application of the
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energy and contacting the cell previously contacted with the delivery
agent with the nucleic acid molecule;
applying ultrasound or electrical energy to a cell and
contacting the cell with the nucleic acid molecule upon conclusion of the
application of the energy;
contacting a cell in the absence of the nucleic acid molecule
with a delivery agent, applying ultrasound or electrical energy to the cell
contacted with the delivery agent, and contacting the cell with a mixture
of the nucleic acid and a delivery agent upon the conclusion of the
application of the energy.
Also provided are methods that include combinations of the above
methods. In general, for any of the methods or combinations of methods,
application of energy to the cells is done prior to introduction of the
nucleic acid molecule. In some instances, however, energy can be
applied in the presence of the nucleic acid molecule can, for example, in
instances when the integrity of the nucleic acid molecule is not
compromised by application of energy in the presence of the nucleic acid
molecule.
The methods provided herein are intended for delivery of large
nucleic acid molecules into cells in a variety of environments for a variety
of purposes. For example, nucleic acid molecules greater than about 0.5,
0.6. 0.7, 0.8, 0.9, 1, 5, 10, 30, 50 and 100 megabase pairs may be
delivered into cells using the methods provided herein. The methods may
be used to deliver the large nucleic acid molecules into cells in vitro or in
vivo.
In in vivo applications of the delivery methods, such as in in vivo
gene therapy, large nucleic acid molecules may be delivered to cells
directly in an animal subject. Such animals include, but are not limited to
mammals. For example, the animal subject may be a human or other
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primate, rodent, rabbit, dog, horse or monkey. Reagents can be
administered locally or systemically (e.g., in the bloodstream) in the
subject. For example, local administration of the nucleic acids, and/or
delivery agents, may be into areas such as joints, the skin, tissues,
tumors and organs. For systemic administration, the nucleic acid
molecules may be targeted to cells or tissues of interest.
The delivery methods provided herein may also be used to deliver
large nucleic acid molecules to a target cell in vitro which is then
introduced into an animal subject, in particular human subjects, such as
may be done, for example, in a method of ex vivo gene therapy. Thus,
also provided herein are methods of in vivo and ex vivo gene therapy
using the methods for delivering large nucleic acid molecules into cells as
provided herein.
In particular embodiments of the methods in which a delivery agent
is used, the delivery agent is a cationic compound. Cationic compounds
include, but are not limited to, a cationic lipid, a cationic polymer, a
mixture of cationic lipids, a mixture of cationic polymers, a mixture of a
cationic lipid and a cationic polymer and a mixture of a cationic lipid and a
neutral lipid, polycationic lipids, non-liposomal forming lipids, activated
dendrimers, ethanolic cationic lipids, cationic amphiphiles and pyridinium
chloride surfactants.
Included among the nucleic acid molecules that may be delivered
into cells using the methods provided herein are artificial chromosomes,
satellite DNA-based artificial chromosomes (SATACs, herein referred to as
ACes) and natural chromosomes or fragments of any of these
chromosomes.
The ultrasound energy can be applied as one continuous pulse or
two or more intermittent pulses. The intermittent pulses of the ultrasound
energy can be applied for substantially the same length of time, at
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substantially the same energy level or can vary in energy level, the length
of time applied, or energy level and the length of time applied. Ultrasound
energy ranges and number of pulses can vary, from methods provided
herein, according to the instrument selected and can be empirically
determined. Typically, ultrasound will be applied for about 30 seconds to
about 5 minutes. The power used is a function of the sonorporator used.
The effects of the ultrasound energy may be enhanced by con-
tacting a cell (in vitro) or administering to a subject (in vivo) a cavitation
compound prior to the application of ultrasound energy. Thus, the
provided methods may include the use of such cavitation compounds.
When electric fields are employed in the methods provided herein,
they are preferably applied to the cells in suspension for about 20 to 50
msec, but the timing and voltage is a function of the instrument used and
the particular parameters. The electrical energy can be applied as one to
five intermittent pulses. As noted, electrical field ranges and number of
pulses can vary according to instrument specification and can be
determined empirically.
Methods are provided for generating transgenic animals, particularly
non-human transgenic animals, by delivering large nucleic acid molecules
into animal cells, in particular non-human animal cells, using delivery
methods provided herein, and exposing the animal cells into which the
large nucleic acid molecules are delivered to conditions whereby a
transgenic animal develops therefrom.
The methods for delivering large nucleic acid molecules into cells
provided herein may also be used in methods of generating transplantable
organs and tissues. Exemplary cells for use in methods of generating
transgenic animals, particularly non-human transgenic animals, or
transplantable organs include, but are not limited to, an embryonic stem
cell, a nuclear transfer donor cell, a stem cell and a cell that is capable of
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the generation of a specific organ. The methods for delivering nucleic
acid molecules into cells provided herein may also be used in methods of
generating cellular protein production cell lines.
Further provided are methods for monitoring delivery of nucleic
acids into a cell. These methods permit the rapid and accurate
measurement of nucleic acid transfer into cells, thus allowing for
screening and optimizing the use of various delivery agents and protocols
for delivery of any nucleic acid into any cell type, in vitro, ex vivo or in
vivo. Further provided are methods to monitor delivery and expression of
nucleic acids in a cell.
In embodiments of the methods for monitoring delivery of nucleic
acids into a cell, labeled nucleic acid molecules, such as DNA, are
delivered into the cell using the delivery agent(s) as described herein, or
using any delivery method known to those of skill in the art. A detection
method, such as flow cytometry, is then used to determine the number of
cells containing the label as an indication of the ability of the delivery
method to facilitate or effect delivery of the nucleic acid molecules.
Other detection methods that may be used in place of or in addition to
flow cytometry include, but are not limited to, fluorimetry, cell imaging,
fluorescence spectroscopy and other such methods known to those of
skill in the art for such detection and, as needed or desired, for
quantitation.
In an exemplary embodiment of the methods for monitoring and
quantifying delivery of nucleic acid molecules, such as DNA, into cells,
the nucleic acid molecule is an artificial chromosome labeled with a
nucleoside or ribonucleoside analog, particularly a thymidine analog, such
as iododeoxyuridine (IdU or IdUrd) and bromodeoxyuridine (BrdU), and the
delivery agent is a cationic compound, which is used alone or in
combination with energy.
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Because of the ease with which numbers of events are collected,
the monitoring methods provided herein, particularly those based on flow
cytometry techniques, provide a method for collection of nucleic acid
molecule delivery data that is statistically superior to previous methods of
evaluating nucleic acid molecules transfer. The positive values are
instrument derived and therefore are not susceptible to judgement errors.
The monitoring methods provided herein permit the rapid, simple
and accurate detection of delivery of small numbers of nucleic acid
molecules into cells. Such small numbers may be sufficient for purposes
of transgenesis, gene therapy, cellular protein production and other goals
of,gene transfer. The monitoring methods also make it possible to rapidly
quantify differences in delivery efficiencies of differing delivery methods
and thus facilitate the development and optimization of methods for the
delivery of nucleic acid molecules, such as DNA, into cells.
These methods can also be used to optimize transfection
efficiencies into cells for which no delivery protocol has been established
or which are not easily transfected. These methods also permit rapid
screening of delivery protocols and agents for their ability to enhance or
permit delivery of nucleic acid molecules, such as DNA, of any size into a
cell.
Methods are also provided that combine methods of monitoring
nucleic acid molecule delivery with methods for monitoring expression of
nucleic acid molecules. It is possible not only to assess the efficiency of
delivery of nucleic acid molecules to cells, but also to monitor the
subsequent expression of the delivered nucleic acid molecules in the same
cell population. Thus, these methods also provide a method for the
mapping of biological events between nucleic acid molecule delivery and
early gene expression, using marker genes, such as, but are not limited
to, fluorescent proteins, such as red, green or blue fluorescent proteins.
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In a particular embodiment of these combined'methods, delivery and
expression of nucleic acid molecules, such as delivery of a chromosome
and expression of genes encoded thereon, are monitored by IdU labeling
of a nucleic acid molecule that contains sequences encoding a green
fluorescent protein.
In particular embodiments, the methods of monitoring delivery and
expression of a nucleic acid molecule include the steps of: introducing
labeled nucleic acid molecules that encode a reporter gene into cells;
detecting labeled cells as an indication of delivery of the nucleic acid into
a cell; and measuring the product of the reporter gene as an indication of
DNA expression in the cell, whereby delivery and expression of nucleic
acid molecules in the cell is detected or determined. The labeled cells
can be detected, for example, by flow cytometry, fluorimetry, cell imaging
or fluorescence spectroscopy. The label, for example, can, be
iododeoxyiuridine (IdU or IdUrd) or bromodeoxyuridine (BrdU), the reporter
gene, for example, can be one that encodes fluorescent protein, enzyme,
such as a luciferase, or antibody. The delivered nucleic acid molecules
include, but are not Iimited to, RNA, including ribozymes, DNA, including
naked DNA and chromosomes, plasmids, chromosome fragments,
typically containing at least one gene or at least 1 Kb,'naked DNA, or
natural chromosomes. The method is exemplified herein by determining
delivery and expression of artificial chromosome expression systems
(ACes). Any types of 'cells, eukaryotic and prokaryotic, including cell
lines, primary celis, primary cell lines, plant cells, and animal cells,
including stem cells, embryonic cells, and other cells into. which delivery
of a nucleic acid molecule can occur, are contemplated, may be used in the
methods
provided herein.
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Thus, in one aspect, the present invention provides
a method for introducing a large nucleic acid molecule into a
cell, comprising: (a) exposing a large nucleic acid molecule
to a first delivery agent in vitro or ex vivo, wherein the
large nucleic acid molecule is greater than 0.6 megabase in
size; (b) exposing the cell in vitro or ex vivo to a second
delivery agent, wherein the second delivery agent is
different from the first delivery agent; and (c) contacting
the cell with the nucleic acid molecule in vitro or ex vivo,
whereby the nucleic acid molecule is delivered into the cell,
wherein steps (a) and (b) are performed sequentially in any
order, provided that if the first or second delivery agent is
energy it is not applied to the nucleic acid molecule and it
is not applied to the cell after contacting the cell with the
nucleic acid molecule.
In another aspect, the present invention provides a
method for delivering a nucleic acid molecule into a cell,
comprising: (a) contacting the cell in the absence of the
nucleic acid molecule with a delivery agent, and applying
ultrasound energy or electrical energy to the cell, wherein
the contacting and applying are performed sequentially or
simultaneously in vitro or ex vivo; and then (b) contacting
the cell with the nucleic acid molecule in vitro or ex vivo,
whereby the nucleic acid molecule is delivered into the cell.
In another aspect, the invention provides a method
for delivering a large nucleic acid molecule into a cell,
comprising: (a) contacting the nucleic acid molecule in vitro
with a cationic lipid composition, wherein the cationic lipid
composition comprises 2,3-dioleyloxy-N-[2(spermine-
carboxamido)ethyl]-N,N-dimethyl-l-
propanaminiumtrifluoroacetate (DOSPA) and
dioleoylphosphatidylethanolamine (DOPE), and the nucleic
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acid molecule is at least 5 megabases; and then
(b) contacting the nucleic acid molecule with a cell
in vitro or ex vivo, wherein steps (a) and (b) are performed
simultaneously or sequentially.
In another aspect, the present invention provides
a method for delivering a nucleic acid molecule into a cell
in a subject comprising: (a) administering a delivery agent
to the subject in the absence of the nucleic acid molecule;
(b) applying ultrasound or electrical energy to the subject
after administering the agent; and (c) administering the
nucleic acid molecule to the subject upon completion of the
application of ultrasound or electrical energy, whereby the
nucleic acid molecule is delivered into the cell.
In another aspect, the present invention provides
a method for delivering a nucleic acid molecule into a cell
in a subject comprising: (a) applying ultrasound or
electrical energy to the subject; and (b) administering to
the subject a nucleic acid molecule and a delivery agent,
upon conclusion of the application of ultrasound or
electrical energy, whereby the nucleic acid molecule is
delivered into the cell, wherein the delivery agent and
nucleic acid are administered sequentially or as a single
composition.
In another aspect, the present invention provides
a method for delivering a nucleic acid molecule into a cell
in a subject comprising: (a) applying ultrasound or
electrical energy to the subject; and (b) administering to
the subject the nucleic acid molecule upon conclusion of the
application of ultrasound or electrical energy, whereby the
nucleic acid molecule is delivered into the cell.
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In another aspect, the present invention provides
a method for ex vivo gene therapy, comprising:
(a) delivering, in vitro, a nucleic acid molecule into a
cell by contacting the cell with the nucleic acid molecule
mixed with a composition that comprises a compound that
delivers nucleic acid molecules into cells; (b) applying
ultrasound or electrical energy to the cell contacted with
the nucleic acid molecule and the compound, whereby the
nucleic acid molecule is delivered into the cell to a
greater extent than in the presence of the compound alone;
and (c) introducing the cell into a subject.
In another aspect, the present invention provides
a method for ex vivo gene therapy, comprising:
(a) contacting, in vitro, a cell in the absence of a nucleic
acid molecule with a composition that comprises a compound
that delivers nucleic acid molecules to a cell;
(b) contacting the cell previously contacted with the
compound with the nucleic acid molecule; (c) applying
ultrasound or electrical energy to the cell contacted with
the compound and the nucleic acid molecule, whereby the
nucleic acid molecule is delivered into the cell to a
greater extent than in the presence of the compound alone;
and (d) introducing the cell into a subject.
In another aspect, the present invention provides
a method for ex vivo gene therapy, comprising: (a) applying,
in vitro, ultrasound or electrical energy to a cell;
(b) contacting the cell with a mixture of a nucleic acid
molecules and a composition that comprises a compound that
delivers nucleic acid molecules to a cell, upon conclusion
of the application of ultrasound or energy, whereby the
nucleic acid molecule is delivered into the cell; and
(c) introducing the cell into a subject.
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In another aspect, the present invention provides
a method for ex vivo gene therapy, comprising: (a) applying,
in vitro ultrasound or electrical energy to a cell;
(b) contacting the cell with a composition that comprises a
composition comprising at least one compound that delivers
nucleic acid molecules into a cell, upon conclusion of the
application of ultrasound or electrical energy; and
(c) contacting the cell previously contacted with the
compound with a nucleic acid molecule, whereby the nucleic
acid molecule is delivered into the cell; and
(d) introducing the cell into a subject.
In another aspect, the present invention provides
a method for ex vivo gene therapy, comprising:
(a) contacting, in vitro a cell with a nucleic acid
molecule; (b) applying ultrasound or electrical energy to
the cell contacted with the nucleic acid molecule, whereby
the nucleic acid molecule is delivered into the cell; and
(c) introducing the cell into a subject.
In another aspect, the present invention provides
a method for ex vivo gene therapy, comprising: (a) applying,
in vitro, ultrasound or electrical energy to the cell;
(b) contacting the cell with a nucleic acid molecule upon
conclusion of the application of ultrasound or electrical
energy, whereby the nucleic acid molecule is delivered into
the cell; (c) introducing the cell into a subject.
In another aspect, the present invention provides
a method for detecting or determining delivery and
expression of a nucleic acid introduced into a cell
comprising; introducing labeled nucleic acid molecules that
encode a reporter gene into cells; detecting labeled cells
as an indication of delivery of the nucleic acid into a
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cell; and measuring the product of the reporter gene as an
indication of DNA expression in the cell, whereby delivery
and expression of nucleic acid molecules in the cell is
detected or determined.
In another aspect, the present invention provides
a method for monitoring the delivery of a nucleic acid
molecule into a cell comprising: (a) labeling the nucleic
acid molecule; (b) delivering the labeled nucleic acid
molecule into a cell; and (c) detecting the labeled nucleic
acid molecule in the cells by flow cytometry, fluorimetry,
cell imaging or fluorescence spectroscopy, as an indication
of delivery of nucleic acid molecule into the cells.
In another aspect, the present invention provides
a method for screening agents for the ability to deliver a
nucleic acid molecule into a cell comprising: (a) delivering
a labeled nucleic acid molecule into the cell in the
presence of the agent; and (b) determining the number of
cells containing the label, as an indication of the ability
of the agent to deliver nucleic acid molecules into the
cell.
In another aspect, the present invention provides
a method for introducing a large nucleic acid molecule into
a synoviocyte, comprising: (a) exposing the nucleic acid
molecule to a delivery agent; (b) exposing the synoviocyte
to a delivery agent; and (c) contacting the synoviocyte with
the nucleic acid molecule, whereby the nucleic acid molecule
is delivered into the synoviocyte, wherein steps (a)-(c) are
performed sequentially in any order or simultaneously.
In another aspect, the present invention provides
a method for delivering a nucleic acid molecule into a
synoviocyte comprising: (a) contacting the synoviocyte in
the absence of the nucleic acid molecule with a delivery
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agent, and applying ultrasound energy or electrical energy
to the synoviocyte, wherein the contacting and applying are
performed sequentially or simultaneously; and then (b)
contacting the synoviocyte with the nucleic acid molecule,
whereby the nucleic acid molecule is delivered into the
synoviocyte.
In another aspect, the present invention provides
a method for delivering a nucleic acid molecule into a
synoviocyte comprising: (a) contacting the synoviocyte with
the nucleic acid molecule and a delivery agent, and applying
ultrasound energy or electrical energy to the synoviocyte,
wherein the contacting and applying are performed
sequentially or simultaneously and whereby the nucleic acid
molecule is delivered into the synoviocyte.
In another aspect, the present invention provides
a method for modulating a rheumatic disease process in a
subject, comprising, introducing a large nucleic acid into
the subject; wherein the large nucleic acid comprises
nucleic acid that is or that encodes an agent that modulates
a rheumatic disease process.
In another aspect, the present invention provides
a method for identifying candidate agents for the treatment
of a connective tissue disease, comprising: introducing a
large nucleic acid molecule comprising nucleic acid that is
or encodes a candidate agent into an animal model of the
disease; and determining if the agent ameliorates one or
more conditions of the disease in the animal.
In another aspect, the present invention provides
a composition comprising cells selected for therapeutic
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treatment of a joint, wherein the cells comprise a large
heterologous nucleic acid.
In another aspect, the present invention provides
a composition comprising cells selected for therapeutic
treatment of a joint, wherein the cells comprise an
artificial chromosome.
In another aspect, the present invention provides
a composition comprising cells selected for therapeutic
treatment of rheumatoid arthritis, wherein the cells
comprise a large heterologous nucleic acid.
In another aspect, the present invention provides
a composition comprising cells selected for therapeutic
treatment of rheumatoid arthritis, wherein the cells
comprise an artificial chromosome.
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DETAILED DESCRIPTION OF THE INVENTION
A. DEFINITIONS
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood'by one of skill
in the art to which this invention belongs.
As used herein, "nucleic acid" refers to a poiynucieotide containing
at least two covalently linked nucleotide or nucleotide analog subunits. A
nucleic acid can be a deoxyribonucleic acid (DNA), a ribonucleic acid
(RNA), or an analog of DNA or RNA. Nucleotide analogs are commercially
available and methods of preparing polynucleotides containing such
nucleotide analogs are known (Lin et al. (1994) Nuc% Acids Res.
22:5220-5234; Jellinek et al. (1995) Biochemistry 34:1 1 363-1 1 372;
Pagratis et al. (1997) Nature Biotechno% 15:68-73). The nucleic acid can
be single-stranded, double-stranded, or a mixture thereof. For purposes
herein, unless specified otherwise, the nucleic acid is double-stranded, or
it is apparent from the context.
The term "nucleic acid" refers to single-stranded and/or double-
stranded polynucleotides, such as deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or
DNA. Also included in the term "nucleic acid" are analogs of nucleic
acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and
other such analogs and derivatives.
As used herein, DNA is meant to include all types and sizes of DNA
molecules including cDNA, plasmids and DNA including modified
nucleotides and nucleotide analogs.
As used herein, nucleotides include nucleoside mono-, di-, and
triphosphates. Nucleotides also include modified nucleotides, such as,
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but are not limited to, phosphorothioate nucleotides and deazapurine
nucleotides and other nucleotide analogs.
As used herein, the term "large nucleic acid molecules" or "large
nucleic acids" refers to a nucleic acid molecule of at least about 0.5
megabase pairs (Mbase) in size, greater than 0.5 Mbase, including nucleic
acid molecules at least about 0.6. 0.7, 0.8, 0.9, 1, 5, 10, 30, 50 and
100, 200, 300, 500 Mbase in size. Large nucleic acid molecules typically
may be on the order of about 10 to about 450 or more Mbase, and may
be of various sizes, such as, for example, from about 250 to about 400
Mbase, about 150 to about 200 Mbase, about 90 to about 120 Mbase,
about 60 to about 100 Mbase and about 15 to 50 Mbase.
Examples of large nucleic acid molecules include, but are not
limited to, natural chromosomes and fragments thereof, especially
mammalian chromosomes and fragments thereof which retain a
centromere and telomeres, artificial chromosome expression systems
(ACes; also called satellite DNA-based artificial chromosomes (SATACs);
see U.S. Patent Nos. 6,025,155 and 6,077,697), mammalian artificial
chromosomes (MACs), plant artificial chromosomes, insect artificial
chromosomes, avian artificial chromosomes and minichromosomes (see,
e.g., U.S. Patent Nos. 5,712,134, 5,891,691 and 5,288,625). The large
nucleic acid molecules may include a single copy of a desired nucleic acid
fragment encoding a particular nucleotide sequence, such as a gene of
interest, or may carry multiple copies thereof or multiple genes or
different heterologous sequences of nucleotides. For example, ACes can
carry 40 or even more copies of a gene of interest. Large nucleic acid
molecules may be associated with proteins, for example chromosomal
proteins, that typically function to regulate gene expression and/or
participate in determining overall structure.
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As used herein, an artificial chromosome is a nucleic acid molecule
that can stably replicate and segregate alongside endogenous
chromosomes in a cell. It has the capacity to act as a gene delivery
vehicle by accommodating and expressing foreign genes contained
therein. A mammalian artificial chromosome (MAC) refers to
chromosomes that have an active mammalian centromere(s). Plant
artificial chromosomes, insect artificial chromosomes and avian artificial
chromosomes refer to chromosomes that include plant, insect and avian
centromeres, respectively. A human artificial chromosome (HAC) refers
to chromosomes that include human centromeres. For exemplary artificial
chromosomes, see, e.g., U.S. Patent Nos. 6,025,155; 6,077,697;
5,288,625; 5,712,134; 5,695,967; 5,869,294; 5,891,691 and
5,721,1 18 and published International PCT application Nos,
WO 97/40183 and WO 98/08964.
As used herein, the term "satellite DNA-based artificial
chromosome (SATAC)" is interchangeable with the term "artificial
chromosome expression system (ACes)". These artificial chromosomes
are substantially all neutral non-coding sequences (heterochromatin)
except for foreign heterologous, typically gene-encoding nucleic acid, that
is interspersed within the heterochromatin for the expression therein (see
U.S. Patent Nos. 6,025,155 and 6,077,697 and International PCT
application No. WO 97/40183). Foreign genes contained in these
artificial chromosome expression systems can include, but are not limited
to, nucleic acid that encodes traceable marker proteins (reporter genes),
such as fluorescent proteins, such as green, blue or red fluorescent
proteins (GFP, BFP and RFP, respectively), other reporter genes, such as
,6-galactosidase and proteins that confer drug resistance, such as a gene
encoding hygromycin-resistance. Other examples of heterologous DNA
include, but are not limited to, DNA that encodes therapeutically effective
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substances, such as anti-cancer agents, enzymes and hormones, and
DNA that encodes other types of proteins, such as antibodies.
As used herein, the terms "heterologous" and "foreign" with
reference to nucleic acids, such as DNA and RNA, are used
interchangeably and refer to nucleic acid that does not occur naturally as
part of a genome or cell in which it is present or which is found in a
location(s) and/or in amounts in a genome or cell that differ from the
location(s) and/or amounts in which it occurs in nature. It can be nucleic
acid that is not endogenous to the cell and has been exogenously
introduced into the cell. Examples of heterologous DNA include, but are
not limited to, DNA that encodes a gene product or gene product(s) of
interest introduced into cells, for example, for purposes of gene therapy,
production of transgenic animals or for production of an encoded protein.
Other examples of heterologous DNA include, but are not limited to, DNA
that encodes traceable marker proteins, such as a protein that confers
drug resistance, DNA that encodes therapeutically effective substances,
such as anti-cancer agents, enzymes and hormones, and DNA that
encodes other types of proteins, such as antibodies.
As used herein, "delivery," which is used interchangeably with
"transfection," refers to the process by which exogenous nucleic acid
molecules are transferred into a cell such that they are located inside the
cell. Delivery of nucleic acids is a distinct process from expression of
nucleic acids.
As used herein, "expression" refers to the process by which
nucleic acid is translated into peptides or is transcribed into RNA, which,
for example, may be translated into peptides, polypeptides or proteins. If
the nucleic acid is derived from genomic DNA, expression may, if an
appropriate eukaryotic host cell or organism is selected, include splicing
of the mRNA. For heterologous nucleic acid to be expressed in a host
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cell, it must initially be delivered into the cell and then, once in the cell,
ultimately reside in the nucleus.
As used herein, cell recovery refers to a "total cell yield" after a
specified time frame, which for purposes herein is twenty-four hours, and
when used with reference to calculation of the clonal fraction
As used herein, cell recovery time refers to a time frame in order
for a cell to equilibrate to new conditions.
As used herein, cell survival refers to cell viability after a cytotoxic
event, such as a delivery procedure.
As used herein, control plating efficiency (CPE) refers to the
fraction of untreated cells, under standard optimal growth conditions for
the particular cells, that survive a plating procedure. Plating efficiency
refers to the fraction of treated cells that survive a plating procedure.
As used herein, clonal fraction is a measurement of cell recovery
after delivery of exogenous nucleic acids into cells and the plating
efficiency of the cells.
As used herein, transfer efficiency is the percentage of the total
number of cells to which nucleic acids are delivered that contain delivered
nucleic acid.
As used herein, transfection efficiency is the percentage of the
total number of cells to which nucleic acids including a selectable marker
are delivered that survive selection.
As used herein, index of potential transfection efficiency means the
theoretical maximum transfection efficiency for a particular cell type under
particular conditions, for example particular concentrations or amounts of
particular delivery agents.
As used herein, the term "cell" is meant to include cells of all
types, of eukaryotes and prokaryotes, including animals and plants.
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As used herein, "delivery agent" refers to compositions, conditions
or physical treatments to which cells and/or nucleic acids may be exposed
in the process of transferring nucleic acids to cells in order to facilitate
nucleic acid delivery into cells. Delivery agents include compositions,
conditions and physical treatments that enhance contact of nucleic acids
with cells and/or increase the permeability of cells to nucleic acids. In
general, nucleic acids are not directly treated with energy, such as
sonoporation.
As used herein, cationic compounds are compounds that have polar
groups that are positively charged at or around physiological pH. These
compounds facilitate delivery of nucleic acid molecules into cells; it is
thought this is achieved by virtue of their ability to neutralize the
electrical
charge of nucleic acids. Exemplary cationic compounds include, but are
not limited to, cationic lipids or cationic polymers or mixtures thereof,
with or without neutral lipids, polycationic lipids, non-liposomal forming
lipids, ethanolic cationic lipids and cationic amphiphiles. Contemplated
cationic compounds also include activated dendrimers, which are
spherical cationic polyamidoamine polymers with a defined spherical
architecture of charged amino groups which branch from a central core
and which can interact with the negatively charged phosphate groups of
nucleic acids (e.g., starburst dendrimers).
Cationic compounds for use as delivery agents also include
mixtures of cationic compounds that include peptides and protein
fragments. The additional components may be non-covalently or
covalently bound to the cationic compound or otherwise associated with
the cationic compound.
As used herein, ultrasound energy is meant to include sound waves
(for external application) and lithotripter-generated shock waves (for
internal application).
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As used herein, electrical energy is meant to include the application
of electric fields to cells so as to open pores in membranes for the
delivery of molecules into the cell, e.g., electroporation techniques.
As used herein, cavitation compound is meant to include contrast
agents that are typically used with ultrasound imaging devices and
includes gas encapsulated and nongaseous agents. These cavitation
compounds enhance the efficiency of energy delivery of acoustic or shock
waves.
As used herein, "pharmaceutically acceptable" refers to
compounds, compositions and dosage forms that are suitable for
administration to the subject without causing excessive toxicity, irritation,
allergic response or other undesirable complication.
As used herein, embryonic stem cells are primitive, immature cells
that are precursors to stem cells.
As used herein, stem cells are primitive, immature cells that are
precursors to mature, tissue specific cells.
As used herein, nuclear transfer donor cells are cells that are the
source of nuclei, which are transferred to enucleated oocytes during the
process of nuclear transfer.
As used herein, the term "subject" refers to animals, plants,
insects, and birds into which the large DNA molecules may be introduced.
Included are higher organisms, such as mammals and birds, including
humans, primates, rodents, cattle, pigs, rabbits, goats, sheep, mice, rats,
guinea pigs, cats, dogs, horses, chicken and others.
As used herein, "administering to a subject" is a procedure by
which one or more delivery agents and/or large nucleic acid molecules,
together or separately, are introduced into or applied onto a subject such
that target cells which are present in the subject are eventually contacted
with the agent and/or the large nucleic acid molecules.
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As used herein, "applying to a subject" is a procedure by which
target cells present in the subject are eventually contacted with energy
such as ultrasound or electrical energy. Application is by any process by
which energy may be applied.
As used herein, gene therapy involves the transfer or insertion of
nucleic acid molecules, and, in particular, large nucleic acid molecules,
into certain cells, which are also referred to as target cells, to produce
specific gene products that are involved in correcting or modulating
diseases or disorders. The nucleic acid is introduced into the selected
target cells in a manner such that the nucleic acid is expressed and a
product encoded thereby is produced. Alternatively, the nucleic acid may
in some manner mediate expression of DNA that encodes a therapeutic
product. This product may be a therapeutic compound, which is
produced in therapeutically effective amounts or at a therapeutically
useful time. It may also encode a product, such as a peptide or RNA,
that in some manner mediates, directly or indirectly, expression of a
therapeutic product. Expression of the nucleic acid by the target cells
within an organism afflicted with a disease or disorder thereby provides a
way to modulate the disease or disorder. The nucleic acid encoding the
therapeutic product may be modified prioi- to introduction into the cells of
the afflicted host in order to enhance or otherwise alter the product or
expression thereof.
For use in gene therapy, cells can be transfected in vitro, followed
by introduction of the transfected cells into the body of a subject. This is
often referred to as ex vivo gene therapy., Alternatively, the cells can be
transfected directly in vivo within the body of a subject.
As used herein, flow cytometry refers to processes that use a laser
based instrument capable of analyzing and sorting out cells and or
chromosomes based on size and fluorescence.
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As used herein, a reporter gene includes any gene that expresses a
detectable gene product, which may be RNA or protein. Preferred
reporter genes are those that are readily detectable. Examples of reporter
genes include, but are.not limited to nucleic acid encoding a fluorescent
protein, CAT (chloramphenicol acetyl transferase) (Alton and Vapnek
(1979), Nature 282: 864-869); luciferase, and other enzyme detection
systems, such as beta-galactosidase; firefly luciferase (deWet et al.
(1987), Mol. Cell. Biol. 7: 725-737); bacterial luciferase (Engebrecht and
Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984),
Biochemistry 23: 3663-3667); and alkaline phosphatase (Toh et al.
(1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. MoI. Appl.
Gen. 2: 101).
As used herein, a reporter gene construct is a DNA molecule that
includes a reporter gene operatively linked to a transcriptional control
sequence. The transcriptional control sequences include a promoter and
other optional regulatory regions, such as enhancer sequences, that
modulate the activity of the promoter, or control sequences that modulate
the activity or efficiency of the RNA polymerase that recognizes the
promoter, or control sequences that are recognized by effector molecules,
including those that are specifically induced by interaction of an
extracellular signal with a cell surface protein. For example, modulation
of the activity of the promoter may be effected by altering the RNA
polymerase binding to the promoter region, or, alternatively, by interfering
with initiation of transcription or elongation of the mRNA. Such
sequences are herein collectively referred to as transcriptional control
elements or sequences. In addition, the construct can include sequences
of nucleotides that alter translation of the resulting mRNA, thereby
altering the amount of reporter gene product.
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As used herein, promoter refers to the region of DNA that is
upstream with respect to the direction of transcription of the transcription
initiation site. It includes the RNA polymerase binding and transcription
imitation sites and any other regions, including, but not limited to
repressor or activator protein binding sites, ca{cium or cAMP responsive
sites, and any such sequences of nucleotides known to those of skill in
the art to alter the amount of transcription from the promoter, either
directly or indirectly.
As used herein, a promoter that is regulated or mediated by the
activity of a cell surface protein is a promoter whose activity changes
when a cell is exposed to a particular extracellular signal by virtue of the
presence of cell surface proteins whose activities are affected by the
extracellular protein.
B. METHODS FOR THE DELIVERY OF DNA INTO CELLS
A variety of methods for delivering nucleic acids, particularly large
nucleic acid molecules, such as artificial chromosomes, including ACes
(formerly designated SATACs), are provided. The methods generally
involve exposing the nucleic acid molecule to an agent that increases
contact between the nucleic acid molecule and the cell, and exposing the
cell to a permeability enhancing agent. Each of the methods provided
herein requires the use of one or both of these agents, which are applied
in different orders. In general, agents, such as energy, which increase
the permeability of a cell, are applied before contacting the cell with a
nucleic acid.
In methods provided herein, large nucleic acid molecules are
delivered using agents, including, but not limited to, delivery agents that
enhance contact between the nucleic acid molecules and the cells and/or
agents and treatments that increase cell permeability. Generally the
nucleic acid molecules are delivered using agents that enhance contact
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between the nucleic acid and cells by neutralizing the charge of the
nucleic acid molecules, and also by using energy to increase permeability
of the cells. The agents can be used individually and in various
combinations and orders of application. In general, energy, such as
sonoporation and electroporation, is not applied to cells after the nucleic
acid molecule is added thereto.
The method selected for delivering particular nucleic acid
molecules, such as DNA, to targeted cells can depend on the particular
nucleic acid molecule being transferred and the particular recipient cell.
Preferred methods for particular nucleic acid molecules, such as DNA, and
recipient cells are those that result in the greatest amount of nucleic acid
molecules, such as DNA, transferred into the cell nucleus with an
acceptable degree of cell survival. Suitable methods for delivery of
particular pairings of nucleic acid molecules, such as DNA, and recipient
cells can be determined using methods of monitoring nucleic acid
molecules, such as DNA, delivery and methods of screening age,nts and
conditions as provided herein or can be determined empirically using
methods known to those of skill in the art.
The method selected requires consideration of a number of
parameters, which are discussed in detail below. A method for detection
of delivered nucleic acid is provided. This method, which can be used for
assessing delivery of any nucleic acid molecule, can be used as a rapid
screening tool to optimize nucleic acid, e.g., chromosome, transfer
conditions.
In particular, delivery methods can first be assessed for the ability
to transfer nucleic acid molecules, such as DNA, into cells and to identify
methods that provide a sufficient number of viable cells that express the
transferred nucleic acid molecules, such as DNA. Once such methods are
identified, they can be optimized using the delivery monitoring methods
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provided herein and then assessed for the ability to provide for expression
of the transferred nucleic acid molecules.
Delivery agents
Delivery agents include compositions, conditions and physical
treatments that enhance contact of nucleic acid molecules, such as DNA,
with cells and/or increase the permeability of cells to nucleic acid
molecules, such as DNA. Such agents include, but are not limited to,
cationic compounds, peptides, proteins, energy, for example ultrasound,
energy and electric fields, and cavitation compounds.
Delivery agents for use in the methods provided herein include
compositions, conditions or physical treatments to which cells and/or
nucleic acid molecules, such as DNA, can be exposed in the process of
transferring nucleic acid molecules, such as DNA, to cells in order to
facilitate nucleic acid molecules, such as DNA, delivery into cells. For
example, compounds and chemical compositions, including, but not
limited to, calcium phosphate, DMSO, glycerol, chloroquine, sodium
butyrate, polybrene and DEAE-dextran, peptides, proteins, temperature,
light, pH, radiation and pressure are all possible delivery agents.
Cationic Compounds
Cationic compounds for use in the methods provided herein are
available commercially or can be synthesized by those of skill in the art.
Any cationic compound can be used for delivery of nucleic acid molecules,
such as DNA, into a particular cell type using the provided methods. One
of skill in the art by using the provided screening procedures can readily -
determine which of the cationic compounds are best suited for delivery of
specific nucleic acid molecules, such as DNA, into a specific target cell
type.
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(a) Cationic Lipids
Cationic lipid reagents can be classified into two general categories
based on the number of positive charges in the lipid headgroup; either a
single positive charge or multiple positive charges, usually up to 5.
Cationic lipids are often mixed with neutral lipids prior to use as delivery
agents. Neutral lipids include, but are not limited to, lecithins; phospha-
tidylethano(amine; phosphatidylethanolamines, such as DOPE
(dioleoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidyl-
ethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine) and
distearoylphosphatidylethanolamine; phosphatidylcholine;
phosphatidylcholines, such as DOPC
(dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine),
POPC (palmitoyloleoylphosphatidylcholine) and distearoyl-
phosphatidylcholine; fatty acid esters; glycerol esters; sphingolipids;
cardiolipin; cerebrosides; and ceramides; and mixtures thereof. Neutral
lipids also include cholesterol and other 3flOH-sterols.
Other lipids contemplated herein, include: phosphatidylglycerol;
phosphatidylglycerols, such as DOPG (dioleo.ylphosphatidylglycerol),
DPPG (dipalmitoylphosphatidylglycerol), and distearoyl-
phosphatidylglycerol; phosphatidylserine; phosphatidyiserines, such as
dioleoyl- or dipaimitoylphosphatidylserine and diphosphatidylglycerols.
Examples of cationic lipid compounds include, but are not limited
to: Lipofectin*(Life Technologies, Inc., Burlington, Ont.)(1:1 (w/w)
formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-
trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanol-
*
amine (DOPE)); LipofectAMINE (Life Technologies, Burlington, Ont., see
U.S. Patent No. 5,334,761) (3:1 (w/w) formulation of polycationic lipid
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-
propanaminiumtrifluoroacetate (DOSPA) and dioleoy.lphosphatidyl-
*Trade-mark
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ethanolamine (DOPE)), LipofectAMINE PLUS (Life'Technologies,
Burlington, Ont. see U.S. Patent Nos. 5,334,761 and 5,736,392; see,
also U.S. Patent No. 6,051,429) (LipofectAmine and Plus reagent),
LipofectAMINE 2000 (Life Technologies, Burlington, Ont.; see also
International PCT application No: WO 00/27795) (Cationic lipid),
Effectene (Qiagen, Inc., Mississauga, Ontario) (Non liposomal lipid
formulation), Metafectene (Biontex, Munich, Germany) (Polycationic lipid),
*
Eu-fectins (Promega Biosciences, Inc., San Luis Obispo, CA) (ethanolic
cationic lipids numbers 1 through 12: CwH,osN504=4CF3CO2H,
C88H178N$O4S2-4CF3CO2H, C40H84N03P=CF3CO2H, C60H103N7O3-4CF3CO2H,
C65HõsN802=6CF3CO2H; Ca9H1oZN6O3-4CFsCO2H, C44H89N503-2CF3CO2H,
C,ooH2o6N1204S2=8CF3COZH, C,s2H3aoN2209=13CF3CO2H, C43HWN4O2=2CF3CO2H,
C43H88N4O3-2CF3CO2H, C41H78NO8P); Cytofectene
(Bio-Rad, Hercules,.CA ) (mixture of a cationic lipid and a neutral lipid),
GenePORTER*(Gene Therapy Systems Inc., San Diego, CA) (formulation
of a neutral lipid (DOPE) and a cationic lipid) and FuGENE 6 (Roche
Molecular Biochemicals, Indianapolis, IN) (Multi-component lipid based
non-liposomal reagent).
(b) Non-lipid cationic compounds
Non-lipid cationic reagents include, but are not limited to
SUPERFECT' (Qiagen, Inc., Mississauga, ON) (Activated dendrimer
(cationic polymer:charged amino groups)) and CLONfectinM (Cationic
amphiphile N-t-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine)
(Clontech, Palo Alto, CA).
Pyridinium amphiphiles are double-chained pyridinium compounds,
which are essentially nontoxic toward cells and exhibit little cellular
preference for the ability to transfect- cells. Examples of a pyridinium
amphiphiles are the pyridinium chloride surfactants such as SAINT-2= (1-
methyl-4-(1-octadec-9-enyi-nonadec-10-enylenyU pyridinium chloride)
*Trade-mark
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(see, e.g., van der Woude et al. (1997) Proc. Natl. Acad. Sci. U.S.A.
94:1160). The pyridinium chloride surfactants are typically mixed with
neutral helper lipid compounds, such as dioleoylphosphatidylethanolamine
(DOPE), in a 1:1 molar ratio. Other Saint derivatives of different chain
lengths, state of saturation and head groups can be made by those of skill
in the art and are within the scope of the present methods.
Energy
Delivery agents also include treatment or exposure of the cell
and/or nucleic acid molecules, but generally the cells, to sources of
energy, such as sound and electrical energy.
Ultrasound
For in vitro and in vivo transfection, the ultrasound source should
be capable of providing frequency and energy outputs suitable for
promoting transfection. For example, the output device can generate
ultrasound energy in the frequency range of 20 kHz to about 1 MHz. The
power of the ultrasound energy can be, for example, in the range from
about 0.05 w/cm2 to 2 w/cm2, or from about 0.1 w/cm2 to about 1
w/cm2. The ultrasound can be administered in one continuous pulse or
can be administered as two or more intermittent pulses, which can be the
same or can vary in time and intensity.
Ultrasound energy can be applied to the body locally or ultrasound-
based extracorporeal shock wave lithotripsy can be used for "in-depth"
application. The ultrasound energy can be applied to the body of a subject
using various ultrasound devices. In general, ultrasound can be
administered by direct contact using standard or specially made
ultrasound imaging probes or ultrasound needles with or without the use
of other medical devices, such as scopes, catheters and surgical tools, or
through ultrasound baths with the tissue or organ partially or completely
surrounded by a fluid medium. The source of ultrasound can be external
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to the subject's body, such as an ultrasound probe applied to the
subject's skin which projects the ultrasound into the subject's body, or
internal, such as a catheter having an ultrasound transducer which is
placed inside the subject's body. Suitable ultrasound systems are known
(see, e.g., International PCT application No. WO 99/21584 and U.S.
Patent No. 5,676,151).
When the cationic compound and nucleic acid molecules, such as
DNA, are administered systemically, the ultrasound can be applied to one
or several organs or tissues simultaneously to promote nucleic acid
molecule delivery to multiple areas of the subject's body. Alternatively,
the ultrasound can be applied selectively to specific areas or tissues to
promote selective uptake of the nucleic acid molecules, such as DNA.
The transfection efficiency of the ultrasound can also be enhanced
by using contrast reagents, which serve as artificial cavitation nuclei,
such as Albunex =(Molecular Biosystems, San Diego, CA), Imagent
(Alliance Pharmaceutical, San Diego, CA), Levovist-SHU (Schering AG,
*
Berlin, Germany), Definity (E.I. du Pont de Nemour, Wilmington, DL),
STUC (Washington University, St Louis, MO) and the introduction of
gaseous microbubbles. A contrast reagent can be introduced local.ly, such
as a joint; introduced systematically, with the enhancement of cavitation
efficiency by focusing lithotripter shock waves at a defined area; or by
targeting a contrast reagent to a particular site and then enhancing
cavitation efficiency by focusing lithotripter shock waves.
Electroporation
Electroporation temporarily opens up pores in a cell's outer
membrane by use of pulsed rotating electric fields. Methods and
apparatus used for electroporation in vitro and in vivo are well known
(see, e.g., U.S. Patent Nos. 6,027,488, 5,993,434, 5,944,710,
*Trade-mark
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5,507,724, 5,501,662, 5,389,069, 5,318,515). Standard protocols may
be employed.
C. TARGET CELLS AND DELIVERY THERETO
The methods provided herein can be used in the delivery of nucleic
acids into any cells, including, but not limited to, any eukaryotic and
prokaryotic cells. Examples of cells that can be used in the methods
include, but are not limited to, cell lines, primary cells, primary cell
lines,
plant cells and animal cells, including stem cells and embryonic cells. For
example, fibroblasts, including lung and skin fibroblasts, fibroblast-like
cells, synoviocytes, fibroblast-like synoviocytes, stem cells, including
embryonic and adult stem cells, such as mesenchymal stem cells,
myoblasts, lymphoblasts, carcinoma and hepatoma cells are among the
many cells into which nucleic acids, and in particular large nucleic acids
and artificial chromosomes, can be delivered and monitored using the
methods provided herein. Particular cells include mammalian cells, for
example, A9 cells (mouse fibroblasts, HPRT-; ATCC Accession no. CCL-
1.4), CHO-S cells and DG44 cells (Chinese hamster ovary cells), V79 cells
(Chinese hamster lung fibroblasts; ATCC Accession no. CCL-39), LMTK"
cells (mouse fibroblasts; ATCC Accession No. CCL-1.3), skin fibroblasts,
L8 cells (rat myoblasts; ATCC Accession No. CRL-1769), CCD1043 SK
cells (human fibroblasts; ATCC Accession No. CRL-2056), adult-derived
mesenchymal stem cells (e.g., derived from human bone marrow;
Cambrex Biosciences, East Rutherford, New Jersey), synoviocytes (rat
and human), Detroit 551 cells (human embryonic skin fibroblasts; ATCC
Accession No. CCL-1 10), NSO (murine myeloma, ECACC Accession No.
851 10503), 293 cells (human embryonic kidney cells transformed by type
5 (Ad 5) DNA (ATCC Accession No. CRL-1 573), P46-Fl (bovine
lymphocyte-like cell line), DT40 (chicken lymphoblasts), EJ30 cells
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(human bladder carcinoma), HepG2 cells (human hepatoma) and murine
and bovine embryos.
In particular embodiments, the methods of delivery of nucleic acids
into cells provided herein can be used in delivering nucleic acids into cells
in order to treat a disease or disorder, e.g., in gene therapy applications.
In gene therapy applications, the nucleic acid to be delivered into a cell
may encode a therapeutic molecule, e.g., a protein. In many instances,
successful gene therapy applications are complicated by a requirement
that large nucleic acids be delivered into cells. It may also be desired to
provide multiple copies of nucleic acid encoding one or more therapeutic
molecules. Compounding the difficulties in gene therapy methods is the
challenge that cells preferred for use in gene therapy applications are
often not readily transfectable. The methods provided herein are
particularly well suited for delivery of large nucleic acids, which may be in
the form of artificial chromosomes or fragments thereof, into cells as may
be used in therapeutic applications.
In Vitro Delivery
'Cationic compounds and nucleic acid molecules, such as DNA, can
be added to cells in vitro either separately or mixed together and with or
without the application of ultrasound or electrical energy. In general, if
energy is applied, it is applied prior to contacting the cells with the
nucleic
acid molecule.
In general, nucleic acid molecules, such as DNA, mixed with
cationic lipids/compounds can be added to a cell as described in the
EXAMPLES. Parameters important for optimization of the delivery of
nucleic acid molecules, such as DNA, into target cells will be apparent to
those of skill in this art. These parameters include, for example, the
cationic compound, cationic compound concentration, the nucleic acid
molecules, such as DNA, the concentration of nucleic acid molecules, the
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cell growth medium, the cell culture conditions, the length of time cells
are exposed to the cationic compound, the toxicity of the cationic
compound to the target cell type, and the amount and time of use of
ultrasound or electroporation among other parameters. It may be
necessary to optimize these parameters for different nucleic acid
molecules, such as DNA, and target cell types. Such optimization is
routine employing the guidance provided herein. In addition, the rapid
screening method can provide direction as to what parameters may need
to be adjusted to optimize delivery (see EXAMPLES). Alteration of culture
conditions, time, reagent concentrations and other parameters, for use
with different combinations of cationic compounds and target cell types
and to optimize delivery, can be empirically determined. If ultrasound
energy is required to be used to enhance transfection efficiency, it can be
appiied as described below and in the EXAMPLES. Electroporation can be
performed as described below or by any suitable protocol known to those
of skill in this art.
The contacting of cells with cationic compounds and nucleic acid
molecules, such as DNA, in separate and distinct steps can be generally
carried out as described in the EXAMPLES. Those of skill in the art can
readily vary the order of the application of the components to the target
cell based on the disclosure herein.
Ex Vivo Gene Therapy
Delivery of nucleic acid molecules, such as DNA, is carried out as
described above in in vitro delivery. After selection has been completed,
cells harboring the nucleic acid molecules, such as DNA, are introduced
into the subject target by a variety of means, including injection, such as
subcutaneous, intramuscular, intraperitoneal, intravascular and
intralymphatic and intra-articular injection. The cells can be administered
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with or without the aid of medical devices such as arthroscopes, other
scopes or various types of catheters.
In Vivo Gene Therapy
In one method for delivering of the nucleic acid molecules, such as
DNA, to target cells in the body of a subject in vivo, the cationic
compound is first delivered to the target area (e.g., tissue, organ, tumor
or joint). After waiting a suitable amount of time, the target area is then
subjected to ultrasound frequency at a suitable energy level for a suitable
time, which will be dependent on the equipment, tissue type and depth of
the target area in the body. Alternatively, electrical energy is delivered to
the target area. The nucleic acid molecule, such as DNA, is then
delivered to the same area. Optionally, this procedure can be repeated so
that the nucleic acid molecules, such as DNA, can be delivered via
multiple injections over time or multiple administrations in different areas
at the same time.
The cationic compound mixed together with the nucleic acid
molecules, such as DNA, can be delivered to the target area. The target
area can then be subjected to ultrasound frequency at a suitable energy
level for a suitable time. Depending on the nucleic acid molecules, such
as DNA, the in vivo location, the cationic compound used and other
variables, it may not be necessary to use ultrasound or electroporation to
achieve suitable transfer efficiency to cells at the target area. Prior to the
application of ultrasound, contrast reagents can be delivered to the target
area to enhance transfer of the nucleic acid molecules, such as DNA.
The nucleic acid molecules, such as DNA, can be delivered to
organs or tissues of the body such as skin, muscle, stomach, intestine,
lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen,
prostate and joints (for example the knee, elbow, shoulder, wrist, hip,
finger, ankle and others). Molecules can be delivered to, for example,
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primary cells and cell lines, such as fibroblast, muscle, stomach, intestine,
lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen,
prostate to mimic in vivo systems.
The cationic compounds and the nucleic acid molecules, such as
DNA, separately or together can be delivered to the target area of the
body by a variety of means, including injection (for example,
subcutaneous, intramuscular, intraperitoneal, intravascular, intra-articular
and intralymphatic injection), instillation, cannulation, slow infusion,
topical application and any other mode of administration. They can be
administered by any suitable mode, including systemically (for example by
intravenous injection), locally, such as by delivery to a specific target area
(tissue or area), using, for example, a catheter or by direct injection.
They can be administered with or without the aid of medical devices such
as arthroscopes, other scopes or various types of catheters.
The cationic compounds can be administered also by coating a
medical device, for example, a catheter, such as an angioplasty balloon
catheter, with a cationic compound formulation. Coating may be
achieved, for example, by dipping the medical device into a cationic lipid
formulation or a mixture of a cationic compound formulation and a
suitable solvent, for example, an aqueous-buffer, an aqueous solvent,
ethanol, methylene chloride, chloroform and other suitable solvent. An
amount of the formulation will naturally adhere to the surface of the
device, which is subsequently administered to a subject, as appropriate.
Alternatively, a lyophilized mixture of a cationic lipid formulation may be
specifically bound to the surface of the device. Such binding techniques
are known (see, e.g., Ishihara et al. (1993) Journal of Biomedical
Materials Research 27:1309-1314).
The cationic compounds and nucleic acid molecules, such as DNA,
can be formulated in pharmaceutically acceptable carriers, such as saline
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or other pharmaceutically acceptable solutions, for delivery in vivo. The
nucleic acid molecules, such as DNA, and cationic compounds, regardless
of the route of administration, are formulated into pharmaceutically
acceptable dosage forms by standard methods known to those of skill in
the art.
For gene therapy, the dosage level of the nucleic acid molecules,
such as DNA, may be varied to achieve optimal therapeutic response for a
particular subject. This depends on a variety of factors including mode of
administration, activity of the nucleic acid molecules, such as DNA,
characteristics of the protein produced, the transfection efficiency of the
target cells (their ability to take up the nucleic acid molecules, such as
DNA), the route of administration, the location of the target cells and
other factors
The dosage to be administered and the particular mode of adminis-
tration will vary depending upon such factors as the age, weight and the
particular animal and region thereof to be treated, the particular nucleic
acid molecule and cationic compound used, the therapeutic or diagnostic
use contemplated, and the form of the formulation, for example,
suspension, emulsion, or liposomal, as will be readily apparent to those
skilled in the art. Typically, dosage is administered at lower levels and
increased until the desirable therapeutic effect is achieved. The amount of
cationic compound that is administered can vary and generally depends
upon the amount of nucleic acid molecules, such as DNA, being
administerecl. For example, the weight ratio of cationic compound to
nucleic acid molecules can be from about 1:1 to about 15:1, including,
for example, a weight ratio of about 5:1 to about 1:1. Generally, the
amount of cationic compound which is administered will vary from
between about 0.1 milligram (mg) to about 1 gram (g). By way of general
guidance, typically for a bodyweight of 70 kg and a composition with
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about 1 to 10 million chromosomes per mi, single dose ranging from 1 to
20 ml is administered as a single or repeated dose.
For localized treatment of diseases, such administration to affected
joints in rheumatoid arthritis, psoriasis and diabetes should be possible, as
well as injection into muscle for treatment of diseases, such as hemophilia
or other genetic diseases. For other than local treatment a targeted
delivery step is needed.
Cells for effecting treatment of disease are also provided. For
example, compositions are provided that contain cells selected for
therapeutic treatment of a joint or rheumatoid arthritis, wherein the cells
contain a large heterologous nucleic acid. For example, the cells of such
a composition may contain an artificial chromosome. In a particular
embodiment of such compositions, the cells can contain an ACes.
Gene Therapy in Connective Tissue and Rheumatic Diseases
Rheumatoid arthritis (RA) is a chronic inflammatory disease
characterized by joint inflammation and progressive cartilage and bone
destruction. Treatment of RA is problematic with current strategies since
relatively high systemic doses are necessary to achieve therapeutic levels
of anti-rheumatic drugs in the joints. In addition, the available treatments
are associated with significant untoward side effects. Gene therapy is
thus a more efficient system for delivery of therapeutic molecules to the
site of inflammation in the treatment of connective tissue diseases,
rheumatic diseases and chronic erosive joint diseases such as RA,
osteoarthritis, ankylosing spondylitis and juvenile chronic arthritis.
In a diarthrodial movable joint, smooth articulation is ensured by
the macromolecular structure of the articular cartilage which covers the
ends of the bones. The cavity or joint space that occurs at the location
of adjacent bones is lined by a tissue referred to as the synovium. The
synovium contains macrophage-like type A cells (presumably derived from
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macrophage/monocyte precursors and exhibiting phagocytic activity) and
fibroblast-like type B cells (more fibroblast in appearance and associated
with production of hyaluronic acid and other components of the joint
fluid). Underlying the synovium is a sparsely cellular subsynovium which
may be fibrous, adipose or areolar in nature. Fibroblast-like synoviocytes
(FLS) are distinguishable from normal fibroblast cells in the subintimal
synovium by differential gene expression patterns. FLS have been shown
to express high levels of uridine diphosphoglucose dehydrogenase
(UDPGD), high levels of vascular cell adhesion molecule-1 (VCAM-1),
intercellular adhesion molecule-1 (ICAM-1) as well as CD44 (hyaluronic
acid receptor), fibronectin receptor and ,63 integrins. Sublining fibroblasts
or fibroblasts from other sources do not express these markers or express
them at lower levels [see, e.g., Edwards (1995) Ann. Rheum. Dis.
54:395-397; Firestein (1996) Arthritis Rheum. 39:1781-1790; Edwards
(2000) Arthritis Res. 2:344-347].
Disease progression in RA involves the thickening of the synovial
lining due to the proliferation of fibroblast-like synoviocytes (FLS) and
infiltration by inflammatory cells (e.g., lymphocytes, macrophages and
mast cells). The normal biology of synoviocytes is also altered in the
pathological process of RA, including invasion and destruction of articular
cartilage and bone. In addition to the production of elastase and
collagenase, synoviocytes mediate the pathophysiological process of RA
by expression of cell surface proteins involved in the recruitment and
activation of lymphocytes and macrophages within the synovium.
Proliferation of synovial cells leads to a pannus tissue that invades and
overgrows cartilage, leading to bone destruction and destruction of joint
structure and function. Proinflammatory cytokines, for example, tumor
necrosis factor-a (TNF-a) and interleukin-1 (IL-1) play key roles in
inflammation and joint damage associated with RA. Pathological effects
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caused by these cytokines include leukocytic infiltration leading to
synovial hyperplasia, cell activation, cartilage breakdown and inhibition of
cartilage matrix synthesis.
Nucleic acid transfer to rheumatoid synovial tissue may result in the
production of mediators that inhibit inflammation or hyperplasia or provide
toxic substances that specifically destroy the diseased synovium.
Retroviral delivery of nucleic acid encoding interleukin-1 receptor
antagonist (IL1-RA) ex vivo and transduction of synoviocytes has been
used in gene therapy of RA in humans to inhibit inflammation [see, e.g.,
Evans (1996) Human Gen. Ther. 7:1261-1280 and Del Vecchio et al.
(2001) Arthritis Res. 3:259-263]. Adenoviral vectors have been proposed
for delivery of nucleic acid encoding an IL-1 receptor antagonist to
synoviocytes in in vivo transduction methods [see, e.g., U.S. Patent No.
5,747,072 and PCT Application Publication No. WO 00/521861.
Artificial chromosomes provide advantages over virus-based
systems for gene therapy. For example, artificial chromosome expression
systems (ACes), and other artificial chromosomes as described in U.S.
Patent Nos. 6,025,155 and 6,077,697 and PCT Application No.
W097/40183, serve as non-integrating, non-viral vectors with a large
capacity for delivering large nucleic acids and/or multiple copies of a
particular nucleotide sequence into cells, such as synoviocytes, both in
vitro and in vivo. Such artificial chromosome systems offer further
advantages in that they allow stable and predictable expression of genes
producing single or multiple proteins over long periods of time.
The methods provided herein may be used to introduce large
nucleic acids, such as, for example, artificial chromosomes, into primary
cells, such as, for example, synoviocytes (e.g., fibroblast-like
synoviocytes) and skin fibroblasts, and skeletal muscle fibroblast cell
lines. Thus, included among the methods provided herein is a method for
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introducing heterologous nucleic acid into a synoviocyte by introducing in
a chromosome, such as for example an artificial chromosome, into the
synoviocyte. In one such embodiment, the artificial chromosome is an
ACes. The synoviocyte can be, for example, a fibroblast-like
synoviocyte.
A particular method provided herein for introducing a large nucleic
acid molecule into a synoviocyte includes steps of exposing the nucleic
acid molecule to a delivery agent and contacting the synoviocyte with the
nucleic acid molecule. In a particular embodiment of this method, the
delivery agent is not energy. In one embodiment, the large nucleic acid
molecule is a chromosome. For example, the nucleic acid can be an
artificial chromosome, such as an ACes. In a particular embodiment, the
synoviocyte is a fibroblast-like synoviocyte. Any delivery agents, such as
described herein, may be used in such methods. For example, the
delivery agent can be one that includes a cationic compound.
Also provided is a method for introducing a nucleic acid molecule
into a synoviocyte that includes steps of exposing the nucleic acid
molecule to a delivery agent, exposing the synoviocyte to a delivery agent
and contacting the synoviocyte with the nucleic acid molecule, whereby
the nucleic acid molecule is delivered into the synoviocyte, and wherein
the steps are performed sequentially in any order or simultaneously. In
some embodiments of the method, if the delivery agent is energy, it is not
applied to the nucleic acid molecule and it is not applied to the
synoviocyte after contacting the synoviocyte with the nucleic acid
molecule. The nucleic acid may be any nucleic acid. In particular
embodiments, the nucleic acid is a large nucleic acid, chromosome,
artificial chromosome or ACes. In a further particular embodiment, the
synoviocyte is a fibroblast-like synoviocyte. Delivery agents, such as
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described herein, may be used in such methods. For example, the
delivery agent can be one that includes a cationic compound.
Another method for delivering a nucleic acid molecule into a
synoviocyte provided herein includes steps of contacting the synoviocyte
in the presence or absence of the nucleic acid molecule with a delivery
agent, and applying ultrasound energy or electrical energy to the
synoviocyte, wherein the contacting and applying are performed
sequentially or simultaneously, and then contacting the synoviocyte with
the nucleic acid molecule, whereby the nucleic acid molecule is delivered
into the synoviocyte. The nucleic acid may be any nucleic acid. In
particular embodiments, the nucleic acid is a large nucleic acid,
chromosome, artificial chromosome or ACes. In a further particular
embodiment, the synoviocyte is a fibroblast-like synoviocyte. Numerous
delivery agents, including agents such as those described herein, may be
used in such methods. For example, the delivery agent can be one that
includes a cationic compound. In one embodiment, the energy is
ultrasound.
Thus, provided herein are methods of delivering nucleic acids, in
particular, large nucleic acids, such as chromosomes, including artificial
chromosomes, e.g., ACes, into primary cells, including synoviocytes and
fibroblasts. These methods may be used in vitro and in vivo.
Also provided herein is a synoviocyte comprising a large
heterologous nucleic acid, a heterologous chromosome or portion thereof,
or an artificial chromosome. In one embodiment, the artificial
chromosome is an ACes. Such synoviocytes include fibroblast-like
synoviocytes. The synoviocytes may be from any species, including, but
not limited to mammalian species. For example, synoviocytes containing
large nucleic acids, such as, for example, artificial chromosomes (e.g.,
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ACes) include primate synoviocytes, as well as rodent, rabbit, monkey,
dog, horse and human synoviocytes.
The ability to achieve delivery of large nucleic acids into such cells
demonstrates the usefulness of the methods in gene therapy applications
as well as in the testing in animal models of disease of possible
therapeutic molecules for use in gene therapy methods. Thus, provided
herein are methods of treating diseases or modulating disease processes
which include steps of introducing a large nucleic acid molecule,
chromosome or portion thereof, or artificial chromosome into a subject
who has the disease.
A method for treating or modulating a rheumatic disease process in
a subject is provided herein. In one embodiment, the method includes
steps of introducing a large nucleic acid into the subject, wherein the
large nucleic acid contains nucleic acid that is or that encodes an agent
that modulates a rheumatic disease process. For example, the the nucleic
acid can be or can encode a molecule that has an anti-rheumatic effect.
Processes associated with rheumatic diseases are known in the art and
are described herein. For example, one such process is an inflammatory
process that includes processes of cell activation, infiltration,
proliferation
and recruitment. In a particular embodiment of this method, the disease
is rheumatoid arthritis. The nucleic acid may be, for example, a
chromosome or portion thereof or an artificial chromosome, e.g., an
ACes. In particular embodiments, the large nucleic acid is introduced into
a site of inflammation in the subject. One possible site of inflammation is
a joint.
Also provided is a method for treating a rheumatic disease in a
subject in which a large nucleic acid is introduced into the subject,
wherein the large nucleic acid contains nucleic acid that is or that
encodes a therapeutic agent. For example, the the nucleic acid can be or
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can encode a molecule that has an anti-rheumatic effect. In a particular
embodiment of this method, the disease is rheumatoid arthritis. The
nucleic acid may be, for example, a chromosome or portion thereof or an
artificial chromosome, e.g., an ACes. In an embodiment of this method,
the large nucleic acid is introduced into a site of inflammation in the
subject. One possible site of inflammation is a joint.
In the methods for modulating a rheumatic disease process or
treating,a rheumatic disease, the method may be practiced in any format,
including ex vivo and in vivo formats. Thus, for example, the nucleic acid
can be introduced into a cell in vitro and then transferred into the subject.
Alternatively, the nucleic acid can be introduced into a cell in vivo. In a
particular embodiment, the nucleic acid is introduced into a synoviocyte,
which can be, for example, a fibroblast-like synoviocyte. The nucleic acid
that is introduced can comprise any nucleic acid that is or that encodes a
molecule that has an anti-rheumatic effect in the subject. For example,
the molecule may alter, counteract or diminish a process of the disease.
The molecule may ameliorate symptoms of the disease. Molecules that
provide anti-rheumatic effects in subjects with RA are known in the art
[see, e.g., Vervoordeldonk and Tak (2001) Best Prac. Res. Clin.
Rheumato% 15:771-788 and WO 00/521861. Such molecules include
anti-inflammatory or immunomodulatory molecules. For example,
interleukin-1 receptor antagonists, soluble interleukin-1 receptor, soluble
tumor necrosis factor receptor, interferon-fl, interleukin-4, interleukin-10,
interleukin-13, transforming growth factor,6, dominant negative IkappaB-
kinase, FasL, Fas-associated death domain protein or CTLA-4 are among
molecules that can have anti-rheumatic effects.
Also provided is a method of identifying, evaluating or testing a
nucleic acid as a potential therapeutic agent in the treatment of a
connective tissue or rheumatic disease by introducing into an animal
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model of a connective tissue or rheumatic disease a large nucleic acid
molecule. The nucleic acid molecule can be one that includes nucleic acid
that is or encodes a candidate therapeutic agent. The method may
include a step of determining if the nucleic acid molecule has any effects,
and in particular any anti-rheumatic effects, on the animal. For example,
in determining if the nucleic acid molecule has any effects on the animal,
it may be evaluated whether one or more conditions of the disease is
effected, such as, for example, amelioration of or reduction in an adverse
condition. In a particular embodiment, the disease is a rheumatic disease,
such as, for example rheumatoid arthritis. The animal is any animal in
which the disease may be modeled. For example, the animal may be a
mammal. In particular embodiments the animal is a monkey, rodent,
rabbit, dog, cat, horse, cow, pig or primate. The large nucleic acid may
be, for example, a chromosome, or portion thereof, or an artificial
chromosome, for example, an ACes. In particular embodiments, the
nucleic acid molecule is in a synoviocyte, such as, for example, a
fibroblast-like synoviocyte. In further embodiments, the nucleic acid is
introduced into a joint of the animal. The nucleic acid molecule may be
introduced into the animal using in vitro or in vivo formats. For example,
the nucleic acid can be introduced into a cell in vitro and then be
transferred into the animal. In another embodiment, the nucleic acid is
introduced into a cell in vivo.
Animal models include, for example, animal models of RA. Several
animal models of RA, and methods for generating such models, are
known in the art. Such models include adjuvant-induced arthritis (AA)
[see, e.g., Kong et al. (1999) Nature 4023:304-309] and collagen type II-
induced arthritis [see, e.g., Tak et al. (1999) Rheumatology 38:362-369;
Han et al. (1998) Autoimmunity 28:197-208; Gerlag et al. (2000) J.
Immunology 165:1652-1658]. For example, experimental induction of
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adjuvant-induced arthritis in Lewis rats leads to severe inflammation in the
bone marrow and soft tissues surrounding joints accompanied by
extensive local bone and cartilage destruction, loss of bone mineral
density and crippling [see, e.g., Bendele et a/. (1999) Arthritis Rheum.
42:498-506].
D. ASSESSING THE DELIVERY OF NUCLEIC ACID INTO CELLS
Microscopic and colony formation analysis methods that may be
used in evaluating stable nucleic acid molecule delivery rely on manual
visualization or measurement of nucleic acid molecules (e.g., a selectable
marker gene) expression, which is a distinct process from delivery. Such
methods are associated with time delays in obtaining an assessment of
the delivery method. Microscopic techniques for visualizing chromosome
or plasmid transfer using bromodeoxyuridine (BrdU) (see. e.g., Pittman et
al. J Immunol Methods 103:87-92 (1987)) are time consuming, restricted
by the large sample size required to detect low levels of transfer and
limited by the necessity of manual scoring. Colony-forming transfection
analysis may require four-to-six weeks to generate and evaluate marker-
expressing transfection colonies.
In contrast, methods provided herein are based on rapid, auto-
mated, sensitive and accurate analysis procedures, such as flow
cytometry, and thus do not involve any time-consuming, laborious and
error-prone steps, such as manual detection of individual transfected cells
by microscopic techniques. The methods make possible the analysis of
nucleic acid molecule delivery data within 48 hours after transfection.
Also, data collected by flow cytometry analysis is statistically superior
due to the ease at which large numbers of events, e.g., nucleic acid
molecule transfer, are collected. The positive values obtained in these
methods are instrument derived and therefore not as susceptible to
judgment errors. Thus, these methods provide for greater accuracy in
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assessing nucleic acid molecule delivery. In contrast, microscopic
analysis is limited by the time involved for scoring positive events and
sample size is restrictive.
Because the methods of monitoring nucleic acid molecule delivery
detect labeled nucleic acid mo(ecu(es, such as DNA, and not a reporter
gene expression product, it is possible to measure absolute values of
nucleic acid molecules transferred, within twenty-four hours, without
being hindered by cell autofluorescence and by the problems of
differentiating wild-type cells from cells expressing low levels of reporter
gene products (see, e.g., Ropp et a/. (1995) Cytometry 21:309-317).
1. Factors to consider in addressing delivery of nucleic acids
Delivery of nucleic acids, including DNA, into cells is a process in
which nucleic acids are transferred to the interior of a cell. Methods for
the delivery of nucleic acids may be assessed in a variety of ways,
including the following.
a. Transfer Efficiency
A delivery method may be assessed by determining the percentage
of recipient cells in which the nucleic acids, including DNA, are present
(i.e., the transfer efficiency). However, when evaluating a delivery
method for the ultimate goal of generating cells that express the
transferred nucleic acid, there are additional factors beyond mere
presence of the nucleic acid in recipient cells that should be considered.
Included among these additional factors is cell viability. When assessing
a proliferating cell population, clonogenicity is the method of choice to
measure viability. When the target cells population is non-dividing or
slow growing, metabolic integrity can be monitored.
b. Clonogenicity
Clonogenicity represents a measure of the survivability of cells with
respect to a delivery procedure, growth conditions and cell manipulations
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(e.g., plating). It is important to assess clonogenicity to determine
whether a delivery procedure results in a sufficient number of viable cells
to achieve a desired number of cells containing the transferred nucleic
acid.
Clonogenicity may be expressed as a clonal fraction. The clonal
fraction is an index that is calculated by multiplying two separate
fractions and normalizing to a control plating efficiency correction factor
(CPE). The two separate fractions that are multiplied in this calculation
are the fraction of cells that survive a delivery procedure (population cell
yield) and the fraction of cells that survive a plating procedure. The
calculation is thus as follows:
Colonal Fraction = # viable colonies after plating x # cells post-transfection
# cells plated # cells transfected x CPE
The values used in this calculation for the number of cells post-
transfection (i.e., post-delivery) and the number of colonies post-plating is
based on cell or colony numbers at certain times in the process. For
instance, the value for the number of cells post-transfection is
representative of the number of cells at a time after nucleic acid delivery
that is sufficient for the delivery process to be completed. This time may
be determined empirically. Typically this time ranges from 4-48 hours and
generally is about one day after transfection. Likewise, the value of the
number of viable colonies post-plating is representative of the number of
colonies at a time after nucleic acid delivery that is sufficient for the non-
viable cells to be eliminated and the viable cells to be established as
colonies. This time may be determined empirically. Typically this time
ranges from that in which the average colony is made up of
approximately 50 cells or generally is a time at which five cell cycles have
passed.
A correction factor is included to take into account the plating
efficiency of control wells, which is the ratio determined by the number of
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colonies counted divided by the number cells initially plated (typically
600-1000 cells). For LM(tk-) and V79-4 cells, the value of the correction
factor typically ranges from about 0.7 to about 1.2 and may be, for
example, 0.9.
The number of cells plated should remain constant at 1000
(simplified plating efficiency assay) done in duplicate, except in the case
where the CPE is below 0.3, then number of cells seeded should be
increas.ed to a range of 5,000-50,000. If the CPE is below 0.1-0.2, then
a viable fraction analysis should be considered.
c. Viable Fraction
If the target cells population is non-dividing or slowly dividing then
reproductive or clonogenicity assays are not relevant. Less direct
measurements of cell viability must be used to measure cell killing that
monitor metabolic death rather than loss of reproductive capacity. These
procedures include, for example: (1) membrane integrity as measured by
dye exclusion, (2) inhibition of nucleic acid synthesis as measured by
incorporation of nucleic acid precursors, (3) radioactive chromium release,
and (4) MTT ASSAY (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
bromide). These methods are different from measurements of loss of
proliferative capacity, as they reflect only immediate changes in
metabolism, which can be reversed or delayed and hence lead to errors in
estimation of cell viability. To minimize these errors, correlation of
duplicate procedures is suggested.
d. Potential Transfection Efficiency (PTE) and
determination of Chromos Index (CI)
In assessing a delivery method used to transfer nucleic acids to
cells with the goal of expression of the nucleic acids, including DNA,
therein, it is desirable to obtain an indication of the theoretical maximum
percentage of cells that are viable and contain the nucleic acid out of the
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total number of cells into which nucleic acids were delivered. This is
referred to as the potential transfection efficiency and may be calculated
from existing or historical experimental data sets and is determined as
follows:
Potential Transfection Efficiency (PTE) = Transfer Efficiency x (Clonal
Fraction or Viable
Fraction) x correction factor (CF)
The Chromos Index (C.I.) is an effective and rapid method to
determine the Potential Transfection Efficiency of a proliferating
population by using experimental values of % labeled nucleic acid, such
as ACes, delivery to measure transfer efficiency and clonal fraction
measured using a simplified clonogenicity assay.
Chromos Index (CI) = % labeled ACes delivery x estimated Clonal fraction x CF
The values of the transfer efficiency and of the clonal fraction and
viable fraction are calculated as described above. The correction factor
(CF) takes into account sample size, sample time and control plating
efficiency. If all these factors are constant for each variable i.e., sampling
time and size then the correction factor will approach the inverse of the
value for the C.P.E., i.e., such that the clonal fraction or transfer
efficiency can still approach 100% even with a low CF, or in other words,
if delivery and viability are 100%, then the maximum potential
transfection efficiency will equal the plating efficiency of the control
cells.
The calculation of C.I. allows for determination of each variable
optimization, with the goal being for parameters, such as transfer
efficiency, clonal fraction, and CF to approach one (or 100%). If sample
size or time varies for either clonal fraction or transfer efficiency, then CF
represents the extrapolated value based on slope or rate of change. An
application of this assessment is provided in the EXAMPLES.
A stable transfection efficiency of about 1% is in the range (1-
100%) that is considered useful for the introduction of large nucleic acid
molecules into target cells. It is possible, using methods provided herein,
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to predict which delivery methods have to be selected for achieving
desired transfection efficiencies without having to grow transfectants for
extended times under selective conditions and determine numbers of cells
surviving selection marker expression. This analysis involves calculation
of the Chromos Index (CI) which integrates a "biological" value (the clonal
fraction) with a measurement of chromosomal "uptake" or transfer
efficiency (percentage of cells containing delivered ACes).
2. Labeling of nucleic acid molecules for transfer
In the methods for monitoring nucleic acid molecule delivery
provided herein, the nucleic acid molecules, such as DNA, to be delivered
are labeled to allow for detection of the nucleic acid molecules in recipient
cells after transfer into the cells. The nucleic acid molecules may be
labeled by incorporation of nucleotide analogs. Any nucleic acid molecule
analog that may be detected in a cell may be used in these methods. The
analog is either directly detectable, such as by radioactivity, or may be
detected upon binding of a detectable molecule to the analog that
specifically recognizes the analog and distinguishes it from nucleotides
that make up the endogenous nucleic acid molecules, such as DNA,
within a recipient cell. Analogs that are directly detectable have intrinsic
properties that allow them to be detected using standard analytical
methods. Analogs may also be detectable upon binding to a detectable
molecule, such as a labeled antibody that binds specifically to the
analogs. The label on the antibody is one that may be detected using
standard analytical methods. For example, the antibody may be
fluorescent and be detectable by flow cytometry or microscopy.
In particular embodiments of these methods, the nucleic acid
molecules, such as DNA, to be transferred is labeled with thymidine
analogs, such as lododeoxyuridine (IdUrd) or Bromodeoxyuridine (BrdU).
In preferred embodiments, IdUrd is used to label the nucleic acid
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molecules, such as DNA, to be transferred. The transferred IdUrd-labeled
nucleic acid molecules, such as DNA, may be immunologically tagged
using an FITC-conjugated anti-BrdU/IdUrd antibody and quantified by flow
cytometry. Thus, the transfer of the labeled nucleic acid molecules, such
as DNA, into recipient cells can be detected within hours after
transfection.
E. STABILITY OF NUCLEIC ACID MOLECULES TO BE DELIVERED
It is also of interest to evaluate the stability of the nucleic acid
molecule, such as DNA, under the selected delivery conditions. Some
delivery conditions and agents may have adverse effects on nucleic acid
molecule structure. Furthermore, the labeling techniques used in certain
methods of monitoring nucleic acid molecules, such as DNA, delivery may
also impact nucleic acid molecules, such as DNA, structure and function.
The effects of delivery conditions on nucleic acid molecules may be
assessed in a variety of ways, including microscopic analysis. In a
particular exemplary analysis of the stability of artificial chromosomes,
e.g., ACes, the chromosomes are exposed to the conditions of interest,
e.g., IdU labeling, and analyzed under a fluorescent microscope for the
ability to remain intact and condensed after incorporation of nucleotide
analogs.
Methods of Monitoring nucleic acid molecule Delivery and
Expression
Methods of monitoring delivery of nucleic acid molecules delivery
provided herein may also be combined with an assessment of nucleic acid
molecule, such as DNA, expression in recipient cells to provide even
further information concerning the overall process of nucleic acid
molecule transfer for purposes of expression.
For example, to facilitate analysis of nucleic acid molecules, such
as DNA, expression, it is desirable to include in the transferred nucleic
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acid molecules, such as DNA, a reporter gene that encodes a readily
detected product. For direct detection, such reporter gene products
include, but are not limited to green fluorescent proteins (GFP), Red
Fluorescent protein (RFP), luciferases, and CAT. For indirect detection,
reporter gene products include, but are not limited, to 6-galactosidase and
cell surface markers.
By using, for example, artificial ch'romosomes such as ACes
containing a GFP reporter gene, such as, but are not limited to, GFP
coding sequences in combination with labeling of the ACes with DNA
analogs, such as IdU, delivery and expression can be rapidly and
accurately monitored. For example, following the delivery of IdU-labeled
GFP gene-containing ACes to target cells by any of the described
methods, the cells containing the ACes are split into two populations.
One population is fixed and stained for ldU and analyzed by flow
cytometry to determine percentage delivery. The other population is
allowed to go through 4-5 cell divisions (approximately 72 hours), and the
GFP fluorescence is measured as an indication of expression.
Such studies have revealed that incorporation of the analog label
does not affect GFP protein expression, which indicates that the methods
may be combined to monitor delivery and early expression of the ACes,
thus providing more information to rapidly evaluate the efficiency of
delivery methods. The combined methods can also be used to map the
biological events between the initial stages of delivery and early gene
expression.
The following examples are included for illustrative purposes only
and are not intended to limit the scope of the invention.
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EXAMPLE 1
Preparation of Artificial Chromosomes
A. GFP Chromosome contained in A9 cell line
Plasmids
Plasmid pIRES-EGFP (see SEQ ID No. 13, plasmid obtained from
Clontech, CA, and is well known, see, e.g., U.S. Patent Nos. 6,034,228,
6,037,133, 5,985,577, 5,976,849, 5,965,396, 5,976,796, 5,843,884,
5,962,265, 5,965,396; see, also, U.S. Patent No. 4,937,190). This
plasmid contains the internal ribosome entry site (IRES; Jackson (1990)
Trends Biochem. 15:477-483; Jang et al. (1988) J. Viro% 62:2636-2643)
of the encephalomyocarditis virus (ECMV) between the MCS and the
enhanced green fluorescent protein (EGFP) coding region. This permits
the gene of interest (cloned into the MCS) and the EGFP gene to be
translated from a single bicistronic mRNA transcript. Plasmid pIRES2-
EGFP is designed for selection, by flow cytometry and other methods, of
transiently transfected mammalian cells that express EGFP and the protein
of interest. This vector can also be used to express EGFP alone or to
obtain stably transfected cell lines without drug and clonal selection.
Enhanced GFP (EGFP) is a mutant of GFP with a 35-fold increase in
fluorescence. This variant has mutations of Ser to Thr at amino acid 65
and Phe to Leu at position 64 and is encoded by a gene with optimized
human codons (see, e.g., U.S. Patent No. 6,054,312). EGFP is a red-
shifted variant of wild-type GFP (Yang et al. (1996) Nuc% Acids Res.
24:4592-4593; Haas et al. (1996) Curr. Bio% 6:315-324; Jackson et al.
(1990) Trends Biochem. 15:477-483) that has been optimized for brighter
fluorescence and higher expression in mammalian cells (excitation
maximum = 488 nm; emission maximum = 507 nm). EGFP encodes the
GFPmutl variant (Jackson (1990) Trends Biochem. 15:477-483) which
contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65
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to Thr. The coding sequence of the EGFP gene contains more than 190
silent base changes which correspond to human codon-usage
preferences (Jang et al. (1988) J. Viro% 62:2636-2643). Sequences
flanking EGFP have been converted to a Kozak consensus translation
initiation site (Huang et al. (1990) Nucleic Acids Res. 78: 937-947) to
further increase the translation efficiency in eukaryotic cells.
Plasmid piRES-EGFP was derived from PIRESneo (originally called
pCIN4) by replacing the neo gene downstream of the IRES sequence with
the EGFP coding region. The IRES sequence permits translation of two
open reading frames from one mRNA transcript. The expression cassette
of pIRES-EGFP contains the human cytomegalovirus (CMV) major
immediate early promoter/enhancer followed by a multiple cloning site
(MCS), a synthetic intron (IVS; Huang et al. (1990) Nucleic Acids Res.
18: 937-947), the EMCV IRES followed by the EGFP coding region and
the polyadenylation signal of bovine growth hormone.
Location of Features (with reference to SEQ ID No. 13):
Human cytomegalovirus (CMV) immediate early promoter: 232-
820;
MCS 909-974;
IVS 974-1269;
IRES of ECMV 1299-1884;
Enhanced green fluorescent protein (EGFP) gene 1905-2621;
fragment containing the bovine polyA signai =2636-2913;
Col El origin of replication 3343-4016; and
Ampicillin resistance gene 5026-4168
Propagation in E. coli
Suitable host strains: DH5a, HB101, and other general
purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 or XL1-Blue .*
Selectable marker: plasmid confers resistance to
kanamycin (30 /tg/mI) to E. cofi hosts.
E. coli replication origin: pUC
Copy number: -500
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Plasmid incompatibility group: pMB1 /ColE1
pCHEGFP2
Plasmid pCHEGFP2 was constructed by deletion of the Nsil/Smal
fragment from pIRES-EGFP. Plasmid piRES-EGFP contains the coding
sequence for a 2.1 kB Nru 1/Xho fragment of pCHEGFP2 containing the
CMV promoter, synthetic intron, EGFP coding sequence and bovine
growth hormone polyadenylation signal.. Digestion of pIRES-EGFP with
Nru 1 and Sma 1, yielded a 2.1 kb fragment. Digested DNA was
fractionated by agarose gel electrophoresis, the separated band was
excised and then eluted from the gel using the Qiaex 11 gel purification
system (Qiagen, Mississauga, Ontario).
pFK161
Cosmid pFK1 61 was obtained from Dr. Gyula Hadlaczky and
contains a 9 kb Notl insert derived from a murine rDNA repeat (see clone
161 described in PCT Application Publication No-. W097/40183 by
Hadlaczky et al. for a description of this cosmid). This cosmid, referred to
as clone 161 contains sequence corresponding to nucleotides 10,232-
15,000 in SEQ ID NO. 16. It was produced by inserting fragments of
the megachromosome (see, U.S. Patent No. 6,077,697 and International
PCT application No. (WO 97/40183); for example, H1D3, which was
deposited at the European Collection of Animal Cell Culture (ECACC)
under Accession No. 96040929, is a mouse-hanister hybrid cell line
*
carrying this megachromosome) into plasmid pWE15 (Stratagene, La
Jolla, California) as follows. Half of a 100 NI low melting point agarose
block (mega-plug) containing isolated SATACs was digested with Notl
overnight at 37 C. Plasmid pWE15 was similarly digested with Notl
overnight. The mega-plug was then melted and mixed with the digested
plasmid, ligation buffer and T4 ligase. Ligation was conducted at 16 C
overnight. Bacterial DH5a cells were transformed with the ligation
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product and transformed cells were plated onto LB/Amp plates. Fifteen to
twenty colonies were grown on each plate for a total of 189 colonies.
Plasmid DNA was isolated from colonies that survived growth on LB/Amp
medium and was analyzed by Southern blot hybridization for the presence
of DNA that hybridized to a pUC19 probe. This screening methodology
assured that all clones, even clones lacking an insert but yet containing
the pWE15 plasmid, would be detected.
Liquid cultures of all 189 transformants were used to generate
cosmid minipreps for analysis of restriction sites within the insert DNA.
Six of the original 189 cosmid clones contained an insert. These clones
were designated as follows: 28 (-9-kb insert), 30 (- 9-kb insert), 60
(-- 4-kb insert), 113 (- 9-kb insert), 157 (-- 9-kb insert) and 161 (-- 9-kb
insert). Restriction enzyme analysis indicated that three of the clones
(113, 157 and 161) contained the same insert.
For sequence analysis the insert of cosmid clone no. 161 was
subcloned as follows. To obtain the end fragments of the insert of clone
no. 161, the clone was digested with Notl and BamHl and ligated with
~.
Notl/BamHl-digested pBluescript KS (Stratagene, La Jolla, California).
Two fragments of. the insert of clone no. 161 were obtained: a 0.2-kb and
a 0.7-kb insert fragment. To subclone the internal fragment of the insert
of clone no. 161, the same digest was ligated with BamHl-digested
pUC19. Three fragments of the insert of clone no. 161 were obtained: a
0.6-kb, a 1.8-kb and a 4.8-kb insert fragment.
The insert corresponds to an internal section of the mouse
ribosomal RNA gene (rDNA) repeat unit between positions 7551-15670
as set forth in GENBANK accession no. X82564, which is provided as
SEQ ID NO. 5. The sequence data obtained for the insert of clone no.
161 is set forth in SEQ ID NOS. 6-12. Specifically, the individual
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subclones corresponded to the following positions in GENBANK accession
no. X82564 (i.e., SEQ ID NO. 5) and in SEQ ID NOs. 6-12:
Subclone Start End Site SEQ ID No.
in X82564
161 k1 7579 7755 Nofl, BamHl 6
161 m5 7756 8494 BamHl 7
161 m7 8495 10231 BamHf 8(shows only sequence corresponding to
nt. 8495-8950),
9 (shows only sequence corresponding to
nt. 9851- 10231)
161 m12 10232 15000 BamHl 10 (shows only sequence corresponding
to nt. 10232-10600),
11 (shows only sequence corresponding
to nt. 14267-15000)
161k2 15001 15676 Notl, BamHl 12
The sequence set forth in SEQ ID NOs. 6-12 diverges in some
positions from the sequence presented in positions 7551-15670 of
GENBANK accession no. X82564. Such divergence may be attributable
to random mutations between repeat units of rDNA.
For use herein, the rDNA insert from the clone was prepared by
digesting the cosmid with Notl and Bg/Il and was purified as described
above. Growth and maintenance of bacterial stocks and purification of
plasmids were performed using standard well known methods (see, e.g.,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory Press), and plasmids were purified
* *
from bacterial cultures using Midi -'and Maxi-preps Kits (Qiagen,
Mississauga, Ontario).
B. PREPARATION OF THE GFP, MURINE A9 CELL LINE
Cell culture and transfection
The murine A9 cell line was obtained from ATCC and cells were
thawed and maintained as described below. Briefly, cells were plated at a
density of 2X106 cells per 15 cm tissue culture dish (Falcon, Becton
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Dickinson Labware, Franklin Lakes, NJ) in growth medium containing of
90% DMEM (Canadian Life Technologies Burlington, ON) and 10% FBS
(Can Sera, Rexdale ON), and were maintained at 37 C, 5% CO2.
Cultures were routinely passaged when cells reached 70%-80%
confluence. Sub culturing was carried out as follows: medium was
removed by aspiration, 10 ml of 1X trypsin-EDTA (Canadian Life
Technologies Burlington, ON) was dispensed onto the cell monolayer and
the dish gently swirled to distribute the trypsin-EDTA. Finally, the bulk of
the trypsin-EDTA was removed by aspiration, and the dish placed at
37 C for 5 minutes. To quench the trypsin-EDTA, 10 ml of growth
medium was added to the dish, and the single cell suspension was
transferred to a 50 ml conical tube. Cell counts were performed using a
cell counting apparatus (Beckman-Coulter, Hialeah FL). The cells were
diluted and re-plated as described above. For cryo-storage, cultures were
harvested by treatment with trypsin-EDTA, counted and the cell
suspension then centrifuged at 500Xg for 5 minutes in a swinging bucket
centrifuge. The cell pellet was resuspended in freezing medium containing
90% DMEM, 20% FBS and 10% DMSO (Sigma-Aldrich, Oakville, ON) at
a density of 1 X10' cells/mi. One ml aliquots of the cell suspension were
then dispensed into cryo-vials (Nunc, Rochester NY), frozen over night in
an isopropanol filled container (NUNC, Rochester NY) and placed at -70 C
and then transferred to the gas phase of a liquid nitrogen freezer for long-
term storage.
A9 cells were transfected using the Ca2PO4 co-precipitation method
(see, e.g., Graham et al. (1978) Virology 52:456-457; Wigier et al.
(1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376; and (1990) Current
Protocols in Molecular Biology, Vol. 1, Wiley Inter-Science, Supplement
14, Unit 9.1.1-9.1.9). One day prior to transfection, A9 cells were plated
at a density of 2 x 106 cells per 10 cm dish and 3 hours before
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transfection the medium was replaced with fresh growth medium.
140 ,ug of the 9 kb rDNA, Notl and 5,ug of the 2.1 kB CMV-EGFP
Xhol/Nrul fragments were mixed, co-precipitated and used to prepare the
Ca2PO4 co-precipitate (Calcium Phosphate Transfection System,
(Canadian Life Technologies Burlington, ON) which was distributed onto 2
10=crrm dishes of subcorifluent A9 cells. The DNA-Ca2PO4 complexes
were left on the celis for 18 hours, after which the precipitate was
removed by aspiration and cells were subjected to glycerol shock for 1.5
minutes. After glycerol shock, the cell monolayers were gently washed
with 2 X 10 mi of dPBS (Canadian Life Technologies Burlington, ON),
followed by addition of 10 ml pre-warmed growth medium. Finally dishes
were returned to the incubator and were maintained at 37 C, 5% C02.
After 3 hours recovery, each dish was passaged onto 3X 15 cm tissue
dishes
GFP fluorescence of cultures was monitored visually during culture
using an inverted microscope equipped with epifluorescence illumination
(Axiovert 25, Zeiss, (North York ON) and #41017 Endow GFP filter set
(Chroma Technologies, Brattleboro, VT). Enrichment of GFP expressing
populations was carried out as described below.
Enrichment of GFP expressing cell populations by
Fluorescence Activated Cell Sorting
*
Cell sorting was carried out using a FACS Vantage flow cytometer
(Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped
~ =
with turbo-sort option and 2 Innova 306 lasers (Coherent, Palo Alto CA).
For cell sorting a 70,vm nozzle was used. The sheath buffer was
-changed to PBS (maintained at 20 p.s.i.). GFP was excited with a 488 nm
Laser beam and excitation detected in FL1 using a 500 EFLP filter.
Forward and side scattering was adjusted to select for viable cells. Only
viable cells were then analyzed for GFP fluorescence. Gating parameters
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were adjusted using wild type A9 cells as negative control and GFP CHO
cells as positive control.
For the first round of sorting, A9 cells were harvested 4 days post-
transfection, resuspended in 10 ml of growth medium and sorted for GFP
expressing populations using parameters described above. GFP positive
cells were dispensed into a volume of 5-10 ml of growth medium
supplemented with 1X penicillin/streptomycin (Canadian Life Technologies
Burlington, ON) while non-expressing cells were directed to waste. The
expressing cells were further diluted to 50 ml using the same medium,
plated onto 2X15 cm dishes and cultured as described in the previous
section. When the sorted populations reached confluence they were re-
sorted to enrich for GFP expressing cells. A total of 4 sequential sorts
were carried out, achieving enrichments of as high as 89% GFP
expressing cells after the final sort. The final GFP expressing populations
were expanded for cryo-preservation and for fluorescence in-situ
hybridization screening (see below). Single cell clones were established
from populations of interest by using the flow cytometer to direct GFP
expressing single cells to individual wells of 96 well plates. These were
cultured as described above.
Fluorescence In-Situ Hybridization
Fluorescence In-Situ Hybridization (FISH) screening was carried out
on GFP enriched populations and single cell clones to detect amplification
and/or artificial chromosome formation. Preparation of metaphase
spreads and hybridizations were performed (see, Telenius et al. (1999)
Chromosome Res 7:3-7). Probes used include pSAT 1, which recognizes
the mouse major repeat (see, e.g., Wong et a/. (1988) Nuc% Acids Res.
16:1 1 645-1 1 661), pFK1 61, which hybridizes to the mouse rDNA-
containing regions and a PCR generated probe against the mouse minor
repeat.
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Thus, in one method provided herein for generating an artificial
chromosome, such as an ACes, heterologous nucleic acid that includes a
selectable marker, e.g., nucleic acid encoding a fluorescent protein or
other protein that may be readily detected using flow cytometry-based
methods or other methods, including, for example, fluorimetry, cell
imaging or fluorescence spectroscopy, is introduced into a cell. For
example, rDNA and DNA encoding enhanced green fluorescent protein
(EGFP) may be introduced into cells, e.g., A9 cells. The transfected cells
may be selected on the basis of properties detectable by flow cytometry-
based methods, or other methods, including, for example, fluorimetry, cell
imaging or fluorescence spectroscopy, e.g., fluorescent properties. For
example, cells containing a fluorescent protein may be isolated from
nontransfected cells using a fluorescence-activated cell sorter (FACS). If
the sorting is conducted prior to chromosomal analysis of the cells for the
presence of artificial chromosomes, it provides a population of transfected
cells that may be enriched for artificial chromosomes and thus facilitates
any subsequent chromosomal analysis of the cells and identification and
selection of cells containing an artificial chromosome, e.g., ACes. For
example, the cells may be analyzed for indications of amplification of
chromosomal segments, the presence of structures that may arise in
connection with amplification and de novo artificial chromosome
formation and/or the presence of artificial chromosomes, such as ACes.
Analysis of the cells typically involves methods of visualizing chromosome
structure, including, but not limited to, G- and C-banding and FISH
analyses using techniques described herein and/or known to those of skill
in the art. Such analyses can employ specific labelling of particular
nucleic acids, such as satellite DNA sequences, heterochromatin, rDNA
sequences and heterologous nucleic acid sequences, that may be subject
to amplification. During analysis of transfected cells, a change in
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chromosome number and/or the appearance of distinctive, for example;
by increased segmentation arising from amplification of repeat units,
chromosomal structures will also assist in identification of cells containing
artificial chromosomes.
C. Purification of artificial chromosomes by Flow Cytometry and
preparation of DNA from flow sorted chromosomes
Artificial chromosomes were purified from the host cell by flow
cytometry (see de Jong (1999).Cytometry 35:129-133). Briefly,
purification was performed on FACS Vantage flow cytometer (Becton
Dickinson lmmunocytometry Systems, San Jose, CA) equipped with a
Turbo Sort Option and two Innova 306 lasers (Coherent, Palo Alto; CA).
The Turbo Sort Option modification include increasing the maximum
system pressure from 20 lb/in2 to 60 lb/in2, increasing the drop drive
frequency from 50,000 drops/s to a maximum of 99,000 drops/s and
increasing the deflection plate voltages from a maximum 6,000 V to
8,000 V. Other modifications are made to the instrument to
accommodate the higher pressures. Hoechst 35258 was excited with the
primary UV laser beam, and excitation detected in FLI by using 420 nm
*
hand-pass filter. Chromomycin A3 was excited by the second laser set at
458 nm and fluorescence detected in FL 4 by using a 475 nm long-pass
filter. Both lasers had an output of 200 mW. Bivariate distributions
(1,024 x 1024 channels) were accumulated during each sort. For all
chromosome sorts, the sheath pressure was set at 30 lb/in2 and a 50 ,um
diameter nozzle was installed. A drop delay profile was performed every
morning and repeated after any major plug. Alignment of the instrument
*
was performed daily by using 3.0,um diameter Sphero rainbow beads
(Spherotech, Libertyville, IL). Alignment was.considered optimized when
a CV of 2.0% or less was achieved for FL1 and FL4.
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Condensing agents (hexylene glycol, spermine and spermidine)
were added to the sheath buffer to maintain condensed chromosomes
after sorting. The sheath buffer contains 15 nM Tris HCI, 0.1 mM EDTA,
20 mM NaCI, 1 % hexylene glycol, 100 mM glycine, 20 1.iM spermine and
50 NM spermidine. The sorted chromosomes were collected in 1.5 rnt
screw-capped Eppendorf tubes at 4 C at a concentration of
approximately 1 x 106 chromosomes/mi, which were then stored at 4 C.
For preparation of purified genomic DNA, sorted chromosome
samples were brought to 0.5% SDS, 50 mM EDTA and 100,ug/riml
Proteinase K, then incubated for 18 hours at 50 C. 1 NI of a 20 mg/mI
glycogen solution (Boehringer Mannheim) was added to each sample,
followed by extraction with an equal volume of Phenol: Chloroform:
Isoamyl Alcohol (25:24:1). After centrifugation at 21,000Xg for 10 min,
the aqueous phases were transferred to fresh microfuge tubes and were
re-extracted as above. 0.2 volumes of .10 M NH40AC, 1/11 of 20 mg/ml
glycogen and 1 volume of iso-propanol were added to the twice extracted
aqueous phases which were then vortexed and centrifuged for 15
minutes at 30,000Xg (at room temperature). Pellets were washed with
200 NI of 70% ethanol and re-centrifuged as above. The washed pellets
were air-dried then resuspended in 5mM Tris-CI, pH 8.0 at 0.5-2X106
chromosome equivalents/NI.
PCR was carried out on DNA prepared from sorted chromosome
samples essentially as described (see, Co et al. (2000) Chromosome
Research 8:183-191) using primers sets specific for EGFP and RAPSYN.
Briefly, 50 /a1 PCR reactions were carried out on genomic DNA equivalent
to 10,000 or 1000 chromosomes in a solution containing 10 mM Tris-Cl,
pH 8.3, 50mM KCI, 200,uM dNTPs, 500 nM of forward and reverse
primers, 1.5 mM MgC12, 1.25 units Taq polymerase (Ampli-Taq, Perkin-
Elmer Cetus, CA). Separate reactions. were carried out for each primer
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set. The reaction conditions were as follows: one cycle of 10 min. at
95 C, then 35 cycles of 1 min. at 94 C, 1 min. at 55 C, 1 min at 72 C,
and finally one cycle of 10 min at 72 C. After completion the samples
were held at 4 C until analyzed by agarose gel electrophoresis using the
following primers (SEQ ID Nos. 1-4, respectively):
EGFP forward primer 5'-cgtccaggagcgcaccatcttctt-3';
EGFP reverse primer 3'-atcgcgcttctcgttggggtcttt-3';
RAPSYN forward primer 5'-aggactgggtggcttccaactcccagacac-3'; and
RAPSYN reverse primer 5'-agcttctcattgctgcgcgccaggttcagg-3'.
All primers were obtained from Canadian Life Technologies,
Burlington, ON.
EXAMPLE 2
Preparation of Cationic vesicles
Vesicles were prepared at a lipid concentration of 700 nmoles/ml
lipid (cationic lipid/DOPE 1:1) as follows. In a glass tube (10mI) 350 nmol
cationic lipid (SAINT-2) was mixed with 350 nmol dioleoyl-
phosphatidylethanolamine (DOPE), both solubilized in an organic solvent
(Chloroform, Methanol or Chloroform/Methanol 1:1, v/v).
Dioleoylphosphatidylethanolamine (DOPE; Avanti Polar Lipids, Alabaster,
AL) forms inverse hexagonal phases in a membrane and weakens the
membrane. Other effectors that may be used are cis-unsaturated
phosphatidylethanolamines, cis-unsaturated fatty acids and cholesterol.
Cis-unsaturated phosphatidylcholines are less effective.
The solvent was evaporated under a stream of nitrogen (15 min/
250,uI solvent at room temperature). The remaining solvent was removed
totally by drying the lipid for 15 min in an desiccator under high vacuum
from a vacuum pump. To the dried mixture was added I ml ultrapure
water. This was vortexed vigorously for about 5 min. The resulting
solution was sonicated in an ultrasonication bath (Laboratory Supplies Inc.
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NY) until a clear solution was obtained. The resulting suspension
contained a population of unilamellar vesicles with a size distribution
between 50 to 100 nm.
EXAMPLE 3
Preparation of Cationic vesicles via alcoholic injection
In a glass tube (10mI) 350 nmol cationic lipid (Saint-2) was mixed
with 350 nmol DOPE, both solubi(ized in an organic solvent (chloroform,
methanol or chloroform/methanol 1/1). The solvent was evaporated under
a stream of nitrogen (15 min/ 250,u1 solvent at room temperature). The
remaining solvent was removed totally by drying the lipid for 15 min
under high vacuum. This was then reconstituted in 100 /aI pure ethanol.
EXAMPLE 4
Transfection of beta ACes into V79-4 cell line
Transfection procedure for various transfection agents
All compounds were tested in a Chinese Hamster lung fibroblast
line (V79-4, ATCC number CCL-39). Approximately 17 hours (2 cell
doublings) prior to transfection, exponentially growing cells were
trypsinized and plated at 250,000 cells per well into a 6 well petri dish
with Dulbecco's Modified Eagle Medium (Life Technologies, Burlington,
ON) and supplemented with 10% FBS (Can Sera, Rexdale ON)). At the
time of transfection, the number of cells per well was estimated to be
approximately 1 million. For transfection, each individual manufacturer's
protocol for complexing to naked DNA was followed, with the exception
that the amount of transfection agent used was varied, to reflect the
different amount and type of DNA present, as well as the different ionic
strength of the complexing. One million ACes (in a volume of 800,u1)
were typically combined with the transfection agent in a wide range of
concentrations (between 5 times and 100 times the lowest manufacturers
suggested concentration). The ACes/transfection mixture was allowed to
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complex for the time recommended by the manufacturer, in volumes
ranging from 0.8 ml to 1:9 ml; some manufacturers recommend adding
media to the complexing reaction. The complexed mixture was then
applied to the recipient cells and transfection allowed to proceed
according to the manufacturer's protocol. Details on the various
conditions used with different agents are presented in Table 1.
Transfection procedure for Superfect agent
*
Superfect was tested in a Chinese Hamster lung fibroblast line (V79-4,
ATCC number CCL-39). Approximately 17 hours (2 cell -doublings) prior to
transfection, exponentially growing cells were trypsinized and plated at
250,000 cells per well into a 6 well petri dish with Dulbecco's Modified
Eagle Medium (Life Technologies, Burlington, ON) and supplemented with
10% FBS (Can Sera, Rexdale ON). One million ACes in 800,u1 of sort
buffer was complexed to 10pl of Superfect reagent. Complex was
incubated at room temperature for 10 minutes. At the time of
transfection, the number of cells per well was estimated to be
approximately 1 million. Media was removed from wells and 6001u1 of
DMEM and 10% FBS was added. Superfect:ACes complex was added to
the wells drop-wise and allowed to incubate for 3 hours at 37 C. After
incubation, transfected cells were trypsinized and transferred to 15 cm
dishes with 25 mI DMEM and 10% FBS and allowed to attach for 24
hours. After 24 hours, selection medium containing of 0.7 mg/ml
hygromycin B was added to each well. The selection medium was
changed every 2-3 days. After 10-12 days colonies were screened for
Beta-galactosidase expression and/or FISHed for detection of intact
chromosome.
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Example of application of the determination of the Chromos Index
Approximately 1 x 106 V79-4 cells were transfected with 1 x 106
IdUrd-labeled ACes complexed with a delivery agent (i.e., Lipofectamine
PLUS and Lipofectamine or Superfect). The transfected cells were then
fixed in ethanol. Fixed cells were denatured and exposed to FITC-
conjugated antibody that specifically binds to BrdU/1dUrd-labeled nucleic
acids.
The percentage of transfected cells containing IdUrd-labeled ACes
was determined using flow cytometry and collecting FITC fluorescence.
.10 Data were accumulated to form bivariate channel distribution showing
forward scatter versus green fluorescence (IdUrd-FITC). The fluorescence
level at which cells were determined to be positive was established by
visual inspection of the histogram of negative control cells such that the
gate for the negative cells was set such that 1 % appeared in the positive
region.
The number of cells recovered at 24 hours post-transfection was
~.
determined by counting an aliquot using a Coulter Counter. To determine
the control plating efficiency of a recipient cell line, the untreated cells
were plated at 600-1000 cells per 10 cm petri dish in growth medium and
left stationary in a 5% CO2 incubator at 37 C for approximately five cell
cycles or until average colony was made up of 50 cells. At this point the
number of viable colonies was determined. The treated cells were seeded
at 1000 cells if the CPE is above 0.1-0.2. If the CPE is low then the
seeding density is increased to 5,000-50,000 cells per dish.
EXAMPLE 5
Ultrasound mediated transfection of LMTK(-) cells with Lipofectamine
LM(tk-) cells were grown at 37 C, 5% C02, in DMEM with
4500 mg/L D-glucose, L-glutamine, pyridoxine hydrochloride and 10%
Fetal Bovine Serum. The corner wells of a 12-well dish were seeded with
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200,000 cells per well (this is to ensure no interference from the
ultrasound waves from other wells) 24 hours before use.
The GFP chromosomes were counted to verify approximately
1 X106 ACes per ml. The chromosomes were resuspended in the tube by
flicking. Ten rul of chromosome suspension was removed and mixed with
an equal volume of 30 mg/ml PI (propidium iodide) stain. Eight NI of the
stained chromosomes was loaded onto a Petroff Hausser counting
chamber and the chromosomes were counted.
The medium was removed from the cells, and the cells were
washed twice with HBSS (without phenol red, Gibco BRL) warmed to
37 C. 500 ,ul of the warmed HBSS was added to each well of cells
(1 ,ul) LipofectAMINE (Gibco BRL) was added to each-well. The plates
were then sealed with parafilm tape and shaken gently at 20 rpm at room
temperature for 30 minutes (Stagger plates -' 10 minutes for ease of
handling).
After incubation, Ultrasound gel (Other-Sonic Generic Ultra sound
transmission gel, Pharmaceutical Innovations, Inc., Newark, NJ) was
applied to the 2.5 cm sonoporator head. Ultrasound was applied with an
ImaRX Sonoporator 100*at an output energy of 2.0 Watt/cm2, for 60
seconds, through the bottom of the plate of cells. After ultrasound of the
well one chromosome per seeded cell (2X105) or 200 jr1 GFP ACes in
sheath buffer (15 nM Tris HCI, 0.1 mM EDTA, 20 mM NaCi, 1%o hexylene
glycol, 100 mM glycine, 20 NM spermine and 50,uM spermidine) are
added immediately to the well. (Repeat until all samples on the plate
requiring ultrasound have been treated). The plate was then sealed once
more with parafilm tape and shaken gently (20 rpm) for 1 hour at room
temperature.
After the incubation, 1 ml (DMEM with 4500 mg/L D-glucose, L-
glutamine and pyridoxine hydrochloride, 10% Fetal Bovine Serum, and a
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lx solution of penicillin and streptomycin from a 10000 units/mI penicillin
and 10000 mg/mi Streptomycin, 100x stock solution) was added to each
well and the cells were incubated 18-24 hours at 37 C.
The cells in the plates were then washed with antibiotic containing
medium and 2 ml of medium was placed in each well. The cells continued
to be incubated at 37 C with 5% CO2 until 48 hours after
transfection/sonoporation. The cells were then trypsinized and
resuspended at a concentration of lx 106 in DMEM to be analyzed by
flow cytometry. ,
Results: Flow analysis was performed on a FACS Vantage (BDIS,
San Jose, CA) equipped with a turbo-sort option and two Inova 305
lasers (Coherent, Palo Alto, CA). The GFP signal excitation is at 488 nm
and the emission detected in FL1 using a 500nm long pass filter. Analysis
of the transfected cells generated populations of GFP positive cells
ranging from 13-27%. Non-sonoporated control value was 5%.
EXAMPLE 6
Ultrasound mediated transfection with Saint-2
A. Ultrasound mediated transfection of CHO-KI cells with
Saint-2
CHO-KI cells were grown at 37 , 5% COZ, in CHO-S-SFM 2
Medium, (Gibco BRL, Paisley, UK). Between 2 x 105 and 5 x 105 cells
were plated onto sterile glass slides in a 12 well plate 24 h before usage.
Transfection of the cells was performed as follows. The medium
was removed from the cells, and the cells were washed twice with HBSS
(Hanks balanced salt solution without Phenol Red (Gibco BRL, UK)) at
370C. Then 500,ul HBSS at 370C was added per well, followed by 10 ,ul
of the freshly prepared vesicle solution (prepared in Example 2) to yield a
final concentration of 23.3 nmol/ml.
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Alternatively, the medium was removed from the cells, and the
cells were washed twice with HBSS. 500,u1 HBSS/lipid solution at 37 C
was added to each well. The HBSS/lipid solution was prepared by adding
1,uI ethanolic lipid solution (prepared as described above) to 500111 HBSS
under vigorous vortexing. The plates were then sealed with parafilm tape
and shaken gently at room temperature for 30 min. After incubation,
ultrasound was applied at an output energy of 0.5 Watt/cm2 for 60 sec
through the bottom of the plate to the cells. The ultrasound was mediated
by an ultrasound gel (Aquasonic 100, Parker, NJ) between transducer and
plate. The ultrasound was applied with an ImaRx Sonoporator 100.
Immediately after applying ultrasound one GFP chromosome per seeded
cell (2 x 105 - 5 x 105) (prepared in Example 1) was added. The plate was
then sealed again and shaken gently for 1 h at room temperature. After
the incubation 1 ml medium (CHO-S-SFM 2 with 10% Fetal Calf Serum,
10000 ,ug/mI Penicillin and 10000 ,ug/mI Streptomycin Gibco BRL, Paisley,
UK) was added to each well and the cells were incubated for 24 h at
37 C. The cells were then washed with medium, 1 ml medium was
added, and the cells were incubated at 370 for another 24 h. Detection
of expressed genes was then assayed by microscopy or detection of the
transferred chromosome by FISH analysis.
The negative control was performed in the same way, but with no
chromosomes added to the cells.
Results
After transfection, using visual inspection, 30% of the cells
remained on the glass slide of which 10% were positive for green
fluorescent protein expression after 48 hours (3% of original population).
After culturing for two weeks, FISH was performed on the cells and 1.4%
of the cells contained an intact artificial chromosome.
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B. Ultrasound mediated transfection of Hep-G2 cells with
Saint-2
Hep-G2 cells were grown at 37 C, 5% C02, in DMEM with 4500
mg/I Glucose, with Pyridoxine/HCI, 10% Fetal Calf Serum, 10000 /ig/ml
Streptomycin and 1000,ug/ml Penicillin. Between 2 x 105 and 5 x 105
cells were plated onto sterile glass slides in a 12 wells plate 24 hours
before usage.
Cells were transfected with GFP chromosomes using the procedure
of Example 6A except that the CHO-KI medium was replaced with Hep-
G2 medium.
Results
After transfection, 30% of the cells remained on the glass slide.
80% of these cells were positive for green fluorescent protein expression.
C. , Ultrasound mediated transfection of A9 cells with Saint-2
A9 cells were grown at 37 C, 5% CO2, in DMEM with 4500 mg/I
Glucose, with Pyridoxine/HCI, 10% Fetal Calf Serum, 10000,ug/mI
Streptomycin and 10000,ug/mI Penicillin (GIBCO BRL, Paisley, UK).
Between 2 x 105 and 5 x 105 cells were plated onto sterile glass slides in
a 12 well plate 24 h before usage.
Cells were transfected with GFP chromosomes using the procedure
of Example 6A except that CHO-KI medium was replaced with A9
medium.
Results
After transfection, 30% of the cells remained on the glass of which
50% were positive for green fluorescent protein expression.
EXAMPLE 7
Delivery of ACes into synoviocytes, skeletal muscle fibroblasts and skin
fibroblasts
A mammalian (murine) ACes artificial chromosome (-60 Mb)
containing primarily murine pericentric heterochromatin, and including a
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reporter gene (/acZ) and a hygromycin B selectable marker gene, prepared
as described in U.S. Patent Nos. 6,025,155 and 6,077,697 and PCT
Application Publication No. WO 97/40183 was delivered into primary rat
fibroblast-like synoviocytes, rat skin fibroblasts and a rat skeletal muscle
fibroblast cell line (L8 cells; ATCC Accession No. CRL-1769). Prior to
delivery, the ACes were labeled with iododeoxyuridine (IdUrd) as
described herein.
Preparation of cells
Primary fibroblast-like synoviocytes and rat skin fibroblasts were
obtained from rats using standard methods [see, e.g., Aupperle et al.
(1999) J. lmmuno% 163:427-433 and Alvaro-Garcia et al. (1990) J. Clin.
Invest. 86: 1790]. Such methods include isolation of synoviocytes from
rodent knees generally by removal of skin and muscle, followed by
mincing of knee joint tissue. The minced tissue is then incubated with
collagenase, filtered through nylon mesh and washed extensively. Cells
can be cultured overnight, after which time non-adherent cells are
removed. Adherent cells can be cultured and passaged by replating at a
dilution when the cultures reach confluence. The cells were plated at
50,000-75,000 cells per 6-well dish in media containing low glucose
DMEM, 1-glutamine, penicillin/streptomycin and 20% FBS. The cells
were grown in a 5% CO2 incubator at 37 C for 3-5 days until
approximately 80% confluence or 500,000 cells per well.
Transfection of cells with ACes
One million IdUrd-labeled ACes were complexed with 2, 5 or 10,11
of Superfect (Qiagen) or Lipofectamine Plus (Life Technologies; Gibco) as
follows. Complexing with Superfect was conducted for 10 minutes at
room temperature. For complexing with Lipofectamine Plus, the indicated
amounts of PLUS reagent were added to 1 million ACes and complexed at
room temperature for 15 minutes. Next the indicated amounts of
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Lipofectamine were added into 200,u1 of low glucose DMEM (no FBS) and
combined with the ACes/PLUS complex for 15 minutes at room
temperature. The complexed ACes were then added dropwise to the cells
in 600 /ul media (final volume of approximately 1.4 ml). After 3 hrs at
37 C in a 5% CO2 incubator, a total volume of 3 ml of culture media (low
glucose DMEM, 1-glutamine, penicillin/streptomycin and 20% FBS) vvas
added. After 24-48 hrs, the -cells were trypsinized to form a. single cell
suspension, centrifuged to remove the supernatant and then fixed in cold
70% ethanol for a minimum of one hour. An aliquot of the fixed cells
was saved for microscopic analysis.
FITC-Conjugated antibody labeling of ACes
Following transfection, the ACes were labeled with FITC-
conjugated antibody that specifically binds to BrdU- or IdUrd-labeled
nucleic acids and the cells were analyzed by FACs for FITC fluorescence
and microscopic staining. Fixed cells were denatured in 2N HCI and
*
0.5% Triton-X for 30 minutes at room temperature. After denaturation,
the cells were neutralized by a series of wash steps at 4 C. To minimize
background staining, the sample was resuspended in PBS and 4%. FBS or
BSA and 0.1 % Triton-X (blocking buffer) for a minimum of 15 minutes.
The cells were then pelleted and exposed for 2 hours at room temperatute
to FITC-conjugated antibody. After the cells were washed with blocking
buffer, the sample was ready for flow cytometry analysis. Samples for
microscopic analysis, were dried on slides and the above staining protocol
was followed, except that BrdU/IdUrd antibody was diluted 1/5 and
exposed to cells for 24 hours.
Results
The delivery of intact ACes was detected within 24 to 48 hours
post transfection. The number of cells recovered at 24 hours post-
transfection was determined by counting an aliquot using a Coulter
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Counter. To determine the control plating efficiency of a recipient cell line
or plating efficiency of the transfected cells, the cells were plated at
1000-10,000 cells per 10 cm petri dish in growth medium and left
stationary in a 5% C02 incubator at 37 C for 10 days. At that point, the
number of viable colonies was determined. The normalized plating
efficiency was calculated as described herein.
When Superfect was used as a delivery agent, the percent delivery
into fibroblast-like synoviocytes as determined by flow cytometry ranged
from -24% to -66.3%. The normalized % plating efficiency was
- 36% when 2,ul of Superfect was used and - 16% when 5lul of
Superfect was used. Higher doses of Superfect were associated with
toxicity and multiple ACes per cell as compared to lower doses. When
Lipofectamine Plus was used as a delivery agent, the percent delivery into
fibroblast-like synoviocytes as determined by flow cytometry ranged from
- 1 1% to - 27% with percent delivery increasing with increasing doses
of agent.
L8 and rat skin fibroblasts (RSF) that had been transfected with
ACes were grown under hygromycin B selection and analyzed for lacZ
expression. While in this example, a hygromycin selection gene was
included in the ACes, there are numerous other selectable marker genes
that may be used in connection with the transfer of heterologous nucleic
acids into cells when it is desirable to include such genes. Such selection
systems are known to those of skill in the art. A choice of selectable
marker gene can, for instance, take into account the level of toxicity of
the selection agent on the host cell for transfection. Identification of an
appropriate selectable marker gene is routine employing the guidance
provided herein.
Clones of L8 cells and RSF cells expressing lacZ were identified.
These results demonstrate that IdUrd-labeled ACes can be delivered
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efficiently into primary cells as well as cell lines and that transgenes
contained in the ACes are expressed in the transfected cells.
EXAMPLE 8
Ex vivo transfer of reporter genes into rat joints
To examine transfer of a heterologous gene into an in vivo
environment and expression of the gene in vivo, L8 cells transfected with
ACes as described above were injected into the ankle joint of rats with
adjuvant-induced arthritis. On day 0, adjuvant induction of arthritis was
performed on Lewis rats. Methods for adjuvant induction of arthritis in
animal models are known in the art [see, e.g., Kong et al. (1999) Nature
4023:304-309]. In one exemplary protocol for adjuvant induction of
arthritis, Lewis rats are immunized at the base of the tail with 1 mg
Mycobacterium tuberculosis H37 RA (Difco, Detroit, Michigan) in 0.1 ml
mineral oil on day 0. Paw swelling typically begins around day 10.
On day 12, intra-articular injection of transfected L8 cells (- 0.7 x
106 cells) or untransfected control cells into the right ankle joint was
performed. On day 14, the rats were sacrificed in order to analyze the
joints for the presence of transplanted transfected L8 cells.
Different tissues of the sacrificed rats were examined by RT-PCR
analysis for the presence of lacZ mRNA. Total mRNA was extracted from
the tissues and RT-PCR was performed using primers specific for the lacZ
gene. The an amplification product was detected only in the synovium,
and not in the other tissues (liver, kidney, heart, spleen and lung).
Synovium from the sacrificed rats was also analyzed by in situ enzymatic
staining X-gal staining for ,6-gal activity. After snap freezing of the
synovium, 8,um and 20 ,um sections were cut, counter-stained with
Mayer's hematoxylin, and analyzed for blue staining of the cells. Staining
was detected in synovium injected with L8 cells that had been
transfected with ACes but not in synovium injected with untransfected
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cells. These results demonstrate successful ex vivo gene transfer in a rat
adjuvant arthritis model using ACes containing a marker gene and thus
the feasibility of treating arthritis and other connective tissue diseases
using ACes as non-viral vectors for gene therapy.
EXAMPLE 9
A flow cytometry technique for measuring delivery of artificial
chromosomes
Production cells lines (see Example 1) were grown in MEM medium
(Gibco BRL) with 10% fetal calf serum (Can Sera, Rexdale ON) with
0.168,ug/mI hygromycin B (Calbiochem, San Diego, CA).
lododeoxyuridine or Bromodeoxyuridine was added directly to culture
medium of the production cell line (CHO E42019) in the exponential
phase of growth. Stock lododeoxyuridine was made in tris base pH 10,
and Bromodeoxyuridine stocks in PBS. Final concentrations of 0.05-1 ,uM
for continuous label of 20-24 hours of 5-50 luM with 15 minute pulse.
After 24 hours, exponentially growing cells were blocked in mitosis with
coichicine (1.0,ug/mI for 7 hours before harvest. Chromosomes were then
isolated and stained with Hoechst 33258 (2.5 /ug/mI) and chromomycin
A3 (50 /ig/mI). Purification of artificial chromosomes was performed using
a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry
systems, San Jose, CA). Chromomycin A3 was excited with the primary
laser set at 457 nm, with emission detected using 475 nm long pass
filter. Hoechst was excited by the secondary UV laser and emission
detected using a 420/44 nm band-pass filter. Both lasers had an output of
150 mW. Bivariate distribution showing cell karyotype was accumulated
from each sort. ACes were gated from other chromosomes and sorted.
Condensing agents (hexylene glycol, spermine, and spermidine) were
added to the sheath buffer to maintain condensed intact chromosome
after sorting. IdU labeling index of sorted chromosomes was determined
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microscopically. An aliquot (2-10 NI) of sorted chromosomes was fixed in
0.2% formaldehyde solution for 5 minutes before being dried on clean
microscopic slide. The microscope sample was fixed with 70% ethanol.
The air-dried slide was denatured in coplin jar with 2N HCI for 30 minutes
at room temperature and washed 2-3 times with PBS. Non specific
binding was blocked with PBS and 4% BSA or serum for minimum of 10
minutes. A 1/5 dilution of FITC conjugated IdU/BrdU antibody (Becton
Dickinson) with a final volume of 60-100 NI was applied to slide. Plastic
strips, Durra seal (Diversified Biotech, Boston, MA) were overlaid on
slides, and slides were kept in dark at 4%C in humidified covered box for
8-24 hours. DAPI (Sigma) 1 Ng/mI in Vectorshield* was used as
counterstain. Fluorescence was detected using Zeiss axioplan 2
microscope equipped for epiflorescence. A minimum of 100 chromosomes
was scored for determining % labeled. Unlabeled chromosomes were
used as negative control.
The day before the transfection, trypsinize V79-4 (Chinese Hamster
Lung fibroblast) cells and plate at 250,000 into a 6 well petri dish in 4
mis DMEM (Dulbecco's Modified Eagle Medium, Life Technologies) and
10% FBS (Can Sera Rexdale ON). The protocol was modified for use
with LM (tk-) cell line by plating 500,000 cells. Lipid or dendrimer reagent
was added to 1 X106 ACes sorted in - 800 N1 sort buffer. Exemplary
protocol variations are set forth in Table 1. Chromosome and transfection
agents.were mixed gently. Complexes were added to cells drop-wise and
plate swirled to mix. Plates were kept at 37 C in a 5% COZ incubator for
specified transfection time. The volume in a well was then made up to 4-
5 ml with DMEM and 10% FBS. Recipient cells were left for 24 hours at
37 C in a 5% CO2 incubator. Trypsinize transfected cells. Samples to be
analyzed for ldU labeled chromosome delivery are fixed in cold 70%
ethanol and stored at -20 C, to be ready for IdU antibody staining.
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Samples to be grown for colony selection are counted and then
transferred to 10-cm dishes at densities of 10,000 and 100,000 cells in
duplicate with remaining cells put in a 15 cm dish. After 24 hours,
selection medium containing of DMEM and 10% FBS with 0.7 mg/mI
hygromycin B, # 400051 (Calbiochem San Diego, CA) is added.
Selection medium is changed every 2-3 days. This concentration of
hygromycin B kills the wild type cells after selection for 7 days. At 10-14
days colonies were expanded and then screened by FISH for intact
chromosome transfer and assayed for beta galactosidase expression.
Table 1: Delivery Transfection Protocols
Agent Dilution Stock Pre treatment Complexing Added to Medium (ml) Transfectio
of ACes time complexes added to wells time (hours
(minutes) before
complexes
2-8 /ug in 20 1.8 ml of 4
CLONFECTIN' NaCI-HEPES serum free
10-20 200%iI of 50% 24
CYTOFECTENE FBS plus
DMEM
ENHANCER + Enhancer 5 10 1.2 3
EFFECTENE minutes
(1:5 ratio)
EU-FECTIN-1 to 5-10 6
11
FUGENE 6 0.5-6 /aI to 15-45 4
final volume
of 100 /aI in
serum free
medium
2.5 /jI added 2-10 2-4
GENEPORTER 2 to 150 /al of
serum free
medium
LIPOFECTAMINE 15 3
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Agent Dilution Stock Pre treatment Complexing Added to Medium (ml) Transfectic
of ACes time complexes added to wells time (hour:
(minutes) before
complexes
LIPOFECTAMINE 20 2.5 5
2000
diluted into 15-45 0.8 6
METAFECTENE 60,u1 serum
free medium
PLUS + PLUS and 200 15 3
LIPOFECTAMINE ,ul of DMEM
(1:1 and 3:2 for 15 minutes
ratio)
SUPERFECT 10 0.6 3
IdU ANTIBODY LABELING
A standard BrdU staining flow cytometry protocol (Gratzer et al.
Cytometry (1981);6:385-393) was used except with some modifications
at the neutralization step, the presence of detergent during denaturation
and the composition of blocking buffer. Between each step samples are
centrifuged at 300 g for 7-10 minutes and supernatant removed. Samples
of 1-2 million cells are fixed in 70% cold ethanol. Cells are then
denatured in 1-2 ml of 2N HCL plus 0.5% triton X for 30 minutes at room
temperature. Sample undergoes 3-4 washes with cold DMEM until
indictor is neutral. Final wash with cold DMEM plus 5% FBS.
Blocking/permeabilization buffer containing PBS, 0.1 % triton X and 4%
FBS is added for 10-15 minutes before pelleting sample by centrifugation.
Add 20 /iI of IdU/BrdU FITC conjugated B44 clone antibody (Becton
Dickinson Immunocytometry Systems, San Jose, CA) to pellet and leave
for 2 hours at room temperature in the dark with agitation every 30
minutes. Wash cells with block/permeabilization buffer and resuspend in
PBS for flow analysis.
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FLOW CYTOMETRY DETECTION OF FLUORESCENT IdUrd
LABELED ACes
Percentage of transfected cells containing IdU labeled ACes was
determined using a flow cytometry with an argon laser turned to 488 nm
at 400 mW. FITC fluorescence was collected through a standard FITC
530/30-nm band pass filter. Cell pop,ulations were gated on the basis of
side scatter versus forward scatter to exclude debris and doublets. Data
was accumulated (15,000 events) to form bivariate channel distribution
showing forward scatter versus green fluorescence (IdU-FITC). The
fluorescence level at which cells were determined to be positive was
established by visual inspection of the histogram of negative control cells,
such that approximately 1 % appeared in the positive region.
Results:
The transfection delivery results of IdU labeled ACes are set forth in
Table 2.
TABLE 2
DOSE DELIVERY
COMPOUND Microliters agent added per 1 % IdU positive (24 hours)
CLONFECTIN 6 0.61
CYTOFECTENE 8 14.67
ENHANCER + EFFECTENE 1.6,10 17.08
(1:5)
EU-FECTIN-1 10 4.57
EU-FECTIN-2 5 0.14
EU-FECTIN-3 10 0.69
EU-FECTIN-4 10 0.24
EU-FECTIN-5 10 0.41
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EU-FECTIN-6 10 0.46
EU-FECTIN-7 10 1.21
EU-FECTIN-8 10 1.58
EU-FECTIN-9 10 0.6
EU-FECTIN-10 10 0.77
EU-FECTIN-1 1 5 1
FUGENE 8 0.49
GENEPORTER 5 22.12
LIPOFECTAMINE 25 17.81
LIPOFECTAMINE 2000 30 10.96
PLUS + LIPOFECTAMINE 12,12 12.2
(1:1)
PLUS + LIPOFECTAMINE 24,16 26.97
(3:2)
METAFECTENE 10 14.14
SUPERFECT 2 27.67
Since modifications will be apparent to those of skill in this art, it is
intended that this invention be limited only by the scope of the appended
claims.
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1
SEQUENCE LISTING
<110> de Jong, Gary
Vanderbyl, Sandra
Hoekstra, Dirk
Oberle, Volker
Drayer, Jan
Tak, Paul Peter
<120> METHODS FOR DELIVERING NUCLEIC ACID
MOLECULES INTO CELLS AND ASSESSMENT THEREOF
<130> 24601-416PC
<140> To Be Assigned
<141> 2002/03/22 (Herewith)
<160> 13
<170> FastSEQ for Windows Version 4.0
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gtccaggag cgcaccatct tctt 24
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<400> 2
atcgcgcttc tcgttggggt cttt 24
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 3
aggactgggt ggcttccaac tcccagacac 30
CA 02441535 2003-11-28
2
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 4
agcttctcat tgctgcgcgc caggttcagg 30
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<213> Mus musculus
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gaattcccct atccctaatc cagattggtg gaataacttg gtatagatgt ttgtgcatta 60
aaaaccctgt aggatcttca ctctaggtca ctgttcagca ctggaacctg aattgtggcc 120
ctgagtgata ggtcctggga catatgcagt tctgcacaga cagacagaca gacagacaga 180
cagacagaca gacagacgtt acaaacaaac acgttgagcc gtgtgccaac acacacacaa 240
acaccactct ggccataatt attgaggacg ttgatttatt attctgtgtt tgtgagtctg 300
tctgtctgtc tgtctgtctg tctgtctgtc tatcaaacca aaagaaacca aacaattatg 360
cctgcctgcc tgcctgcctg cctacacaga gaaatgattt cttcaatcaa tctaaaacga 420
cctcctaagt ttgccttttt tctctttctt tatctttttc ttttttcttt tcttcttcct 480
tccttccttc cttccttcct tccttccttt ctttctttct ttctttcttt cttactttct 540
ttctttcctt cttacattta ttcttttcat acatagtttc ttagtgtaag catccctgac 600
tgtcttgaag acactttgta ggcctcaatc ctgtaagagc cttcctctgc ttttcaaatg 660
ctggcatgaa tgttgtacct cactatgacc agcttagtct tcaagtctga gttactggaa 720
aggagttcca agaagactgg ttatattttt catttattat tgcattttaa ttaaaattta 780
atttcaccaa aagaatttag actgaccaat tcagagtctg ccgtttaaaa gcataaggaa 840
aaagtaggag aaaaacgtga ggctgtctgt ggatggtcga ggctgcttta gggagcctcg 900
tcaccattct gcacttgcaa accgggccac tagaacccgg tgaagggaga aaccaaagcg 960
acctggaaac aataggtcac atgaaggcca gccacctcca tcttgttgtg cgggagttca 1020
gttagcagac aagatggctg ccatgcacat gttgtctttc agcttggtga ggtcaaagta 1080
caaccgagtc acagaacaag gaagtataca cagtgagttc caggtcagcc agagtttaca 1140
cagagaaacc acatcttgaa aaaaacaaaa aaataaatta aataaatata atttaaaaat 1200
ttaaaaatag ccgggagtga tggcgcatgt ctttaatccc agctctcttc aggcagagat 1260
gggaggattt ctgagtttga ggccagcctg gtctgcaaag tgagttccag gacagtcagg 1320
gctatacaga gaaaccctgt cttgaaaact aaactaaatt aaactaaact aaactaaaaa 1380
aatataaaat aaaaatttta aagaatttta aaaaactaca gaaatcaaac ataagcccac 1440
gagatggcaa gtaactgcaa tcatagcaga aatattatac acacacacac acacagactc 1500
tgtcataaaa tccaatgtgc cttcatgatg atcaaatttc gatagtcagt aatactagaa 1560
gaatcatatg tctgaaaata aaagccagaa ccttttctgc ttttgttttc ttttgcccca 1620
agatagggtt tctctcagtg tatccctggc atccctgcct ggaacttcct ttgtaggttt 1680
ggtagcctca aactcagaga ggtcctctct gcctgcctgc ctgcctgcct gcctgcctgc 1740
ctgcctgcct gcctgcctca cttcttctgc cacccacaca accgagtcga acctaggatc 1800
tttatttctt tctctttctc tcttctttct ttctttcttt ctttctttct ttctttcttt 1860
ctttctttct ttcttattca attagttttc aatgtaagtg tgtgtttgtg ctctatctgc 1920
tgcctatagg cctgcttgcc aggagagggc aacagaacct aggagaaacc accatgcagc 1980
tcctgagaat aagtgaaaaa acaacaaaaa aaggaaattc taatcacata gaatgtagat 2040
atatgccgag gctgtcagag tgctttttaa ggcttagtgt aagtaatgaa aattgttgtg 2100
tgtcttttat ccaaacacag aagagaggtg gctcggcctg catgtctgtt gtctgcatgt 2160
agaccaggct ggccttgaac acattaatct gtctgcctct gcttccctaa tgctgcgatt 2220
aaaggcatgt gccaccactg cccggactga tttcttcttt tttttttttt tggaaaatac 2280
ctttctttct ttttctctct ctctttcttc cttccttcct ttctttctat tctttttttc 2340
tttctttttt cttttttttt ttttttttaa aatttgccta aggttaaagg tgtgctccac 2400
aattgcctca gctctgctct aattctcttt aaaaaaaaac aaacaaaaaa aaaaccaaaa 2460
cagtatgtat gtatgtatat ttagaagaaa tactaatcca ttaataactc ttttttccta 2520
CA 02441535 2003-11-28
3
aaattcatgt cattcttgtt ccacaaagtg agttccagga cttaccagag aaaccctgtg 2580
ttcaaatttc tgtgttcaag gtcaccctgg cttacaaagt gagttccaag tccgataggg 2640
ctacacagaa aaaccatatc tcagaaaaaa aaaaagttcc aaacacacac acacacacac 2700
acacacacac acacacacac acacacacac acacacacag cgcgccgcgg cgatgagggg 2760
aagtcgtgcc taaaataaat atttttctgg ccaaagtgaa agcaaatcac tatgaagagg 2820
tactcctaga aaaaataaat acaaacgggc tttttaatca ttccagcact gttttaattt 2880
aactctgaat ttagtcttgg aaaagggggc gggtgtgggt gagtgagggc gagcgagcag 2940
acgggcgggc gggcgggtga gtggccggcg gcggtggcag cgagcaccag aaaacaacaa 3000
accccaagcg gtagagtgtt ttaaaaatga gacctaaatg tggtggaacg gaggtcgccg 3060
ccaccctcct cttccactgc ttagatgctc ccttcccctt actgtgctcc cttcccctaa 3120
ctgtgcctaa ctgtgcctgt tccctcaccc cgctgattcg ccagcgacgt actttgactt 3180
caagaacgat tttgcctgtt ttcaccgctc cctgtcatac tttcgttttt gggtgcccga 3240
gtctagcccg ttcgctatgt tcgggcggga cgatggggac cgtttgtgcc actcgggaga 3300
agtggtgggt gggtacgctg ctccgtcgtg cgtgcgtgag tgccggaacc tgagctcggg 3360
agaccctccg gagagacaga atgagtgagt gaatgtggcg gcgcgtgacg gatctgtatt 3420
ggtttgtatg gttgatcgag accattgtcg ggcgacacct agtggtgaca agtttcggga 3480
acgctccagg cctctcaggt tggtgacaca ggagagggaa gtgcctgtgg tgaggcgacc 3540
agggtgacag gaggccgggc aagcaggcgg gagcgtctcg gagatggtgt cgtgtttaag 3600
gacggtctct aacaaggagg tcgtacaggg agatggccaa agcagaccga gttgctgtac 3660
gcccttttgg gaaaaatgct agggttggtg gcaacgttac taggtcgacc agaaggctta 3720
agtcctaccc ccccccccct tttttttttt tttcctccag aagccctctc ttgtccccgt 3780
caccgggggc accgtacatc tgaggccgag aggacgcgat gggcccggct tccaagccgg 3840
tgtggctcgg ccagctggcg cttcgggtct tttttttttt tttttttttt ttttcctcca 3900
gaagccttgt ctgtcgctgt caccgggggc gctgtacttc tgaggccgag aggacgcgat 3960
gggccccggc ttccaagccg gtgtggctcg gccagctgga gcttcgggtc tttttttttt 4020
tttttttttt tttttttctc cagaagcctt gtctgtcgct gtcaccgggg gcgctgtact 4080
tctgaggccg agaggacgcg atgggtcggc ttccaagccg atgtggcggg gccagctgga 4140
gcttcgggtt tttttttttc ctccagaagc cctctcttgt ccccgtcacc gggggcgctg 4200
tacttctgag gccgagagga cgtgatgggc ccgggttcca ggcggatgtc gcccggtcag 4260
ctggagcttt ggatcttttt tttttttttt cctccagaag ccctctcttg tccccgtcac 4320
cgggggcacc ttacatctga gggcgagagg acgtgatggg tccggcttcc aagccgatgt 4380
ggcggggcca gctggagctt cgggtttttt ttttttcctc cagaagccct ctcttgtccc 4440
cgtcaccggg ggcgctgtac ttctgaggcc gagaggacgt gatgggcccg ggttccaggc 4500
ggatgtcgcc cggtcagctg gagctttgga tcattttttt ttttccctcc agaagccctc 4560
tcttgtcccc gtcaccgggg gcaccgtaca tctgaggccg agaggacacg atgggcctgt 4620
cttccaagcc gatgtggccc ggccagctgg agcttcgggt cttttttttt ttttttcctc 4680
cagaagcctt gtctgtcgct gtcacccggg gcgctgtact tctgaggccg agaggacgcg 4740
atgggcccgg cttccaagcc ggtgtggctc ggccagctgg agcttcgggt cttttttttt 4800
tttttttttt ttcctccaga aaccttgtct gtcgctgtca cccggggcgc ttgtacttct 4860
gatgccgaga ggacgcgatg ggcccgtctt ccaggccgat gtggcccggt cagctggagc 4920
tttggatctt tttttttttt ttttcctcca gaagccctct cttgtccccg tcaccggggg 4980
caccttacat ctgaggccta gaggacacga tgggcccggg ttccaggccg atgtggcccg 5040
gtcagctgga gctttggatc tttttttttt ttttcttcca gaagccctct tgtccccgtc 5100
accggtggca ctgtacatct gaggcggaga ggacattatg ggcccggctt ccaatccgat 5160
gtggcccggt cagctggagc tttggatctt attttttttt taattttttc ttccagaagc 5220
cctcttgtcc ctgtcaccgg tggcacggta catctgaggc cgagaggaca ttatgggccc 5280
ggcttccagg ccgatgtggc ccggtcagct ggagctttgg atcttttttt ttttttttct 5340
tttttcctcc agaagccctc tctgtccctg tcaccggggg ccctgtacgt ctgaggccga 5400
gggaaagcta tgggcgcggt tttctttcat tgacctgtcg gtcttatcag ttctccgggt 5460
tgtcagggtc gaccagttgt tcctttgagg tccggttctt ttcgttatgg ggtcattttt 5520
gggccacctc cccaggtatg acttccaggc gtcgttgctc gcctgtcact ttcctccctg 5580
tctcttttat gcttgtgatc ttttctatct gttcctattg gacctggaga taggtactga 5640
cacgctgtcc tttccctatt aacactaaag gacactataa agagaccctt tcgatttaag 5700
gctgttttgc ttgtccagcc tattcttttt actggcttgg gtctgtcgcg gtgcctgaag 5760
ctgtccccga gccacgcttc ctgctttccc gggcttgctg cttgcgtgtg cttgctgtgg 5820
gcagcttgtg acaactgggc gctgtgactt tgctgcgtgt cagacgtttt tcccgatttc 5880
cccgaggtgt cgttgtcaca cctgtcccgg ttggaatggt ggagccagct gtggttgagg 5940
gccaccttat ttcggctcac tttttttttt tttttttctc ttggagtccc gaacctccgc 6000
tcttttctct tcccggtctt tcttccacat gcctcccgag tgcatttctt tttgtttttt 6060
ttcttttttt tttttttttt ttggggaggt ggagagtccc gagtacttca ctcctgtctg 6120
CA 02441535 2003-11-28
4
tggtgtccaa gtgttcatgc cacgtgcctc ccgagtgcac ttttttttgt ggcagtcgct 6180
cgttgtgttc tcttgttctg tgtctgcccg tatcagtaac tgtcttgccc cgcgtgtaag 6240
acattcctat ctcgcttgtt tctcccgatt gcgcgtcgtt gctcactctt agatcgatgt 6300
ggtgctccgg agttctcttc gggccagggc caagccgcgc caggcgaggg acggacattc 6360
atggcgaatg gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420
cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc cggcccgacg ctgccccgcg 6480
cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540
tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600
ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660
gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720
gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780
tgtctgagaa gcccgtgaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840
tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900
ggtcgcggct ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960
gagaggcctg gctttcgggg gggaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020
aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080
ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcggtg gccggggctc 7140
gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200
ggaaagggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc ccctggaggg 7260
ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320
ggcctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380
ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440
gcacaccccc gcgtgcgcgt actttcctcc cctcctgagg gccgccgtgc ggacggggtg 7500
tgggtaggcg acggtgggct cccgggtccc cacccgtctt cccgtgcctc acccgtgcct 7560
tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620
ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680
ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740
cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800
gttctgtggg agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860
gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920
cggtgtcgcc tcctcgggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980
ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt cccgccggtt ttgcctcgcg 8040
tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100
gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160
ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220
acccgtggcc gtgctgtcgg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280
cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcgggag 8340
gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400
gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460
agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520
ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580
accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640
gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcggccctcc 8700
cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760
catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820
tcgccggggg ctggccgctg tccggtctct cctgcccgac ccccgttggc gtggtcttct 8880
ctcgccggct tcgcggactc ctggcttcgc ccggagggtc agggggcttc ccggttcccc 8940
gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000
gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgc ggtcagttgg gccctggcgt 9060
tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120
gacgggtggc ctatccaggg ctcgcccccg ccgacccccg cctgcccgtc ccggtggtgg 9180
tcgttggtgt ggggagtgaa tggtgctacc ggtcattccc tcccgcgtgg tttgactgtc 9240
tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300
cggcccggtg cggtcgacgt tccggctctc ccgatgccga ggggttcggg atttgtgccg 9360
gggacggagg ggagagcggg taagagaggt gtcggagagc tgtcccgggg cgacgctcgg 9420
gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480
tccggccgca tgcactctcc cgttccgcgc gagcgcccgc ccggctcacc cccggtttgt 9540
cctcccgcga ggctctccgc cgccgccgcc tcctcctcct ctctcgcgct ctctgtcccg 9600
cctggtcctg tcccaccccc gacgctccgc tcgcgcttcc ttacctggtt gatcctgcca 9660
ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720
CA 02441535 2003-11-28
agtgaaactg cgaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780
ctacttggat aactgtggta attctagagc taatacatgc cgacgggcgc tgacccccct 9840
tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900
ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960
acgccccccg tggcggcgac gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020
tcgccgtgcc taccatggtg accacgggtg acggggaatc agggttcgat tccggagagg 10080
gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140
cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200
ggaatgagtc cactttaaat cctttaacga ggatccattg gagggcaagt ctggtgccag 10260
cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320
tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380
ccccttgcct ctcggcgccc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440
gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctgga taccgcagct 10500
aggaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560
taagagggac ggccgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620
gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca agaacgaaag 10680
tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740
cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800
ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860
ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920
cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980
ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040
ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100
gcgttcagcc acccgagatt gagcaataac aggtctgtga tgcccttaga tgtccggggc 11160
tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220
aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280
gaattcccag taagtgcggg tcataagctt gcgttgatta agtccctgcc ctttgtacac 11340
accgcccgtc gctactaccg attggatggt ttagtgaggc cctcggatcg gccccgccgg 11400
ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460
agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520
ctgtggagga gcggcggcgt ggcccgctct ccccgtcttg tgtgtgtcct cgccgggagg 11580
cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640
gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cctccgattt cccctccccc 11700
tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760
gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820
ggtttttgac ccgtcccggg ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880
cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940
ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000
gtcctccccg ctcctgtccc gggtacctag ctgtcgcgtt ccggcgcgga ggtttaagga 12060
ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120
cggtcgttcg ggcggctctc cctcagactc catgaccctc ctccccccgc tgccgccgtt 12180
cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctcgggc gccgtggggg 12240
cccgacccgc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300
gggttcccgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360
cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420
cctctctctc cggggagagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480
cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540
tgcgtcgatg aagaacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600
catcgacact tcgaacgcac ttgcggcccc gggttcctcc cggggctacg cctgtctgag 12660
cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720
ctcgcagggc caacccccca acccgggtcg ggccctccgt ctcccgaagt tcagacgtgt 12780
gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840
cgcgctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900
gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960
tgggaaccca ccgcgccccc gtggcgcccg ggggtgggcg cgtccgcatc tgctctggtc 13020
gaggttggcg gttgagggtg tgcgtgcgcc gaggtggtgg tcggtcccct gcggccgcgg 13080
ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140
gtttgggtct tgcgctgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200
ggcgccgcgc accctccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260
gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320
CA 02441535 2003-11-28
6
cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc 13380
cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440
ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500
gggagggaga gggcctcgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560
ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagat cctccacccc cgccgccctc 13620
ccctcccgcc ggcctctcgg ggaccccctg agacggttcg ccggctcgtc ctcccgtgcc 13680
gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740
tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800
attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860
gaagagccca gcgccgaatc cccgccgcgc gtcgcggcgt gggaaatgtg gcgtacggaa 13920
gacccactcc ccggcgccgc tcgtgggggg cccaagtcct tctgatcgag gcccagcccg 13980
tggacggtgt gaggccggta gcggccccgg cgcgccgggc tcgggtcttc ccggagtcgg 14040
gttgcttggg aatgcagccc aaagcgggtg gtaaactcca tctaaggcta aataccggca 14100
cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160
tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag 14220
gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280
ccccgttcct cccgacccct ccacccgcgc gtcgttcccc tcttcctccc cgcgtccggc 14340
gcctccggcg gcgggcgcgg ggggtggtgt ggtggtggcg cgcgggcggg gccgggggtg 14400
gggtcggcgg gggaccgccc ccggccggcg accggccgcc gccgggcgca cttccaccgt 14460
ggcggtgcgc cgcgaccggc tccgggacgg ccgggaaggc ccggtgggga aggtggctcg 14520
gggggggcgg cgcgtctcag ggcgcgccga accacctcac cccgagtgtt acagccctcc 14580
ggccgcgctt tcgccgaatc ccggggccga ggaagccaga tacccgtcgc cgcgctctcc 14640
ctctcccccc gtccgcctcc cgggcgggcg tgggggtggg ggccgggccg cccctcccac 14700
ggcgcgaccg ctctcccacc cccctccgtc gcctctctcg gggcccggtg gggggcgggg 14760
cggactgtcc ccagtgcgcc ccgggcgtcg tcgcgccgtc gggtcccggg gggaccgtcg 14820
gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatgtcg gctacccacc 14880
cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940
gaaagccgcc gtggcgcaat gaaggtgaag ggccccgccc gggggcccga ggtgggatcc 15000
cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060
aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgggcaggg 15120
cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180
cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240
tttccctcag gatagctggc gctctcgctc ccgacgtacg cagttttatc cggtaaagcg 15300
aatgattaga ggtcttgggg ccgaaacgat ctcaacctat tctcaaactt taaatgggta 15360
agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420
ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480
gacgctcatc agaccccaga aaaggtgttg gttgatatag acagcaggac ggtggccatg 15540
gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600
aatggatggc gctggagcgt cgggcccata cccggccgtc gccgcagtcg gaacggaacg 15660
ggacgggagc ggccgcgggt gcgcgtctct cggggtcggg ggtgcgtggc gggggcccgt 15720
cccccgcctc ccctccgcgc gccgggttcg cccccgcggc gtcgggcccc gcggagccta 15780
cgccgcgacg agtaggaggg ccgctgcggt gagccttgaa gcctagggcg cgggcccggg 15840
tggagccgcc gcaggtgcag atcttggtgg tagtagcaaa tattcaaacg agaactttga 15900
aggccgaagt ggagaagggt tccatgtgaa cagcagttga acatgggtca gtcggtcctg 15960
agagatgggc gagtgccgtt ccgaagggac gggcgatggc ctccgttgcc ctcggccgat 16020
cgaaagggag tcgggttcag atccccgaat ccggagtggc ggagatgggc gccgcgaggc 16080
cagtgcggta acgcgaccga tcccggagaa gccggcggga ggcctcgggg agagttctct 16140
tttctttgtg aagggcaggg cgccctggaa tgggttcgcc ccgagagagg ggcccgtgcc 16200
ttggaaagcg tcgcggttcc ggcggcgtcc ggtgagctct cgctggccct tgaaaatccg 16260
ggggagaggg tgtaaatctc gcgccgggcc gtacccatat ccgcagcagg tctccaaggt 16320
gaacagcctc tggcatgttg gaacaatgta ggtaagggaa gtcggcaagc cggatccgta 16380
acttcgggat aaggattggc tctaagggct gggtcggtcg ggctggggcg cgaagcgggg 16440
ctgggcgcgc gccgcggctg gacgaggcgc cgccgccctc tcccacgtcc ggggagaccc 16500
cccgtccttt ccgcccgggc ccgccctccc ctcttccccg cggggccccg tcgtcccccg 16560
cgtcgtcgcc acctctcttc ccccctcctt cttcccgtcg gggggcgggt cgggggtcgg 16620
cgcgcggcgc gggctccggg gcggcgggtc caaccccgcg ggggttccgg agcgggagga 16680
accagcggtc cccggtgggg cggggggccc ggacactcgg ggggccggcg gcggcggcga 16740
ctctggacgc gagccgggcc cttcccgtgg atcgcctcag ctgcggcggg cgtcgcggcc 16800
gctcccgggg agcccggcgg gtgccggcgc gggtcccctc cccgcggggc ctcgctccac 16860
ccccccatcg cctctcccga ggtgcgtggc gggggcgggc gggcgtgtcc cgcgcgtgtg 16920
CA 02441535 2003-11-28
7
gggggaacct ccgcgtcggt gttcccccgc cgggtccgcc ccccgggccg cggttttccg 16980
cgcggcgccc ccgcctcggc cggcgcctag cagccgactt agaactggtg cggaccaggg 17040
gaatccgact gtttaattaa aacaaagcat cgcgaaggcc cgcggcgggt gttgacgcga 17100
tgtgatttct gcccagtgct ctgaatgtca aagtgaagaa attcaatgaa gcgcgggtaa 17160
acggcgggag taactatgac tctcttaagg tagccaaatg cctcgtcatc taattagtga 17220
cgcgcatgaa tggatgaacg agattcccac tgtccctacc tactatccag cgaaaccaca 17280
gccaagggaa cgggcttggc ggaatcagcg gggaaagaag accctgttga gcttgactct 17340
agtctggcac ggtgaagaga catgagaggt gtagaataag tgggaggccc ccggcgcccg 17400
gccccgtcct cgcgtcgggg tcggggcacg ccggcctcgc gggccgccgg tgaaatacca 17460
ctactctcat cgttttttca ctgacccggt gaggcggggg ggcgagcccc gaggggctct 17520
cgcttctggc gccaagcgtc cgtcccgcgc gtgcgggcgg gcgcgacccg ctccggggac 17580
agtgccaggt ggggagtttg actggggcgg tacacctgtc aaacggtaac gcaggtgtcc 17640
taaggcgagc tcagggagga cagaaacctc ccgtggagca gaagggcaaa agctcgcttg 17700
atcttgattt tcagtacgaa tacagaccgt gaaagcgggg cctcacgatc cttctgacct 17760
tttgggtttt aagcaggagg tgtcagaaaa gttaccacag ggataactgg cttgtggcgg 17820
ccaagcgttc atagcgacgt cgctttttga tccttcgatg tcggctcttc ctatcattgt 17880
gaagcagaat tcaccaagcg ttggattgtt cacccactaa tagggaacgt gagctgggtt 17940
tagaccgtcg tgagacaggt tagttttacc ctactgatga tgtgttgttg ccatggtaat 18000
cctgctcagt acgagaggaa ccgcaggttc agacatttgg tgtatgtgct tggctgagga 18060
gccaatgggg cgaagctacc atctgtggga ttatgactga acgcctctaa gtcagaatcc 18120
gcccaagcgg aacgatacgg cagcgccgaa ggagcctcgg ttggccccgg atagccgggt 18180
ccccgtccgt cccgctcggc ggggtccccg cgtcgccccg cggcggcgcg gggtctcccc 18240
ccgccgggcg tcgggaccgg ggtccggtgc ggagagccgt tcgtcttggg aaacggggtg 18300
cggccggaaa gggggccgcc ctctcgcccg tcacgttgaa cgcacgttcg tgtggaacct 18360
ggcgctaaac cattcgtaga cgacctgctt ctgggtcggg gtttcgtacg tagcagagca 18420
gctccctcgc tgcgatctat tgaaagtcag ccctcgacac aagggtttgt ctctgcgggc 18480
tttcccgtcg cacgcccgct cgctcgcacg cgaccgtgtc gccgcccggg cgtcacgggg 18540
gcggtcgcct cggcccccgc gcggttgccc gaacgaccgt gtggtggttg ggggggggat 18600
cgtcttctcc tccgtctccc gaggacggtt cgtttctctt tccccttccg tcgctctcct 18660
tgggtgtggg agcctcgtgc cgtcgcgacc gcggcctgcc gtcgcctgcc gccgcagccc 18720
cttgccctcc ggccttggcc aagccggagg gcggaggagg gggatcggcg gcggcggcga 18780
ccgcggcgcg gtgacgcacg gtgggatccc catcctcggc gcgtccgtcg gggacggccg 18840
gttggagggg cgggaggggt ttttcccgtg aacgccgcgt tcggcgccag gcctctggcg 18900
gccggggggg cgctctctcc gcccgagcat ccccactccc gcccctcctc ttcgcgcgcc 18960
gcggcggcga cgtgcgtacg aggggaggat gtcgcggtgt ggaggcggag agggtccggc 19020
gcggcgcctc ttccattttt tcccccccaa cttcggaggt cgaccagtac tccgggcgac 19080
actttgtttt ttttttttcc cccgatgctg gaggtcgacc agatgtccga aagtgtcccc 19140
cccccccccc ccccccggcg cggagcggcg gggccactct ggactctttt tttttttttt 19200
tttttttttt ttaaattcct ggaaccttta ggtcgaccag ttgtccgtct tttactcctt 19260
catataggtc gaccagtact ccgggtggta ctttgtcttt ttctgaaaat cccagaggtc 19320
gaccagatat ccgaaagtcc tctctttccc tttactcttc cccacagcga ttctcttttt 19380
tttttttttt tttggtgtgc ctctttttga cttatataca tgtaaatagt gtgtacgttt 19440
atatacttat aggaggaggt cgaccagtac tccgggcgac actttgtttt tttttttttt 19500
tccaccgatg atggaggtcg accagatgtc cgaaagtgtc ccgtcccccc cctccccccc 19560
ccgcgacgcg gcgggctcac tctggactct tttttttttt tttttttttt tttaaatttc 19620
tggaacctta aggtcgacca gttgtccgtc tttcactcat tcatataggt cgaccggtgg 19680
tactttgtct ttttctgaaa atcgcagagg tcgaccagat gtcagaaagt ctggtggtcg 19740
ataaattatc tgatctagat ttgtttttct gtttttcagt tttgtgttgt tttgtgttgt 19800
tttgtgttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt 19860
tttgtgttgt gttgtgttgt gttgtgttgg gttgggttgg gttgggttgg gttgggttgg 19920
gttgggttgg gttgggttgt gttgtttggt tttgtgttgt ttggtgttgt tggttttgtt 19980
ttgtttgctg ttgttttgtg ttttgcgggt cgaacagttg tccctaaccg agtttttttg 20040
tacacaaaca tgcacttttt ttaaaataaa tttttaaaat aaatgcgaaa atcgaccaat 20100
tatccctttc cttctctctc ttttttaaaa attttctttg tgtgtgtgtg tgtgtgtgtg 20160
tgtgtgtgtg tgcgtgtgtg tgtgtgtgtg cgtgcagcgt gcgcgcgctc gttttataaa 20220
tacttataat aataggtcgc cgggtggtgg tagcttcccg gactccagag gcagaggcag 20280
gcagacttct gagttcgagg ccagcctggt ctacagagga accctgtctc gaaaaatgaa 20340
aataaataca tacatacata catacataca tacatacata catacataca tacatatgag 20400
gttgaccagt tgtcaatcct ttagaatttt gtttttaatt aatgtgatag agagatagat 20460
aatagataga tggatagagt gatacaaata taggtttttt tttcagtaaa tatgaggttg 20520
CA 02441535 2003-11-28
8
attaaccact tttccctttt taggtttttt tttttttccc ctgtccatgt ggttgctggg 20580
atttgaactc aggaccctgg caggtcaact ggaaaacgtg ttttctatat atataaatag 20640
tggtctgtct gctgtttgtt tgtttgcttg cttgcttgct tgcttgcttg cttgcttgct 20700
tgcttttttt tttcttctga gacagtattt ctctgtgtaa cctggtgccc tgaaactcac 20760
tctgtagacc agcctggcct caatcgaact cagaaatcct cctgcctctt gtctacctcc 20820
caattttgga gtaaaggtgt gctacaccac tgcctggcat tattatcatt atcattatta 20880
attttattat tagacagaac gaaatcaact agttggtcct gtttcgttaa ttcatttgaa 20940
attagttgga ccaattagtt ggctggtttg ggaggtttct tttgtttccg atttgggtgt 21000
ttgtggggct ggggatcagg tatctcaacg gaatgcatga aggttaaggt gagatggctc 21060
gatttttgta aagattactt ttcttagtct gaggaaaaaa taaaataata ttgggctacg 21120
tttcattgct tcatttctat ttctctttct ttctttcttt ctttcagata aggaggtcgg 21180
ccagttcctc ctgccttctg gaagatgtag gcattgcatt gggaaaagca ttgtttgaga 21240
gatgtgctag tgaaccagag agtttggatg tcaagccgta taatgtttat tacaatatag 21300
aaaagttcta acaaagtgat ctttaacttt tttttttttt tttctccttc tacttctact 21360
tgttctcact ctgccaccaa cgcgctttgt acattgaatg tgagctttgt tttgcttaac 21420
agacatatat tttttctttt ggttttgctt gacatggttt ccctttctat ccgtgcaggg 21480
ttcccagacg gccttttgag aataaaatgg gaggccagaa ccaaagtctt ttgaataaag 21540
caccacaact ctaacctgtt tggctgtttt ccttcccaag gcacagatct ttcccagcat 21600
ggaaaagcat gtagcagttg taggacacac tagacgagag caccagatct cattgtgggt 21660
ggttgtgaac cacccaccat gtggttgcct gggatttgaa ctcaggatct tcagaagacg 21720
agtcagggct ctaaaccgat gagccatctc tccagccctc ctacattcct tcttaaggca 21780
tgaatgatcc cagcatggga agacagtctg ccctctttgt ggtatatcac catatactca 21840
ataaaataat gaaatgaatg aagtctccac gtatttattt cttcgagcta tctaaattct 21900
ctcacagcac ctccccctcc cccacactgc ctttctccct atgtttgggt ggggctgggg 21960
gaggggtggg gtgggggcag ggatctgcat gtcttcttgc aggtctgtga actatttgcg 22020
atggcctggt tctctgaact gttgagcctt gtctatccag aggctgactg gctagttttc 22080
tacctgaagt ccctgagtga tgatttccct gtgaattc 22118
<210> 6
<211> 175
<212> DNA
<213> Mus Musculus
<400> 6
ctcccgcgcg gcccccgtgt tcgccgttcc cgtggcgcgg acaatgcggt tgtgcgtcca 60
cgtgtgcgtg tccgtgcagt gccgttgtgg agtgcctcgc tctcctcctc ctccccggca 120
gcgttcccac ggttggggac caccggtgac ctcgccctct tcgggcctgg atccg 175
<210> 7
<211> 755
<212> DNA
<213> Mus musculus
<400> 7
ggtctggtgg gaattgttga cctcgctctc gggtgcggcc tttggggaac ggcggggtcg 60
gtcgtgcccg gcgccggacg tgtgtcgggg cccacttccc gctcgagggt ggcggtggcg 120
gcggcgttgg tagtctcccg tgttgcgtct tcccgggctc ttgggggggg tgccgtcgtt 180
ttcggggccg gcgttgcttg gcttacgcag gcttggtttg ggactgcctc aggagtcgtg 240
ggcggtgtga ttcccgccgg ttttgcctcg cgtctgcctg ctttgcctcg ggtttgcttg 300
gttcgtgtct cgggagcggt ggtttttttt tttttcgggt cccggggaga ggggtttttc 360
cgggggacgt tcccgtcgcc ccctgccgcc ggtgggtttt cgtttcgggc tgtgttcgtt 420
tccccttccc cgtttcgccg tcggttctcc ccggtcggtc ggccctctcc ccggtcggtc 480
gcccggccgt gctgccggac ccccccttct gggggggatg cccgggcacg cacgcgtccg 540
ggcggccact gtggtccggg agctgctcgg caggcgggtg agccagttgg aggggcgtca 600
tgcccccgcg ggctcccgtg gccgacgcgg cgtgttcttt gggggggcct gtgcgtgcgg 660
gaaggctgcg cacgttgtcg gtccttgcga gggaaagagg cttttttttt ttagggggtc 720
gtccttcgtc gtcccgtcgg cggtggatcc ggcct 755
CA 02441535 2003-11-28
9
<210> 8
<211> 463
<212> DNA
<213> Mus musculus
<400> 8
ggccgaggtg cgtctgcggg ttggggctcg tccggccccg tcgtcctccg ggaaggcgtt 60
tagcgggtac cgtcgccgcg ccgaggtggg cgcacgtcgg tgagataacc ccgagcgtgt 120
ttctggttgt tggcggcggg ggctccggtc gatgtcttcc cctccccctc tccccgaggc 180
caggtcagcc tccgcctgtg ggcttcgtcg gccgtctccc cccccctcac gtccctcgcg 240
agcgagcccg tccgttcgac cttccttccg ccttcccccc atctttccgc gctccgttgg 300
ccccggggtt ttcacggcgc cccccacgct cctccgcctc tccgcccgtg gtttggacgc 360
ctggttccgg tctccccgcc aaaccccggt tgggttggtc tccggccccg gcttgctctt 420
cgggtctccc aacccccggc cggaagggtt cgggggttcc ggg 463
<210> 9
<211> 378
<212> DNA
<213> Mus musculus
<400> 9
ggattcttca ggattgaaac ccaaaccggt tcagtttcct ttccggctcc ggccgggggg 60
ggcggccccg ggcggtttgg tgagttagat aacctcgggc cgatcgcacg ccccccgtgg 120
cggcgacgac ccattcgaac gtctgcccta tcaactttcg atggtagtcg atgtgcctac 180
catggtgacc acgggtgacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 240
ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga cccggggagg 300
tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtccac 360
tttaaatcct ttaagcag 378
<210> 10
<211> 378
<212> DNA
<213> Mus musculus
<400> 10
gatccattgg agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta 60
tattaaagtt gctgcagtta aaaagctcgt agttggatct tgggagcggg cgggcggtcc 120
gccgcgaggc gagtcaccgc ccgtccccgc cccttgcctc tcggcgcccc ctcgatgctc 180
ttagctgagt tgtcccgcgg ggcccgaagc gtttactttg aaaaaattag agttgtttca 240
aagcaggccc gagccgcctg gataccgcca gctaggaaat aatggaatag gaccgcggtt 300
cctattttgt ttggttttcg gaactgagcc catgattaag ggaaacggcc gggggcattc 360
ccttattgcg ccccccta 378
<210> 11
<211> 719
<212> DNA
<213> Mus musculus
<400> 11
ggatctttcc cgctccccgt tcctcccggc ccctccaccc gcgcgtctcc ccccttcttt 60
tcccctctcc ggaggggggg gaggtggggg cgcgtgggcg gggtcggggg tggggtcggc 120
gggggaccgc ccccggccgg caaaaggccg ccgccgggcg cacttcaacc gtagcggtgc 180
gccgcgaccg gctacgagac ggctgggaag gcccgacggg gaatgtggct cggggggggc 240
ggcgcgtctc agggcgcgcc gaaccacctc accccgagtg ttacagccct ccggccgcgc 300
tttcgcggaa tcccggggcc gaggggaagc ccgatacccg tcgccgcgct tttcccctcc 360
ccccgtccgc ctcccgggcg ggcgtggggg tgggggccgg gccgcccctc ccacgcccgt 420
ggtttctctc tctcccggtc tcggccggtt tggggggggg agcccggttg ggggcggggc 480
CA 02441535 2003-11-28
ggactgtcct cagtgcgccc cgggcgtcgt cgcgccgtcg ggcccggggg gttctctcgg 540
tcacgccgcc cccgacgaag ccgagcgcac ggggtcggcg gcgatgtcgg ctacccaccc 600
gacccgtctt gaaacacgga ccaaggagtc taacgcgtgc gcgagtcagg ggctcgcacg 660
aaagccgccg tggcgcaatg aaggtgaagg gccccgtccg ggggcccgag gtgggatcc 719
<210> 12
<211> 685
<212> DNA
<213> Mus musculus
<400> 12
cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgtcgggg 60
aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ctgggcaggg 120
cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 180
cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 240
tttccctcag gatagctggc gctctcgcaa ccttcggaag cagttttatc cgggtaaagg 300
cggaatggat taggaggtct tggggccgga aacgatctca aactatttct caaactttaa 360
atgggtaagg aagcccggct cgctggcgtg gagccgggcg tggaatgcga gtgcctagtg 420
ggccactttt ggtaagcaga actggcgctg cgggatgaac cgaacgccgg gttaaggcgc 480
ccgatgccga cgctcatcag accccagaaa aggtgttggt tgatatagac agcaggacgg 540
tggccatgga agtcggaatc cgctaaggag tgtgtaacaa ctcacctgcc gaatcaacta 600
gccctgaaaa tggatggcgc tggagcgtcg ggcccatacc cggccgtcgc cggcagtcgg 660
aacgggacgg gacgggagcg gccgc 685
<210> 13
<211> 5162
<212> DNA
<213> Artificial Sequence
<220>
<223> Chimeric bacterial plasmid
<400> 13
gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900
gagctcggat cgatatctgc ggccgcgtcg acggaattca gtggatccac tagtaacggc 960
cgccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc 1020
ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat 1080
ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa 1140
aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact 1200
tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac 1260
tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa 1320
cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgtgattttc 1380
caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 1440
gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 1500
CA 02441535 2003-11-28
11
gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 1560
caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 1620
agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1680
aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1740
accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1800
gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1860
acgatgataa gcttgccaca acccgggatc caccggtcgc caccatggtg agcaagggcg 1920
aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1980
acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 2040
agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 2100
cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 2160
agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 2220
actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 2280
tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2340
acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2400
tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2460
acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2520
ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2580
ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cctagagctc 2640
gctgatcagc ctcgactgtg cctctagttg ccagccatct gttgtttgcc cctcccccgt 2700
gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2760
tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2820
caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2880
ttctgaggcg gaaagaacca gctggggctc gagtgcattc tagttgtggt ttgtccaaac 2940
tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 3000
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 3060
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 3120
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3180
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3240
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3300
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3360
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3420
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3480
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3540
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3600
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3660
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3720
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3780
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3840
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3900
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3960
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4020
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4080
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4140
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4200
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4260
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 4320
cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4380
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4440
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4500
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4560
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4620
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4680
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4740
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4800
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4860
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4920
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4980
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5040
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5100
CA 02441535 2003-11-28
12
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5160
tc 5162
