U.S. patent application number 13/535267 was filed with the patent office on 2012-11-15 for gene delivery.
Invention is credited to Christopher D. BATICH, Jon DOBSON.
Application Number | 20120288505 13/535267 |
Document ID | / |
Family ID | 34639915 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120288505 |
Kind Code |
A1 |
DOBSON; Jon ; et
al. |
November 15, 2012 |
GENE DELIVERY
Abstract
The present invention relates to a method of delivery of a
therapeutic agent to a target cell, the method comprising targeting
particles comprising the therapeutic agent to the cell using
magnetic means to apply a magnetic force to said particles so as to
tend to move said particles towards said magnetic means and at the
same time moving said magnetic means.
Inventors: |
DOBSON; Jon;
(Stoke-on-Trent, GB) ; BATICH; Christopher D.;
(Gainesville, FL) |
Family ID: |
34639915 |
Appl. No.: |
13/535267 |
Filed: |
June 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11912198 |
Jun 12, 2008 |
8232102 |
|
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PCT/GB2006/001477 |
Apr 21, 2006 |
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13535267 |
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Current U.S.
Class: |
424/135.1 ;
424/130.1; 514/1.1; 514/44A; 514/44R |
Current CPC
Class: |
A61N 2/00 20130101; A61P
1/18 20180101; A61P 19/04 20180101; A61P 15/00 20180101; A61N 2/004
20130101; A61N 2/02 20130101; A61P 1/00 20180101; A61P 1/16
20180101 |
Class at
Publication: |
424/135.1 ;
514/44.R; 514/44.A; 514/1.1; 424/130.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/395 20060101 A61K039/395; A61K 38/02 20060101
A61K038/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2005 |
GB |
0508110.4 |
Claims
1. A method of treatment of a disease, the method comprising
delivery of a therapeutic agent into target cells in vivo, wherein
the method comprises targeting magnetic particles comprising the
therapeutic agent into the target cells in vivo by using magnetic
means to apply a magnetic force to said particles so as to move
said particles towards said magnetic means, and at the same time
oscillating said magnetic means, thereby delivering the therapeutic
agent to said cells.
2. The method of claim 1, wherein said magnetic means are used to
apply the magnetic force to said magnetic particles so as to move
said magnetic particles in a first direction towards said magnetic
means and at the same time moving said magnetic means relative to
said magnetic particles in a second direction at an angle greater
than 0.degree. and less than 180.degree. to said first
direction.
3. The method of claim 2, wherein said angle is greater than
0.degree. and less than 90.degree. to said first direction.
4. The method of claim 1, wherein the direction of oscillation of
said magnetic means is substantially perpendicular to the direction
of attraction of the magnetic particles toward said magnetic
means.
5. The method of claim 1, wherein the magnetic force applied to
said magnetic particles is a translational force.
6. The method of claim 1, wherein the magnetic means is a magnet or
array of magnets.
7. The method of claim 1, wherein the magnetic means is an
electromagnet.
8. The method of claim 1, wherein the magnetic means is a magnetic
field.
9. The method of claim 1, wherein the magnetic means oscillates
with a frequency in the range of 1 to 100 Hz.
10. The method of claim 1, wherein the amplitude of oscillation of
the magnetic means is in the nanometer to millimeter range.
11. The method of claim 1, wherein the magnetic particles have a
mean size of between 10 .mu.m and 5 nm.
12. The method of claim 1, wherein the magnetic particles are made
from a magnetizable material.
13. The method of claim 1, wherein the magnetic particles are made
from a magnetizable material selected from the group consisting of:
elemental iron, chromium, manganese, cobalt, nickel, and compounds
thereof.
14. The method of claim 13, wherein the magnetizable material is an
iron salt.
15. The method of claim 14, wherein the iron salt is selected from
the group consisting of: magnetite (Fe.sub.3O.sub.4), maghemite
(.gamma.Fe.sub.2O.sub.3), greigite (Fe.sub.3S.sub.4), and
combinations thereof.
16. The method of claim 1, wherein the cells are mammalian or human
cells.
17. The method of claim 1, wherein the cells are lung cells kidney
cells, nerve cells, mesenchymal cells, muscle cells
(cardiomyocyte), liver cells, red or white blood cells,
erythrocytes, lymphocytes, monocytes, macrophages, leukocytes,
pancreatic .beta. cells; epithelial cells, lung, gastric cells,
endothelial cells, bone cells, skin cells, gastrointestinal cells,
bladder cells, reproductive cells, sperm cells, egg cells, cells of
the uterus, prostate or endocrine gland, pituitary cells, embryonic
stem (ES) cells, embryonal germ (EG) cells, tumor cells, or cancer
cells.
18. The method of claim 1, wherein the therapeutic agent is DNA,
RNA, interfering RNA (RNAi), a peptide, a polypeptide, an antibody,
a single chain antibody fragment, an aptamer, or a small
molecule.
19. The method of claim 1, wherein the therapeutic agent is a
polynucleotide, RNA or DNA, and the method involves the genetic
transformation of the target cell.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/912,198, filed Jun. 12, 2008, which is the
US national phase entry of International Patent Application No.
PCT/GB2006/001477, filed Apr. 21, 2006, which claims priority to UK
Patent Application No. 0508110.4, filed Apr. 22, 2005.
FIELD OF INVENTION
[0002] The present invention relates to methods for the delivery of
therapeutic agents to target cells.
BACKGROUND
[0003] Cystic fibrosis causes the body to produce thick secretions
that affect the lungs and digestive tract. One in every ten babies
born with cystic fibrosis undergoes an operation within the first
few days of life due to a bowel obstruction. Children and adults
suffer from repeated chest infections and problems with pancreas
function. The latter complication makes it difficult for cystic
fibrosis sufferers to digest food. This can lead to malnutrition,
poor growth, physical weakness and delayed puberty. In older
patients insulin production can become deficient due to increasing
pancreatic disease thus resulting in diabetes. Cystic fibrosis can
also cause blockages of liver ducts. This occurs in approximately
8% of sufferers however the health risk is so severe that liver
transplants are necessary. While the disease has serious effects on
the gut, pancreas, liver and reproductive tract the effect it has
on the lungs are the most severe. Repeated cycles of infection lead
to continuous inflammation and damage to the lungs which ultimately
leads to respiratory failure and death.
[0004] Cystic fibrosis is a genetic disease caused by a mutation
within a single gene, CFTR (Cystic Fibrosis Trans-membrane
Conductance Regulator). Thus by treating patients using gene
therapy it is possible to treat the underlying cause of the disease
and not the symptoms. Introduction of CFTR has been shown to
correct the cystic fibrosis defect in vitro. Gene therapy has been
tested on humans using viruses and liposomes as transfection
vectors. Recombinant viruses used for gene transfer need to be able
to infect both dividing and non-dividing cells, integrate into the
host genome and give long term gene expression. Of all of the viral
vectors tested so far (adenovirus, retrovirus, adeno-associated
virus and sendai virus) non have all of these features. Viral
vectors used as gene delivery systems also have potential safety
issues and are ineffective long term due to a triggering of the
immune response. Similar transfection problems apply to a wide
variety of genetic diseases.
[0005] The present invention addresses the need for a non-viral
gene transfection agent which mitigates the disadvantages
associated with recombinant viral vectors. Non-viral agents are
non-infectious, relatively non-immunogenic, have low toxicity, can
carry larger DNA plasmids and can be produced cheaply on a large
scale. One type of agent is DNA-coated magnetic particles.
[0006] Current magnetic based transfection systems have a low
efficiency of transformation. The present inventors have developed
a magnetic particle based delivery system which has surprisingly
been shown to have a transformation efficiency 10 times greater
than the current systems based on initial in vitro studies.
SUMMARY
[0007] According to a first aspect of the invention there is
provided a method of delivery of a therapeutic agent to a target
cell the method comprising targeting particles comprising the
therapeutic agent to the cell using magnetic means to apply a
magnetic force to the particles so as to tend to move the particles
towards or away from the magnetic means and at the same time moving
the magnetic means.
[0008] In a preferred aspect of the invention the magnetic means
are used to apply a magnetic force to the particles so as to tend
to move the particles in a first direction towards or away from the
magnetic means and at the same time moving the magnetic means
relative to the particles in a second direction at an angle to the
first direction.
[0009] The movement of the magnetic means in a second direction is
generally at a non-zero angle to the first direction, for example
at an angle between 0 and 180.degree., such as at an angle of
between 0 and 90.degree., to the first direction.
[0010] Preferably the movement of the magnetic means in a second
direction is an oscillating movement. The oscillation frequency at
which the magnet(s) is driven will usually be varied and will
generally be in the range of 0 up to 100 Hz although values outside
this range may be used.
[0011] In a preferred aspect of the invention the movement of the
magnetic means in a second direction is substantially perpendicular
to the first direction in which the particles tend to move.
[0012] The magnetic force applied to the particles so as to move
the particles towards or away from the magnetic means may be
described as a translational force. The translational force is
produced by a magnetic field with a gradient. Preferably the
direction of the translational force is towards the magnet.
[0013] In a preferred aspect of the invention the magnetic means is
a magnet or array of magnets. The magnet may be an
electromagnet.
[0014] The particles may be attracted to, or repelled from, the
magnetic means. Preferably the particles are attracted to the
magnetic means.
[0015] In a further preferred aspect of the invention the particle
is a magnetic particle. Preferably the particle is made from a
magnetizable material. The magnetizable particle may be inherently
magnetic or may be one which reacts in a magnetic field.
[0016] Generally, any magnetic material may be used, however, by
the term magnetic we mean, for example, a material which is
paramagnetic superparamagnetic, ferromagnetic, and/or
antiferromagnetic, examples of which include elemental iron,
chromium manganese, cobalt, nickel, or a compound thereof. The iron
compound may be an iron salt which may be selected from the group
which includes magnetite (Fe.sub.3O.sub.4), maghemite
(.gamma.Fe.sub.2O.sub.3) and greigite (Fe.sub.3S.sub.4), or any
combination thereof. The chromium compound may be chromium
dioxide.
[0017] The particles may be provided within the pores of a polymer.
Alternatively, the particles may comprise a magnetic core with a
biocompatible coating. The biocompatible coating may comprise a
polymer, e.g. dextran, polyvinyl alcohol (PVA), polyethylenimine
(PEI) or silica.
[0018] In a further preferred aspect of the invention the particles
have a mean size of between 10 .mu.m and 5 nm, for example between
1 .mu.m and 10 nm.
[0019] Preferably the particles are nanoparticles.
[0020] Larger, magnetically blocked particles (>30 nm for
magnetite) will experience a torque in the oscillating field as the
field vector changes its angle with respect to the magnetization
vector of the particles according to the equation:
.tau.=.mu.B sin .theta.
where .tau. is the torque, .mu. is the magnetic moment, B is the
magnetic flux density and .theta. is the angle between the applied
field and the particle's magnetization vector. This twisting,
wedging and pulling enhances the movement of the
particle/therapeutic agent complex resulting in the improved uptake
in the cells.
[0021] In the method of the invention, the cell may be a bacterial
cell, plant cell or animal cell. The animal cell may be a mammalian
cell, for example a human cell.
[0022] In the method of the invention, the cell may be a lung cell,
kidney cell, nerve cell, mesenchymal cell, muscle cell
(cardiomyocyte), liver cell, red or white blood cell (e.g.
erythrocyte, lymphocyte, monocyte, macrophage, leukocyte),
pancreatic .beta. cell; epithelial cell (e.g. lung, gastric),
endothelial cell, bone cell, skin cell, gastrointestinal cell,
bladder cell, reproductive cell (sperm or egg cell), cells of the
uterus, prostate or endocrine gland (e.g. pituitary); embryonic
stem (ES) cells; embryonal germ (EG) cells, tumor cell, cancer
cell.
[0023] The method of the invention may be an ex vivo or in vivo
method. Preferably the method is carried out in vivo.
[0024] The method described herein has application in the treatment
of a wide range of disorders including. Thus the method has
application as a method for the treatment or prevention of clinical
disorders and diseases.
[0025] In the method of the invention the therapeutic agent may be
a pharmaceutical, nutraceutical or agrochemical agent. The
pharmaceutical agent may include DNA, RNA, interfering RNA (RNAi),
a peptide, polypeptide, an antibody (e.g. antibody fragment such as
a single chain antibody fragment), an aptamer, a small molecule.
Small molecules may include, but are not limited to, peptides,
peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,
polynucleotides, polynucleotide analogs, nucleotides, nucleotide
analogs, organic or inorganic compounds (i.e., including
hetero-organic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0026] In a preferred method of the invention the therapeutic agent
is DNA. In a further preferred method of the invention, the
therapeutic agent is the gene encoding the Cystic Fibrosis
Trans-membrane Conductance Regulator.
[0027] A further aspect of the invention provides the use of
moveable magnetic means in the manufacture of a system for
targeting particles comprising a therapeutic agent to a target
cell. Preferably the magnetic means are in motion, preferably still
in constant motion.
[0028] In a preferred use according to the invention the magnetic
means are used to apply a magnetic force to the particles so as to
tend to move the particles towards or away from the magnetic means
and at the same time moving the magnetic means.
[0029] In a further preferred use according to the invention the
magnetic means are used to apply a magnetic force to the particles
so as to tend to move the particles in a first direction towards or
away from the magnetic means and at the same time moving the
magnetic means relative to the particles in a second direction at
an angle to the first direction.
[0030] Where the use is in vivo, the magnetic means may be moved
external to the body. The movement of the magnetic means may be
controlled by a motor or a magnet. The movement of the magnetic
means may be remotely controlled.
[0031] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of the words, for
example "comprising" and "comprises", means "including but not
limited to", and is not intended to (and does not) exclude other
moieties, additives, components, integers or steps.
[0032] Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0033] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith.
[0034] The invention will be described by way of example only with
reference to the following figures:
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a schematic representation of the magnet array
drive system and sample holder for in vitro cell culture and
Air/Liquid Interface (tissue) studies;
[0036] FIG. 2 shows GFP expression in HEK293T cells transfected
with 150 nm magnetic nanoparticles coated with pEGFPC 1 DNA in
response to magnetic field;
[0037] FIG. 3 is a histogram showing luciferase activity in HEK293
T cells transfected with 150 nm magnetic nanoparticles coated with
pCIKLux luciferase reporter; and
[0038] FIG. 4 is a histogram showing luciferase activity in
NC1-H292 human lung epithelial cells transfected with OzBiosciences
Polymag.RTM. particles coated with pCIKLux luciferase reporter
construct in response to static and oscillating magnetic fields.
All transfections were performed in 96 well tissue culture plates
using 0.1 .mu.g DNA/well. Genejuice (GJ) and Lipofectamine 2000
(LF2000) transfections were carried out according to the
manufacturer's recommended protocol. Data shown as mean.+-.SEM (n=6
for all groups). Magnet diameter=6 mm.
EXAMPLE
[0039] The reporter genes Green Fluorescent Protein (GFP) and
luciferase were attached to commercially available magnetic
nanoparticles. The particles generally consisted of a magnetic core
(magnetite--Fe.sub.3O.sub.4 and/or its oxidation product
maghemite--.gamma.Fe.sub.2O.sub.3) with a polymer coating, such as
dextran, PVA or silica, and ranged in size from .about.10 nm to
.about.1 .mu.m. Magnetite is a naturally occurring iron oxide and
is found in many organs in the human body. In addition magnetite is
FDA-approved for MRI contrast enhancement and thus is suitable for
clinical trials.
[0040] Magnetic nano-particles coated with 1800 branched
polyethyleneimine (PEI) were incubated with DNA in order to bind
the reporter genes to the particles. The gene/particle complex was
then introduced into mono-layer cultures of HEK293T kidney cells
within the incubator. Culture dishes were positioned on a
custom-built holder above the magnet array, housed within the
incubator.
[0041] The reporter gene/particle complex is targeted to cells via
a high gradient rare earth (NdFeb) magnet which are focused over
the target site. These magnets produce a translational force on the
particles due to the high field strength/gradient product according
to the equation:
F mag = ( x 2 - x 1 ) V 1 .mu. o B ( .gradient. B )
##EQU00001##
where x.sub.2 is the volume magnetic susceptibility of the magnetic
particle, x.sub.1 is the volume magnetic susceptibility of the
surrounding medium, .mu..sub.o is the magnetic permeability of free
space, B is the magnetic flux density in Telsa (T) (Pankhurst et
al. 2003). This translational force `pulls` the particles towards
the magnet.
[0042] The particles are delivered using a high precision
oscillating horizontal drive system which is controlled by a
computer and custom designed control software. The amplitude of the
array's drive system can vary between a few nanometers to
millimeters and the frequency can vary from static up to 100's of
Hz depending upon the parameters for the target.
[0043] HEK293T cells were seeded in 96 well plates at
5.times.10.sup.3 cells/well. The cells were transfected with 5
ug/well of 150 nm dextran/magnetite composite nanoparticles coated
with PEI, loaded with pCIKlux DNA (binding capacity approx 0.2 ug
DNA/ug particles). The cells were exposed to magnetic fields as
shown for 24 hr post transfection, using a stack of 3.times.NdFeB 4
mm magnets per well. The cells exposed to moving field were exposed
for 2 hrs at 2 Hz using a 200 .mu.m displacement and then the
magnets left in place for 22 hrs in static position.
[0044] Data shown in FIGS. 2 and 3 as average +/-SEM (n=12 for each
group).
* * * * *