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FIELD OF THE INVENTION
The present invention relates to genetic transformation. In particular, the
present invention relates to the insertion of foreign genetic material
into mammalian hematopoietic and unattached cells.
BACKGROUND OF THE INVENTION
Mammalian blood cells are an attractive target for manipulation by genetic
engineering. Many blood diseases are caused by defects in single genes,
and these diseases could be treated through gene therapy by insertion of a
single correct gene copy in appropriate cells. Examples of single gene
defects are hemophilias, such as Factor IX deficiency and Factor VIII
deficiency, and immuno-deficiencies, such as adenosine deaminase (ADA)
deficiency. Manipulation of blood cells by the addition of a normal or
corrective gene copy would provide a therapeutic strategy for treatment of
these diseases. Blood cells are one of the ideal candidates for delivery
of peptides or proteins systemically since they can secrete these products
into the blood circulation. Genetic manipulation of blood cells in
non-human animals may also be useful by providing experimental animal
models for development of clinical protocols.
Lymphocytes have previously been the subject of genetic manipulation.
Lymphocytes arise from the lymphoid system and comprise 20% of all
leucocytes (white blood cells). During exposure to an antigen, specific
lymphocytes are stimulated by the antigen. Stimulated lymphocytes may
proliferate and produce antibodies to the antigen or may become part of a
cellular immune response. The two major types of lymphocytes are T cells,
which become helper or killer cells and are responsible for cellular
immune response, and B cells, which produce antibodies.
In one prior example of genetic manipulation of lymphocytes, tumor
infiltrating lymphocytes (TIL) have been isolated from melanoma tumors,
infected with a retrovirus vector, and returned to patients. Rosenberg, et
al., N. Eng. J. Med. 323: 570-578 (1990). The TILS were infected with the
retrovirus simply to mark them so that their fate in the patient could be
monitored. The study determined that the infused TILS persisted in the
patient and produced no adverse effect. Recently, genetically transformed
T and B cells have been proposed as a treatment for ADA deficiency. The T
and B cells from ADA deficient patients would be infected with a
retrovirus vector encoding an ADA gene, and these infected cells returned
to the patient. Canto, et al., Proc. Natl. Acad. Sci. USA 83:6563-6567
(1986).
Bone marrow cells are another attractive target for genetic manipulation.
Hematopoietic stem cells found in the bone marrow produce all the cells
present in blood--lymphocytes, erythrocytes, platelets, granulocytes,
macrophages and monocytes. Mitotic division of the stem cells produces two
daughter cells, which either return to the stem cell pool or differentiate
into a specific type of blood cell. Differentiation of stem cells involves
consecutive cell differentiation and ends with the creation of various
defined blood cell populations which live for up to a few months and then
die.
The hematopoietic system is an attractive target for gene transfer for
several reasons. First, well-developed procedures exist for bone marrow
transplantation. Second, hematopoietic cells develop into many different
kinds of cells, and there are many genetic diseases that affect these
blood cells.
Gene transfer into cells at different stages in the hematopoietic system
will have different results. Transformed differentiated cells will express
the gene transiently in a certain type of cell for a limited time--until
the cell dies. Transformation of a stem cell can result in a continued
stable expression of the gene, in all of the cells derived from that stem
cell, for the life of the animal.
Several research groups have demonstrated gene transfer into hematopoietic
stem cells of mice by procedures different than those of the present
invention. A. D. Miller, Blood 76[2]: 271-278 (1990), describes a typical
stem cell experiment. Donor animals were first treated with 5-fluorouracil
to kill differentiated blood cells. This treatment was intended to induce
the mitotic division of stem cells. Retrovirus vectors, which are
effective for transformation only in dividing cells, were then exposed to
the cells. The putatively transformed bone marrow was then injected into
recipient animals. Recently, several groups have shown long-term
expression of both the human beta-globin and the ADA gene in mice using
the retrovirus procedure.
Miller (above, at 273) details some of the current problems in bone marrow
genetic transformation. One particular problem is that "much of the
repopulating ability of marrow is lost during the infection procedure."
Miller points out that in applications where donors are limited, such as
in humans, such losses may be a considerable practical obstacle to gene
therapy.
What is needed in the art of gene transfer is an effective method of
transforming unattached cells such as blood and hematopoietic cells.
Previously, the vast majority of efforts directed at transformation of
unattached cells have used retrovirus transformation vectors or
electroporation. The apparatus used for the transformation technique of
the present invention is based on a quite different method of transporting
the foreign DNA into the genome of the target cells. As disclosed by Klein
et al., Nature, 327: 70-73 (1987), an instrument for the acceleration of
very small particles of metal, coated with DNA, is effective in causing
transient expression in plant cells in vivo. The transforming DNA is
coated onto very small particles which are shot as ballistic projectiles
into the tissues to be transformed. While the apparatus described by
Klein, et al. has been demonstrated to have utility in transforming plant
cells in culture, this particular apparatus has the disadvantage that the
force of particle impact is not readily adjustable. Thus, it is a
difficult apparatus to use for transformation of different cells and
organisms, because a wide range of kinetic energies of particle propulsion
are not available. Yang, et al. (Proc. Natl. Acad. Sci. 87: 9568-9572
(December, 1990)) disclose a method of transforming solid tissue mammalian
somatic cells in situ via particle bombardment. Yang, et al. employed a
particle acceleration device with an adjustable voltage and transformed
cell cultures and liver, skin and muscle tissues. A similar device is
illustrated as effective in germ line transformation of plants in U.S.
Pat. No. 5,015,580.
SUMMARY OF THE INVENTION
The present invention is a method of transforming unattached mammalian
cells via particle bombardment. Cells are first isolated, suspended in
liquid, and placed on a target surface. The amount of moisture on the
target surface must be controlled, either by spreading the cell suspension
into a thin film or by placing the cell suspension onto a porous surface.
Particles are coated with copies of a nucleic acid construct, and the
coated particles are accelerated into the supported unattached cells. The
treated cells are then assayed for the presence or expression of the
nucleic acid.
It is an object of the present invention to create transformed unattached
cells.
It is another object of the present invention to create transformed
lymphocytes capable of infusion or transplantation.
It is another object of the present invention to create transformed bone
marrow cells capable of infusion.
An advantage of the present invention is that unattached cells are
transformed easily and quickly. Because the method of the present
invention is flexible and adaptable, the method is applicable to a wide
variety of cells.
Another advantage of the present invention is that unattached cells are
transformed in such a manner that they are still viable and proliferative.
Another advantage of the present invention is that the nucleic acid
construct is delivered to the target cell by purely physical means.
Other objects, advantages and features will become apparent from the
following specification, drawings and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an exploded schematic view of the preferred embodiment of a
particle accelerator constructed to perform the method of the present
invention.
FIG. 2 is a horizontal cross sectional view of the particle accelerator of
FIG. 1.
FIG. 3 is a diagram of plasmid pWRG1601.
FIG. 4 is a diagram of plasmid CMV-LUX.
FIG. 5 is a diagram of plasmid CMV-Bgal.
FIG. 6 is a diagram of plasmid pWRG1602.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed toward the transformation of unattached
mammalian cells. By "unattached cells," it is meant cells that function
independently in mammalian bodies and are not inherently structurally
connected to other cells or a cellular matrix. Cells found in blood, such
as lymphocytes and erythrocytes, and blood cell progenitors, such as bone
marrow cells, are unattached cells as the term is used herein. Unattached
cells useful in the present invention may be maintained as long-term
primary cultures or as cell lines in culture. Cells may be isolated from
these cultures and used in the method of the present invention. Unattached
cells may also be directly isolated from a mammalian body.
By "transformation," we mean incorporation of a nucleic acid construct into
a cell. The incorporation may be permanent or transient. The term
"transformation" is used here only in the sense of genetic transformation
through the insertion of a foreign nucleic acid construct and is not
intended to describe the process of onset of malignancy in a cell which is
also sometimes referred to as a transformation.
In brief, the method of the present invention involves first preparing
copies of a nucleic acid construct and coating these construct copies onto
biologically inert carrier particles. Mammalian unattached cells are
isolated, suspended in liquid medium, and placed on a target surface. It
is essential to the success of the present invention that the amount of
moisture on the target surface is controlled. In one embodiment of the
present invention, this is achieved by placing the liquid cell suspension
on a porous surface. In another embodiment, the liquid cell suspension is
spread out in a thin layer. These cells are "bombarded" with the
construct-coated particles. The bombardment consists of physically
accelerating the construct-coated particles into the cells on the target
surface with an appropriate amount of force so that the particles lodge in
the interior of at least some of the cells. As a final step, the
expression of the construct or the existence of the construct in the cells
is verified.
The invention is directed toward the introduction of exogenous, often
chimeric, nucleic acid constructs into unattached cells. Such exogenous
nucleic acid constructs consist of DNA or RNA from another organism,
whether of the same or a different species. By the term "nucleic acid
construct" we mean to include populations of RNA or DNA, as well as
isolated and manipulated fragments.
The exogenous DNA construct would normally include a coding sequence for a
transcription product or a protein of interest together with flanking
regulatory sequences effective to cause the expression of the protein or
the transcription product in the transformed cells of an organism.
Examples of flanking regulatory sequences are a promoter sequence
sufficient to initiate transcription and a terminator sequence sufficient
to terminate the gene product whether by termination of transcription or
translation. Suitable transcriptional or translational enhancers can be
included in the exogenous gene construct to further assist the efficiency
of the overall transformation process and expression of the encoded
protein.
Gene products other than proteins may be expressed by the inserted nucleic
acid construct. For example, the inserted construct could express a
negative-strand RNA effective either to suppress the expression of a
native gene or to inhibit a disease pathology. The construct could be RNA,
as an alternative to DNA, if only transient expression of a gene product
is desired.
The present invention makes particular use of an apparatus for using an
adjustable electric discharge to physically accelerate nucleic acid coated
onto small particles into the genetic material of unattached cells. A
suitable apparatus for use within the present invention is illustrated in
FIGS. 1 and 2. The apparatus consists of a spark discharge chamber 12 into
which are inserted two electrodes 14 which are spaced apart by a distance
of approximately 1-2 mm. The spark discharge chamber is a horizontally
extended rectangle having two openings 16 and 18 out its upward end. One
opening 16 is covered by an access plate 20. The other opening, located
opposite from the electrodes 14 is intended to be covered by a carrier
sheet 22.
The electrodes 14 are connected to a suitable adjustable source of electric
discharge voltage. Such a source of electric discharge voltage would
preferably include suitable electric switching connected to a capacitor of
the 1 to 2 microfarad size range. Preferably, the voltage of the charge
introduced into the capacitor is adjustable, such as through the use of an
autotransformer, through a range of 1 to 50,000 volts. Suitable switching
is provided so that the capacitor can be discharged through the electrodes
14 safely and conveniently by a user.
The carrier sheet 22 intended to be placed upon the opening 18 on the spark
discharge chamber 12 is preferably a sheet of aluminized Saran-coated
mylar. The carrier sheet 22 should be planar and relatively light. A
retaining screen 24 is placed approximately 5-10 millimeters above the
opening in the discharge chamber. A target surface 26 is placed
approximately 5-25 millimeters above the retaining screen 24.
The nucleic acid construct intended to be transformed into the unattached
cells is prepared by suitable DNA preparation techniques well known to one
of ordinary skill in the art. The construct is coated onto small particles
of a durable, dense, biologically inert material such as gold, the
particles typically being 0.2 to 3 microns in size. One source of
particularly suitable gold particles is Elicit Industries, Inc. (New York,
N.Y.). Preferably, particles are 0.8-1.2 microns.
A PEG (polyethylene glycol) precipitation method is one method used to coat
the DNA onto the particles, although other methods would also be suitable.
In one tube, 10 mg gold particles, 25 mg DNA and H.sub.2 O are mixed to a
total volume of 100 .mu.l. 100 .mu.l of 50% PEG 4000 in H.sub.2 O, 100
.mu.l 1M CaCl.sub.2 and 200 .mu.l H.sub.2 O are mixed in a second tube.
The contents of the first tube are added to the second tube with rapid
mixing. The nucleic acid-coated gold particles settle out, and the
supernatant is removed.
Alternatively, particles of microcrystalline gold are placed into a tared
microfuge tube and weighed. Typically, approximately 10 mg of gold
particles are coated at a time. Five volumes (.mu.l/mg) of 0.1M spermadine
are added to the gold. Plasmid DNA is added to the mixture to give 0.5-25
.mu.g of DNA per mg of gold particles. The concentration of the plasmid
DNA solution is sufficient that the volume of DNA solution added is less
than or equal to 0.4 of the volume of spermadine used. The spearmint, DNA
and gold are mixed and allowed to incubate at room temperature for 10-15
minutes. Five volumes (.mu.l/mg based on the initial mass of gold used) of
2.5M CaCl.sub.2 is added dropwise with constant mixing. The mixture is
incubated for 3 minutes at room temperature, then centrifuged for 10-15
seconds to collect the coated particles. Following centrifugation, the
supernatant is removed and discarded.
After the particles are coated, they are washed with 100% ethanol and
resuspended to the desired "particle loading rate" in 100% ethanol.
"Particle loading rate" describes the amount of coated carrier particles
placed on the carrier sheet 22. Preferable particle loading rates are 0.05
to 0.5 mg coated particles per cm.sup.2 carrier sheet. The coated carrier
particles are then placed upon the carrier sheet 22 which is inserted on
top of the spark discharge chamber 12. The coated particles are placed on
the planar carrier sheet so as to have an even horizontal distribution.
This even distribution is important for successful transformation of large
numbers of small cells in a statistically significant way. Preferably, an
ethanol suspension of coated particles is pipette onto the carrier sheet
22. The coated particles are allowed to settle and most of the ethanol is
drained. The residual ethanol is allowed to evaporate, thus leaving a
thin, even layer of coated particles.
Unattached mammalian cells are placed on the target surface 26. Suitable
cells may come from a cell culture line, or be directly isolated from a
mammalian body, as illustrated by the examples below. These cells must be
in a liquid suspension. In the examples below, the cells are suspended in
culture medium.
It is pivotal to the success of the method of the present invention that
the amount of moisture on the target surface 26 be controlled. We have
found that efficient transformation is achieved only when the cell/liquid
ratio is within certain parameters. In our Examples, we have demonstrated
two ways to efficiently control this moisture. One method involves plating
cells on a porous surface. Examples of suitable porous surfaces are filter
paper (Whatman No. 1, Whatman Paper, Ltd.) and polycarbonate membranes
(2-5 .mu.m pore size, Poretics Corp., Livermore, Calif.) Other porous
materials may be suitable, but the material must be compatible with cell
viability. By "porous" we mean material that will allow excess media to
drain away from the target cells, but will retain the cells and allow
enough media to remain on the surface of the material to keep the target
cells moist and viable. Another method of controlling moisture involves
placing an aliquot of the cell suspension (approximately 10-25 .mu.l,
containing 1-5 million unattached cells) on the target surface 26 and
spreading this liquid into a thin film such that the cell/liquid ratio is
suitable for efficient particle bombardment.
A small droplet of water, approximately 2-4 microliters in volume, is
placed bridging between the ends of the electrodes 14. The access plate
cover 20 is then placed over the top of the discharge chamber 12.
Preferably, the atmosphere between the carrier sheet 22 and the target 26
is largely replaced with helium by enclosing the apparatus and target and
introducing helium in the enclosure in sufficient quantity to largely
displace the atmospheric gases.
Since the carrier sheet 22 is light and rapidly accelerated, it is very
flexible. Accordingly, the method propelling the carrier sheet 22 becomes
important because a single point force would distort the sheet and not
achieve the desired result of a uniform layer of particles travelling into
the cells on the target surface 26. A gaseous shock wave is the means
employed to impact the carrier sheet 22 and lift it in a planar movement
in which the carrier sheet 22 travels across the distance to the retaining
screen 24 without losing its shape. The apparatus of FIGS. 1 and 2
achieves this effect through the use of an electric spark discharge.
However, there are other means to achieve a similar gaseous shock wave.
At this point a spark discharge between the electrodes 14 is initiated by
the use of appropriate electronic switching. The force of the electric
discharge bridges the spark discharge gap between the electrodes 14 and
instantly vaporizes the small droplet of water placed between the
electrodes. The force of the vaporization of that water creates a shock
wave within the spark discharge chamber 12 which radiates outward in all
directions. The shock wave propels the carrier sheet 22 upwards with great
velocity until the carrier sheet 22 contacts the retaining screen 24. The
presence of the helium provides less drag on the flight of the carrier
sheet and carrier particles as well as less viscous medium to lessen
propagation of the shock wave to the target 26.
The carrier sheet 22 is retained at the retaining screen 24, and the coated
particles fly off of the carrier sheet and travel freely on toward the
target surface. The particles proceed into the target surface and enter
the cells. The momentum of the particles as they impact the surface of the
target cells is adjustable based on the voltage of the initial electric
discharge applied to the electrodes 14. By variations in the amount of the
electric energy discharged through the electrodes 14 the velocity by which
the particles impact the target can be adjusted. Thus, the depth of
penetration of the particles into the cells of a target can be
continuously adjusted over the range of adjustment of the electric
discharge. Penetration of the particles can also be adjusted by altering
the particle size (larger particles usually penetrate further) and shape
(spear-shaped particles penetrate further than spherical particles).
After bombardment with the nucleic acid-coated particles, the cells are
cultured in appropriate media. The cells are assayed to verify the
presence and/or expression of the nucleic acid construct. Suitable assays
are disclosed in the examples below. Easily assayed genes, such as
disclosed in the examples, may be coupled in tandem with a gene of
therapeutic interest. Alternatively, a selection agent, such as an
antibiotic resistance gene, could be transformed in tandem with a gene of
therapeutic interest. Upon culture with the antibiotic, resistant cells
would continue to grow while nonresistant cells would die. Ultimately,
these transformed blood cells and blood-cell-progenitor cells would be
infused into a patient by procedures known in the art.
Although the unattached cells of some of the examples below seem to be only
transiently transformed, the method of the present invention can also
result in stable transformation of these cells as shown in another example
below. Yang, et al., Proc. Natl. Acad. Sci., USA 87: 9568 (1990)
demonstrated that two cell lines were stably transformed at a rate of
2.times.10.sup.-3 -6.times.10.sup.-4 when subjected to particle
bombardment. Additionally, when a variety of plant and bacterial cells
were subjected to particle-mediated transformation methods, 0.1%-5% of the
transiently expressing transformants proved to be stable transformants.
Detection of these stable transformants required screening a large number
of transient transformants. Similarly, stable transformation of unattached
cells have been detected when statistically large samples of transformed
cells are examined. Populations of cells, some of which are stably
transformed, can be selected so that the transformed cells can be
selectively propagated. This process can lead to a stable population or
culture of stably expressing cells which can be returned to the host
mammal's body for therapeutic value. Thus, cells can be removed,
transformed with a gene for a therapeutic protein (and a selectable
marker), selected and proliferated, and then returned to the body to
deliver the therapeutic protein.
EXAMPLES
A. TRANSFORMATION OF LYMPHOCYTES
1. Preparation of Lymphocytes
B-cells and T-cells are the two major classes of lymphocytes. In order to
investigate the applicability of the present invention to all lymphocytes,
we chose to transform cells representative of each type. We chose mouse
CTTL-2 cells (ATCC TIB214), which are cytotoxic T-lymphocyte cells and
human WIL2-NS cells (ATCC LRL8155), which are B-lymphoblast cells.
(Lymphoblasts are immature lymphocytes.) These cells were obtained from
ATCC (American Type Culture Collection) and grown in culture medium
suggested by the supplier in "Catalogue of Cell Lines and Hybridomas," 6th
Ed., 1988, ATCC.
2. Preparation of Nucleic Acid Constructs
The lymphocyte cells were transformed with plasmid pWRG1601. FIG. 3 is a
diagram of pWRG1601. Transformation with pWRG1601 leads to expression and
secretion of human growth hormone in a wide range of mammalian cell types.
Plasmid pWRG1601 includes a chimeric gene comprising the human
cytomegalovirus (CMV) immediate-early promoter (M. Poser et al. (1985)
Cell 41: 521-530) and the transcribed region and downstream flanking
region from the human growth hormone (HUGH) gene (R. F. Shelden et al.
(1986) Mol. Cell. Biol. 6: 3173-3179). In addition to the CMV-HuGH gene,
pWRG1601 contains regions from Epstein-Barr virus (EBV) that include an
origin of replication (ORI P) and a chimeric nuclear antigen 1 (EBNA1)
gene, and the bacterial plasmid vector pGEM3 (Promega). The EBV regions
provide functions sufficient for autonomous plasmid replication in human
and some other mammalian cells, but not in mouse cells. Thus, these
regions are nonessential to the present experiments. The pGEM3 regions
provide for replication and selection of the plasmid in E. coli.
Plasmids were propagated in standard E. coli host strains, and plasmid DNA
was isolated via conventional methods. Plasmid DNA was coated onto
microcrystalline gold particles of 0.8-1.2 microns as described above. The
gold particles were coated at 0.5-25 .mu.g of DNA per mg gold particles.
3. Target Preparation
An aliquot of cells in culture medium was counted on a hemocytometer. The
cells were then harvested from culture by centrifugation (300.times.g for
7 minutes) and resuspended in fresh culture medium. An aliquot containing
10.sup.6 -10.sup.7 cells was then pipetted onto either a sterile filter
paper (Whatmann No. 1) or polycarbonate membrane overlying a sterile
filter paper that was prewet with culture medium. The excess culture
medium was wicked away by the filter paper.
The target was then bombarded at 10 kV with 163 .mu.g of gold particles
coated with pWRG1601. Immediately after blasting, culture medium was added
to the target. The cells and medium were then removed and cultured under
standard suspension culture conditions as described in the ATCC catalogue
(above).
4. Production and Secretion of HuGH by Transformed Lymphocytes
Culture medium from bombarded cells and nonbombarded cells (controls) was
analyzed for human growth hormone using a commercial immunoassay kit
(Nichols Institute Diagnostics, San Juan Capistrano, Calif.) that
specifically detects human growth hormone. At three and seven days after
bombardment, lymphocytes were harvested as described above and resuspended
in fresh medium. The spent medium was then assayed for HUGH. The medium
from nonbombarded cells was used as a control sample to establish
background levels for the growth hormone assays.
In all cases, a background level was determined and was essentially the
same as the level of samples in which HuGH was not present. Expression and
secretion of human growth hormone was detected in both the B-lymphoblasts
and the T-lymphocytes that had been subjected to bombardment with
particles coated with pWRG1601. By "expression and secretion" we mean that
greater than 5 ng of HuGH per 10.sup.6 cells per day was measured.
Transformed B-lymphoblast cultures that expressed and secreted human
growth hormone during the culture period were identified for subsequent
infusion into mice.
5. Infusion of Mice with Transformed Cells
These experiments have been successfully performed with B-lymphoblasts.
Eight days after bombardment, three groups of cells--one control group
(nonbombarded WIL2-NS cells) and two groups of WIL2-NS cells transformed
with pWRG1601--were counted and divided into aliquots containing
approximately 3.times.10.sup.6 cells each. The two groups of bombarded
cells were called UF10B and F12A.
The cells were collected from the culture medium by centrifugation at
300.times.g for 7 minutes. The supernatant medium was removed by
aspiration and retained for human growth hormone assay. The cells were
resuspended in fresh medium at approximately 10.sup.7 -10.sup.8 cells/ml.
The growth hormone assays indicate the transformed lymphoblasts were
secreting human growth hormone at rates of 10-20 ng/10.sup.6 cells/24 hrs.
In all cases control cultures showed no production of human growth
hormone.
The lymphoblast suspensions (both control groups and transformed groups)
were injected into BALB/c mice intravenously, intraperitoneally or
subcutaneously. 50 microliters were injected intravenously in the tail
vein, 100 microliters were injected intraperitoneally, and 100 microliters
were injected subcutaneously and intradermally. Blood samples were
collected from the mice two hours after injection of the lymphoblasts. The
mice were sacrificed 24 hours after injection, and a final blood sample
collected. Analysis of human growth hormone levels in the blood serum gave
the results shown below.
TABLE 1
______________________________________
Route of Time post- Serum HuGH
Injection Sample injection (ng/ml).sup.1
______________________________________
subQ UF10B 2 hr 88.6 .+-. 76*.sup.2
subQ F12A 2 hr 0.1 .+-. 0.4*
IP UF10B 2 hr 5.0 .+-. 0.2
IP F12A 2 hr 4.8 .+-. 0.6
IV UF10B 2 hr 5.1 .+-. 3.6
subQ UF10B 24 hr <0.1 .+-. 0.05*
IP UF10B 24 hr 0*
IP F12A 24 hr <0.2 .+-. 0.02*
IV UF10B 24 hr <0.1 .+-. 0.01*
______________________________________
*These values are not significant.
.sup.1 Average value of two assays .+-. standard deviation.
.sup.2 One of the assays indicated a high HuGH level while the other did
not, resulting in a high standard deviation.
The results show that both intravenous and intraperitoneal injection of
transfected lymphoblasts expressing the human growth hormone gene resulted
in transient appearance of human growth hormone in circulating blood. One
assay from subcutaneous injection gave a significant HuGH reading, but the
duplicate assay indicated essentially background levels.
B. TRANSFORMATION OF BONE MARROW CELLS
1. In General
The general protocol of our bone marrow transformation was as follows: Bone
marrow cells were flushed from the tibias and femurs of a male Holtzman's
rat (approximately 5 weeks old). Bone marrow cell isolation is described
in Mishell and Shiigi, Selected Methods in Cellular Immunology, p. 11,
publ. Freeman and Co., N.Y., 1980. In brief, the rat is first killed and
dipped in alcohol. The tibia and femur were separated from the skin and
muscle and transferred to a buffer-containing culture dish. The bones were
punctured at both ends with a needle and the marrow was expelled by
pushing buffer through the bone. The marrow is drawn in and out of the
needle to obtain a single-cell suspension.
The bone marrow cells were purified away from red blood cells by
centrifugation on Ficoll-Hypaque. Mishell and Shiigi (above, page 205),
describe this method. In brief, the method depends on cells of a certain
density passing through dense medium and forming a pellet during
centrifugation. The pellet contains red blood cells, dead cells, and cell
debris. The interface and the Ficoll-Hypaque contain the other cells. The
method is as follows: 12 parts of a 14% Ficoll solution are mixed with 5
parts of a 32.8% Hypaque solution and sterilized. The mixture should have
a density of 1.09 grams per cm.sup.3. The chilled cell solution is
prewarmed to 20.degree. C. and layered on top of a 4 ml Ficoll-Hypaque
solution in a centrifuge tube. The tubes are placed in a prewarmed
centrifuge and centrifuged at 2000.times.g for 20 minutes. The partially
purified bone marrow cells were purified away from the Ficoll-Hypaque by
rinsing three times in Hank's basal medium and resuspended in a small
volume of culture medium.
Alternatively, red blood cells were partially removed by lysis via
hypotonic shock in Example B-1. Red blood cell lysis is described in
Mishell and Shiigi (above, p. 22). This procedure is as follows: 0.1 ml of
packed cells are diluted with 0.1 ml of diluent (Hank's basal medium) and
the cell pellet is resuspended. 1-2 ml of 1/10.times.diluent is added and
mixed into the cells. Cells are exposed for 15 seconds of hypotonic shock,
and full strength (1.times.) diluent is added and mixed. The cell mixture
is centrifuged at 200.times.g for 10 minutes.
The bone marrow cells, purified by either method, were spread in an
18.times.18 mm pattern on the surface of a 35 mm cell culture dish. This
spreading is done by pipetting 10 .mu.l-25 .mu.l of cell suspension onto
the center of a 35 mm petri dish and spreading the liquid into a
18.times.18 mm square with a disposable cell scraper (Baxter McGaw Park,
Ill.). We preferably placed 5.times.10.sup.6 cells in 10 .mu.l of medium
on each target. We have used 1.times.10.sup.6 -5.times.10.sup.7 cells in
volumes of 5-50 .mu.l per target with 10 .mu.l being the optimum volume.
To coat the particles, plasmid DNA was prepared via standard methods. We
chose to bombard the bone marrow cells with the plasmids pCMV-LUX (FIG. 4)
and pCMV-Bgal (FIG. 5). The pCMV-LUX plasmid encodes firefly luciferase;
the pCMV-Bgal plasmid encodes E. coli B-galactosidase.
The cells were bombarded with DNA-coated particles at a voltage of 5-19 kV.
Example B-4 demonstrated that the optimum bombardment voltage was 6 kV.
After bombardment, 1-2 ml of culture medium was added and the bone marrow
cells were cultured overnight by incubation in a 5% CO.sub.2 : 95% air
atmosphere at 37.degree. C. The next day, the cells were assayed for the
expression of reporter genes, luciferase and B-galactosidase.
The assay for luciferase was performed as described in deWet, et al., Mol.
Cell. Biol. 7: 725-737 (1987). In general, the assay measures the
oxidation of luciferin, catalyzed by luciferase. The oxidized luciferin
emits a photon that we measured using a luminometer (A.L.L. Monolight
2001). The rate of photon production, hence light intensity, provides a
measure of luciferase concentration. Extraction buffer (100 mM KPO.sub.4,
pH 7.5, 100 .mu.g/ml bovine serum albumin, 0.62 mg/ml leupeptin, 2.5 mM
phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 mM dithiothreitol) was
added to the cells at equal weight:volume. Extraction was performed by
triton lysis and sonication. The samples were spun in a refrigerated
centrifuge to remove cell debris and the cell-free extract was assayed
immediately. In a reaction cuvette, 80 .mu.l of 5.times.reaction buffer
was mixed with distilled water, and 1-50 .mu.l samples of the cell free
extracts were added (total volume should equal 400 .mu.l). The mixture was
vortexed and 100 .mu.l of 0.5 mM luciferin was added. The amount of
luciferase activity was expressed as relative light units (RLU) per
bombarded target. 5.times.reaction buffer is 70 mM glycylglycine, 70 mM
magnesium chloride and 50 mg/ml bovine serum albumin, pH adjusted to 7.8.
The assay for B-galactosidase depends on the conversion of
4-methyl-umbelliferyl-beta-D-galactoside (MUG), a non-fluorescent
galactoside, to D-galactose and the highly fluorescent
methylumbelliferone. The fluorescent product was measured using a
fluorometer (excitation set at 350 nm and fluorescence emission read at
450 nm). This assay is extremely sensitive and only a few thousand cells
are required for accurate determination of B-galactosidase activity.
The B-galactosidase assay is described in detail in McGregor, et al. Som.
Cell Molec. Genet., 13; 253-265 (1987). In brief, cells were resuspended
in Z-buffer (60 mM Na.sub.2 PO.sub.4.7H.sub.2 O; 40 mM NaH.sub.2
PO.sub.4.H.sub.2 O; 10 mM KCl; 1 mM MgSO.sub.4.7 H.sub.2 O, pH to 7.0 with
NaOH or HCl.). Up to 105 .mu.l of the cell suspension was deposited on the
well of a microtiter dish. 15 .mu.l of 1% Triton X-100 was added to each
well. The sample was incubated for 5-10 minutes to solublize the cells. 30
.mu.l of 3 mM MUG was added to each well. A stop solution (300 mM glycine,
50 mM EDTA, pH 11.2) was added to each well after 90 minutes. The
resulting solution was placed in a fluorometer and the fluorescence
intensity was measured.
2. Transformation of Bone Marrow: Luciferase
Bone marrow cells from two rats were harvested as in our standard protocol
and pooled. In this Example, we compared two ways of partially purifying
the bone marrow. We wanted to remove the red blood cells, but wanted to
leave the marrow cell population as crude as possible beyond that. Most
workers use Ficoll-Hypaque (F/H) purified cells. However, some bone marrow
cells are lost to the pellet during centrifugation in this method. We
tried lysis of red blood cells by hypotonic shock as an alternative.
Hypotonic shock was performed as described above.
The purified bone marrow cells were concentrated by centrifugation
(300.times.g, 5 minutes) resuspended in a small volume of medium, and
spread onto target surfaces as described above. The "hypotonic shocked"
cells were concentrated to 6.times.10.sup.6 cells/10 .mu.l. The
ficoll/hypaque-purified cells were concentrated to 2.6.times.10.sup.6
cells/10 .mu.l. 10 .mu.l of each sample was spread on the target surface.
The cells were then bombarded at 10 kV with pCMV-LUX and culture medium
was immediately added. The results of this experiment, Table 2, indicated
similar transformation frequency for either method of partial purification
of the bone marrow cells.
TABLE 2
______________________________________
Particle
Loading
Sample #
Rate Isolation Results
______________________________________
5 0.2 mg/cm.sup.2
F/H 12884 RLU/target
6 0.2 mg/cm.sup.2
Hypotonic shock
46232 RLU/target
7 0.4 mg/cm.sup.2
F/H 21632 RLU/target
8 0.4 mg/cm.sup.2
Hypotonic shock
40790 RLU/target
______________________________________
3. Transformation of Bone Marrow
In this Example, we tested different cell concentrations on the target
surface to find which concentration gave the highest transient activity
without wasting cells. The cells were bombarded at 10 kV, 0.3 mg/cm.sup.2
particle loading rate on the carrier sheet, using pCMV-Bgal. We plated 10
.mu.l of cell suspension onto the target surface. Table 3 tabulates the
results, which show that a certain cell density is required for optimal
gene transfer.
TABLE 3
______________________________________
Relative MUG Activity/
Sample # Cells per target
10.sup.6 cells
______________________________________
1 2.5 .times. 10.sup.6
1
2,3 5 .times. 10.sup.6
4.5
4,5 7.5 .times. 10.sup.2
4.75
6,7 1 .times. 10.sup.7
6.75
8,9 2.5 .times. 10.sup.7
3.5
______________________________________
The values obtained with the MUG fluorometric assay have been normalized to
the 1.times.10.sub.6 cell level. The results indicate that
l.times.10.sup.7 cells per 10 .mu.l per target was the most efficient use
of cells.
4. Transformation of Bone Marrow: Agglutinin Treatment
Cells in this Example were extracted as per our protocol. We used a soybean
agglutinin (SBA) treatment to fractionate the bone marrow cells into two
populations based on ability of the cells to be agglutinated by SBA, a
lectin that binds specific residues on the surface of more mature lymphoid
cells. Undifferentiated bone marrow stem cells should not bind SBA.
Soybean agglutinin treatment is as described in Mishell and Shiigi (above,
page 226). In brief, cells are mixed with an equal volume of
SBA-containing solution and incubated for 5-10 minutes at room
temperature. The cells are layered on top of 40 ml of buffer containing 2%
bovine serum albumin and incubated at room temperature for 15-30 minutes
allowing separation of agglutinated and non-agglutinated cells. The top
and bottom layers of cells were removed separately and transferred to
centrifuge tubes with Pasteur pipettes. The bottom layer of cells was
suspended in 0.2M galactose and incubated at room temperature. The cells
were pelleted and washed twice with the galactose solution and once with
buffer before using.
Before bombarding, the cells in the SBA+ #2 sample were placed on a 5 .mu.m
polycarbonate membrane placed in a petri dish.
Results shown in Table 4, indicate luciferase expression in both samples.
TABLE 4
______________________________________
Sample Support Results
______________________________________
SBA+ #2 5 .mu.M polycarbonate
91817 RLU/target
SBA+ #4 plastic culture dish
88610 RLU/target
______________________________________
5. Transformation of Bone Marrow: Power Study
This Example was a calibration of the kV charge used to bombard the cells.
We found that 6 kV is the optimum kV for the bone marrow cells using the
other parameters we had developed (1.times.10.sup.7 cells/10 .mu.l/target,
Particle loading rate=0.1 mg/cm.sup.2). Table 5 shows these results.
TABLE 5
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