Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND DEVICE FOR MICROINJECTION OF MACROMOLECULES INTO NON-ADHERENT CELLS
This application claims the benefit of U.S) Provisional Application No.:
60/033,820,
filed December 20, 1996.
The present invention relates to the field of molecular transfer of materials
into
living cells, and to improved methods for accomplishing same, particularly in
the
incorporation of molecular materials into cells that can be made to exist in
an adherent state
in vitro. The invention further relates to the field of gene therapy, as a
technique for
introducing genetic material into a cell is provided. The invention also
relates to the field
of mechanical apparatus for micro-injecting molecular materials into living
cells, devices
for introducing materials into a cell with improved cell viability and
retention/expression
of incorporated materials therein, also being provided in the present
disclosure.
BACKGROI1ND ART
Microinjection of macromolecules (e.g., antibodies, mRNA, DNA) into living
cells
has proven to be a powerful approach for studying the biology of cells at the
molecular
level. Manual microinjection methods have been developed independently by
Diacumakos
and Graessmann [Diacumakos, E. ( 1973); Graessmann, A. ( 1970)], twc~r
pioneers of
microinjection technology. The methods Employ a micromanipulator that is used
in
directing a glass microinjection needle into a living cell. The microinjection
needle is
connected to a syringe assembly that forces the sample out of the needle. The
flow of the
sample solution into the cell is typically visually monitored employing a
phase-contrast
microscope. In this manner, a change in phase contrast indicates that the
sample has been
injected into the cell. These methods require extensive training of the
personnel who
perform the injections; this training period lasts for months before
acceptable consistent
injections can be accomplished. These manual microinjection approaches are
time-
consuming, permitting maximal injection rates in the range of 300-500 cells
per hour.
Semi-automated (Eppendorf) and automated (Zeiss) microinjection systems have
also been described that include an electronic: interface between the
micromanipulator and
the injection system. Such systems require less technician training and skill,
and are thus
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preferred to purely manual microinjection apF~roaches. In addition, they
enable more rapid
injection of cells (> 1,000 cells per hour).
Microinjection has been described for the incorporation of genetic material
into
adherent cells with some degree of success 1;85-95% short-term expressing,
viable cells,
up to 30% stably expressing cells). Manual microinjection of larger adherent
cell types
(e.g., fibroblasts) has been reported to yield satisfactory survival (85-
100%).
Microinjection technology has even been successfully applied to injecting
macromolecules into the pronuclei of non-adherent egg cells (100 microns
diameter) as
part of the protocol used in producing transgenic animals. Such technology has
also been
used to inject sperm into eggs when performing the intracytoplasmic sperm
injection
(ICSI) procedure when treating human infertility. However, both the holding
pipettes ( 10
microns) and microinjection needles (1-3 microns) used for these injections
are much too
large to be useful for injecting smaller celils (eg. hematopoietic cells which
are ~5-10
micron diameter).
Small, normally non-adherent cells (e.g., primary hematopoietic cells) manual
microinjection has generally yielded only a small percentage of surviving
micro-injected
cells. For example, in Graessmann, et al. (PNAS 77:433-436, 1980), although
the Raji
human B cell line could be anchored to tissue culture plates coated with anti-
lymphocyte
IgG, phytohemagglutinin (PHA), or Concanavalin A (Con A), this cell line, due
to its small
size and fragility, could not be efficiently micro-injected with DNA.
Microinjection was
only possible when larger polykaryons were formed by cell fusion.
Diacumakos et al. (Exp. Cell Res. 1981, ~, 73-77) relate to a method for
attaching mouse erythroleukemia (MEL) cells to glass coverslips. This is
accomplished
by first coating cover slips with collagen, and then subsequently coating the
cover-slip with
concanavalin A and 1-cyclohexyl-3-(2-morpholinoethyl) carbo-diimide p-toluene-
sulfonate
methyl ester. Using this approach, the microinjection of inducing chemicals
into MEL
cells was reported. Since Con A can affect cells in a variety of ways [eg. low
doses induce
blast transformation and mitosis in normal lymphocytes; higher doses inhibit
growth of
both lymphocytes and proliferating lymphoid cells (McClain and Edelman, 1976);
plating
of mitotic HeLa cells onto Con A treated coverslips delays entry into S phase
of the cell-
cycle (Brown, et al., 1985)], its influence on stem/progenitor cells must be
evaluated.
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M. Graessmann and A. Graessrnann (in Microinjection and Organelle
Transplantation Techniques, Academic Press Incorporated, London, 1986)
describe a
general microinjection procedure using glass <;apillaries (Methods in
Enzymology; 1 O 1:48).
These glass capillaries are described as having an outer diameter of about 0.5
microns.
Attachment of the Friend leukemia cells, a cell type that typically does not
grow in an
adherent state, is accomplished through use of concanavalin A and a linker
molecule, such
as glutaraldehyde. In this manner, attachment of these cells to a plastic
Petri dish is
reported.
Y. Mori et al. (European Patent Application EP0463508 A1) relates to a method
for inj ecting substances into cells using a temperature-responsive polymeric
compound and
a cell adhesive substance to immobilize the target cell onto a plastic or
glass plate. A
microcapillary pipette having a diameter of :l to 2 microns at its tip is
employed. The cell
adhesive substances included the following molecules: gelatin, lectins, bridge
oligopeptides, adhesive proteins, positively charged polymers, collagen,
fibronectin,
laminin, proteoglycan, glycosaminoglycan and thrombospondin.
There are two primary reasons for the great difficulty in micro-inj ecting
hematopoietic cells, both primary cells [defnnition: cells directly
transferred from an in vivo
setting (e.g., in human, mouse, etc.) to in vitro culture without any
transforming event] and
transformed cells [definition: cells which have an unlimited potential for
further cell
division-the transformation event either occ;urnng in vivo (e.g., the cells
are leukemic) or
in vitro]: ( 1 ) there is significant difficulty in immobilizing hematopoietic
cells in a manner
that does not significantly affect the biological properties of the attached
cells. For
example, although attachment of hematopoietic cells has been described via
lectins such
as PHA, Con A, or pokeweed mitogen, as mentioned above, these agents are well
known
for their mitogenic, or in some cases inhibitory, effects on hematopoietic
cells. Thus, the
immobilized cells are likely biologically modified by the immobilization step.
(2) the
small size of hematopoietic cells, particularly for primary hematopoietic stem
cells (5-7
micron diameter), makes them extremely difficult to inject. Since commercially
available
microinjection needles typically have inner diameters of approximately 0.5
micron- and,
therefore, outer diameters in the range of 0.7-1.0 micron- it is not
unexpected that
hematopoietic cells are adversely affected by injection with such needles.
There have been
some reports of microinjection needles with outer diameter of 0.1-0.3 micron.
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Gene transfer to non-cycling hematopoietic stem cells with acceptable long-
term
expression in progeny cells continues to be a major technical challenge in the
art of gene
therapy approaches. Even though a wide variety of methods including
electroporation,
liposomes, retroviruses, and adeno-associated virus have been described and
explored, such
continue to present limitations that preclude the wide-scale, routine use of
macro- and
micro molecular transfer to living cells. l3oth electroporation and liposome-
mediated
delivery have been hampered by the inability to establish stable maintenance
of the
introduced genes in a significant fraction of cells, while recombinant C-type
retroviral
vectors and adeno-associated vectors are inefficient in both transduction of
non-cycling
cells and maintenance of long-term cell tyI>e specific gene expression.
Some have reported retrovirus-mediated genetic modification of cycling human
progenitor cells and long-term culture initiating cells (LTC-ICs), these
hematopoietic cells
reportedly being capable of sustaining hematopoiesis in vivo for a finite
period (reportedly
about 2 to 6 months). For long-term benefit of the gene therapy, this
technique likely
requires repeated treatment of the patient with modified stem cell
populations, as progeny
cells from modified progenitor cells are relatively quickly replaced with
unmodified
(transduced) progeny of unmodified stem cells.
Clinical trials involving retrovirus-mediated transduction of drug resistance
genes
(e.g., the human mufti-drug resistance-1 gene, MDR-1, which protects cells
from several
chemotherapeutic agents such as Vincristine, Taxol and Doxorubicin) into
marrow or
peripheral blood CD34+ cells have been reported. The CD34+ antigen marks all
currently
assayable human hematopoietic stem and progenitor cells. However, retroviral
(type C)
vector-mediated transduction requires that the cell be cycling in order to
effect
transduction. Quiescent, i.e., non-cycling, hematopoietic stem cells, which
typically
constitute a portion of the population of cells being treated, are not
effectively transduced.
Thus, retroviral mediated gene transfer is a fairly inefficient technique for
transfer of
genetic material into stem cells. Relatively low frequency (0.1-1.0%) of gene
marked
peripheral leukocytes has been reported. using this technique to treat CD34+
cells for
transplantation in human trials. Other studies report that retroviral
transduction protocols
used deplete human stem cell reconstituting activity.
The expression of retrovirus-transduced transgenes is frequently silenced in
the
progeny of transduced human or primate progenitors, and even in progeny of
transduced
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mouse stem/progenitors (Challita and Kohn, 1994; Akkina et al., 1994; Lu, et
al. 1994).
As a consequence, hematopoietic cells which are retrovirally genetically
modified with
drug resistance gene may not efficiently display the drug resistance
phenotype. This
problem, while significant for retrovirus vectors (maximum of 8kb), is even
more acute for
adeno-associated virus (AAV) vectors (maa;imttm of 4kb).
A need continues to exist in the medical arts for improved methods of
introducing
foreign materials into non-adherent cells.) particularly into non-adherent
cells using
microinjection techniques, and the use of these methods in treatment of
physiological
disorders.
Vii, integrins are members of the integrin gene family of cell adhesion
receptors that
recognize particular ligands such as fibrone:ctin, laminin, collagen,
epiligrin, invasin, and
vascular cell adhesion molecule-1 (VCAM-1; Stuiver & O=Toole Stem Cells 13:
250-262,
October 1995). p, integrins mediate cell-cell and cell extracellular matrix
interactions
(Stuiver & O=Toole, 1995). The ~3 subunit of integrins contributes to both
ligand binding
and the transduction of intracellular signals. The ligand binding affinity of
several
integrins from the (3,(a, (3,, as (3,, and ab ~3,), (3z(aLp2 anda,"~i~, and
~i3 subfamily a,~p3)
can be regulated by a variety of stimuli (Re;f.: Schwartz, M.A., Schaller,
M.D., Ginsberg,
M.H. ( 1995) Annu. Rev. Cell Dev. Biol. 11:549-599. (Integrins: Emerging
Paradigms of
Signal Transduction)). In turn, the integrins can also affect different
cellular functions. For
example, monoclonal antibodies directed against the Vii, subunit induce
negative signaling
effects on T cell proliferation (Schwartz et al., 1995).
Various cell types express cell-surface molecules of the integrin family which
are
used in attachment of cells to various substrates including extracellular
matrix molecules
and other cells. Several methods have been reported for activation of cells
expressing
integrins to attach to particular substrates. These methods include incubation
of cells with
anti-integrin antibodies, cytokines, extracellular matrix proteins (e.g.,
flbronectin) and
peptides or fragments thereof, lipids, divalent cations, and phorbol esters.
A variety of primary and transfornned cell lines have reportedly been
immobilized
to surfaces via integrin activating antibodies. The primary cells have
included PHA
stimulated peripheral blood T-cell blasts (Wayner and Kovak, 1992) and resting
peripheral
blood lymphocytes (Arroyo, et al. ). Transformed cell lines that have
reportedly been
immobilized include K562 (human erytlvroleukemic; Kovach et al., 1992), U937
(human
<>
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myelomonocytic; Wayner & Kovak), M07e (Levesque), TF 1 (human erythroleukemic;
Levesque), A375 (melanoma cells; Arroyo et al. ), Jurkat (human T
lymphoblastoid;
Wayner and Kovach, 1992), Ramos (human B lymphoblastoid; Wayner and Kovach,
1992), and ST-1 (human B lymphoblastoid; Wayner and Kovach, 1992).
While some investigators have reponted the ability of anti-integrin antibodies
and
other agents such as peptides to activate a cell to become adherent and to
thus adhere to a
substrate, there has been no application of tlhis technology to microinjection
of cells that
do not grow in an adherent state, such as hematopoietic cells. This technical
difficulty,
among others, has precluded the use of microinjection of important cell types
that do not
grow in an adherent state, most notably hem~atopoietic stem cells. The use of
cell-surface
integrins to immobilize hematopoietic cells would overcome this difficulty and
allow these
cells to be micro-injected.
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In one aspect, the present invention provides for improved methods of
immobilizing non-adherent cells to a surface sufficient to facilitate the
efficient
introduction of macro- and micro-molecules, including genetic material,
proteins, peptides
and immunoglobulins into living cells. The present invention contemplates that
a variety
of immobilization techniques can be employed for immobilizing a non-adherent
cell to a
surface. In the present invention, a non-adherent cell is defined as: 1 ) a
cell that is routinely
manntained in suspension culture in vitro; or 2) a cell which is routinely
maintained in an
adherent state in culture, but is intentionally detached and allowed to be
maintained for a
defined period of time in suspension for the purpose of experimentation or
manipulation.
In some embodiments, the method comprises attaching non-adherent cells having
cell surface-expressed adhesion molecules, such as integrins (Fig. l A & 1 B),
to a surface,
the method comprising: treating said surface with adhesive molecules, and
treating said
non-adherent cell having cell surface-expressed molecules such as integrins or
CD44, with
an activating molecule such as an antibody which activates the cell surface
expressed
molecules to bind to a surface treated with adlhesive molecules. This
procedure results in
an otherwise non-aulherent cell being attached to a substrate surface su~cient
for
performing a microinjection procedure (Fig.lB).
In some embodiments the non-adherent cells can also be attached to a surface
comprised of adhesive molecules without th.e use of activating antibodies
(Fig. l C). This
depends on the cell type and the adhesive chosen. The present invention, in
particular
aspects, provides for immobilization of cells to substrates [e.g., fibronectin
or portions of
the fibronectin molecule derived either by protease digestion, recombinant
expression (eg.
Retronectin; CH-296), or synthesized peptif~esJ without additional stimuli of
cell surface
expressed adhesion molecules, i.e., attachment of some cell types to
fibronectin
peptides/fragments may be sufficient for microinjection without prior
activation (e.g.,
U937, HUT-78 cells, CEM cells, hematopo~ietic stem/progenitor cells). Some
cell types
(e.g., hematopoietic stem/progenitor cells) attach to the carboxyl end of
fibronectin with
sufficient strength to withstand microinjection.
Examples of adhesive molecules include by way of example and not exclusion:
fibronectin, collagen, laminin, epiligrin, invasin, osteospondin,
thrombospondin,
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proteoglycan, glycosaminoglycan, ICAM and VCAM-1, and fragments thereof
derived by
protease digestion or recombinant expression; synthesized peptides (with or
without
chemical modification), or oligosaccharide fragments thereof. Such is also
anticipated to
include both linear and circularized fragments; of said proteins and peptides,
or fragments
thereof.
In another aspect, the invention provides for a method for the delivery of
material
into a non-adherent cell. In one embodiment, the method comprises:
immobilizing the
non-adherent cell to an adhesive surface, and micro-injecting a solution
containing said
foreign material into said immobilized non-adherent cell through a
microinjection needle
(Fig.12B). The microinjection needle may be fiuther described as having an
outer diameter
in the range of about 0.05 to about 1 micron, or about 0.05 to about 0.5
microns.
In other embodiments, the method of immobilizing non-adherent cells to a
surface
comprises: immobilizing the non-adherent cell with a holding pipette having a
distal end
with an inner bore diameter of about 0.5 to about 2.5 microns; and applying a
vacuum with
a syringe assembly to the holding pipette, driven either manually (e.g.,
Eppendorf or
Zandler) or with a powered device (e.g., World Precision Instruments), to said
holding
pipette while said distal end of said holding pipettes is in close proximity
to said non-
adherent cell (Fig.l2A). In some embodiments, the holding pipettes may be made
using
a DeFonbrune microforge. According to one method for preparing the pipettes,
appropriate
bends are made in a glass capillary so that it: fits in a chuck assembly
holder, such that a
1-5 gram weight can be hung from the capillary positioned inside a heating
filament. Heat
is then applied softening the glass and resulting in the weight pulling the
glass capillary to
an about 1 to an about 5 micron diameter, at which point the piece of glass
capillary from
which the weight is suspended breaks away. 'This results in a holding pipette
with an about
1 to an about 5 micron diameter tip. The tip is then brought close to the
heating filament,
and the tip is heat polished. This provides a. smooth tip with an opening of
an about 0.5
to an about 2.5 microns. This holding pipette can then be attached to a
syringe assembly
that can be used to create a vacuum that will hold the cell in place for the
microinjection
procedure.
When making such a holding pipeae, one is not limited to the DeFonbrune
microforge. For example, any of the connmercially available needle pulleys
(Suffer
Instruments, Narishige, KopfJ can be used to pull a capillary to provide a
pipette as
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described here as part of the invention. For most embodiments of the
invention, the tip
should be heat polished with a microforge (DeFonbrune or Narishige).
The microinjection method of the invention is directed at providing a
population
of modified non-adherent cells, especially hematopoietic stem cells that
include a desired
molecule without significant loss of living cells or biological function. The
estimated
number of stem cells that will need to survive the microinjection procedure
for a gene
therapy procedure to is estimated to be about 1 SO to about 5,000 stem cells.
The present invention also contemplal:es that microinjection-mediated delivery
of
transgenes or transgenes with accessory protE;ins, such as integration
proteins, directly to
the nuclei of primitive cord blood stem cells I;Fig. 8), will be more
efficient than delivery
of same with retroviruses and AAV vectors. lft is further contemplated that
larger regions
of DNA, such as those containing regulatory elements and intron/exon
structure, will be
deliverable by the present microinjection method. Such will also avert the
dysregulated
expression and silencing frequently observed in the progeny of transduced stem
cells.
The present invention also contemplates using other methods for delivery of
transgenes or transgenes with accessory proteins. For example, iontophoresis,
particle
bombardment, electroporation, cationic liposome-mediated transfection, peptide-
mediated
gene delivery (e.g. polylysine), receptor-mediated endocytosis, red blood cell-
mediated
transfection, hypotonic swelling, micropriclting, and addition of nucleic
directly to the
medium surrounding the cells.
In another aspect, the invention provides a gene therapy method for treating
physiological disorders (Figs. 5, 6, & 7). By way of example, such
physiological disorders
include thalassemia and cancer.
Another aspect of the invention provides a specifically designed apparatus for
delivering foreign material into a non-adherent cell. In some embodiments, the
apparatus
comprises a microinjection needle having ar.~ outer diameter in the range of
about 0.05 to
about 0.5 microns.
In some embodiments, the apparatus comprises a microinjection needle having an
outer diameter in the range of about 0.05 to about 0.5 microns with a beveled
tip. Internal
opening dimensions maybe employed that are tailored to accommodate the
thickness of the
walls of the glass capillary used in making the needle in some embodiments of
the present
invention. In particular, the needle will be made using a glass capillary with
a sufficient
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wall thickness that when used in making the injection needle will result in a
needle that has
a tip with thick enough walls to not easily break when coming in contact with
a plastic
material when the needle is used in a cell injection procedure. In some
embodiments, the
needle will satisfy the above requirements and when used in the injection
procedure results
in minimal damage to the cells, i.e., will have the best observed cell
viability following the
injection procedure.
In some embodiments of the injection needles provided in the present
invention,
the glass capillary will have a sufficient wall thickness such that the needle
tip will have
a thickness such that it does not easily break v~rhen coming in contact with a
material more
pliable than plastic. The needle tip may be further defined as being coated
with a non-
sticky compound (e.g., silicon).
These embodiments will provide a needle that will not stick to intracellular
components, thus decreasing the chance of damaging the cell via the
microinjection
procedure. The needle in other embodiments may be coated both externally and
internally
with a non-sticky compound (e.g., silicon). These needles will be particularly
efficacious
in the delivery of viscous, concentrated, or ;>ticky macromolecules from the
needles, in
particularly those needles with very small outer diameters (.OS- 0.5 microns).
The invention, in still another aspect, provides a method for the protection
of
hematopoietic stem cells and their progeny :from chemotherapy. Such cells, by
way of
example, will be treated such that they gain resistance to such chemotherapy
agents as
alkylating agents, anthracyclines, vinca alkaloids, Etoposide, Taxol and the
like, or any
combination. The protection of the hematopoietic stem cell and its progeny
from
chemotherapy will be provided by microinjection of said cells with multiple
drug
resistance genes, such as the MGMT gene and/or MDR-1 gene, and delivery of
these
modified cells to a patient. The MGMT gene has been isolated and described in
the
literature, which teachings are specifically incorporated herein by reference
(Wang, et al.,
1996; Moritz, et al, 1995; Preuss, et al, 1996). The MDR-1 gene is also
described in the
literature, and in particular in I. Roninson, et al., which reference is
specifically
incorporated herein by reference. (Koc, et a,l., 1996).
Preparations comprising a population of cells enriched with modified human
hematopoietic stem cells having nucleic acid. material introduced into them
comprise yet
another aspect of the invention.
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Kits useful for the immobilization andl microinjection of foreign materials
into non-
adherent cells are also provided. These kits in some embodiments comprise: a
carrier
means adapted to contain at least two container means; a container means
comprising a
microinjection needle having an outer diameter in the range of about 0.05 to
about 1
S microns, and a container means comprising; a microinjection needle having
all of these
characteristics. The kit can comprise an immobilization surface (available
both uncoated
or adhesive-coated), microinjection needles} and, optionally, a cloning ring.
The adhesive
when not coated directly on the immobilization surface can be provided in a
separate
container. When required, activators of cell attachment (e.g., antibodies that
activate cell
surface integrins) can be provided in a sepaJrate container. The said kit may
also include
reagents (eg. divalent cations, peptides, etc.) used for the detachment of
cells from the
adhesive after microinjection.
It should be understood that application of the methods and apparati provided
by
this invention as opposed to those disclosed by the prior art will ( 1 )
facilitate
microinj ection of a wider range of primary and transformed non-adherent
cells; and (2)
permit immobilization; microinjection and recovery of micro-injected cells
with less
adverse effects upon the biological activity (e.g., in vivo reconstitution of
the hematopoietic
system by genetically modified stem cells) of the cell; and (3) permit
injection of non-
adherent cells with semi-automated or automated microinjection instruments
capable of
injecting more cells per hour.
In another embodiment, the present invention provides a kit for the microinj
ection
of foreign material into non-adherent cells having cell surface-expressed
integrins, the kit
comprising: an immobilization surface coated with an adhesive, and anti-
integrin
monoclonal antibody (or other equivalent integrin activation) and a
microinjection needle
having an outer diameter in the range of about 0.1 to about 0.15 microns.
Other
embodiments of the kit may include microinjection needles with other outer and
inner
diameter sizes, or combinations of various aizes, as described herein.
It is contemplated that the methods and apparati disclosed herein will be
useful for
the transfer of foreign material into human cells as an approach to gene
therapy and
somatic cell therapy.
Thus, in accordance with one aspect of the present invention, there is
provided a
composition comprising hematopoietic stem cells, or more generally non-
adherent cells,
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which are genetically engineered with DNA which encodes a therapeutic agent of
interest.
The genetically engineered cells are subsequently employed as a therapeutic
agent.
Accordingly, the present invention also provides a gene therapy method for the
treatment of physiological disorders responsive to gene therapy comprising:
administering
to a patient parenterally a composition enriched for genetically modified
human
hematopoietic stem cells, the stem cells containing genetic material delivered
by
microinjection (Fig. 7). It is contemplated that a composition of cells that
includes at least
about 150 to about 5,000 viable genetically modified cells, potentially
supplemented with
a sufficient number of genetically modified or unmodified hematopoietic
progenitor cells
for short-term hematopoietic reconstitution, will provide treatment and or
therapy of the
targeted disorder.
In accordance with another aspect of the present invention, there are provided
hematopoietic stem cells, which have been genetically engineered by
microinjection to
include DNA which encodes a marker or therapeutic agent, with such cells
expressing the
encoded product in vivo. In this particular embodiment, the DNA cures a
genetic
deficiency of the cells and the expression product is either not secreted from
the cells (for
example, hemoglobin) or secreted from the cells (for example, Factor VIII).
The invention is also directed to a method of enhancing the therapeutic
effects of
hematopoietic stem cells (hSCs).
In accordance with another embodiment, there is provided a process for
treating a
patient with a therapeutic agent by providing; the patient with hSCs
genetically engineered
with DNA encoding such therapeutic agent.
In accordance with another embodiment, a patient is provided with hSCs which
are
genetically engineered with DNA encoding a therapeutic agent whereby such
therapeutic
agent may be expressed in vivo. As hereinabove indicated, such genetically
engineered
cells may be provided by administering to t:he patient hSCs which have been
genetically
engineered ex vivo by hSCs microinjection.
In accordance with a further aspect of the present invention, there is
provided a
composition comprising (i) hSCs genetically engineered with DNA encoding a
therapeutic
agent and (ii) a pharmaceutically acceptable carrier suitable for
administration to a patient.
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FIG. 1 A and FIG. 1 B. FIG. 1 A demonstrates potential mechanisms for
activating
the integrins present on the surface of various non-adherent cells. In FIG. 1
A, CD34'
S stem/progenitor cells are shown to express both the a4 Vii, integrin as well
as the a5 Vii,
integrin on their surface. The adhesive molecules used as an example here is
fibronectin.
The a4 p, is shown to bind to the LDV-containing peptide sequences, while the
as (3, binds
to the RGD-containing peptide. The cells shovvn with a "rounded" suspension
morphology
when they attach to a substrate, such as fibronectin, are generally attached
only in a weak
tethered form. The tethering of cells to a substrate is not sufficient for
microinjection of
cells employing a rapid scale method because they are easily dislodged by the
microinjection needle. The activation of integ;rins by various mechanisms
including those
enumerated here leads to very tight attaclvnent of cells to fibronectin by
converting
integrins from a state with low affinity for the ligand to a state with high
affinity (Fig.
1 A)for the ligand, and to support the spreading of the cells. Cells that are
more avidly
bound do withstand the semi-automated and automated microinjection process.
Fig. 1 B
demonstrates that, in some cases, attachment ~md spreading of a cell on an
adhesive surface
can occur without additional activating agents added prior to, or concurrent
with cell
attachment. FIG. 1 C demonstrates the ability of cells to attach and spread,
in the absence
of any activating agent to carboxyl terminal fi~agments of fibronectin
(containing the RDV
and LDV recognition sites). (Kimizuka et al,., 1991 ).
FiG. 2A and 2B. Methods by which cells immobilized on a surface may be
released from the surface immobilization. Cells immobilized on a surface with
fibronectin
(Fig. 2A) may be released with a variety of methods including competition with
peptides,
inhibition of integrin mediated attachment by calcium, chelation,
trypsinization, or
disruption by pipetting. The cells can evenlually be recovered in an non-
adherent form
where they lose their spread, adherent mophology and return to a more rounded,
non-
adherent shape (Fig. 2B).
FIG. 3 shows retention of both myeloid and erythroid colony forming activity
by
CD34+ cells that have been immobilized via an activated integrin/fibronectin
attachment
(forward slashed bar). Colony forming activity by concanavalin A immobilized
CD34+
cells is also shown (back slashed bar). Shown on the left is erythroid burst
(BFU-E) colony
13
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formation. Both methods of attachment showed no significant difference from
that of the
control non-immobilized cells (open bar). Myeloid (CFU-GM) colony formation
showed
no significant decline in colony formation for the integrin/fibronectin
immobilized cells
in comparison with the control. In contrast, with concanavalin A
immobilization, the
myeloid colony formation was only about 50°.% of the control non-
immobilized cells. The
small standard deviation indicates this is a significant difference both from
that of the
control and the integrin/fibronectin sample.
FIG. 4 shows a time course evaluation of the number of fluorescent cells as a
percentage of micro-injected cells. CD34+ ceills (-~- and -1'- ) or
CD34+/CD38'/Thy-1'°
cells (-1- and -1-) were attached with the integrin/fibronectin method
outlined in the
examples. Cells were injected with needles having 0.2 micron O.D. tips and
FITC-Dextran
material of 150,000 molecular weight was injected. Shown at the less than 1
hour time
point is the assessment of the percentage of good injections giving rise to
fluorescent cells
immediately after injection. These samples vvere then followed for various
time points, 4
hours, 24 hours, 48 hours or greater after thc~ injection and the number of
cells that were
fluorescent is shown as the percentage of the total number of good injections.
'The decline
in this number reflects: ( I ) quenching of fluorescence by subsequent
fluorescent
microscopy; (2) death of cells due to microinjection-mediated injury; and/or
(3) possible
toxicity of FITC-Dextran for hematopoietic stem/progenitor cells. Shown are 2
representative studies for each cell type.
FIG. 5 shows a schematic of the hematopoietic system with all of the various
lineages proceeding eventually from the pluripotent stem cells. The CD34+
cells
containing all measurable human stem/progenitors constitute 0.5 to 1 % of the
total
mononuclear cells in cord blood or bone mancow. Also shown is a phenotype that
was used
to describe the pluripotent stem cell. Those are cells that are CD34+, CD38-,
CD45Ra-f°,
CD71-f°, Thy-f° and a rough estimate of their frequency is
expressed as a percentage of
mononuclear cells.
FIG. 6 shows, in schematic form, the complete hematopoietic system of
individuals
with defective genes, deletions or mutations in their chromosomal DNA (* =
mutation in
chromosomal DNA sequences).
FIG. 7 shows the outcome of the introduction of genetically modified stem
cells
into the patient (* = mutation in chromosomal DNA sequences; ~ = corrected
gene). If
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the correcting DNA is put into the pluripotent stem cell and the introduced
DNA is retained
in progeny cells as the hematopoietic system i,s allowed to develop,
genetically corrected
cells will comprise the complete hematopoiE;tic system (i.e., all lineages and
stages of
maturation).
FIG. 8 shows an advantage of the mic:roinjection system-that is, the ability
to co-
deliver DNA (rectangles) together with proteins (circles) into the same cell.
In this
example, the proteins were chosen to facilitatc: the integration of the DNA
sequences into
the chromosomal DNA. Shown here are examples where the sequences and proteins
are
selected either from a marine leukemia virus (a retrovirus) or the adeno-
associated virus.
FIG. 9 shows the DNA sequences which are intended to correct a genetic defect
(*)
in the chromosomal DNA, and in this case is proposed that proteins ( circles)
active in
homologous recombination will be co-injected together with the correct DNA
sequences.
The object is to replace the deleted or mutated sequences with their correct
copies supplied
via microinjection~ventually giving rise to a cell which would be corrected at
the
previously defective allele.
FIG. l0A and FIG. l OB show CD34+ stem progenitor cells plated onto
fibronectin
in the absence of any antibodies (i.e., simply in media, FIG. l0A) and via
their attachment
to fibronectin in the presence of the anti Vii, activating integrin antibody,
TS 2/16.2.1 (FIG.
l OB). As can be seen from FIG. 10A, cells attached in the absence of antibody
are only
loosely tethered. They maintain their round morphology, and the refractile
nature of the
outside surface of the cells indicates that they are not attached flat to the
surface; rather
they are simply tethered at a point. This is to be contrasted from the
extremely flat
morphology of the integrin attached cells (FTG. l OB). These cells are tightly
attached to
the surface, frequently have podia emanating from the cell, and are highly
spread in
comparison with either the non-immobilized or tethered cells.
FIG. 11 shows an ethidium bromide atained agarose electrophoresis gel of
lambda
wild type DNA digested with HindIII/EcoltI either pre- or post-filtration
through a 0.1
micron filter. There was no evidence for loss of material of sizes at least up
to 21 kb in
length. Since all fragments (including the 2lkb fragment) successfully passed
through
filter pores of 0.1 micron size; this strongly suggests that linear fragments
of at least 21 kb
in length will successfully pass through injection needles of inner diameter
greater than or
equal to about 0.1 micron inner diameter.
*rB
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FIG. 12A and FIG. 12B - Microinjection Approaches. FIG. 12A demonstrates an
injection technique employing a holding piper:es to stabilize the cell. Once
stabilized with
the holding pipette, (via vacuum suction) the c ell may be inj ected with the
microinj ection
needle. The microinjection needle is represented by the needle having a label
of 0.12p.
The holding pipette is represented by the appendage marked with a 2-4p. (Both
of these
dimensions (i.e., 0.12, 2-4u) are chosen for example purposes only). FIG. 12B
represents
an injection technique for microinjection of cells that are immobilized by
treating a surface
of a culture plate with an adhesive molecule. Cells attached through the
methods described
in this application withstand the microinjection process.
FIG. 13 - Attachment/detachment/spreading of U937 cells on commercially
available fibronectin-coated dishes following treatment with lmg mAb
TS2/16.2.1/mI
(-~- = cells attached; -~- = cells with miicropseudopodia, projections or
extensions;
-~- =cells loosely attached).
FIG. 14 - Proliferation of U937 cells following attachment to commercially
available fibronectin coated dishes(-~- = U937 cells treated with 1 mg mAb
TS2/16.2.1/ml; -~- = U937 cells untreated) viability U937 cells treated with
mAb
TS2/16.2.1 = 96.4%; U937 cells untreated = 95.8%.
FIG. 15 - Attachment and spreading of CD34+ cells isolated from cord blood on
commercial coated fibronectin dishes following treatment with TS2/16.2.1 nnAb
(-~- =cells attached; -~- = cells with micropseudopodia, projections or
extensions;
-~- =cells loosely attached).
FIG. 16 - Proliferation and viability o~f CD34+ cells isolated from cord blood
in the
presence or absence of lmg mAb TS2/16.2.1/ml. (-~- = presence of mAb; -~- -
presence of control IgG mAb). Viability of CD34+ cells treated with mAb
TS2/16.2.1
- -~-; untreated = -~-.
FIG. 17 - CD34+ cell attachment to fibronectin (-~- and -O- ) versus
retronectin
(-O- and -~-} in the absence (-O- and -~-) or presence (-~- and -O ) of 1 mg
mAb
TS2/16.2.1/ml.
FIG. 18 - U937 cells quartzJborosilicate needle. Percent survival U937 cells 2
hours, 24 hours, and 48 hours after injection with the quart~Jborosilicate
needle (hatched
bars = quartz; open bars = borosilicate).
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WO 98128406 PCT/US97/23781
FIG. 19 - CD34+ cells quartz/borosilicate needle. Percent survival of cells at
2
hours, 24 hours, and 48 hours after injection (hatched bars = quartz; open
bars =
borosilicate).
FIG. 20 - Size distribution data for borosilicate needle version 1.OB;
frequency
versus outer diameter (in microns) of borosil:icate needle.
FIG. 21 - Size distribution data for quartz needle version 1.OQ. Frequency
versus
outer diameter (in microns) of quartz needle.
FIG. 22A, FIG. 22B, & 22B' - FIG. ;?2A - flow chart demonstrating loading of a
siliconized needle. Use of a pressure device to load a sample into the needle
barrel of a
siliconized needle. FIB 22B & B' - air or otter gas bubble is expelled from
the tip of the
silicon needle after loading of the sample. Thus may be accomplished by use of
a glass, or
other similar material, filament to the tip of the sample-loaded needle.
FIG. 23 - gridded cell plate. An elastomeric stamp or manifold device may be
used
to lay dovim defined islands of adhesive material in the desired spacial
orientation to
accommodate the number of cells to be injeci:ed in a single batch. By way of
example, the
defined islands of adhesive may be laid down with a pre-determined spacial
orientation to
accomunodate about 1,000 cells. Each cell will be spaced every about 10 to
about 20
microns. In that example, the stamp will have an overall dimension of lmun x
lmm. In
some embodiments, the stamps may be arn~nged to conform to the organization of
the
needles to be used in the injection procedure. One or more needles may be
configured to
move from cell to cell, with one needle injecting the cells in each of the lmm
islands. In
some embodiments, the geometry of the surface area covered by the adhesive
material can
be controlled to limit cell spreading in order to regulate cell function (eg.
proliferation,
differentiation, secretion of cell products, etc.).
FIG. 24A-24B - Injection manifold. FIG. 24A demonstrates the top view of one
embodiment of the injection manifold. The :injection needles 1 extend from the
mianifold
2. The number of needles and spatial arrangement of the needles may vary
according to
the desired arrangement of the user and to :provide a needle injection profile
that would
correspond to the grid pattern arrangement ~of the girded cell plate wells
used. FIG. 24B
demonstrates a side view of the injection ma~ufold. An inlet 3 is connected to
the manifold
2. A single or multiple needle arrangement of needles 1 extend from the
manifold 2.
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FIG. 25 - Microinjection assembly unit. The needle 1 is positioned to provide
contact with a tissue culture dish S. Metal ~Eilaments 2 contact a resistivity
monitor 4,
which is in turn in contact with a sound system 3.
FIG. 26 - Viability of U937 cells after microinjection using borosilicate
(hatched
bars) or quartz needles (speckled bars), versions 1.0B and 1.OQ, respectively.
FIG. 27 - Viability of CD34+ cells after microinjection using borosilicate
(hatched
bars) or quartz needles (speckled bars), versions 1.OB and 1.OQ, respectively.
FIG. 28A & FIG. 28B - Scanning electron micrographs of A. Version I.OB
borosilicate injection needle. FIG 28 B. Version I .0 Q quartz injection
needle.
BEST MODES OF CARRYING OUT THE IIWENTION
The microinjection method practiced according to the invention in some
embodiments employs non-adherent cells and microinjection needles with outer
diameters
of about 0.05 microns to about 0.5 microns. '.Che method in some applications
provides for
immobilization of a non-adherent cell onto a surface, followed by
microinjection of the cell
to include a desired foreign material. The invention further provides for the
removal of
modified cells from a culture surface with minimal damage and/or loss of cell
viability.
The modified cells as provided according to the present invention can be used
in
a variety of applications, including: (a) in laboratory studies, (b) for
production of desired
proteins (e.g., in vitro production of monoclonal antibodies), and (c) to
treat a physiological
disorder. In this regard, the techniques disclosed herein may be used in gene
therapy. In
another aspect, the invention provides for a preparation of cells enriched in
genetically
modified cells. Such preparations may also be used to administer parenterally
to a patient
suffering from a gene therapy responsive physiological disorder wherein the
genetically
modified non-adherent cell and its progeny naay express a therapeutic agent,
thus treating
the patients physiological disorder.
Microinjection Method
As used herein, the terms "microinjection" or "micro-injecting" refer to the
delivery
of foreign material into a non-adherent cell. In some embodiments, this method
employs
a microinjection needle as described below.
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In some embodiments of the method, the microinjection process of the invention
proceeds generally as follows. A cloning ring is affixed to an irnmobilization
surface, such
as a tissue culture plate, and the surface of the plate enclosed by the
cloning ring is coated
with an adhesive, such as fibronectin. The coated immobilization surface is
then exposed
to a mixture of non-adherent cells to be immobilized in the presence of an
activating anti-
integrin monoclonal antibody for a period of tame and at a temperature
sufficient to permit
immobilization of the non-adherent cells to the immobilization surface. In
other instances,
the non-adherent cells will be added to the said immobilization surface in the
absence of
an activating antibody or other activator of attachment. An ideal needle is
loaded with a
sample solution containing an effective amount or concentration of foreign
material. The
tissue culture plate containing the immobilized non-adherent cells is placed
on the stage
of a microscope, and the microinjection needle is placed in a micromanipulator
such as that
sold by Narishige, or an electronically controlled manipulator such as that
sold by
Eppendorf, mounted on the same microscope. A device, such as an SAS 10/2 air
screw
actuated microinjection/aspiration syringe or an automated Eppendorf 5246
Transjector,
provides the pressure necessary for delivery of the sample solution (possibly
containing
a fluorescent marker) from the microinjection needle into the nucleus of the
cell.
Following insertion of the microinjection needle into the nucleus of the non-
adherent cell,
an effective amount of the sample solution is injected into the cell. Delivery
may be
monitored via a phase contrast microscope. However, it may be advantageous to
deliver
such small volumes that no observable change in the cell will be detected via
light
microscopy. After retraction of the microinj~ection needle, nuclear delivery
of the foreign
fluorescent material may be confirmed with a microscope equipped with a
fluorescence
detector and the modified cells are subsequently detached from the
immobilization surface.
The microinjection process can be done manually, semi-automatically, or
automatically according to the equipment employed. The manual microinjection
approach
involves using a micromanipulator to direct .a glass micropipette (loaded with
an injection
sample) into a living cell's nuclear or cytopl;asmic compartment, all viewed
with a phase-
contrast microscope. The injection needle is connected to a syringe assembly
that provides
the pressure which continuously forces the: sample out of the needle. The
needle tip is
inserted into the cell, and the lightening of tree phase contrast caused by
the flow of sample
solution into the cell is visually monitored. The change in phase contrast
indicates
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WO 98/28406 PCT/US97/23781
injection of the sample into the cell, whereupon the injection needle is
removed from the
cell. The semi-automatic microinjection proceas proceeds as follows: The
microinjection
needle is directed into the nucleus of the cell using the manual settings.
This is done to set
the Z value, i.e., the vertical position that the; needle will return to when
performing the
automatic inj ections. Upon setting the Z value, the needle is pulled out of
the nucleus and
positioned above the nucleus of the cell to be injected. The automated system
is then
activated. The needle is directed into the cell :nucleus at the same time a
pulse of pressure
expels the injection sample into the nucleus. After the injection is
completed, the needle
returns to the position directly above the injected cell. A new cell is then
located and the
procedure repeated.
The volume of solution containing foreign material which is micro-injected
into the
non-adherent cells can be optimized as desired. Generally, the volume injected
will not
exceed about 2% to about 5% by volume of the non-adherent cell nucleus
receiving the
solution. The concentration of foreign material and the physical properties of
the solution
being injected into the non-adherent cell can impact the success of the
microinjection.
According to the invention, other variables such as temperature, speed of
microinjection
needle penetration into and retraction fra~m the non-adherent cell, inner and
outer
microinjection needle tip diameter, length of time the needle has an increased
internal
pressure (for expelling the injection sample), angle of needle when
penetrating the cell, and
the internal pressure in the needle both during and after the injection
procedure must be
optimized for each cell type other parameters possibly requiring optimization.
The
temperature used during microinjection cm be varied from about room
temperature
(22 ° C) to about 37 ° C using the heated stage that is part of
the microscope used for the
injection procedure. The volume of solution injected into the non-adherent
cell will vary,
among other things, according to the volume of the non-adherent cell.
The pressure used to micro-inject sample solution into the cell will vary
according
to inner diameter and tip geometry (e.g., l:aper length and flare) of the
microinjection
needle and sample solution concentration and viscosity. The pressure should be
sufficiently low to maintain sample solution flow rate and delivered volume
below that
which is deternzined to provide maximum cell viability following the injection
procedure.
As used herein, the term "microinjection needle" is taken to mean a
microcapillary
comprised of borosilicate, alumina silicate;, or quartz glass, or other
suitable material,
CA 02275474 1999-06-18
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which is used to inject foreign material into non-adherent cells. The
microinjection needles
of the invention can be prepared from conventional glass capillaries with an
automated
pipette puller such as the P-87, P-97, or P;Z000 models from Suffer
Instruments. The
microinjection needles can also be prepared manually by employing a microforge
or other
similar device. The microinjection needles of the invention will have an outer
diameter
less than about 0.5 microns, preferably less than about 0.25 microns and more
preferably
in the range of about 0.05 microns to about 0.5 microns. The inner bore of the
microinjection needles will be sufficiently large enough to permit passage of
macromolecules from a reservoir through the microinjection needle tip and into
the non-
adherent cell and will generally be about 0.02-0.4 microns in diameter.
The microinjection needles contemplated by the present invention can be
prepared
generally by optimizing the particular heating filament, the type of capillary
(glass
composition and inner/outer diameter), and equipment settings (e.g., heat,
pull strength,
and velocity of pull) employed. The final size and geometry of the
microinjection needle
can be determined by a variety of methods, such as scanning electron
microscopy (SEM),
resistivity measurement, or bubble pressure assay.
The final tip outer diameter (O.D.), taper length, and flare of the
microinjection
needle can be controlled by employing the method described herein.
Considerations such
as filament or laser temperature, size of filament, velocity of pull, pull
rate, initial capillary
outer diameter, initial capillary wall thickness, initial capillary bore inner
diameter (LD.),
capillary composition, period of exposure to heat and annealing rate can all
be optimized
as needed to yield microinjection needles having the desired characteristics.
When practiced according to the present invention, a non-adherent cell, having
been
micro-injected with a solution containing a foreign material, will remain
viable for an
extended period of time and will retain and possibly express the foreign
material, i.e., a
non-adherent cell receiving foreign DNA vvill be able to replicate the DNA and
express a
protein associated with that DNA.
The process of the present invention is not limited to microinjection of
foreign
materials solely into the nucleus of non-adherent cells. For example, the
foreign material
can be injected into the cytoplasm or various other cellular organelles.
As used herein, the term "foreign material" refers to materials such as intact
virions,
DNA, RNA, proteins, small organic molecules, metabolites, macromolecules,
organelles,
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WO 98128406 PCT/US97/23781
plasmid vectors, enzymes, inorganic substances, chromosomes, artificial
chromosomes,
episomal plasmids and other materials which are external to the non-adherent
cell being
micro-injected.
A "non-adherent cell" is one that grows as a suspension culture as opposed to
one
that grows attached in tissue culture. Examples of non-adherent cell types
contemplated
by the present invention include mouse, human, primate and canine cells such
as primary
and transformed B-cells (B-lymphocytes), 'h-cells (T-lymphocytes),
hematopoietic stem
cells, granulocytes/neutrophils, myeloblasts, erythroblasts and others.
Primary
stem/progenitor cells contemplated in the invention also include CD34+,
CD34+/CD38-,
CD34+/CD38+, CD34+/liri/Thy-1'°, CD34-'/CD45Ra"°/CD71-
'°/Thy-1'°, CD34+/c-kit'°,
CD34+/CD38-/CD33-/CD19'/CD45Ra-/c-kit", CD34+/CD38'/CD45Ra-~'°/CD71-
"°/Thy-I'°,
and CD34+/CD38'/Thyl'°. Transformed hematopoietic cells useful in the
invention include
a variety of cells such as U937 and KG-1. A primary cell is one which is
directly removed
from its in vivo source; i.e., the cell has not been manipulated or
transformed to provide for
I S indefinite growth in culture.
In practicing the microinjection metr~od of the invention, the following four
criteria
should be considered: (a) a non-adherent cell should be attached sufficiently
so as to
minimize dislodgement due to microinjection, (b) the micro-injected non-
adherent cell
should be removable from the surface to which it is attached with minimal
damage or
reduction in biological activity, (c) the immobilization should generally not
induce cell
activation and/or differentiation. Such could potentially interfere with
subsequent
biological activity of the non-adherent cell, e.g., loss of stem cell
activity. An additional
consideration is that the microinjection process itself should not adversely
impair the
viability or biological function of the cell.
As used herein, the term "immobili2:ation surface" is taken to mean a plastic,
glass,
quartz or other surface onto which a non-adherent cell can be immobilized or
attached.
Such surfaces may include slides, Petri <iishes, plastic/tissue culture plates
or dishes,
coverslips, chromatographic resins, porou:o membranes, holding pipettes and
the like.
By "immobilizing" or "immobilization" is meant the process by which a non-
adherent cell is attached to or held by an immobilization surface with
sufficient strength
to permit microinjection. Such processes include the retention of a non-
adherent cell by
a holding pipette having a reduced pressure or vacuum, attachment of the non-
adherent cell
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to a surface by way of lectins or a linking agent, attachment of the cells to
a surface via
antigen-specific monoclonal antibodies eithe:r directly or indirectly, or
activation of cell
surface expressed adhesion molecules such as integrins or other adhesion
proteins on the
non-adherent cell by way of various activating agents, activating anti-
integrin antibodies,
S cytokines, or activating cations to include div~~lent cations (e.g.,
Mn'~'~). Alternatively, cells
expressing adhesive molecules such as integrins may not require additional
activation for
strong attachment. A non-adherent cell attached to or held by an
immobilization surface
is referred to as "an immobilized non-adherent cell."
By "lectin" is meant materials such. as PHA and Con A. Lectins are plant and
animal proteins known to interact with specific carbohydrate structures on the
surface of
cells, thereby, facilitating attachment. By "linking agent" is meant materials
such as
glutaraldehyde and collagen.
As used herein, the terms "substrate" or "adhesive" are taken to mean
materials
such as fibronectin, collagen, lamirun, VCAIVIs, ICAMs, epiligrin, invasin,
osteospondin,
thrombospondin, hyaluronic acid, proteoglyc;an, glycosaminoglycan, or
fragments thereof
(to include recombinant molecules) or peptidles (unmodified or chenucally
modified). Cell
surface expressed integrins on non-adherent cells bind with the substrate or
adhesive either
in the native state or once the integrin has been activated with agents such
as anti-integrin
monoclonal antibodies. The substrate or adhesive attaches directly to an
immobilization
surface (or in some cases a linker is used to attach the matrix molecule to a
particular
surface) thereby permitting immobilization of a non-adherent cell onto the
immobilization
surface. When the adhesive is attached to the immobilization surface, the
resultant is
termed an "adhesive-surface couple".
Integrins are proteinaceous molecules expressed on the surface of various
cells and
serve a variety of biological functions such as mediating adherence to various
matrices/cells. A cell surface expressed integrin is an integrin that is
produced by a cell
and is associated with the cell membrane or cell surface and generally has at
least a part of
itself disposed external to the cell. Such cell surface expressed integrins
include for
example VLA-4 (a4~i,) and VLA-S (a5~3,).
N. L. Kovack, et al. in J. Cell Biol. 116:499-509, 1992 discloses that cell
surface
expressed integrins can be activated by an 2u~ti-integrin antibody so that the
activated cell
surface expressed integrin will aid in cellular adhesion to a variety of
substrates.
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As used herein, the term "anti-integrin antibody" refers to an antibody
capable of
binding to an integrin. According to the present invention, the anti-integrin
antibody can
include, by way of example and without limitavtion, the monoclonal antibodies
(Mabs) anti-
~i,, , 8A2, TS2/16.2.1, anti-(32, anti-~i3, and anti-Vii,, as well as
polyclonals.
The cell surface expressed integrins may also be activated by other methods
including antibodies, cytokines, divalent cations and peptides.
When practicing one of the immobilization methods of the invention, an
appropriate combination of cell surface expressed adhesion molecule activators
and
adhesive should be selected. Integrin and adhesive combinations known to bind
are shown
in Table 1.
Table 1
~3 subunit a subunit Adhesive
a, c:ollagens, laminin
a2 c:ollagens, laminin
a3 laminin, fibronectin, epiligrin,
collagen
a4 VCAM-1, fibronectin
as f ibronectin
ab laminin
a, laminin
a" vitronectin, fibronectin
(3Z aL ICAM-1, ICAM-2, ICAM-3
a~ iC3b, IAM-l, fibrinogen, factor
X
aX fibrinogen, iC3b
X33 atm i:ibrinogen, fibronectin,
vWF, vitronectin
a" thrombospondin
vitronectin, fibrinogen, fibronectin
VWF,
thrombospondin, osteospondin
(34 ab laminin, basement membrane
protein
(35 a,, vitronectin
(36 a" i:~bronectin
(3, a4 'VCAM-1, fibronectin, MAdCAM-1
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As discussed above, non-adherent cells can be attached to a surface via
antigen-
specific Mabs, which themselves are directly attached (i.e., covalently bound)
to the
surface. Such antigen-specific Mabs include, for example, anti-CD34, anti-CD4,
and anti
VLA-4, anti-VLA-5.
The method of detachment of immobilized cells from a surface will be selected
according to the immobilization method employed. When the method of cell
surface
expressed integrin mediated immobilization onto an immobilization surface
coated with
an adhesive is employed, cellular detachment is accomplished by competition
with
peptides) (e.g., from fibronectin), release by protease treatment (e.g.,
trypsin), release with
cell disassociation buffer, polyanions which may interfere with the heparin
binding site,
cations such as Ca++ which interfere with integrin function, or simple
disruption by
pipetting. When a cell is immobilized by b nding to an antibody which is
directly, i.e.,
covalently, bound to an immobilization surface, such as with glutaraldehyde,
cell
detachment is accomplished by either the methods mentioned above or
competition by
excess molecule to which the antibody binds, or excess antibody or antibody
fragments
(such as Fabs).
When practicing another embodiment of the non-adherent cell immobilization
method of the invention, a holding pipette is employed. As used herein, the
term "holding
pipette" refers to a microcapillary having an inner bore diameter (opening) of
about 0.5
microns to about 2.5 microns which is capable of holding a non-adherent cell
without
puncturing or otherwise damaging the non-adherent cell surface. The bore of
the holding
pipette is under a reduced pressure (vacuum) which provides the force by which
a non-
adherent cell is drawn to and immobilized onto a distal end of the holding
pipette. The
holding pipette of the invention is made according to Example 13. The holding
pipette can
be made using a DeFonbrune microforge. Appropriate bends are made in a glass
capillary
so that it fits in a chuck assembly holder such that an about 1 to about 5
gram weight can
be hung from the capillary positioned inside a heating filament. Heat is
applied softening
the glass resulting in the weight pulling the glass capillary to an about 1 to
an about 3
micron diameter at which point the piece of glass capillary from which the
weight is
suspended breaks away leaving a holding pipette with an about 1 to an about 3
micron
diameter tip. The tip is then brought close to the heating filament and the
tip is heat
CA 02275474 1999-06-18
WO 98/28406 PCT/US97/23781
polished resulting in a smooth tip with an opening of about 0.5 to about 2.5
microns. This
holding pipette can then be attached to a syringe assembly that can be used to
create a
vacuum that will hold the cell in place during the microinjection procedure.
When making such a holding pipette, one is not limited to the DeFonbrune
S microforge. For example, any of the connmercially available needle pullers
(Suffer
Instruments, Narishige, Kopf) can be used to pull a capillary to the above
dimensions.
However, the tip must still be heat polished v~ith a microforge (DeFonbrune or
Narishige).
Generally, the holding pipette used in the invention will have an O.D. less
than
about 2.5 microns such that a non-adherent: cell held by the holding pipette
will not be
aspirated into the pipette. In order to mininnize penetration of the cell wall
by the holding
pipette, the holding pipette tip will have to be fire-polished or suitably
smoothed. Either
the Eppendorf or Zandler syringe assemblies; have been designed to produce a
vacuum in
the holding pipette that will hold the cell in place during the injection
procedure, but not
rupture the membrane of the cell in the process.
Barbs should be removed from t:he holding pipette tip. This is generally
accomplished by fire-polishing. Care should be taken not to seal the holding
pipette during
fire-polishing. This can be accomplished by passing a gentle stream of air (or
an inert gas)
through the holding pipette while heating tree tip to about .5 to about 2.5
microns, which
is sufficient to anneal but not melt the holding pipette tip.
To facilitate monitoring of cell viat~ility post-microinjection, non-adherent
cells
micro-injected with foreign material such ass DNA can be co-micro-injected
with either
fluorochrome- (e.g., FITC, Oregon green, P;hodamine) coupled dextran (MW of
various
molecular weights, e.g., 150,000), or a vital fluorescent DNA-stain (e.g.,
Hoechst 33342
yoyo dye), Green Fluorescent Protein (GFP), or DNA encoding the GFP reporter
gene.
Depending upon cell culture conditions used pre- and post-microinjection,
micro-injected
non-adherent cells can be driven into cycle v~rith hematopoietic cytokines or
growth factors
such as IL-3, IL-6, SCF and Flt-3 ligand or with other agents, such as
neutralizing anti-
TGF-~i antibody. Alternatively, it may be preferable to maintain cells in
culture conditions
which enable survival without inducing cycling or differentiation. It should
be noted that
post-microinjection, non-adherent cells that have been detached from the
immobilization
surface will regain their non-adherent properties and can be grown again in
suspension as
non-adherent cells.
26
CA 02275474 1999-06-18
WO 98128406 PCTIUS97I23781
The microinjection method and apparati of the invention are described in
Example 1.
A stock solution containing foreign material, indicated as "Sample" in Table
2, was
typically buffered.
A stock solution of foreign material, indicated as "Sample" in Table 2,
typically
included phosphate-buffered saline (PBS), water and Tris-EDTA (TE). The stock
solution
was then either directly micro-injected into the recipient cells or diluted
with a second
buffer solution prior to microinjection into the recipient cells. The second
buffer solution
typically contained Hepes (50 mM), KCl (100 mM) and NaH2P04 (5 mM) and had a
pH
of about 7.2. The concentration, in mglml, of foreign material ("Sample")
actually micro-
injected into the cells is indicated as "Cone" i.n Table 2. The sample
solution was typically
centrifuged at about 10,000 to 15,000 rpm using a table-top Eppendorf micro-
centrifuge,
or filtered through a 0.02 mm or 0.1 mm membrane and/or dialyzed prior to
microinjection
into the cells.
The cells receiving the sample solution by microinjection included primary
cells
or transformed cells (e.g., U937, TF-1 cells) (CD34+, CD34+/CD38-,
CD34+/CD38+, and
CD34+/CD38-/Thy'°}. CD34-expressing cells are primary human
stem/progenitor cells
immunomagnetically purified from umbilical cord blood. The CD34+ /CD38' and
CD34+/
CD38- /Thy-1'° cell populations were isolated by fluorescent activated
cell sorting (FACS).
The CD38' subpopulation of the CD34+ cells comprises a more primitive subset
of cells.
3T3 denotes the Swiss 3T3 fibroblast cell line, which is an adherent mouse
fibroblast cell
line. U937 is a generally non-adherent human myelomonocytic cell line.
The sample foreign material micro-injected into the cells included DNAs
(pCMV-(i, ph-GFP, pGreen Lantern ("pGref;n"), pCMV-(3/ph-GFP in a 1:1
concentration
ratio), fluorescent conjugates ( PE-RAM, FI7."C-GAM, rhodamine-dextran, FITC-
dextran),
or mixtures thereof. Various combinations o~f these and other foreign
materials previously
described can be micro-injected in the indicated cell lines.
The cells were immobilized onto a siuface, typically a tissue culture plate or
Petri
dish, by a variety of methods by treating the surface with a molecule
"adhesive" (e.g.,
fibronectin, "Fn") (Table 2). In some cases, "activators" (e.g., anti-p,,
integrin monoclonal
antibodies) 8A2 Ab (8A2 from N. Kovach) and TS2 sup (conditioned supernatant
from the
TS2/16.2.1 hybridoma cell line, T. Spring;er, ATCC no. HB-243) are anti-~3t
integrin
27
CA 02275474 1999-06-18
WO 98/28406 PCTIUS97/23781
monoclonal antibodies which were used for activating cell surface-expressed
integrins on
respective cells for their immobilization onto fibronectin-coated plates. Con
A
(concanavalin A) was used to immobiliize respective cells by first treating
the
immobilization surface with glutaraldehyde and then exposing the derivatized
surface first
to concanavalin A and then to respective cells. Biotin CD34 immobilization was
attempted
by exposing glutaraldehyde derivatized plates to biotinylated anti-CD34
antibody and
respective cells.
The number of cells plated in the cloning ring prior to microinjection
generally
ranged from about 3 00 to about 2000. Only a fraction of these were actually
micro
injected. The number of cells micro-injected per experiment, indicated as
"Cells (#)" in
Table 3, ranged from about 2 to about 150 and the microinjections were
identified as
"good" (g) or "not acceptable" (na). "Good" microinjections provided cells: (
1 ) that
showed a mild swelling of the nucleus during microinjection; (2) which volume
of sample
solution received was not so great as to destroy the cell; and (3) that were
not immediately
destroyed by the penetration or retraction of the microinjection needle.
Microinjections
failing any of these criteria were identified as "not acceptable".
The cells were assayed or visually monitored immediately after microinjection
and
at four hours, 12-18 hours, 48 hours and greater than 72 hours after
microinjection. The
number, size, viability status and/or color and intensity of fluorescence of
the micro-
injected cells was monitored by phase <;ontrast and fluorescent microscopy.
Red
fluorescence was from rhodamine micro-injE;cted into the cell and green
fluorescence was
from FITC-dextran or GFP expression by the micro-injected cell. Occasionally,
the
expression of beta-gal by the micro-injected cell was monitored by x-gal
straining.
Although the microinjections were generally conducted on a heated (about
37°C)
microscope stage, some were conducted at .ambient temperature (about 20
°-24 ° C).
Following immobilization of the primary cells or transformed cultures onto the
immobilization surface coated with an adhesive, aliquots of stock solutions
containing
varying concentrations of foreign material were micro-injected into the
immobilized cells.
Microinjection needles having an O.D. in the ranges of about 0.9 to about 1.1
microns
(indicated as Eppendorf "Femtotips"), about 0.45 to about 0.60 microns
(indicated as
"fine"), or specific values (as indicated in Table 2) were prepared and
employed. The
results are summarized in Tables 2 and 3.
28
CA 02275474 1999-06-18
WO 98/28406 PCT/LTS97/23781
w
w '.:
a o
~
A
G b G L7 D G
z o ~: w ~: ~; ~ ~-
L
d d
a
~ ~
0 0
0 0
i
..,
H
W W ~ w W W
z
0
H
U
A
A + + + + +
~r et et
" M M U U U U U
O
A A
U U
w
U
.~ ...,
0 0 0 0
v ,x
N N O N N N N
v ~ O O m O O O O
y ~ ..~ ..r ~i
~ ~ ~
GL
O . . .
G C
Q
U U U U
a. c, a, c.
w ~ _; ~ ~ L7 a a ~ c1
a a a
G~ G~ G7 G7
c a
va . . w c. ca. cs~ a.
a:
W .. N ~ N N N N
29
CA 02275474 1999-06-18
WO 98/28406 PCT/US97123781
w
.~
A N N N N N N N N
z O c c O O c O O c
N N N N C CS N N
H H H H z z ~ H
g ~ A A
f~ f~ f~ f~ a tai U w w
O +
a, i,
~r H ~ ~r ~r
z oo H U
o0 00
x U
w
U U U U U U
M M
U M U A
~
a ~ ~ ~ ~ a ~ a
.r w
r r a" = = e.; z.;
r r
N N N O N N N N
+r
O
L
z
w w w
x V EU-~ V ~ H '
H H H
~i -o w ~, ~. c r= r= G c~
O --~ N
W oho ono ~ ~ ~ O O O
CA 02275474 1999-06-18
WO 98/28406 PCT/US97/23781
w
.a
N
W N N N, N
z o 0 0 0 0 0
a a a
z z H H H
d d
v ~ f~ f~
u f
r
z
0
U
A
M M M M V
A A A A
U U U U
U
w
U
p, G~ C.
.,
~
V ~ ~7 V'7 O
7
N N N N t!7
O
4.
L~
w ~ ~ w
.
A a a A a
U U U
U U
b w w w w w
o- M ~ v~
o
31
CA 02275474 1999-06-18
WO 98/28406 PCT/US97I23781
w
C~
d
0 0 0 0 0 0 0
a~a~a~a~a~a~a~ a~a~a~a~
0 0 0 0 o G ~ ~ a~
w
E-
d
W
c~ c~f
by by
~ ~
r ~ e~
N >, ~ ~ ~ z z z z z z
_ "~ _
E..
M
Z V1 V1
V O O
W
O
~ A ~ A ~ ~ A A A A A ~ A A
.3 o z z z O M N ~fiM ~ N ~ z z z z z z z z
w
a
M
U ~ ~1 f~ A A h N A C~Ca
N M ~, ~ ~ ~ M N ~
~ z z z z N ~ z z z
;
d
H
O
~
~ z z ~ z z z z ~ N h z ''~ ' h ' '~ M
--
N
M
d'
dl
r
~
V
y ~ ~ 00V~~ bybyd0bAd0byd0 dp~ bD
~
a o h o op~ ~ N o 0 0 0 o v,~no 0 0 0 0
N
h .~~ ~ M ~O~nh N N N N M N ,.")~n~n
M ~
~1O
~i
O --~N M ~t~nN M O~O ~'N M etM ~t
N ~1 0 0 0 0 0
N N N N o00oO~OvO~,..,.-,.-..~ .-,.-..-~.-.'
32
CA 02275474 1999-06-18
WO 98/28406 PCT/US97/23781
The results indicate that non-adherent cells and, in particular, primary
primitive
hematopoietic cells from the CD34+ stem/progc;nitor population, can be
successfully micro-
injected with various macromolecules including; DNA by employing the novel
microinjection
method and apparati of the invention. The genetically modified cells
successfully express
the respective product of the genetic foreign material micro-injected into
them.
In some embodiments of the invention, a suflacient amount (generally less than
5-10% by
volume of the cell nucleus being micro-injected) of a stock solution
containing a nucleic acid
foreign material is micro-injected into a non-adherent cell, more preferably a
hematopoietic
stem cell or other hematopoietic cell, immobilized onto an immobilization
surface by way
of an adhesive and possibly an activating agent that activates cell surface-
expressed integrins
on the non-adherent cell. The microinjection is accomplished by employing a
microinjection
needle having an outer diameter less than about 0.20 microns and in some
embodiments
between about 0.05 to about 0. I S microns.
Gene Therapy
The present invention also provides a method for the transduction of
hematopoietic
stem cells (hSCs) and thus an alternative strate:gry for their direct genetic
modification by: ( 1 )
direct delivery of DNA sequences into the nuclei of hSCs by microinjection;
(2) integration
of micro-injected transgene sequences in the chromatin of hSCs or
extrachromosomal
maintenance of the transgene sequences on episomal vectors or artificial human
chromosomes and persistence of those sequences in the progeny of said hSCs;
and (3)
microinjection of sufficiently large (15-25 Kb) transgenic DNA constructs
containing
regulatory elements, such as promoters, enhancers and locus control regions
(LCRs), and
intron/exon structure necessary for appropriate; long-term, cell type-specific
expression of the
introduced transgenes; and 4) microinjection of DNA/protein mixtures with the
proteins)
included in the injection sample which aid in l;ene integration andlor
targeting (Figs. 8 & 9).
Genetically modified hSCs prepared according to the methods of the present
invention can be employed for gene therapy applications once said modified
hSCs have been
delivered to humans for long-term reconstitution.
According to the present invention, he:matopoietic stem cells that have been
modified
by microinjection of foreign material can be used to treat a variety of
physiological disorders
?~3
CA 02275474 1999-06-18
WO 98/28406 PCTIUS97/23781
such as, by way of example and without limitation, AIDS, cancer, thalassemia,
anemia, sickle
cell anemia, adenosine deaminase deficiency., Fanconi Anemia, Gaucher disease,
Hurler
Syndrome, immune deficiencies, and metabolic diseases.
The physiological disorders contemplated within the invention will be
responsive to
gene therapy. By "responsive to gene therapy" is meant that a patient
suffering from such
disorder will enjoy a therapeutic or clinical benefit such as improved
symptomatology or
prognosis.
As indicated above, one aspect of the present invention relates to the use of
modified
hSCs, as cellular vehicles for gene transfer. The genes, or transgenes, can be
any gene having
clinical usefulness, for example, therapeutic or marker genes or genes
correcting gene defects
(e.g., mutant hemoglobin genes in thalassemia or sickle cell anemia) in blood
cells.
Preferably, the primary human cells are blood. cells. The term "blood cells"
as used herein
is meant to include all forms of blood cells as well as progenitors and
precursors thereof, as
hereinabove described.
Thus, in one embodiment, the invention is directed to a method of enhancing
the
therapeutic effects of hSCs, comprising: (i) mic;ro-injecting into the hSCs of
a patient a DNA
segment encoding a product that enhances the therapeutic effects of the human
primary cells;
and (ii) introducing the genetically modified hSCs into the patient.
The DNA produces the agent in the patients body and, in accordance with such
embodiment, the agent is expressed at the tissue site itself. Similarly, as
hereinabove
indicated, hSCs which are genetically engineered need not be targeted to a
specific site and,
in accordance with the invention, such engineered hSCs and their progeny
function as a
systemic therapeutic; e.g., a desired therapeutic; agent can be expressed and
secreted from the
cells systemically.
More specifically, there is provided a nnethod of enhancing the therapeutic
effects of
hSCs that are infused in a patient, comprising: (i) micro-injecting into the
hSCs of a patient
a DNA segment encoding a product that enhances the therapeutic effects of the
blood cells;
and (ii) introducing cells resulting from step (i) into the patient.
When the modified hSCs are not "targeted," the genes are inserted in such a
manner
that the patients transformed blood cells which are progeny of the modified
hSCs will
produce the agent in the patient's body (Fig. '7).
34
CA 02275474 1999-06-18
WO 98/28406 PCT/US97/23781
The primary human blood cells that are the progeny of modified hSCs and which
can
be used in the present invention include, by way of example, leukocytes,
granulocytes,
monocytes, macrophages, lymphocytes, and erythroblasts. For example, stem-
cells from
thalassemic or sickle cell anemia patients that we genetically modified with
the appropriate
hemoglobin gene may give rise to genetically corrected red blood cells.
The DNA earned by the hSCs can be: any DNA having clinical usefulness, for
example, any DNA that directly or indirectly enhances the therapeutic effects
of the cells.
Alternatively, the DNA carried by the hSCs can be any DNA that allows the hSCs
to exert
a therapeutic effect that the hSCs would not normally exert. Examples of
suitable DNA that
can be used for genetically engineering, for example, blood cells, include
those that encode
cytokines such as tumor necrosis factor (TNF), interleukins (for example,
interleukins 1-12),
globin genes, DNA-repair genes, drug-resistance genes, Fanconi Anemia genes
and anti-HIV
(Human Immunodeficiency Virus) resistance l;enes.
The DNA which is used for transducing the human cells can be one whose
expression
product is secreted from the cells. Alternatively, it may encode for gene
products retained
within the cell. The human cells can also b~e genetically engineered with DNA
which
functions as a marker, as hereinafter described in more detail.
In one aspect, the inserted genes are marker genes which permit determination
of the
traffic and survival of the transformed cells in vivo. Examples of such marker
genes include
the neomycin resistance (neon) gene, mufti-drug resistant gene, thymidine
kinase gene, ~i-
galactosidase, dihydrofolate reductase (DHFR) and chloramphenicol acetyl
transferase.
The hSCs are genetically engineered in vitro. For example, cells may be
removed
from a patient and stem cells isolated; genetically engineered in vitro with
DNA encoding
the therapeutic agent, with such genetically engineered hSCs being
readministered along with
a pharmaceutically acceptable carrier to the patient. Such a treating
procedure is sometimes
referred to as an ex vivo treatment.
In some embodiments, the progeny of tree modified hSCs are primary human cells
and
more preferably are primary human nucleated t~lood cells which express in the
appropriated
progeny cells the product of the genetic foreign material micro-injected into
the parent hSCs.
The pharmaceutically acceptable cattier may be a liquid carrier (for example,
a saline
solution) or a solid carrier; for example, an implant of a biocompatible and
non-immunogenic
material. In employing a liquid carrier, the engineered cells may be
introduced parenterally,
3 ~~
CA 02275474 1999-06-18
WO 98/28406 PCT/ITS97/23781
e.g., intravenously, sub-cutaneously, intramL~scularly, intraperitoneally,
intralesionly, or
directly into the bone marrow.
Results from mouse, large animal, and. human studies permit a reasonable
estimate
of the number of stem cells that need to be delivered to humans for long term
reconstitution.
Since the genetic therapies under consideration will frequently be directed to
children,
estimates herein are based on their smaller body weight. Furthermore, although
a significant
number of unmarked, short-term reconstituting cells may need to be co-
delivered to ensure
rapid engraftment and survival, our focus is on. the much smaller number of
gene-modified,
long-term reconstituting stem cells.
Three independent mouse studies have reported long-term reconstitution with as
few
as about 100 marrow cells (Spangrude et al., 1988), 10 marrow cells (Jones et
al., 1996), or
even 1 marrow cell (20% of mice reconstituting (Osawa, et al., 1996). If
direct scaling by
weight alone is appropriate, an average reconstitution requirement of about S
cells for mice
would extrapolate into approximately about a. 5,000 marrow cell requirement
for a human
child. Whether one needs to also scale for thE; increased human life span is
not yet clear.
In human marrow transplantation, the; minimal dose typically delivered is 1 x
108
nucleated cells per kg body weight, equivalent to 2.5 x 109 cells for a 25 kg
child. Reported
experimental data and modeling of feline hematopoiesis indicate that the stem
cell frequency
is approximately 1 in 1.7 x 106 marrow cells (Abkowitz et al., 1996). If this
same frequency
holds for human marrow, delivery of 2.5 x 1 X09 cells corresponds to delivery
of 1450 stem
cells.
Children reportedly reconstitute with as little as 30 mls of transplanted cord
blood,
likely due to the significant proliferative poteni:ial of primitive
hematopoietic cord blood cells
(Kurtzberg et al., 1996). Assuming approximately 1.5-3 x 108 nucleated cells
in this volume,
with a stem cell frequency of 1 in 105 to 106 thds translates into successful
engraftment with
as few as 1 SO-3,000 stem cells.
Reported evidence from engraftment of human cord blood cells in NOD/SCID mice
suggests a very close relationship between NOD/SCID reconstituting cells
(SRCs) and
human stem cells. SRCs are present at a frequency of ~ 1 in 1 f' CD34' cells
(Serrano et al.,
1996). Thus, 30 mls of cord blood (with approximately 3 x 10' mononuclear
cells, 1 % of
which are CD34+) contain approximately 30 SRCs. Even if the seeding efficiency
of SRCs
in NOD/SCID mice is only 10%, this translates into 300 SRCs.
36
CA 02275474 1999-06-18
WO 98/28406 PCT/US97I23781
Thus, all four calculations suggest the required number of stem cells may be
in the
range of about 150 to about 5,000. Since succe:>sful reconstitution depends
not only on stem
cell delivery, but rapid engraftrnent by short-tenor progenitors, it is
possible that the stem cell
requirements are even less than those calculated above.
Clinical benefit for genetic diseases will. generally require connection in a
significant
fraction of the stem cells present in vivo. This rnay be accomplished by in
vitro selection for
modified hSCs prior to engraftment (so that only the successfully transduced
cells are
transplanted into the patient) and/or by subsequent in vivo selection for
modified hSCs (to
enrich for modified hSCs at the expense of endogenous, unmodified hSCs). This
may require
transduction of hSCs with two independently regulated genes present on the
same DNA
construct: the selectable gene targeted for expression in stem/progenitor
cells and the
therapeutic gene (e.g., ADA or globin) targeted for expression in the required
cell type.
Transduction of hSCs with the human O6-methylguanine DNA methyltransferase
(MGMT) gene may enable in vivo selection of surviving, modified hSCs by
briefly treating
patients with alkylating agents of the nitrosourea class (e.g., 1,3-bis (2-
chloroethyl)-1
nitrosourea; BCNU). Whereas most anti-neoFdastic drugs (e.g., Taxol) are toxic
to cycling
hematopoietic progenitors, sparing the quiescent hematopoietic stem cells,
nitrosoureas such
as BCNU also exert their DNA-damaging and toxic effects directly on the stem
cells.
MGMT, which reportedly removes O6-alkylguanine induced in DNA by various
alkylating
agents (Mitra et al., 1993), is reportedly normally expressed at very low
levels in
hematopoietic stem/progenitor cells (Wang et a~l.,1996; Moritz et al., 1995).
However, when
exogenously expressed in cells, MGMT reportedly confers cell resistance to
BCNU, CCNU,
dacarbazine, N-methyl-N=-nitro-N'-nitrosoguanidine, temozolomide, and
streptozotocin
(Preuss et al., 1996; Spain et al., 1992). For e~;ample, mice expressing MGMT
in their stem
cells were reportedly resistant to BCNU-induced hematosuppression (Maze et
al., 1996).
Although the human multiple drug resistance gene (MDR-1 ) has been proposed
for in vivo
selection of transduced stem cells, the fact: that human stem cells already
reportedly
constitutively express MDR-1 (Chaudhary ei' al., 1991) suggests that any
enrichment for
transduced cells by MDR-1 resistant drugs (e.~;., taxol) may occur at the
level of progenitors
but not stem cells. As is true for any proposed in vivo selection (e.g., CCNU,
taxol) for
marked hematopoietic cells, it will be imperative to minimize drug toxicity
for other organs
and cells. Finally, MGMT transgene expression, by itself, should, as
previously reported,
3'7
CA 02275474 1999-06-18
WO 98lZ8406 PCT/US97/23781
confer resistance in hematopoietic cells to agents such as BCNU employed in
high-dose or
repetitive chemotherapy for breast and other cancers (Chabner et al., 1993).
In one aspect, gene therapy employing genetically modified hematopoietic stem
cells
may include the following elements. Apoproximately 1-10 x 103 highly enriched
hematopoietic stem cells are obtained from, human cord blood and are
temporarily
immobilized. Microinjection of these cells delivers a reproducible volume-
containing DNA
and possible integration enzymes) - such that 1-3 copies of the DNA are
successfully
integrated per cell. Microinjected DNAs of 15-25 kb in size, containing two
independently
regulated transgenes, are integrated without arrangement. One transgene,
targeted for
expression in stem cells, provides for in vitro (e;.g., rsGFP, or truncated
nerve growth factor
receptor; tNGF-R) or in vivo (e.g., MGMT) selection of transduced stem cells.
The
therapeutic transgene (e.g., ADA for ADA SC:ID, globin for hemoglobinopathies,
MDR-1
for chemoresistance) is targeted for expression in the appropriate
hematopoietic cells.
The present invention may be employed for introducing relatively large
fragments of
nucleic acid into a cell. For example, nucleic acid sequences of DNA having
molecular
weights of between 20 kb and 24 kb have been iintroduced through an opining of
0.1 microns.
Hence, it is anticipated that the microneedles of the present invention having
an opinion of
0.1 microns may be used with such relatively large molecules.
The relatively large-sized DNA can be filtered through a 0.1 micron filter. In
addition, the present studies demonstrate that the passage of these relatively
large molecules
was achieved without loss of integrity of the molecule (Fig. 11).
The following abbreviations have been used in the preparation of this
disclosure.
pCMV-~i DNA plasmid expressing the p-gal reporter gene under
control of the; cytomegalovirus (CMV) promoter/enhancer
sequences.
PE-RAM Phycoerythrin (a fluorochrome) labeled rabbit anti-mouse
Immunoglobulins
FITC GAM Fluorescein :fsothiocyanate (a fluorochrome) labeled goat
anti-mouse Immunoglobulins
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phGFP DNA plasmid expressing the humanized red shifted green
fluorescent protein (GFP) reporter gene under control of
the CMV promoter/enhancer sequences (from CIonTech)
pGreen DNA plasmid (pGreen Lantern) expressing the humanized
red shifted GFP reporter gene under control of the CMV
promoter/enhancer sequences (from GIBCO-BRL)
rhodamine-Dextran Rhodamine (a fluorochrome) coupled to dextran
FITC-Dextran FITC coupled to dextran
CD34+CD38-Thy-1+ the CD38-T'hy-1+ (actually Thy-1 ~' subpopulation of
CD34+ cells isolated by FACS.
line
U937 a human myelomonocytic cell line, normally non-
adherent.
8A2 Ab and TS2 sup anti-p, integrin-mediated attachment to fibronectin-coated
plates. 8A2 (N. Kovach) and TS2/16.2.1 (T. Springer,
ATCC) are ~murine monoclonal antibodies specific for
human [i, integrin.
hSC hematopoietic stem cell
Materials used herein were obtained as :follows: PE-RAM {B-D, Beckton
Dickinson),
phGFP (ClonTech), pGreen (Gibco), rhodamine-dextran (Molecular Probes), FITC-
Dextran
(Sigma Chemical Co. and Molecular Probes). pCMV-(3 and FITC GAM were prepared
as
described in the present disclosure, and are available in the inventor's
laboratory. Some
3T3 Swiss 3T3 fibroblasts-an adherent mouse fibroblast cell
materials were obtained from Sigma Chemical Company. Cell types described
herein were
obtained from the American Type Culture Collection.
The foregoing will be better understood with reference to the following
examples
which detail certain procedures according to the present invention. All
references made to
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these examples are for the purposes of illustration. They are not to be
considered limiting as
to the scope and nature of the present invention.
EXAMPLE 1
SET 1VERY OF FITC-DEXTRAN TO IMMOBILIZED CD34* CELLS
The present example demonstrates the utility of the invention for introducing
a
molecule of relatively small size into a living cell, wherein the cell retains
viability and is
provided in an immobilized state during the time the molecule in being
introduced into the
cell.
Purification and Culturing of CD34+ Cells
The CD34* antigen, present on approximately 0.5-1.0% of mononuclear bone
marrow
and umbilical cord blood cells, marks all measurable human hematopoietic stem
and
progenitor cells (Fig. 5). Umbilical cord blood cells were obtained from
normal human fetal
deliveries, and mononuclear cells were purified by centrifugation over Ficoll-
hypaque.
CD34* cells were isolated by immunomagnetic selection with the Miltenyi
MiniMACS CD34
Multisort Isolation Kit (involves ( 1 ) incubation. of cells with anti-CD34
antibody coupled via
dextran to ixnmunomagnetic particles, (2) isolation of magnetically-labeled
cells by passing
through a column attached to a magnet, (3) release of cells from magnetic
particles by
cleavages with dextranase, (4) separation of cells from magnetic particles by
passing through
column attached to a magnet). Subsequent FACS analysis, with another anti-CD34
antibody
recognizing a different CD34 epitope, demonstrated that the cells were 85-95%
pure for
CD34 expressing cells. Purified cells were maintained overnight ( 18 hrs) in
serum free
medium (Iscoves Modified Dulbecco's Medium (IMDM, Gibco) supplemented with
bovine
serum albumin (2%, StemCell Technology), 'insulin (10 micrograms/ml,),
transferrin (200
microgram/ml, ICN), 2-mercaptoethanol (0.05 mM, Sigma), low-density
lipoprotein (40
microgram/ml, Sigma), and pen-strep (100 units and 50 microgram/ml,
respectively)
containing 20 ng/ml human Flt-3 ligand (Peprotesh), 20 ng/ml human Interleukin-
3 (IL-3,
Peprotech), and 20 ng/ml human Stem Cell Factor (SCF, Peprotech) [IMDM/F-3-S]
at 37°C
with 6% CO2.
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~reyaration of Fibronectin-Coated Surface
A 6 mm glass cloning ring was attached via Vaseline(R) to a 35 mm tissue
culture
dish (Corning). The dish surface enclosed by the cloning ring was coated with
fibronectin
by adding 30-50 microliters of a 50 microgram/ml fibronectin solution
(Boehringer
Mannheim, #1051-407) in phosphate buffered saline (PBS, Sigma), and incubating
overnight
at 4 ° C (alternatively, can be for 45 min. at room temperature).
Excess fibronectin-
containing solution was removed from the cloning ring immediately prior to
addition of cells.
Attachment of CD34+ Cells to Fibronectin-Gated Dish
After overnight culture, cells were prepared at a concentration of 8 x I04
cells/ml in
IMDM/F-3-S. This cell-containing media {f,5 microiiters containing
approximately 2000
cells) was mixed with 25 microliters of media {IMDM) conditioned 2 days by the
TS2/16.2.1
hybridoma cell line (ATCC #HB-243 which produces antibody reactive with
Integrin ~i,-
human CD29). The 50 microliters of cell/antibody mixture was placed into a 6
mm glass
cloning ring enclosing the fibronectin-coated surface. Cells, in the presence
of antibody,
were allowed to attach to fibronectin for greater than 30 min, at 37 °C
in the presence of 6%
COZ. Figure l0A shows an example of cells incubated on a fibronectin surface
without the
addition of the activating TS2/16.2.1 antibody. Cells are loosely attached and
will not
withstand microinjection. Figure l OB shows an example of cells incubated on a
fibronectin
surface in the presence of the activating TS2/16.2.1 antibody. The cells are
more spread with
the presence of numerous microspikes andl will withstand the microinjection
process.
Subsequently, 1 ml of IMDM/F-3-S was added outside the cloning ring, and the
35 mm plate
containing cells and cloning ring was spun at Ei00 rpm for 5 min (Beckman low-
speed GS-6R
centrifuge, swinging bucket rotor, brake off).
~!Iicroinj~ection of CD34+ Cells with FITC-D
Fine glass microinjection needles were prepared from thin-walled borosilicate
glass
capillaries (Sutter, 1.2 mm O.D., 0.94 mm LD~.) with an automated pipette
puller (Suffer, P-
87, 3 mm box filament). Scanning Electron Microscopy (SEM) was used to
determine the
outer diameter of microinjection needles pulled with the identical program;
O.D.s between
0.17 and 0.22 micron were obtained. F~ ITC-dextran ( 150,000 M. W., Sigma) at
a
concentration of 0.25% (weight per volume) in 50 mM Hepes (pH 7.2/100 mM KCl/5
mM
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].'LE 2
DELIVERY OF FIT'C-DEXTRAN TO
IMMOBILIZED CD34+/CD38' ~~'THY-1'° PRIMITIVE CELLS
The present example demonstrates the utility of the present invention for the
stable
incorporation of a foreign molecule into a cell, and particularly immature,
undifferentiated
cells.
Pnrifica_t;_nn of P_ri__m__i_tiye CD34+/CD38'/ Thy-1 ~_°_Cells
CD34+/CD38-/Thy-1'° cells comprise approximately 1-4% of CD34+ cells,
and exhibit
properties consistent with that of highly primitive hematopoietic cells
(highly enriched in
long term culture initiating cells (LTC-ICs)). As such, they represent a
candidate population
of stem cells. These cells were purified by first immunomagnetically isolating
CD34+ cells
(Miltenyi Minimacs, see Example 1 ) followed by fluorescence activated cell
sorting (FACS)
with PerCP-CD34 (Becton-Dickinson), PE-(:D38 (Becton-Dickinson), and FITC-Thy-
1
(Pharmingen) antibodies. Cells were sorted with Becton-Dickinson FACSVantage
with
automated cell deposition unit. T'he primitive nature of these cells was
further confirmed by
the vast majority of them expressing the CD45Ra'/CD71- phenotype.
Culturing of cells, attachment of fibronectin-coated dishes, microinjection of
cells
with FITC-dextran, monitoring and subsequent culture were performed as
described in
Example 1. Cells were well attached to the fibronectin, in that they were not
dislodged
during microinjection. Of 32 CD34+/CD38~/Thy-1'° cells (Experiment 84,
Tables 2-3; Fig.
4), each micro-injected with an estimated 2-10 femtoliters of 0.25% FITC-
dextran, 9 cells
were positive for fluorescence 30 min. post-microinjection. Three fluorescent
cells were still
present 24 hrs. post-microinjection.
EXA~V(PLE 3
F,XPRESSION OF RED-SHIFTED GREEN FLUORESCENT
PROTEIN BY MICROT~1JECTE~. IMMOBILIZED CD34+ CELLS
The present example demonstrates thf; utility of the present invention for the
stable
incorporation of a foreign nucleic acid sequence encoding a protein into a
cell, and the
successful expression of that protein by the: modified cell. In doing same,
the present
example also demonstrates the utility of the present invention as a gene
therapy technique.
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Using 0.45 micron microinjection needles, Green Fluorescent Protein (GFP)
reporter
gene expression, 24-48 hrs. post-microinjectior.~, in 5-15% of immobilized
CD34+ cells (6-8
micron diameter) was obtained. Cells werf; micro-injected with approximately
20-40
femtoliters of 50 ng/microliter pGreen Lantern DNA plasmid (Gibco BRL) which
expresses
the humanized red shifted Green Fluorescent 1?rotein under control of the
cytomegalovirus
(CMV) promoter/enhancer. The remainder of the cells were killed by
microinjection-pipette
induced cell damage or delivery of too much volume.
By employing microinj ection needles having an outer diameter of about 0.2
microns,
GFP expression 24-48 hrs. post-injection in 5%-15% of immobilized CD34+ cells
was
obtained.
F_X_AMPL~
~~STON OF RED-SHIFTED CiRFFN FLUORESCENT PROTEIN
BY MICROTNJFCTED IMMOBILIZED CD34+/CD38'/Thv-1'° CELLS
CD34+/CD38-/Thy-1'° cells were isolated and attached to fibronectin as
described in
Example 2. Seventy cells were micro-injected with an estimated 2-10
femtoliters of a 100
ng/microliter solution containing pGreen LantE;rn DNA in microinjection buffer
(Experiment
93, Tables 2-3). Microinjection needles of 0.17-0.22 micron O.D. were
employed. GFP
expression was observed in 8 cells 5 hours post-microinjection. At 24 hours
post-
microinjection, 5 cells were positive for GFP expression.
~,KAryIpLE 5
f'lIMPARiC(1N f1F TNTT~(TRIT1/FTRRONECTIN. CONCANAVALIN A
AND ANTI-CD34 MEDIATED ATTACHMENT
The above methods of immobilization were compared as described below for their
ability to strongly but reversibly immobilize non-adherent cells, especially
CD34+, and to
withstand the force of microinjection.
Four conditions were examined in this study:
{1) ICH-3 (biotinylated) anti-CD3~4 mAb [100 micrograms/ml] (very weak
attachment, manual microinjection difficult);
(2) Con A [100 micrograms/ml] (strong attachment, manual microinjection
possible,
withstands force of automated microinjection);
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(3) ~~Keweed mitoee~ [100 micrograms/lril] (poor attachment);
{4) anti-b 1 integrin attachment to fibronectin (strong attachment, manual
microinjection possible, withstands force of automated microinjection).
In two studies, attachment of CD34+ cells to plates coated with biotinylated
anti
s CD34 monoclonal antibody was examined. In two studies, plates were first
precoated with
glutaraldehyde before addition of biotinylated anti-CD34 mAb (100 microgram/ml
in PBS;
ICH-3 antibody, CalTag). In one study, there was no precoating with
glutaraldehyde. Cells
attached minimally B demonstrating a type of attachment described as
"tethered" . These cell
types were relatively difficult to micro-inject.
Similar results were obtained for lectin phytoloacca americana (pokeweed
mitogen;
100 microgram/ml in PBS, Sigma) precoated with glutaraldehyde (as described
below for
Con A). CD34' cells were minimally attached, and could not be micro-injected.
There was strong attachment of CD34+ cells to plates coated with Con A. Plates
coated with Con A were prepared as follows:
1. Precoat dish with glutaraldehyde:
2. Add 2.5% glutaraldehyde into cloning ring attached to issue culture dish,
let sit
overnight at 4°C,
3. Wash 4 times with sterile water,
4. Add Con A, 9100 micrograms/ml in PE9S (Sigma) to dish, at 37° C for
1 hour.
Remove Con A.
5. Add cells in the colony ring, incubated. 37° C for 30', then spun
plate, or to
fibronectin coated plates-in the presence of anti-beta 1 integrin activating
antibody
(TS2/I 6.2.1 hybridoma cell line; ATCC #HB-243; produces antibody reactive
with integrin
[i,-human CD29).
Biotinvlated ICH-3
Results indicated a weak attachment of CD34+ cells to the immobilization
surface
making manual microinjection difficult, but possible.
Con A
Results indicated a strong attachment of C1734+ cells to the immobilization
surface
making both manual and automated microinjection feasible.
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~~eweed Mitogen
Results indicated weak attachment of CD34+ cells to the immobilization
surface.
Anti-b~ intP~~ antibody
Results indicated a strong attachment of CD34+ cells to the immobilization
surface
making both manual and automated microinjection feasible.
T,~~rr~rnnm mATION OF UnMONOCYTIC CELL
The ability of the described integrin/fi.bronectin immobilization method to
attach a
transformed leukemic cell line, specifically tlhe human U937 myelomonocytic
cell line is
demonstrated in the present example. These cells normally grow continuously in
culture as
suspension cells. When U937 cells were simply added to fibronectin coated
plates
(fibronectin plates prepared according to our standard protocol} in the
absence of antibody;
there was some attachment of cells to the fibronectin, and some manual
microinjection was
possible. When U937 cells were incubated 'with either the 8A2 or TS2/16.2.1
antibodies
during culture with fibronectin coated plate:., a significant improvement in
the quality of
attachment was noted. Again, manual microinjection was successfully
demonstrated.
25
~IPLE 7
ATTACHl~~ENT AND SPR~T~'~TNCi OF SUSPENSION
TT 'r'rJRED CELLS TO INTACT HUN[~xr FTRRnNFr'~TtN IN THE PRESENCE
OF TS2/16.2.1 MONOCLONAL ANTIBODY
The kinetics of attachment and subsequent spreading of three suspension-grown
cell
types to intact human fibronectin (Fn) in the presence or absence of the
activating anti-
integrin monoclonal antibody (mAb} TS2/115.2.1 were investigated. The cell
types studied
thus far are U937, human myelomonocytic cell line; CEM, human lymphoblastic
leukemia
cell line; and hSC, human primary hematopoietic CD34+ cord blood stem cells.
Commercially available culture dishes pre-coated with human Fn (Becton-
Dickinson,
product #40457) were used for this series of experiments. The Fn provided is
in a denatured
state due to the fact that the matrix is dried onto the dishes. U937 cells
were plated into
cloning rings placed on the dishes in the presence of the mAb at 37°C
and allowed to attach
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WO 98/28406 PCT/US97I23781
and spread for various times. The number of attached cells was determined by
directly
counting the number of cells in the cloning rings. In the presence of the mAb,
U937 cells
attached rapidly to Fn. Approximately 100% of the cells attached within 20
min. of mAb
addition. Cellular projections, extensions, and micropseudopodia could be
detected almost
immediately (Fig. 13). The presence of cellular extensions correlated with
firm attachment,
not simple tethering, allowing for successful rnicroinjection.
When CEM cells and hSCs (Fig. 15) were attached to the Fn coated dishes in the
presence of the TS2/16.2.1 mAb, virtually 100'% of the cells were attached
within 30 and 90
minutes; respectively.
Thus far, these cell types did not readily attach to Fn in the absence of the
activating
mAb. This is the case regardless of the confbrmational state of the Fn. For
example, as
mentioned above, dishes pre-coated with dried denatured Fn have also been
examined. Fn
in an aggregated, though non-denatured, state was also prepared by coating
dishes with Fn
in PBS. A coating procedure which retains the native conformation of Fn was
also employed
in which the Fn is coated in a carbonate buffer.
;PLE 8
ATTACHMENT AND SPREADING OF SUSPENSION GROWN CEL .~
TO COMMERCIALLY AVAILABLE ~,F~TI1ESBASED ON FN ~EQ NCES
The present example demonstrates the utility of the present invention for the
use of
peptides and mixtures of peptides ("cocktail") to promote the attachment of
cells to a surface
with reduced disruption to the cells. Rather than utilizing an entire adhesive
molecule (e.g.,
fibronectin), portions of the molecule (produced by recombinant expression or
protease
digestion), a cocktail (mixture) of synthetic adhesive peptides that bind cell
surface adhesion
molecules may be used to coat the surface of a plate to which cells attached.
Various fragments of Fn were tested for their ability to attach suspension-
grown cells.
Retronectin (MW = 62,613; Rn) is a commercially available (Pan Vera, Corp;
product
#TAKT100A) recombinant human Fn fragment containing an RGD cell binding domain
(type III repeat), a high affinity heparin-binding site, and the CS-1 site
within the
alternatively spliced IIICS region which contains the LDV amino acid sequence
(Kimizuka,
F. et al. ( 1991 ). The RGD and LDV amino acid sequenced are known to bind the
a4[31 and
47
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WO 98/28406 PCT/LTS97I23781
a5~i 1 integrins, respectively. Rn has also been used to enhance the
efficiency of retroviral
gene transfer (Hanenberg, H., et al., 1996). .
Culture dishes were coated with Rn in either PBS or carbonate buffer. U937
were
plated onto the adhesive surfaces within cloning rings and the kinetics of
attachment,
spreading, and detachment (after mAb removal) were measured as described for
the
experiments on intact Fn. U937 cells bound to Rn in the presence of the
TS2/16.2.1 mAb
with more rapid attachment and spreading kinetics than to intact Fn. Cell
attachment to and
spreading on Rn occurred in the absence of activating mAb. The present study
suggests that
attachment and spreading on Rn was more efficient in the absence of the mAb.
Similar
results were obtained when hSC's were allowed to attach and spread on Rn (Fig.
17).
For attachment of cells expressing alpha 4/beta 1 or alpha 5/beta 1 integrins,
dishes
would also be coated with peptides (unmodifie;d or perhaps chemically modified
to increase
the affinity for cell surface expressed adhesion molecules or to facilitate
coating of culture
dishes) containing amino acid sequences that are targets for attachment (e.g.,
sequences
containing the LDV sequence of amino acids iiom the CS-1 region of fibronectin
bind alpha
4/beta 1, and sequences containing the RGD amino acid sequence of fibronectin
bind alpha
S/beta 1).
EXAMPLE 9
RETENTION OF HEMATOPOIETIC COLONY FOR_M_ING ACTIVITY
BY INTEGRIN/FIBRONECTIN IMMOBILIZED rD34+ CELLS
The present example demonstrates the utility of the invention for providing an
effective mechanism for genetically modifying undifferentiated cell types,
such as CD34+
cells. In this manner, the present application also demonstrates the utility
of the present
invention for a method to provide gene therapy using cells that are provided
to an animal, the
cells being provided to the animal in an undifferentiated state and containing
a particular gene
or gene fragment thereof.
CD34+ cells were immunomagnetically purified as described in Example 1 above.
In a first study, the colony-forming ability of integrin/fibronectin
immobilized CD34+ cells
{maintained overnight in an immobilized condition and subsequently released
for colony
assays) was compared with CD34+ cells maintained overnight under similar
culture
conditions where the cells were not immobilized with integrin/fibronectin
(Fig. 3). For
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WO 98/28406 PCTIUS97123781
integrin/fibronectin immobilization, 2000 CD:34+ cells in IMDM/F-3-S were
mixed with an
equal volume of IMDM media conditioned 2 Pays by the TS2/16.2.1 hybridoma cell
line, and
plated into a well of a 96 well plate previously coated with fibronectin. The
control well
contained 2000 CD34+ cells in IMDM/F-3-S alone. On the following day, cells
(immobilized
or control) were recovered from each well and plated in duplicate in 35 mm
dishes containing
1 ml MethoCult GF culture media (containing 0.9% Methylcellulose, 30% fetal
bovine
serum, 1 % bovine serum albumin, 10-4M 2-Mercaptoethanol, 2 mM L-glutamine, 50
ng/ml
human SCF,10 mg/ml human GM-CSF,10, ng/ml human IL-3 and 3 units/ml
Erythropoietin
(StemCell Technologies Inc.); 16 days post plating, the number of CFU-derived
colonies
were assayed. For integrin/fibronectin immohilized cells, 70 BFU-E, I 1 CFU-
GM, 5 CFU-
GEMM,10 CFU-CFU-GM, 7 CFU-GEMM, 11 CFU-G, and 2 CFU-M per 1000 plated cells
were likewise generated. The total number o:f CFU-GM, CFU-G, and CFU-M are
totaled to
give Amyeloid colonies, and both erythroid colony (BFUE-E) and myeloid colony
results
from immobilized cells are expressed as a percentage of the non-immobilized
control (Fig.
3 ). In a second experiment, 3 wells of a f6 well plate were cultured
overnight at 4 ° C,
followed by 4 x washing with water, and then coated with 100 microgram/ml
concanavalin
A 2 hours at 37 ° C. Excess fibronectin and Con A were then removed.
2000 cells in 25
microliters IMDM/F-3-S were mixed with 2.'i microliters TS2/ 16.2.1
supernatant and added
to each of the 3 fibronectin coated wells. 2:000 cells in 50 microliters
IMDM/F-3-S were
added to each of 3 untreated wells (control). .After overnight incubation,
cells were recovered
from each of two wells from each condition (i.e., integrin/fibronectin, Con A,
control), and
were plated in 1 ml methylcellulose-containing integrin/fibronectin
immobilized cell: 183
BFU-E, 19 CFU-GM, 11 CFU-GEMM, 13 CFU-G, 24 CFU-M. For Con A immobilized
cells: 155 BFU-E, 11 CFU-GM, 8 CFU-GEIvIM, 15 CFU-G, 17 CFU-M. For control
cells:
169 BFU-E, 20 CFU-GM, 8 CFU-GEMM, 29 CFU-G, 27 CFU-M.
A third study was performed similarly to the second study. Non-immobilized
cells
were compared to integrin/fibronectin and Con A attached cells:
integrin/fibronectin: SO
BFU-E, 35 CFU-G, GM, or M, 1.5 CFU-G)~;MM; Con A: 65 BFU-E, 26 CFU-G, GM, or
M,
CFU-GEMM; Control: 52 BFU-E, 50 CFU'-G, GM, or M, 0.5 CFU-GEMM.
'The results of the three integrin/fibronectin immobilization studies
demonstrate no
significant effect (either inhibitory or stimulatory) on the number or size of
colonies derived
from immobilized vs. control non-immobilized cells. Cells immobilized via Con
A
49
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CA 02275474 1999-06-18
WU 98/Z8406 PCT/US97123781
demonstrated granulocyte/macrophage type colonies reduced approximately 50%
with
respect to control non-immobilized cells (Fig.. 3).
~X~iPLE I U
EFFECT OF EXPOSL~~O TS2/16.2.1 ON
SFT.FCTED CELL11T.AR FUNCTIONS
The effect of mAb exposure on three different cellular parameters (viability,
proliferation, and retention of primitive stem cell activity) was
investigated. U937 cells were
seeded onto commercially available Fn-coated dishes in the presence or absence
of the mAb.
Viability was determined by trypan blue dye exclusion and cell proliferation
was determined
by direct cell counting with a hemocytomete;r. Treatment with the mAb did not
affect cell
viability. There was no significant differencE; in the viability of U937 cells
in the presence
(96.4%) or absence (95.8%) of the mAb. Simiilar results were obtained with CEM
and hSC's
(Fig. 16). Proliferation of U937 cells and CEM cells were unaffected by mAb
exposure. The
rate in the increase of cell number was similar in the presence or absence of
mAb (Fig. 14).
Preliminary studies indicate that hSC's can also proliferate in the presence
of the mAb at the
same rate as controls when stimulated with the appropriate cytokine cocktail
(Fig. 16).
The effect of mAb exposure and subsequent attachment to and release from Fn
and
Rn on primitive hematopoietic stem cell activity in hSC's using the NOD/SCID
reconstitution assay was also investigated. Tlle present study indicated that
attachment either
to Fn after exposure to the activating mAb or to Rn without an activating mAb
allowed for
the retention of NOD/SCID reconstituting activity.
~1~F OF VARIOUS REA(iE T~1 S FOR DETA~'HMFNT OF CELLS FROM AN
~p;~SUBSTR ,A-TE.
Reamirements for a cell detachment nrotocc~
1) is not toxic or damaging to cells; 2) does not trigger differentiation or
other
biological effects, causing the cells to lose stem cell activity; and 3) must
be relatively rapid
and efficient, releasing the majority of the cells in a short period of time
(Fig. 2). This is
particularly important where stem cells that are modified to include a nucleic
acid sequence
CA 02275474 1999-06-18
WO 98/28406 PCT/US97123781
of potential therapeutic action are to be transferred from a cell plate into
an animal. By
minimizing the damaging effects from physical and/or chemical disruption, the
number of
cells that remain viable is optimized, and hence the number of cells provided
in the treatment
is enriched.
S~nontarneous detachment of cei_ls:
Since it is important to detach the cells with the least amount of cellular
disruption,
kinetics of cell detachment were investigated after simply removing the
activating antibody.
Detachment of the U937 cells from commercially prepared Fn-coated dishes
(denatured Fn)
occurred spontaneously after the mAb was removed by washing with buffer (Fig.
13).
Approximately 50% of the cells detached within 24 hours of mAb removal and
virtually
100% were detached by 48 hours. A more rapid rate of release could be achieved
by gently
applying a stream of buffer directly on the cells via a pipette. When U937
cells were plated
on native or aggregated Fn the detachment occurred more slowly.
Detachment of either the U937 cell or hSC's did not readily occur on Rn after
simply
removing the mAb. 'This effect was seen whether Rn was coated in PB S or in
the carbonate
buffer.
hSC's were more resistant to spontaneous release after mAb removal. Only 40%
of
the cells spontaneously released from the Fn by 48 hours post-mAb removal
(Fig. 15).
However, the cells could be effectively released by pipetting, as described
for the U937
above.
It is anticipated that peptide sequences (unmodified or perhaps chemically
modified
to increase the affinity for cell surface expressed adhesion molecules)
corresponding to
targeted amino acid sequences in the adhesive (e.g., peptides containing the
LDV or RGD
sequences) may be employed as releasing agents to detach cells from an
immobilized state.
Again, it is anticipated that cell immobilization involving several
adhesion/adhesive
interactions may be best competed by a cocktail of peptides. By way of
example, where
retronectin is used to promote the attachment of cells, the following
compositions alone or
in combination may be used to detach the cells: 1. LDV sequences - provides
interference
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with a4 (i, binding to fibronectin; 2. RGD sf;quences - provides interference
with as (3,
binding to fibronectin.
Studies by the inventors have demonstrated that peptides containing LDV or RGD
sequences promote the detachment of hSC's from both Rn and Fn.
Detachment of cells from an adhesive surface may also be accomplished by
antibodies which interfere with the normal interaction between adhesion
molecules and the
adhesive (e.g., anti-~i 1, anti-a 4, anti-VLA-4, anti-CS-1 antibodies).
Detachment may also
be accomplished by mono- or poly -sacchar:ides (or other charged molecules,
e.g., lipids,
polyanions and polycations) which may interfere with the normal interactions)
between
adhesion molecules and the adhesive.
l~Ptar~ent of cells using various reagents:
Various reagents can be used to detacln cells from an adhesive surface. For
example,
1. polyanions - may disrupt binding to the heparin binding site (eg. heparin,
heparin sulfate,
hyaluronan, other polysaccharides, etc.); 2. certain divalent cations (eg.
calcium) - are known
to disrupt integrin function; 3. certain chelating agents (eg. EDTA or EGTA) -
are known to
disrupt integrin function; 4) disintegrins - are naturally occurring soluble
proteins, originally
described fiom snake venom (Blobel, C.P., 1997), which contain integrin
binding sequences;
5. combinations of the above.
~;1 PM LE 12
METHOD OF PREPARING MICROINJECTION NEEDLES
The present example outlines a method that may be used in the preparation of
the
microinjection needles of the present invention. As will be readily
appreciated by one of
ordinary skill in the art, this procedure may be employed to prepare a number
of different
diameter needles without the exercise of an undue amount of experimentation.
Fine glass microinjection needles of approximately 0.2 +/- 0.02 micron outer
diameter
were prepared from thin-walled borosilicate ;glass capillaries (Sutter, 1.2 mm
O.D., 0.94 mm
LD.) with an automated pipette puller (Suffer, P-87, 3 mm box filament). Finer
glass
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microinjection needles of average outer diameter 0.12 micron, 0.15 micron, or
0.17 micron
were prepared from thick-walled borosilicate glass capillaries (Suffer, 1.2 mm
O.D., 0.6 mm
LD.) with the Suffer P-87 pipette puller. Parameters for temperature, pull
rate, and pressure
were optimized for each needle size desired. In order to rapidly quality
control the O.D. of
needles immediately after pulling, we perfonr~ed resistivity measurements on
sample needles
filled with electrolyte. By comparing the measured resistivity against a
calibration curve, one
can obtain a reasonable estimate of needle tip diameter. By SEM, verification
was obtained
that automatically pulled needles exhibit stru<:tural integrity and uniformity
of tip geometry.
EXAMPLE 13
~V~F~'HOD OF PRFPA~1NG A HOLDING PIPET
The holding pipette was prepared using a DeFonbrune microforge. Appropriate
bends
are made in a glass capillary so that it fits in a chuck assembly holder such
that a 1-5 gram
weight can be hung from the capillary positioned inside a heating filament.
Heat is applied,
softening the glass, resulting in the weight pulling the glass capillary to a
1-3 micron
diameter, at which point the piece of glass capillary from which the weight is
suspended
breaks away leaving a holding pipette with a 'l-3 micron diameter tip. The tip
is then brought
close to the heating filament, and the tip is heat polished resulting in a
smooth tip with an
opening of .5 to 2.5 microns. This holding pipette can then be attached to a
syringe assembly
that can be used to create a vacuum which urill hold the cell in place for the
microinjection
procedure.
When making such a holding pipette, one is not limited to the DeFonbrune
microforge. For example, any of the commercially available needle pulleys
(Suffer
Instruments, Narishige, Kopf) can be used to pull a capillary to the above
dimensions.
However, the tip must still be heat polished and appropriate bends made in the
holding
pipette using a microforge (DeFonbrune or lVarishige).
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~IPLE 14
MICROINJECTION METHOD EMPLOYING
MICROINJECTIQN NEEDL~AND HOLDING PIPET
The present example details one method by which the present microinjection
technique may be employed to introduce nucleic acid into the chromosomal DNA
of a cell.
In particular, the technique employs holding, pipettes that stabilize cells
through use of a
vacuum. In some embodiments, the technique; described here and variations
thereof may be
automated so as to provide a more rapid production of genetically modified
cells.
As an alternative, an inj ection chamber that can have a vacuum behind a
material
with pores sufficient to hold numerous cells in place for subsequent injection
may be
employed. This chamber in some embodiments will be used in conjunction with an
inverted microscope using phase contrast microscopy.
~P~
Viability of U937 Cells After Microinjection Usine
Borsilicate oy~~~artz Needles
1. Quartz Needles
Table 4
Total Cells Cells AliveCells AliveCells Alive
Injected After 2h After 24h After 48h
Study One 57 35 30 27
50 25 20 20
50 32 29 25
Study Two 50 36 30 23
62 48 41 36
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50 29 21 20
Total Survival319 205 171 151
ercentage 100 64% 54% 47%
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2. Borosilicate Needles
Talble 5
Total Cells Cells AliveCells AliveCells Alive
Injected After 2h After 24h After 48h
Study One 36 30 25 23
29 17 7 6
68 42 14 10
Study Two 56 36 27 20
30 19 18 18
51 29 11 10
Study Three 52 26 20 20
60 27 21 20
Total Survival382 226 143 127
ercentage 100 59% 37% 33%
EXAMPLE 16
Viability of Stem CD3~4+ After Microiniection
Using Borosi]js;ate; or O,uartz Needles
The present example demonstrates the utility of the present invention for
providing
enriched populations of viable, genetically rr~odified cells, by
microinjection.
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1. Quartz Needles
Table 6
Total Cells Cells Cells Alive Cells Alive
Injected Alive After After 48h
After 24h
2h
Study One 50 12 9 7
50 16 10 Ring Moved
50 18 15 15
17 14 14 9
Study Two 46 32 11 7
50 26 18 17
50 21 16 14
Study Three 50 17 14 12
50 22 16 12
Total Survival263 178 123 93
ercentage 100 68% 47% 44%
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2. Borosilicate Needles
Tahle 7
Total Cells Cells AliveCells Alive Cells Alive
Injected After
Alter 2h 24h After 48h
Study One S1 34 14 10
54 32 16 10
Total Survival105 66 30 20
ercentage 100 X63% 29% 19%
Percent viability data obtained using quartz injection needles (Version I.OQ-
hSCs:
0.07 micron O.D.) and borosilicate injection needles (Version 1.OB-hSCs: 0.25
micron O.D.)
injection needles. Such needles were used to inject U937 (Figs. 18 & 26) and
primary human
hematopoietic CD34+ cord blood stem cells (Figs. 19 & 27). Such cells were
prepared for
injection as described in Example I. The stem cells were injected
intranuclearly with Oregon
Green conjugated with Dextran (a detectable fluorescent molecule) and percent
viability
determined at 2, 24, and 48 hours. See Figure:. 18 and 19 for the viability
studies and Figures
20 and 21 for the data regarding the outer diameter of the tips (as measured
using a scanning
electron microscope) of the needles used to perform the experiments. See
Tables 4, 5, 6, &
7 above for the data used to produce the graphs shown in Figures 26 and 27.
Figures I 8 and
19 present data obtained in the first run study. Figures 26 and 27 present the
cumulative data
obtained in these and subsequent studies conducted under the same conditions.
The Version 1.OB and Version 1.OQ needles yield consistently higher
viabilities than
the data shown in Tables 2 and 3. It should be; noted that these new data
support and extend
the data shown in Tables 2 and 3. All data shown in Tables 2 and 3 was
collected while
performing manual nuclear injections into CD34+ cells, while the new data was
collected
while performing semi automatic nuclear injections into both CD34+ and U937
cells.
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It is important to note that for the most efficient gene therapy, it is
important to
demonstrate that the cells are attached sufficiently to use a semi-automatic
or automatic
microinjection system without disrupting the cells or significantly affecting
cell viability, as
this will allow for a large number of cells to be; injected in a short time
span. The data shown
in Figures 18 and 19 support our previous studies and supports our claim that
a semi-
automatic or automatic injection system can be used to deliver DNA and
proteins into the
nucleus of CD34+ cells as part of a gene therapy protocol.
An improvement in the Version I.OB and Version 1.OQ needles as regards flow
has
been noted, when compared to the borosilicate needles used to generate the
data shown in
Tables 2 and 3 (early prototype needles made. either on a DeFonbrune
microforge or pulled
using a different needle pulling program than has been developed for pulling
the new
needles). Although it was possible to inject cells using the early prototype
needles, as seen
by the data shown in Tables 2 and 3, routinely as many as 5 needles were
required to perform
approximately 50 injections. This prototype needle had flowing problems and
plugged very
easily. With the Version 1.OB and Version 1.OQ needles, the flow is improved
and the
Version 1.OB needle can be routinely used to inject greater than 50
cells/needle. The Version
l .OQ needle may be routinely used to inject greater than 25 cells/needle.
EXAMPLE 17
Per pa_ration of Needles for Sca_nning~lectron Microsconv:
Needle tips were sputter coated with. gold-palladium in a SCD004 Bal-Tec
sputter
coater for 120 seconds at lSmA. The needle tips were then scanned and
photographed using
a Phillips 525M scanning electron microscope at 15KV. Outer diameter
measurements were
determined and the .046 micron coat value subtracted as the correction factor.
Figure 28
represents photographs of scanning electron microscopic images of the 1.OB
(28A) and 1.OQ
(28B) needles.
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Table 8
Scanning EM Data for Wersion 1.0 Q-hSC's Needle
Column 1
1. .104
2. .129
3. . .154
4. .104
5. .I54
6. .054
7. .054
8. .054
9. .030
10. .030
11. .054
12. .054
13. .030
14. .004
15. .054
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16. .154
17. .129
1 g, .054
19. .054
20. .104
21. .029
22. .054
23, .029
24. .029
25. .054
26. .054
27. .079
28. .079
29. .054
The statistics generated from the above measurements appear below
Mean Std. Dev.Std. ErrorVariance Coef. Var.Count
0.07 0.042 0.008 0.002 60.103 29
Minimum Maximum Range Sum Sum of
Sqr
0.004 0.154 0.15 2.019 0.19
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TABLE 9 (Scanning EM Data for Version 1.OB-hSC's Needle)
Needle I~'am~( icrons)
1 0.254
2 0.304
3 0.304
4 0.179
0.179
0.179
7 0.204
The statistics from the above measurements appear below:
Mean Std. Dev.Std. Error Variance Coe Var.Count
0.229 0.058 0.022 0.003 25.212 7
Minimum Maximum Range Sum Sum
of
Sqr
0.179 0.304 0.125 1.603 ~
0.387
EXAMPLE 1818
jMPROVEMENTS TO MICROT_NJECTIO]~T TECHNO .O Y.
Monitoringysuccessful inj~ tec ions:
A. Resistivity measurements throughout the injection procedure accomplished
by monitoring flow from the needle throughout the injection process modifying
the needle
holder as shown in Figure 25.
If the needle is flowing properly, we should observe a specific resistivity
measurement based on the needle tip diameter, until the needle penetrates the
cell at which
time we expect to see (Brown, K.T., and D.G. Flaming: Advanced Micropipette
Techniques
for Cell Physiology, John Wiley and Sons, 1!92, p. 157.).
B. For monitoring nuclear injections, include an expression vector that upon
injection into the nucleus expresses a detecmble protein that does not hurt
the cell and can
be detected in the living cell without perturbing cellular function (e.g.,
pCMV-GEP
expression vector).
C. For monitoring either nuclear ~or cytoplasmic injections detectable
molecules
(e.g., Oregon Green conjugated with Dextran) can be injected and followed with
time.
D. A sound device could be incorporated into the assembly that will produce an
audible signal to indicate a successful injection (Figure 25).
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~lnnrovements of injection needles:
A. In some cases, the injection sample will be very sticky to the quartz or
borosilicate and will immediately plug the: needle, or coat the needle
diluting out the
molecule that you want to inj ect into the cell. We have worked out a
procedure that allows
us to siliconize these small needles including the Quartz Version with an
average O.D. of .07
microns. We are following a procedure used to siliconize much larger injection
needles used
in generating transgenic animals (DePamph.ilis, M.L., Herman, S.A., Martinez-
Salas, E.,
Chalifour, L.E., Wirak, D.O., Cupo, D.Y. 2md Miranda, M.: "Microinjecting DNA
into
Mouse Ova to Study DNA Replication and Gene Expression and to Produce
Transgenic
Animals" BioTechniques: 6: 662-680, 1988).
Injection pipettes are siliconized for 2-4 days in a desiccator with vapor
from a small
beaker of hexamethyidisilazane (Pierce). Tlus procedure produces a
monomolecular layer
of silicon coating the total injection needle. l:Jnfortunately, in the small
needles that we are
1 S using, this procedure negates the capillary filling of the injection
needle resulting in a large
air bubble in the end of the needle that cannot be expelled. We have found
that by making
a small glass filament, we can suck the air bubble out, resulting in a flowing
needle (Figure
22).
B. It is possible that using the Sutter Micropipette Beveler, a bevel may be
applied to these needles again improving their % viability when used to inject
the small stem
cells.
C. In the case of some embodiments of small needles, Version I.OQ and 1.OB
(see Figure below), the flare of the needle is important as regards both
loading of the needle
and flow from the needle. The P2000 needle puller was used to pull the quartz
needles. This
resulted in both a smaller needle tip that had. a greater flare.
Resistivity measurements from the Version 1.OB (O.D., 0.25 microns) needle
have
been obtained that equal those for the Version 1.OQ needle (O.D., 0.07
microns). The
1.OQ needle has a considerably smaller outer diameter. The Version 1.OQ needle
has
greater flare than that of the Version 1.OB arid flows when appropriate
pressure is applied,
whereas a borosilicate needle having a similar outer diameter (0.07 microns)
requires
pressures that exceeds those that can be produced by this particular injection
equipment.
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Flare tip
:
D1 -~~~ Ve:rsion 1 .OQ ;~-D2
1-- Defined Flare Region --r
Version 1.OB ;~--D2
Flare is an important characteristic that governs flow through the middle. As
can be seen
by looking at Table 10 and 12, respectively, the quartz injection needles
(Version 1.OQ)
have a greater flare than the borosilicate injection needles (Version 1.OB).
The D 1:D2
ratio for the borosilicate needles is in some embodiments an average of about
1:1.8 to
about 1:3. The quartz needles (Version 1.OQ) has a D1:D2 ratio that is, in
some
embodiments, 1:3 to about 1:18. "L" as defined in the diagram is the distance
(length)
between the flare tip, D1, and the diameter, I)2. In the actual needles
measured for Table
2, the length between D l and D2 was 1.3 microns.
Table 10 - Flare of Quartz Microinjection Needles, Version 1.OQ
D1 D2
D 1: D2
Ratio
1 0.10 4 1:6.800
v
2 0. 4 I :5.50
3 0.154 0.754 I:4.
4 0.104 .704 1: .8 0
5 0.1 0. 1:4.20
54
6 0.054 0.404 1:7. 0
7 . 54 . 54 1: . 00
0.05 0.2 1:4.700
9 0.030 0.454 1:15.1 0
10 0.03 0. 1:8.500
54
11 0. 54 0. 1:4.700
54
12 0.054 0.604 1:11.200
3 5 3 0.03 .204 1: 6.
14 0.004 0. 1:63.500
5
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1 S 0. S4 0.3~ 1: . 0
16 .154 .~8( 1:5.2
4--
17 .I2 0.7 1:5. 0
i 8 ~. S4 . S4 1:6. 0
S 19 O.OS 0. 1:5. 00
0 0.104 0. S4 1:3. 00
21 0. 29 0.~3 1:12.2
4
22 0.0 .~ 1:9.300
4
23 0.02 . S4 1:1 . 0
24 0.029 . 04 1:17. 0
2S 0. 4 0.454 1: . 0
26 _ .S 1: . 00
~4
27 0. 7 .S 1:6.400
8 0.07 0.45 1: .700
29 O.OS O.S 1: .300
Table 11
X, : D2:D 1 Ratio
Mean: Std. Dev.: Std. Error: Variance: Coef Var.: Count:
9.79 10.908 2.026 118.98 111.422 29
Minimum: Maximum: Range: Sum: Sum of Sqr.: # Missing:
3.4 63.5 60.1 283.9 6110.73 I4
2S
Table 12 - Flare of Borosilicate Mficroinjection Needles, Version 1.OB
D1 D2
D 1:D2 Ratio
1 .254 .604 1:2.300
2 .304 .SS4 1:1.000
3 .304 .654 1:2.150
4 .179 .504 1:2.800
5 .179 .504 1:2.800
6 .179 .SS4 1:3.100
7 .204 .504 1:2.400
8 .224 .604 1:2.700
9 .204 .404 1:2.000
10 .304 .SS4 1:1.800
11 .254 .SS4 1:2.200
12 .254 .654 1:2.600
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13 .254 .554 1:2.200
14 .279 .604 1:2.200
15 .329 .704 1:2.100
16 .304 .554 1:1.000
17 .254 .504 1:2.000
18 .204 .504 1:2.500
19 .204 .554 1:2.700
20 .254 .554 1:2.200
21 .304 .604 1:2.000
22 .254 .554 1:2.200
23 .229 .504 1:2.200
24 .254 .254 1:2.400
A ratio of D 1 to D2 in the range of about l :about 1.5 to about l :about 20,
or about
1: about 2 to about 1: about 10 provide the D~ 1:D2 width ratios according to
some
embodiments of the invention. In other aspects, the invention provides for
widths on the
average of about 1: about 7, or about 1: aboul: 6, or about 1: about 5, or
about 1: about 4,
or about 1: about 3, or about 1: about 2.5, or onally, in the range of about
1:1.5 to about
1:2.5 are within the range of the present invention.
Table 13
D2:D 1 lEtatio
Mean: Std. Dev.: Std. Error Variance: Coef Var.: Count:
2.298 .346 .071 .12 15.054 24
Minimum: Maximum: Range: Sum: Sum of Sqr.: # Missing
1.8 3.1 1.3 55.15 129.482 0
D. The capillaries used in making the injection needles have a internal
filament. This internal filament, among other things, helps the sample flow
into the tip of
the needle during the loading procedure. The loading process may also be aided
by the
filament increasing the capillary action. In loading the injection needles
with small outer
diameters, the needle should be loaded slowly. This will, among other things,
introduce
3 S as few air bubbles into the tip of the needle as possible. If air bubbles
are introduced, they
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cannot be expelled form the needle and the needle must be replaced. The
greater the
D 1:D2 ratio, the higher the number of needles that load effectively, having
only minimal
air bubbles introduced.
E. The hydration state of the small needles is also a factor to consider for
proper loading of the needles. Optimally, the needle should be used the same
day that it
is pulled. In preparation for pulling the needle, the capillary may be
attached to a vacuum
and solutions drawn through the capillary. B;y way of example, the following
fluids may
be drawn through the capillary.
I . DriCote (Fisher) to make the glass hydrophobic,
2. Acetone
3. Filtered 70% Ethanol-dehydration
Other solutions well known to those of ordinary skill in the art may also be
drawn up into
the capillary and used in the preparation of the needles, given the present
disclosure.
The capillaries are then baked at 200 ° C for up to an hour, and then
pulled using
either the P87 puller or the P2000 puller (botla from Suffer instruments). The
needles used
the same day that they are pulled work most consistently as regards flow. With
increasing
time the needles decrease in usefulness. The needles do not flow after 2 days
of storage. This
may be due to particulate matter that was becoming attached through
electrostatic interactions
with time, thus clogging the needle. Although this is true in some cases, the
majority of the
needles are believed to lose their capacity t~o flow, at least in part because
they become
hydrated (especially in humid conditions). With time, when attempting to load
the needle
with injection sample, numerous air bubbles form in the tip of the needle.
This will result
in a needle that does not flow. With the present technology, one cannot
achieve high enough
pressures to expel the air bubbles. However, by simply baking the needle at
200 ° C for 1
hour, one can restore the ability to load the needle with minimal air bubbles,
thus restoring
the flow.
~ LP E 19
C~,LL INJECTION WORK STATION
The present example defines the work: station of the present invention for use
in the
microinjection of cells. While many differf;nt configurations of the work
station will be
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appreciated from the one described herein, the present description defines one
particular
configuration of the station as envisioned by the present inventors. The work
station as
provided in one embodiment is illustrated in FIG. 25. The work station
comprises a
microscope stage
One of the challenges in microinjection technology using the automatic
injector is that
the injector must define how far the needle penetrates the cell (set a Z
value), without going
through the cell, and contacting the hard tissue culture plastic. Sometimes,
due to
inconsistency in the surface of the tissue cull:ure dish, the needle comes in
contact with the
tissue culture plastic, breaking the needle. There are several possible ways
to get around this
problem.
1. Place a pressure sensor in the micromanipulator that would indicate that
the
needle has contacted something harder than 'the cell.
2. Install a laser, sonar or radar device in the microinjection system that
would
monitor the needle tip position relative to the tissue culture dish, stopping
the
micromanipulator's motion whenever the tip comes close to the tissue culture
dish surface.
3. Apply a transparent coating to~ the tissue culture dish that is of a
composition
that the needle could penetrate without breaking. We may actually be able to
use the cell
attachment molecules as part of this transpa~~ent coating.
~;MPLE 20
MICROINJF;CTOR PLATES
Example A: Singhvi, R., A. Kumar, (J.P. Lopez, G.N. Stephanopoulos, D.LC.
Wang,
G.M. Whitesides and D.E. Ingber: "Engineering Cell Shape and Function" Science
264: 696-
698 (1994).
The technique utilizes an elastomeric stamp (polydimethyl-siloxane) to imprint
gold
surfaces with pre-defined patterns (micrometer-sized) of self assembled
monolayers of
alkanethiols. There may be a problem associated with this approach unless the
stamp can be
made transparent (See Example C; Fig. 23}.
Example B: A manifold, containing 5-10 micron O.D. needles arranged in the
same
defined pattern as the manifold containing the injection needles, would be
used to deposit
microdroplets of solutions containing adhesion molecules onto to a culture
surface (Fig. 24).
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Cells could then be plated onto these adhesiion islands, and the manifold
containing the
injection needles then used to inject the cells.
Example C: Mrksich, M., L.E. Dike, G.M. Whitesides: "Using Microcontact
Printing
to Pattern the Attachment of Mammalian Cells to Self Assembled Monolayers of
S Alkanethiolates on Transparent Films of Gold and Silver" Exp. Cell Res.
235:305 (1997).
This technique utilizes the same principles as in Example A except that now
transparent films
of gold and silver are used, thus making it possible to use phase contrast
microscopy to
monitor the injections.
E~~IPLE 21
GENE THERAPY METHOD
The present example demonstrates the utility of the present invention for use
in gene
therapy protocols together with the herein described injection compositions
and
microinjection methodology. Employing same, an alternative method for the
introduction
of nucleic acid into the nucleic acid of a cell v~rithout the use of a
carrier, such as a retrovirus,
adenovirus, or other carrier cell will be provided.
Gene therapeutic applications of stenn cell microinjection to include the
following
elements: Approximately 1-10 x 103 highly enriched stem cells will be obtained
from blood,
and will be temporarily immobilized. Microinjection of these cells will
deliver a
reproducible volume-containing DNA and possibly integration enzymes) - such
that 1-3
copies of the DNA are successfully integrated. per cell. Microinjected DNAs of
15-25 kb in
size, containing two independently regulated transgenes, will be integrated
without
rearrangement. One transgene, targeted for expression in stem cells, will
provide for in vitro
(e.g., rsGFP, or truncated nerve growth factor receptor; tNG-R) or in vivo
(e.g., MGMT)
selection of transduced stem cells. The therapeutic transgene (e.g., DNA for
ADA SCID,
globin for hemoglobinopathies, MDR-I for chemoresistance) will be targeted for
expression
in the appropriate hematopoietic cells.
The above is a detailed description of particular embodiments of the
invention. Those
of skill in the art should, in light of the present disclosure, appreciate
that obvious
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modifications of the embodiments disclosed herein can be made without
departing from the
spirit and scope of the invention. All of the compositions and methods
disclosed and claimed
herein can be made and executed without undue experimentation in light of the
present
disclosure. The full scope of the invention is set out in the claims that
follow and their
equivalents. Accordingly, the claims and specification should not be construed
to unduly
narrow the full scope of protection to which l:he present invention is
entitled.
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SEQ. ID NO. 1:
H-Glu-lle-Leu-Asp-Val-Pro-Ser-Thr-OH
SEQ. ID NO. 2:
H-Arg-Gly-Asp-Ser-OH
71
*rB
CA 02275474 1999-06-18
WO 98128406 PCT/US97123781
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