Note: Descriptions are shown in the official language in which they were submitted.
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FIBRIN SEALANT AS A TRANSFECTION/TRANSFORMATION VEHICLE
FOR GENE THERAPY
This application relates to the use of fibrin polymers as vehicles for
delivering
genetic material to a cell or tissue.
Fibrin sealants are used to create solid formulations of polymerized fibrin or
other
fibrin-related molecules. The form of the polymerization is typically
initially the
formation of stabile non-covalent associations between the fibrin molecules.
In many
cases. the non-covalent associations are supplemented by subsequent covalent
cross-links
that form due to the action of activated Factor XIII. These solid formulations
are often
used to stop or reduce fluid leakage after injury, such as air leakage from
the lung or
blood leakage.
One form of fibrin sealant overcomes concerns with the safety of blood
products
and the enzymes typically used to convert fibrinogen to a form capable of
polymerization
by using recombinant or autologous fibrinogen and keeping snake-derived
converting
enzymes well segregated from the step at which the sealant is applied to a
patient. See,
e.g., Edwardson et al., U.S. Patent 5,739,288. Autologous fibrinogen is
practical through
the technology of Edwardson et al., since preparation of the sealant is
conducted within
minutes from a small volume of blood; and segregation of the converting
enzymes is
possible by stabilizing the fibrin in soluble form while affinity binding
techniques are
used to segregate the enzymes away from the sealant.
In gene therapy, one seeks to transfect or transform cells of a certain cell
type,
such as liver cells, pancreatic cells, lung cells, muscle cells, leucocytes
and the like, to
insert an gene to correct a genetic defect or otherwise provide a helpful
function. Such a
gene can include a nucleic acid construct that expresses an antisense RNA to
interfere in
the expression of a certain mRNA or one or more constructs that express two
complementary strands designed to interfere in the expression of a certain
mRNA.
Similarly, nucleic acid-based vaccines seek to induce a percentage of cells to
produce immune-reaction inducing polypeptides, to induce an antibody-based or
cellular-based immune response.
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Where viral vectors are used in gene therapy, tissue specificity can be
provided
by the cell surface markers utilized by the virus to gain entry into the
cells. However,
this avenue is only helpful where a virus that targets a given tissue exists
and can be
practically utilized as a vector. Moreover, where viral vectors are known to
be favorable
for gene therapy, such as the adenovirus, their preferred cellular targeting
mechanism
may not be appropriate for the desired target cell types. Thus, further tools
are needed to
help increase specificity for the desired target cell type, and to overcome
vector
preferences for alternative cell targets.
Now provided are compositions of fibrin sealants that incorporate recombinant
vectors for delivery to a tissue or cell against which the sealant will be
polymerized and,
typically, adhered. By use of such compositions, the vectors can be maintained
at a
locally at high concentration in the solid gel produced by the sealant,
thereby increasing
the efficiency of transfection or transformation of cells. Moreover, the
fibrin gel is
tolerant of a number of agents used as adjuvants in the transfection or
transformation of
cells. Thus, such fibrin sealant compositions can be used to deliver vectors
to cells or
tissues, whether or not the sought for transfection or transformation is in
connection with
traditional concepts of gene therapy. The method can be used, for example, to
create
cells, such as plant cells, that produce a desirable product (such as a
protein or a small
molecule produced as a result of the transforming event).
Summary of the Invention
In one embodiment, the invention provides a method of transforming a cell
comprising the steps of: applying a transformation effective amount of a
nucleic acid to
the cell; applying a fibrin gel to the cell so as to entrap a transformation
effective amount
of the nucleic acid; and transforming the cell with the nucleic acid. In one
aspect, the
nucleic acid is applied in admixture with a fibrin or fibrinogen composition
that forms
the fibrin gel.
In another embodiment, the invention provides a method of conducting gene
therapy comprising: conducting the steps outlined above; and implanting the
transformed cells into an animal. In one aspect, the cell to which the nucleic
acid is
applied is a precursor of a more specialized cell type, and the method further
comprises:
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maturing the cell to the specialized cell type either ire vitro or irz vivo
following the
implanting.
In another embodiment, the invention provides a method of conducting gene
therapy comprising the steps of: applying a transformation effective amount of
a gene
therapy effective nucleic acid to a tissue; applying a fibrin gel to the
tissue so as to
entrap a transformation effective amount of the nucleic acid; and transforming
cells of
the tissue with the nucleic acid. In one aspect, the method further comprises:
surgically
exposing the tissue to allow for the applying steps.
In still another embodiment, the invention provides a method of conducting
surgery on an animal comprising: surgically exposing an internal tissue;
applying a
transformation effective amount of a nucleic acid to a tissue; applying a
fibrin gel to the
tissue so as to entrap a transformation effective amount of the nucleic acid;
and
transforming cells of the tissue with the nucleic acid, wherein the nucleic
acid encodes
antigens or contains peptides that induce an antibody or cytotoxic T
lymphocyte response
to infection by a pathogenic microbe. In one aspect, the nucleic acid encodes
antigens or
contains peptides that induce an antibody or cytotoxic T lymphocyte response
to
infection by a pathogenic microbe that is a member of the genus Streptococcus,
Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria,
Haemophilus,
Actinonrycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,
Pasturella,
Moraxella, Acinetobacter, Erysipelothrix, Br-anhamella, Actinobacillus,
Streptobacillus,
Listeria, Calymmatobacteriunr, Brucella, Bacillus, Clostridium, Treponema,
Escherichia,
Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira,
Spirillum,
Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,
Chlamydia,
Borrelia, Mycoplasma, Helicobacler, Saccharonryces, Kluveronryces, Candida, or
Pneumocytis.
In yet another embodiment, the invention provides a kit comprising: (a) a
first
composition for forming a fibrin gel comprising one of (i) fibrin monomer,
(ii)
fibrinogen or another fibrin precursor or (ii) a fibrin-analog; (b) a second
composition
for forming a fibrin gel comprising ( 1 ), where the first composition is
pursuant to (i), an
agent that reverses the conditions which stabilize fibrin as the monomer, (2),
where the
first composition is pursuant to (ii), an agent that converts the fibrinogen
or
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fibrin-precursor to fibrin or (3), where the first composition is pursuant to
(iii), a
fibrin-related molecule that forms a gel with the fibrin-analog; and (c)
composed
separately in a third composition or incorporated into the first or second
composition, a
gene therapy effective amount of nucleic acid, wherein the fibrin gel formed
of the first
and second compositions is effective to entrap the nucleic acid in the
vicinity of a cell or
tissue. In one aspect, the nucleic acid is composed with a separate adjuvant
for
increasing the efficacy with which the nucleic acid transforms or transfects
cells.
In yet still another embodiment, the invention provides a method of conducting
gene therapy comprising: transforming or transfecting cells with a nucleic
acid to create
recombinant cells; implanting the recombinant cells into an animal; and
applying a
fibrin gel to entrap recombinant cells at a desired location within the
animal. In one
aspect, the method further comprises: surgically exposing the tissue to allow
for the
implanting and applying steps.
Fibrin and Blood Clotting
One mechanism for hemostasis, i.e., prevention of blood loss, is the formation
of
a blood clot. Clot formation in humans occurs by means of a complex cascade of
reactions with the final steps being the conversion of fibrinogen by thrombin,
calcium
ions and activated Factor XIII to form ultimately cross-linked fibrin II
polymer,
alternatively known as insoluble fibrin II polymer, which is the insoluble
fibrin clot.
Fibrinogen represents about 2 to 4 grams/liter of the blood plasma protein and
is a
complex protein consisting of three pairs of disulfide-linked polypeptide
chains
designated (Aa)z, (B/3)2, and y2. "A" and "B" represent two small amino
terminal
peptides, known as fibrinopeptide A and fibrinopeptide B, respectively. The
six
polypeptide chains of fibrinogen are folded into at least three globular
domains in a
linear disposition, two terminal "D-domains" and a central "E-domain". The E-
domain is
believed to contain all six N-terminal residues of the polypeptide chains in
fibrinogen
molecule. Each D-domain contains the C-terminal sequence from one a-chain, one
(3-chain. and one y-chain.
The formation of insoluble fibrin clots (e.g., cross-linked fibrin II polymer)
is
believed to begin with fibrinogen being converted by thrombin to fibrin I
monomer.
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This conversion involves thrombin-mediated cleavage of the 16 amino acid
fibrinopeptide A (G I -R 16) from each the two Aa-chains of fibrinogen,
producing two
a-chains each with a new N-terminal having the amino acid sequence G 17-P-R-
V20-.
The fibrin I monomer, it is believed, can spontaneously polymerize with other
fibrin I or
fibrin II monomers due to intermolecular interactions (i.e., non-covalent
bonds) between
the E-domain of the converted fibrin monomer, which now has accessible non-
covalent
bonding sites, and a D-domain of a different fibrin I or fibrin II monomer.
Each
D-domain of a fibrin monomer carries a polymerization site capable of stably
interacting
with an E-domain of a fibrin I or fibrin II monomer.
Contacts between the two E-domain polymerization sites of one fibrin I monomer
with two complementary D-domain polymerization sites, each from two different
fibrin I
monomers, are believed to result in linear fibrin fibrils (i.e., polymers)
with half
staggered overlapping molecular contacts. The fibrin I polymer so formed is
sometimes
referred to as soluble fibrin I polymer because, by treatment with appropriate
chemical
means, the fibrin I polymer can be depolymerized and reconverted to fibrin I
monomers.
The next step in the formation of fibrin clots involves the conversion of
fibrin I
monomer to fibrin II monomer. This step involves the thrombin-mediated
cleavage of
the fibrinopeptide B from each of the two B/3-chains of fibrin I. The removal
of the 14
amino acid fibrinopeptide B produces (3-chains, each having a N-terminal
sequence of
G-H-R-. Fibrin II monomers, like fibrin I monomers, can spontaneously
polymerize with
other fibrin II or fibrin I monomers due to intermolecular interaction sites
in the E-
domain of one fibrin II monomer, which are made accessible by the cleavage
reaction,
with the D-domain of another fibrin II or fibrin I monomer. Like fibrin I
polymer, fibrin
II polymer is also sometimes referred to as soluble fibrin II polymer because
by use of
appropriate chemical treatments it can be depolymerized and reconverted to
fibrin II
monomers. The exposure of the ~3-chain N-terminal sequences in the E-domain is
important to fibrin clot formation as it facilitates covalent crosslinking by
activated
Factor XIII of adjacent fibrin II monomers in the fibrin II polymer. Although
activated
Factor XIII is also capable of crosslinking fibrin I monomers in a fibrin
polymer, the
reaction is less efficient due to the presence of fibrinopeptide B on fibrin
I. Cross-linked
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fibrin II polymer is sometimes referred to as insoluble fibrin II polymer
because it cannot
be depolymerized and reconverted to fibrin II monomers .
In addition to thrombin and Factor XIII, calcium ions are believed to be
important
in the formation of fibrin clots and have a number of important roles. Calcium
ions are
believed necessary for the activation of prothrombin to thrombin, and since
thrombin
activates Factor XIII, calcium ions are indirectly necessary for Factor XIII
activation.
Further. active Factor XIII is believed to be a calcium-dependent enzyme that
cannot
cross-link fibrin polymers in the absence of calcium ions. Calcium ions also
directly
bind to polymeric fibrin and change the opacity and mechanical properties of
the fibrin
polymeric strands. For reviews of the mechanism of blood coagulation and the
components of a fibrin clot, see C.M. Jackson, Ann. Rev. Biochem., 49:765-811,
1980,
and B. Furie and B.C. Furie, Cell, 53:505-518, 1988.
Fibrin Sealants
A fibrin sealant is a biological adhesive whose effects imitate the stages of
coagulation to form a fibrin polymer. The sealant can be designed so that the
fibrin
monomer will be converted to insoluble fibrin polymer. One type of fibrin
sealant uses
fibrinogen and consists of two components. One component comprises
concentrated
human fibrinogen, bovine aprotinin and Factor XIII. The second component
comprises
calcium chloride and an enzyme, such as thrombin, that converts fibrinogen to
fibrin.
Application of this type of sealant is generally carried out with a double-
barreled syringe,
which permits simultaneous delivery of both components to the desired site of
the fibrin
clot formation. The mixing of the two components at the target site produces a
fibrin clot
via the sequence of reactions described above.
The fibrinogen component of this type of fibrin sealant is typically prepared
from
pooled human plasma. The fibrinogen can be concentrated from the human plasma
by
cryoprecipitation and precipitation using various reagents, e.g.,
poly(ethylene glycol),
diethyl ether, ethanol, ammonium sulfate or glycine. For reviews of this type
of fibrin
sealants, see M. Brennan, Blood Reviews 5:240-244, 1991; J.W. Gibble and P.M.
Ness,
Transfusion 30:741-747, 1990; H. Matras, J. Oral Maxillofac. Surg. 43:605-611,
1985
and R. Lerner and N. Binur, J. ofSurgical Research 48:165-181, 1990.
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A second, newer type of fibrin sealant uses compositions consisting primarily
of
fibrin I or fibrin II monomers. See European Patent Application No. 0 592 242,
published April, 1994. In these types of sealants, fibrin I monomers or fibrin
II
monomers or desBB fibrin monomers are prepared in advance of sealant
application
from fibrinogen using an appropriate proteolytic enzyme, such as thrombin or
batroxobin. The fibrin monomers are maintained in soluble form using an
appropriate
buffer. Useful buffers include those that have a low pH or a chaotropic agent.
The fibrin
I monomers, fibrin II monomers or desBB fibrin monomers in such solutions can
be
converted to fibrin polymers by mixing the solution with a second solution to
produce a
mixture with conditions that permit the spontaneous polymerization of the
fibrin
monomers to form a fibrin clot.
Fibrin I, fibrin II and desBB fibrin monomer-based sealants have several
advantages over fibrinogen-based sealants. Notably, fibrin monomer-based
sealants do
not include bovine or human thrombin. The use of such sealants, when the
fibrin
monomer is prepared from the autologous source (i.e., the patients
themselves),
introduces no foreign proteins into the recipient and thereby avoids
complications arising
from immunological reactions and risk of blood-borne infections. The fibrin
monomer-
based sealants can be conveniently prepared. Soluble fibrin polymer can be
dissolved
using a weak acidic solution. In some embodiments, the resulting fibrin
monomers are
lyophilized to fine powders. Such powders can easily be re-dissolved in a weak
acid and
induced to re-polymerize by the addition of an alkali buffer. Alternatively,
the powdered
fibrin monomers can be dissolved in a chaotropic solution, e.g., urea, to a
high
concentration (> 150 mg/ml) and induced to re-polymerize by the addition of
water.
A further advantage of fibrin monomer-based sealants is that as they generally
use autologous components, their use poses a lower risk of exposure to blood-
transmitted
infectious agents such as hepatitis (including hepatitis B, and non-A, non-B
hepatitis)
and acquired immune deficiency virus (AIDS). See L.E. Silberstein et al.,
Transfusion,
28:319-321, 1988; K. Laitakari and J. Luotonen, Laryngoscope 99:974-976, 1989;
and
A. Dresdale et al., Annals of Thoracic Surgefy 40:385-387, 1985. Diseases
caused by
such agents can be transmitted by conventional fibrinogen-based sealants
because the
fibrinogen component is typically prepared from pooled human plasma. Moreover,
the
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use of fibrin-based sealants can also avoid the risks associated with the
bovine thrombin
component of fibrinogen-based sealants. Bovine thrombin preparations can carry
the
infectious agent bovine spongiform encephalitis (BSE) as well as viral
pathogens of
mammals. Also, bovine thrombin is a potent antigen, which can cause adverse
immunological reactions in humans. For further discussions of these types of
complications that are associated with fibrinogen-based sealants, see Taylor,
J. Hospital
Infection 18 (Supplement A):141-146, 1991 and Prusiner et al., Cornell Vet
81:85-96,
1991.
Recombinant Fibrinogen and Fibrin
Genetic engineering can produce fibrinogen and fibrin monomers in
comparatively high yields, in substantially pure form, and in the absence of
pathogenic
viruses such as hepatitis and HIV. Heterologous expression of fibrinogen and
fibrin
chains also allows the construction of mutations which can mimic naturally
occurring
fibrin variants, and the isolation and study of these proteins without a need
for patients
with these rare genetic defects.
Each of the three different polypeptide chains (Aa,, B~3 and y) of fibrinogen
is
coded by a separate gene. The cDNAs for each of these chains have been
prepared
(Chung et al., Ann. N. Y. Acad. Sci. 408:449-456, 1983; Rixen et al.,
Biochemisdy
22:3237-3244, 1983; Chung et al., Biochemistry 22:3244-3250, 1983; Chung et
al.,
Biochemistry 22:3250-3256, 1983) and expressed in prokaryotic organisms.
Furthermore, each human fibrinogen chain has been introduced separately (Huang
et al.,
J. Biol. Chenz 268:8919-8926, 1993; Roy et al., J. Biol. Cheer. 267:23151-
23158, 1992;
Roy et al., J. Biol. Cheer. 266:4758-4763, 1991 ) or in combination (Hartwig
and
Danishefsky, J. Biol. Chem. 266:6578-6585, 1991; Huang et al., J. Biol. Chem.
268:8919-8926, 1993; Roy et al., 1991, J. Biol. Cheer., 266:4758-4763; Redman
and
Samar, U.S Patent Application 07/663,380, filed March, 1991, available from
Natl.
Technology Information Service No. PAT-APPL07663 380INZ) into expression
plasmids and transfected into eukaryotic cells.
Most of the plasmids used in expressing recombinant human fibrinogen are
derived from those constructed by Dr. D. Chung, University of Washington,
Seattle and
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are based on cDNA clones (Rixen et al., Biochemistry 22:3237-3244, 1983; Chung
et al.,
BIOCl7e1)7l.S~rl~ 22:3244-3250, 1983; Chung et al., Biochemistry 22:3250-3256,
1983).
The expression of recombinant fibrinogen chains was first achieved in E.coli
(Bolyard
and Lord. Gene 66:183, 1988; Bolyard and Lord, Blood, 73:1202-1206, 1989; Lord
and
Fowlkes, Blood. 73:166-171. 1989). The individually expressed chains show
antigenic
similarities with fibrinogen and display thrombin cleavable sites similar to
those found in
native fibrinogen (Bolyard and Lord, Blood, 73:1202-1206, 1989; Lord and
Fowlkes,
Blood. 73:166-171, 1989). Fibrinopeptides A and B can be released from
recombinant
fibrinogen (Bolyard and Lord, Blood, 73:1202-1206, 1989; Lord and Fowlkes,
Blood,
73:166-171.1989).
Eukaryotic cells carrying appropriate expression plasmids encoding individual
fibrinogen chains have been shown to synthesize the encoded fibrinogen chains
and to
result in the intracellular formation of dimeric chain molecules, e.g. Aa2,
B(3, or y2
dimers (Roy et al., J. Biol. Cheat., 265:6389-6393, 1990; Zhang and. Redman,
J. Biol.
1 ~ Che»z. 267:21727-21732. 1992). Furthermore, when appropriate plasmids
containing
genes encoding for all three human fibrinogen chains are transferred into the
same cell,
then not only are all three chains expressed but the polypeptide chains
associate in pairs
and intact fibrinogen is secreted into the surrounding medium (Roy et al., J.
Biol. Che»z.,
266:478-4763, 1991; Hartwig and Danishefsky, J. Biol. Che»r. 266:6578-6585,
1991).
Like natural fibrinogen, the secreted recombinant fibrinogen consists of three
pairs of
non-identical polypeptide chains and is functional in forming fibrin polymers.
Fibrinogen is naturally synthesized by liver, and megakaryocyte cells and
transformed liver cells maintained in culture are able to continue fibrinogen
synthesis
and secretion (See Otto et al., J. Cell. Biol. 105:1067-1072, 1987; Yu et al.,
Thrornb. Res.
46:281-293, 1987; Alving et al., Arclz Biochem. Biophys. 217:19, 1982). One
such cell
line is the Hep G2 cells (Drs. Knowles and Aden, blister Institute,
Philadelphia). This
line synthesizes an excess of Aa- and y-chains over the Bb-chains resulting in
non-
productive dimeric complexes of Aa- and y-chains (e.g., Aa2y2). The
introduction of
an additional expression vector encoding B(3-chains resulted in the formation
of trimeric
complexes (AaB(3y) which adopt the correct folding and intrachain disulfide
bonding
patterns (Roy et al., J. Biol. Clze»i., 265:6389-6393, 1990). The mechanism of
this
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folding is unknown and may involve ancillary proteins and enzymes (Roy et al.,
J. Biol.
Chem., 267:23151-23158, 1992). These studies demonstrated not only the correct
transcription of B~3 cDNA but also that the excess B(3-chain enhanced the
assembly and
secretion of intact fibrinogen.
In Hep G2 cells, the Aa,B(3y trimeric complexes associate in pairs to form
intact
fibrinogen molecules, which become glycosylated and are actively secreted from
the cell
(Huang et al., J. Biol. Chenz 268:8919-8926, 1993). Indeed only correctly
assembled
fibrinogen molecules are secreted. Thus, Hep G2 cells have the synthetic and
secretory
apparatus for the assembly of fibrinogen.
Subsequent experiments have introduced fibrinogen chain encoding cDNA
plasmids into eukaryotic cells that do not normally synthesize fibrinogen.
These
experiments successfully produced functional fibrinogen, demonstrating that
the factors
needed for fibrinogen assembly and secretion are not unique to liver-derived
cells like
Hep G2. Eukaryotic cells known to be capable of assembling and secreting
recombinant
fibrinogen include baby hamster kidney cells (BHK), COS cells and Chinese
hamster
ovary cells (CHO) (Roy et al., J. Biol. Chem. 266:4758-4763, 1991; Hartwig and
Danishefsky, J. Biol. Chem. 266:6578-6585, 1991; Farrell et al., Biochemistry
30:9414-
9420, 1991 ).
Intact functional fibrinogen secreted by stably transformed eukaryotic cells
results in the accumulation of fibrinogen levels of around 1-2 pg/ml. Methods
are
known for increasing the output of recombinant proteins from transfected cells
like CHO
cells such that the expression levels can approach a thousand fold the basal
secretory
level.
Additional description of methods of recombinantly producing fibrin-related
molecules can be found in PCT/LJS95/05527.
Gene Therapy
A lesson of the last 20 plus years in which scientists have begun actively
considering methods to introduce genetic material into appropriate target
tissues to
overcome a genetic disease has been that the effort is more complex than was
initially
anticipated. Some of the goals needed to be met to create successful gene
therapy tools
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include: ( 1 ) efficient transduction of the target cells; (2) long-term
expression of the
gene; (3) lack of a disabling immune response to the vector or transduced
cell; and (4)
absence of toxicity. See, Samulski et al., "Adeno-associated Viral Vectors" in
Developmefzt of Human Gene Therapy, Cold Spring Harbor Laboratory Press, 1998,
pp.
131-172. (This article, along with this entire treatise on gene therapy, is
incorporated by
reference herein.) All the above listed goals, especially the first three,
identify areas that
have given rise to substantial barriers to efficient gene therapy. Vectors
typically
transduce only a percentage of the cells to which they are applied. The
transducing gene
is often maintained on an episome and is therefore often not a stably
incorporated and
maintained genetic element. Moreover, incorporation into the chromosomal DNA
is
often dependent on cell division, thereby limiting the scope of target tissues
to replicating
tissues. Viral vectors often carry the nucleic acid encode proteins that
induce immunity,
thereby carrying the seeds for the destruction of the transduced cells.
Certain viral
vectors overcome some of these problems but otherwise create at least an
implication of
danger. For example, non-replicating forms of the human immunodeficiency virus
are
being engineered for use as gene therapy vectors that allow for the
incorporation of the
genetic material into genomic DNA. Such vectors must maintain the genetic
tools by
which to facilitate genomic incorporation, but must lack enough of the gene
products that
create infectivity, such that in this case for AIDS there is no chance that
recombination
events will regenerate an infective particle. See, Naldini et al., "Lentiviral
Vectors" in
Development of Human Gene Therapy, Cold Spring Harbor Laboratory Press, 1998,
pp.
47-60.
The good news is that all of these problems are now well-recognized, and the
viral vectors used in gene therapy have improved to address such problems.
Moreover,
gene therapy can be conducted without viral vectors. Also, in other genetic
transformations the problems of toxicity and immune response do not come to
fore to the
same degree. In nucleic acid-based vaccines, for example, an immune response
is
desirable, as can be a process by which expression of the transforming gene
attenuates so
that production of the immuno-stimulant attenuates over time.
Viral vectors have also been subject to engineering to change their target
cell
preference, for instance by binding or incorporating antibodies. For instance,
Valsesia-
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Wittmann et al. modified the cell-surface binding characteristics of avian
leukosis virus.
J. Yirol. 68: 4609-4619, 1994. Erythropoietin, which of course binds its
cognate
receptor, has been incorporated into Moloney murine leukemia virus (Mo-MLV).
Kasahara et al., Science 266: 1373-1376, 1994. A tumor-targeting single-chain
antibody
has been incorporated into spleen necrosis virus. Chu and Dornburg, J. Virol.
69: 2659
2663. 1995. HIV envelop protein has been incorporated into murine leukemia
viral
vectors. Mammamo et al., J. Virol. 71: 3341-3345, 1997. Such targeting methods
with
respect to adenoviral vectors are reviewed by Reynolds and Curiel, "Strategies
to Adapt
Adenoviral Vectors for Gene Therapy Applications: Targeting and Integration,"
in
Development of Human Gene Therapy, Cold Spring Harbor Laboratory Press, I 998,
pp.
111-130.
As reviewed in Development ofHuman Gene Therapy, Cold Spring Harbor
Laboratory Press, 1998, a wide variety of viral vectors have been selected or
engineered
for gene therapy. Moreover, nucleic acid can be delivered successfully without
the use
of viral vectors. For example, an early-developed method for increasing
transfection
efficiency was to use calcium phosphate-precipitated nucleic acid. The
transfection
potential of nucleic acid is increased by compacting it with polycationic
polymers such
as DEAF dextran (Veheri et al., Virology 27: 434-436, 1965), polylysine (Wu et
al., J.
Biol. Chem. 266: 14338-14342 1991), cationic peptides (Wadhwa et al.,
Bioconjugate
Chenz 8: 81-88, 1997; and Niidome et al., J. Biol. Che»z 272: 15307-15312
1997),
polyethyleneimine (Boussiff et al., Proc. Natl. Acad Sci USA 92: 7297-7301,
1995), a
glucaramide-based polyamino polymer (Goldman et al., Nat. Biotechnol. 15: 462-
466,
1997), polyamidoamine dendrimers (Dielinska et al., Biochim. Biophys. Acta
1353: 180-
190, 1997). Other polymers useful as enhancers of nucleic acid uptake include
erodable
2~ microspheres (Mathiowitz et al., Nature 386: 410-412, 1997) and polyvinyl
pyrrolidone
(Mumper et al., Phar»z Res. 13: 701-709, 1996). Other enhancers include
cationic
liposomes into which the nucleic acid is incorporated. Felgner et al., 1987;
Felgner and
Ringold, 1989. Such liposomes, or "lipoplexes," are believed to insert the
nucleic acid
into a target cell by a membrane fusion mechanism. Illustrative of the many
cationic
lipid formulations now available (see, Felgner et al., "Synthetic Delivery
Systems," in
Deoelopment of Human Gene Therapy, Cold Spring Harbor Laboratory Press, 1998,
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pp. 241-260), is DOTMA (N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium).
Other such cationic lipid formulations include LipofectinT"', a 1:1 (w/w)
liposome
formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-
trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine
(DOPE), LipofectAMINET"', a 3:1 (w/w) liposome formulation of the polycationic
lipid
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-
propanaminiumtrifluoroacetate (DOSPA) and the neutral lipid dioleoyl
phosphatidylethanolamine (DOPE) in membrane-filtered water, and
LipofectACE'~"', a
1:2.5 (w/w) liposome formulation of the cationic lipid dimethyl
dioctadecylammonium
bromide (DDAB) and dioleoyl phosphatidylethanolamine (DOPE) in membrane-
filtered
water (all from Life Technologies, Rockville, MD). Moreover, gene transfer can
also be
achieved without such adjuvants. Targeting techniques can also be employed
which bind
or affix targeting molecules to the nucleic acid or nucleic acid complex to be
used for
transfection. Cotton and Wagner, "Receptor-mediated Gene Delivery Strategies,"
in
Development ofHuman Gene Therapy, Cold Spring Harbor Laboratory Press, 1998,
pp. 261-277.
As will be recognized by those of ordinary skill, the nucleic acid sought to
be
introduced into cells will often include, in addition to the portion conveying
the primary
genetic characteristic of interest, a portion encoding a substance that is
itself, or gives rise
to, a molecule that is readily detectable. This "reporter" molecule serves as
a surrogate
for determining or estimating success in introducing the primary genetic
characteristic.
Where cells in culture are being transformed, a portion of the nucleic acid
can encode a
substance required for the cells to survive in the face of an appropriate
challenge.
The nucleic acid can be single or double-stranded, though non-virally mediated
techniques that seek to express a portion of the nucleic acid will typically
use
double-stranded nucleic acid.
Preparation of a Preferred Sealant Composition
First, blood is collected and a plasma is isolated. Added to the plasma is an
enzyme that converts fibrinogen to fibrin. In converting fibrinogen to fibrin,
preferably
care is taken to prevent the formation of cross-links between fibrin molecules
via the
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transaminase activity of factor XIIIa. This can be done by a number of
techniques
including for example the use of factor XIIIa inhibitors such as heavy metals
(such as
mercury). thiomerosal {[(o-carboxyphenyl) thioJethyl mercury sodium salt},
inhibitory
antibodies. or calcium chelators (since calcium is a necessary cofactor for
the enzyme).
Calcium chelators include, but are not limited to, EGTA (ethylenglycolbis-(2-
aminoethylether)tetra-acetic acid), and the like. For example, the converting
enzyme is
batroxobin, ("Btx"), a proteinase from the snake venom of snakes of the genus
Bothrops,
used at a concentration of about 0.1 pg/ml to about 100 pg/ml, preferably to a
concentration of about 0.5 pg/ml to about 50 pg/ml.
Other proteinases of appropriate specificity can also be used. Snake venom
proteinases are particularly suitable, including without limitation the venom
enzymes
from Agkistrodon acutus. Agkistrodon contortrix contortrix, Agkistrodon halys
pallas,
Agkistrodon (Calloselasma) rhodostoma, Bothrops asper, Bothrops atrox,
Bothrops
insularis, Bothrops jararaca, Bothrops Moojeni, Lachesis. ntuta muta,
C'rotalus
adamanteus, Crotalus durissus terrifrcus, Trimeresurus flavorviridis,
Trimeresurus
grarnineus and Bitis gabonica.
The fibrinogen-converting enzyme is favorably coupled to a converting enzyme
binding partner which is used in an affinity procedure to reduce the
concentration of the
enzyme in a preparation. In the example, the converting enzyme binding partner
is
biotin. a member of the biotin-avidin binding pair, a pair of molecules that
bind with
extremely high affinity. An amino acid sequence for avidin is described in
Dayhoff,
Atlas of Protein Sequence, Vol. 5, National Biomedical Research Foundation,
Washington, DC, 1972 (see also, DeLange and Huang, J. Biol. Chem. 246: 698-
709,
1971 ), and an amino acid sequence for Streptavidin is described in Argarana
et al., Nucl.
Acid Res. 14:1871-1882, 1986. Nucleic acid sequences are available, for
example, as
follows: (1) chicken mRNA for avidin, Gene Bank Acc. No. X05343, Gore et al.,
Narcl.
Acid Res. 15: 3595-3606, 1987; (2) chicken, strain White Leghorn gene for
avidin, Gene
Bank Acc. No. L27818 (3) streptavidin from Strep. avidinii, Gene Bank Acc. No.
X03591, Argarana et al., Nucl. Acid Res.14:1871-1882, 1986; (4) synthetic gene
for
streptavidin from Strep. avidinii, Gene Bank Acc. No. A00743, Edwards,
W089/03422;
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and (5) synthetic gene for streptavidin, Gene Bank Acc. No. X65082, Thompson
et al.,
Gene 136: 243-246, 1993.
Avidin and Streptavidin are preferably used in a tetrameric form, although
monomers can be used. Other binding pairs that bind with high affinity include
an
antibody specific for a polypeptide or other molecule, any polypeptide to
which an
antibody is available or can be prepared, thioredoxin, which binds
phenylarsine oxide
(expression vectors include, for example, the thioredoxin fusion protein
vector pTrxFus
available from Invitrogen, Carlsbad, CA), poly-His sequences that bind to
divalent
cations such as nickel II (expression vectors include, for example, the
pThioHis vectors
A, B and C available from Invitrogen), glutathione-S-transferase vectors that
bind to
glutathione (vector for example available from Pharmacia Biotech, Piscataway,
NJ).
Methods of producing such antibodies are available to those of ordinary skill
in light of
the description herein of polypeptide expression systems and of known antibody
production methods. For antibody preparation methods, see, for example,
Ausubel et al.,
Short Protocols in Molecular Biology, John Wiley & Sons, New York, 1992. Very
high
affinity binding characteristics, while highly convenient, are not essential.
Any affinity
that can be used in an affinity-binding procedure to reduce the concentration
of
converting enzyme in a preparation can be used in this context. Note that if
the affinity
procedure simply uses an antibody against the converting enzyme, then this
aspect of the
invention does not require a coupled converting enzyme binding partner, since
the
enzyme itself comprises the converting enzyme binding partner.
Unless the process is designed to prevent polymerization of fibrin monomer
during the enzymatic conversion from fibrinogen to fibrin, the fibrin formed
will
polymerize into fibrin polymer, and thereby form a fibrin clot. After the
solids are
isolated, fibrin monomer is recovered from the fibrin clot. Fibrin monomer is
recovered,
for example, by adding a solubilizing agent to the fibrin clot. Such
solubilizing agents
can include, for example, acid solutions such as aqueous solutions having pH
of about 5
or less, or chaotropic agents, such as urea, sodium bromide, guanidine
hydrochloride,
potassium cyanide, potassium iodide or potassium bromide. The solubilizing
agents can
be used at near the minimum concentration effective to maintain fibrin monomer
(i.e., a
fibrin-solubilizing effective amount). A number of conditions for forming
fibrin
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monomer are described in Edwardson et al., European Patent Application No. EP
592,242.
A solid material having bound thereto a second binding partner, which is the
complementary binding partner to the converting enzyme binding partner, is
then added
the fibrin monomer preparation to bind any converting enzyme as may continue
to be
found in the preparation. The solids, which, depending on the protocol used,
can include
the solid material or any residual fibrin clot material, is then removed, for
instance by
filtration or centrifugation.
The processed material can be stored in liquid form, for instance at about
4°C or
less. in frozen form, or as a dried form such as a lyophilizate. Lyophilizates
are formed
by standard methods. These lyophilizates are generally reconstituted in
purified water or
in a buffered aqueous solution. For the fibrin monomer, generally, the same
solution
composition of solubilizing agent previously used in the process can be used
to
reconstitute the lyophilizate. Or, if the user desires the fibrin to
polymerize on
reconstitution, an aqueous solution, which either (a) lacks a solubilizing
agent or (b) is
capable of reversing any solubilizing conditions carried in the lyophilizate,
is employed.
As illustrated, to form fibrin sealants (i.e., clots) the fibrin monomer and a
non-
enzymatic polymerizing agent can be mixed together. The polymerizing agent is
any
reagent effective to reverse the conditions that prevent the polymerization of
fibrin
monomer. For example, if fibrin monomer is in an acidic solution, such as a
0.2 M
sodium acetate, pH 4.0 solution, the polymerizing agent can be a basic
solution, such as,
without limitation, a solution of HEPES (N-[2-hydroxyethyl)piperazine-N~-
[ethanesulfonic acid]), sodium hydroxide, potassium hydroxide, calcium
hydroxide,
bicarbonate buffers such as sodium bicarbonate and potassium bicarbonate, tri-
metal
salts of citric acid, salts of acetic acid and salts of sulfuric acid.
Preferred alkaline
buffers include: carbonate/bicarbonate; glycine; bis hydroxeythylaminoethane
sulphonic
acid (BES); hydroxyethylpiperazine propane sulphonic acid (EPPS); Tricine;
morpholino
propane sulphonic acid (MOPS); trishydroxymethyl aminoethane sulphonic acid
(TES);
cyclohexylaminoethane sulphonic acid (CHES); trishydroxymethyl aminoethane
sulphonic acid (TES). The amount of alkaline buffer that is utilized should be
enough to
allow polymerization of the fibrin. It is preferred that about 10 parts to
about one part of
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composition comprising fibrin monomer be mixed with about 1 part alkaline
buffer. It is
even more preferred that such ratio be about 9:1. The preferred ratio depends
on the
buffer, its concentration and pH, and the desired concentration of the fibrin
polymer.
Where acidic pH is used as the solubilizing agent, the fibrin solubilization
can occur in
the presence of calcium ions, such as at a concentration of about 20 mM.
Incorporating Nucleic Acid into tire Fibrin Gel
In one preferred embodiment, three streams of aqueous preparations are mixed
to
initiate a rapid clot formation process. These preparations can be, for
example, a fibrin
monomer preparation, a composition comprising the nucleic acid for
transforming or
transfecting cells ("transforming composition" or "TC"), and a non-enzymatic
polymerizing agent. Or, in another example, the preparations are fibrinogen, a
fibrinogen-converting enzyme and the TC. To allow the resulting gel-forming
mixture to
remain pliable for period of time, the sealant mixture is generally formed
either during
the process by which the sealant is applied to its recipient surface, or
within a few
minutes prior to application. Generally, the sealant mixture remains
conveniently pliable
for about 30 seconds or less.
In another preferred embodiment, the three streams are sprayed so that they
converge and mix. Suitable spray heads are described in US Patent Nos.
5,605,541,
5,376,079, and 5,520,658 and PCT Application 97/20585. Where the spray heads
utilized are designed to spray only one solution, additional spray heads can
be aligned to
deliver other solutions to the site of delivery. For example, where the spray
head delivers
two concentric rings of sprayed solution or suspension, and uses gas outlets
to shape and
merge the streams, a second such spray head can be used to deliver a third
solution.
Instead of three streams, the TC can, where appropriate, be incorporated into
one
of the other two preparations.
Alternatively, the TC can be mixed with the sealant after the polymerization
process has been initiated but while the composition remains pliable. Or, the
TC can be
applied to cells or tissue, and the sealant can be applied to fix the TC in
place. Such a
subsequently applied sealant would preferably be applied concurrent with or
soon after
the process which polymerizes the sealant is initiated.
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Other types of fibrin sealant useful in the invention, other than that
described in
some detail above, are described in, for example: Wadtrom, U.S. Patent
5,631,01 l;
Cochrum, U.S. Patent 5,510,102; Pines et al., U.S. Patent 5,330,974; Matras,
J. Oral
Maxillofacial Surgery 13: 605-611, 1985; and Brennan, Blood Reviews 5: 240-
244,
1991. Another device for spraying a fibrin sealant is described, for example,
in Avoy,
U.S. Patent 4,902,281.
llTiscella»eo»s Aspects
When body fluids are used as the source for fibrin, in many cases it will be
desirable to isolate with the fibrin ancillary factors such as factor XIII or
factor XIIIa and
thrombin. When purification techniques are used that isolate fibrin via the
reversible
formation of a fibrin polymer, it is believed that the fibrin polymer has
affinity for a
number of such ancillary factors, such that the isolated product will retain
these factors.
In some cases, it will be desirable to limit the amount that non-fibrin
materials are
washed out of the fibrin polymer, for instance, by limiting the degree to
which the fibrin
polymer is compressed in the course of a method according to the invention, in
order to
assure the co-isolation of sufficient amounts of ancillary factors.
The present invention can be used for treating any animal having a fibrin-
based
system for controlling bleeding, but is preferably used for treating mammals,
most
preferably humans.
Def»itio»s
The following terms shall have, for the purposes of this application, the
respective meaning set forth below.
O cell precursor of a more specialized cell type. A precursor cell is a cell,
typically
referred to as a "stem" cell or a "pluripotent" cell, which has the potential
to, but has not
yet, differentiated into a more specialized cell.
O fibrin. One of a number of derivatives of fibrinogen {e.g., fibrin I (i.e.,
desAA-
fibrin). fibrin II (i.e., desAAdesBB fibrin) or des BB fibrin} that can
polymerize to form
a precipitate of fibrin polymer. The derivatives are typically created by
cleaving the A or
B fibrinopeptides from fibrinogen.
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O fibrin analog. A form of fibrin monomer is an engineered version of fibrin,
or "fibrin
analog," which will not self polymerize, but will polymerize with another
fibrin-related
molecule such as fibrinogen. Such an engineered fibrin is described in
Cederholm-
Williams et al., "Recombinant Fibrin Chains, Fibrin and Fibrin-Homologs," PCT
Application No. PCT/LJS95/05527, filed May 2, 1995.
O fibrin chain precursor. Precursor of a fibrin a-chain or (3-chain containing
a
N-terminal peptide that can be cleaved to yield the fibrin chain effective in
a fibrin to
allow polymerization.
O fibrin clot-forming effective amount. An effective amount of clot-forming
fibrin is
that quantity or concentration (if in a liquid form) of a fibrin (for example
fibrin
monomer) which forms sufficient clot material to be of utilized as a fibrin
sealant.
O fibrin monomer. Fibrin monomer is fibrin that is held in soluble form and
prevented
from clotting. for instance by the presence of a polymerization inhibitor such
as acidic
pH or a chaotropic agent or by being kept in a form which prevents
polymerization, such
as a sufficiently dehydrated form or a frozen form.
O fibrin polymer. Fibrin molecules, in the absence of conditions that prevent
polymerization of fibrin monomer, interact noncovalently to form polymers,
here termed
"fibrin polymers", which - when sufficient mass is achieved - form a visible
adherent
precipitate with clot-like properties. By the action of factor XIIIa, fibrin
polymer can be
covalently crosslinked. Prior to the crosslinking action of factor XIIIa,
fibrin polymer
can be reversibly converted to fibrin monomer. Even when some initial such
crosslinking has occurred, it is believed that fibrin polymer can be
reversibly converted
to fibrin monomer.
O fibrin precursor. A Precursor of fibrin comprises one or more fibrin chain
precursors
which must be processed to yield a form of fibrin that polymerizes with
corresponding
fibrin molecules. A fibrin precursor contains one or more leader peptides on
its
constituent chains. The leader peptides) can be processed in vivo or in vitro
from the
fibrin precursor to yield fibrin.
O gene therapy. As used herein, "gene therapy" includes any intervention in an
animal
(preferably a mammal, more preferably a human) that (i) causes a cell in the
animal to
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express (as RNA or protein) a recombinant nucleic acid, whether such
expression is
transient or stable, or (ii) causes a change in the cell's genome, such as an
insertion, that
changes the cell's pattern of gene expression. Hence, gene therapy includes
uses of
nucleic acid-based vaccines.
O high affinity binding. High affinity binding between a first substance and a
second
substance is binding of sufficient avidity to allow for the first or second
substance to be
used as an affinity ligand for the isolation of the other substance.
Typically, high affinity
binding is reflected in an association constant of about 105 M-' or more,
preferably 106
M-' or more, yet more preferably 10' M-' or more.
O Or. The conjunction "or" is used to express that at least one of the recited
alternatives linked by or is applicable in a given context and to include the
conjunctive
sense , joining two or more of the recited alternatives. In other words,
unless the context
indicates a contrary meaning, "or" includes the meaning sometimes expressed as
"and/or."
O Polyucleotide or nucleic acid. The terms polynucleotide(s) or nucleic acids)
(herein
"polynucleotide(s)") generally refer to any polyribonucleotide or
polydeoxyribonucleotide,
which can be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)"
include, without limitation, single- and double-stranded DNA, DNA that is a
mixture of
single- and double-stranded regions or single-, double- and triple-stranded
regions, single-
and double-stranded RNA, and RNA that is mixture of single- and double-
stranded regions,
hybrid molecules comprising DNA and RNA that can be single-stranded or, more
typically,
double-stranded, or triple-stranded regions, or a mixture of single- and
double-stranded
regions. In addition, "polynucleotide" as used herein refers to triple-
stranded regions
comprising RNA or DNA or both RNA and DNA. One of the molecules of a triple-
helical
region often is an oligonucleotide. As used herein, the term
"polynucleotide(s)" also includes
DNAs or RNAs as described above that contain one or more modified bases. Thus,
DNAs or
RNAs with backbones modified for stability or for other reasons are
"polynucleotide(s)" as
that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases,
such as
inosine, or modified bases, such as tritylated bases, to name just two
examples, are
polynucleotides as the term is used herein. It will be appreciated that a
great variety of
modifications have been made to DNA and RNA that serve many useful purposes
known to
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those of skill in the art. The term "polynucleotide(s)" as it is employed
herein embraces such
chemically, enzymatically or metabolically modified forms of polynucleotides,
as well as the
chemical forms of DNA and RNA characteristic of viruses and cells, including,
for example,
simple and complex cells. "Polynucleotide(s)" also embraces short
polynucleotides often
referred to as oligonucleotide(s).
O transformed cell. A cell is transformed if a nucleic acid is recombinantly
introduced
into it or its ancestor so as to temporarily or stably (1) cause the cell to
express a
polypeptide or RNA in an amount not otherwise expressed by the cell or (2)
interfere with
the translation or transcription of a nucleic acid normally found in the cell.
O transforming composition. A transforming composition is a composition
containing
a gene therapy effective amount of a nucleic acid.
All publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their
entirety as if each individual publication or reference were specifically and
individually
indicated to be incorporated by reference herein as being fully set forth. Any
patent
application to which this application claims priority is also incorporated by
reference
herein in its entirety in the manner described above for publications and
references.
While this invention has been described with an emphasis upon preferred
embodiments, it will be obvious to those of ordinary skill in the art that
variations in the
preferred devices and methods may be used and that it is intended that the
invention may
be practiced otherwise than as specifically described herein. Accordingly,
this invention
includes all modifications encompassed within the spirit and scope of the
invention as
defined by the claims that follow.
