U.S. patent application number 12/566831 was filed with the patent office on 2011-02-10 for methods for treating blood coagulation disorders.
This patent application is currently assigned to Samuel WADSWORTH. Invention is credited to Abraham SCARIA, Samuel WADSWORTH.
Application Number | 20110034539 12/566831 |
Document ID | / |
Family ID | 26935536 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110034539 |
Kind Code |
A1 |
WADSWORTH; Samuel ; et
al. |
February 10, 2011 |
METHODS FOR TREATING BLOOD COAGULATION DISORDERS
Abstract
The present invention relates to a method of treating an
individual having a blood coagulation defect (e.g., hemophilia A,
hemophilia B), comprising administering to the individual an
effective amount of a DNA vector encoding modified Factor VII
(FVII), wherein the modified Factor VII leads to generation of
Factor VIIa in vivo. In a particular embodiment, the invention
pertains to a method of treating an individual having a blood
coagulation defect comprising administering to the individual an
effective amount of a nucleic acid encoding a modified FVII wherein
the modified FVII comprises a signal which codes for precursor
cleavage by furin at the activation cleavage site of the modified
FVII. The invention also relates to a method of treating an
individual having a blood coagulation disorder comprising
administering to the individual an effective amount of a nucleic
acid encoding the light chain of human FVII and a nucleic acid
encoding the heavy chain of human FVII operably linked to a leader
sequence. Compositions, expression vectors and host cells
comprising nucleic acid which encodes a modified Factor VII,
wherein the modified Factor VII leads to generation of Factor VIIa
in vivo is also encompassed by the present invention.
Inventors: |
WADSWORTH; Samuel;
(Framingham, MA) ; SCARIA; Abraham; (Framingham,
MA) |
Correspondence
Address: |
GENZYME CORPORATION;LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Assignee: |
WADSWORTH; Samuel
Framingham
MA
SCARIA; Abraham
Framingham
MA
|
Family ID: |
26935536 |
Appl. No.: |
12/566831 |
Filed: |
September 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10057620 |
Oct 25, 2001 |
7615537 |
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12566831 |
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60243046 |
Oct 25, 2000 |
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60307492 |
Jul 24, 2001 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C12N 2799/021 20130101;
C12Y 304/21021 20130101; C07K 2319/02 20130101; A61K 48/00
20130101; A61P 7/02 20180101; C12N 9/6437 20130101 |
Class at
Publication: |
514/44.R |
International
Class: |
A61K 31/713 20060101
A61K031/713; A61P 7/02 20060101 A61P007/02 |
Claims
1. A method of promoting blood coagulation in an individual having
a blood coagulation defect and in need thereof, comprising
administering to the individual a blood coagulation enhancing
effective amount of a DNA vector encoding a Factor VII polypeptide
that can be converted to Factor VIIa when expressed in said
individual, said Factor VII polypeptide comprising an enzymatic
cleavage site susceptible to cleavage by furin, whereby cleavage by
furin produces Factor VII heavy chain and Factor VII light chain
molecules.
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Application Ser. Nos.
60/243,046 filed Oct. 25, 2000 and 60/307,492 filed Jul. 24, 2001
respectively. The contents of these applications are hereby
incorporated by reference into the present disclosure.
BACKGROUND OF THE INVENTION
[0002] Hemophilia is an X-linked bleeding disorder that results
from a deficiency in coagulation factor VIII (hemophilia A) or
factor IX (hemophilia B). Patients are conventionally treated by
protein replacement therapies using plasma-derived or recombinant
factor VIII or factor IX. Gene therapies for both hemophilia A and
B are in various stages of pre-clinical and clinical trails.
However, 25% of hemophilia A patients develop inhibitors (e.g.,
antibodies) to factor VIII and about 5% of hemophilia B patients
generate inhibitors to factor IX. These inhibitors lead to the
ineffectiveness of protein replacement or gene replacement
therapies.
[0003] It is known that basal levels of Factor VIIa in plasma are
greatly reduced in patients with hemophilia B (Factor IX
deficiency) and, to a lesser extent, patients with hemophilia A
(Factor VIII deficiency). Wildgoose et al., Blood 1:25-28 (1992).
In the absence of activated FVIIa, the intrinsic blood clotting
pathway involving FVIII and FIX, is severely limited in effective
coagulation. Recently, recombinant activated Factor VII (rFVIIa,
NovoSeven, Novo, Nordisk) has been shown to have therapeutic value
to bypass or correct the coagulation defects in hemophilia A and B
patients with inhibitors, especially in patients with inhibitors
who were undergoing surgical procedures. However, recombinant FVIIa
is expensive to manufacture. Anther critical problem is the short
half life (2 hours) of recombinant FVIIa. Therefore, recombinant
FVIIa therapy requires an intravenous infusion of high doses of the
protein every 2 hours.
[0004] A need exists for alternative therapies for blood
coagulation disorders such as hemophilia.
SUMMARY OF THE INVENTION
[0005] In the methods of the present invention, activated Factor
VII is provided to a patient suffering from a coagulation defect,
such as hemophilia. The Factor VII is delivered via DNA vectors,
which may be viral or non-viral in origin. In one preferred
embodiment, the activated Factor VII is provided using a DNA vector
encoding a modified FVII. This modified FVII comprises a cleavage
site, such as a furin cleavage site or other appropriate cleavage
site, such that the modified Factor VII molecule is cleaved to form
the light chain and heavy chain of Factor VII, which can then form
suitable disulfide bonds to form activated Factor VII. In other
preferred embodiments, activated Factor VII is supplied using DNA
vectors which separately encode the light chain of Factor VII and
the heavy chain of Factor VII, such that no cleavage is necessary,
and the individual chains are both present and can form suitable
disulfide bonds to form activated Factor VII. The individual DNA
vectors which separately encode the light chain of Factor VII and
the heavy chain of Factor VII may be provided on the same plasmid,
either as two separate expression cassettes with separate
regulatory sequences, or as part of a single polycistronic
expression cassette. Alternatively, the individual DNA vectors
which separately encode the light chain of Factor VII and the heavy
chain of Factor VII may be provided on separate plasmids or
vehicles which may be co-transformed into a single cell, so that
both individual chains are present and can form suitable disulfide
bonds to form activated Factor VII. In certain embodiments of the
present invention, surrounding conditions, such as pH, temperature
and electrovalent charges in the medium can be adjusted to
optimally promote proper disulfide bonding.
[0006] The present invention further relates to method of treating
an individual having a blood coagulation defect (e.g., hemophilia
A, hemophilia B), comprising administering to the individual an
effective amount of a DNA vector expressing modified Factor VII
(FVII), wherein the modified Factor VII leads to generation of
Factor VIIa in vivo. In one embodiment, the modified Factor VII
comprises an amino acid sequence which codes for a signal for
precursor cleavage by the protease furin at the activation cleavage
site of the modified Factor VII. For example, the amino acid signal
in the modified FVII can comprise an Arg149-X150-Lys151-Arg152
signal sequence or an Arg149-X150-Arg141-Arg152 signal sequence,
such as an Arg149-Gln150-Lys151-Arg152 sequence. In another
embodiment, the DNA vector encoding modified Factor VII is
administered as a combination of two compositions wherein the first
composition comprises the light chain (from about amino acid 1 to
about amino acid 152) of human Factor VII and the second
composition comprises the heavy chain from about (amino acid 153 to
about amino acid 406) of human Factor VII and (operably linked to)
a leader sequence (e.g., derived from a cytokine or a clotting
factor). The DNA encoding modified Factor VII of the present
invention can be administered as any gene transfer vector, such as
viral vectors, including adenovirus, AAV, retrovirus and
lentivirus, as well as plasmid DNA with or without a suitable lipid
or polymer carriers, and is administered under conditions in which
the nucleic acid is expressed in vivo. Alternatively, the DNA
encoding modified FVII can be administered as naked DNA or in
association with an amphiphilic compound, such as lipids or
compounds, or with another suitable carrier.
[0007] The present invention also relates to methods of treating
hemophilia in an individual, comprising administering to the
individual an effective amount of a DNA vector encoding modified
Factor VII wherein the modified Factor VII leads to generation of
Factor VIIa in vivo. In one embodiment, the present invention
relates to a method of treating hemophilia in an individual who has
developed an inhibitor of Factor VIII, comprising administering to
the individual an effective amount of a DNA vector encoding
modified Factor VII wherein the modified Factor VII leads to
generation of Factor VIIa in vivo. In another embodiment, the
invention relates to a method of treating hemophilia in an
individual who has developed an inhibitor of Factor IX, comprising
administering to the individual an effective amount of a DNA vector
encoding modified Factor VII wherein the modified Factor VII leads
to generation of Factor VIIa in vivo.
[0008] In a particular embodiment, the invention pertains to a
method of treating an individual having a blood coagulation defect
comprising administering to the individual an effective amount of a
DNA vector comprising a nucleic acid encoding a modified FVII
wherein the modified FVII comprises a signal which codes for
precursor cleavage by furin at the activation cleavage site of the
modified FVII.
[0009] The invention also relates to a method of treating an
individual having a blood coagulation disorder comprising
administering to the individual an effective amount of a DNA vector
comprising a nucleic acid encoding the light chain of human FVII
and a nucleic acid encoding the heavy chain of human FVII operably
linked to a leader sequence.
[0010] Compositions comprising DNA vectors encoding a modified
Factor VII, wherein the modified Factor VII leads to generation of
Factor VIIa in vivo is also encompassed by the present invention.
In one embodiment, the modified Factor VII comprises an amino acid
sequence which codes for a signal for precursor cleavage by furin
at the activation cleavage site of the modified Factor VII.
[0011] The present invention also relates to an expression vector
comprising nucleic acid encoding a modified Factor VII, wherein the
modified Factor VII leads to generation of Factor VIIa in vivo. In
one embodiment, the nucleic acid sequence encodes an amino acid
sequence which includes a signal for precursor cleavage by furin at
the activation cleavage site of the modified Factor VII. In another
embodiment, the nucleic acid construct comprises two expression
constructs which encode a modified Factor VII wherein the first
expression construct comprises amino acids 1-152 of human Factor
VII and the second expression comprises amino acids 153-406 of
human Factor VII and a leader sequence.
[0012] The present invention also relates to host cells comprising
a DNA vector comprising a nucleic acid which encodes a modified
Factor VII, wherein the modified Factor VII leads to generation of
Factor VIIa in vivo. In one embodiment, the nucleic acid sequence
encodes an amino acid sequence which includes a signal for
precursor cleavage by furin at the activation cleavage site of the
modified Factor VII. In another embodiment, the nucleic acid
construct comprises two expression constructs which encode a
modified Factor VII wherein the first expression construct
comprises amino acids 1-152 of human Factor VII and the second
expression comprises amino acids 153-406 of human Factor VII and a
leader sequence.
[0013] Host cells comprising a DNA vector encoding a modified
Factor VII in accordance with the present invention may be cultured
ex vivo and administered to or implanted into an individual
suffering from a blood coagulation defect or disease such as
hemophilia A, hemophilia B or Factor VII deficiency.
[0014] Thus, the present invention provides for an alternative
treatment of blood clotting defects, such as hemophilia A or
hemophilia B, in an individual, particularly where the individual
has developed inhibitors to conventional treatment (e.g.,
inhibitors against FVIII and/or FIX).
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 illustrates the intrinsic and extrinsic pathways for
fibrin clot formation and the mechanism by which FVIIa can act.
[0016] FIG. 2 illustrates examples of mutations to the FVII amino
acid sequence which can be engineered at the nucleotide level in
order to create a furin cleavage site at the activation site of
FVII.
[0017] FIG. 3 illustrates examples of mutations to the FVII amino
acid sequence which can be engineered at the nucleotide level in
order to create an SK1 cleavage site at the activation site of
FVII.
[0018] FIG. 4 illustrates clotting time of 293 cells [FIG. 4A] and
Hep3B cells FIG. 4B] untransfected, and transfected with FVII and
FVIIa.
[0019] FIG. 5 illustrates clotting time in 293 cell supernates from
normal, FVIII-.
[0020] FIG. 6 illustrates clotting time in a modified aPTT
assay.
[0021] FIG. 7 illustrates clotting time in Beige/SCID mice
transfected with FVII and FVIIa with CMV and liver-specific
promoters [LSP].
[0022] FIG. 8 illustrates clotting time in FVIII knockout mice
transfected with FVIIa.
[0023] FIG. 9 illustrates clotting time in a PTT assay of FVIII
knockout mice.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Factor VII is a single chain glycoprotein (mol. wt. 50,000)
of 406 amino acids that is secreted into the blood where it
circulates in a zymogen form. In vitro, FVII can be proteolytically
activated to activated Factor FVII, or FVIIa, by the action of
activated coagulation factors Factor X (FXa), Factor IX (FIXa),
Factor XII (FXIIa) or Factor II (FIIa). FVIIa does not promote
coagulation by itself, but can complex with tissue factor (TF)
exposed at the site of injury. The FVIIa/TF complex can convert FX
to FXa, thereby inducing local hemostasis at the site of injury.
Activation of FVII to FVIIa involves proteolytic cleavage at a
single peptide bond between Arg-152 and Ile-153, resulting in a
two-chain molecule consisting of a light chain of 152 amino acid
residues and a heavy chain of 254 amino acid residues held together
by a single disulfide bond. Hemophilia patients have normal levels
of FVII, however, they suffer from a relative deficiency in FVIIa
and other activated clotting factors.
[0025] The present invention further relates to DNA expression
vectors and constructs, which may be useful for gene therapy by
providing an effective amount of activated Factor VII to the
plasma, or to a suitable depot organ, such as liver or lung, within
a patient. The DNA vectors may comprise nucleic acid encoding a
modified Factor VII, wherein the modified Factor VII leads to
generation of Factor VIIa in vivo. Various embodiments of the
invention are possible, each of which is capable of producing an
effective amount of activated FVII in a patient who is otherwise
lacking sufficient clotting factors to achieve blood coagulation.
The present invention in various embodiments thus comprises (1)
administering DNA vectors which encode activated FVII; (2)
administering DNA vectors which encode a modified FVII such that
FVII will be cleaved to form activated FVII; (3) administering DNA
vectors which encode FVII, together with administration of an
activator [e.g., FIXa, FXa or FXIIa] such that FVII will be cleaved
to form activated FVIIa; (4) administering DNA vectors which
separately encode the light chain of FVII and the heavy chain of
FVII, such that both chains are present in a cell and can
associate, form disulfide bonds to form activated FVII.
[0026] Suitable DNA vectors for modified Factor VII may have been
modified to create an activation cleavage site, such as a furin or
other subtilisin cleavage site, at an appropriate position within
the Factor VII DNA sequence. For example, a modification may be
made in the area of about amino acid 147 through about 154 of human
Factor VII to create an appropriate cleavage site. The DNA vector
may be a viral vector such as an adenovirus vector, a
partially-deleted adenovirus vector, a fully-deleted adenovirus
vector, an adeno-associated virus vector, a pseudoadenovirus, a
retrovirus vector and a lentivirus vector. An example of an
alternate cleavage enzyme which may be suitable for use in the
present invention is SK1. Seidah et al. (1999) PNAS, Vol. 96,
1321-1326.
[0027] Alternative DNA expression vectors and constructs for use in
the present invention include more than one DNA vector which
separately encode the light chain of Factor VII, which begins at
about amino acid 1 and continues until about amino acid 147 to
about amino acid 152 of human Factor VII; and the heavy chain of
Factor VII, which begins from about amino acid 147 to amino acid
154 and continues to about amino acid 406 human Factor VII. The DNA
vector encoding the heavy chain of Factor VII may preferably be
designed to include a separate leader sequence, such as a leader
sequence of a protein selected from the group consisting of: a
cytokine, growth factor, colony stimulating factor and a clotting
factor. In addition, the present invention comprises nucleic acid
constructs comprising polycistronic expression cassettes, wherein
the expression cassette comprises (a) nucleic acids encoding the
light chain of Factor VII and (b) nucleic acids encoding the heavy
chain of Factor VII, and wherein (a) and (b) are separated by an
internal ribosome entry site or other suitable spacer for
expression of polycistronic messages.
[0028] The present invention further includes methods of treating
hemophilia and methods of promoting blood coagulation by
administering a DNA vector which encodes human Factor VII. Such
methods may use viral or non-viral vectors, such as adenovirus,
adeno-associated viruses, retroviruses, lentiviruses, and
recombinant versions of the above, as well as naked plasmid DNA,
and DNA in conjunction with a suitable compound, such as a cationic
lipid or amphiphilic polymers. In preferred embodiments, the method
may further include co-administering of an activating amount of an
activator protein, such as FIXa, Fxa or FXIIa. The activator may be
administered in the form of a suitable protein formulation, or may
be administered using a DNA vector which encodes the activator. The
activator may be administered immediately prior to, simultaneously
with, or subsequently to administration of the DNA vector which
encodes Factor VII.
[0029] In one embodiment, the present invention provides DNA
vectors encoding a modified version of clotting Factor VII such
that it leads to generation of (or can be converted to) activated
Factor VII (FVIIa) in vivo. Accordingly, the present invention
provides a method of treating an individual having a blood
coagulation defect comprising administering to the individual an
effective amount of a DNA vector encoding the modified FVII
described herein, wherein the modified FVII leads to generation of
(is converted to) FVIIa in vivo.
[0030] The DNA vectors useful in the present invention include both
viral and non-viral vectors. The viral DNA vectors useful in the
present invention may include adenoviral, AAV, retroviral and
lentiviral vectors. The non-viral DNA vectors may include
amphiphilic compounds, polymers and lipids, as well as `naked DNA`
vectors.
[0031] Blood coagulation defects associated with defects in one or
more of the clotting factors can be treated using the methods
described herein. Examples of blood clotting defects which can be
treated using the methods described herein include hemophilia
(e.g., hemophilia A, hemophilia B) and blood clotting defects
associated with the presence of inhibitors (e.g., antibodies) of a
(one or more) clotting factor (e.g., FVII, FVIII, FIX) in an
individual. The modified FVII of the present invention is suitable
to administer to a variety of individuals, such as mammals, and
particularly, humans.
[0032] In one embodiment, a signal (sequence) for precursor
cleavage by a protease is introduced into the activation cleavage
site of FVII, wherein FVIIa is produced upon cleavage of the signal
by the protease. Preferably, the signal is cleaved by a protease
that is present in cells into which the modified FVII is
introduced. Any suitable signal for precursor cleavage by a
protease which, when cleaved results in generation of FVIIa, can be
introduced into the activation cleavage site of the modified FVII
of the present invention. For example, a signal which is cleaved by
furin [also known as PACE, see U.S. Pat. No. 5,460,950], other
subtilisins [including PC2, PC1/PC3, PACE4, PC4, PC5/PC6 and
LPC/PC7/PC8/SPC7; Nakayama, Biochem. J., 327:625-635 (1997)]
enterokinase [see U.S. Pat. No. 5,270,181 or chymotrypsin may be of
use, and can be introduced into the cleavage activation site of
FVII for use in the present invention. The disclosure of each of
the above documents is hereby incorporated herein by reference.
[0033] In a particular embodiment, the modified FVII comprises an
amino acid sequence which codes for a signal for precursor cleavage
by furin. Furin is a ubiquitously expressed protease that resides
in the trans-golgi and processes protein precursors before their
secretion. Furin cleaves at the COOH-terminus of its consensus
recognition sequence, Arg-X-Lys-Arg or Arg-X-Arg-Arg, and to a
lesser extent, Arg-X-X-Arg. The amino acid (aa) sequence at
position 149-152 of human FVII is Pro-Gln-Gly-Arg. An example of
this embodiment is one in which the nucleotide sequence of FVII is
modified such that Pro-149 is changed to Arg-149 and Gly-151 is
changed to Lys-151. The resulting amino acid sequence
Arg-Gln-Lys-Arg is a signal for precursor cleavage by the protease
furin. Other examples for producing a furin cleavage site in the
nucleotide sequence of FVII include substituting amino acids 147
through 150, 148 through 151, 150 through 153 or amino acids 151
through 154 with suitable amino acids to produce a furin cleavage
site with the sequence Arg-X-Lys-Arg or Arg-X-Arg-Arg.
[0034] In another preferred embodiment, the DNA vector encoding the
modified FVII containing a furin cleavage site may be co-expressed
with a DNA vector encoding furin. In this manner, FVIIa could be
produced in cells that would not ordinarily express furin, and thus
which would not ordinarily cleave the modified FVII product to form
FVIIa.
[0035] In the modified FVII coding DNA vectors described herein, in
place of furin, other proteases, such as those of the subtilisin
family, can be used. These include PC2, PC1/PC3, PACE4, PC4,
PC5/PC6 and LPC/PC7/PC8/SPC7. See Nakayama, Biochem. J.,
327:625-635 (1997) and the references cited therein for their
disclosure of the amino acid sequences and coding DNA sequences for
these subtilisin convertase proteins. The disclosure of these
publications is hereby incorporated herein by reference.
[0036] In other embodiments of the present invention, the DNA
vectors encoding the heavy chain and light chain of FVII can be
separated and introduced into the same cell(s). In one particular
embodiment, nucleic acid (e.g., cDNA) encoding human FVII is split
into two expression cassettes. The first cassette encodes the light
chain (from about amino acid 1 to about amino acid 152) of human
FVII which includes the pre-pro leader sequence of human FVII. The
second cassette encodes the heavy chain of human FVII (from amino
acid 153 to about amino acid 406) with a pre-pro leader sequence
from any well secreted protein fused to the N-terminus. For
example, the pre-pro leader sequence can be derived from a cytokine
(e.g., interleukins, including IL-1, IL-2, IL-3, IL-4, IL-6, IL-8,
IL-9, IL-11, IL-12, IL-13, IL-14, IL-15 and IL-16), colony
stimulating factors such as G-CSF, GM-CSF and M-CSF, growth
factors, such as IGF, KGF, BGF, FGF, hormones and clotting factors
(e.g., Factors I through Factors XIII, including FV, FVII, FVIII,
FIX and FX). The two expression cassettes may be cloned into the
same or different regions of a vector, such as adenovirus (e.g., E1
and/or E3 regions), partially-deleted adenovirus or fully-deleted
adenovirus. In another embodiment of the present invention, the
heavy and light chains may be introduced into the same cell using
two different vectors, such as through co-transformation. In yet
another embodiment of the present invention, the light chain and
heavy chain can be introduced into a cell on a single,
polycistronic expression cassette. The coding sequences of the
light and heavy chain in such a polycistronic cassette are
preferably driven by a single promoter and are preferably separated
by an internal ribosome entry site ["IRES"]. By means of the above
embodiments, the light chain and heavy chain of FVIIa are thus
expressed in the same cell in vivo upon introduction of this vector
via intravenous, intramuscular, intraportal or other route of
administration.
[0037] Additional, modified versions of clotting Factor VII which
generate (or are converted to) activated Factor VII (FVIIa) in vivo
similar to those described herein, can be prepared by those of
skill in the art. Such modified versions of FVII can be assessed
for their ability to convert to FVIIa in vivo using a variety of
known assays for FVIIa activity.
[0038] The DNA molecules encoding FVII for use in the present
invention can be derived from any suitable mammalian source and
modified as described herein. For example, the FVII can be of human
origin (U.S. Pat. No. 4,784,950) or of bovine origin (Takeya, et
al., J. Biol. Chem., 263:14868-14872 (1988)), as well as other
species' origin, and may be chimeric, for example including domains
of human and non-human FVII [see, for example, by analogy U.S. Pat.
Nos. 5,364,771 and 5,563,045 (FVIII)]. The modifications described
herein can be introduced into other mammalian FVII as they are
identified, and the ability of the resulting modified FVII to
produce FVIIa in vivo, can be assessed using known methods. In
addition, the DNA encoding modified FVII described herein can be
obtained from commercial sources, recombinantly produced or
chemically synthesized. Sequence modifications of the modified FVII
described herein can be accomplished using a variety of techniques.
For example site-directed mutagenesis and/or enzymatic cleavage can
be used.
[0039] In the methods of the present invention, activated Factor
VII is provided to a patient suffering from a coagulation defect,
such as hemophilia. The Factor VII is delivered via DNA vectors,
which may be viral or non-viral in origin. In one preferred
embodiment, the activated Factor VII is provided using a DNA vector
encoding a modified FVII. This modified FVII comprises a cleavage
site, such as a furin cleavage site or other appropriate cleavage
site, such that the modified Factor VII molecule is cleaved to form
the light chain and heavy chain of Factor VII, which can then form
suitable disulfide bonds to form activated Factor VII. In other
preferred embodiments, activated Factor VII is supplied using DNA
vectors which separately encode the light chain of Factor VII and
the heavy chain of Factor VII, such that no cleavage is necessary,
and the individual chains are both present and can form suitable
disulfide bonds to form activated Factor VII. The individual DNA
vectors which separately encode the light chain of Factor VII and
the heavy chain of Factor VII may be provided on the same plasmid,
either as two separate expression cassettes with separate
regulatory sequences, or as part of a single polycistronic
expression cassette. Alternatively, the individual DNA vectors
which separately encode the light chain of Factor VII and the heavy
chain of Factor VII may be provided on separate plasmids or
vehicles which may be co-transformed into a single cell, so that
both individual chains are present and can form suitable disulfide
bonds to form activated Factor VII. In certain embodiments of the
present invention, surrounding conditions, such as pH, temperature
and electrovalent charges in the medium can be adjusted to
optimally promote proper disulfide bonding.
[0040] The modified FVII of the present invention can be
administered by introducing nucleic acid (e.g., DNA, cDNA, RNA)
encoding the modified FVII into the individual wherein the nucleic
acid is expressed and FVIIa is expressed in vivo. Alternatively,
the nucleic acid encoding the modified FVII can be administered ex
vivo to cells (e.g., hepatocytes, myoblasts, fibroblasts,
endothelial cells, keratinocytes, hematopoietic cells) of the
individual and then transferred into the individual wherein the
modified FVII is expressed and FVIIa is generated in vivo. For
example, the nucleic acid (e.g., cDNA) encoding modified FVII can
be cloned into an expression cassette that has a promoter
(constitutive or regulatable) to drive transgene expression and a
polyadenylation sequence downstream of the nucleic acid. Suitable
promoters include the cytomegalovirus [CMV] promoter, and
conditional promoters such as the dimerizer gene control system,
based on the immunosuppressive agents FK506 and rapamycin, the
ecdysone gene control system and the tetracycline gene control
system. Also useful in the present invention may be the
GeneSwitch.TM. technology [Valentis, Inc., Woodlands, Tex.]
described in Abruzzese et al., Hum. Gene Ther. 1999 10:1499-507,
the disclosure of which is hereby incorporated herein by
reference.
[0041] In preferred embodiments, the DNA vectors used, whether they
encode Factor VIIa, a modified Factor VII or separately the light
chain of Factor VII and the heavy chain of Factor VII, may be
introduced under the control of a regulatable promoter. The
advantages of such a system are that the DNA vectors may be
administered to the patient, and the serum levels of Factor VIIa
may be closely monitored, as well as phenotypic parameters which
indicate whether sufficient levels of blood coagulation are being
achieved. With inducible or regulatable promoters, the clinician
may exert additional optimization of the methods of the present
invention, such that optimal levels of activated FVII are achieved
for blood coagulation.
[0042] The expression cassette is then inserted into a vector such
as adenovirus, partially-deleted adenovirus, fully-deleted
adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus,
naked plasmid, plasmid/liposome complex, etc. for delivery to the
host via intravenous, intramuscular, intraportal or other route of
administration. Expression vectors which can be used in the methods
and compositions of the present invention include, for example,
viral vectors. One of the most frequently used methods of
administration of gene therapy, both in vivo and ex vivo, is the
use of viral vectors for delivery of the gene. Many species of
virus are known, and many have been studied for gene therapy
purposes. The most commonly used viral vectors include those
derived from adenoviruses, adeno-associated viruses [AAV] and
retroviruses, including lentiviruses, such as human
immunodeficiency virus [HIV].
[0043] Adenoviral vectors for use to deliver transgenes to cells
for applications such as in vivo gene therapy and in vitro study
and/or production of the products of transgenes, commonly are
derived from adenoviruses by deletion of the early region 1 (E1)
genes (Berkner, K. L., Curr. Top. Micro. Immunol. 158L39-66 1992).
Deletion of E1 genes renders such adenoviral vectors replication
defective and significantly reduces expression of the remaining
viral genes present within the vector. However, it is believed that
the presence of the remaining viral genes in adenoviral vectors can
be deleterious to the transfected cell for one or more of the
following reasons: (1) stimulation of a cellular immune response
directed against expressed viral proteins, (2) cytotoxicity of
expressed viral proteins, and (3) replication of the vector genome
leading to cell death.
[0044] One solution to this problem has been the creation of
adenoviral vectors with deletions of various adenoviral gene
sequences. In particular, pseudoadenoviral vectors (PAVs), also
known as `gutless adenovirus` or mini-adenoviral vectors, are
adenoviral vectors derived from the genome of an adenovirus that
contain minimal cis-acting nucleotide sequences required for the
replication and packaging of the vector genome and which can
contain one or more transgenes (See, U.S. Pat. No. 5,882,877 which
covers pseudoadenoviral vectors (PAV) and methods for producing
PAV, incorporated herein by reference). Such PAVs, which can
accommodate up to about 36 kb of foreign nucleic acid, are
advantageous because the carrying capacity of the vector is
optimized, while the potential for host immune responses to the
vector or the generation of replication-competent viruses is
reduced. PAV vectors contain the 5' inverted terminal repeat (ITR)
and the 3' ITR nucleotide sequences that contain the origin of
replication, and the cis-acting nucleotide sequence required for
packaging of the PAV genome, and can accommodate one or more
transgenes with appropriate regulatory elements, e.g. promoter,
enhancers, etc.
[0045] Other, partially deleted adenoviral vectors provide a
partially-deleted adenoviral (termed "DeAd") vector in which the
majority of adenoviral early genes required for virus replication
are deleted from the vector and placed within a producer cell
chromosome under the control of a conditional promoter. The
deletable adenoviral genes that are placed in the producer cell may
include E1A/E1B, E2, E4 (only ORF6 and ORF6/7 need be placed into
the cell), pIX and pIVa2. E3 may also be deleted from the vector,
but since it is not required for vector production, it can be
omitted from the producer cell. The adenoviral late genes, normally
under the control of the major late promoter (MLP), are present in
the vector, but the MLP may be replaced by a conditional
promoter.
[0046] Conditional promoters suitable for use in DeAd vectors and
producer cell lines include those with the following
characteristics: low basal expression in the uninduced state, such
that cytotoxic or cytostatic adenovirus genes are not expressed at
levels harmful to the cell; and high level expression in the
induced state, such that sufficient amounts of viral proteins are
produced to support vector replication and assembly. Preferred
conditional promoters suitable for use in DeAd vectors and producer
cell lines include the dimerizer gene control system, based on the
immunosuppressive agents FK506 and rapamycin, the ecdysone gene
control system and the tetracycline gene control system. Also
useful in the present invention may be the GeneSwitch.TM.
technology [Valentis, Inc., Woodlands, Tex.] described in Abruzzese
et al., Hum. Gene Ther. 1999 10:1499-507, the disclosure of which
is hereby incorporated herein by reference.
[0047] The partially deleted adenoviral expression system is
further described in WO99/57296, the disclosure of which is hereby
incorporated by reference herein.
[0048] Adenoviral vectors, such as PAVs and DeAd vectors, have been
designed to take advantage of the desirable features of adenovirus
which render it a suitable vehicle for delivery of nucleic acids to
recipient cells. Adenovirus is a non-enveloped, nuclear DNA virus
with a genome of about 36 kb, which has been well-characterized
through studies in classical genetics and molecular biology
(Hurwitz, M. S., Adenoviruses Virology, 3.sup.rd edition, Fields et
al., eds., Raven Press, New York, 1996; Hitt, M. M. et al.,
Adenovirus Vectors, The Development of Human Gene Therapy,
Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York
1999). The viral genes are classified into early (designated E1-E4)
and late (designated L1-L5) transcriptional units, referring to the
generation of two temporal classes of viral proteins. The
demarcation of these events is viral DNA replication. The human
adenoviruses are divided into numerous serotypes (approximately 47,
numbered accordingly and classified into 6 groups: A, B, C, D, E
and F), based upon properties including hemaglutination of red
blood cells, oncogenicity, DNA and protein amino acid compositions
and homologies, and antigenic relationships.
[0049] Recombinant adenoviral vectors have several advantages for
use as gene delivery vehicles, including tropism for both dividing
and non-dividing cells, minimal pathogenic potential, ability to
replicate to high titer for preparation of vector stocks, and the
potential to carry large inserts (Berkner, K. L., Curr. Top. Micro.
Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64
1994).
[0050] PAVs have been designed to take advantage of the desirable
features of adenovirus which render it a suitable vehicle for gene
delivery. While adenoviral vectors can generally carry inserts of
up to 8 kb in size by the deletion of regions which are dispensable
for viral growth, maximal carrying capacity can be achieved with
the use of adenoviral vectors containing deletions of most viral
coding sequences, including PAVs. See U.S. Pat. No. 5,882,877 of
Gregory et al.; Kochanek et al., Proc. Natl. Acad. Sci. USA
93:5731-5736, 1996; Parks et al., Proc. Natl. Acad. Sci. USA
93:13565-13570, 1996; Lieber et al., J. Virol. 70:8944-8960, 1996;
Fisher et al., Virology 217:11-22, 1996; U.S. Pat. No. 5,670,488;
PCT Publication No. WO96/33280, published Oct. 24, 1996; PCT
Publication No. WO96/40955, published Dec. 19, 1996; PCT
Publication No. WO97/25446, published Jul. 19, 1997; PCT
Publication No. WO95/29993, published Nov. 9, 1995; PCT Publication
No. WO97/00326, published Jan. 3, 1997; Morral et al., Hum. Gene
Ther. 10:2709-2716, 1998.
[0051] Since PAVs are deleted for most of the adenovirus genome,
production of PAVs requires the furnishing of adenovirus proteins
in trans which facilitate the replication and packaging of a PAV
genome into viral vector particles. Most commonly, such proteins
are provided by infecting a producer cell with a helper adenovirus
containing the genes encoding such proteins.
[0052] However, such helper viruses are potential sources of
contamination of a PAV stock during purification and can pose
potential problems when administering the PAV to an individual if
the contaminating helper adenovirus can replicate and be packaged
into viral particles.
[0053] It is advantageous to increase the purity of a PAV stock by
reducing or eliminating any production of helper vectors which can
contaminate preparation. Several strategies to reduce the
production of helper vectors in the preparation of a PAV stock are
disclosed in U.S. Pat. No. 5,882,877, issued Mar. 16, 1999; U.S.
Pat. No. 5,670,488, issued Sep. 23, 1997 and International Patent
Application No. PCT/US99/03483, incorporated herein by reference.
For example, the helper vector may contain mutations in the
packaging sequence of its genome to prevent its packaging, an
oversized adenoviral genome which cannot be packaged due to size
constraints of the virion, or a packaging signal region with
binding sequences that prevent access by packaging proteins to this
signal which thereby prevents production of the helper virus.
[0054] Other strategies include the design of a helper virus with a
packaging signal flanked by the excision target site of a
recombinase, such as the Cre-Lox system (Parks et al., Proc. Natl.
Acad. Sci. USA 93:13565-13570, 1996; Hardy et al., J. Virol.
71:1842-1849, 1997, incorporated herein by reference); or the phage
C31 integrase [see Calos et al., WO 00/11555]. Such helper vectors
reduce the yield of wild-type levels.
[0055] Another hurdle for PAV manufacturing, aside from the
problems with obtaining helper vector-free stocks, is that the
production process is initiated by DNA transfections of the PAV
genome and the helper genome into a suitable cell line, e.g., 293
cells. After cytopathic effects are observed in the culture
indicating a successful infection, which may take up to from 2 to 6
days, the culture is harvested and is passaged onto a new culture
of cells. This process is repeated for several additional passages,
up to 7 times more, to obtain a modes cell lysate containing the
PAV vector and any contaminating helper vector. See Parks et al.,
1996, Proc. Natl. Acad. Sci. USA 93:13565-13570; Kochanek et al.,
1996, Proc. Natl. Acad. Sci. USA 93:5731-5736. This lengthy process
is not optimal for commercial scale manufacturing. Additionally,
this process facilitates recombination and rearrangement events
resulting in the propagation of PAV genomes with unwanted
alterations. The use of adenoviruses for gene therapy is described,
for example, in U.S. Pat. No. 5,882,877; U.S. patent, the
disclosures of which are hereby incorporated herein by
reference.
[0056] Adeno-associated virus (AAV) is a single-stranded human DNA
parvovirus whose genome has a size of 4.6 kb. The AAV genome
contains two major genes: the rep gene, which codes for the rep
proteins (Rep 76, Rep 68, Rep 52, and Rep 40) and the cap gene,
which codes for AAV replication, rescue, transcription and
integration, while the cap proteins form the AAV viral particle.
AAV derives its name from its dependence on an adenovirus or other
helper virus (e.g., herpesvirus) to supply essential gene products
that allow AAV to undergo a productive infection, i.e., reproduce
itself in the host cell. In the absence of helper virus, AAV
integrates as a provirus into the host cell's chromosome, until it
is rescued by superinfection of the host cell with a helper virus,
usually adenovirus (Muzyczka, Curr. Top. Micor. Immunol.
158:97-127, 1992).
[0057] Interest in AAV as a gene transfer vector results from
several unique features of its biology. At both ends of the AAV
genome is a nucleotide sequence known as an inverted terminal
repeat (ITR), which contains the cis-acting nucleotide sequences
required for virus replication, rescue, packaging and integration.
The integration function of the ITR mediated by the rep protein in
trans permits the AAV genome to integrate into a cellular
chromosome after infection, in the absence of helper virus. This
unique property of the virus has relevance to the use of AAV in
gene transfer, as it allows for a integration of a recombinant AAV
containing a gene of interest into the cellular genome. Therefore,
stable genetic transformation, ideal for many of the goals of gene
transfer, may be achieved by use of rAAV vectors. Furthermore, the
site of integration for AAV is well-established and has been
localized to chromosome 19 of humans (Kotin et al., Proc. Natl.
Acad. Sci. 87:2211-2215, 1990). This predictability of integration
site reduces the danger of random insertional events into the
cellular genome that may activate or inactivate host genes or
interrupt coding sequences, consequences that can limit the use of
vectors whose integration of AAV, removal of this gene in the
design of rAAV vectors may result in the altered integration
patterns that have been observed with rAAV vectors (Ponnazhagan et
al., Hum Gene Ther. 8:275-284, 1997).
[0058] There are other advantages to the use of AAV for gene
transfer. The host range of AAV is broad. Moreover, unlike
retroviruses, AAV can infect both quiescent and dividing cells. In
addition, AAV has not been associated with human disease, obviating
many of the concerns that have been raised with retrovirus-derived
gene transfer vectors.
[0059] Standard approaches to the generation of recombinant rAAV
vectors have required the coordination of a series of intracellular
events: transfection of the host cell with an rAAV vector genome
containing a transgene of interest flanked by the AAV ITR
sequences, transfection of the host cell by a plasmid encoding the
genes for the AAV rep and cap proteins which are required in trans,
and infection of the transfected cell with a helper virus to supply
the non-AAV helper functions required in trans (Muzyczka, N., Curr.
Top. Micor. Immunol. 158:97-129, 1992). The adenoviral (or other
helper virus) proteins activate transcription of the AAV rep gene,
and the rep proteins then activate transcription of the AAV cap
genes. The cap proteins then utilize the ITR sequences to package
the rAAV genome into an rAAV viral particle. Therefore, the
efficiency of packaging is determined, in part, by the availability
of adequate amounts of the structural proteins, as well as the
accessibility of any cis-acting packaging sequences required in the
rAAV vector genome.
[0060] One of the potential limitations to high level rAAV
production derives from limiting quantities of the AAV helper
proteins required in trans for replication and packaging of the
rAAV genome. Some approaches to increasing the levels of these
proteins have included placing the AAV rep gene under the control
of the HIV LTR promoter to increase rep protein levels (Flotte, F.
R., et al., Gene Therapy 2:29-37, 1995); the use of other
heterologous promoters to increase expression of the AAV helper
proteins, specifically the cap proteins (Vincent, et al., J. Virol.
71:1897-1905, 1997); and the development of cell lines that
specifically express the rep proteins (Yang, Q., et al., J. Virol.,
68:4847-4856, 1994).
[0061] Other approaches to improving the production of rAAV vectors
include the use of helper virus induction of the AAV helper
proteins (Clark, et al., Gene Therapy 3:1124-1132, 1996) and the
generation of a cell line containing integrated copies of the rAAV
vector and AAV helper genes so that infection by the helper virus
initiates rAAV production (Clark et al., Human Gene Therapy
6:1329-1341, 1995).
[0062] rAAV vectors have been produced using replication-defective
helper adenoviruses which contain the nucleotide sequences encoding
the rAAV vector genome (U.S. Pat. No. 5,856,152 issued Jan. 5,
1999) or helper adenoviruses which contain the nucleotide sequences
encoding the AAV helper proteins (PCT International Publication
WO95/06743, published Mar. 9, 1995). Production strategies which
combine high level expression of the AAV helper genes and the
optimal choice of cis-acting nucleotide sequences in the rAAV
vector genome have been described (PCT International Application
No. WO97/09441 published Mar. 13, 1997).
[0063] Current approaches to reducing contamination of rAAV vector
stocks by helper viruses, therefore, involve the use of
temperature-sensitive helper viruses (Ensigner et al., J. Virol.,
10:328-339, 1972), which are inactivated at the non-permissive
temperature. Alternatively, the non-AAV helper genes can be
subcloned into DNA plasmids which are transfected into a cell
during rAAV vector production (Salvetti et al., Hum. Gene Ther.
9:695-706, 1998; Grimm, et al., Hum. Gene Ther. 9:2745-2760, 1998;
WO97/09441). The use of AAV for gene therapy is described, for
example, in U.S. Pat. No. 5,753,500, the disclosures of each of the
above are hereby incorporated herein by reference.
[0064] Retrovirus vectors are a common tool for gene delivery
(Miller, Nature (1992) 357:455-460). The ability of retrovirus
vectors to deliver an unrearranged, single copy gene into a broad
range of rodent, primate and human somatic cells makes retroviral
vectors well suited for transferring genes to a cell.
[0065] Retroviruses are RNA viruses wherein the viral genome is
RNA. When a host cell is infected with a retrovirus, the genomic
RNA is reverse transcribed into a DNA intermediate which is
integrated very efficiently into the chromosomal DNA of infected
cells. This integrated DNA intermediate is referred to as a
provirus. Transcription of the provirus and assembly into
infectious virus occurs in the presence of an appropriate helper
virus or in a cell line containing appropriate sequences enabling
encapsidation without coincident production of a contaminating
helper virus. A helper virus is not required for the production of
the recombinant retrovirus if the sequences for encapsidation are
provided by co-transfection with appropriate vectors.
[0066] Another useful tool for producing recombinant retroviral
vectors are packaging cell lines which supply in trans the proteins
necessary for producing infectious virions, but those cells are
incapable of packaging endogenous viral genomic nucleic acids
(Watanabe & Termin, Molec. Cell. Biol. (1983) 3(12):2241-2249;
Mann et al., Cell (1983) 33:153-159; Embretson & Temin, J.
Virol. (1987) 61(9):2675-2683). One approach to minimize the
likelihood of generating RCR in packaging cells is to divide the
packaging functions into two genomes, for example, one which
expresses the gag and pol gene products and the other which
expresses the env gene product (Bosselman et al., Molec. Cell.
Biol. (1987) 7(5):1797-1806; Markowitz et al., J. Virol. (1988)
62(4):1120-1124; Danos & Mulligan, Proc. Natl. Acad. Sci.
(1988) 85:6460-6464). That approach minimizes the ability for
co-packaging and subsequent transfer of the two-genomes, as well as
significantly decreasing the frequency of recombination due to the
presence of three retroviral genomes in the packaging cell to
produce RCR.
[0067] In the event recombinants arise, mutations (Danos &
Mulligan, supra) or deletions (Boselman et al., supra; Markowitz et
al., supra) can be configured within the undesired gene products to
render any possible recombinants non-functional. In addition,
deletion of the 3' LTR on both packaging constructs further reduces
the ability to form functional recombinants.
[0068] The retroviral genome and the proviral DNA have three genes:
the gag, the pol, and the env, which are flanked by two long
terminal repeat (LTR) sequences. The gag gene encodes the internal
structural (matrix, capsid, and nucleocapsid) proteins; the pol
gene encodes the RNA-directed DNA polymerase (reverse
transcriptase) and the env gene encodes viral envelope
glycoproteins. The 5' and 3' LTRs serve to promote transcription
and polyadenylation of the virion RNAs. The LTR contains all other
cis-acting sequences necessary for viral replication. Lentiviruses
have additional genes including vit vpr, tat, rev, vpu, nef, and
vpx (in HIV-1, HIV-2 and/or SIV). Adjacent to the 5' LTR are
sequences necessary for reverse transcription of the genome (the
tRNA primer binding site) and for efficient encapsidation of viral
RNA into particles (the Psi site). If the sequences necessary for
encapsidation (or packaging of retroviral RNA into infectious
virions) are missing from the viral genome, the result is a cis
defect which prevents encapsidation of genomic RNA. However, the
resulting mutant is still capable of directing the synthesis of all
varion proteins.
[0069] Lentiviruses are complex retroviruses which, in addition to
the common retroviral genes gag, pol and env, contain other genes
with regulatory or structural function. The higher complexity
enables the lentivirus to modulate the life cycle thereof, as in
the course of latent infection. A typical lentivirus is the human
immunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo,
HIV can infect terminally differentiated cells that rarely divide,
such as lymphocytes and macrophages. In vitro, HIV can infect
primary cultures of monocyte-derived macrophages (MDM) as well as
HeLa-Cd4 or T lymphoid cells arrested in the cell cycle by
treatment with aphidicolin or gamma irradiation. Infection of cells
is dependent on the active nuclear import of HIV preintegration
complexes through the nuclear pores of the target cells. That
occurs by the interaction of multiple, partly redundant, molecular
determinants in the complex with the nuclear import machinery of
the target cell. Identified determinants include a functional
nuclear localization signal (NLS) in the gag matrix (MA) protein,
the karyophilic virion-associated protein, vpr, and a C-terminal
phosphotyrosine residue in the gag MA protein. The use of
retroviruses for gene therapy is described, for example, in U.S.
Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, the disclosures of
which are hereby incorporated herein by reference.
[0070] Other methods for delivery of DNA to cells do not use
viruses for delivery. For example, cationic amphiphilic compounds
can be used to deliver the nucleic acid of the present invention.
Because compounds designed to facilitate intracellular delivery of
biologically active molecules must interact with both non-polar and
polar environments (in or on, for example, the plasma membrane,
tissue fluids, compartments within the cell, and the biologically
active molecular itself), such compounds are designed typically to
contain both polar and non-polar domains. Compounds having both
such domains may be termed amphiphiles, and many lipids and
synthetic lipids that have been disclosed for use in facilitating
such intracellular delivery (whether for in vitro or in vivo
application) meet this definition. One particularly important class
of such amphiphiles is the cationic amphiphiles. In general,
cationic amphiphiles have polar groups that are capable of being
positively charged at or around physiological pH, and this property
is understood in the art to be important in defining how the
amphiphiles interact with the many types of biologically active
(therapeutic) molecules including, for example, negatively charged
polynucleotides such as DNA.
[0071] Examples of cationic amphiphilic compounds that have both
polar and non-polar domains and that are stated to be useful in
relation to intracellular delivery of biologically active molecules
are found, for example, in the following references, which contain
also useful discussion of (1) the properties of such compounds that
are understood in the art as making them suitable for such
applications, and (2) the nature of structures, as understood in
the art, that are formed by complexing of such amphiphiles with
therapeutic molecules intended for intracellular delivery.
[0072] (1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84,
7413-7417 (1987) disclose use of positively-charged synthetic
cationic lipids including
N->1(2,3-dioleyloxy)propyl!-N,N,N-trimethylammonium chloride
("DOTMA"), to form lipid/DNA complexes suitable for transfections.
See also Feigner et al., The Journal of Biological Chemistry,
269(4), 2550-2561 (1994).
[0073] (2) Behr et al., Proc. Natl. Acad. Sci. USA, 86, 6982-6986
(1989) disclose numerous amphiphiles including
dioctadecylamidologlycylspermine ("DOGS").
[0074] (3) U.S. Pat. No. 5,283,185 to Epand et al. describes
additional classes and species of amphiphiles including
3.beta.>N--(N.sup.1,N.sup.1-dimethylaminoethane) carbamoyl!
cholesterol, termed "DC-chol".
[0075] (4) Additional compounds that facilitate transport of
biologically active molecules into cells are disclosed in U.S. Pat.
No. 5,264,618 to Felgner et al. See also Felgner et al., The
Journal Of Biological Chemistry, 269(4), pp. 2550-2561 (1994) for
disclosure therein of further compounds including "DMRIE"
1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide.
[0076] (5) Reference to amphiphiles suitable for intracellular
delivery of biologically active molecules is also found in U.S.
Pat. No. 5,334,761 to Gebeyehu et al., and in Felgner et al.,
Methods (Methods in Enzymology), 5, 67-75 (1993).
The use of compositions comprising cationic amphiphilic compounds
for gene delivery is described, for example, in U.S. Pat. No.
5,049,386; U.S. Pat. No. 5,279,833; U.S. Pat. No. 5,650,096; U.S.
Pat. No. 5,747,471; U.S. Pat. No. 5,767,099; U.S. Pat. No.
5,910,487; U.S. Pat. No. 5,719,131; U.S. Pat. No. 5,840,710; U.S.
Pat. No. 5,783,565; U.S. Pat. No. 5,925,628; U.S. Pat. No.
5,912,239; U.S. Pat. No. 5,942,634; U.S. Pat. No. 5,948,925; U.S.
Pat. No. 6,022,874;U.S. Pat. No. 5,994,317; U.S. Pat. No.
5,861,397; U.S. Pat. No. 5,952,916; U.S. Pat. No. 5,948,767; U.S.
Pat. No. 5,939,401; and U.S. Pat. No. 5,935,936, the disclosures of
which are hereby incorporated herein by reference.
[0077] In addition, nucleic acid encoding modified FVII of the
present invention can be delivered using "naked DNA". Methods for
delivering a non-infectious, non-integrating DNA sequence encoding
a desired polypeptide or peptide operably linked to a promoter,
free from association with transfection-facilitating proteins,
viral particles, liposomal formulations, charged lipids and calcium
phosphate precipitating agents are described in U.S. Pat. No.
5,580,859; U.S. Pat. No. 5,963,622; U.S. Pat. No. 5,910,488; the
disclosures of which are hereby incorporated herein by
reference.
[0078] Gene transfer systems that combine viral and nonviral
components have also been reported. Cristiano et al., (1993) Proc.
Natl. Acad. Sci. USA 90:11548; Wu et al. (1994) J. Biol. Chem.
269:11542; Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099;
Yoshimura et al. (1993) J. Biol. Chem. 268:2300; Curiel et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8850; Kupfer et al. (1994)
Human Gene Ther. 5:1437; and Gottschalk et al. (1994) Gene Ther.
1:185. In most cases, adenovirus has been incorporated into the
gene delivery systems to take advantage of its endosomolytic
properties. The reported combinations of viral and nonviral
components generally involve either covalent attachment of the
adenovirus to a gene delivery complex or co-internalization of
unbound adenovirus with cationic lipid: DNA complexes.
[0079] As described herein, an effective amount of DNA vector
encoding Factor VIIa, a modified FVII, or FVII light chain and FVII
heavy chain is administered to the individual. An "effective
amount" of DNA vectors encoding the FVIIa, modified FVII or the
light and heavy chains of FVII, is an amount such that when
administered, it produces biologically active FVII molecule, which
results in enhanced blood clotting in the individual to whom it is
administered relative to blood clotting when an effective amount of
these vectors capable of producing activated FVII protein is not
administered. In addition, the amount of modified FVII administered
to an individual will vary depending on a variety of factors,
including the size, age, body weight, general health, sex and diet
of the individual, and the time of administration, duration or
particular qualities of the blood clotting defect. In the
particular embodiments wherein adenoviral or AAV vectors are used,
the dose of the DNA encoding modified FVII can be delivered via
adenoviral or AAV particles, generally in the range of about
10.sup.6 to about 10.sup.15 particles, more preferably in the range
of about 10.sup.8 to about 10.sup.13 particles. In the particular
embodiments wherein retroviral or lentiviral vectors are used, the
dose of the DNA encoding modified FVII can be delivered via
retroviral or lentiviral particles, generally in the range of about
10.sup.4 to about 10.sup.13 particles, more preferably in the range
of about 10.sup.6 to about 10.sup.11 particles. When DNA is
delivered in the form of plasmid DNA, a useful dose will generally
range from about 1 ug to about 1 g of DNA, preferably in the range
from about 100 ug to about 100 mg of DNA. The skilled clinician may
also determine the suitable dosage based upon expression levels
geared to meet particular plasma concentration levels of FVII.
Normal plasma concentration levels are approximately 500
nanograms/ml. However, it is known that significant amounts of
coagulation can be achieved with only a fraction of this
concentration. Accordingly, the dosage of DNA encoding modified
FVII to be used in the present invention may be tailored in order
to achieve a FVII plasma concentration level of about 5
nanograms/ml to about 1000 nanograms/ml. Methods for measuring the
plasma concentration levels of FVII are known in the art, and can
be used to monitor and/or tailor the dosage regimen
appropriately.
[0080] The DNA vector encoding modified FVII can be administered
using a variety of routes of administration. For example, the
modified FVII can be administered intravenously, parenterally,
intramuscularly, subcutaneously, orally, nasally, by inhalation, by
implant, by injection and/or by suppository. The composition can be
administered in a single dose or in more that one dose over a
period of time to confer the desired effect.
[0081] The present invention also provides compositions (e.g.,
pharmaceutical compositions) comprising the DNA vectors encoding
the modified FVII described herein. In one embodiment, the Factor
VII comprises an amino acid sequence which codes for a signal for
precursor cleavage by furin at the activation cleavage site of the
modified Factor VII. The compositions described herein can also
include a pharmaceutically acceptable carrier. The terms
"pharmaceutically acceptable carrier" or "carrier" refer to any
generally acceptable excipient or drug delivery device that is
relatively inert and non-toxic. Exemplary carriers include calcium
carbonate, sucrose, dextrose, mannose, albumin, starch, cellulose,
silica gel, polyethylene glycol (PEG), dried skim milk, rice flour,
magnesium strearate and the like.
[0082] Other suitable carriers (e.g., pharmaceutical carriers)
include, but are not limited to sterile water, salt solutions (such
as Ringer's solution), alcohols, gelatin, carbohydrates such as
lactose, amylose or starch, talc, silicic acid, viscous paraffin,
fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone,
etc. Such preparations can be sterilized and, if desired, mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, coloring and/or aromatic substances and
the like which do not deteriously react with the DNA vector
encoding modified FVII. A carrier (e.g., a pharmaceutically
acceptable carrier) is preferred, but not necessary to administer
the DNA vector encoding modified FVII. Suitable formulations and
additional carriers are described in Remington's Pharmaceutical
Sciences (17.sup.th Ed., Mack Publ. Co., Easton, Pa.), the
teachings of which are incorporated herein by reference in their
entirety.
[0083] The present invention also relates to an expression vector
comprising nucleic acid encoding a modified Factor VII, wherein the
modified Factor VII leads to generation of Factor VIIa in vivo. In
one embodiment, the nucleic acid sequence encodes an amino acid
sequence which includes a signal for precursor cleavage by furin at
the activation cleavage site of the modified Factor VII. In another
embodiment, the nucleic acid construct comprises two expression
constructs which encode a modified Factor VII wherein the first
expression construct comprises amino acids 1-152 of human Factor
VII and the second expression comprises amino acids 153-406 of
human Factor VII and a leader sequence.
[0084] The present invention also relates to host cells comprising
nucleic acid which encodes a modified Factor VII, wherein the
modified Factor VII leads to generation of Factor VIIa in vivo. In
one embodiment, the nucleic acid sequence encodes an amino acid
sequence which includes a signal for precursor cleavage by furin at
the activation cleavage site of the modified Factor VII. In another
embodiment, the nucleic acid construct comprises two expression
constructs which encode a modified Factor VII wherein the first
expression construct comprises amino acids 1-152 of human Factor
VII and the second expression comprises amino acids 153-406 of
human Factor VII and a leader sequence.
EXAMPLES
Materials & Methods
Cloning of FVII:
[0085] The factor VII cDNA was cloned by PCR (Perkin Elmer, 25
cycles) from a human liver cDNA library (Clontech) using primer
5432JS (5'-CTAGCCTAGG CCACCATGGT CTCCCAGGCC CTCAGGCTC-3' and primer
5433JS (5'-CCTTAATTAA CTAGGGAAAT GGGGCTCGCA GGAG-3'. The PCR
product was cloned into a pCR-Blunt-II TOPO vector (Invitrogen),
sequenced for accuracy and then subcloned into pCMV expression
vector, which has the CMV promoter/enhancer and an SV40 poly A.
Cloning of FVII Light Chain (LC):
[0086] The FVII light chain was cloned by PCR from the plasmid
pCMV/hFVII using primer 5432JS shown above and primer 5479JS
(5'-GCTAGCCTAT CGGCCTTGGG G-3'). This construct contains the FVII
leader sequence and amino acids #1 (Ala) to #152 (Arg). The PCR
product has been cloned into the pCR-Blunt-II TOPO vector,
sequenced for accuracy and then subloned into the pCMV expression
vector.
Cloning of FVII Heavy Chain (HC):
[0087] The FVII heavy chain was cloned by three primer PCR from the
plasmid pCMV/hFVII. The three primers used were 5432JS, 5433JS and
primer 5480JS (5'-TGCACCGGCG CCGGCGCATT GTGGGGGGCA AGGTGT-3'). This
construct contains the FVII leader sequence followed by amino acids
#153 (Ile) to #406 (Pro). The PCR product has been cloned into
pCR-Blunt-II TOPO vector, sequenced and then subcloned into the
pCMV expression vector.
Mutagenesis to Create Furin Cleavage Site in FVII:
[0088] The cleavage site for the conversion of FVII to FVIIa has
been mutated to a furin recognition site using three primer PCR
mutagenesis method. The original amino acid sequence #149 (Pro) and
#151 (Gly) has been changed to #149 (Arg) and #151(Lys) to generate
the furin recognition site Arg.sup.149-Gln-Lys-Arg.sup.152. The PCR
product has been cloned into pCR-Blunt-II TOPO vector, sequenced
and then subcloned into the pCMV expression vector.
In Vitro Transcription/Translation:
[0089] All clones mentioned above were tested by in vitro
transcription/translation. The three clones were found to produce
proteins of the expected sizes, that is, light chain=24 kd, heavy
chain=34 kd and full length FVII=50 kd.
(6) Western Blot Analysis:
[0090] All plasmids were transfected into Hep3B cells (human
hepatoma cell line) and FVII expression was measured at 24 hours by
western blot analysis of the cell lysates using antibody to FVII
obtained from Haematologic Technologies. All of the FVII clones
expressed FVII proteins.
Membrane Contact Site Mutations:
[0091] It has been shown that certain mutations in the membrane
contact site for factor VII can increase the membrane affinity of
the protein (Shah et al (1998) PNAS, Vol. 95, 4229-4234) and
increase the rate of autoactivation with soluble tissue factor
compared to wild type factor VII. In one example of the present
invention, we have combined these mutations (Pro-10 mutated to Gln
and Lys-32 mutated to Glu) along with our mutations around amino
acid 152 to generate a version of factor VII that are recognized by
furin or SKI-1 and is more potent at FVII specific clotting. See
FIG. 2.
(8) Generation of SKI-1 Recognition Sites for FVII Processing:
[0092] SKI-1 is a proprotein convertase that is present in the
golgi of most tissues and cells and has a unique cleavage
specificity (Seidah et al. (1999) PNAS, Vol. 96, 1321-1326). In one
example of the present invention the cDNA for FVII is modified
around amino acid 152 to create a recognition site for SKI-1, such
that SKI-1 cleaves FVII to generate active FVIIa. See FIG. 3.
Cloning & Mutagenesis of Human FVII
[0093] Human FVII cDNA was PCR amplified from a human liver Quick
Clone cDNA (Clontech) and cloned into pCMV, a plasmid containing
the CMV promoter and SV40 polyA and pLSP, a plasmid containing the
AAT promoter and BGH polyA. The endogenous FVII cleavage site was
mutated to contain a furin recognition site using the primer 5' AGC
AAA CGC CAA AAG CGA ATT GTG GGG GGC AAG 3' which mutates Pro149 to
an Arg and Gly151 to a Lys.
In Vitro Expression & Proteolytic Processing by Furin
[0094] 293 and Hep3B cells were transfected with 10 ug pCMV/FVII or
pCMV/FVIIa using either the Profection CaPO.sub.4 Transfection Kit
(Promega) or Lipofectamine 2000 (Gibco), respectively. In vitro
expression of secreted FVIIa was detected using a FVIIa specific
clotting assay described below. To demonstrate proteolytic
processing of FVII by furin, an in vitro transcription/translation
reaction was done on the pCMV/FVII and pCMV/FVIIa plasmids. FVII
was immunoprecipitated from the lysate using 2 ug of a polyclonal
sheep anti-human FVII antibody (Haematologic Technologies)
overnight at 4.degree. C. A furin digest was done directly on the
protein A sepharose beads in 100 mM Hepes, 0.5% Triton X-100, 1 mM
CaCl.sub.2, 1 mM 2-mercaptoethanol and 2 U of furin enzyme (NEB) at
30.degree. C. for 1 hour. Following furin digestion, 20 uL of
2.times.SDS loading dye was added to the reaction and the entire
sample was run on a 14% SDS PAGE gel.
Immunoprecipitation
[0095] 293 cells were transfected with 10 ug pCMV/FVII and
pCMV/FVIIa plasmids. 48 hours after transfection, cells were
labeled with 35S-Met/Cys for 4 hours. FVII was immunoprecipitated
from the cell lysate and media using 2 ug of a polyclonal sheep
anti-human FVII antibody. Samples were run on a 14% SDS PAGE
gel.
FVIIa Specific Clotting Assay
[0096] A FVIIa specific clotting assay, Staclot VIIa-rTF, was
purchased from Diagnostica Stago. Clotting time was determined by
adding 50 uL human FVII deficient plasma+50 uL rTF/pL+either 50 uL
of supernatant from transfected cells or 50 uL of mouse plasma
diluted 1:500 and incubating at 37.degree. C. for 3 minutes.
Clotting was initiated by adding 50 uL 0.025M CaCl.sub.2 and the
clotting time was measured on a Start 4 Clot Detection System
(Diagnostica Stago). Modified aPTT (Activated Partial
Thromboplastin Time) A modified APTT assay was performed by adding
50 uL of either human FVIII or FIX deficient plasma (George King
Biomedical)+50 uL APTT reagent (Diagnostica Stago)+10 uL rTF/pL
diluted 1:1000+50 uL of supernatant from Hep3B cells transfected
with either pCMV/FVII or pCMV/FVIIa and incubating at 37.degree. C.
for 3 minutes. Clotting was initiated by adding 50 uL 0.025M
CaCl.sub.2 and the clotting time was measured on Start 4 Clot
Detection System (Diagnostica Stago). See FIG. 5.
Diluted Partial Thromboplastin Time (PTT) Assay
[0097] Diluted partial thromboplastin time was determined by
incubating 50 uL of either human FVIII or FIX deficient plasma or
normal human plasma+50 uL thromboplastin with calcium (Sigma)
diluted 1:100 in 0.154M NaCl+50 uL media from 293 cells transfected
with pCMV/FVII or pCMV/FVIIa and incubating at 37.degree. C. for 3
minutes. Clotting was initiated by adding 50 uL 0.025M CaCl.sub.2
and the clotting time was measured on Start 4 Clot Detection System
(Diagnostica Stago). See FIG. 6.
In Vivo Studies in Normal & FVIII k/o Mice
[0098] Beige/SCID and FVIII k/o mice were injected with 10 ug FVII
or FVIIa plasmid via the tail vein using a high volume injection
technique. Plasma was collected out to 5 weeks post injection.
FVIIa was measured using the Staclot VIIa-rTF assay. See FIG.
7.
Prothrombin Time (PT) Assay
[0099] Prothrombin time was determined by incubating 50 uL of FVIII
KO mouse plasma at 37.degree. C. for 1 minute. Clotting was
initiated by adding 100 uL thromboplastin with calcium (Sigma
Diagnostics) and the clotting time measured on Start 4 Clot
Detection System (Diagnostica Stago).
[0100] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that numerous
modifications and changes in form and details and optimization of
parameters may be made therein without departing from the scope of
the invention encompassed by the appended claims. Such
modifications, changes and optimizations constitute part of the
present invention.
[0101] The disclosure of all of the publications which are cited in
this specification are hereby incorporated herein by reference for
the disclosure contained therein.
Sequence CWU 1
1
2018PRTHomo sapiens 1Ser Lys Pro Gln Gly Arg Ile Val1 528PRTHomo
sapiens 2Ser Lys Arg Gln Lys Arg Ile Val1 5313PRTHomo sapiens 3Ser
Lys Pro Gln Gly Arg Arg Arg Arg Ala Asn Ile Val1 5 10412PRTHomo
sapiens 4Ser Lys Pro Gln Gly Arg Arg Arg Arg Ser Ile Val1 5
1059PRTHomo sapiens 5Ser Lys Arg Gln Lys Arg Ala Ile Val1
5611PRTHomo sapiens 6Ser Lys Arg Gln Arg Arg Ala Asn Gly Gly Lys1 5
1078PRTHomo sapiens 7Arg Lys Arg Gln Lys Arg Ile Val1 584PRTHomo
sapiensmisc_feature(2)..(3)Xaa can be any naturally occurring amino
acid 8Arg Xaa Xaa Arg198PRTHomo sapiensmisc_feature(1)..(1)Xaa can
be any naturally occurring amino acid 9Xaa Lys Xaa Gln Xaa Arg Ile
Val1 5104PRTHomo sapiens 10Arg Gln Lys Arg1114PRTHomo sapiens 11Pro
Gln Gly Arg11239DNAArtificial Sequenceoligonucleotide primer
12ctagcctagg ccaccatggt ctcccaggcc ctcaggctc 391334DNAArtificial
Sequenceoligonucleotide primer 13ccttaattaa ctagggaaat ggggctcgca
ggag 341421DNAArtificialOligonucleotide primer 14gctagcctat
cggccttggg g 211536DNAArtificial SequenceOligonucleotide primer
15tgcaccggcg ccggcgcatt gtggggggca aggtgt 361633DNAArtificial
SequenceOligonucleotide primer 16agcaaacgcc aaaagcgaat tgtggggggc
aag 33174PRTHomo sapiensmisc_feature(2)..(2)Xaa can be any
naturally occurring amino acid 17Arg Xaa Lys Arg1184PRTHomo
sapiensmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 18Arg Xaa Arg Arg1196PRTHomo sapiensmisc_feature(1)..(2)Xaa
can be any naturally occurring amino acid 19Xaa Xaa Arg Xaa Xaa
Arg1 5206PRTHomo sapiensmisc_feature(1)..(5)Xaa can be any
naturally occurring amino acid 20Xaa Xaa Xaa Xaa Xaa Gly1 5
* * * * *