U.S. patent application number 10/310398 was filed with the patent office on 2006-08-24 for nucleic acid injected into hapatic vein lumen and delivered to primate liver.
Invention is credited to Vladimir G. Budker, James E. Hagstrom, Julia Hegge, Jon A. Wolff.
Application Number | 20060189553 10/310398 |
Document ID | / |
Family ID | 32468028 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060189553 |
Kind Code |
A9 |
Wolff; Jon A. ; et
al. |
August 24, 2006 |
Nucleic acid injected into hapatic vein lumen and delivered to
primate liver
Abstract
Processes are described for obtaining high levels of gene
expression in primates after injection of nucleic acid to the liver
via the lumen of the hepatic vein. The described process results in
high level of gene expression with transient increases in liver
enzymes.
Inventors: |
Wolff; Jon A.; (Madison,
WI) ; Hegge; Julia; (Monona, WI) ; Hagstrom;
James E.; (Middleton, WI) ; Budker; Vladimir G.;
(Middleton, WI) |
Correspondence
Address: |
MIRUS CORPORATION
505 SOUTH ROSA RD
MADISON
WI
53719
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20040110697 A1 |
June 10, 2004 |
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Family ID: |
32468028 |
Appl. No.: |
10/310398 |
Filed: |
December 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09447966 |
Nov 23, 1999 |
6627616 |
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10310398 |
Dec 5, 2002 |
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09391260 |
Sep 7, 1999 |
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09447966 |
Nov 23, 1999 |
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08975573 |
Nov 21, 1997 |
6265387 |
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09391260 |
Sep 7, 1999 |
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08571536 |
Dec 13, 1995 |
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08975573 |
Nov 21, 1997 |
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Current U.S.
Class: |
514/44R ;
424/93.2; 435/456 |
Current CPC
Class: |
A61K 48/0075
20130101 |
Class at
Publication: |
514/044 ;
435/456; 424/093.2 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86 |
Claims
1. A process for delivering a polynucleotide to a primate liver
cell, comprising: inserting the polynucleotide in a solution into
the lumen of a hepatic vein.
2. The process of claim 1 wherein the solution is injected at a
rate of 18 ml/kg/min or greater.
3. The process of claim 1 wherein the solution is injected at a
rate of 36 ml/kg/min or greater.
4. The process of claim 1 wherein the solution is injected at a
rate of 48 ml/kg/min or greater.
5. The process of claim 1 wherein the injection of the solution
results in portal vein pressure of 10 mm Hg or greater.
6. The process of claim 1 wherein the injection of the solution
results in portal vein pressure of 45 mm Hg or greater.
7. The process of claim 1 wherein the injection of the solution
results in portal vein pressure of 105 mm Hg or greater.
8. The process of claim 1 wherein the polynucleotide consists of
naked DNA.
9. The process of claim 1 wherein the polynucleotide is selected
from the group consisting of a viral vectors and a non-viral
vectors.
10. The process of claim 1 wherein the polynucleotide consists of a
blocking polynucleotide for preventing gene expression.
11. The process of claim 10 wherein the blocking polynucleotide
consists of anti sense.
12. The process of claim 10 wherein the blocking polynucleotide
consists of siRNA.
Description
FIELD OF THE INVENTION
[0001] The invention generally relates to techniques for
transferring genes into mammalian parenchymal cells in vivo. More
particularly, a method is provided for transfecting hepatic cells
with polynucleotides delivered intravascularly under pressure.
BACKGROUND OF THE INVENTION
[0002] It was first observed that the in vivo injection of plasmid
DNA into muscle enabled the expression of foreign genes in the
muscle (Wolff, J A, Malone, R W, Williams, P, et al. Direct gene
transfer into mouse muscle in vivo. Science 1990;247: 1465-1468.).
Since that report, several other studies have reported the ability
for foreign gene expression following the direct injection of DNA
into the parenchyma of other tissues. Naked DNA was expressed
following its injection into cardiac muscle (Acsadi, G., Jiao, S.,
Jani, A., Duke, D., Williams, P., Chong, W., Wolff, J. A. Direct
gene transfer and expression into rat heart in vivo. The New
Biologist 3(1), 71-81,1991.), pig epidermis (Hengge, U. R., Chan,
E. F., Foster, R. A., Walker, P. S., and Vogel, J. C. Nature
Genetics 10:161-166 (1995)), rabbit thyroid (M. Sikes, B. O'Malley,
M. Finegold, and F. Ledley, Hum. Gene Ther. 5, 837 (1994), lung by
intratracheal injection (K. B. Meyer, M. M. Thompson, M. Y. Levy,
L. G. Barron, F. C. Szoka, Gene Ther. 2, 450 (1995)), into arteries
using a hydrogel-coated angioplasty balloon (R. Riessen et al,
Human Gene Ther. 4, 749 (1993)) (G. Chapman et al. Circ. Res. 71,
27 (1992)), melanoma tumors (R. G. Vile and I. R. Hart, Cancer Res.
53, 962 (1993)) and rat liver [(Malone, R. W. et al. JBC
269:29903-29907 (1994)) (Hickman, M. A. Human Gene Therapy
5:1477-1483 (1994))].
[0003] Another important target tissue for gene therapy is the
mammalian liver, given its central role in metabolism and the
production of serum proteins. A variety of techniques have been
developed to transfer genes into the liver. Cultured hepatocytes
have been genetically modified by retroviral vectors [(Wolff, J. A.
et al. PNAS 84:3344-3348 (1987) (Ledley, F. D., Darlington, G. J.,
Hahn, T. and Woo, S.C.L. PNAS 84:5335-5339 (1987)] and re-implanted
back into the livers in animals and in people [(J. R. Chowdhury et
al. Science 254, 1802 (1991) (M. Grossman et al. Nature Genetics 6,
335 (1994)]. Retroviral vectors have also been delivered directly
to livers in which hepatocyte division was induced by partial
hepatectomy [(Kay, M. A. et al Hum Gene Ther. 3:641-647 (1992)
(Ferry, N., Duplessis, O., Houssin, D., Danos, O. and Heard, J. -M.
PNAS 88:8377-8381 (1991) (Kaleko, M., Garcia, J. V. and Miller, A.
D. Hum Gene THer. 2:27-32 (1991)]. The injection of adenoviral
vectors into the portal or systemic circulatory systems leads to
high levels of foreign gene expression that is transient [(L. D.
Stratford-Perricaudet, M. Levrero, J. F. Chasse, M. Perricaudet, P.
Briand, Hum. Gene Ther. 1, 241 (1990) (H. A. Jaffe et al. Nat.
Genet. 1, 372 (1992) (Q. Li, M. A. Kay, M. Finegold, L. D.
Stratford-Perricaudet, S. L. C. Woo, Hum. Gene Ther. 4, 403
(1993)]. Non-viral transfer methods have included polylysine
complexes of asialoglycoproteins that are injected into the system
circulation [Wu, G. Y. and Wu, C. H. J. Biol. Chem. 263:14621-14624
(1988)].
[0004] Foreign gene expression has also been achieved by
repetitively injecting naked DNA in isotonic solutions into the
liver parenchyma of animals treated with dexamethasone [(Malone, R.
W. et al. JBC 269:29903-29907 (1994) (Hickman, M. A. Human Gene
Therapy 5:1477-1483 (1994)]. Plasmid DNA expression in the liver
has also been achieved via liposomes delivered by tail vein or
intraportal routes [(Kaneda, Y., Kunimitsu, I. and Uchida, T. J.
Biol. Chem. 264:12126-12129 (1989) (Soriano, P. et al. PNAS
80:7128-7131 (1983) Kaneda, Y., Iwai, K. and Uchida, T. Science
243:375-378 (1989)].
[0005] Despite this progress, there is still a need for a gene
transfer method that can efficiently and safely cause the
expression of foreign genes in the liver in a and/or repetitive
manner.
SUMMARY OF THE INVENTION
[0006] The present invention provides for the transfer of
polynucleotides into parenchymal cells within tissues in situ and
in vivo. An intravascular route of administration enables a
prepared polynucleotide to be delivered to the parenchymal cells
more evenly distributed and more efficiently expressed than direct
parenchymal injections. The efficiency of polynucleotide delivery
and expression was increased substantially by increasing the
permeability of the tissue's blood vessel. This was done by
increasing the intravascular hydrostatic (physical) pressure and/or
increasing the osmotic pressure. Expression of a foreign DNA was
obtained in mammalian liver by intraportally injecting plasmid DNA
in a hypertonic solution and transiently clamping the hepatic
vein/inferior vena cava. Optimal expression was obtained by
clamping the portal vein and injecting the hepatic vein/inferior
vena cava.
[0007] A process is described for delivering a polypeptide into a
parenchymal cell in a mammal, comprising, transporting the
polynucleotide into a vessel communicating with the parenchymal
cell of the mammal such that the polynucleotide is transfected into
the parenchymal cell.
[0008] A process for delivering a coded polynucleotide into a
parenchymal cell of a mammal for expression of a protein,
comprising, transporting the polynucleotide to a vessel containing
a fluid and having a permeable wall; and, increasing the
permeability of the wall for a time sufficient to complete delivery
of the polynucleotide.
DETAILED DESCRIPTION
A. Definitions
[0009] The term, naked polynucleotides, indicates that the
polynucleotides are not associated with a transfection reagent or
other delivery vehicle that is required for the polynucleotide to
be delivered to the parenchymal cell. A transfection reagent is a
compound or compounds used in the prior art that bind(s) to or
complex(es) with polynucleotides and mediates their entry into
cells. The transfection reagent also mediates the binding and
internalization of polynucleotides into cells. Examples of
transfection reagents include cationic liposomes and lipids,
calcium phosphate precipitates, and polylysine complexes.
Typically, the transfection reagent has a net positive charge that
binds to the polynucleotide's negative charge. The transfection
reagent mediates binding of polynucleotides to cell via its
positive charge (that binds to the cell membrane's negative charge)
or via ligands that bind to receptors in the cell. For example,
cationic liposomes or polylysine complexes have net positive
charges that enable them to bind to DNA. Other vehicles are also
used, in the prior art, to transfer genes into cells. These include
complexing the polynucleotides on particles that are then
accelerated into the cell. This is termed biolistic or gun
techniques. Other methods include eletroporation in which a device
is used to give an electric charge to cells. The charge increases
the permeability of the cell.
[0010] The term polynucleotide is a term of art that refers to a
string of at least two base-sugar-phosphate combinations.
Nucleotides are the monomeric units of nucleic acid polymers. The
term includes deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA) in the form of an oligonucleotide messenger RNA, anti-sense,
plasmid DNA, parts of a plasmid DNA or genetic material derived
from a virus. A polynucleotide is distinguished, here, from a
oligonucleotide by containing more than 120 monomeric units.
Anti-sense is a polynucleotide that interferes with the function of
DNA and/or RNA. A polynucleotide is considered in this
specification to include a non-natural polynucleotide (not
occurring in nature), for example: a derivative of natural
nucleotides such as phosphothionates or peptide nucleic acids.
[0011] A polynucleotide can be delivered to a cell in order to
produce a cellular change that is therapeutic. The delivery of
polynucleotides or other genetic material for therapeutic purposes
(the art of improving health in an animal including treatment or
prevention of disease) is gene therapy. The polynucleotides are
coded to express a whole or partial protein, or may be anti-sense,
and can be delivered either directly to the organism in situ or
indirectly by transfer to a cell that is then transplanted into the
organism. The protein can be missing or defective in an organism as
a result of genetic, inherited or acquired defect in its
genome.
[0012] For example, a polynucleotide may be coded to express the
protein dystrophin that is missing or defective in Duchenne
muscular dystrophy. The coded polynucleotide is delivered to a
selected group or groups of cells and incorporated into those
cell's genome or remain apart from the cell's genome. Subsequently,
dystrophin is produced by the formerly deficient cells. Other
examples of imperfect protein production that can be treated with
gene therapy include the addition of the protein clotting factors
that are missing in the hemophilias and enzymes that are defective
in inborn errors of metabolism such as phenylalanine
hydroxylase.
[0013] A delivered polynucleotide can also be therapeutic in
acquired disorders such as neurodegenerative disorders, cancer,
heart disease, and infections. The polynucleotide has its
therapeutic effect by entering the cell. Entry into the cell is
required for the polynucleotide to produce the therapeutic protein,
to block the production of a protein, or to decrease the amount of
a RNA.
[0014] Additionally, a polynucleotide can be delivered to block
gene expression. Such polynucleotides can be anti-sense by
preventing translation of a messenger RNA or could block gene
expression by preventing transcription of the gene. Small
inhibiting RNA (siRNA) can also be used to inhibit gene expression.
Preventing both RNA translation as well as DNA transcription is
considered preventing expression. Transcription can be blocked by
the polynucleotide binding to the gene as a duplex or triplex. It
could also block expression by binding to proteins that are
involved in a particular cellular biochemical process.
[0015] Polynucleotides may be delivered that recombine with genes.
The polynucleotides may be DNA, RNA, hybrids and derivatives of
natural nucleotides. Recombine is the mixing of the sequence of a
delivered polynucleotide and the genetic code of a gene. Recombine
includes changing the sequence of a gene.
[0016] Delivery of a polynucleotide means to transfer a
polynucleotide from a container outside a mammal to within the
outer cell membrane of a cell in the mammal. The term transfection
is used herein, in general, as a substitute for the term delivery,
or, more specifically, the transfer of a polynucleotide from
directly outside a cell membrane to within the cell membrane. If
the polynucleotide is a primary RNA transcript that is processed
into messenger RNA, a ribosome translates the messenger RNA to
produce a protein within the cytoplasm. If the polynucleotide is a
DNA, it enters the nucleus where it is transcribed into a messenger
RNA that is transported into the cytoplasm where it is translated
into a protein. The polynucleotide contains sequences that are
required for its transcription and translation. These include
promoter and enhancer sequences that are required for initiation.
DNA and thus the corresponding messenger RNA (transcribed from the
DNA) contains introns that must be spliced, poly A addition
sequences, and sequences required for the initiation and
termination of its translation into protein. Therefore if a
polynucleotide expresses its cognate protein, then it must have
entered a cell.
[0017] A therapeutic effect of the protein in attenuating or
preventing the disease state can be accomplished by the protein
either staying within the cell, remaining attached to the cell in
the membrane or being secreted and dissociating from the cell where
it can enter the general circulation and blood. Secreted proteins
that can be therapeutic include hormones, cytokines, growth
factors, clotting factors, anti-protease proteins (e.g.
alpha-antitrypsin) and other proteins that are present in the
blood. Proteins on the membrane can have a therapeutic effect by
providing a receptor for the cell to take up a protein or
lipoprotein. For example, the low density lipoprotein (LDL)
receptor could be expressed in hepatocytes and lower blood
cholesterol levels and thereby prevent atherosclerotic lesions that
can cause strokes or myocardial infarction. Therapeutic proteins
that stay within the cell can be enzymes that clear a circulating
toxic metabolite as in phenylketonuria. They can also cause a
cancer cell to be less proliferative or cancerous (e.g. less
metastatic). A protein within a cell could also interfere with the
replication of a virus.
[0018] The delivered polynucleotide can stay within the cytoplasm
or nucleus apart from the endogenous genetic material.
Alternatively, the polynucleotide could recombine (become a part
of) the endogenous genetic material. For example, DNA can insert
into chromosomal DNA by either homologous or non-homologous
recombination.
[0019] Parenchymal cells are the distinguishing cells of a gland or
organ contained in and supported by the connective tissue
framework. The parenchymal cells typically perform a function that
is unique to the particular organ. The term "parenchymal" often
excludes cells that are common to many organs and tissues such as
fibroblasts and endothelial cells within the blood vessels.
[0020] In a liver organ, the parenchymal cells include hepatocytes,
Kupffer cells and the epithelial cells that line the biliary tract
and bile ductules. The major constituent of the liver parenchyma
are polyhedral hepatocytes (also known as hepatic cells) that
presents at least one side to an hepatic sinusoid and apposed sides
to a bile canaliculus. Liver cells that are not parenchymal cells
include cells within the blood vessels such as the endothelial
cells or fibroblast cells.
[0021] In striated muscle, the parenchymal cells include myoblasts,
satellite cells, myotubules, and myofibers. In cardiac muscle, the
parenchymal cells include the myocardium also known as cardiac
muscle fibers or cardiac muscle cells and the cells of the impulse
connecting system such as those that constitute the sinoatrial
node, atrioventricular node, and atrioventricular bundle.
[0022] In a pancreas, the parenchymal cells include cells within
the acini such as zymogenic cells, centroacinar cells, and basal or
basket cells and cells within the islets of Langerhans such as
alpha and beta cells.
[0023] In spleen, thymus, lymph nodes and bone marrow, the
parenchymal cells include reticular cells and blood cells (or
precursors to blood cells) such as lymphocytes, monocytes, plasma
cells and macrophages.
[0024] In the nervous system which includes the central nervous
system (the brain and spinal cord) peripheral nerves, and ganglia,
the parenchymal cells include neurons, glial cells, microglial
cells, oligodendrocytes, Schwann cells, and epithelial cells of the
choroid plexus.
[0025] In the kidney, parenchymal cells include cells of collecting
tubules and the proximal and distal tubular cells. In the prostate,
the parenchyma includes epithelial cells.
[0026] In glandular tissues and organs, the parenchymal cells
include cells that produce hormones. In the parathyroid glands, the
parenchymal cells include the principal cells (chief cells) and
oxyphilic cells. In the thyroid gland, the parenchymal cells
include follicular epithelial cells and parafollicular cells. In
the adrenal glands, the parenchymal cells include the epithelial
cells within the adrenal cortex and the polyhedral cells within the
adrenal medulla.
[0027] In the parenchyma of the gastrointestinal tract such as the
esophagus, stomach, and intestines, the parenchymal cells include
epithelial cells, glandular cells, basal, and goblet cells.
[0028] In the parenchyma of lung, the parenchymal cells include the
epithelial cells, mucus cells, goblet cells, and alveolar
cells.
[0029] In fat tissue, the parenchymal cells include adipose cells
or adipocytes. In the skin, the parenchymal cells include the
epithelial cells of the epidermis, melanocytes, cells of the sweat
glands, and cells of the hair root.
[0030] In cartilage, the parenchyma includes chondrocytes. In bone,
the parenchyma includes osteoblasts, osteocytes, and
osteoclasts.
[0031] An intravascular route of administration enables a
polynucleotide to be delivered to parenchymal cells more evenly
distributed and more efficiently expressed than direct parenchymal
injections.
[0032] Intravascular herein means within a hollow tubular structure
called a vessel that is connected to a tissue or organ within the
body. Within the cavity of the tubular structure, a bodily fluid
flows to or from the body part. Examples of bodily fluid include
blood, lymphatic fluid, or bile. Examples of vessels include
arteries, arterioles, capillaries, venules, sinusoids, veins,
lymphatics, and bile ducts. The intravascular route includes
delivery through the blood vessels such as an artery or a vein.
[0033] Polypeptide refers to a linear series of amino acid residues
connected to one another by peptide bonds between the alpha-amino
group and carboxy group of contiguous amino acid residues. Protein
refers to a linear series of greater than 50 amino acid residues
connected one to another as in a polypeptide.
[0034] Vectors are polynucleic molecules originating from a virus,
a plasmid, or the cell of a higher organism into which another
nucleic fragment of appropriate size can be integrated without loss
of the vectors capacity for self-replication; vectors introduce
foreign DNA into host cells, where it can be reproduced. Examples
are plasmids, cosmids, and yeast artificial chromosomes; vectors
are often recombinant molecules containing DNA sequences from
several sources. A vector includes a viral vector: for example,
adenovirus (icosahedral (20-sided) virus that contains; there are
over 40 different adenovirus varieties, some of which cause the
common cold) DNA; adenoassociated viral vectors (AAV) which are
derived from adenoassociated viruses and are smaller than
adenoviruses; and retrovirus (any virus in the family Retroviridae
that has RNA as its nucleic acid and uses the enzyme reverse
transcriptase to copy its genome into the DNA of the host cell's
chromosome; examples include VSV G and retroviruses that contain
components of lentivirus including HIV type viruses).
[0035] Afferent blood vessels of organs are defined as vessels
which are directed towards the organ or tissue and in which blood
flows towards the organ or tissue under normal physiologic
conditions. Conversely, the efferent blood vessels of organs are
defined as vessels which are directed away from the organ or tissue
and in which blood flows away from the organ or tissue under normal
physiologic conditions. In the liver, the hepatic vein is an
efferent blood vessel since it normally carries blood away from the
liver into the inferior vena cava. Also in the liver, the portal
vein and hepatic arteries are afferent blood vessels in relation to
the liver since they normally carry blood towards the liver.
B. Delivery of Polynucleotides
[0036] In a preferred embodiment of the present invention, a naked
polynucleotide is delivered into a liver blood vessel at distal or
proximal points. A liver blood vessel includes the portal venous
system which transports blood from the gastrointestinal tract and
other internal organs (e.g. spleen, pancreas and gall bladder) to
the liver. Another liver blood vessel is the hepatic vein. The
hepatic vein may also be reached via the inferior vena cava or
another blood vessel that ultimately connects to the liver. A
needle or catheter is used to inject the polynucleotide into the
vascular system. The injection can be performed under direct
observation following an incision and visualization of the tissues
blood vessels. Alternatively, a catheter can be inserted at a
distant site and threaded so that it resides in the vascular system
that connects with the target tissue. In another embodiment, the
injection could be performed by using a needle that traverses the
intact skin and enters a vessel that supplies or drains from the
target tissue.
[0037] In a preferred embodiment, the liver and portal vein of mice
(25 g, 6-week old ICR mice) are visualized through a ventral
midline incision. Anesthesia was obtained from intramuscular
injections of 1000 .mu.g of ketamine-HCl (Parke-Davis, Morris
Plains, N.J.) in 1 ml of normal saline and methoxyflurane
(Pitman-Moore, Mudelein, Ill. USA) which was administered by
inhalation as needed. Plasmid DNA in 1 ml of various solutions
containing heparin to prevent clotting was injected into the portal
vein using a needle over approximately 30 sec. At various times
after the injection, the animals were sacrificed by cervical
dislocation and the livers (average weight of 1.5 g) were divided
into six sections composed of two pieces of median lobe, two pieces
of left lateral lobe, the right lateral lobe, and the caudal lobe
plus a small piece of right lateral lobe. Each of the six sections
were placed separately into an homogenizing buffer. The homogenates
were centrifuged and the supernatant analyzed for the foreign gene
product. If the gene product is secreted then blood is obtained
from the retro-orbital venous sinus and the level of the secreted
protein is assayed in the blood. For example, the expression of the
human growth hormone gene can be detected by measuring the amount
of human growth hormone in the mouse serum using a radioimmune
assay (RIA) (HGH-TGES 100T kit from Nichols Institute, San Juan
Capistrano, Calif., USA). Alternatively, the foreign gene could
produce an enzyme that corrects an abnormality in the disease
state. For example, the phenylalanine hydroxylase gene could be
used to normalize the elevated phenylalanine blood levels in a
genetic mouse model of phenylketonuria.
[0038] In the liver, the hepatic vein is an efferent blood vessel
since it normally carries blood away from the liver into the
inferior vena cava. Also in the liver, the portal vein and hepatic
arteries are afferent blood vessels in relation to the liver since
they normally carry blood towards the liver. In a preferred
embodiment, plasmid DNA may be efficiently expressed if delivered
by a retrograde route into the efferent vessel of the liver (i.e.
the hepatic vein). As demonstrated in the examples that follow,
injections were directed into the inferior cava which was clamped
in two locations; proximal and distal to the entry of the hepatic
vein into the inferior vena cava. Specifically, the downstream
inferior vena cava clamp was placed between the diaphragm and the
entry point of the hepatic vein. The upstream inferior vena cava
clamp was placed just upstream of the entry point of the renal
veins. Since the veins of other organs such as the renal veins
enter the inferior vena cava at this location, not all of the
injection fluid went into the liver. In some of the animals that
received retrograde injections in the inferior vena cava, the
hepatic artery, mesenteric artery, and portal vein were clamped
(occluded).
C. Permeability
[0039] The efficiency of the polynucleotide delivery and expression
was increased substantially by increasing the permeability of a
blood vessel within the target tissue. Permeability is defined here
as the propensity for macromolecules such as polynucleotides to
move through vessel walls and enter the extravascular space. One
measure of permeability is the rate at which macromolecules move
through the vessel wall and out of the vessel. Another measure of
permeability is the lack of force that resists the movement through
the vessel wall and out of the vessel. Vessels contain elements
that prevent macromolecules from leaving the intravascular space
(internal cavity of the vessel). These elements include endothelial
cells and connective material (e.g. collagen). Increased
permeability indicates that there are fewer of these elements that
can block the egress of macromolecules and that the spaces between
these elements are larger and more numerous. In this context,
increased permeability enables a high percentage of polynucleotides
being delivered to leave the intravascular space; while low
permeability indicates that a low percentage of the polynucleotides
will leave the intravascular space.
[0040] The permeability of a blood vessel can be increased by
increasing the intravascular hydrostatic pressure. In a preferred
embodiment, the intravascular hydrostatic pressure is increased by
rapidly (from 10 seconds to 30 minutes) injecting a polynucleotide
in solution into the blood vessel which increases the hydrostatic
pressure. In another preferred embodiment, hydrostatic pressure is
increased by obstructing the outflow of the injection solution from
the tissue for a period of time sufficient to allow delivery of a
polynucleotide. Obstructing means to block or impede the outflow of
injection fluid, thereby transiently (reversibly) blocking the
outflow of the blood. Furthermore, rapid injection may be combined
with obstructing the outflow in yet another preferred embodiment.
For example, an afferent vessel supplying an organ is rapidly
injected and the efferent vessel draining the tissue is ligated
transiently. The efferent vessel (also called the venous outflow or
tract) draining outflow from the tissue is also partially or
totally clamped for a period of time sufficient to allow delivery
of a polynucleotide. In the reverse, an efferent is injected and an
afferent vessel is occluded.
[0041] In another preferred embodiment, the intravascular pressure
of a blood vessel is increased by increasing the osmotic pressure
within the blood vessel. Typically, hypertonic solutions containing
salts such as NaCl, sugars or polyols such as mannitol are used.
Hypertonic means that the osmolality of the injection solution is
greater than physiologic osmolality. Isotonic means that the
osmolality of the injection solution is the same as the
physiological osmolality (the tonicity or osmotic pressure of the
solution is similar to that of blood). Hypertonic solutions have
increased tonicity and osmotic pressure similar to the osmotic
pressure of blood and cause cells to shrink.
[0042] The permeability of the blood vessel can also be increased
by a biologically-active molecule in another preferred embodiment.
A biologically-active molecule is a protein or a simple chemical
such as histamine that increases the permeability of the vessel by
causing a change in function, activity, or shape of cells within
the vessel wall such as the endothelial or smooth muscle cells.
Typically, biologically-active molecules interact with a specific
receptor or enzyme or protein within the vascular cell to change
the vessel's permeability. Biologically-active molecules include
vascular permeability factor (VPF) which is also known as vascular
endothelial growth factor (VEGF). Another type of
biologically-active molecule can also increase permeability by
changing the extracellular connective material. For example, an
enzyme could digest the extracellular material and increase the
number and size of the holes of the connective material.
EXAMPLES
[0043] The following examples are intended to illustrate, but not
limit, the present invention.
Example 1
Intraportal Injections of Plasmid DNA:
Methods:
[0044] After the livers of 25 g, 6-week old mice were exposed
through a ventral midline incision, solutions containing pBS.CMVLux
plasmid DNA (described below) were manually injected over
approximately 30 sec into the portal vein using a 30-gauge,
1/2-inch needle and 1-ml syringe. In some animals, a 5.times.1 mm,
Kleinert-Kutz microvessel clip (Edward Weck, Inc., Research
Triangle Park, N.C.) was applied during the injection at the
junction of the hepatic vein and caudal vena cava. Anesthesia was
obtained from intramuscular injections of 1000 .mu.g of
ketamine-HCl (Parke-Davis, Morris Plains, N.J.) in 1 ml of normal
saline and methoxyflurane (Pitman-Moore, Mudelein, Ill. USA) which
was administered by inhalation as needed was purchased from Sigma.
Heparin was purchased from LyphoMed (Chicago, Ill).
Reporter Genes and Assays
[0045] The pBS.CMVLux, plasmid DNA was used to express luciferase
from the human immediate early cytomegalovirus (CMV) promoter (I.
Danko, et al., Gene Therapy 1, 114 (1994) incorporated herein by
reference). At two days after injection, the livers were assayed
for luciferase expression as previously reported (J. A. Wolff, et
al., Science 247, 1465 (1990)) except modified as below. The
animals were sacrificed by cervical dislocation and the livers
(average weight of 1.5 g) were divided into six sections composed
of two pieces of median lobe, two pieces of left lateral lobe, the
right lateral lobe, and the caudal lobe plus a small piece of right
lateral lobe. Each of the six sections were placed separately into
200 .mu.l of lysis buffer (0.1% Triton X-100, 0.1M K-phosphate, 1
mM DTT pH 7.8) that was then homogenized using a homogenizer PRO
200 (PRO Scientific Inc., Monroe Conn.). The homogenates were
centrifuged at 4,000 rpm for 10 min. at 4.degree. C. and 200 .mu.l
of the supernatant were analyzed for luciferase activity. Relative
light units (RLU) were converted to pg of luciferase using
standards from Analytic Luminescence Laboratories (ALL, San Diego,
Calif.). Luciferase protein
(pg)=5.1.times.10.sup.-5.times.RLU+3.683 (r.sup.2=0.992). Total
luciferase/liver was calculated by adding all the sections of each
liver and multiplying by 23 to account for dilution effects. For
each condition, the mean total luciferase/liver and the associated
standard deviation are shown.
Results:
[0046] After the livers of 25 g, 6-week old mice were exposed
through a ventral midline incision, 100 .mu.g of pBS.CMVLux,
plasmid DNA in 1 ml of solutions was injected into the portal vein
via a 30-gauge, 1/2-inch needle over approximately 30 sec. Two days
after injection, a mean of only 0.4 ng of total luciferase/liver
was produced when the DNA was delivered intraportally in an
isotonic solution without ligation of the hepatic vein (Table 1).
Inclusion of 20% mannitol in the injection solution increased the
mean total luciferase/liver over ten-fold to 4.8 ng (Table 1).
[0047] In order to prevent the DNA's rapid transit and to increase
the intraportal hydrostatic pressure, the hepatic vein was clamped
for two min after injection. Luciferase production increased
another three-fold to 14.7 ng (Table 1).
[0048] When the DNA was injected in a hypertonic solution
containing 0.9% saline, 15% mannitol and 2.5 units/ml of heparin to
prevent microvascular thrombosis and with the hepatic vein clamped,
luciferase expression increased eight-fold to 120.3 ng/liver (Table
1). These results are also shown in Table 7 (no dexamethasone
condition) in Example 3 below for each individual animal. If the
mannitol was omitted under these conditions, luciferase expression
was ten-fold less (Table 1).
[0049] These results indicate that hypertonicity, heparin and
hepatic vein closure are required to achieve very high levels of
luciferase expression. TABLE-US-00001 TABLE 1 Mean total luciferase
in the liver following the intraportal injection (over 30 seconds)
of 100 .mu.g pBS.CMVLux in 1 ml of different solutions with no
clamp or with the hepatic vein and inferior vena cava clamped for
two minutes. Mean Luciferase Number of Condition (total ng/liver)
Standard Error Livers no clamp, normal saline 0.4 0.7 n = 6
solution (NSS) no clamp, 20% mannitol 4.8 8.1 n = 3 clamp, 20%
mannitol 14.6 26.3 n = 9 clamp, 2.5 units heparin/ 11.8 12.5 n = 4
ml in NSS clamp, 15% mannitol and 120.3 101.5 n = 12 2.5 units
heparin/ml in NSS
[0050] Luciferase activities in each liver were evenly distributed
in six divided sections assayed (Table 2). All six parts of each
liver from all three animals had substantial amounts of luciferase.
This is in marked contrast to the direct interstitial, intralobar
injection of DNA in which the expression is restricted to the site
of injection (R. W. Malone et al., J. Biol. Chem 269, 29903 (1994);
M. A. Hickman, et al., Hum. Gene Ther. 5, 1477 (1994) incorporated
herein by reference). TABLE-US-00002 TABLE 2 The distribution of
luciferase expression over the six liver sections in animals
injected intraportally (over 30 seconds) with 100 .mu.g of
pBS.CMVLux in 1 ml of normal saline solution plus 15% mannitol and
2.5 units heparin/ml and with the hepatic vein clamped for 2
minutes. Total luciferase/Liver (ng/Liver/mouse) Liver Section
Mouse #1 Mouse #2 Mouse #3 1/2 of median lobe 496.5 66.9 304.5
other 1/2 of median 177.0 126.1 241.4 lobe 1/2 of left lateral
763.8 208.7 325.2 lobe other 1/2 of left 409.4 160.4 218.9 lateral
lobe right lateral lobe 527.8 129.7 216.2 caudal lobe + small 374.1
149.7 240.8 piece of right lateral lobe Total 2,748.6 841.5 1,547.0
Mean 458.1 140.3 257.8 Range 177-763 67-209 216-325 Standard
Deviation 194.0 46.6 45.9
Conclusions:
[0051] 1. High levels of luciferase expression were obtained from
injecting 100 .mu.g of pBS.CMVLux intraportally.
[0052] 2. The highest levels of luciferase expression were obtained
when the animals were injected intraportally over 30 seconds with
100 .mu.g of pBS.CMVLux in 1 ml of normal saline solution plus 15%
mannitol and 2.5 units heparin/ml and with the hepatic vein clamped
for 2 minutes.
[0053] 3. These high levels of expression were consistently
obtained in dozens of mice.
[0054] 4. The luciferase expression was evenly distributed
throughout the liver.
Example 2
[0055] The effects of other factors on expression were explored
using the same methods for the intraportal injection of
pBS.CMVLux.
Methods:
[0056] Unless otherwise specified, the intraportal injections and
luciferase assays were done as in Example 1.
Results:
[0057] Compared to the results with 100 .mu.g of pBS.CMVLUX,
luciferase expression was not greater with 500 .mu.g of plasmid DNA
(Table 3). Luciferase expression was approximately 7-fold less if
20 .mu.g of pBS.CMVLux DNA was injected instead of 100 .mu.g.
TABLE-US-00003 TABLE 3 Total luciferase expression in each liver of
each animal injected intraportally (over 30 sec) with 20 .mu.g, 100
.mu.g, or 500 .mu.g of pBS.CMVLux in 1 ml of normal saline solution
plus 15% mannitol and 2.5 units heparin/ml and with the hepatic
vein occluded for 2 min. Total luciferase/Liver (ng/Liver/mouse)
Mouse Number 100 .mu.g pBS.CMVLux 500 .mu.g pBS.CMVLux 1 1,023 15 2
178 82 3 108 23 4 140 340 Mean 362 115 Standard 441 153
Deviation
[0058] The times for which the hepatic vein was occluded were
varied from 2 min to 4 min and to 6 min. In Table 4, one can see
that the time of occlusion did not have a large effect on
expression. TABLE-US-00004 TABLE 4 Effect of time of hepatic vein
occlusion on luciferase expression in animals injected
intraportally with 100 .mu.g of pBS.CMVLux in 1 ml of normal saline
solution plus 15% mannitol and 2.5 units heparin/ml. Total
luciferase/Liver (ng/Liver/mouse) Mouse Number 2 min 4 min 6 min 1
4.6 1.9 32.7 2 44.9 11.5 6.4
[0059] The times over which the injections were done were varied
from 30 seconds to 1 minute and 2 minutes. In Table 5, one can see
that injecting the 1 ml of the DNA solution (100 .mu.g pBS.CMVLux)
over 30 seconds enabled the highest levels of luciferase
expression. Longer times of injection led to lower levels.
TABLE-US-00005 TABLE 5 Effect of length of injection (time it took
to inject all of the 1 ml) on luciferase expression in animals
injected intraportally with 100 .mu.g of pBS.CMVLux in 1 ml of
normal saline solution plus 15% mannitol and 2.5 units heparin/ml
and with the hepatic vein occluded for 2 min. Total
luciferase/Liver (ng/Liver/mouse) Mouse Number 30 sec 1 min 2 min 1
2,697 188 21.6 2 790 13.4 19.9 3 1,496 141.1 11.8 Mean 1,662 114 18
Standard 964 91 5 Deviation
[0060] If the total volume of the injection fluid was 0.5 ml
instead of 1.0 ml, luciferase expression decreased 70-fold (Table
6) suggesting that 0.5 ml was not sufficient to fill the
intravascular space and distribute the DNA throughout the
parenchyma. TABLE-US-00006 TABLE 6 Total luciferase expression in
each liver of each animal injected intraportally (over 30 sec) with
100 .mu.g of pBS.CMVLux in either 0.5 or 1 ml of normal saline
solution plus 15% mannitol and 2.5 units heparin/ml and with the
hepatic vein occluded for 2 min. Total luciferase/Liver
(ng/Liver/mouse) Mouse Number 0.5 ml 1 ml 1 1.6 51.9 2 4.7 124.8 3
0.4 266.9 Mean 2.3 147.9 Standard 2.3 109.4 Deviation
Conclusions:
[0061] 1. The optimal conditions are in fact the conditions first
described in example 1: the animals were injected intraportally
over 30 seconds with 100 .mu.g of pBS.CMVLux in 1 ml of normal
saline solution plus 15% mannitol and 2.5 units heparin/ml and with
the hepatic vein clamped for 2 minutes.
[0062] 2. Use of 500 .mu.g of pBS.CMVLux did not enable greater
levels of expression but expression was approximately7-fold less if
20 .mu.g of DNA was used.
[0063] 3. Occluding the hepatic vein for longer than 2 minutes did
not increase expression.
[0064] 4. Injecting the pBS.CMVLux over 30 seconds gave the highest
luciferase levels as compared to injection times longer than 30
seconds.
[0065] 5. Injecting the pBS.CMVLux in 1 ml gave higher luciferase
levels than injecting the pBS.CMVLux in 0.5 ml.
Example 3
[0066] The intraportal injections and luciferase assays were
performed as in Example 1 except that some animals received daily
subcutaneous injections of 1 mg/kg of dexamethasone (Elkins-Sinn,
Cherry Hill, N.J.) starting one day prior to surgery. The
conditions for the injections were intraportal injections over 30
seconds with 100 .mu.g of pBS.CMVLux in 1 ml of normal saline
solution plus 15% mannitol and 2.5 units heparin/ml and with the
hepatic vein clamped for 2 minutes.
Results:
[0067] Under the conditions described above (i.e., hypertonic
solution containing heparin and hepatic vein closure) into animals
that had been injected with daily injections of dexamethasone
starting the day prior to plasmid injection, luciferase expression
was three-fold greater than the expression without dexamethasone
(Table 7). TABLE-US-00007 TABLE 7 The effect of dexamethasone
injections on luciferase expression after the intraportal injection
of pBS.CMVLux. Total luciferase/Liver (ng/Liver/mouse) Mouse Number
NO Dexamethasone WITH Dexamethasone 1 51.9 1,181.1 2 124.8 364.7 3
266.9 82.8 4 73.7 120.5 5 52.6 1,022.9 6 7.3 178.1 7 146.1 107.6 8
231.4 140.2 9 271.2 10 8.7 11 8.3 12 201.1 Mean 120.3 399.8
Standard 101.4 444.1 Deviation
[0068] Dexamethasone could have increased the production of
luciferase and the expression of other genes by several mechanisms.
They include increasing the amount of plasmid DNA that enters the
liver cells by modifying the state of the liver cells. It could
also help the liver cells withstand the increased pressure.
However, the most likely mechanism is that dexamethasone directly
stimulates the CMV promoter and thereby directly increases
expression of luciferase by stimulating transcription of the
luciferase messenger RNA.
[0069] The use of dexamethasone demonstrates that using a readily
available pharmaceutical, the levels of expression can be
substantially increased and regulated.
Conclusion:
[0070] 1. Dexamethasone administration increased luciferase
expression from intraportally-injected pBS.CMVLux plasmid DNA
three-fold.
[0071] 2. This demonstrates that the expression from the liver can
be regulated using a commonly-used pharmaceutical.
Example 4
Methods:
[0072] The intraportal injections were performed using the
previously stated technique of injections over 30 seconds with 100
.mu.g of plasmid DNA in 1 ml of normal saline solution plus 15%
mannitol and 2.5 units heparin/ml and with the hepatic vein clamped
for 2 minutes. The mice also received daily subcutaneous injections
of 1 mg/kg of dexamethasone (Elkins-Sinn, Cherry Hill, N.J.)
starting one day prior to surgery.
[0073] The plasmids pBS.CMVLacZ and pBS.CMVnLacZ were used to
express a cytoplasmic and nuclear .beta.-galactosidase protein,
respectively, from the CMV promoter (Picard, D. & Yamamoto, K.
EMBO J. 6:3333-3340, 1987; incorporated herein by reference). They
were constructed by placing either a 3.5-kg-HindIII/XbaI
B-galactosidase sequence from pSDKLacZpa (Danko, I. et al. Gene
Therapy 1:114-121, 1994; incorporated herein by reference) or a
sequence encoding a nuclear-localizing -galactosidase (Picard, D.
& Yamamoto, K. EMBO J. 6:3333-3340, 1987; incorporated herein
by reference) into pBlueCMV (Danko, I. et al. Gene Therapy 1:
114-121, 1994; incorporated herein by reference).
[0074] Two days after intraportal injection, the livers were
perfused with 1% paraformaldehyde and 1.25% glutaraldehyde in
phosphate buffered saline (PBS) and then kept in this solution for
one day. After the livers were stored in 30% sucrose, they were
cryosectioned. The sections were mounted on slides and stained for
1 hour to one day with a PBS solution (pH 7.5) containing 400
.mu.g/ml X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside)
(Sigma), 5 mM potassium ferricyanide, 5 mM ferrocyanide, and 1 mM
MgCl.sub.2. After washing, the sections were then counter-stained
with hematoxylin and eosin. In the livers injected with the
nuclear-localizing .beta.-galactosidase vector, the washing step
after hematoxylin incubation was omitted to decrease its nuclear
staining.
Results:
[0075] Having defined the optimal conditions, the types and
percentages of transfected cells were determined. After injections
of a 100 .mu.g of the cytoplasmic (pBS.CMVLacZ) or the nuclear
(pBS.CMVnLacZ) .beta.-galactosidase expression vectors into
dexamethasone-treated animals, liver cryosections 10- to 30-.mu.m
thick were stained for .beta.-galactosidase using X-gal at pH 7.5
to prevent background staining. Intense blue staining was observed
in approximately 1% of the liver cells and was evenly distributed
throughout the liver. X-gal incubations for only 1 hour resulted in
intensely blue cells; suggesting that the transfected cells
expressed relatively large amounts of the foreign genes. Control
livers injected with 100 .mu.g of pBS.CMVLux did not contain any
positively-stained cells. Necrosis was observed in approximately
10% of the sections. However, some livers with high
.beta.-galactosidase expression did not contain any sections with
necrosis.
[0076] The hepatocytes were identified by their characteristic
morphology. For example, many of the cells in the livers injected
with the nuclear .beta.-galactosidase vector, pBS.CMVnLacZ, had
blue staining in two nuclei, which is a trait only of hepatocytes.
Although the majority of the positively-stained cells were
hepatocytes a few small, non-hepatocyte cells contained blue
staining.
Conclusion:
[0077] 1. Approximately 1% of the liver cells were transfected with
the B-galactosidase gene throughout the entire liver.
[0078] 2. Almost all of the transfected liver cells were
hepatocytes.
Example 5
Methods:
[0079] Luciferase expression in the liver was compared to that in
cultured HepG2 hepatocytes in 35-mm plates. Transfections were done
using 3 .mu.g of pBS.CMVLux/plate and either 3 .mu.g of Lipofectin
(Life Technologies, Bethesda, Md.) or 6 .mu.g of LipofectAMINE
(Life Technologies, Bethesda, Md.) per manufacturer's instructions.
Two days after transfection, 200 ul of lysis buffer was added to
the cultures and 20 ul of the supernatant were analyzed for
luciferase activity as in Example 1.
Results:
[0080] The efficiency of luciferase expression using this technique
was compared to other methods of gene transfer both in vitro and in
vivo. Transfections performed under optimal conditions with
pBS.CMVLUX and Lipofectin or LipofectAMINE (Life Technologies Inc.)
in HepG2 hepatocytes in culture (n=8) yielded a mean total of
3.7.+-.4.5 ng luciferase/35-mm plate and 2.8.+-.2.0 ng
luciferase/35-mm plate. Thus the efficiency of transfection
(without dexamethasone) in terms of ng of luciferase/.mu.g of
pBS.CMVLUX DNA was approximately 1 ng/.mu.g both in vitro and in
vivo.
[0081] The published procedure of repetitively and directly
injecting naked plasmid DNA into a rat liver lobe was reduced
proportionately for mouse liver (R. W. Malone et al., J. Biol. Chem
269, 29903 (1994); M. A. Hickman, et al., Hum. Gene Ther. 5, 1477
(1994); incorporated herein by reference). A total of 100 .mu.g of
pBS.CMVLUX in a total volume of 200 ul of normal saline was
injected within five different sites (40 ul/site) into the left
lateral lobe of 30 g mice treated with dexamethasone. A mean total
of only 0.1 ng/liver (4 livers; 0.001 ng luciferase/.mu.g DNA) was
obtained and the luciferase expression was only present in the
injected lobe. Approximately 30-fold more luciferase expression was
obtained if the direct intralobar injections were done using 1 ml
of injection fluid and clamping the hepatic vein. In the previous
studies involving the multiple injections of a total of 500 .mu.g
of pCMVL into a liver lobe of dexamethasone-treated rats, a mean of
9.87 ng of luciferase/liver (0.02 ng/.mu.g DNA) was expressed (R.
W. Malone et al., J Biol. Chem 269, 29903 (1994); M. A. Hickman, et
al., Hum. Gene Ther. 5, 1477 (1994)).
[0082] With regard to muscle, we typically inject 10 .mu.g of
pBS.CMVLUX or pBS.RSVLUX ((Danko, I. et al. Gene Therapy 1:114-121,
1994)) in normal saline into 6-8 mouse quadriceps muscle per
experiment. In dozens of experiments, mean total luciferase per
muscle was 0.4-1 ng (.+-.0.5-1.2) and the efficiency was 0.04-0.1
ng luciferase/.mu.g DNA. TABLE-US-00008 TABLE 8 Comparison of
efficiency of gene transfer in terms of luciferase expressed per
.mu.g of pBS.CMVLux plasmid DNA used for the method (ng
luciferase/.mu.g DNA). Amount of Efficiency Mean pBS. (ng Total
Yield Of CMVLux Luciferase/ Method of Gene Transfer Luciferase (ng)
Used (.mu.g) .mu.g DNA) Intraportal Mouse Liver 120.3 .+-. 101.5
100 1.2 (above optimal conditions- n = 12 Table 1) hepatic vein
clamped HepG2 In Vitro with 3.7 .+-. 4.5 3 1.2 Lipofectin (n = 8)
HepG2 In Vitro with 2.8 .+-. 2.0 3 0.9 LipofectAMINE (n = 8)
Intralobar Mouse Liver 0.1 .+-. 0.1 100 0.001 (20 ul/site .times. 5
sites) n = 4 hepatic vein not clamped Intralobar Mouse Liver 2.8
.+-. 5.6 100 0.028 (1 ml/1 site) n = 4 hepatic vein clamped
Intralobar Rat Liver 9.87 500 0.02 (5 sites) from published data
(Journal of Biologic Chemistry 269: 29903, 1994; Human Gene Therapy
5: 1477, 1994) Intramuscular 0.4-1 .+-. 10 0.04-0.1 0.5-1.2 n >
50
Conclusions:
[0083] 1. The intraportal delivery of naked DNA was more than an
order of magnitude more efficient than interstitial delivery into
either liver or muscle and more evenly distributed.
Example 6
Methods:
[0084] The intraportal injections were done using the above optimal
injections which are intraportal injections over 30 seconds with
100 .mu.g of pCMVGH in 1 ml of normal saline solution plus 15%
mannitol and 2.5 units heparin/ml and with the hepatic vein clamped
for 2 minutes. Some animals received daily intramuscular injections
of 100 mg/kg of cyclosporine (Sandimmune, Sandoz) or daily
subcutaneous injections of 1 mg/kg of dexamethasone (Elkins-Sinn,
Cherry Hill, N.J.), or both starting one day prior to surgery.
[0085] The previously described pCMVGH plasmid DNA was used to
express human growth hormone (hGH) (C. Andree, et al., Proc. Natl.
Acad. Sci. U.S.A. 91, 12188 (1994); incorporated herein by
reference). Blood obtained from the retro-orbit sinus was analyzed
for serum concentration of hGH using the radioimmune assay (RIA),
HGH-TGES 100T kit from Nichols Institute (San Juan Capistrano,
Calif.).
Results:
[0086] Human growth hormone (hGH) was used as a marker gene to
assess the ability of this gene transfer technique to produce a
therapeutic serum protein (Table 9). Two days after the intraportal
injection of 100 .mu.g of pCMVGH under the above optimal
conditions, the mean hGH serum concentration was 57.+-.22 ng/ml
(n=12) with a range of 21-95 ng/ml. Neither dexamethasone nor
cyclosporine pre-treatment significantly affected these initial hGH
levels. In two animals injected with pBS.CMVLUX, background hGH
levels were 0.3.+-.0.1 ng/ml for 4 weeks afterwards.
[0087] In humans, normal pulsatile levels of GH peak at
approximately 20 ng/ml above baseline values of approximately 1
ng/ml and can attain concentrations of 10-180 ng/ml after growth
hormone releasing hormone (GHRH) stimulation (A. Favia, J. D.
Veldhuis, M. O. Thorner, M. L. Vance, J. Clin. Endocrinol. Metab.
68, 535 (1989); R. W. Holl, M. L. Hartman, J. D. Veldhuis, W. M.
Taylor, M. O. Thorner, J. Clin. Endocrinol. Metab. 72, 854 (1991);
W. S. Evans et al., Am. J. Physiol. 252, E549 (1987); F. P. Alford,
H. W. G. Baker, H. G. Burger, J. Clin Endocrinol. Metab. 37,
515(1973); incorporated herein by reference). The half-life of hGH
is approximately 20 min in humans and 4.5 min in mice; hence these
serum levels could translate into much higher levels for more
stable proteins (S. Peeters and H. G. Friesen, Endocrinol. 101,
1164 (1977); A. Favia, J. D. Veldhuis, M. O. Thorner, M. L. Vance,
J. Clin. Endocrinol. Metab. 68, 535 (1989); incorporated herein by
reference). For example, if a protein such as alpha-antitrypsin has
a half-life that is ten times longer than human GH, then the
circulating blood levels should be at more than ten times higher
given the same efficiency of protein production. Another example is
that for hemophilia which requires levels of factor VIII or IX in
the range of approximately 1 82 g of the clotting factor/ml of
blood. Given the increased stability of these clotting factors,
then the 0.1 .mu.g/ml of hGH that we can achieve after intraportal
injection of the respective gene means that we would be able to
obtain therapeutic levels of clotting factors to prevent bleeding
in patients with hemophilia. In summary, these results demonstrate
that the intraportal naked DNA technique could be used to produce
therapeutic levels of a circulating blood protein.
[0088] Serial measurements of hGH serum levels enabled the
stability of expression in individual mice to be assessed (Table
9). In untreated animals, hGH expression was unstable as in
previous studies in which the plasmid DNA was delivered to
non-hepatectomized livers using polylysine complexes or intralobar
injections of naked DNA.
[0089] An immune response could kill hepatocytes expressing the
human protein. To test the hypothesis that expression was unstable
because of an immune response, hGH levels were followed in animals
that received cyclosporine with or without dexamethasone
administration (Table 9). After an acute drop off, hGH levels
remained at 6-11 ng/ml for four weeks in animals that received both
dexamethasone and cylcosporine. In animals that received
dexamethasone alone or cyclosporine alone, hGH expression was
prolonged as compared to the non-treated animals but not to the
same extent as the animals that received both agents. The ability
for this gene transfer method to enable expression of a foreign
gene should increase its utility. TABLE-US-00009 TABLE 9 Mean serum
levels (ng/ml of serum) of human growth hormone (hGH) following
intraportal administration of pCMVGH under optimal conditions in
mice (2 to 3 animals for each timepoint) receiving various
treatments. Optimal conditions are defined as the use of 0.9%
saline, 15% mannitol, 2.5 units/ml heparin solution that was
intraportally injected with the hepatic vein closed. DAYS AFTER
INJECTION NONE CSA alone DEX alone CSA + DEX 2 69 43 72 51 4 11 8
14 14 8 3 6 7 13 12 0 4 7 15 15 0 3 5 13 21 0 1.5 2.6 9.7 28 0 1
2.2 7.9
Conclusions:
[0090] 1. These results demonstrate that the intraportal naked DNA
technique could be used to produce therapeutic levels of a
circulating blood protein that is currently used to treat
humans.
[0091] 2. The levels of the circulating blood protein (i.e. hGH)
remained elevated for at least one month after a single
injection.
Example 7
Methods:
[0092] After the portal veins of 25 g, 6-week old mice were exposed
through a ventral midline incision, 100 .mu.g of pBS.CMVLux plasmid
DNA in 0.5 ml or 1 ml of normal saline solution plus 15% mannitol
and 2.5 units heparin/ml were manually injected over 30 seconds
into the portal vein near the junction of the splenic vein and
portal vein. The portal vein had two clamps placed distal and
proximal to the point of injection so as to direct the injection
fluid into only the splenic vein and to prevent the injection fluid
from going to the liver or intestines. The injections were done
using a 30-gauge, 1/2-inch needle and 1-ml syringe. 5.times.1 mm,
Kleinert-Kutz microvessel clips (Edward Weck, Inc., Research
Triangle Park, N.C.) were used. Anesthesia was obtained from
intramuscular injections of 1000 .mu.g of ketamine-HCl
(Parke-Davis, Morris Plains, N.J.) and methoxyflurane
(Pitman-Moore, Mudelein, Ill. USA) which was administered by
inhalation as needed. was purchased from Sigma. Heparin was
purchased from LyphoMed (Chicago, Ill.).
[0093] Two days after injection the spleens and pancreas were
removed and placed in 500 ul of lysis buffer and 20 ul were
analyzed for luciferase expression as described above.
Results:
[0094] Substantial amounts of luciferase activity were obtained in
the spleen and pancreas of all four mice with both injection fluids
of 0.5 ml and 1 ml. TABLE-US-00010 TABLE 10 Luciferase expression
after the intravascular-administration of pBS.CMVLux into the
splenic vein via the portal vein. Total luciferase/Organ
(pg/organ/mouse) Injection Volume Spleen Pancreas 0.5 ml 814.4 97.2
0.5 ml 237.3 88.7 1 ml 168.7 109.4 1 ml 395.0 97.7 Mean 403.9 98.3
Standard 289.6 8.5 Deviation
Conclusion:
[0095] 1. Intravascularly-administered plasmid DNA can express
efficiently in spleen and pancreas.
Example 8
Methods:
[0096] 100 .mu.g of pBS.CMVLux in 10 ml of normal saline solution
plus 15% mannitol was injected into the femoral artery of adult
rats with the femoral vein clamped. One to four days after
injection, the quadricep was removed and cut into 10 equal
sections. Each sections were placed into 500 ul of lysis buffer and
20 ul were assayed for luciferase activity as described above.
Results:
[0097] Substantial amounts of luciferase expression were expressed
in the quadriceps following the intravascular delivery of plasmid
DNA. TABLE-US-00011 TABLE 11 Luciferase expression in the quadricep
of a rat after the injection of 100 .mu.g of pBS.CMVLux into the
femoral artery and with the femoral vein clamped. Total Luciferase
Rat Number (pg/quadriceps) 1 157.5 2 108.8 3 139.2 4 111.3 Mean
129.2 Standard Deviation 23.4
Conclusion:
[0098] 1. Intravascularly-administered plasmid DNA can express
efficiently in muscle.
Example 9
[0099] The previous examples involved injections into the afferent
blood vessels of organs. In the liver, the hepatic vein is an
efferent blood vessel since it normally carries blood away from the
liver into the inferior vena cava. Also in the liver, the portal
vein and hepatic arteries are afferent blood vessels in relation to
the liver since they normally carry blood towards the liver.
[0100] These set of experiments were designed to determine whether
plasmid DNA could be efficiently expressed if delivered by a
retrograde route into the efferent vessel of the liver (i.e. the
hepatic vein).
[0101] Since another luciferase expression vector was used, pCILuc,
the results obtained with the hepatic vein injections were directly
compared to results using the above technique of injecting the
portal vein.
Methods:
[0102] 100 .mu.g of pCILuc in 1 ml of normal saline solution plus
15% mannitol and 2.5 units heparin/ml were injected over 30 seconds
into hepatic vein via the inferior vena cava. Since it was
difficult to directly inject the hepatic vein in rodents, the
injections were directed into the inferior cava which was clamped
in two locations; proximal and distal (i.e. downstream and
upstream) to the entry of the hepatic vein into the inferior vena
cava. Specifically, the downstream inferior vena cava clamp was
placed between the diaphragm and the entry point of the hepatic
vein. The upstream inferior vena cava clamp was placed just
downstream of the entry point of the renal veins. Therefore, the 1
ml of the injection fluid entered the hepatic vein and the liver.
Since the veins of other organs such as the renal veins enter the
inferior vena cava at this location, not all of the 1 ml of
injection fluid goes into the liver.
[0103] In some of the animals that received retrograde injections
in the inferior vena cava, the hepatic artery, mesenteric artery,
and portal vein were clamped (occluded) for approximately five
minutes immediately before and then after the injections.
Specifically, the order of placing the clamps were as follows:
first on hepatic artery, then portal vein, then downstream vena
cava, and then upstream vena cava. It took about three minutes to
place all these clamps and then the injections were done. The
clamps were left in place for an additional two minutes from the
time that the last clamp (upstream vena cava clamp) was placed.
[0104] The intraportal injections were performed as stated using
optimal intraportal injections over 30 seconds with 100 .mu.g of
pCILuc in 1 ml of normal saline solution plus 15% mannitol and 2.5
units heparin/ml and with the hepatic vein clamped for 2
minutes.
[0105] Some of the mice also received daily subcutaneous injections
of 1 mg/kg of dexamethasone (Elkins-Sinn, Cherry Hill, N.J.)
starting one day prior to surgery.
[0106] The pCILuc plasmid expresses a cytoplasmic luciferase from
the CMV promoter. It was constructed by inserting the cytoplasmic
luciferase CDNA into the pCI (Promega Corp., Madison, Wis.) CMV
expression vector. Specifically, a NheI/EcoRI restriction digestion
fragment containing the cytoplasmic luciferase cDNA was obtained
from pSPLuc (Promega Corp.) and inserted into pCI plasmid DNA that
was digested with NheI and EcoRI, using conventional recombinant
DNA techniques (Sambrook, J., Fritsch, E. F., and Maniatis, T.
(1989) in Molecular Cloning Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.).
[0107] Two days after the injections, the luciferase activity was
measured as above in six liver sections composed of two pieces of
median lobe, two pieces of left lateral lobe, the right lateral
lobe, and the caudal lobe plus a small piece of right lateral
lobe.
Results:
[0108] A. Inferior Vena Cava/Hepatic Vein Injections with the
Portal Vein and Hepatic Artery Clamped (*Injections in animal #3
were not optimal since the fluid leaked during the injections.)
Injections were done in 6-week old animals that received
dexamethasone. TABLE-US-00012 Luciferase Activity (ng) Sections
Animal #1 Animal #2 Animal #3* 1 5,576.7 4,326.4 1,527.4 2 8,511.4
4,604.2 1,531.6 3 5,991.3 5,566.1 2,121.5 4 6,530.4 9,349.8 1,806.3
5 8,977.2 4,260.1 484.2 6 9,668.6 6,100.2 1,139.3 total liver
45,255.5 34,206.9 8,610.4 Mean 29,357.6 standard deviation
18,797.7
[0109] B. Inferior Vena Cava/Hepatic Vein Injections with the
Portal Vein and Hepatic Artery not Clamped. Injections were done in
6-week old animals that did not receive dexamethasone.
TABLE-US-00013 Luciferase Activity (ng) Sections Animal #1 Animal
#2 1 360.6 506.2 2 413.5 724.7 3 463.0 626.0 4 515.5 758.6 5 351.6
664.8 6 437.8 749.6 total liver 2,542.0 4,029.8 Mean 3,285.9
standard deviation 1,052.1
[0110] C. Portal Vein Injections with the Hepatic Vein Clamped in 6
month old mice that received dexamethasone. TABLE-US-00014
Luciferase Activity (ng) Sections Animal #1 Animal #2 Animal #3 1
287.4 417.0 129.2 2 633.7 808.1 220.5 3 689.8 1,096.5 328.2 4 957.8
1,056.9 181.6 5 660.7 1,487.4 178.6 6 812.4 1,276.4 233.4 total
liver 4,041.8 6,142.2 1,271.5 Mean 3,818.5 standard deviation
2,443.0
[0111] D. Portal Vein Injections with the Hepatic Vein Clamped in 6
week old mice that received dexamethasone. TABLE-US-00015
Luciferase Activity (ng) Sections Animal #1 Animal #2 Animal #3 1
352.9 379.1 87.0 2 667.5 373.9 108.2 3 424.8 1,277.9 178.4 4 496.3
1,308.6 111.9 5 375.2 296.4 162.3 6 434.7 628.7 123.0 total liver
2,751.4 4,264.7 770.9 Mean 2,595.7 standard deviation 1,752.1
[0112] E. Summary Table Comparing the Luciferase Expression
Obtained Using the Above Conditions. TABLE-US-00016 Injection Mean
Total Luciferase/Liver Times Condition@ (.mu.g/liver) Condition D.
Condition A 29.4 11.3 X Condition B 3.3 1.27 X Condition C 3.8 1.46
X Condition D 2.6 1.00 X
[0113] Condition A=Inferior Vena Cava/Hepatic Vein Injections with
the Portal Vein and Hepatic Artery Clamped in 6 week-old animals
that received dexamethasone.
[0114] Condition B=Inferior Vena Cava/Hepatic Vein Injections with
the Portal Vein and Hepatic Artery not Clamped in 6-week old
animals that did not receive dexamethasone.
[0115] Condition C=Portal Vein Injections with the Hepatic Vein
Clamped in 24 week old mice that received dexamethasone.
[0116] Condition D=Portal Vein Injections with the Hepatic Vein
Clamped in 6 week old mice that received dexamethasone.
Conclusions:
[0117] 1. Retrograde delivery of plasmid DNA into the efferent
vessels of the liver via the hepatic vein/inferior vena cava leads
to high levels of gene expression.
[0118] 2. The highest levels were achieved using this retrograde
approach if the afferent vessels to the liver (portal vein and
hepatic artery) were occluded.
[0119] 3. The CILuc plasmid enabled much higher levels of
luciferase expression than the pBS.CMVLux plasmid (see above
examples) using the portal vein approach in both 6-week old and
6-month old mice.
[0120] 4. Under all conditions, luciferase expression was evenly
distributed throughout all six liver sections.
Example 10
[0121] Animals that received injections into the inferior vena cava
were assayed for luciferase to determine whether retrograde
delivery into the efferent vessels (veins) of other organs enable
gene expression.
Methods:
[0122] In the same animals that were injected using condition A
above (Inferior Vena Cava/Hepatic Vein Injections with the Portal
Vein and Hepatic Artery Clamped in 6 week-old animals that received
dexamethasone), the kidneys were removed and assayed for luciferase
as described above.
[0123] In the same animals that were injected under condition B
above (Inferior Vena Cava/Hepatic Vein Injections with the Portal
Vein and Hepatic Artery NOT Clamped in 6-week old animals that did
not receive dexamethasone), the adrenal gland and diaphragm muscle,
abdominal muscles, and back muscles were removed for luciferase
analysis.
Results:
[0124] A. Luciferase Activity in Kidneys in Animals Injected Under
Condition A. TABLE-US-00017 Total Luciferase Activity/ Kidney
(pg/kidney) Animal #1 Animal #2 Animal #3* Right Kidney 10,827.8
7,662.3 636.3 Left Kidney 733.1 753.8 479.7 *Injection fluid
leaked.
[0125] B. Luciferase Activity in Adrenals and Various Muscles
Injected Under Condition B. TABLE-US-00018 Total Luciferase
Activity/ Tissue (pg/tissue) Animal #7 Animal #8 Animal #9 right
adrenal not assayed 82.0 49.9 left adrenal not assayed 48.4 42.2
Diaphragm 41.9 67.9 117.6 Abdomen 40.4 43.9 44.0 Back 37.7 40.1
40.9
Conclusions:
[0126] 1. Retrograde delivery of plasmid DNA into the efferent
vessels of several different tissues led to substantial levels of
foreign gene expression in the tissues.
[0127] 2. These tissues include the adrenal glands (suprarenal
glands), the diaphragm muscle, back muscles and abdominal
muscles.
[0128] 3. Foreign gene expression in the diaphragm would be
especially useful for Duchennes muscular dystrophy since humans
with this disorder die from respiratory failure due to fibrosis of
the diaphragm muscle
Example 11
[0129] This example explores the use of more accessible vessels
such as the hepatic vein and the bile duct, for delivering the
naked pDNA in mice. Efficient gene expression was obtained using
these efferent delivery routes. Occlusion of other vessels to
restrict outflow of the injection solutions enhanced but was not
critical for efficient expression. Repetitive injections into the
bile duct were also accomplished. Preliminary results are also
presented in larger animals, the rat and dog. The incorporation of
these findings into laboratory and clinical protocols is
discussed.
Materials and Methods:
[0130] Plasmid Constructs: The pCILuc plasmid expresses a
cytoplasmic luciferase from the human CMV immediately early (hCMV
ID) promoter. It was constructed by inserting the luc+ gene, an Nhe
I-EcoR I luc+ fragment from pSLuc+ (Promega, Madison, Wis.), into
the pCI expression vector (Promega). pCILux expresses peroxisomal
luciferase under control of the hCMV IE promoter. It was
constructed by inserting the luciferase gene (Hind III-Bam HI
fragment from pBlueCMVLux) into the Sma I site of pCI. pCILacZ was
constructed by placing the E. coli LacZ gene (Pst I-Apa I fragment
pBS-RSV-LacZ) into the pCI vector (Sma I site). The pCMVGH
construct was previously described (Andree, et al., 1994).
[0131] Injection Methods: Plasmid delivery into the hepatic vessels
was performed in 6-week old ICR mice, 2.5-6.25 month-old, 200-300
gram Sprague-Dawley rats, and beagle dogs. Ventral midline
incisions were performed to expose the liver and the associated
vessels. The mice were anesthetized with intramuscular injections
of 1000:g of ketamine-HCl (Parke-Davis, Morris Plains, N.J.) and
methoxyflurane (Pitman-Moore, Mudelein, Ill.) which was
administered by inhalation as needed. The rats were anesthetized
with ether and the dogs were anesthetized with halothane by
inhalation. The pDNA was injected in solutions containing 2.5
units/ml or heparin (Lypho-Med, Inc., Chicago, Ill.) (Qian et al.,
1991) and either normal saline (0.9% NaCl) or 15% mannitol in
normal saline (Sigma Chemical Co., St. Louis, Mo.). All animals
received humane care in compliance with institutional (IACUC)
guidelines.
[0132] In mice, the intraportal injections were performed as
previously described (Budker, et al., 1996). 100:g of pDNA in 1 ml
were manually injected over 30 see using a 30-gauge, 1/2-inch
needle and 1-ml syringe without occluding the portal vein upstream
from the point of injection. In some animals, a 5.times.1 mm,
Kleinert-Kutz microvessel clip (Edward Week, Inc., Research
Triangle Park, N.C.) was applied during the injection at the
junction of the hepatic vein and caudal vent cava.
[0133] DNA was delivered in mice to the hepatic vein via an
occluded IVC. Clamps (6.times.1 mm, Kleinert-Kutz curved
microvessel clip (Edward Week, Inc., Research Triangle Park, N.C.)
were applied downstream (toward the heart) of the hepatic vein and
upstream (towards the legs) of the hepatic and renal veins.
Injections were done upstream of the hepatic vein. In some of the
injections, the portal vein and hepatic artery were clamped using
6.times.1 mm, Kleinert-Kutz curved microvessel clips. The IVC mouse
injections were also performed with 100:g of pDNA in 1 ml that were
manually injected over -30 sec with a 30-gauge, 1/2-inch needle and
1-ml syringe.
[0134] The bile duct injections in mice were performed using manual
injections with a 30-gauge, 1/2-inch needle and 1-ml syringe. A
5.times.1 mm, Kleinert-Kutz microvessel clip was used to occlude
the bile duct downstream from the point of injection in order to
prevent flow to the duodenum and away from the liver. The
gallbladder inlet was not occluded. In some of the bile duct
injections, the junction of the hepatic vein and caudal vena cava
clamped as above. In yet other injections, the portal vein and
hepatic artery were clamped in addition to the occlusion of the
hepatic vein.
[0135] In mice, repetitive injections into the bile duct were done
by placing a polyethylene tube (I.D. 0.28 mm, O.D. 0.61 mm;
Intramedic Clay Adams Brand, Becton Dickinson Co., Sparks, Md.,
USA) catheter into the bile duct after making a hole with a 27
gauge needle. The tubing was secured by a suture around the bile
duct and tubing; thereby occluding the bite duct. The other end of
the tubing was placed outside the skin of the animal's back so that
surgery was not required for repeat injections. No blood vessel
occlusions were done for these repetitive administrations. After
completion of the studies, anatomical examination indicated that
the catheter remained in the bile duct.
[0136] In rats, the intraportal, IVC, and bile duct injections were
done as in mice but with the following modifications. The
injections were done through a 25-g butterfly needle using a
peristaltic pump (Preston varistaltic power pump, Manostat Corp.,
New York, N.Y.) over 1 or 3 minutes. The downstream IVC clamps in
the IVC injections were done downstream of the kidneys. For the
portal vein injections, the portal vein and hepatic artery were
clamped. The outflow through the hepatic vein was restricted in
some animals by clamping the upstream and downstream IVC. In some
animals the livers were first flushed with normal saline prior to
DNA injection. Rat bile duct injections were done the same as mice.
The rat doesn't have a gallbladder.
[0137] In some of the rat portal vein injections, a 25 G needle
connected to a pressure gauge (Gilson Medical Electronics, Model
ICT-1 1 Unigraph), was inserted into the liver parenchyma to
determine the peak pressure within the liver during the injections.
The statistical relationship between pressure and luciferase was
done using Spearman's rank correlation. A smoothing spline
regression model describing the relationship between log luciferase
and pressure was estimated using generalized additive model
methodology (Hastie, 1992). Akaike's information criteria (Akaike,.
1973) indicated that a smoothing spline with 2 degrees of freedom
resulted in the best fit over all integer degrees of freedom
between 0 and 5.
[0138] The injections in dogs were done as in rats except that an
18 gauge, 2 inch angiocath (Becton Dickinson, San Jose, Calif.) was
used. All dogs except dog #1 were females. Table I indicates the
injection conditions. For the bile duct injections, a suture was
applied to occlude transiently the bile duct downstream from the
point of injection. A DeBakey multipurpose vascular clamp was
applied to the cystic duct during injection to prevent the
injectate from entering the gallbladder. In dogs, the DNA was
pCILux.
[0139] Protein Assays: The luciferase assays were done as
previously reported (Wolff, et al., 1990). One day after pCILuc
injections, the animals were sacrificed and the rodent livers were
divided into 6 sections composed of right lateral lobe, caudate
lobe, two pieces of median lobe and two pieces of left lateral
lobe. For each of the six pieces, 0.7 ml of lysis buffer (0.1%
Triton X-100, O.1 M potassium phosphate, 1 mM DTF pH 7.8) was used
for mice and four ml of lysis buffer were used for rat liver. For
the dog livers, approximately 10% of each lobe was divided into
five to 20 pieces and placed into two ml of lysis buffer. The
samples were homogenized using a PRO 200 homogenizer (PRO
Scientific Inc., Monroe, Conn.) and centrifuged at 4,000 rpm for 10
min. at 4EC. Twenty:1 of the supernatant was analyzed for
luciferase activity. Relative light units (RLU) were converted to
pg of luciferase using standards from Analytic Luminescence
Laboratories (ALL, San Diego, Calif.). Luciferase protein
(pg)=5.1.times.10.sup.-5.times.RLU+3.683 (r.sup.2=0.992).
[0140] Ten-.mu.m thick tissue sections were stained for
.E-backward.-galactosidase expression as previously described using
1-4 hour X-GAL incubations (Budker, et al., 1996). Hematoxylin was
used for the counterstain but the alkaline step was omitted so that
the hematoxylin stain remained red. The percent of blue-stained
cells in the liver sections was determined by counting .about.3000
cells in three sections and averaging.
[0141] Blood obtained from the retro-orbit sinus was analyzed for
serum concentration of hGH using the RIA, HGH-TGES 100T kit from
Nichols Institute (San Juan Capistrano, Calif.). Serum ALT and GGT
levels were done using EKTACHEM DT slides and a KODAK EKTACHEM DT
60 ANALYZER as recommended by the manufacturer (Kodak, Rochester,
N.Y.).
[0142] Mice Luciferase Experiments: Our previous studies used the
pBS.CMVLux plasmid for evaluating the optimal conditions for naked
pDNA expression following importal injection. These optimal
conditions were intraportal injections of 100:g of pDNA in 1 ml of
15% mannitol and 2.5 units heparin/ml in normal saline solution.
The injections were done over 30 seconds with the hepatic vein and
IVC occluded. In this study, 100:g of pCILuc injected under similar
conditions yielded a mean total luciferase protein/liver of
3.73:g/liver, approximately 30- times greater than that obtained
with pBS.CMVLux. Part of this increase could be attributed to
greater operator experience with these injection techniques.
Injections with pCILuc under these conditions without clamping the
hepatic vein yielded approximately 750-fold less luciferase.
[0143] The hepatic veins (via the IVC) of another set of mice were
injected with 100:g of pCILuc in 1 ml of 15% mannitol and 2.5 units
heparin/ml in normal saline solution. A mean total luciferase
protein/liver of 17.34:g/liver was obtained when the portal vein
was clamped as compared to a mean total luciferase protein/liver of
2.83:g/liver without occluding the portal vein.
[0144] Similar results were also obtained when bile ducts were
injected with 100:g of pCILuc in 1 ml of 15% mannitol and 2.5 units
heparin/ml in normal saline solution. A mean total luciferase
protein/liver of 15.39:g/liver was obtained when the hepatic vein
was clamped as compared to a mean total luciferase protein/liver of
1.33:g/liver without occluding the hepatic vein. If mannitol was
omitted then the bile duct injections without clamping any blood
vessels yielded approximately 15-fold less luciferase
(0.086:g/liver.+-.0.06, n=25). Clamping the hepatic artery, and
portal vein in addition to the hepatic vein did not improve
expression beyond what was obtained when only the hepatic vein was
clamped (data not shown).
[0145] Serum ALT and GGT assays were performed on mice one and
eight days after each of the above injections with pCILuc (4 mice
for each condition). No increases in GOT were observed after any of
the injections including the bile duct injections. Serum ALT levels
increased to 200-400 U/L one day after portal vein and bile duct
injections. One day after IVC injections serum ALT levels increased
to -1500 U/L in half of the mice but was only -250 U/L in the other
half. By eight days after injection, serum ALT levels decreased to
baseline levels in all animals. For positive control purposes, a
non-lethal intraperitoneal injection of 40:1 of 50%, carbon
tetrachloride in mineral oil was performed. An average of 25,900
U/L (n=4) was observed one day after injection.
[0146] Rat and Dog Luciferase Experiments: Similar injections into
the portal vein, IVC (to the hepatic vein), and bile duct were done
in rats. For the portal vein injections, the injection volumes were
increased 15-times over that used in mice because the rat livers
are -15-times larger than mouse livers. One day after 750 of:g of
pCILuc in 15 ml of 15% mannitol and 2.5 units heparin/ml in normal
saline solution were injected into the portal vein while occluding
the hepatic vein, an average of 53.5:g of luciferase/liver was
obtained. The efficiency of gene transfer in the rat was compared
to that in mice two ways. In terms of efficiency as defined by the
mg of luciferase per mg of tissue weight, the levels of expression
for portal injections were 3.6:g of luciferase/g of tissue in rats
as compared to 3.7:g of luciferase/g of tissue in mice.
Alternatively, in terms of efficiency as defined by the ng of
luciferase per:g of pDNA delivered, the efficiencies of expression
for portal injections were 71 ng of luciferase/:g DNA in rats as
compared to 37 ng of luciferase/:g DNA in mice.
[0147] Less but still substantial luciferase expression was
obtained when the injections of pCILuc were done into the efferent
vessels of rats such as the IVC or bile duct. Injections of 750:g
of pClLuc in 15 ml into the hepatic vein (via the IVC) while
occluding the portal vein yielded an average of 1.5:g of
luciferase/liver. Injections of 750:g of pCILuc in 5 to 8 ml into
the bile duct without any outflow obstruction yielded an average of
1.3:g of luciferase/liver.
[0148] Parenchymal pressures of 12-50 mm Hg were measured in 23 rat
livers during the injection of 750:g pCILuc in 15 or 20 ml into the
portal vein while occluding the IVC. Spearman's rank correlation
between pressure and luciferase expression was 0.76 (two-side
p-value <0.001) indicating that pressure and luciferase were
significantly positively associated. It appeared that pressures of
over 40 mm Hg did not result in increased expression. The necessity
of the non-linear component over and above a simple linear fit was
verified by an approximate full versus reduced F-test (p-value=0.0
14), indicating that the observed plateau effect is real.
Examination of both the residual quantile-quantile and the residual
versus prediction plots further reveals that there are no serious
violations of the regression model assumptions and therefore the
regression models and p-values are valid (Fisher and van Belle,
1993).
[0149] Preliminary experiments explored the ability of naked pCILux
(not pCILuc) to be expressed in dogs (Table I). In mice, in the
case of intraportal injection, pCILux provided 3.times. lower
expression than pCILuc (data not shown). In five dogs, various
amounts of DNA were injected into either the bile duct with or
without blocking outflow by occluding the IVC. In one animal, the
DNA was injected into the IVC without any outflow blockage. All the
dogs survived the procedure except animals that had the IVC
occluded recovered more slowly post-operatively. The animals were
sacrificed one day after the injections and dozens of tissue
samples from each liver lobe were analyzed for luciferase. The
luciferase expression was evenly distributed over all the lobes in
each liver except in one lobe of one dog. Routine histological
analysis in dog #5 (Table I) indicated that the tissue architecture
was substantially disrupted suggesting that the injection volumes
were too large.
[0150] Decreasing the volume of injection in dog #6 to 200 ml
resulted in the best expression (Table I).
[0151] Mice and Rat .beta.-Galactosidase Results: The
.E-backward.-galactosidase expression vector was used to determine
the percent and type of cells that were transfected. As previously
noted for portal vein injections (Budker, et al., 1996), the vast
majority of the blue-stained cells appeared to be hepatocytes on
morphological grounds but a few appeared to be endothelial or other
types of cells. A preponderance of hepatocytes were also stained
blue after the bile duct or IVC injections in mice or rats.
[0152] Under the various injection conditions, the percent of cells
paralleled the levels of luciferase expression. In mice, the IVC
injections gave the highest percentage of
.beta.-galactosidase-positive cells in which 7% of hepatocytes were
positive. In rats, the portal vein injections gave the highest
percentage of .beta.-galactosidase-positive cells in which 7% of
hepatocytes were positive. In some animals, liver cell damage was
evident in less than 5% of the cells. Of note in the rat livers
injected into the portal vein, almost all of the positively-stained
cells were periacinar with few positive cells around the central
vein.
[0153] Repeat Bile Duct Injections: The bile ducts of mice were
cannulated and 100:g of pCMVhGH in 1 ml of 15% mannitol in normal
saline were injected once a week. Serum levels of hGH increased one
day after the fast injection and then decreased to background
levels by seven days after injection. One day after the second
injection, hGH levels again increased and then were back to
background levels by seven days after the second injection. Only
minimal increases in hGH levels occurred after the third injection.
Mice that had the highest levels after the first injection had the
lowest levels after the second injection (mice 3 and 6) and vice
versa (mice 1, 2, and 4). In another set of animals (4 mice), the
bile duct injections were repeated four times with pCMVhGH and then
pCILuc was injected. The first three pCMVhGH injections led to
similar increases in hGH serum levels. Although there were only
minimal raises in hGH serum levels following the fourth injection,
injection of pCILuc yielded an average of 29.2 ng/liver (.+-.7.1,
n=3). The liver in one of the four mice was grossly yellow and
scarred as a result of the bile duct ligation and did not express
any luciferase.
Discussion
[0154] This report extends the findings of the previous study
showing pDNA expression following afferent intraportal delivery and
demonstrates efficient plasmid expression following delivery via
efferent vessels such as the hepatic vein or bile duct. Expression
of luciferase or galactosidase was evenly distributed throughout
the entire liver when either of the three vessels were injected.
Combining these surgical approaches with improved plasmid vectors
enabled uncommonly high levels of foreign gene expression in which
over 15:g of luciferase protein/liver was produced in mice and over
50:g in rats. Equally high levels of .beta.-galactosidase
expression were obtained in that 7% of the hepatocytes had intense
blue staining with only a one hour X-GAL incubation. These levels
of foreign gene expression are among the highest levels obtained
with nonvital vectors and approach what can be achieved with vital
vectors.
[0155] Using the portal vein administration route, occlusion of the
outflow is critical for expression. Outflow occlusion increases the
expression with the efferent administrative routes, but substantial
amounts of expression were obtained even when the hepatic vein was
not blocked. Most likely the natural direction of blood flow
provides a sufficient impetus to retard the egress of injection
fluid and raise the hydrostatic pressure. The use of these efferent
vessels simplifies the administration for potential human
applications since they are easier to access by non-invasive
methods. If no occlusion is used then only one vessel has to be
reached. These efferent routes should also be considered for the
administration of viral and non-viral vectors as has been done with
the delivery of adenoviral vectors into the bile duct [Vrancken
Peeters, et al., 1996b; Vrancken Peeters, et al., 1996a; Yang et
al., 1993).
[0156] The mechanism of pDNA uptake is not known but may involve
native cellular uptake processes (Budker, et al., 1996). It is of
interest that high levels of luciferase expression could
occasionally be obtained when the DNA was injected into the bile
duct in small volumes of isotonic solutions without occluding the
IVC. Increased osmolar and hydrostatic pressure may not be critical
for uptake of the pDNA by hepatocytes as they are not in muscle
cells (Wolff, et al., 1990; Wolff, et al., 1991; Wolff, et al.,
1992a). This would suggest that the mechanism of pDNA uptake may in
fact involve endogenous cellular pathways. Increased hydrostatic
and osmotic pressures may raise expression by enhancing these
cellular internalization processes (Haussinger, 1996). For example,
hepatocyte shrinkage stimulates replication of the duck hepatitis B
virus (Offensperger, et al., 1994).
[0157] The raised pressures could also increase the delivery of the
pDNA to the hepatocyte surface not only for the blood vessel
administrations (through the sinusoidal fenestrae) but for the bile
duct injections as well. The increased pressures could transiently
attenuate bile secretion thereby decreasing the clearance of the
pDNA (Stieger, et al., 1994). For example, the hyperosmolar
mannitol should induce cell shrinkage that is known to inhibit
taurocholate excretion into bile (Haussinger, et al., 1992).
[0158] Hepatocytes are functionally polarized cells in which the
basal and apical membranes have different exocytic, endocytic and
transcytotic functions (Hubbard, et al., 1994). The success with
either blood vessel or bile duct routes could indicate that both
the basal (sinusoidal) and apical (bile canalicular) membranes
share a common pathway for the cellular entry of pDNA. However, the
passage of DNA between the basal-lateral and bile canalicular
spaces cannot be excluded even under the more gentle injection
conditions. Plasmid DNA injected into the bile duct could be taken
up from the basal-lateral surface of hepatocytes after passage
pancellularly. If only the basal-lateral surface takes up the pDNA
then the increased hydrostatic and osmotic pressures may enhance
pDNA expression by disrupting the tight junctions between
hepatocytes and thereby increase the flow of pDNA between the
canalicular and basal-lateral spaces (Desmet and De Vos, 1982).
[0159] The bile duct was cannulated to determine whether repeat
injections could be done. Substantial hGH levels were obtained
alter the first two injections. The hGH levels dropped considerably
by one week after each injection presumably because of an immune
response to the foreign protein. The inability to detect hGH after
the third or fourth injections was most likely due to a more rapid
response of the immune response sensitized to hGH. The ability to
subsequently obtain luciferase expression argues against an immune
response against the pDNA that prevents expression. Previous
studies have failed to detect anti-DNA antibodies following the
administration of naked pDNA (Nabel, et al., 1992; Jiao, et al.,
1992). The ability to repetitively administer naked pDNA without
inducing an immune response against the vector is a distinct
advantage of naked pDNA over viral and some types of non-viral
vectors.
[0160] Other studies in progress in our laboratory suggest that
suppression of the immune system enables more persistent
expression. In post-mitotic myofibers, plasmids can persist
extrachromosomally and express for at least two years; presumably
because the pDNA is not being lost as result of cell division
(Wolff, et al., 1992b; Herweijer, et al., 1995). Quite possibly
pDNA would be lost slowly in hepatocytes which have a half-life of
up to a year in rodents and humans (Webber, et al., 1994; Leffert,
et al., 1988). If so then liver-based genetic disorders such as
hemophilia could be treated by injections every six months. The
bile duct could be accessed repeatedly by upper gastrointestinal
endoscopy. Similarly, the hepatic vein could be non-invasively
accessed via peripheral or central veins. In addition, gene
transfer could be delivered to newborns via the umbilical cord
vessels to get them over a newborn metabolic crisis as occurs in
the organic acidunas and the urea cycle defects.
[0161] Often gene transfer techniques that work in mice do not work
in larger animals. Our results demonstrate that the technique works
in rats that are approximately ten-fold bigger than mice. The dog
results indicate that the liver of larger non-rodent mammal can
express naked pDNA. Although substantial levels of luciferase
activity were obtained, further optimization of the injection
conclidons is required to increase the efficiency of expression so
that they are comparable to those in rodents. The studies to
determine the relationship between intraparenchymal pressure and
luciferase expression in rats are a first step towards this goal.
Minimal liver cell damage occurred in the rodents as evident by
serum chemistries and histology but the injections were more
disruptive to the hepatocytes in dogs. Presumably, the key factor
is the efficient delivery of the pDNA to the hepatocyte surface
with minimal cellular or tissue disruption.
[0162] In the research laboratory, the described techniques will
enable rodents to be used just as immortalized and primary liver
cell cultures are now used for gene and cellular studies of liver
function. The transfer of genes into cells in culture have been a
critically important tool for deciphering the function of genes and
for studying the effect of expressed proteins on cellular
processes. Typically, the gene under study is placed within a
plasmid vector and transiently transfected into the appropriate
cell in culture. Isoforms and mutant forms of the gene under study
can be quickly placed into plasmid expression vectors and studied.
Our findings indicate that a similar plasmid-based approach could
be used to study the effects of gene function in hepatocytes in
situ. Given that the high levels of expression are transient in
this system, it would be best if these effects occurred within a
few days. The use of pDNA avoids the laborious steps necessary for
the production of vital vectors or generation of transgenic mice
and thereby enables many different genes and their related mutated
forms to be quickly studied. It will permit are mechanism of gene
expression and their effects on liver function to be expeditiously
probed within the context of a complete mammalian organism.
Example 12
[0163] The intravascular delivery of naked pDNA to muscle cells
also has attraction. Muscle has a high density of capillaries
(Browning, 1996) and the capillaries are in close contact with the
myofibers (Lee 1995). However, the endothelium in muscle
capillaries is of the continuous, non-fenestrated type and has low
solute permeability especially to large macromolecules (Taylor, A.
E., and D. N. Granger. Exchange of macromolecules across the
microcirculation. In: Handbook of Phusiology. The Cardiovascular
System. Microcirculation.. Bethesda, Md.: Am. Physiol. Soc., 1984,
sect. 2, vol. IV, chapt. 11, p. 467) The mechanism of macromolecule
transendothelial transport is poorly understood. Cell biologists
have proposed that this transport occurs by transcytosis involving
plasmalemmal vesicles or convective transport through transient
transendothelial channels formed by the fusion of vesicles. (Michel
C. C. Cardiovascular Res. 1996). Physiologists have modeled the
muscle endothelium as having a large number of small pore with
radii of 4 nm and a very low number of large pores with radii of
20-30 nm. (Rippe B. Physiological Rev, 1994). Although the radius
of gyration of 6 kb pDNA is .about.100 nm (Fishman, D. M.,
Biopolymers, 38, 535-552), supercoiled DNA in plectonomic form has
superhelix dimensions of approximately 10 nm (Rybenkov V. V. J.
Mol. Biol. 267, 299-311 1997). This implies that pDNA has a
possibility to traverse microvascular walls by stringing through
their large pores. We hypothesized that the rate of pDNA
extravasation could be increased by enhancing fluid convection
through these large pores by raising the transmural pressure
difference in selective regions. This report demonstrates that
intravascular pDNA injections under increased pressure can in fact
lead to high levels of foreign gene expression in muscles
throughout the selected hindlimb of an adult rat.
Hydrostatic Pressure
[0164] 475 ug of pCILux in normal saline solution (NSS) was
injected into the femoral arteries of adult Sprague-Dawley rats
while blood inflow and outflow were blocked. Injection of pCILux, a
luciferase expression vector utilizing the CMV promoter (LIVER II
promoter accepted for HGT), was done after both the femoral artery
and vein was occluded for 10 min. Two days after pDNA injections,
the luciferase activities were measured in all the muscles of the
hindlimb. The highest levels of luciferase expression were achieved
when the pCILux was injected in 9.5 ml of normal saline within 10
sec. Injection volumes less than 9.5 ml resulted in substantially
less expression. Injection times more than 10 sec also resulted in
much less expression. This critical dependence on volume and speed
of injection indicates that either increased hydrostatic pressure
or rapid flow is required for efficient expression. Artery
injections performed without occluding the femoral vein resulted in
approximately 200-fold less expression (1.8.+-.1.2 ng of luciferase
per all hindlimb muscles).
Other Factors
[0165] Further studies were performed to determine the effect of
ischemia on the expression of the intravascularly injected pCILux.
The time of ischemia was adjusted by varying the time that both the
femoral artery and vein were occluded prior to pDNA injection.
Although the highest level of expression was obtained with 10 min
of ischemia, the expression levels were only a few-fold lower at
shorter or longer ischemia times. This suggests that ischemia is
not a critical factor for enabling egress of the pDNA out of the
intravascular space. However, the blood flow for the zero ischemia
time point is disrupted for 30 sec and this may be sufficient to
affect vascular permeability. Ischemia could increase expression
either by capillary recruitment and vasodilatation) or augmenting
permeability (Mathieu-Costello O. Int J Microcirc 1995, 15,
231-237). In addition, ischemia could possibly increase pDNA
expression by affecting transcription or translation. Ischemia can
be tolerated by muscle for two to three hours (Gidlof A., Int. J.
Microcirc. Clin. Exp. 1987, 7,67-86). Histologic analysis of the
muscle did not reveal any hemorrhage or myofiber damage.
[0166] Other factors were explored to increase the level of
expression. Hypotonic or hypertonic injection solutions resulted in
less expression. Although, the effect of the hypertonic injection
solution (normal saline solution with 15% mannitol) may have been
mitigated by the slower rate of injection (over 30 sec instead of
10 sec) that results from its increased viscosity. The
pre-injection with collagenase resulted in a 3.5-fold increase to
1,332 ng/total muscles. The collagenase was used to disrupt the
basement membrane of the capillaries and thereby increase pDNA
egress. It could have also increased expression by disrupting the
extracellular matrix within the muscle. Further studies are in
progress to determine the optimal conditions for collagenase
administration without causing hemorrhage.
Distribution of Foreign Gene Expression
[0167] The luciferase expression was distributed among all muscle
groups in the leg (Table 1). The lower levels in the lower anterior
leg and foot are probably due to their high content of tendons and
small amount of muscles. The levels after intravascular injection
was up to 40 times higher than the levels after intramuscular
injection.
[0168] The type and percentage of the transfected cells in the
muscle were determined using the .beta.-galactosidase reporter
system. Using the best injection condition, approximately 10-50% of
the myofibers expressed .beta.-galactosidase.
[0169] These expression results provide indirect evidence that pDNA
extravasation occurred. More direct evidence was obtained using
fluorescently-labeled DNA injected into the femoral artery. The
labeled DNA was distributed extravascularly in all the limb muscles
and surrounded most of the myofibers (data not shown).
Conclusion
[0170] These results demonstrate that the intraarterial delivery of
pDNA to muscle can be greatly enhanced when injected rapidly, in a
large volume and when all blood vessels leading into and out of the
hindlimb are occluded. These conditions presumably increase the
intravascular hydrostatic pressure and thereby increase the
convective outflow that transports the pDNA into contact with
myofibers. The increased intravascular pressure may increase the
number, size and permeability of the microvascular pores (Rippe, B.
Acta Physiol. Scand. 125, 453-459, 1985) (Wolf, M. B. Am. J.
Physiol. 257, H2025-H2032,1989). Preliminary studies using
collagenase suggest that enzymatic disruption of the vessels
basement membrane or muscle extracellular matrix may also increase
the delivery of pDNA to the myofibers. Ischemia also increased
expression moderately. Furthermore, it is expected that other
enzymes such as hylaronidase will perform similarly to
collagenase.
[0171] This study shows that the intravascular approach increases
expression in muscle up to 40-fold as compared to intramuscular
injection. This is the first demonstration that naked plasmid DNA
can be efficiently expressed in muscles of adult animals larger
than mice. It is also the first report of an intravascular
non-viral gene transfer approach for muscle. Further studies in
larger animals will determine the clinical relevance of this
study.
Description of Injections
[0172] A 4 cm long abdominal midline excision was performed in
150-200 g, adult Sprague-Dawley rats anesthetized with 80 mg/mg
ketamine and 40 mg/kg xylazine. Microvessel clips (Edward Weck,
Inc., Research Triangle Park, N.C.) were placed on external iliac,
caudal epigastric, internal iliac and deferent duct arteries and
veins to block both outflow and inflow of the blood to the leg. A
27 g butterfly needle was inserted into the external iliac artery
and the DNA solution was injected by hand. TABLE-US-00019 TABLE 1
Distribution of luciferase expression among the different hindlimb
muscle groups two days following intraarterial injection with 475
.mu.g of pCILux in 9.5 ml of NSS over 10. Means and their standard
deviations are shown for 6 animals. Total Luciferase Luciferase
Conc. Injection type Muscle group (ng) (ng/g tissue) Intravascular
Upper Leg Anterior 149.1 .+-. 51.2 109.2 .+-. 37.5 Upper Leg
Posterior 74.6 .+-. 36.0 43.4 .+-. 20.9 Upper Leg Medial 88.7 .+-.
63.7 61.6 .+-. 44.5 Lower Leg Posterior 114.5 .+-. 89.7 66.0 .+-.
51.7 Lower Leg Anterior 3.0 .+-. 1.0 5.4 .+-. 1.7 Foot 0.5 .+-. 0.2
4.1 .+-. 1.4 Intramuscular Upper Leg Anterior 3.7 .+-. 0.9 2.8 .+-.
0.8
Example 13
[0173] Adenoviral vectors can be delivered to muscle parenchymal
cells by an intravascular route.
[0174] Methods: the same methods that were used to deliver naked
plasmid DNA to muscle in rats were also used. The adenoviral vector
CMVLacZ that expresses the E. coli beta-galactosidase from the
immediate early promoter of the human cytomegalovirus (commonly
called the "CMV promoter") was prepared as previously described
(Yang, T., K. U. Jooss, Q. Su, H. C. J. Ertl and J. M. Wilson:
Immune responses to viral antigens versus transgene product in the
elimination of recombinant adenovirus-infected hepatocytes in vivo,
Gene Therapy, 3(2): 137-144, 1996) The rat iliac artery was
preinjected with 0.5 mg of papaverine and 40 ng of collagenase in 3
ml of saline while blocking the iliac artery and vein.
5.times.10.sup.8 particles of the adenoviral vector CMVLacZ in 10
ml of saline was injected in about 10 sec and 2 minutes later the
clamps were opened. Two days after injection the leg muscle were
assayed for luciferase as above.
[0175] Results: TABLE-US-00020 TABLE Distribution of luciferase
activity following the intraarterial injection of adenovirus
CMVLacZ. Muscle Group Luciferase (ng) Upper Leg Anterior 59.04
Upper Leg Posterior 18.33 Upper Leg Medial 4.44 Lower Leg Posterior
11.04 Lower Leg Anterior 5.33 Foot 0.22 Total 98.40
Example 14
[0176] Surgical Description--Cynomolgus monkeys (n=6) weighing 2.5
to 3.3 kg and were sedated with ketamine (10-15 mg/kg IM). After
sedation, animals were intubated and anesthesia was maintained with
1.0 to 2.0% isoflurane. An intravenous catheter was inserted into
the cephalic vein for administering fluids and EKG electrodes were
attached to the limbs for monitoring heart rate. The surgical area
was prepped and draped for surgery using aseptic technique. A
femoral cutdown was performed and a 5 cm segment of the femoral
vein was dissected free of connective tissue. The vein was ligated
distally and a 6F introducer was inserted into the vein and secured
with a vessel tourniquet. A 4F injection catheter was inserted
through the introducer and advanced into the inferior vena cava
(IVC). An abdominal incision was made extending from just below the
xyphoid to the pubis. A retractor was placed inside the abdominal
cavity and moist sponges were used to retract and hold the
intestines. The infra hepatic IVC was exposed and the exact
placement of the injection catheter within the IVC was adjusted so
that the tip of the catheter was adjacent to the hepatic vein. A
vessel tourniquet was placed loosely around the infra hepatic IVC
to prevent backflow during the nucleic acid injection. The supra
hepatic IVC was dissected free of connective tissue and ligament
attachments. In some animals (n=3), a catheter (20 gauge) was
inserted into the portal vein and attached to a pressure transducer
to measure pressure changes during the injection. Immediately
before the injection, a vascular clamp was placed on supra hepatic
IVC and the vessel tourniquet was tightened around the infra
hepatic IVC. The IVC remained occluded during the injection and for
2 minutes post injection. After the injection, the clamps were
removed and the portal vein pressure catheter was pulled. The
abdominal cavity was closed in 3 layers with 3-0 PDS suture. The
catheter and introducer were pulled from the femoral vein, the
vessel was ligated and the incision was closed in 2 layers with 3-0
PDS suture. Prior to the completion of the surgery, the animal was
given buprenorphine (0.005-0.01 mg/kg IM) as an analgesic. Blood
samples were collected on days 0, 1, 4, 7, 14 and 21 for measuring
reporter gene expression (secreted alkaline phosphatase), liver
enzymes and a complete blood count.
[0177] Injection description--Two 60 ml syringes were filled with a
total volume of 120 ml of injection solution containing 0.9% NaCl,
7.5% mannitol and 10 mg of CMV-SEAP. Syringes were attached to a
syringe pump (Harvard Instrument) and connected to the injection
catheter via an extension line with a 3-way stopcock. The injection
flow rate was set at either 60, 120 or 160 ml/min. In addition to
using the syringe pump, hand injections were performed on two
animals. The flow rate was continuous during the injection except
for one animal that had a preset pause (5 seconds) half way through
the injection. During the injection, the entire liver swelled and
patchy areas were blanched from the injection solution. In five of
the six animals, it was noted that the liver swelled to the point
where fluid would begin to leak out the exterior of the liver
capsule by the end of the injection.
[0178] Pressure--The portal vein pressure during injection was
measured at two injection flow rates. An injection rate of 120
ml/min produced a transient elevation in portal vein pressure to 45
mm Hg while a flow rate of 160 ml/min resulted in a pressure
increase to 105 mm Hg. This pressure increase lasted for the length
of the injection and rapidly returned to preinjection levels.
[0179] Toxicity--All animals recovered and regained normal activity
within 24 hours after surgery.
[0180] Liver enzymes were elevated in all animals after surgery
with the highest levels on day 1. Enzyme levels gradually declined
thereafter and returned to normal levels around day 14. Day one
levels of alanine aminotransferase (ALT) varied from 4960-338 U/L.
The animals with the highest ALT levels were also the animals with
the highest level of gene expression. One of the animals that
showed both high reporter gene expression and elevated liver
enzymes was sacrifice at 21 days to harvest tissue for histology.
H+E slides of the right and left lobes showed normal tissue with no
significant pathology.
[0181] Reporter Gene Expression--In all the animals in this study,
reporter gene expression peaked at day 2. The first animal (animal
#1) in this study was a hand injection and resulted in the highest
gene expression (day 2=2414 ng/ml SEAP) in this study. This hand
injection delivered .about.120 ml of solution/minute. Using a
syringe pump we found that 60 ml/min (animal #3) and 120 ml/min
(animal #2) resulted in a low level of gene expression on day 2 (99
and 71 ng/ml SEAP respectively) but a flow rate of 160 ml/min
(animal #5) dramatically improved expression (day 2=1991 ng/ml
SEAP). This injection rate was repeated in a second animal (animal
#6) but with a preset pause (5 seconds) halfway through the
injection to determine if continuous flow was important for high
gene expression. The expression in this animal was also high (day
2=985 ng/ml) but lower than the continuous syringe pump injection.
In one additional animal, the pump failed during the injection and
had to be helped by hand (animal #4). This injection delivered 120
ml in approximately one minute and resulted in a SEAP level of 350
ng/ml on day 2. TABLE-US-00021 TABLE Hepatic Vein Delivery of
Nucleic Acid to Primate Liver. Expression of plasmid CMV-SEAP in
liver and analysis of toxicity. day animal 0 1 2 4 7 14 21 SEAP
expression (ng/ml) 1 2300.7 2414.7 1605.1 1144.1 504.2 0.7 2 70.9
70.8 43.4 18.0 3.4 1.6 3 63.9 98.8 85.5 49.5 13.4 9.2 4 206.7 350.1
184.9 59.3 55.7 2.5 5 1686.1 1990.7 1737.8 831.9 371.7 155.8 6
740.9 985.1 864.4 634.6 232.5 106.7 ALT 1 3148 1758 669 239 92 71 2
22 338 198 90 45 39 21 3 25 885 468 209 95 44 28 4 39 725 499 224
112 57 40 5 51 438 207 57 41 6 54 4960 2116 767 148 70 white blood
cell count 1 4.1 4.5 8.5 6.3 5.5 2 15.8 13.1 17.9 16.6 14.0 17.0
14.0 3 15.4 10.2 9.6 11.6 12.4 15.5 10.3 4 6.7 11.8 8.9 11.6 10.4
9.7 8.9 5 8.1 12.8 8.1 10.2 8.2 15.3 13.0 6 8.5 17.6 12.0 8.4 8.6
7.9 ALKP 1 1122 1353 896 566 219 148 2 164 287 288 218 188 237 210
3 182 301 372 272 249 223 196 4 134 250 303 235 195 161 140 5 165
1147 1407 1036 831 443 295 6 251 560 931 632 392 322 CPK 1 2987 918
380 208 131 233 2 361 1180 246 164 138 235 3 1253 3769 1250 188 210
91 318 4 411 1358 554 227 107 99 184 5 487 1600 787 421 112 680 6
789 12800 451 382 2027 333 AST 1 973 169 47 36 37 39 2 29 102 48 26
32 40 31 3 29 360 101 27 33 28 20 4 26 156 69 38 36 26 24 5 32 515
122 47 50 36 43 6 32 6617 90 50 56 31 GGT 2 59 71 65 56 57 60 3 83
105 97 80 85 84 78 4 76 73 69 72 73 74 5 42 88 78 68 72 57 57 6 137
119 154 146 153 158
[0182] The foregoing is considered as illustrative only of the
principles of the invention. Further, since numerous modifications
and changes will readily occur to those skilled in the art, it is
not desired to limit the invention to the exact construction and
operation shown and described.
* * * * *