U.S. patent application number 10/615518 was filed with the patent office on 2004-01-22 for long term expression of gene products.
This patent application is currently assigned to ARCH Development Corporation. Invention is credited to Leiden, Jeffrey M..
Application Number | 20040013650 10/615518 |
Document ID | / |
Family ID | 21820973 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040013650 |
Kind Code |
A1 |
Leiden, Jeffrey M. |
January 22, 2004 |
Long term expression of gene products
Abstract
A process for increasing the circulating levels of self gene
products in a mammal for an extended period of time is provided. In
accordance with that process, muscle cells of the mammal are
transformed with an expression vector that contains a
polynucleotide that encodes the gene product and which vector
drives expression in the muscle.
Inventors: |
Leiden, Jeffrey M.;
(Chicago, IL) |
Correspondence
Address: |
HALE AND DORR LLP
300 PARK AVENUE
NEW YORK
NY
10022
US
|
Assignee: |
ARCH Development
Corporation
Chicago
IL
60637
|
Family ID: |
21820973 |
Appl. No.: |
10/615518 |
Filed: |
July 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10615518 |
Jul 8, 2003 |
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09091134 |
Jun 23, 1998 |
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6613319 |
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09091134 |
Jun 23, 1998 |
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PCT/US97/14764 |
Aug 22, 1997 |
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60024511 |
Aug 23, 1996 |
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Current U.S.
Class: |
424/93.2 |
Current CPC
Class: |
A61K 38/27 20130101;
C12N 2799/022 20130101; C07K 14/505 20130101; A61K 48/0075
20130101; A61K 38/1816 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/93.2 |
International
Class: |
A61K 048/00 |
Claims
I claim:
1. A process for increasing the circulating levels of a self
protein in the blood stream of an immunocompetent animal which
comprises delivering a viral vector in vivo to muscle cells of said
animal by intramuscular injection in an amount sufficient to obtain
expression of and increase the circulating level of said self
protein in the bloodstream of said animal for a period greater than
about 30 days, wherein said self protein is a polypeptide hormone
and undergoes secretion, diffusion or transport to the circulation
upon expression in vivo.
2. The process of claim 1 wherein the animal is a primate.
3. The process of claim 2 wherein the primate is a human.
4. The process of claim 1 wherein the viral vector is a
replication-defective adenoviral vector or a retroviral vector.
5. The process of claim 1 wherein the self protein is a cytokine,
colony stimulating factor, nerve growth factor, insulin, glucagon,
rennin, parathyroid hormone, growth hormone, growth factor or
erythropoietin.
6. The process of claim 1 wherein the circulating level of the self
protein is increased for a period of time greater than about 60
days.
7. The process of claim 1 wherein the circulating level of the self
protein is increased for a period of time greater than about 90
days.
8. The process of claim 1 wherein the circulating level of the self
protein is increased for a period of time greater than about 120
days.
9. The process of claim 1 wherein the circulating level of the self
protein is increased for a period of time ranging from about 90
days to about 365 days.
10. The process of claim 1 wherein the muscle cells are cardiac
muscle cells or skeletal muscle cells.
11. The process of claim 1, wherein said immunocompetent animal is
being treated with an immunosuppressant.
12. A process for increasing the circulating levels of a self
protein in the blood stream of an immunocompetent animal which
comprises transforming muscle cells in vivo with a viral vector
encoding a self protein, wherein the expression vector is delivered
to said animal by intramuscular injection in an amount sufficient
to obtain expression of and increase the circulating level of said
self protein in the bloodstream of said animal for a period greater
than about 30 days, wherein said self protein is a polypeptide
hormone and undergoes secretion, diffusion or transport to the
circulation upon expression in vivo.
13. The process of claim 12 wherein the animal is a primate.
14. The process of claim 13 wherein the primate is a human.
15. The process of claim 12 wherein the expression vector is a
viral vector.
16. The process of claim 15 wherein the viral vector is a
replication-defective adenoviral vector or a retroviral vector.
17. The process of claim 12 wherein the polypeptide is a cytokine,
colony stimulating factor, nerve growth factor, insulin, glucagons,
rennin, parathyroid hormone, growth hormone, growth factor or
erythropoietin.
18. The process of claim 12 wherein the circulating level of the
self protein is increased for a period of time greater than about
60 days.
19. The process of claim 12 wherein the circulating level of the
self protein is increased for a period of time greater than about
90 days.
20. The process of claim 12 wherein the circulating level of the
self protein is increased for a period of time greater than about
120 days.
21. The process of claim 12 wherein the circulating level of the
self protein is increased for a period of time ranging from about
90 days to about 365 days.
22. The process of claim 12 wherein the muscle cells are cardiac
muscle cells or skeletal muscle cells.
23. The process of claim 12, wherein said immunocompetent animal is
being treated with an immunosuppressant.
24. A process for increasing the circulating levels of a self
protein in the blood stream of an immunocompetent animal which
comprises transforming muscle cells of said animal ex vivo with an
expression vector encoding a self protein; and delivering said
transformed muscle cells to said animal by intramuscular injection
in an amount sufficient to obtain expression of and increase the
circulating level of said self protein in the bloodstream of said
animal for a period greater than about 30 days, wherein said self
protein is a polypeptide hormone and undergoes secretion, diffusion
or transport to the circulation upon expression in vivo.
25. The process of claim 24 wherein the animal is a primate.
26. The process of claim 25 wherein the primate is a human.
27. The process of claim 24 wherein the expression vector is a
plasmid.
28. The process of claim 24 wherein the expression vector is a
viral vector.
29. The process of claim 28 wherein the viral vector is a
replication-defective adenoviral vector or a retroviral vector.
30. The process of claim 24 wherein the self protein is a cytokine,
colony stimulating factor, nerve growth factor, insulin, glucagons,
rennin, parathyroid hormone, growth hormone, growth factor or
erythropoietin.
31. The process of claim 24 wherein the circulating level of the
self protein is increased for a period of time greater than about
60 days.
32. The process of claim 24 wherein the circulating level of the
self protein is increased for a period of time greater than about
90 days.
33. The process of claim 24 wherein the circulating level of the
self protein is increased for a period of time greater than about
120 days.
34. The process of claim 24 wherein the circulating level of the
self protein is increased for a period of time ranging from about
90 days to about 365 days.
35. The process of claim 24 wherein the muscle cells are cardiac
muscle cells or skeletal muscle cells.
36. The process of claim 24, wherein said immunocompetent animal is
being treated with an immunosuppressant.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
09/091,134, filed Jun. 23, 1998, which is the national phase
application of PCT/US97/14764, filed Aug. 22, 1997, which is a
continuation-in-part of and claims benefit to U.S. Provisional
Patent Application Serial No. 60/024,511, filed Aug. 23, 1996, the
disclosures of which are each incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The field of this invention is gene expression. More
particularly, this invention pertains to a process for increasing
the circulating levels of gene products over an extended period of
time.
BACKGROUND OF THE INVENTION
[0003] A large number of inherited and acquired serum protein
deficiencies including hemophilia A, diabetes mellitus and the
erythropoietin-responsi- ve anemias are currently treated by
repeated intravenous or subcutaneous injections of purified or
recombinant proteins. Although largely effective, such therapies
are both expensive and inconvenient. Moreover, in diseases such as
hemophilia A, there is not sufficient recombinant protein available
to allow a comprehensive program of prophylactic therapy. Given
these problems, there has been considerable interest in developing
novel gene-based therapies for such serum protein deficiencies. An
initial series of studies demonstrated that skeletal myoblasts
genetically modified in vitro could be reimplanted by intramuscular
injection and would subsequently produce stable, physiological
levels of recombinant proteins in the systemic circulation of adult
immunocompetent mice. Subsequently, several groups have
demonstrated the stable production of recombinant serum proteins
following a single intramuscular (IM) injection of
replication-defective adenovirus (RDAd) vectors. Despite these
initial successes, both myoblast transplantation and IM injection
of RDAd vectors have thus far been associated with problems that
may preclude their widespread clinical application.
[0004] The studies reported to date have all been done on rodents
such as mice. Those data may not reflect and may not be predictive
of results in larger animals such as primates. It is well known in
the art, for example, that physiological or therapeutic doses
observed in rodents are not necessarily predictive of effective
doses in larger mammals. Still further, the amount of vector needed
in large mammals may preclude their utility. For example, the mass
of vector needed in primates may be so large that their injection
results in either adverse reactions to the injection (e.g.,
anaphylactic shock), generation of an immune response or secondary
infection resulting from the use of large numbers of viral
particles. Still further, the data from previous reports do not
address the question of whether there is any correlation between
the amount or dose of transforming vectors and increases in the
levels of gene products. There continues to be a need in the art,
therefore, for processes for increasing the circulating levels of
gene products in large mammals such as primates.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides a process of increasing the
circulating levels of a gene product in the blood stream of a
mammal for a period of time greater than about 30 days. The process
includes the step of transforming muscle cells of the mammal with a
polynucleotide that encodes the gene product, wherein the
expression vector drives long-term expression of the
polynucleotide.
[0006] A preferred mammal is an animal used for food such as a cow,
domesticated animals such as dogs and cats and primates. A
preferred primate is a human. A process of the present invention
can be used to increase circulating levels of any gene product.
Exemplary such gene products are RNA molecules, single-stranded DNA
molecules and polypeptides. Polypeptides are particularly
preferred. Especially preferred polypeptides are polypeptide
hormones such as growth hormone and erythropoietin.
[0007] A process of the present invention can use any muscle such
as smooth muscle, cardiac muscle and skeletal muscle. Cardiac and
skeletal muscle are preferred. The use of skeletal muscle is most
preferred.
[0008] Any suitable expression vector can be used in the present
process. Exemplary and preferred such vectors are plasmids and
replication defective adenoviral vectors. The muscle cells can be
transformed either in vivo or ex vivo. When transformed in vivo,
the expression vector is directly injected into a muscle mass of
the mammal. When transformed ex vivo, muscle cells are removed from
the mammal, transformed ex vivo and the transformed muscle cells
reimplanted into the mammal.
[0009] A process of the present invention is useful for increasing
the circulating levels of gene products over an extended period
time. Using a process of this invention, those levels can be
increased for periods of time ranging from greater than about 60,
90 or 120 days and even for as long as one year.
[0010] The safety and efficacy of IM injection of adenoviral
vectors encoding Epo in both mice and non-human primates has been
determined. In an initial series of experiments, the relationship
between the dose of vector injected and the corresponding
elevations in serum Epo levels and hematocrits in both species were
studied. The results demonstrated that there is a threshold dose in
both mice and monkeys (approximately 2.5-8.times.10.sup.7 pfu/gm
body weight) that is required to obtain long-term Epo expression
and polycythemia. A single TM injection of mice with 10.sup.9 pfu
of vector resulted in elevations in hematocrits from control values
of 49% to treated values of 81% which were stable for more than one
year. Similarly, a single IM injection of a monkey with
4.times.10.sup.11 pfu of an adenoviral vector encoding simian Epo
(AdsEpo) resulted in elevations of hematocrits from control levels
of 40% to treated levels of =70% which were stable for 84 days. IM
injection of monkeys with vector was determined to be safe in that
no abnormalities in chest X-rays, serum chemistries, hematologic or
clotting profiles (except for elevated hematocrits) or organ
pathology were seen during the 84 day time course of the
experiment.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention provides a process of increasing the
circulating level of a gene product in the blood stream of a mammal
for a period of time greater than about 30 days. The process
includes the step of transforming muscle cells of the mammal with a
polynucleotide that encodes the gene product, wherein the
expression vector drives long-term expression of the
polynucleotide.
[0012] As is well known in the art, gene products include
polynucleotides such as DNA and RNA and polypeptides. As is also
well known in the art, those gene products can be secreted from the
cells where they are made into the extracellular fluid compartment
of the organism. From there, those products diffuse into the blood.
The process of the present invention can be used to increase the
levels of those gene products in the blood over an extended period
of time. A process of this invention is particularly useful in
increasing the levels of polypeptide gene products. Preferably, the
polypeptide is secretory product of a cell. Exemplary such products
are cytokines, colony stimulating factors, nerve growth factors and
the like. Exemplary and preferred such secretory products are
polypeptide or protein hormones. Such hormones are well known in
the art. Exemplary polypeptide hormones are insulin, glucagon,
renin, parathyroid hormone, growth hormone, erythropoietin and the
like.
[0013] A process of the present invention can be used to increase
the circulating level of a gene product in any mammal. The process
is particularly useful in large mammals such as domestic pets,
those used for food production and primates. Exemplary large
mammals are dogs, cats, horses, cows, sheep, deer and pig.
Exemplary primates are monkeys, apes and humans. The use of the
present process in humans is particularly preferred.
[0014] The present invention discloses that increased circulating
levels of gene products can be realized by transforming muscles
cells of the mammal with a polynucleotide that encodes that gene
product. As is well known in the art, mammals contain three types
of muscle cells: smooth muscle, cardiac muscle and skeletal muscle.
Any one of these muscle types can be used in a present process.
Because of the accessibility of large masses of cardiac and
skeletal muscle, use of these muscle types is preferred. In an
especially preferred embodiment, a process of this invention uses
skeletal muscle.
[0015] As used herein, the phrase "expression vector" means any
vehicle for delivering an encoding polynucleotide to a cell such
that the polynucleotide is expressed and a gene product is formed.
Expression vectors are well known in the art. As is also well known
in the art, particular vectors are especially suitable for
transforming mammalian cells. For use in the present invention,
plasmids and viral vectors are preferred. Exemplary viral vectors
include retroviral vectors and adenoviral vectors. The vectors,
especially the adenoviral vectors, are made replication defective
using standard procedures well known in the art. The choice of a
particular vector depends inter alia on the nature of the gene
product to be produced.
[0016] Replication-defective adenoviruses represent an efficient
and safe method of in vivo gene transfer. These vectors can be
prepared at high titer (up to 10.sup.11 pfu/ml) and infect many
replicating and non-replicating cell types in vivo. Adenoviruses
are common and relatively benign human pathogens that have not been
associated with persistent infections or neoplasias in humans.
Wild-type adenoviruses have been used previously for human
vaccination. As disclosed herein, a single IM injection of a
replication-defective adenovirus encoding hEpo can be used to
produce dose-dependent elevations in serum Epo levels and
hematocrits which were stable over the 120 day time course of these
experiments. The injected adenovirus remains localized at the site
of administration and does not cause muscle pathology. Taken
together, these results show that IM injection of
replication-defective adenoviruses is useful for the treatment of a
number of acquired and inherited human serum protein
deficiencies.
[0017] Thus, in one embodiment, the expression vector is a
replication defective adenoviral vector. A single IM injection of
immunocompetent mice with 10.sup.9 to 3.times.10.sup.9 plaque
forming units (pfu) of an E1- and E3-deleted replication-defective
adenovirus vector encoding murine Epo (AdmEpo) resulted in
elevations of serum Epo levels and hematocrits from control values
of approximately 45% to treated values of approximately 80% which
were stable for at least 112 days ( Tripathy et al., Nature Med. 2,
545-550, 1996).
[0018] As disclosed herein, an adenoviral vector, RdAd, encoding
self Epo can be used to produce sustained and significant
elevations in hematocrits in both mice and non-human primates.
Unlike previous observations in immunocompromised SCID animals, the
persistence of transgene expression in immunocompetent animals
appears to be critically dependent upon the dose of virus
administered, with a threshold dose of approximately
2.5-8.times.10.sup.7 pfu/gm in both mice and monkeys required to
obtain persistent transgene expression. From a safety standpoint,
there was no evidence of pulmonary or hepatic toxicity and there
was no demonstrable long term organ pathology in monkeys injected
once IM with 4.times.10.sup.10-4.times.10.sup.11 pfu of AdsEpo.
Taken together, these results show that IM injection of RdAd
encoding human Epo can be used to safely and effectively treat
patients with Epo-responsive anemias.
[0019] Previous studies in immunocompromised animals demonstrated
that IM injection of SCID mice with as little as 10.sup.7 pfu of
Epo-encoding RdAd resulted in stable elevations in hematocrits.
Increasing the viral dose to 10.sup.8 or 10.sup.9 pfu produced
further increases in both serum Epo levels and hematocrits
(Tripathy et al., Proc. Natl. Acad. Sci. USA 91, 11557-11561,
1994). Thus, in SCID animals there appeared to be a simple linear
relationship between the dose of virus administered and the
resulting levels of Epo in the serum. The data disclosed herein in
immunocompetent mice and monkeys clearly demonstrate a more complex
relationship between viral dose and hematocrit. IM injection of
immunocompetent mice and monkeys with low doses of virus resulted
in only transient increases in hematocrit, whereas injection with
higher doses of virus led to sustained elevations in serum Epo
levels and hematocrits. Moreover, there appeared to be a threshold
dose (2.5-8.times.10.sup.7 pfu/gm) in both species that was
required for persistent transgene expression.
[0020] The observed differences between the SCID and
immunocompetent animals clearly implicated the immune system as the
critical determinant of these different dose-response
relationships. However, there are several possible alternative
mechanisms that might explain these differences. First, it is
possible that the immune system eliminates a significant fraction
of the Epo-expressing myocytes, independent of the dose of virus
administered. In this case, initial infection of a relatively large
number of myocytes might be required to end up with a sufficient
number of Epo-producing cells to produce physiologic elevations in
serum Epo levels. Such a model is supported by our measurements of
serum Epo levels following IM injection of immunocompetent mice
with AdmEpo. Peak serum Epo levels observed 1 week following viral
infection subsequently declined by approximately 70% by 4 weeks
after injection and then stabilized. In the animals injected with
10.sup.8 pfu of AdmEpo this decline resulted in serum Epo levels
that fell to or below endogenous circulating Epo levels and
therefore did not result in a polycythemia. In contrast, in the
animals injected with 10.sup.9 pfu of AdmEpo, this decline still
resulted in serum Epo levels that were approximately 10-fold above
pre-injection levels, thereby leading to significant elevations in
hematocrit.
[0021] Alternatively, it is possible that the type of immune
response elicited by IM injection of the AdmEpo vectors is
critically dependent upon the dose of vector administered. It has
recently been shown that exposure of mice to high doses of antigen
leads predominantly to Th2 responses, whereas administration of
lower doses of antigen leads to Th1. Therefore, it is possible that
Th1 responses to the low dose of AdmEpo in the animals receiving
4.times.10.sup.10 pfu of virus led to CTL-mediated elimination of
virus infected cells, whereas Th2 responses to high doses of vector
allowed persistence of larger numbers of Epo-producing myocytes in
the animals that received 4.times.10.sup.11 pfu of virus. These two
mechanisms are, of course, not mutually exclusive.
[0022] The dose of virus (in pfu/gm) required to produce sustained
transgene expression in mice and monkeys was quite similar. This
finding indicates that it is possible to predict human doses based
upon our rodent and primate data. From the mouse data, a single
injection of 10.sup.9 pfu of AdmEpo resulted in a sustained 30
point elevation in hematocrit (from approximately 50% to 80%).
Thus, it requires approximately 1.33.times.10.sup.6 pfu/gm body
weight to produce a 1 point elevation in hematocrit. For example,
to produce a 15 point increase in hematocrit in a 70 kg human with
Epo-responsive anemia (e.g., to increase the hematocrit from 23% to
38%) it would require a single IM injection of (1.33.times.10.sup.6
pfu/gm).times.15 (% increase in hematocrit).times.70,000
gm=1.4.times.10.sup.12 pfu of AdhEpo. A previously reported
adenoviral vector, AdhEpo, which is identical to AdmEpo and AdsEpo
except that it contains the human Epo cDNA, has been shown to drive
the expression and secretion of high levels of human Epo following
both in vitro infection of myocytes and IM injection of SCID mice
(Tripathy et al., Proc. Natl. Acad. Sci. USA, 91,
11557-11561,1994).
[0023] The lack of toxicity observed in Cynomologus monkeys
following IM injection of 4.times.10.sup.11 pfu of AdsEpo, a dose
sufficient to produce significant elevations in hematocrits, also
augurs well for the safety of human gene therapy using IM injection
of Epo-encoding vectors. This relative lack of toxicity as compared
to previous experiments involving intravenous or inhaled routes of
administration may reflect the fact that relatively little vector
infects the liver or lung following IM administration. In addition,
because previous experiments have demonstrated that immune
responses to foreign transgenes play an important role in the
inflammatory responses to RDAd-infected cells (Tripathy et al.,
Nature Med. 2, 545-550,1996), the use of a self trangene (to which
the animal is tolerant) may have significantly reduced immune
responses to the vector-infected cells in these animals. The
present finding of excessive polycythemia in the monkey injected
with 4.times.10.sup.11 pfu of AdsEpo suggested that it will be
essential to begin human trials with low doses of vector in order
to carefully assess dose-response relationships in humans. However,
the fact that injected muscle can be removed to terminate therapy
adds a relative safety factor to therapies involving IM as opposed
to systemic or pulmonary administration of RDAd. The use of such
vectors will significantly increase the safety of
adenovirus-mediated gene therapy for Epo-responsive anemias.
Finally, the finding of significant levels of anti-adenoviral
antibodies in mice and monkeys following a single IM injection of
RDAd will likely make readministration of the vector difficult or
impossible. Indeed, recent studies in mice have demonstrated that
it is not possible to readminister AdsEpo to mice even 9 months
after an initial IM injection. Modifications of the vector or
transient immunosuppresive regiments will therefore likely be
necessary to obviate this problem.
[0024] In another embodiment, the expression vector is a plasmid.
The IM injection of plasmid DNA has a number of distinct advantages
as compared to the use of RDAd vectors. First, plasmid DNA vectors
are easier to construct, can accept large cDNA inserts, and can be
prepared as pure chemical solutions without the risk of
contamination with wild-type infectious particles. In addition, IM
injection of adult immunocompetent animals with RDAd has been
associated with immune responses that eliminate virus infected
cells in 14-28 days, thereby producing only transient recombinant
gene expression in vivo. Of equal importance, previous infection
with wild-type adenovirus results in a neutralizing antibody
response which may preclude administration of an RDAd vector. In
contrast, the present disclosure demonstrates long-term Epo
expression following a single IM injection of plasmid DNA even in
adult immunocompetent animals. Moreover, because there were no
detectable antibodies against mEpo in the sera of mice 90 days
after injection with pVRmEpo, it is possible to readminister
plasmid DNA by IM injection if repeated therapy or dose escalation
is required.
[0025] The present invention discloses the construction and
characterization of a novel plasmid vector that produces high level
expression and secretion of erythropoietin (Epo) following IM
injection into adult-immunocompetent mice. A single IM injection of
as little as 10 mg of this plasmid produced physiologically
significant levels of mEpo in the systemic circulation of adult
immunocompetent mice and resulted in significant elevations in
hematocrits that were stable for at least 90 days. The injected
plasmid DNA remained localized at the site of injection and the
amount of Epo production (as reflected by the elevated hematocrits)
was proportional to the dose of plasmid DNA injected. Thus, IM
injection of plasmid DNA represents a feasible approach to the
treatment of serum protein deficiencies.
[0026] As shown in detail hereinafter in the Examples, IM injection
of as little as 10 mg of pVRmEpo (a novel plasmid expression
vector, pVRmEpo, which directs high level production and secretion
of mEpo from skeletal myocytes in vitro) into adult immunocompetent
mice resulted in dose-dependent elevations in hematocrits that
remained stable for at least 90 days. The increased hematocrits
observed in the pVRmEpo-injected mice reflected persistent
production of mEpo from the injected muscle and secretion of this
protein into the systemic circulation. Finally, the injected DNA
remained predominantly localized to muscle at the site of
injection. The data disclosed herein represent the first
demonstration of the delivery of physiologically significant levels
of recombinant protein to the systemic circulation following the IM
injection of a plasmid DNA expression vector.
[0027] The expression vector drives expression of the
polynucleotide that encodes the particular gene product. Thus, the
vector needs to contain those expression elements necessary for
expression. For example, a polynucleotide of an expression vector
of the present invention is preferably operatively associated or
linked with an enhancer-promoter. A promoter is a region of a DNA
molecule typically within about 100 nucleotide pairs in front of
(upstream of) the point at which transcription begins. That region
typically contains several types of DNA sequence elements that are
located in similar relative positions in different genes. As used
herein, the term "promoter" includes what is referred to in the art
as an upstream promoter region or a promoter of a generalized RNA
polymerase transcription unit.
[0028] Another type of transcription regulatory sequence element is
an enhancer. An enhancer provides specificity of time, location and
expression level for a particular encoding region (e.g., gene). A
major function of an enhancer is to increase the level of
transcription of a coding sequence in a cell that contains one or
more transcription factors that bind to that enhancer. Unlike a
promoter, an enhancer can function when located at variable
distances from a transcription start site so long as the promoter
is present.
[0029] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer-promoter is operatively linked to a coding sequence
that encodes at least one gene product. As used herein, the phrase
"operatively linked" or its grammatical equivalent means that a
regulatory sequence element (e.g. an enhancer-promoter or
transcription terminating region) is connected to a coding sequence
in such a way that the transcription of that coding sequence is
controlled and regulated by that enhancer-promoter. Means for
operatively linking an enhancer-promoter to a coding sequence are
well known in the art.
[0030] An enhancer-promoter used in an expression vector of the
present invention can be any enhancer-promoter that drives
expression in a host cell. By employing an enhancer-promoter with
well known properties, the level of expression can be optimized.
For example, selection of an enhancer-promoter that is active in
specific cells (e.g., muscle cells) permits tissue or cell specific
expression of the desired product. Still further, selection of an
enhancer-promoter that is regulated in response to a specific
physiological signal can permit inducible expression.
[0031] A coding sequence of an expression vector is operatively
linked to a transcription terminating region. RNA polymerase
transcribes an encoding DNA sequence through a site where
polyadenylation occurs. Typically, DNA sequences located a few
hundred base pairs downstream of the polyadenylation site serve to
terminate transcription. Those DNA sequences are referred to herein
as transcription-termination regions. Those regions are required
for efficient to polyadenylation of transcribed messenger RNA
(mRNA). Enhancer-promoters and transcription-terminating regions
are well known in the art. The selection of a particular
enhancer-promoter or transcription-terminating region will depend,
as is also well known the art, on the cell to be transformed.
[0032] The muscle cells can be transformed either in vivo or ex
vivo. When transformed in vivo, the expression vector can be
directly injected into muscle cells of the mammal. Alternately, the
vector can be delivered to the muscle cells by infusing the vector
into an artery or vein that perfuses the target muscle. Means for
transforming smooth, cardiac and skeletal muscle in vivo are well
known in the art. In a preferred embodiment, the vectors are
directly injected into cardiac or skeletal muscle.
[0033] When transformed ex vivo, muscle cells are removed from the
mammal, transformed ex vivo and the transformed muscle cells
reimplanted into the primate. When ex vivo procedures are used, the
use of skeletal muscle is preferred.
[0034] A process of the present invention is used to increase the
circulating level of a gene product. As used herein, the term
"increase" means to raise the circulating level above the
pre-transformation level. Thus, the present process can be used to
enhance levels above the normal physiological level of that gene
product or can be used to correct abnormal deficiencies in the
level of that product. Further, as shown hereinafter in the
Examples, the circulating level of a gene product can be increased
in a dose-dependent fashion. Preferably, the pre-transformation
circulating levels can increased at least 10 percent with use of
the present process. Even more preferably, the circulating levels
can be increased at least 20 percent, 50 percent, 100 percent or
even greater. The data set forth hereinafter in the Examples also
show that the physiological activity (e.g., hematocrit) of the gene
product (e.g., erythropoietin) can also be increased with use of
the present process.
[0035] The Examples that follow illustrate preferred embodiments of
the present invention and are not limiting of the specification and
claims in any way.
EXAMPLE 1
[0036] Plasmid Vectors
[0037] pVRhEpo contains an 840 by Not I human erythropoietin (hEpo)
cDNA fragment from pAdEF1hEpo cloned into the Not I site of the
plasmid vector, pVR1012 (24). pVRmEpo contains a 620 by Sal I/Bgl
II murine erythropoietin cDNA fragment obtained by PCR of
pAdEF1mEpo (25) with sense and antisense primers
(5'-GGGGTCGACGGCGGGGAGATGGGGGTGCCCG [SEQ ID NO: 1],
5'-GGGAGATCTAGTTCACC TGTCCCCTCTCCTGC [SEQ ID NO: 2]) and cloned
into the Sal I and Bgl II sites of pVR1012. pVRbgal contains the
bacterial lacZ gene cloned into the multiple cloning site (MCS) of
pVR1012 and pVR1902 contains the canine factor IX cDNA cloned into
the MCS of pVR1012.
[0038] Transfections--C2C12 myoblasts (10.sup.6 cells in a 10 cm
tissue culture dish) were transfected with 15 mg of pVRmEpo using
the lipofectarnine reagent (Gibco BRL, Gaithersburg, Md.).
Approximately 16 hours later, the transfected cells were placed in
fusion medium (DMEM, 2% horse serum, 1% penicillin/streptomycin)
and the cells were allowed to fuse into myotubes overnight. Media
was harvested at the times indicated after fusion and assayed for
mEpo using a radioimmunoassay.
[0039] IM infection of plasmid DNA--Purified plasmid DNA was
resuspended in sterile PBS -/- (Gibco BRL, Gaithersburg, Md.) at a
concentration of 3 mg/ml. Mice were injected IM into the tibialis
anterior or rectus femoris muscles with 50-100 .mu.l of DNA
solution per site containing 10-100 mg of DNA. Hematocrits were
measured on blood collected by tail vein or orbital
venipuncture.
[0040] Epo assays--Tissues were harvested from mice 90 days after
injection with plasmid DNA and homogenized in approximately 1 ml of
Epo specimen diluent buffer (R and D Systems, Minneapolis, Minn.),
centrifuged at 10,000.times. g for 10 minutes, and supernatants
collected. Murine Epo levels were measured in cell culture
supernatants, serum, and tissue lysates using a radioimmunoassay
that is specific for mEpo.
[0041] PCR--Southern assays--Total cellular DNA was isolated from
mouse tissues. For SCID mice, approximately 1 mg of total cellular
DNA from each tissue was subject to the PCR using primers
corresponding to sequences within the hEpo cDNA
(5'-CCAGACCCCGAAGCATGG) (SEQ ID NO: 3) and the pVR plasmid
(5'-GGAAGACTTAAGGCAGCG) (SEQ ID NO: 4). For CD-1 and BALB/c mice,
approximately 100 ng of cellular DNA from each tissue was subjected
to the PCR using primers corresponding to sequences within the mepo
cDNA (5'-GAAGTCAGGCTACGTAGACCACTG) (SEQ ID NO: 5) and the pVR1012
plasmid (5'-GTCTGAGCAGTACTCGTTGC) (SEQ ID NO: 6). The resulting PCR
products were fractionated on a 1% agarose gel and analyzed by
Southern blotting using a radiolabeled Bam HI/Pvu II fragment of
the hEpo cDNA or a radiolabeled Bgl II/Sac I fragment of the mEpo
cDNA as probes. All cellular DNA samples were also subjected to the
PCR using primers specific for the cardiac troponin C (cTnC) gene
and the products were visualized in ethidium-bromide stained
agarose. PCR conditions were 35 cycles (94.degree. C. for 1 min.,
72.degree. C. for 1 min.) followed by a 10 minute extension at
72.degree. C.
EXAMPLE 2
[0042] In Vitro Analysis of pVRmEpo
[0043] In order to construct a plasmid expression vector that could
program high level production and secretion of recombinant proteins
from skeletal myofibers in vivo, the human (hEpo) and murine (mEpo)
erythropoietin cDNA were cloned into pVR1012, a plasmid vector
which contains a eukaryotic expression cassette controlled by the
CMV IE promoter, and the CMV IE 5' untranslated and intron A
sequences followed by the bovine growth hormone polyadenylation
signal. PVR1012 was used in these experiments because this plasmid
backbone has been shown to program high level luciferase expression
following IM injection into immunocompetent mice.
[0044] In initial experiments, C2C12 skeletal myoblasts were
transfected with pVRmEpo and allowed to fuse into multinucleated
myotubes. Culture supernatants from these transfected myotubes were
assayed for mEpo at various times after transfection. C2C12 cells
transiently transfected with pVRmEpo produced approximately 2000
mU/hr/10.sup.6 cells of mEpo (4800 mU/ml.times.10 ml/24 hours).
Supernatants from control (pVRbgal-transfected) C2C12 cells did not
contain detectable mEpo levels. Thus, pVRmEpo programmed high level
mEpo expression and secretion following transfection into cultured
skeletal myocytes. Similar high-level hEpo expression and secretion
was observed following transfection of C2C12 cells with
pVRhEpo.
EXAMPLE 3
[0045] IM Injection of Epo Expression Vector DNA Produces
Physiologically Significant Levels of Epo in the Systemic
Circulation of Mice
[0046] To determine whether IM injection of a plasmid Epo
expression vector could produce physiologically significant levels
of Epo in the systemic circulation of adult mice, adult SCID; adult
mice were injected IM with 300 mg of pVRhEpo or the control
plasmid, pVR1012 which does not contain a cDNA insert. Hematocrits
of the pVRhEpo-injected mice rose from pre-injection values of
48.+-.1.2% to values of 682.+-.2.4% within 14 days of
injectionandremained elevatedat this level for the 90 day time
course ofthe experiment. These elevated hematocrits were
significantly different from those of control mice injected with
identical amounts of pVR1012 (P<0.006). Injection of adult
immunocompetent CDI mice with 300 mg of pVRmEpo produced similar
elevations in hematocrits (74.+-.2.4% in the pVRmEpo-injected
animals vs. 47.+-.1% in control injected animals; P<0.01) which
were also sustained over the 90 day time course of the
experiment.
EXAMPLE 4
[0047] Elevated Serum and Muscle Epo Levels in the pVRmEpo-Injected
Mice
[0048] To demonstrate directly that the increased hematocrits
observed in the pVRmEpo-injected mice reflected persistently
elevated serum mEpo levels in these animals, serum mEpo levels were
assayed 90 days after injection using a radioimmunoassay (RIA) that
can detect mEpo. Serum mEpo levels in the pVRmEpo-injected animals
were significantly elevated as compared to mEpo levels in serum
from control mice (52 mU/ml in the pVRmEpo-injected mice vs. 8
mU/ml in the control mice; P<0.03). To determine the site of
mEpo production in the pVRmEpo-injected mice, tissue lysates
prepared from liver, kidney, and muscle at the site of IM injection
were assayed for mEpo by RIA. Murine Epo levels in lysates from the
pVRmEpo-injected muscle were significantly elevated as compared to
levels in control uninjected muscle lysate (130 mU/ml in the
pVRmEpo-injected muscle vs. 2 mU/ml in the uninjected muscle;
P<0.0001). There were no significant differences in the mEpo
levels detected in the other tissue lysates tested from the
pVRmEpo-injected or uninjected animals. Thus, the elevated
hematocrits observed in the pVRmEpo-injected animals reflected
persistent production and secretion of recombinant mEpo from the
pVRmEpo-injected muscle.
[0049] A dose-response relationship between the amount of DNA
injected and the subsequent elevation in hematocrit--To determine
directly if the level of polycythemia observed following IM
injection of pVRmEpo was proportional to the amount of DNA
injected, BALB/c mice were injected IM with 10, 100, or 300 mg of
pVRmEpo, and hematocrits were measured during the 90 days following
injection. IM injection of as little as 10 mg of pVRmEpo resulted
in stable elevations in hematocrits from pre-injection values of
48.+-.0.4% to post-injection levels of 64.+-.3.3% at 45 days after
injection. Injections of 100 or 300 mg of DNA caused further
increases in hematocrits to levels of 79.+-.3.3% at 45 days after
injection which declined to 67.+-.4.7% at 90 days after injection.
The hematocrits observed in each treatment group were significantly
elevated at each time point from those observed in control mice
injected with 300 mg of pVR1012 (P<0.004). Thus, the observed
levels of polycythemia were proportional to the amount of pVRmEpo
DNA injected at least over the range of 10-100 mg of injected
DNA.
[0050] Plasmid DNA remains localized at the site of IM
injection--To determine the distribution of plasmid DNA following
IM injection with pVRmEpo or pVRhEpo, mice were sacrificed 90 days
after injection and total cellular DNA from a number of tissues was
assayed for the presence of pVRmEpo DNA using a PCR assay that
could detect as little as 0.00001 copies of the plasmid per cell.
In both SCID mice injected with pVRhEpo and CD1 and BALB/c mice
injected with pVRmEpo, plasmid DNA could be detected in lysates
prepared from the injected muscle. In some animals, a barely
detectable signal was also observed in the liver. Taken together
with the studies data set forth above, the results show that the
preponderance of plasmid DNA remained localized at the site of IM
injection.
EXAMPLE 5
[0051] Long-Term Expression
[0052] Material and Methods Adenovirus Vectors. AdBglII is an
"empty" E1- and E3-deleted RDAd that does not express a transgene
(Barr et al., Gene Ther. 1, 51-58, 1994). AdmEpo is an E1- and
E3-deleted RDAd containing the murine Epo cDNA under the
transcriptional control of the elongation factor 1 (EF1) promoter
and 4F2 heavy chain (4F2HC) first intron enhancer. The construction
and characterization of this vector has been described previously
(Tripathy et al., Nature Med. 2, 545-550, 1996). AdsEpo, an E1- and
E3-deleted first generation replication-defective adenovirus vector
containing the cynomolgus simian erythropoietin (sEpo) cDNA under
the transcriptional control of the EF1 promoter and 4F2HC first
intron enhancer was constructed as follows: PCR primers
(5'-GGGGGGATCCGCACCTGGTCATCTGTCC-3' (SEQ ID NO: 7) and
(5'-GGGAAGCTTCCCGGCCAGGCGCGGAGATGG-3') (SEQ ID NO: 8) were designed
to amplify a 603 bp sEpo cDNA fragment from pMKE83 (obtained from
American Type Culture Collection) with Hind III and Bam HI
compatible ends. The resulting PCR product was subcloned into Hind
III+Bam HI-digested pAdEFI (Tripathy et al., Proc. Natl. Acad. Sci.
USA, 91,11557-11561, 1994) to produce pAdsEpo. The fidelity of the
sEpo cDNA PCR product was confirmed by dideoxy DNA sequence
analysis. pAdsEpo was then used to generate the AdsEpo adenovirus
by transfection of 293 cells as described previously (Tripathy et
al., Proc. Natl. Acad. Sci. USA, 91, 11557-11561,1994). The
resulting virus was plaque-purified 3 times prior to preparation of
a master seed stock. All adenovirus preparations were produced by
infection of 293 cells with master seed stocks that were free of
detectable replication-competent helper virus as determined using
a. PCR assay (Tripathy et al., Proc. Natl. Acad. Sci. USA.
91,11557-11561,1994). Adenoviruses were purified by centrifugation
in CsCl density gradients, desalted by dialysis in storage buffer
(10 mM Tris, pH 7.4, 1 MM MgCl.sub.2, 10% glycerol) and frozen in
aliquots at -70.degree. C. Virus titers were determined by plaque
assay on 293 cells.
[0053] Intramuscular Injection of RDAd. For the mouse experiments,
a total volume of 50 mil containing 10.sup.8 or 10.sup.9 pfu of
AdBglII or AdmEpo was injected IM via a 26 gauge needle into a
single site in the right tibialis anterior muscle of adult
immunocompetent CD1 mice. For the monkey experiments, adult
Cynomolgus monkeys were anesthetized with ketamme and atropine,
intubated and ventilated with Halothane during adenoviral
injections. The skin above the injection site was shaved and
tattooed to mark the site of injection. The monkeys were then
injected IM with a total dose of 4.times.10.sup.11 pfu of AdBglII
or AdsEpo (10 injections of 1 ml each of virus at a concentration
of 4.times.10.sup.10 pfu/ml into 10 different sites) or with
4.times.10.sup.10 pfu of AdsEpo (2 injections of 1 ml each of virus
at a concentration of 2.times.10.sup.10 pfu/ml into 2 different
sites). Buprenorphine was used over the following 48 hours to
manage animal discomfort.
[0054] Hematocrits and Epo Assay. Blood was obtained from mouse
tail veins by tail bleeds and monkey femoral veins by venipuncture.
Hematocrits were determined by centrifugation of whole blood. mEpo
assays were performed using a radioimmunoassay as described
previously (Tripathy et al., Nature Med. 2, 545-550, 1996). sEpo
assays were performed using the Quantikine IVD rhEpo ELISA kit
(R&D Systems, Minneapolis, Minn.) according to the
manufacturer's instructions.
[0055] Detection of Anti-adenoviral Antibodies. Titers of
anti-adenoviral antibodies were determined by ELISA as described
previously (Tripathy et al., Nature Med. 2, 545-550, 1996) except
that peroxidase-conjugated rabbit anti-monkey IgG (Accurate
chemical & Scientific, Westbury, N.Y.) was used instead of
alkaline phosphatase conjugated goat anti-mouse IgG.
EXAMPLE 6
[0056] Dose-Response Relationship, of Long-Term Epo Expression
Following IM Injection of Mice with AdmEpo.
[0057] Previous studies have demonstrated stable dose-dependent
elevations of hematocrits and serum Epo levels following a single
IM injection of 10.sup.7, 10.sup.8, or 10.sup.9 pfu of Epo-encoding
RDAd in immuno-compromised SCID mice (Tripathy et al., Proc. Natl.
Acad. Sci. USA, 91, 11557-11561, 1994). To determine if a similar
dose-response relationship existed in adult immunocompetent CD1
mice, and whether the persistence of Epo transgene expression was
dependent upon the dose of virus administered, we injected adult
CD1 mice IM with 10.sup.8 or 10.sup.9 pfu of AdnEpo or with
10.sup.9 pfu of a control virus, AdBgl II, that lacks the mEpo
transgene. As expected, mice injected with the AdBgl II control
virus showed no significant change in their hematocrits from the
normal pre-injection values of 52.+-.1.5%. In contrast, mice
injected with 10.sup.9 pfu AdmEpo displayed significant elevations
in hematocrits from pre-injection values of 49.+-.0.9% to levels of
81.+-.3% 84 days after injection (p<0.0001). Interestingly,
unlike SCID mice which displayed stable elevations in hematocrits
after injection of 10.sup.7, 10.sup.8 or 10.sup.9 pfu of AdEpo
vectors (Tripathy et al., Proc. Natl. Acad. Sci. USA. 91,
11557-11561, 1994), immunocompetent CD1 mice injected with 10.sup.8
pfu of AdmEpo showed only a transient rise in hematocrit to
65.+-.1.6% at 14 days after injection followed by a return to
baseline values of approximately 51.+-.1.0% by 21 days
post-injection. In additional experiments using a range of virus
doses, we demonstrated that it is necessary to inject mice with at
least 7.times.10.sup.8pfu of AdmEpo to produce persistent Epo
expression in the blood for 90 days. Increasing doses of AdmEpo
between 7.times.10.sup.8 pfu and 3.times.10.sup.9 pfu resulted in
dose-dependent elevations in hematocrits in CD1 mice.
[0058] To ensure that the observed changes in hematocrits
accurately reflected changes in serum Epo levels in these mice, a
radioimmunoassay was used to directly measure serum Epo levels in
animals from each of the three experimental groups. Mice injected
with 10.sup.8 pfu of AdmEpo displayed transient elevations of serum
Epo levels to 13 mU/ml one week after injection followed by a
return to baseline levels (<5 mU/ml) at 14 days post-injection.
In contrast, serum Epo levels in the mice injected with 10.sup.9
pfu AdmEpo peaked at 91 mU/ml one week after injection, then
declined to 26 mU/ml at 14 days post injection with persistence of
this level throughout the remainder of the 84 day experiment. Thus,
the changes in hematocrits observed in these mice reflect
persistently elevated serum Epo levels. Because the half-life of
Epo in the serum is approximately 4 hours, these results also
demonstrated the persistence of Epo expression for at least 84 days
following a single IM injection of 10.sup.9 pfu of AdmEpo. When
taken together, these experiments demonstrated that unlike SCID
mice, which displayed dose-dependent elevations in hematocrits
following injection with as little as 10.sup.7 pfu of Epo-encoding
vectors (Tripathy et al., Proc. Natl. Acad. USA. 91, 11557-11561,
1994), the immunocompetent.CD1 animals required higher doses of
virus to produce sustained, levels of transgene-encoded Epo
expression in the systemic circulation.
EXAMPLE 7
[0059] Persistent Expression of Physiological Levels of mEpo in the
Systemic Circulation of Mice for More than One Year Following a
Single IM Injection of AdmEpo.
[0060] Previous studies have demonstrated persistent elevations in
hematocrits in CD1 mice for at least 112 days following a single IM
injection of 1-3.times.10.sup.9 pfu of AdmEpo. Given the potential
difficulties with re-administration of adenovirus to vectors
following an initial infection (due to the generation of
neutralizing antibodies) (Barr et al., Gene Ther. 2, 151-155, 1995;
Kass et al., Gene Ther. 1, 395-402, 1994; Mastrangeli et al., Hum.
Gene Ther. 7, 79-87, 1996; Tripathy et al., Nature Med. 2, 545-550,
1996; Yang et al. 1996), it was of interest to determine the
longevity of transgene expression following a single IM injection
of AdmEpo. Moreover, given the differences in immune responses in
different strains of inbred mice, it was important to evaluate the
persistence of transgene expression in different mouse strains.
Accordingly, adult C57BL/6, C3H, CD1, and BalbC mice were injected
once IM with 2-3.times.10.sup.9 pfu of AdmEpo and hematocrits were
followed serially for one year. All strains of mice demonstrated
persistent elevations in hematocrits which were stable for at least
one year. In contrast, control mice injected IM with a vector
lacking the mEpo transgene displayed no significant changes in
hematocrits over the time course of the experiment. These
experiments show that a single IM injection of an RDAd encoding a
self transgene (mEpo) can result in stable expression of
physiological levels of Epo in multiple strains of mice for at
least one year.
EXAMPLE 8
[0061] Elevations of Hematocrits and Serum Epo Levels Following a
Single IM Injection of AdsEpo in Cynomolgus Monkeys.
[0062] The results of the mouse experiments described above
demonstrated long term Epo expression in a small rodent model
following a single IM injection of an RDAd encoding a self
transgene (mEpo). However, before this approach could be considered
for use in human gene therapy, it was important to confirm its
efficacy and safety in a large animal model, preferably a
primate.
[0063] It is essential to use RDAd encoding self transgenes in
pre-clinical trials in order to avoid cellular and humoral immune
responses directed against foreign transgene products (Tripathy et
al. 1996a). Accordingly, for trials in non-human primates, AdsEpo,
an E1- and E3-deleted RDAd containing the cynomolgus simian Epo
cDNA under the transcriptional control of the EF1 promoter and 4F2
HC first intron enhancer was made. AdsEpo was shown to program high
level expression of sEpo following infection of C2C12 myoblasts in
vitro. Adult Cynomolgus monkeys were injected once IM with
4.times.10.sup.10 or 4.times.10.sup.11 pfu of AdsEpo. These doses
were calculated based upon the ratios of the body weights of mice
and monkeys: the average weight of a mouse is 25 g and the weight
of our monkeys was approximately 5 kg. Therefore, a dose of
2.times.10.sup.9 pfu in a mouse would be equivalent to a dose of
4.times.10.sup.11 in a monkey while a dose of 2.times.10.sup.8 in a
mouse is equivalent to a dose of 4.times.10.sup.10 in a monkey. A
control monkey received a single IM injection of 4.times.10.sup.11
pfu of the AdBgl II virus.
[0064] The control monkey which was injected with the AdBglII virus
did not demonstrate a significant change in hematocrit from the
pre-injection values of 40%. The monkey injected with
4.times.10.sup.10 pfu of AdsEpo showed a rise in hematocrit to a
peak level of 61% at one month post-injection and a subsequent
gradual decline to 49% by day 84, the end of the experiment. In
contrast, the monkey injected with 4.times.10.sup.11 pfu of AdsEpo
displayed an elevation of hematocrit to 71% by 56 days post
injection. In order to avoid cerebral thrombotic events, the
protocol specified weekly phlebotomy of all animals with
hematocrits of =65%. Accordingly, this animal was phlebotomized 50
ml (approximately 10% of its blood volume) at days 56, 63, 70, and
77. Despite this repeated phlebotomy, the animal demonstrated
stable hematocrits of =65% throughout the remainder of the
experiment. Measurement of serum Epo levels at the end of the
experiment were in accord with the observed hematocrits: the animal
injected with 4.times.10.sup.10 pfu of AdsEpo whose hematocrit was
mildly elevated had a serum Epo level of 4.4.+-.0.3 mU/ml, 13-fold
elevated over the control level of 0.34.+-.0.15 mU/ml. In contrast,
the animal injected with 4.times.10.sup.11 pfu of AdsEpo had a
serum Epo level of 52.7.+-.2 mU/ml, 150-fold higher than the animal
injected with AdBglII (p<0.0001).
EXAMPLE 9
[0065] Safety of AdsEpo Infection in Non-Human Primates.
[0066] Intravenous and inhaled administration of first generation
adenovirus vectors have been associated with hepatic toxicity and
pulmonary inflammation, respectively (Brody et al., Hum. Gene Ther.
5, 821-836, 1994; Yang et al., Proc. Natl. Acad. Sci. USA 91,
4407-4411, 1994; Yang et al., J. Immunol. 155, 2564-2570, 1995). To
assess the safety of IM injection of AdsEpo, chest X-rays, serum
chemistries, hematologic profiles, and clotting parameters were
monitored in the monkeys receiving IM injections of both AdsEpo and
AdBglII. In addition, all three animals underwent necropsy at the
end of the experiment which was performed by an independent
pathologist. Chest x-rays were normal in all animals at 1 and 4
weeks after infection as were lung histologies at necropsy. Thus,
there was no evidence of pulmonary toxicity in any of the animals.
The monkey that received 4.times.10.sup.11 pfu of AdBglII displayed
a rise in ALT to 2-4-fold over the normal range between 1 and 4
weeks after injection. This elevated serum ALT level returned to
normal by the end of the 84 day experiment and was not accompanied
by elevations of GGT, bilirubin, or alkaline phosphatase. Moreover,
no significant elevations in LFTs were seen in either of the
monkeys injected with AdsEpo. Consistent with these findings, none
of the monkeys displayed liver pathology at necropsy 84 days after
injection. However, it should be noted that these studies might
have missed hepatic inflammation occurring at earlier times after
injection, an important consideration given previous findings that
hepatic inflammation is maximal 7-14 days after IV injection of
first generation RDAd (Yang et al. 1994). Nevertheless, the lack of
significant elevations of liver enzymes throughout the experiment
when taken together with the normal liver histologies seen 84 days
after infection show that IM injection of RDAd is not associated
with significant long-term hepatotoxicity. Histological examination
of muscle at the site of adenovirus injection did not reveal
significant pathology 84 days after. Consistent with this
observation, serum CPKs were normal in all three animals at 1 and
12 weeks after injection. Thus, the protocol was not associated
with detectable muscle toxicity. With the exception of a single
elevated platelet count seen in the monkey injected with
4.times.10.sup.10 of AdsEpo, abnormalities in serum chemistries,
hematologic profiles (aside from elevated hematocrits) or clotting
parameters were not detected in any of the animals. In both animals
injected with AdsEpo, necropsy revealed an increase in the bone
marrow cellularity of the erythroid series. There were no other
significant findings in any of the animals on necropsy. Taken
together, these experiments demonstrated the relative safety of IM
injection of AdsEpo, at least up to the dose of 4.times.10.sup.11
pfa in a 5 kg monkey.
EXAMPLE 10
[0067] Anti-Adenoviral Antibody Titers Following AdsEpo
Injection.
[0068] Previous studies in rodents have demonstrated that mice
injected IM or IV with first-generation RDAd develop high titers of
neutralizing antibodies that preclude repeated administration of
the viral vector for at least 60-90 days (Barr et al., Gene Ther.
2, 151-155, 1995; Kass et al., Gene Ther. 1, 395-402, 1994;
Mastrangeli et al., Hum. Gene Ther. 7, 79-87, 1996; Tripathy et
al., Nature Med. 2, 545-550, 1996; Yang et al., Gene Ther. 3,
137-144, 1996). Indeed, an IM injection of 10.sup.9 PFU of AdmEpo
into mice that had received 10.sup.8 pfu of AdmEpo 9 months
previously was unable to produce further increases in hematocrits.
To determine the extent of the anti-adenoviral humoral immune
response following IM injection of Cynomolgus monkeys with AdsEpo,
sera from all three animals were assayed for the presence of
anti-adenoviral antibodies by ELISA. In the animal injected with
4.times.10.sup.11 pfu of AdBgl or AdsEpo, the titer of
anti-adenoviral antibodies increased 200-fold from pre-injection
values. In contrast, the animal injected with 4.times.10.sup.10 pfu
of AdsEpo displayed a 20-fold rise in titer of anti-adenoviral
antibodies. Thus, based upon small numbers of primates, there
appeared to be a dose-dependent humoral response to the injected
adenovirus.
Sequence CWU 1
1
8 1 31 DNA Artificial Sequence primer 1 ggggtcgacg gcggggagat
gggggtgccc g 31 2 32 DNA Artificial Sequence primer 2 gggagatcta
gttcacctgt cccctctcct gc 32 3 18 DNA Artificial Sequence primer 3
ccagaccccg aagcatgg 18 4 18 DNA Artificial Sequence primer 4
ggaagactta aggcagcg 18 5 24 DNA Artificial Sequence primer 5
gaagtcaggc tacgtagacc actg 24 6 20 DNA Artificial Sequence primer 6
gtctgagcag tactcgttgc 20 7 28 DNA Artificial Sequence primer 7
ggggggatcc gcacctggtc atctgtcc 28 8 30 DNA Artificial Sequence
primer 8 gggaagcttc ccggccaggc gcggagatgg 30
* * * * *