U.S. patent application number 09/174508 was filed with the patent office on 2001-07-05 for methods of administering adenoviral vectors.
Invention is credited to BRUDER, JOSEPH T., KOVESDI, IMRE.
Application Number | 20010006947 09/174508 |
Document ID | / |
Family ID | 22636422 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006947 |
Kind Code |
A1 |
BRUDER, JOSEPH T. ; et
al. |
July 5, 2001 |
METHODS OF ADMINISTERING ADENOVIRAL VECTORS
Abstract
The present invention provides methods for administering an
adenoviral gene transfer vector comprising an exogenous gene to an
animal. One method involves utilizing systemic neutralizing
antibodies to neutralize the adenoviral gene transfer vector
outside a targeted muscle. Another method involves the repeat
administration of an adenoviral gene transfer vector to a skeletal
muscle.
Inventors: |
BRUDER, JOSEPH T.;
(FREDERICK, MD) ; KOVESDI, IMRE; (ROCKVILLE,
MD) |
Correspondence
Address: |
JOHN KILYK
LEYDIG VOIT & MAYER LTD
SUITE 4900 TWO PRUDENTIAL PLAZA
180 NORTH STETSON
CHICAGO
IL
606016780
|
Family ID: |
22636422 |
Appl. No.: |
09/174508 |
Filed: |
October 16, 1998 |
Current U.S.
Class: |
514/44R ;
424/184.1; 424/93.1; 435/320.1 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 2039/505 20130101; C07K 16/081 20130101; A61P 37/02 20180101;
A61P 43/00 20180101 |
Class at
Publication: |
514/44 ;
424/93.1; 424/184.1; 435/320.1 |
International
Class: |
A61K 048/00; A61K
031/70 |
Claims
What is claimed is:
1. A method of targeting a gene product to a particular muscle of
an animal, said method comprising (a) inducing in an animal
systemic neutralizing antibodies to an adenoviral gene transfer
vector, (b) administering said adenoviral gene transfer vector
comprising an exogenous gene encoding a gene product to a
particular muscle of said animal such that said exogenous gene is
expressed and said gene product is produced in said particular
muscle of said animal, and (c) neutralizing said adenoviral gene
transfer vector outside of said particular muscle of said
animal.
2. The method of claim 1, wherein said animal is a mammal.
3. The method of claim 2, wherein said mammal is a human.
4. The method of claim 1, wherein said systemic neutralizing
antibodies to said adenoviral gene transfer vector are produced by
administration of an antigen.
5. The method of claim 4, wherein said antigen is the same as said
adenoviral gene transfer vector, except that it does not contain an
exogenous gene encoding a gene product.
6. The method of claim 4, wherein said antigen is the same as said
adenoviral gene transfer vector.
7. The method of claim 4, wherein said antigen is administered to
said animal systemically.
8. The method of claim 1, wherein said neutralizing of said
adenoviral gene transfer vector outside said particular muscle of
said animal is a result of the presence of said neutralizing
antibodies.
9. The method of claim 1, wherein said neutralizing of said
adenoviral gene transfer vector outside said particular muscle of
said animal is such that said production of said gene product is at
least 90% less than the production of said gene product outside
said particular muscle of a naive animal of the same species as
said animal after administration of said adenoviral gene transfer
vector.
10. The method of claim 9, wherein said neutralizing of said
adenoviral gene transfer vector outside said particular muscle of
said animal is such that said production of said gene product is at
least 99% less than the production of said gene product outside
said particular muscle of a naive animal of the same species as
said animal after administration of said adenoviral gene transfer
vector.
11. The method of claim 10, wherein said neutralizing of said
adenoviral gene transfer vector outside said particular muscle of
said animal is such that said production of said gene product is at
least 99.9% less than the production of said gene product outside
said particular muscle of a naive animal of the same species as
said animal after administration of said adenoviral gene transfer
vector.
12. A method of producing a gene product in a skeletal muscle of an
animal, said method comprising (a) administering an adenoviral
vector to said skeletal muscle of said animal, and (b) at least
seven days after said administration, administering an adenoviral
gene transfer vector comprising an exogenous gene encoding a gene
product to said skeletal muscle of said animal such that said
exogenous gene is expressed and said gene product is produced in
said skeletal muscle of said animal.
13. The method of claim 12, wherein said animal is a mammal.
14. The method of claim 13, wherein said mammal is a human.
15. The method of claim 12, wherein said adenoviral vector in step
(a) is said adenoviral gene transfer vector comprising an exogenous
gene encoding a gene product in step (b).
16. The method of claim 15, wherein production of said gene product
in said skeletal muscle of said animal as a result of said second
intramuscular administration of said adenoviral gene transfer
vector is at least 10% of the production of said gene product as a
result of said first intramuscular administration of said
adenoviral gene transfer vector comprising an exogenous gene
encoding a gene product.
17. The method of claim 16, wherein production of said gene product
in said skeletal muscle of said animal as a result of said second
intramuscular administration of said adenoviral gene transfer
vector is at least 50% of the production of said gene product as a
result of said first intramuscular administration of said
adenoviral gene transfer vector comprising an exogenous gene
encoding a gene product.
18. The method of claim 17, wherein production of said gene product
in said skeletal muscle of said animal as a result of said second
intramuscular administration of said adenoviral gene transfer
vector is at least 80% of the production of said gene product as a
result of said first intramuscular administration of said
adenoviral gene transfer vector comprising an exogenous gene
encoding a gene product.
19. The method of claim 18, wherein production of said gene product
in said skeletal muscle of said animal as a result of said second
intramuscular administration of said adenoviral gene transfer
vector is at least substantially the same as the production of said
gene product as a result of said first intramuscular administration
of said adenoviral gene transfer vector comprising an exogenous
gene encoding a gene product.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to methods useful in the
administration of gene products to animals using adenoviral
vectors.
BACKGROUND OF THE INVENTION
[0002] Modified viruses have proven convenient vector systems for
investigative and therapeutic gene transfer applications, and
adenoviral vector systems present several advantages for such uses.
Adenoviruses are generally associated with benign pathologies in
humans, and the 36 kb of the adenoviral genome has been extensively
studied. Adenoviral vectors can be produced in high titers (e.g.,
about 10.sup.13 pfu), and such vectors can transfer genetic
material to nonreplicating, as well as replicating, cells (in
contrast with, for example, retroviral vectors which only transfer
genetic material to replicating cells). The adenoviral genome can
be manipulated to carry a large amount of exogenous DNA (up to
about 8 kb), and the adenoviral capsid can potentiate the transfer
of even longer sequences (Curiel et al., Hum. Gene Ther., 3,
147-154 (1992)). Additionally, adenoviruses generally do not
integrate into the host cell chromosome, but rather are maintained
as a linear episome, thus minimizing the likelihood that a
recombinant adenovirus will interfere with normal cell function.
Aside from being a superior vehicle for transferring genetic
material to a wide variety of cell types, adenoviral vectors
represent a safe choice for gene transfer, a particular concern for
therapeutic applications.
[0003] A variety of recombinant adenoviral vectors have been
described. Most of the vectors in use today derive from the
adenovirus serotype 5 (Ad5), a member of subgroup C. An exogenous
gene of interest typically is inserted into the early region 1 (E1)
of the adenovirus. Disruption of the E1 region decreases the amount
of viral proteins produced by both the early regions (DNA binding
protein) and late regions (penton, hexon, and fiber proteins),
preventing viral propagation. These replication deficient
adenoviral vectors require growth in either a complementary cell
line or in the presence of an intact helper virus, which provides,
in trans, the essential E1 functions (Berker et al., J. Virol., 61,
1213-1220 (1987); Davidson et al., J. Virol., 61, 1226-1239 (1987);
Mansour et al., Mol. Cell Biol., 6, 2684-2694 (1986)). More
recently, adenoviral vectors deficient in both E1 and the early
region 4 (E4) have been used to substantially abolish expression of
viral proteins. In order to insert the larger genes (up to 8 kb)
into the adenoviral genome, adenoviral vectors additionally
deficient in the nonessential early region 3 (E3) are used.
Multiply deficient adenoviral vectors are described in published
PCT patent application WO 95/34671.
[0004] One limitation of adenoviral vector systems is the ability
of the adenoviral vector to transduce a wide variety of
proliferating and quiescent cells (Michou et al., Gene Ther., 4,
473-482 (1997)). This ability, while a benefit in transducing the
target area, is a limitation when the adenoviral vector "leaks" out
of the targeted area and transduces other cells it contacts.
Tranduction of the surrounding cells is a severe problem when the
gene product encoded by the adenoviral vector is harmful, toxic, or
otherwise undesirable with respect to these non-targeted areas.
[0005] Another limitation of the adenoviral vector system is the
cellular and humoral immune response generated within the host
animal. Initial administration elicits a reaction from both
CD8.sup.+ and CD4.sup.+ T cell lymphocytes which eliminate virus
infected cells within 28 days after infection, limiting the
duration of the transgene expression. In addition, neutralizing
antibodies produced by B lymphocytes in cooperation with CD4.sup.+
cells inhibit the effectiveness of a repeat administration of the
adenoviral vector. Proliferation and specificity of the antibodies
is achieved through interactions between the adenoviral vector,
B-cell surface immunoglobulins and activated CD4.sup.+ surface
proteins (particularly CD40Li, which binds CD40 on the surface of
the B cell) (Yang et al., J. Virol., 69, 2004 (1995)).
[0006] Attempts to circumvent the humoral immune response to allow
repeat administration of the adenoviral vector have met with
limited success. These attempts have been focused in two areas,
immunosuppression and alteration of the adenoviral vector. Several
groups have experimented with various immunosuppressant drugs or
antibodies specific for CD4.sup.+, CD40 ligand, or CTLA4Ig to
reduce the adenovirus-specific humoral immune response (Lee et al.,
Hum. Gene Ther., 7, 2273 (1996) (CD4.sup.+); Yang et al., J.
Virol., 70, 6370 (1996) (CD40 ligand); Kay et al., Nature Gen., 11,
191 (1995) (CTLA4Ig)). Although some of these results have been
encouraging, there is a substantial risk associated with systemic
immune suppression in a clinical setting.
[0007] In another study, subretinal administration of an adenoviral
vector containing the bacterial .beta.-galactosidase gene resulted
in minimal circulating antibodies specific to the adenoviral
vector. This was most likely a reflection of the immune privileged
status of the retina. Although there was minimal retinal toxicity
to the adenovirus, several of the animals injected developed
localized granulomatous infiltrate at the injection site (Bennett
et al., Hum. Gene Ther., 7, 1763-1769 (1996)). Subretinal
administration is not an option for many applications where
adenoviral vectors are employed.
[0008] Alteration of the adenoviral vector is time consuming and
has not been entirely successful in sufficiently attenuating the
immune response. Limited readministration of the adenoviral vector
has been accomplished when adenoviral vectors of different
serotypes within the same subgroup are used; however, persistence
of expression of the transgene was not comparable to the initial
administration (Mack et al., Hum. Gene Ther., 8, 99-109
(1997)).
[0009] Accordingly, there is a need for improved methods of
administering adenoviral vectors to animals, particularly, to
prevent leakage of the adenoviral vector from the target area and
to circumvent the humoral immune response elicited by adenoviral
vectors. The present invention provides such methods. This and
other advantages of the present invention, as well as additional
inventive features, will be apparent from the description of the
invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides a method of targeting a gene
product in a particular muscle of an animal. The method utilizes
systemic neutralizing antibodies to neutralize an adenoviral gene
transfer vector containing an exogenous gene outside the particular
muscle. The adenoviral gene transfer vector is administered such
that the exogenous gene is expressed and the gene product is
produced only in the particular muscle of administration.
[0011] The present invention further provides a method of producing
a gene product in a skeletal muscle of an animal. The method
comprises a first intramuscular administration of an adenoviral
vector to the skeletal muscle of an animal, and a second
administration of an adenoviral gene transfer vector containing an
exogenous gene encoding a gene product. Administration is such that
the exogenous gene is expressed and the gene product is produced in
the skeletal muscle of the animal.
[0012] The invention may best be understood with reference to the
accompanying drawings and in the following detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram depicting the original
adenovirus used to derive the adenoviral vectors AdCMVNull and
AdCMV.Z, the regions of addition and deletion of the original
adenovirus, and the expression cassettes of the adenoviral vectors
AdCMVNull and AdCMV.Z.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides methods useful in the
administration of gene products to animals using adenoviral gene
transfer vectors. The ability to target an adenoviral vector and to
repeatedly administer a therapeutic adenoviral vector in a clinical
setting is useful in improving treatment efficacy and in enabling
the treatment of diseases. This invention provides a method to
limit the infection of non-target tissue following administration
of an adenoviral vector to a particular muscle of an animal. The
vector targeting potential is useful for cardiac, particularly,
endocardial, administration, as the risk of misinjection of the
adenoviral vector is high. As adenoviral vectors cannot be
readministered systemically, the present invention also provides a
method for repeat administration of an adenoviral gene transfer
vector comprising an exogenous gene to the skeletal muscle of an
animal.
[0015] The term "exogenous gene", as it is used herein, refers to
any gene in an adenoviral gene transfer vector which is not native
to the adenovirus which comprises the adenoviral vector. The gene
includes a nucleic acid sequence encoding a gene product operably
linked to a promoter. Any portion of the gene can be non-native to
the adenovirus which comprises the adenoviral vector. For example,
the gene can comprise a non-native nucleic acid sequence encoding a
gene product which is operably linked to a native promoter. It
should be appreciated that the exogenous gene can be any gene
encoding an RNA or protein of interest to the skilled artisan.
Therapeutic genes, genes encoding a protein that is to be studied
in vitro and/or in vivo, genes encoding anti-sense RNA's, and
modified viral genes are illustrative of possible exogenous
genes.
[0016] The term "adenoviral gene transfer vector", as it is used
herein, refers to any replication incompetent adenoviral vector
with an exogenous gene encoding a gene product inserted into its
genome. The vector must be capable of replicating and being
packaged when any deficient essential genes are provided in trans.
An adenoviral vector desirably contains at least a portion of each
terminal repeat required to support the replication of the viral
DNA, preferably at least about 90% of the full ITR sequence, and
the DNA required to encapsidate the genome into a viral capsid.
Many suitable adenoviral vectors have been described in the
art.
[0017] In one embodiment, the present invention provides a method
of targeting a gene product to a muscle of an animal using an
adenoviral gene transfer vector containing an exogenous gene
encoding a gene product. Systemic neutralizing antibodies to a
particular adenoviral gene transfer vector are first induced in the
animal. The adenoviral vector is then administered to a particular
muscle of an animal such that the exogenous gene encoded by the
adenoviral vector is expressed and the gene product produced in the
particular muscle of the animal. In addition, the adenoviral vector
is neutralized outside the muscle of administration.
[0018] The present invention can be practiced with any suitable
animal, preferably a mammal, more preferably, a human.
Additionally, the adenoviral vector can be administered to any
suitable muscle of the animal; however, it is preferably
administered to the heart.
[0019] Any suitable method can be used to induce systemic
neutralizing antibodies to the adenoviral vector. Desirably, an
antigen is administered to the animal. This antigen can be the
adenoviral gene transfer vector, but preferably, it is an identical
adenoviral vector, except without an exogenous gene (an AdCMVNull
vector, an example of which can be found in FIG. 1). The antigen
can also be administered by any suitable method. Depending on the
antigen, administration can be to any suitable area of the animal.
In order to induce the systemic neutralizing antibodies, the
antigen can be administered any number of suitable times, e.g.,
once, twice, or more.
[0020] Using the AdCMVNull vector administration, the antigen can
be administered systemically (rather than to the target muscle) to
prevent any damage to the particular muscle. Systemic
administration can be accomplished through intravenous injection,
either bolus or continuous, or any other suitable method. An added
benefit of systemic administration is that it requires a much
smaller amount of antigen to produce the same levels of circulating
antibodies as administration to any muscle of the animal.
[0021] Administration of the antigen produces circulating
neutralizing antibodies. While not wishing to be bound by any
particular theory, it is believed that when the adenoviral gene
transfer vector is administered to the particular muscle of the
animal, some of the adenoviral particles escape the muscle. These
adenoviral particles are then neutralized by the antibodies
circulating throughout the animal such that significantly less (and
preferably substantially no) gene product is produced outside the
particular muscle. The amount of exogenous gene product produced
outside the area of administration is preferably at least 10% less
(more preferably at least 50% less, and most preferably at least
80% less) than production of the gene product outside the
particular muscle of administration in a naive animal, which does
not have circulating neutralizing antibodies to the adenoviral gene
transfer vector.
[0022] Neutralization of adenoviral particles outside of the
particular muscle prevents production of the exogenous gene carried
in the adenoviral gene transfer vector. This is extremely useful in
situations where the exogenous gene is harmful, or toxic, to the
animal when present in areas other than the particular muscle of
administration. An example of this is vascular endothelial growth
factor (VEGF protein), which mediates vascular growth. While
vascular growth is desirable in the heart to repair damaged cardiac
muscle, growth outside the heart can lead to severe problems,
including blindness, and increased aggressiveness of tumor
cells.
[0023] In another embodiment, the present invention provides a
method of producing a gene product in a skeletal muscle. An
adenoviral vector is first administered to the skeletal muscle of
an animal. An adenoviral vector containing an exogenous gene
encoding a gene product is then administered to the same skeletal
muscle such that the exogenous gene is expressed and the gene
product is produced in the skeletal muscle. Any suitable animal can
be used; however, preferably, the animal is a mammal, more
preferably, a human.
[0024] After the second or subsequent administration of the
adenoviral gene transfer vector, production of the gene product in
the muscle of the animal is desirably at least 1% of (such as at
least 10% of, preferably at least 50% of, more preferably at least
80% of, and most preferably, substantially the same as) production
of the gene product after a first or preceding administration with
the same adenoviral gene transfer vector containing the exogenous
gene encoding the gene product. While not wishing to be bound by
any particular theory, it is believed that the level of gene
product produced in the skeletal muscle of an animal after the
second or subsequent administration to the muscle can be
substantially similar to that of the first or preceding
administration because neutralizing antibodies, which are produced
by the first or preceding administration, cannot readily penetrate
the muscle and destroy the adenoviral gene transfer vector. This
holds true even when the neutralizing antibody response is boosted
with two or more initial administrations before the final or
subsequent intramuscular administration of the adenoviral gene
transfer vector containing the exogenous gene encoding the gene
product.
[0025] To facilitate the administration of adenoviral vectors, they
can be formulated into suitable pharmaceutical compositions.
Generally, such compositions include the active ingredient (i.e.,
the adenoviral vector) and a pharmacologically acceptable carrier.
Such compositions can be suitable for delivery of the active
ingredient to a patient for medical application, and can be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0026] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in a conventional manner using
one or more pharmacologically or physiologically acceptable
carriers comprising excipients, as well as optional auxiliaries
which facilitate processing of the active compounds into
preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen. Thus, for
injection, the active ingredient can be formulated in aqueous
solutions, preferably in physiologically compatible buffers. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art. For oral administration, the active
ingredient can be combined with carriers suitable for inclusion
into tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like. For administration by
inhalation, the active ingredient is conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant. The active
ingredient can be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion. Such
compositions can take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and can contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Other pharmacological excipients are known in the art.
[0027] The present inventive methods are useful in the context of
the treatment of animals, e.g., medical treatment. In addition, the
present inventive methods are useful in the production of gene
products, e.g., in vivo protein production (which can entail
subsequent protein recovery) as well as in research, e.g.,
investigation of gene expression, adenoviral targeting, and the
like.
EXAMPLE
[0028] The present invention is further described in the following
example. This example serves only to illustrate the invention and
is not intended to limit the scope of the invention in any way.
[0029] This example illustrates use of the present inventive method
of targeting production of a gene product to a particular muscle in
an animal, as well as the present inventive method of repeat
administration to produce a gene product in a skeletal muscle of an
animal. In particular, systemic neutralizing antibodies to an
adenoviral vector were induced in an animal, and then the
adenoviral vector comprising an exogenous gene encoding a gene
product was administered to a particular muscle of the animal such
that the exogenous gene was expressed and the gene product was
produced in the particular muscle of the animal. In addition, the
adenoviral vector was neutralized outside of the particular muscle
of the animal such that there was limited expression of the
exogenous gene resulting in production of the gene product outside
of the particular muscle of the animal.
[0030] For the purposes of this experimental work, C57B16 mice were
used as the test animals because their immune system is able to
recognize adenoviral vectors as foreign antigens and mount a
sufficient immune response to destroy the adenoviral vectors,
thereby preventing expression of an exogenous gene forming a part
of the adenoviral vector. The mice were separated into three
groups. Systemic neutralizing antibodies were induced in the mice
of group 1 with an adenoviral vector which did not contain an
exogenous gene encoding a gene product (AdCMVNull). A similar
adenoviral vector (AdCMV.Z), with a gene expression cassette
encoding a reporter gene product (i.e., a gene product that could
be readily detected), was administered to the mice of groups 1 and
2 to determine whether production of the reporter gene product
.beta.-galactosidase (.beta.-gal) was limited to the right
gastrocnemius muscle or could be detected in other areas of the
mice, particularly the liver inasmuch as adenoviral vectors are
known to localize in the liver after entering the bloodstream of an
animal (Jaffee et al., Nat. Genet., 1, 372-78 (1992)). The mice of
group 2 were treated as a naive group. Only the adenoviral vector
AdCMV.Z, with a gene expression cassette encoding the reporter gene
product .beta.-gal, was administered intrajugularly to the mice of
group 2, i.e., no adenoviral vector was administered to induce
systemic neutralizing antibodies in the mice before the
administration of the adenoviral vector AdCMV.Z. The mice of group
2 otherwise were treated in the same manner as the mice of group 1.
Finally, a control group, group 3, which did not receive any
administration of adenoviral vectors, was included.
[0031] The AdCMVNull vector was a replication-deficient adenoviral
vector with deletions in the E1 and E3 regions. An expression
cassette was inserted in the E1-deleted region of the adenoviral
vector that included an SV40 polyA sequence and a cytomegaloviral
promoter (CMV). The AdCMVNull vector is depicted in FIG. 1.
[0032] The AdCMV.Z vector was also a replication-deficient
adenoviral vector similar to the AdCMVNull vector, except that the
expression cassette included a nucleic acid sequence encoding the
reporter gene product .beta.-gal operably linked to the CMV
promoter, from left to right, relative to the viral vector. The
AdCMV.Z vector is also depicted in FIG. 1.
[0033] The protocol for administration of the AdCMVNull and AdCMV.Z
vectors to the mice of the two groups was as follows: the mice of
group 1 were immunized with an intramuscular injection of
1.times.10.sup.10 pu of AdCMVNull on day 1 of the experiment, and
received a subsequent intramuscular injection of 1.times.10.sup.10
pu of AdCMV.Z on day 14. The mice of group 2 (the naive mice)
received an injection of 1.times.10.sup.10 pu of AdCMV.Z on day 14.
The mice of group 3 did not receive any injections.
[0034] On day 15, the mice in all three groups were sacrificed. The
.beta.-gal activity in the mice was determined in the liver and
right gastrocnemius muscle. Neutralizing antibody titers also were
determined in the mice. The results of these analyzes are set forth
below in Table 1.
1 TABLE 1 .beta.-galactosidase Neutralizing Activity (RLU/mg
protein) Antibodies Right Gastrocnemius (reciprocal Muscle Liver
dilution) Group 1 1.4447 .times. 10.sup.6 8.0697 .times. 10.sup.3
32 (AdCMVNull) Group 2 4.0748 .times. 10.sup.6 5.2022 .times.
10.sup.6 1.0 (Naive) Group 3 1.0683 .times. 10.sup.4 7.898 .times.
10.sup.3 n/a (Control)
[0035] As is apparent from the experimental results set forth
above, the mice in the first two groups had essentially the same
levels of .beta.-gal activity in the right gastrocnemius muscle,
about 10.sup.6 RLU/mg. The mice of group 3 (the control group) had
a .beta.-gal activity level of about 10.sup.4 RLU/mg. The results
demonstrate that there was gene expression in the targeted muscle,
even in the mice of group 1, which were the subject of the repeat
administration. Moreover, the mice of group 1, in which systemic
neutralizing antibodies were induced, had significantly less
.beta.-gal activity in the liver, about 10.sup.4 (or a hundred-fold
less than measured in the target muscle and approximately the same
as the control), thereby demonstrating that there was localization
of the targeted gene product to the targeted muscle in accordance
with the present invention. In distinct contrast, the mice of group
2, in which neutralizing antibodies were not induced, had
essentially the same level of .beta.-gal activity in the liver,
about 10.sup.6 RLU/mg, as in the targeted muscle, thereby
indicating that in the absence of the present inventive method,
when there is undesirable leaking of the adenoviral vector outside
the targeted muscle, there is wide-spread production of the gene
product of interest.
[0036] All of the references cited herein, including patents,
patent applications, and publications, are hereby incorporated in
their entireties by reference.
[0037] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the claims below.
* * * * *