U.S. patent application number 12/761310 was filed with the patent office on 2010-08-05 for use of rapamycin to inhibit immune response and induce tolerance to gene therapy vector and encoded transgene products.
This patent application is currently assigned to Genzyme Corporation. Invention is credited to Abraham Scaria.
Application Number | 20100196401 12/761310 |
Document ID | / |
Family ID | 26916076 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100196401 |
Kind Code |
A1 |
Scaria; Abraham |
August 5, 2010 |
USE OF RAPAMYCIN TO INHIBIT IMMUNE RESPONSE AND INDUCE TOLERANCE TO
GENE THERAPY VECTOR AND ENCODED TRANSGENE PRODUCTS
Abstract
Disclosed are methods for transient co-administration of
rapamycin together with a gene therapy vector encoding a transgene.
The present invention is directed to inhibiting the immune response
of a host to the administered gene therapy vector and encoded trans
gene product, thus allowing persistent trans gene expression and
repeated administration of the gene therapy product to the host.
The present invention is also of relevance in genetic disease
patients that mount immune responses to protein replacement
therapies in which case the present invention provides for
transient co-administration of rapamycin together with protein
replacement therapy. In a further aspect of the invention,
co-administration of rapamycin could inhibit a secondary immune
response in a host that has been pre-immunized with the gene
therapy vector or pre-immunized with the protein product encoded by
the transgene.
Inventors: |
Scaria; Abraham;
(Framingham, MA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Genzyme Corporation
|
Family ID: |
26916076 |
Appl. No.: |
12/761310 |
Filed: |
April 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11924949 |
Oct 26, 2007 |
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12761310 |
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11312299 |
Dec 20, 2005 |
7307068 |
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11924949 |
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10135164 |
Apr 30, 2002 |
7045508 |
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11312299 |
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09911782 |
Jul 24, 2001 |
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10135164 |
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09876574 |
Jun 7, 2001 |
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09911782 |
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60221738 |
Jul 31, 2000 |
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Current U.S.
Class: |
424/172.1 ;
424/130.1; 514/44R |
Current CPC
Class: |
A61K 38/47 20130101;
A61K 31/435 20130101; A61P 43/00 20180101; A61K 38/37 20130101;
A61K 31/375 20130101; A61K 31/70 20130101; A61K 38/465 20130101;
A61K 31/015 20130101; A61K 38/465 20130101; C12N 2799/021 20130101;
A61K 38/47 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 48/00 20130101; A61K 2300/00 20130101; A61K
31/015 20130101; A61K 38/4846 20130101; A61K 31/435 20130101; A61K
31/375 20130101; A61K 38/4846 20130101; A61K 39/395 20130101; A61K
39/395 20130101; A61K 31/4745 20130101; A61K 38/37 20130101; A61K
31/70 20130101; A61P 37/06 20180101 |
Class at
Publication: |
424/172.1 ;
514/44.R; 424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7088 20060101 A61K031/7088; A61P 37/06
20060101 A61P037/06 |
Claims
1. A method for inhibiting the immune response of a host to a gene
therapy vector and encoded transgene product, said method
comprising co-administering the gene therapy vector with an
effective amount of rapamycin.
2. The method of claim 1, wherein the method further comprises
administering an effective amount of an agent which blocks a
co-stimulation pathway.
3. The method of claim 2, wherein the agent which blocks a
co-stimulation pathway is selected from the group consisting of
CTLA4-Ig, antibodies to B7-I, antibodies to B7-2, antibodies to
CD28, antibodies to CD40L; and combinations of the above.
4. The method of claim 3, wherein the gene therapy vector is an
adenoviral vector containing a deletion of adenoviral gene
sequences.
5. The method of claim 3, wherein the trans gene encodes a protein
selected from the group consisting of glucocerebrosidase,
alpha-galactosidase A, beta-galactosidase, sphingomyelinase,
iduronate sulfatase, alpha-glucosidase and alpha-iduronidase.
6. The method of claim 3, wherein the trans gene encodes a protein
selected from the group consisting of Factor VIIA, Factor VII or
Factor IX.
7. A method for allowing persistent expression of a trans gene,
said method comprising co-administering the gene therapy vector
with an effective amount of rapamycin.
8. The method of claim 7, wherein the method further comprises
administering an effective amount of an agent which blocks a
co-stimulation pathway.
9. The method of claim 8, wherein the agent which blocks a
co-stimulation pathway is selected from the group consisting of
CTLA4-Ig, antibodies to B7-I, antibodies to B7-2, antibodies to
CD28, antibodies to CD40L; and combinations of the above.
10. The method of claim 9, wherein the gene therapy vector is an
adenoviral vector containing a deletion of adenoviral gene
sequences.
11. The method of claim 9, wherein the transgene encodes a protein
selected from the group consisting of glucocerebrosidase,
alpha-galactosidase A, beta-galactosidase, sphingomyelinase,
iduronate sulfatase, alpha-glucosidase and alpha-iduronidase.
12. The method of claim 9, wherein the transgene encodes a protein
selected from the group consisting of Factor VIIA, Factor VIII or
Factor IX.
Description
[0001] The present application is a continuation of co-pending U.S.
Ser. No. 09/911,782 filed on Jul. 24, 2001 which is a continuation
in part of U.S. Ser. No. 09/876,574 filed on Jun. 7, 2001 which
claims priority to U.S. Provisional Application No. 60/221,738,
filed Jul. 31, 2000. The contents of the above-referenced
applications are hereby incorporated by reference into the present
disclosure.
BACKGROUND
[0002] Immunosuppressant drugs have been used for purposes of
preventing adverse immune responses, either a rejection of a
transplanted organ, or an attack on the patients own body by its
own immune system caused by an autoimmune disease, without unduly
suppressing the ability of the patient's immune system to combat
infection. Such immunosuppressants have included rapamycin [U.S.
Pat. No. 5,694,950]; FK 506 [U.S. Pat. No. 5,365,948]; and
cyclosporine.
[0003] With the advent of gene therapy, a need exists for methods
of repeat administration of gene therapy vectors, such as viral
vectors, exists. Methods are needed which are able to effectively
overcome the body's normal immune response to gene therapy vectors
such as viral vectors. In order to overcome the immunologic
problems associated with repeat administration of adenoviral
vectors, the use of broad immunosuppressants (Engelhardt et al.,
Proc. Natl. Acad. Sci. USA 91:6196-6200 (1994)) and cytoablative
agents (Dai et al., Proc. Natl. Acad. Sci. USA 92:1401-1405 (1995))
to overcome the immune response of the host to first generation Ad
vectors have been tested. Transient co-administration of an
immunoglobulin, CTLA4-Ig, along with an intravenous injection of Ad
vector expressing a non-immunogenic transgene product (human oc-I
anti-trypsin) has been shown to lead to persistent transgene
expression from mouse liver (Kay et al., Nat. Genetics 11:191-197
(1995)). CTLA4-Ig blocks the B7-CD28 pathway of T cell
co-stimulation, which is required for optional activation of T
cells. (Jenkins et al., Immunity 1:443-446 (1994); Lenschow et al.,
Ann. Rev. Immunol. 14:233-258 (1996)). Although adenoviral-specific
antibody levels were reduced in CTLA4-Ig treated mice, the
inhibition was not sufficient to allow secondary gene transfer via
repeat administration of the vector under the conditions tested
(Kay et al., Nat. Genetics 11:191-197 (1995)). Thus, many
immunosuppressant molecules are not effective for gene therapy
purposes in which persistent expression of a foreign transgene is
desired. Accordingly, a need exists for methods of employing
immunosuppressant drugs which are effective when used with gene
therapy vectors.
SUMMARY OF INVENTION
[0004] The present invention provides for transient
co-administration of rapamycin together with a gene therapy vector
encoding a transgene. The present invention is directed to
inhibiting the immune response of a host to the administered gene
therapy vector and to the encoded transgene product, thus allowing
persistent transgene expression and repeated administration of the
gene therapy product to the host. The present invention is also of
relevance in genetic disease patients that mount immune responses
to protein replacement therapies, in which case the present
invention provides for transient co-administration of rapamycin
together with protein replacement therapy. In a further aspect of
the invention, co-administration of rapamycin could inhibit a
secondary immune response in a host that has been pre-immunized
with the gene therapy vector or pre-immunized with the protein
product encoded by the trans gene. In preferred embodiments, the
present invention relates to methods and compositions for blocking
signal 2, but not signal 1, of the interaction between major
histocompatibility complex [MHC] on antigen presenting cells [APC]
binding to T-cell receptor [TCR], while at the same time blocking
one or more of the co-stimulation pathways: B7-CD28 and CD40-CD40
ligand. Thus, compositions of the present invention comprise (1) an
agent which blocks signal 2, but not signal I, of the MHCTCR
interaction pathway; (2) an agent which blocks a co-stimulation
pathway; and (3) a therapeutic agent. The agent which blocks signal
2, but not signal 1, of the MHC-TCR interaction is preferably
rapamycin, but may also be a rapamycin analog, an antibody which
binds to the MHC, blocking interaction with TCR, or an antibody to
TCR, provided such antibody to TCR is antagonistic, and does not
activate the T cell to which it binds. The agent which blocks a
co-stimulation pathway is preferably selected from the group
consisting of CTLA4-Ig, antibodies to B7-1, antibodies to B7-2,
antibodies to CD28, and antibodies to CD40L. One antibody to CD40L
which may be used in the present invention as the agent blocking
co-stimulation is MR1. The therapeutic agent is preferably a gene
therapy vector which encodes a therapeutic gene. Suitable gene
therapy vectors include viral vectors, such as adenovirus,
adeno-associated virus, retrovirus, including lentivirus vectors.
Other gene therapy vectors include cationic or amphiphilic
compounds, such as lipids, as well as polymers, liposomes and naked
DNA. Useful therapeutic genes include those encoding lysosomal
storage enzymes, such as glucocerebrosidase, alpha-galactosidase A,
sphingomyelinase, iduronate sulfatase, alpha-glucosidase,
galactosamine-6-sulfatase, beta-galactosidase, galactosamine-4
sulfatase (arylsulfatase B), alpha-glucosidase, and
alpha-iduronidase. Other preferred therapeutic genes include those
useful for the production of hemophilic proteins, most preferably
Factor VIIA, Factor VII and Factor IX. In other embodiments, the
therapeutic agent may be a polypeptide, or a combination of
polypeptide and gene therapy vectors.
BRIEF DESCRIPTION OF THE FIGURES
[0005] FIG. 1 shows the anti-.alpha.-gal antibody titers for
experiments in which Ad2 was co-administered with anti-CD40L and/or
Rapamycin.
[0006] FIG. 2 shows the anti-.alpha.-Ad2 antibody titers for
experiments in which Ad2 was co-administered with anti-CD40L and/or
Rapamycin.
[0007] FIG. 3 shows the transgene expression for experiments in
which Ad2 was co administered with anti-CD40L and/or Rapamycin.
[0008] FIG. 4 shows the anti-.alpha.-gal antibody titers for
experiments in which Ad2 was co-administered with anti-B7-1 and
anti-B7-2 and/or Rapamycin.
[0009] FIG. 5 shows the anti-.alpha.-Ad2 antibody titers for
experiments in which Ad2 was co-administered with anti-B7-1 and
anti-B7-2 and/or Rapamycin.
DETAILED DESCRIPTION OF THE INVENTION
[0010] Rapamycin is a natural product derived from a soil
microorganism, which was originally described as an antibiotic and
subsequently found to possess some immunosuppressive properties.
Rapamycin has recently been approved for use in patients for kidney
transplantation in combination with cyclosporine and
corticosteroids. Recent reports in the literature claim that
rapamycin in combination with co-stimulation blockade can induce
tolerance to allografts in mice.
[0011] The present invention provides for transient
co-administration of rapamycin or an analog or derivative thereof,
together with a gene therapy vector encoding a transgene. The
present invention is directed to inhibiting the immune response of
a host to the administered gene therapy vector and encoded trans
gene product, thus allowing persistent transgene expression and
repeated administration of the gene therapy product to the host.
The present invention is also of relevance in genetic disease
patients that mount immune responses to protein replacement
therapies in which case the present invention provides for
transient co-administration of rapamycin together with protein
replacement therapy. In a further aspect of the invention,
co-administration of rapamycin could inhibit a secondary immune
response in a host that has been pre-immunized with the gene
therapy vector or pre-immunized with the protein product encoded by
the transgene.
[0012] Wherever the present application refers to "rapamycin", in
addition to naturally occurring forms of rapamycin, the present
invention includes the use of rapamycin analogs and derivatives.
Many such analogs and derivatives are known in the art, for
example, including but not limited to those described in U.S. Pat.
Nos. 6,015,809; 6,004,973; 5,985,890; 5,955,457; 5,922,730;
5,912,253; 5,780,462; 5,665,772; 5,637,590; 5,567,709; 5,563,145;
5,559,122; 5,559,120; 5,559,119; 5,559,112; 5,550,133; 5,541,192;
5,541,191; 5,532,355; 5,530,121; 5,530,007; 5,525,610; 5,521,194;
5,519,031; 5,516,780; 5,508,399; 5,508,290; 5,508,286; 5,508,285;
5,504,291; 5,504,204; 5,491,231; 5,489,680; 5,489,595; 5,488,054;
5,486,524; 5,486,523; 5,486,522; 5,484,791; 5,484,790; 5,480,989;
5,480,988; 5,463,048; 5,446,048; 5,434,260; 5,411,967; 5,391,730;
5,389,639; 5,385,910; 5,385,909; 5,385,908; 5,378,836; 5,378,696;
5,373,014; 5,362,718; 5,358,944; 5,346,893; 5,344,833; 5,302,584;
5,262,424; 5,262,423; 5,260,300; 5,260,299;5,233,036; 5,221,740;
5,221,670; 5,202,332; 5,194,447; 5,177,203; 5,169,851; 5,164,399;
5,162,333; 5,151,413; 5,138,051; 5,130,307; 5,120,842; 5,120,727;
5,120,726; 5,120,725; 5,118,678; 5,118,677; 5,100,883; 5,023,264;
5,023,263; 5,023,262; all of which are incorporated herein by
reference.
[0013] In the sections which follow, detailed therapeutic regimens
are provided for combination therapy of eight specific LSDs (i.e.
Gaucher's, Fabry's, Niemann-Pick B, Hunter's, Morquio's,
Maroteaux-Lamy, Pompe's, and Hurler's-Scheie's in its various
clinical manifestations), as well as hemophilic factors Factor
VIIA, Factor VIII and Factor IX.
1. Gaucher's
[0014] Gaucher's disease is caused by inactivation of
glucocerebrosidase and accumulation of glucocerebroside.
2. Fabry'S
[0015] Fabry's disease is caused by inactivation of
alpha-galactosidase A and accumulation of GL-3. The enzymatic
defect leads to systemic deposition of glycosphingolipids with
terminal alpha-galactosyl moieties, predominantly
globotriaosylceramide and, to a lesser extent, galabiosyl ceramide
and blood group B substances. In addition to assay for specific
activity of alpha-galactosidase A and accumulation of GL-3, assay
for deposition of glycosphingolipid substrates in body fluids and
in lysosomes of vascular endothelial, perithelial and smooth muscle
cells of blood vessels. Other manifestations which can be useful
for assay include proteinuria and other signs of renal impairment,
such as red cells or lipid globules in the urine, and elevated
erythrocyte sedimentation rate. Also, anemia, decreased serum iron
concentration, high concentration of beta-thromboglobulin, and
elevated reticulocyte counts or platelet aggregation. Desnick et
al., in Scriver et al., Metabolic and Molecular Bases of Inherited
Disease, (7th ed. 1995) p. 2741-2784.
3. Niemann-Pick B
[0016] Niemann-Pick B disease is caused by inactivation of
sphingomyelinase and accumulation of sphingomyelin.
4. Hunter's
[0017] Hunter's disease (a.k.a. MPSII) is caused by inactivation of
iduronate sulfatase and accumulation of dermatan sulfate and
heparan sulfate. Hunter's disease presents clinically in severe and
mild forms.
5. Morquio's
[0018] Morquio's syndrome (a.k.a. MPS IV) results from accumulation
of keratan sulfate due to inactivation of either of two enzymes. In
MPS IVA the inactivated enzyme is galactosamine-'6sulfatase and in
MPS IVB the inactivated enzyme is beta-galactosidase.
6. Maroteaux-Lamy
[0019] Maroteaux-Lamy syndrome (a.k.a. MPS VI) is caused by
inactivation of galactosamine4-sulfatase (arylsulfatase B) and
accumulation of dermatan sulfate.
7. Pompe's
[0020] Pompe's disease is caused by inactivation of
alpha-glucosidase and accumulation of glycogen. Hers first proposed
the concept of inborn lysosomal disease based on his studies of
type II glycogen storage disease (a.k.a. Pompe's disease, GAA or
acid maltase deficiency (AMD); see H. G. Hers, 1965,
Gastroenterology 48, 625). An assay for accumulated intra lysosomal
accumulation of glycogen granules, particularly in myocardium,
liver and muscle fibers, or serum elevation of CK is described in
Hirschorn, in Scriver et al., Metabolic and Molecular Bases of
Inherited Disease, (7.sup.th ed. 1995) p. 2443-2464.
8. Hurler's-Scheie's
[0021] Hurler's-Scheie's disease, also known as MPS1, is caused by
inactivation of alpha-iduronidase and accumulation of dermatan
sulfate and heparin sulfates, In addition to enzyme assay or by
accumulation of the dermatan and heparan sulfates, assay for the
disease can be by excessive urinary dermatan and heparan sulfate
excretion. Nuefeld and Muenzer, in Scriver et al., Metabolic and
Molecular Bases of Inherited Disease, (7th ed. 1995) p.
2465-2494.
9. Hemophilic Factors
[0022] Other preferred transgenes included genes encoding Factor
VIIA, Factor VII or Factor IX. The Factor VIII gene may be
full-length (see, e.g., U.S. Pat. No. 4,965,199; U.S. Pat. No.
5,618,789); B-domain deleted (see, e.g., U.S. Pat. No. 4,868,112
and U.S. Pat. No. 5,661,008) or a chimeric hybrid (see, e.g., U.S.
Pat. No. 5,563,045; U.S. Pat. No. 5,888,974 and U.S. Pat. No.
5,859,204). The Factor IXgene is preferably of human origin (see,
e.g., U.S. Pat. No. 4,994,371 and U.S. Pat. No. 5,521,070). The
Factor VIIA gene is preferably of human origin (see, e.g., U.S.
Pat. No. 4,456,591; U.S. Pat. No. 4,784,950; U.S. Pat. No.
5,190,919; U.S. Pat. No. 5,254,672 and U.S. Pat. No.
6,039,944).
[0023] Other preferred transgenes include full length cystic
fibrosis transmembrane receptor (CFTR), dystrophin, ornithine
transcarbamylase (OTC), alpha.1I-antitrypsin (A1AT), Rb, and
p53.
[0024] Adenoviral vectors are attractive vehicles for gene transfer
to a wide variety of dividing and non-dividing cells in vivo,
including liver, muscle, lung, brain, heart, etc. However,
transgene expression is usually transient in nature due to the
generation of cellular and humoral immune responses to both Ad
vector proteins and transgene products. The immune response to
adenoviral vector, encoded proteins can be reduced or circumvented
by using deleted partially adenoviral vectors or pseudoadenoviral
vectors (PAV) that are completely deleted of adenoviral genes (also
referred to as fully deleted Ad vectors, mini-adenoviral vectors,
helper dependent Ad vectors or gutless Ad vectors). However, the
problem remains of neutralizing antibodies to Ad capsid proteins
that prevent re-administration of Ad vector of the same serotype.
Adenoviral vectors, such as pseudoadenoviral vectors, retroviral
vectors, adeno associate virus (AAV) vectors or lentiviral vectors
do not encode any viral proteins, however, this does not address
the issue of immunogenicity of the trans gene product, which could
potentially be a neo-antigen in patients with genetic disease that
we wish to treat with these vectors.
Viral Vectors
[0025] One of the most frequently used methods of administration of
gene therapy, both in vivo and ex vivo, is the use of viral vectors
for delivery of the gene. Many species of virus are known, and many
have been studied for gene therapy purposes. The most commonly used
viral vectors include those derived from adenoviruses,
adeno-associated viruses [AAV) and retroviruses, including
lentiviruses, such as human immunodeficiency virus [HIV].
Adenovirus
[0026] Adenoviral vectors for use to deliver transgenes to cells
for applications such as in vivo gene therapy and in vitro study
and/or production of the products of trans genes, commonly are
derived from adenoviruses by deletion of the early region 1 (E1)
genes (Berkner, K. L., Curro Top. Micro. Immunol. 158:39-66, 1992).
Deletion of E1 genes renders such adenoviral vectors replication
defective and significantly reduces expression of the remaining
viral genes present within the vector. However, it is believed that
the presence of the remaining viral genes in adenoviral vectors can
be deleterious to the transfected cell for one or more of the
following reasons: (1) stimulation of a cellular immune response
directed against expressed viral proteins, (2) cytotoxicity of
expressed viral proteins, and (3) replication of the vector genome
leading to cell death.
[0027] One solution to this problem has been the creation of
adenoviral vectors with deletions of various adenoviral gene
sequences. In particular, partially deleted adenoviral vectors
["DeAd" vectors), and pseudoadenoviral vectors (PAVs), also known
as `gutless adenovirus` or mini adenoviral vectors, are adenoviral
vectors derived from the genome of an adenovirus that contain
minimal cis-acting nucleotide sequences required for the
replication and packaging of the vector genome and which can
contain one or more transgenes (See, U.S. Pat. No. 5,882,877 which
covers pseudoadenoviral vectors (PAV) and methods for producing P A
V, incorporated herein by reference). Such PAVs or DeAd vectors,
which can accommodate up to about 36 kb of foreign nucleic acid,
are advantageous because the carrying capacity of the vector is
optimized, while the potential for host immune responses to the
vector or the generation of replication-competent viruses is
reduced. P A V and DeAd vectors contain the 5' inverted terminal
repeat (ITR) and the 3' ITR nucleotide sequences that contain the
origin of replication, and the cis-acting nucleotide sequence
required for packaging of the adenoviral genome, and can
accommodate one or more trans genes with appropriate regulatory
elements, e.g. promoters, enhancers, etc.
[0028] Other adenoviral vectors have been created with the deletion
of certain specific genes, which may be some or all of the
adenoviral early genes, including E1, E2a, E2b [terminal peptide
and DNA polymerase], E3, most or all of the E4 genes, and may also
include some or all of the adenoviral late genes, L1 through
L5.
[0029] Adenoviral vectors, such as DeAd vectors and PAVs, have been
designed to take advantage of the desirable features of adenovirus
which render it a suitable vehicle for delivery of nucleic acids to
recipient cells. Adenovirus is a non-enveloped, nuclear DNA virus
with a genome of about 36 kb, which has been well-characterized
through studies in classical genetics and molecular biology
(Hurwitz, M. S., Adenoviruses Virology, 3rd edition, Fields et al.,
eds., Raven Press, New York, 1996; Hitt, M. M. et al., Adenovirus
Vectors, The Development of Human Gene Therapy, Friedman, T. ed.,
Cold Spring Harbor Laboratory Press, New York, 1999). The viral
genes are classified into early (designated E1-E4) and late
(designated L1-L5) transcriptional units, referring to the
generation of two temporal classes of viral proteins. The
demarcation of these events is viral DNA replication. The human
adenoviruses are divided into numerous serotypes (approximately 47,
numbered accordingly and classified into 6 groups: A, B, C, D, E
and F), based upon properties including hemaglutination of red
blood cells, oncogenicity, DNA and protein amino acid compositions
and homologies, and antigenic relationships.
[0030] Recombinant adenoviral vectors have several advantages for
use as gene delivery vehicles, including tropism for both dividing
and non-dividing cells, minimal pathogenic potential, ability to
replicate to high titer for preparation of vector stocks, and the
potential to carry large inserts (Berkner, K. L., Curr. Top. Micro.
Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64,
1994).
[0031] PAVs and DeAd vectors have been designed to take advantage
of the desirable features of adenovirus which render it a suitable
vehicle for gene delivery. While adenoviral vectors can generally
carry inserts of up to 8 kb in size by the deletion of regions
which are dispensable for viral growth, maximal carrying capacity
can be achieved with the use of adenoviral vectors containing
deletions of most viral coding sequences, including PAVs. See U.S.
Pat. No. 5,882,877 of Gregory et al.; Kochanek et al., Proc. Natl.
Acad. Sci. USA 93:5731-5736, 1996; Parks et al., Proc. Natl. Acad.
Sci. USA 93:13565-13570, 1996; Lieber et al., J. Virol.
70:89448960, 1996; Fisher et al., Virology 217:11-22, 1996; U.S.
Pat. No. 5,670,488; PCT Publication No. WO 96/33280, published Oct.
24, 1996; PCT Publication No. WO 96/40955, published Dec. 19, 1996;
PCT Publication No. WO 97/25446, published Jul. 19, 1997; PCT
Publication No. WO 95/29993, published Nov. 9, 1995; PCT
Publication No. WO 97/00326, published Jan. 3, 1997; Morral et al.,
Hum. Gene Ther. 10:2709-2716, 1998.
[0032] Since P A V s and DeAd vectors are deleted for part or most
of the adenovirus genome, production of PAVs, requires the
furnishing of adenovirus proteins in trans which facilitate the
replication and packaging of a PAV genome into viral vector
particles. Most commonly, such proteins are provided by infecting a
producer cell with a helper adenovirus containing the genes
encoding such proteins. However, such helper viruses are potential
sources of contamination of a PAV or DeAd virus stock during
purification and can pose potential problems when administering the
adenovirus to an individual if the contaminating helper adenovirus
can replicate and be packaged into viral particles.
[0033] It may be advantageous to increase the purity of a PAV or
DeAd virus stock by reducing or eliminating any production of
helper vectors which can contaminate preparation. Several
strategies to reduce the production of helper vectors in the
preparation of a DeAd virus or P A V stock are disclosed in U.S.
Pat. No. 5,882,877, issued Mar. 16, 1999; U.S. Pat. No. 5,670,488,
issued Sep. 23, 1997 and International Patent Application No.
PCT/US99/03483, incorporated herein by reference. For example, the
helper vector may contain mutations in the packaging sequence of
its genome to prevent its packaging, an oversized adenoviral genome
which cannot be packaged due to size constraints of the virion, or
a packaging signal region with binding sequences that prevent
access by packaging proteins to this signal which thereby prevents
production of the helper virus.
[0034] Other strategies include the design of a helper virus with a
packaging signal flanked by the excision target site of a
recombinase, such as the Cre-Lox system (Parks et al., Proc. Natl.
Acad. Sci. USA 93:13565-13570, 1996; Hardy et al., J. Virol.
71:1842-1849, 1997, incorporated herein by reference). Such helper
vectors reduce the yield of wild-type levels.
[0035] Another hurdle for DeAd virus or PAV manufacturing, aside
from the problems with obtaining helper vector-free stocks, is that
the production process is initiated by DNA transfections of the
DeAd virus or PAV genome and the helper genome into a suitable cell
line, e.g., 293 cells. After cytopathic effects are observed in the
culture indicating a successful infection, which may take up to
from 2 to 6 days, the culture is harvested and is passaged onto a
new culture of cells. This process is repeated for several
additional passages, up to 7 times more, to obtain a modest cell
lysate containing the PAV or DeAd vector and any contaminating
helper vector. See Parks et al., 1996, Proc. Natl. Acad. Sci. USA
93:13565-13570; Kochanek et al., 1996, Proc. Natl. Acad. Sci. USA
93:5731-5736. This lengthy process is not optimal for commercial
scale manufacturing. Additionally, this process facilitates
recombination and rearrangement events resulting in the propagation
of PAV or DeAd viral genomes with unwanted alterations. The use of
adenoviruses for gene therapy is described, for example, in U.S.
Pat. No. 5,882,877; US patent, the disclosures of which are
incorporated herein by reference.
Adeno-Associated Virus [AAV]
[0036] Adeno-associated virus (AAV) is a single-stranded human DNA
parvovirus whose genome has a size about of 4.6 kb. The AA V genome
contains two major genes: the rep gene, which codes for the rep
proteins (Rep 76, Rep 68, Rep 52 and Rep 40) and the cap gene,
which codes for AAV structural proteins (VP-1, VP-2 and VP-3). The
rep proteins are involved in AAV replication, rescue, transcription
and integration, while the cap proteins form the AAV viral
particle. AAV derives its name from its dependence on an adenovirus
or other helper virus (e.g., herpes virus) to supply essential gene
products that allow AAV to undergo a productive infection, i.e.,
reproduce itself in the host cell. In the absence of helper virus,
AAV integrates as a provirus into the host cell's chromosome, until
it is rescued by superinfection of the host cell with a helper
virus, usually adenovirus (Muzyczka, Curr. Top. Micro. Immunol.
158:97-127, 1992).
[0037] Interest in AAV as a gene transfer vector results from
several unique features of its biology. At both ends of the AAV
genome is a nucleotide sequence known as an inverted terminal
repeat (ITR), which contains the cis-acting nucleotide sequences
required for virus replication, rescue, packaging and integration.
The integration function of the ITR mediated by the rep protein in
trans permits the AAV genome to integrate into a cellular
chromosome after infection, in the absence of helper virus. This
unique property of the virus has relevance to the use of AAV in
gene transfer, as it allows for a integration of a recombinant AAV
containing a gene of interest into the cellular genome. Therefore,
stable genetic transformation, ideal for many of the goals of gene
transfer, may be achieved by use of rAAV vectors. Furthermore, the
site of integration for AAV is well-established, and has been
localized to chromosome 19 of humans (Kotin et al., Proc. Natl.
Acad. Sci. 87:2211-2215, 1990). This predictability of integration
site reduces the danger of random insertional events into the
cellular genome that may activate or inactivate host genes or
interrupt coding sequences, consequences that can limit the use of
vectors whose integration is random, e.g., retroviruses. However,
because the rep protein mediates the integration of AA V, removal
of this gene in the design of rAAV vectors may result in the
altered integration patterns that have been observed with rAAV
vectors (Ponnazhagan et al., Hum. Gene Ther. 8:275-284, 1997).
[0038] There are other advantages to the use of AAV for gene
transfer. The host range of AAV is broad. Moreover, unlike
retroviruses, AAV can infect both quiescent and dividing cells. In
addition, AAV has not been associated with human disease, obviating
many of the concerns that have been raised with retrovirus-derived
gene transfer vectors.
[0039] Standard approaches to the generation of recombinant rAAV
vectors have required the coordination of a series of intracellular
events: transfection of the host cell with an rAAV vector genome
containing a trans gene of interest flanked by the AAV ITR
sequences, transfection of the host cell by a plasmid encoding the
genes for the AA V rep and cap proteins which are required in
trans, and infection of the transfected cell with a helper virus to
supply the non-AAV helper functions required in trans (Muzyczka,
N., Curf. Top. Micro. Immunol. 158: 97-129, 1992). The adenoviral
(or other helper virus) proteins activate transcription of the AAV
rep gene, and the rep proteins then activate transcription of the
AAV cap genes. The cap proteins then utilize the ITR sequences to
package the rAAV genome into an rAAV viral particle. Therefore, the
efficiency of packaging is determined, in part, by the availability
of adequate amounts of the structural proteins, as well as by the
accessibility of any cis-acting packaging sequences required in the
rAAV vector genome.
[0040] One of the potential limitations to high level rAAV
production derives from limiting quantities of the AAV helper
proteins required in trans for replication and packaging of the
rAAV genome. Some approaches to increasing the levels of these
proteins have included placing the AAV rep gene under the control
of the HIV LTR promoter to increase rep protein levels (Flotte, F.
R. et al., Gene Therapy 2:29-37, 1995); the use of other
heterologous promoters to increase expression of the AAV helper
proteins, specifically the cap proteins (Vincent et al., J. Virol.
71:1897-1905, 1997); and the development of cell lines that
specifically express the rep proteins (Yang, Q. et al., J. Virol.
68:4847-4856, 1994).
[0041] Other approaches to improving the production of rAAV vectors
include the use of helper virus induction of the AAV helper
proteins (Clark et al., Gene Therapy 3:1124-1132, 1996) and the
generation of a cell line containing integrated copies of the rAAV
vector and AAV helper genes so that infection by the helper virus
initiates rAAV production (Clark et al., Human Gene Therapy
6:1329-1341, 1995).
[0042] rAAV vectors have been produced using replication-defective
helper adenoviruses which contain the nucleotide sequences encoding
the rAAV vector genome (U.S. Pat. No. 5,856,152 issued Jan. 5,
1999) or helper adenoviruses which contain the nucleotide sequences
encoding the AAV helper proteins (PCT International Publication
WO95/06743, published Mar. 9, 1995). Production strategies which
combine high level expression of the AAV helper genes and the
optimal choice of cis-acting nucleotide sequences in the rAAV
vector genome have been described (PCT International Application
No. WO97/09441 published Mar. 13, 1997).
[0043] Current approaches to reducing contamination of rAAV vector
stocks by helper viruses, therefore, involve the use of
temperature-sensitive helper viruses (Ensinger et al., J. Virol.
10:328-339, 1972), which are inactivated at the non-permissive
temperature. Alternatively, the non-AAV helper genes can be sub
cloned into DNA plasmids which are transfected into a cell during
rAAV vector production (Salvetti et al., Hum. Gene Ther. 9:695-706,
1998; Grimm et al., Hum. Gene Ther. 9:2745-2760, 1998). The use of
AAV for gene therapy is described, for example, in U.S. Pat. No.
5,753,500; US patent, the disclosures of which are hereby
incorporated herein by reference.
Retroviruses
[0044] Retrovirus vectors are a common tool for gene delivery
(Miller, Nature (1992) 357:455-460). The ability of retrovirus
vectors to deliver an unrearranged, single copy gene into a broad
range of rodent, primate and human somatic cells makes retroviral
vectors well suited for transferring genes to a cell.
[0045] Retroviruses are RNA viruses wherein the viral genome is
RNA. When a host cell is infected with a retrovirus, the genomic
RNA is reverse transcribed into a DNA intermediate which is
integrated very efficiently into the chromosomal DNA of infected
cells. This integrated DNA intermediate is referred to as a
provirus. Transcription of the provirus and assembly into
infectious virus occurs in the presence of an appropriate helper
virus or in a cell line containing appropriate sequences enabling
encapsidation without coincident production of a contaminating
helper virus. A helper virus is not required for the production of
the recombinant retrovirus if the sequences for encapsidation are
provided by co-transfection with appropriate vectors.
[0046] Another useful tool for producing recombinant retroviral
vectors are packaging cell lines which supply in trans the proteins
necessary for producing infectious virions, but those cells are
incapable of packaging endogenous viral genomic nucleic acids
(Watanabe & Temin, Molec. Cell. Biol. (1983) 3(12):2241-2249;
Mann et al., Cell (1983) 33:153-159; Embretson & Temin, J.
Viroi. (1987) 61(9):2675-2683). One approach to minimize the
likelihood of generating RCR in packaging cells is to divide the
packaging functions into two genomes, for example, one which
expresses the gag and pol gene products and the other which
expresses the env gene product (Bosselman et al., Molec. Cell.
Biol. (1987) 7(5):1797-1806; Markowitz et al, J. Viroi. (1988)
62(4): 1120-1124; Danos & Mulligan, Proc. Natl. Acad. Sci.
(1988) 85:6460-6464). That approach minimizes the ability for
co-packaging and subsequent transfer of the two-genomes, as well as
significantly decreasing the frequency of recombination due to the
presence of three retroviral genomes in the packaging cell to
produce RCR.
[0047] In the event recombinants arise, mutations (Danos &
Mulligan, supra) or deletions (Boselman et al., supra; Markowitz et
al., supra) can be configured within the undesired gene products to
render any possible recombinants non-functional. In addition,
deletion of the 3' LTR on both packaging constructs further reduces
the ability to form functional recombinants.
[0048] The retroviral genome and the proviral DNA have three genes:
the gag, the pol, and the env, which are flanked by two long
terminal repeat (LTR) sequences. The gag gene encodes the internal
structural (matrix, capsid, and nucleocapsid) proteins; the pol
gene encodes the RNA-directed DNA polymerase (reverse
transcriptase) and the env gene encodes viral envelope
glycoproteins. The 5' and 3' LTRs serve to promote transcription
and polyadenylation of the virion RNAs. The LTR contains all other
cis-acting sequences necessary for viral replication. Lentiviruses
have additional genes including vit vpr, tat, rev, vpu, nef, and
vpx (in HIV-1, HIV-2 and/or SIV). Adjacent to the 5' LTR are
sequences necessary for reverse transcription of the genome (the
tRNA primer binding site) and for efficient encapsidation of viral
RNA into particles (the Psi site). If the sequences necessary for
encapsidation (or packaging of retrovirual RNA into infectious
virions) are missing from the viral genome, the result is a cis
defect which prevents encapsidation of genomic RNA. However, the
resulting mutant is still capable of directing the synthesis of all
virion proteins.
[0049] Lentiviruses are complex retroviruses which, in addition to
the common retroviral genes gag, pol and env, contain other genes
with regulatory or structural function. The higher complexity
enables the lentivirus to modulate the life cycle thereof, as in
the course of latent infection. A typical lentivirus is the human
immunodeficiency virus (HIV, the etiologic agent of AIDS. In vivo,
HIV can infect terminally differentiated cells that rarely divide,
such as lymphocytes and macrophages. In vitro, HIV can infect
primary cultures of monocyte-derived macrophages (MDM) as well as
HeLa-Cd4 or T lymphoid cells arrested in the cell cycle by
treatment with aphidicolin or gamma irradiation. Infection of cells
is dependent on the active nuclear import of HIV preintegration
complexes through the nuclear pores of the target cells. That
occurs by the interaction of multiple, partly redundant, molecular
determinants in the complex with the nuclear import machinery of
the target cell. Identified determinants include a functional
nuclear localization signal (NLS) in the gag matrix (MA) protein,
the karyophilic virion-associated protein, vpr, and a C-terminal
phosphotyrosine residue in the gag MA protein. The use of
retroviruses for gene therapy is described, for example, in U.S.
Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, the disclosures of
which are incorporated herein by reference.
Non-Viral Vectors
[0050] Other methods for delivery of DNA to cells do not use
viruses for delivery. These include the use of compounds, such as
cationic amphiphilic compounds; as well as DNA in the absence of
viral or non-viral compounds, known as "naked DNA."
Cationic Amphiphilic Compounds:
[0051] Because compounds designed to facilitate intracellular
delivery of biologically active molecules must interact with both
non-polar and polar environments (in or on, for example, the plasma
membrane, tissue fluids, compartments within the cell, and the
biologically active molecule itself), such compounds are designed
typically to contain both polar and non-polar domains. Compounds
having both such domains may be termed amphiphiles, and many lipids
and synthetic lipids that have been disclosed for use in
facilitating such intracellular delivery (whether for in vitro or
in vivo application) meet this definition. One particularly
important class of such amphiphiles is the cationic amphiphiles. In
general, cationic amphiphiles have polar groups that are capable of
being positively charged at or around physiological pH, and this
property is understood in the art to be important in defining how
the amphiphiles interact with the many types of biologically active
(therapeutic) molecules including, for example, negatively charged
polynucleotides such as DNA.
[0052] Examples of cationic amphiphilic compounds that have both
polar and non-polar domains and that are stated to be useful in
relation to intracellular delivery of biologically active molecules
are found, for example, in the following references, which contain
also useful discussion of (1) the properties of such compounds that
are understood in the art as making them suitable for such
applications, and (2) the nature of structures, as understood in
the art, that are formed by complexing of such amphiphiles with
therapeutic molecules intended for intracellular delivery.
[0053] (1) Feigner, et al., Proc. Natl. Acad. Sci. USA, 84,
7413-7417 (1987) disclose use of positively-charged synthetic
cationic lipids including
N->1(2,3-dioleyloxy)propyll-N,N,N-trimethylammonium chloride
("DOTMA"), to form lipid/DNA complexes suitable for transfections.
See also Feigner et al., The Journal of Biological Chemistry,
269(4), 2550-2561 (1994).
[0054] (2) Behr et al., Proc. Natl. Acad. Sci. USA, 86, 6982-6986
(1989) disclose numerous amphiphiles including
dioctadecylamidologlycylspermine ("DOGS").
[0055] (3) U.S. Pat. No. 5,283,185 to Epand et al. describes
additional classes and species of amphiphiles including
3.beta.>N-(N.sup.1,N.sup.1-dimethylaminoethane)carbamoyll
cholesterol, termed "DC-chol".
[0056] (4) Additional compounds that facilitate transport of
biologically active molecules into cells are disclosed in U.S. Pat.
No. 5,264,618 to Feigner et al. See also Feigner et al., The
Journal Of Biological Chemistry, 269(4), pp. 2550-2561 (1994) for
disclosure therein of further compounds including "DMRIE"
1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide.
[0057] (5) Reference to amphiphiles suitable for intracellular
delivery of biologically active molecules is also found in U.S.
Pat. No. 5,334,761 to Gebeyehu et al., and in Feigner et al.,
Methods (Methods in Enzymology), 5, 67-75 (1993).
[0058] The use of compositions comprising cationic amphiphilic
compounds for gene delivery is described, for example, in U.S. Pat.
No. 5,049,386; U.S. Pat. No. 5,279,833; U.S. Pat. No. 5,650,096;
U.S. Pat. No. 5,747,471; U.S. Pat. No. 5,767,099; U.S. Pat. No.
5,910,487; U.S. Pat. No. 5,719,131; U.S. Pat. No. 5,840,710; U.S.
Pat. No. 5,783,565; U.S. Pat. No. 5,925,628; U.S. Pat. No.
5,912,239; U.S. Pat. No. 5,942,634; U.S. Pat. No. 5,948,925; U.S.
Pat. No. 6,022,874; U.S. Pat. No. 5,994,317; U.S. Pat. No.
5,861,397; U.S. Pat. No. 5,952,916; U.S. Pat. No. 5,948,767; U.S.
Pat. No. 5,939,401; and U.S. Pat. No. 5,935,936, the disclosures of
which are hereby incorporated by reference.
"Naked DNA" Transfer
[0059] Methods for delivering a non-infectious, non-integrating DNA
sequence encoding a desired polypeptide or peptide operably linked
to a promoter, free from association with transfection-facilitating
proteins, viral particles, liposomal formulations, charged lipids
and calcium phosphate precipitating agents is described in U.S.
Pat. No. 5,580,859; U.S. Pat. No. 5,963,622; U.S. Pat. No.
5,910,488; the disclosures of which are hereby incorporated by
reference.
Combined Viral and Non-Viral Gene Transfer Systems
[0060] Gene transfer systems that combine viral and nonviral
components have been reported. Cristiano et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 11548; Wu et al. (1994) J. Biol. Chern. 269:
11542; Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6099;
Yoshimura et al., (1993) J. Biol. Chem. 268: 2300; Curiel et al.
(1991) Proc. Natl. Acad. Sci USA 88: 8850; Kupfer et al. (1994)
Hum. Gene Ther. 1437; and Gottschalk et al. (1994) Gene Ther.
1:185. In most cases, adenovirus has been incorporated into the
gene delivery systems to take advantage of its endosomolytic
properties. The reported combinations of viral and nonviral
components generally involve either covalent attachment of the
adenovirus to a gene delivery complex or co-internalization of
unbound adenovirus with cationic lipid: DNA complexes.
[0061] In a specific example, we have co-administered rapamycin
together with Ad2/.alpha.-gal vector. The rapamycin treatment (70
ug per mouse) was given by I.P. injections daily for 7 days. The
Ad2/.alpha.-gal vector was delivered I.V. As seen in the figures,
transient treatment with rapamycin was able to significantly
inhibit CTL responses to Ad vector and Ad/.alpha.-gal vector. The
antibody responses to both Ad vector and a-galactosidase protein
was also significantly inhibited.
Dosage of Rapamycin
[0062] The optimal dosage of rapamycin may be derived by methods
known in the clinical arts, including, but not limited to linear
equations based on population parameters such as age, weight or
sex; non-linear least squares modeling methods; Bayesian analysis
which employs specific data about the medical status of a
particular patient; pharmacokinetics compartment modeling; and the
trial-and-error method, in which a patient could be given
incremental dosages of rapamycin, and the patient's reaction
thereto could be observed and used to determine the frequency and
quantity of subsequent dosages. Preferred methods for dosing are
the methods described in U.S. Pat. No. 5,694,950, the disclosure of
which is hereby incorporated herein by reference. In general, a
preferred dosage of rapamycin is expected to be from about 0.05 to
about 0.15 milligrams of rapamycin per kilogram of the patient's
body weight. This dosage may be increased in order to obtain
additional efficacy.
[0063] The dosage of gene vector may be determined by one who is
expert in the field. For viral gene therapy, the dosage will
generally be in the preferred ranges from about 10e9 to about 10e13
particles per kg/body weight for adenovirus; from about 10e9 to
about 10e14 particles per kg/body weight for AAV; and from about
10e6 to about 10e10 transducing units/kg/body weight for retrovirus
or lentivirus.
Rapamycin+Anti-CD40L Experiments
[0064] Balb/c mice were injected intravenously with 6e10
particles/mouse of Ad2/CMV.alpha.-gal vector on day 0. Mice were
given intraperitoneal injections of 500 ug/mouse of MR-1, an
anti-CD40L antibody, on days -I, +2, +7 and +13. Rapamycin was
injected I.P. at 2.5 mg/kg daily from day 0 to +13. One control
group of mice received Ad2/CMV.alpha.-gal vector alone. Treatment
groups received (1) Ad vector+Rapamycin; (2) Ad vector+anti-CD40L;
or (3) Ad vector+Rapamycin+anti-CD40L. Results are shown in FIG. 1
[anti-.alpha.-gal antibody titers] and FIG. 2 [anti-Ad2 antibody
titers]. All groups of mice were bled at the indicated time points
to assay for antibodies to Ad2 and .alpha.-galactosidase protein
expression levels in the serum. As can be seen from the results,
anti-.alpha.-gal antibody and anti-Ad2 antibody titers were
significantly lower in the treatment groups than in the control
group. As seen in. FIG. 3, transient treatment with anti-CD40L and
Rapamycin led to persistent transgene expression for periods as
long as 160 days.
Rapamycin+Anti-B7 Experiments
[0065] Balb/c mice were injected intravenously with 7e10
particles/mouse of Ad2/CMVHI.alpha.-gal vector on day 0. Mice were
given intraperitoneal injections of 100 ug/mouse of anti-B7-1, and
100 ug/mouse of anti-B7-2 on days -I, +2 and +7. Rapamycin was
injected I.P. at 2.5 mg/kg daily from day 0 to +13. One control
group of mice received Ad2/CMVHI.alpha.-gal vector alone. Treatment
groups received (1) Ad vector+anti-B7-1+anti-B7-2; or (2) Ad
vector+anti-B7-1+anti-B7-2+Rapamycin. Results are shown in FIG. 3
[anti-.alpha.-gal antibody] and FIG. 4 [anti-Ad2 antibody titers].
All groups of mice were bled at the indicated time points to assay
for antibodies to Ad2 and .alpha.-galactosidase protein expression
levels in the serum. As can be seen from the results,
anti-.alpha.-gal antibody and anti-Ad2 antibody titers were
significantly lower in the treatment groups than in the control
group.
[0066] The above examples are non-limiting, and are included for
illustrative purposes only. The skilled artisan, having read the
disclosure contained herein, will readily appreciate that many
modifications, additions and improvements are possible. Such
modifications, additions and improvements are part of the present
invention.
[0067] The disclosure of each and every publication mentioned in
this specification is hereby incorporated by reference for the
teachings contained therein.
* * * * *