WO2008069942A2

WO2008069942A2 - Novel methods of enhancing delivery of a gene therapy vector using steroids

Info

Publication number: WO2008069942A2
Application number: PCT/US2007/024526
Authority: WO
Inventors: Xinzhong Wang; Michael Parr
Original assignee: Biogen Idec Ma Inc.
Priority date: 2006-12-05
Filing date: 2007-11-29
Publication date: 2008-06-12
Also published as: WO2008069942A3

Abstract

The invention provides methods for enhancing the delivery of a therapeutic gene product into a subject by administering a viral vector comprising a nucleic acid encoding the therapeutic gene product along with a steroid or a fragment, variant, derivative, or analog thereof that reduces the level of an inflammatory response and/or that modulates Kupffer cell function.

Description

NOVEL METHODS OF ENHANCING DELIVERY OF A GENE THERAPY VECTOR USING STEROIDS

Background of the Invention

Field of the Invention.

[0001] This invention relates to gene therapy. Specifically, the invention relates to' methods for enhancing the delivery and expression of a therapeutic gene product in a subject. Enhanced delivery of a viral vector comprising a nucleic acid encoding the therapeutic gene product and enhanced expression of the therapeutic gene product is achieved by administering a steroid, or a fragment, variant, derivative, or analog thereof, that reduces the level of an inflammatory response and/or that modulates Kupffer cell function.

Background of the Invention.

[0002] Viral vectors, including replication-defective vectors, are being used as gene delivery vehicles for a wide range of transgenes in pre-clinical and clinical studies for many pathological indications. Administration of viral vectors can occur through systemic or local routes. Moreover, routes of administration including intravenous, intraperitoneal, and subcutaneous administration can result in the transduction of hepatocytes followed by expression of a viral vector-encoded transgene and detectable circulating levels of a secreted transgene product.

[0003] Delivery of large amounts of viral vectors, including recombinant adenoviral vectors, encoding a therapeutic transgene can result in high expression levels of the transgene. However, large amounts of viral vectors can lead to complications such as liver-toxicity and induction of an inflammatory response. It is therefore desirable to achieve high levels of transgene expression without the toxicity and inflammation that can accompany administration of large amounts of viral vectors.

[0004] Delivery of small amounts of viral vectors, including recombinant adenoviral vectors, encoding a therapeutic transgene can lead to low or undetectable expression levels of the transgene. In contrast, small increases in the amount of viral vectors administered to a subject can lead to disproportionately large--/, e., non-linear— increases in expression levels of the transgene encoded by the viral vector (Tao et al, Molecular Therapy 3: 28-35 (2001), involving the human IFN-β transgene). Furthermore, transgene expression from a viral vector can be dramatically increased by coadministering a viral vector lacking the transgene (Id.). The enhancement of transgene expression has also been observed in mice treated with liposomal doxorubicin, which depletes liver macrophages known as Kupffer cells (Id.). Other agents have also been shown to modulate Kupffer cell function, including steroids (See Ziegler et al, Human Gene Therapy 13: 935-945 (2002) and Wolff e* al., J. Virology 71: 624-629 (1997)).

[0005] Another complication that may be associated with delivery of large amounts of viral vectors in gene therapy is the generation of a host inflammatory response, potentially resulting in clinical toxicity and impairment of gene transfer efficacy. Acute inflammatory responses have been observed, for example, in many animal models after a high-dose administration of adenoviral vectors, and vigorous cytokine release is likely the cause of acute toxic reactions seen in some human trials (Crystal et al, Nature Genetics 5:42-51 (1994); McElvaney et al, Nature Medicine 1: 182- 184 (1995)).

[0006] Several strategies have been pursued to decrease viral-induced inflammatory responses. One strategy involves diminishing cytotoxic T lymphocyte (CTL) responses by minimizing viral gene expression through use of second- and third-generation adenoviral vectors in which multiple viral genetic loci are deleted or rendered defective by mutation (Christ et al, Hum. Gene Therapy 11: 415-427 (2000); Gao et al, J. Virol. 70: 8934-8943 (1996)). For the latter, adenoviral vectors with El- and E4-deleted regions have been shown to express less viral protein and to exhibit substantially less toxicity in terms of vector-induced hepatitis (Gao et al, J. Virol. 70: 8934-8943 (1996); Wang et al, Gene Ther. 4: 393-400 (1997)). Another strategy involves the use of systemic corticosteroids (Sterman et al, Cancer Gene Therapy 7: 1511- 1518 (2000)).

[0007] Accordingly, there is a need in the art of gene therapy for better control of transgene expression and inflammatory responses in subjects treated with recombinant viral vectors. Brief Summary of the Invention

This invention provides a novel method of enhancing the delivery and expression of a therapeutic gene product by administering to a subject a viral vector comprising a nucleic acid encoding the therapeutic gene product in combination with a steroid or a fragment, variant, derivative, or analog thereof. In some embodiments, the steroid, or fragment, variant, derivative, or analog thereof, reduces the level of an inflammatory response and/or modulates Kupffer cell function. In certain embodiments, the steroid may be prednisolone, cortisone, corticosterone, dexamethasone, or a combination of two or more of these steroids. In certain embodiments, the steroid is prednisolone. In certain embodiments, the steroid may reduce the level of an inflammatory response by lowering a subject's production of cytokines and/or chemokines. In certain embodiments, the steroid may be administered prior to administering the viral vector. For example, the steroid may be administered 24 hours or less prior to administering the viral vector, 1 hour or less prior to administering the viral vector, or five minutes or less prior to administering the viral vector. In certain embodiments, the steroid may be administered concurrently with the viral vector. In certain embodiments, the inflammatory response is only transiently reduced by the steroid. In certain embodiments, the inflammatory response is reduced for a prolonged period by the steroid. In certain embodiments, the subject is a rodent. In certain embodiments, the subject is a primate. In certain embodiments, the primate is a human. In certain embodiments, the viral vector is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of these routes of administration. In certain embodiments, the steroid is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of these routes of administration. In certain embodiments, the viral vector is an adenovirus vector. In certain embodiments, the viral vector is a replication- defective viral vector. In certain embodiments, the therapeutic gene product is selected from LIGHT, interferon-β, herpes simplex virus thymidine kinase, p53, and a combination of two or more of these gene products. In certain embodiments, the therapeutic gene product is LIGHT. In certain embodiments, the therapeutic gene product is interferon-β. In certain embodiments, the therapeutic gene product is human interferon-β. The invention also relates to methods for modulating delivery of a virally encoded nucleic acid, where the methods allow for a near linear correlation between viral dose and expression of a therapeutic gene product encoded by the nucleic acid. In certain embodiments, a novel method is provided for modulating delivery of a virally encoded transgene in a subject by identifying a dosage inflection point where the dosage curve becomes nonlinear for expression of a transgene and administering a steroid or a fragment, variant, derivative, or analog thereof that reduces the level of an inflammatory response and/or modulates Kupffer cell function such that a near linear response is obtained between viral dose and expression of the therapeutic gene product. In certain embodiments, the steroid may be prednisolone, cortisone, corticosterone, dexamethasone, or a combination of two or more of these steroids. In certain embodiments, the steroid is prednisolone. In certain embodiments, the steroid may reduce the level of an inflammatory response by lowering a subject's production of cytokines and/or chemokines. In certain embodiments, the steroid may be administered prior to administering the viral vector. For example, the steroid may be administered 24 hours or less prior to administering the viral vector, 1 hour or less prior to administering the viral vector, or five minutes or less prior to administering the viral vector. In certain embodiments, the steroid may be administered concurrently with the viral vector. In certain embodiments, the subject is a rodent. In certain embodiments, the subject is a primate. In certain embodiments, the primate is a human. In certain embodiments, the viral vector is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of these routes of administration. In certain embodiments, the steroid is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of these routes of administration. In certain embodiments, the viral vector is an adenoviral vector. In certain embodiments, the viral vector is a replication-defective viral vector. In certain embodiments, the therapeutic gene product is selected from LIGHT, interferon-β, herpes simplex virus thymidine kinase, p53, and a combination of two or more of these gene products. In certain embodiments, the therapeutic gene product is LIGHT. In certain embodiments, the therapeutic gene product is interferon-β. In certain embodiments, the therapeutic gene product is human interferon-β. The invention also relates to kits for delivering a viral vector encoding a therapeutic gene product, comprising the viral vector encoding the therapeutic gene product, a steroid or a fragment, variant, derivative, or analog thereof, and an instruction manual for use, wherein the steroid or fragment, variant, derivative, or analog thereof reduces the level of an inflammatory response and/or modulates Kupffer cell function in a subject receiving the viral vector. In certain embodiments, the steroid may be prednisolone, cortisone, corticosterone, dexamethasone, or a combination of two or more of these steroids. In certain embodiments, the steroid is prednisolone. In certain embodiments, the steroid may reduce the level of an inflammatory response by lowering a subject's production of cytokines and/or chemokines. In certain embodiments, the viral vector is an adenoviral vector. In certain embodiments, the viral vector is a replication-defective viral vector. In certain embodiments, the therapeutic gene product is selected from LIGHT, interferon-β, herpes simplex virus thymidine kinase, p53, and a combination of two or more of these gene products. In certain embodiments, the therapeutic gene product is LIGHT. In certain embodiments, the therapeutic gene product is interferon-β. In certain embodiments, the therapeutic gene product is human interferon-β.

Brief Description of the Figures

[0011] FIG. 1 is a graph depicting the linear dose response of hTFN-β expressed from an adenoviral vector ("AdhlFN-β") due to pretreatment with increasing amounts of steroid 6 hours prior to administration of the vector.

[0012] FIG. 2 is a graph comparing the enhancement of hlFN-β expression from the AdhlFN-β vector by pretreatment with either prednisolone, cortisone, or corticosterone 4 hours prior to intravenous administration of AdhlFN-β vector.

[0013] FIG. 3 is a graph depicting enhanced expression of hlFN-β as a result of pretreatment with dexamethasone ("DEX") for either 4 or 24 hours prior to intravenous administration of the AdhlFN-β vector.

[0014] FIG. 4 is a graph comparing the enhancement of hlFN-β expression from the AdMFN- β vector by pretreatment with an adenoviral vector expressing the lacZ reporter gene ("AdlacZ") or pretreatment with steroids.

[0015] FIG. 5A-5B are graphs depicting the effects in mice of pretreatment with dexamethasone for various time points on expression of hIFN-β from subsequently administered AdhlFN-β vector. FIG. 5B is a magnified view of the data in FIG. 5A.

[0016] FIG. 6A-6B are graphs depicting mouse strain-specific differences in transgene expression from the AdhlFN-β vector after pretreatment with dexamethasone, depicting expression in athymic nude mice (FIG. 6A) or Balb/c mice (FIG. 6B).

[0017] FIG. 7A-7C are graphs showing that pretreatment with dexamethasone for 4 hours prior to administration of an adenoviral vector expressing luciferase ("AdLUX") results in a reduced inflammatory response to administered viral vector as measured by levels of the inflammatory markers IL-6 (FIG. 7A), MCP-I (FIG. 7B), and TNF-α (FIG. 7C) at the indicated time points after administration of AdLUX.

Detailed Description of the Invention

Definitions.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs, hi the case of conflict, the present application, including definitions, will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication, patent application, patent or reference were specifically and individually indicated to be incorporated by reference.

[0019] Although materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable materials and methods are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

[0020] The methods and techniques of the present invention are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general or specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989), Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane., eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1990), the disclosures of which are herein incorporated by reference. Standard techniques are used for the preparation, formulation, and delivery of pharmaceuticals as well as in the treatment of patients. [0021] For techniques related to adenovirus, see, e.g., Feigner and Ringold,

Nature 337: 387-388 (1989), Berkner and Sharp, Nucl. Acid Res. 11: 6003- 6020 (1983), Graham, EMBO J. 3: 2917-2922 (1984), and Bett et a!., Proc. Natl. Acad. Sd. USA 91: 8802-8806 (1994), the disclosures of which are herein incorporated by reference.

[0022] In order to further define this invention, the following terms and definitions are provided.

[0023] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0024] It is to be noted that the term "a" or "an" entity refers to one or more of that entity. As such, the terms "a" or "an", "one or more," and "at least one" can be used interchangeably.

[0025] As used herein, the term "administration" refers to systemic and/or local administration. The term "systemic administration" refers to non- localized administration such that an administered substance may affect several organs or tissues throughout the body or such that an administered substance may traverse several organs or tissues throughout the body in reaching a target site. For example, administration into a subject's circulation may result in expression of a therapeutic product from an administered vector in more than one tissue or organ, or may result in expression of a therapeutic product from an administered vector at a specific site, e.g., due to natural tropism or operable linkage of the therapeutic nucleic acid to tissue-specific promoter elements. One of skill in the art would understand that various forms of administration are encompassed by systemic administration, including those forms of administration encompassed by parenteral administration such as intravenous, intramuscular, intraperitoneal, and subcutaneous administration. In some embodiments, systemic administration can be used to elicit a systemic effect associated with treatment of a local or systemic disease or condition. A systemic effect may be desirable for the treatment of a local disease or condition, for example, to prevent the spread of said disease or condition. The term "local administration" refers to administration at or near a specific site. One of skill in the art would understand that various forms of administration are encompassed by local administration, such as direct injection into or near a specific site. In some embodiments, local administration is associated with treatment of a disease or condition where a local effect is desired (e.g. administration to the lung for the treatment of lung cancer). A local effect may be desired in association with either local or systemic diseases or conditions. A local effect may be desired in association with a systemic disease or condition to treat a local aspect of a systemic disease or condition.

[0026] Throughout the specification and claims, the term "comprise" and variations such as "comprises" or "comprising" indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers in the specified method, structure, or composition.

[0027] Throughout the specification and claims, the term "consists of and variations such as "consist of or "consisting of indicate the inclusion of any recited integer or group of integers but that no additional integer or group of integers may be added to the specified method, structure, or composition.

[0028] Throughout the specification and claims, the term "consists essentially of and variations such as "consist essentially of or "consisting essentially of indicate the inclusion of any recited integer or group of integers and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure, or composition.

[0029] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

[0030] Polypeptides as described herein may include fragment, variant, or derivative molecules thereof without limitation. The terms "fragment," "variant," "derivative" and "analog" when referring to a polypeptide include any polypeptide which retains at least some biological activity. Polypeptide fragments may include proteolytic fragments, deletion fragments, and fragments which more easily reach the site of action when delivered to an animal. Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three-dimensional epitopes. Polypeptide fragments may comprise variant regions, including fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally, such as an allelic variant. By an "allelic variant" is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Polypeptide fragments of the invention may comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Variant polypeptides may also be referred to herein as "polypeptide analogs." Polypeptide fragments of the present invention may also include derivative molecules. As used herein a "derivative" of a polypeptide or a polypeptide fragment refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[0031] As used herein, "fusion protein" means a protein comprising a first polypeptide linearly connected, via peptide bonds, to a second polypeptide. The first polypeptide and the second polypeptide may be identical or different, and they may be directly connected, or connected via a peptide linker. As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining together of two more elements or components by any means including chemical conjugation or recombinant means. An "in- frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence. A "linker" sequence is a series of one or more amino acids separating two polypeptide coding regions in a fusion protein. As used herein, the term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). A polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the untranslated 5' and 3' sequences, the coding sequences, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0033] As used herein, the term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. The terms "isolated" nucleic acid or "isolated" polynucleotide refers to a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0034] As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide or polypeptide fragment of the present invention. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0035] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide may normally include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0036] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0037] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or ERES, also referred to as a CITE sequence).

[0038] In other embodiments, a polynucleotide of the present invention is

RNA, for example, in the form of messenger RNA (mRNA).

[0039] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide is used or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0040] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, a RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes, without limitation, transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is biochemical, expression includes the creation of that biochemical and any precursors. [0041] As used herein, the terms "treat" and "treatment" refers to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to having the condition or disorder or those in which the condition or disorder is to be prevented.

[0042] As used herein, the term "steroid" means any of numerous naturally occurring or synthetic fat-soluble organic compounds containing a 17-carbon 4-ring system including the sterols, various hormones (including adrenal and sex hormones), certain glycosides, bile acids, and the precursors of certain vitamins.

[0043] The terms "fragment," "variant," "derivative," and "analog" when referring to a steroid include any steroid which retains at least some biological activity. A steroid analog is any compound duplicating the effect of the steroid of interest.

[0044] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans; domestic animals; farm animals; zoo animals; sport animals; pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. [0045] As used herein, a "therapeutically effective amount" or "therapeutic dose" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutic result may be, e.g., lessening of symptoms, prolonged survival, improved mobility, and the like. A therapeutic result need not be a "cure".

[0046] As used herein, a "prophylactically effective amount" or "prophylactic dose" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, a prophylactically effective amount will be less than a therapeutically effective amount for treatment of an advanced stage of disease.

[0047] As used herein, the term "gene therapy" refers to a procedure in which a disease phenotype is corrected through the introduction of genetic information into the affected organism. Gene therapy includes "ex vivo gene therapy," in which cells are removed from a subject and cultured in vitro. In ex vivo gene therapy a polynucleotide such as a functional gene is introduced into the cells in vitro, the modified cells are expanded in culture, and then reimplanted in the subject. Gene therapy also includes "in vivo gene therapy," in which target cells are not removed from the subject. Rather, the transferred polynucleotide (e.g., a gene encoding, upon expression, for an interferon) is introduced into cells of the recipient organism in situ, that is, within the recipient. In vivo gene therapy has been examined in several animal models, and recent publications have reported the feasibility of direct gene transfer in situ into organs and tissues. As used herein, gene therapy includes any condition amenable to gene therapy such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (i.e., a pathological condition which is not attributable to an inborn defect), cancers, and nascent or early stage conditions. Gene therapy can be used in further combination with other treatments including, but not limited to, surgical tumor debulking procedures, radiation therapy, or other chemotherapies.

[0048] As used herein, the term "tumor" refers, in part, to any undesirable proliferation of cells, including malignant and non-malignant tumors, solid or fluid tumors, carcinomas, myelomas, sarcomas, leukemias, lymphomas, and other cancerous, neoplastic, or tumorigenic diseases.

Method of Enhancing Delivery of Therapeutic Gene Products.

[0049] The invention provides methods for enhancing expression of a viral vector-encoded therapeutic gene product by delivering to a subject the viral vector in conjunction with a steroid. It is believed that the steroid may function by reducing the inflammatory response produced by introduction of the viral vector, and/or by modulating, e.g. reducing or blocking, Kupffer cell function in the treated subject. Modulation of Kupffer cell function is described in US 2004-0086486 Al, the contents of which are herein incorporated by reference.

[0050] In general, the method can be used to deliver a viral vector comprising a nucleic acid encoding any therapeutic gene product to a subject. Examples of therapeutic gene products encoded by such nucleic acids include polypeptides, antisense nucleic acids, small interfering RNAs, ribozymes, and components of a spliceosome. In the case where the therapeutic gene products are polypeptides, the encoded polypeptide can be, e.g., a cytokine such as: a tumor necrosis superfamily member such as LIGHT, an interferon such as interferon-alpha, interferon-beta, and interferon-gamma, and an interleukin such as interleukin- 1, interleukin-2, interleukin-4, interleukin-8, and interleukin- 12; a growth factor such as: erythropoietin, human growth hormone, insulin, granulocyte colony stimulating factor ("G-CSF"), and granulocyte-macrophage colony stimulating factor ("GM-CSF"); and a clotting factor such as: factor VIII and factor IX.

[0051] hi certain embodiments, the nucleic acid is provided in a vector that allows for encapsulation of the gene of the encoded therapeutic product into a particle, hi certain embodiments the particle can be taken up by a Kupffer cell. A suitable particle is a viral particle, e.g., an adenovirus particle, hi certain embodiments, the nucleic acid is not part of a viral vector.

[0052] Any method known in the art for the insertion of polynucleotide sequences into a vector may be used. Such methods are described in, e.g., Sambrook et al. and Ausubel et al, both of which are herein incorporated by reference. Vectors may include appropriate transcriptional and translational control signals operatively linked to the polynucleotide sequence for a particular therapeutic gene. Promoters and enhancers may also be used to control expression of therapeutic proteins or gene products. Promoter activation may be tissue specific or inducible by a metabolic product or administered substance. Such promoters and enhancers include, but are not limited to, the native E2F promoter, the cytomegalovirus immediate-early promoter and enhancer (Karasuyama et al., J. Exp. Med. 169: 13 (1989)), the human beta-actin promoter (Gunning et al., Proc. Nat. Acad. Sd. USA 84: 4831 (1987)), the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al, MoI Cell. Biol. 4: 1354 (1984)), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al, eds., RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1985)), the SV40 early region promoter (Bernoist and Chambon, Nature 290: 304 (1981), the promoter of the Rous sarcoma virus (RSV) (Yamamoto et al, Cell 22: 787 (1980)); the herpes simplex virus (HSV) thymidine kinase promoter (Wagner et al, Proc. Nat. Acad. ScL USA 78: 1441(1981)), and the adenovirus promoter (Yamada et al, Proc. Nat. Acad Sci. USA 82: 3567 (1985)).

[0053] Expression vectors compatible with mammalian host cells for use in gene therapy include, for example, plasmids and viral vectors.

[0054] Specific viral vectors for use in gene transfer systems are now well established and include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, and herpes simplex viral vectors. See for example: Madzak et al, J. Gen. Virol. 73: 1533-36 (1992): papovavirus SV40; Moss et al, Curr. Top. Microbiol. Immunol. 158: 25-38 (1992): vaccinia virus; Margulskee, Curr. Top. Microbiol. Immunol. 158: 67-93 (1992): herpes simplex virus (HSV) and Epstein-Barr virus (EBV); Miller, Curr. Top. Microbiol. Immunol. 158: 1-24 (1992): retrovirus; Brandyopadhyay et al, MoI Cell. Biol. 4: 749-754 (1984): retrovirus; Miller et al, Nature 357: 455-450 (1992): retrovirus; Anderson, Science 256: 808-813 (1992): retrovirus; herpes viruses (for example, herpes simplex virus based vectors); and parvoviruses (for example, "defective" or non-autonomous parvovirus based vectors); Hofinann, et al, PNAS 92: 10099-10103 (1995): baculovirus; Boyce, et al, PNAS 93: 2348-2352 (1996): baculovirus; all of which are herein incorporated by reference. In various embodiments, recombinant viral vectors designed for use in gene therapy are used in the invention. See, e.g., Hu and Pathak, Pharmacol Rev. 52: 493-512 (2000); Somia and Verma, Nature Rev. 1: 91-99 (2000); van Beusechem et al, Gene Ther. 7: 1940-1946 (2000); Glorioso et al, Nature Med. 7: 33-40 (2001). Additionally, viral vectors may be administered in combination with transient immunosuppressive or immunomodulatory therapies. See, e.g., Jooss et al, Hum Gene Ther. 7: 1555-1566 (1996); Kay et al, Pro. Nat. Acad. Sd. USA 94: 4686-4691.

[0055] In other embodiments, viral serotypes, e.g., the general adenovirus types 2 and 5 (Ad2 and Ad5, respectively) may be administered, possibly on an alternating dosage schedule where multiple treatments will be administered. Specific dosage regimens may be administered: over the course of several days, when an immune response against the viral vector is anticipated, or both. In non-limiting examples of specific embodiments, Ad5-based viral vectors may be used on day 1 , Ad2-based viral vectors may be used on day 2, or vice versa.

[0056] In some embodiments, nucleic acids are additionally provided in replication-defective recombinant viruses or viral vectors. These can be generated in packaging cell lines that produce only replication-defective viruses. See, e.g., Sections 9.10-9.14, Ausubel et al. In certain embodiments, the nucleic acid encoding a therapeutic gene product is not part of a viral vector.

[0057] It is further contemplated in this invention that the viral vector comprising a nucleic acid encoding a therapeutic gene product may act synergistically with an agent that reduces the level of an inflammatory response and/or that modulates Kupffer cell function in the subject to enhance the delivery of a viral vector comprising said nucleic acid. As used herein, the term "synergistic" or "synergistically" refers to the combined effect being greater than the sum of the individual effects. For example, the viral vector and steroid may be formulated such that the individual components if dosed separately would comprise a sub-therapeutic dose. As used herein, "sub- therapeutic dose" refers to an amount of an active agent that would not provide treatment, prevention, or amelioration of the condition to be treated or prevented if dosed alone, without other active agents. In certain embodiments, the nucleic acid encoding a therapeutic gene product is not part of a viral vector.

Adenoviral vectors.

[0058] In some embodiments, a vector for delivering a nucleic acid is an adeno virus-based vector. See, e.g., Berkner et al., Curr. Top. Microbiol. Immunol. 158: 39-61 (1992). In some embodiments, the adenovirus-based vector is an Ad-2 or Ad-5 based vector. See, e.g., Muzyczka, Curr. Top. Microbiol. Immunol. 158: 97-123(1992); AIi et al, Gene Therapy 1: 367-384 (1994); and U.S. Pat. Nos. 4,797,368 and 5,399,346.

[0059] Adenoviruses can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. For example, the general adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are currently being developed for clinical trials, including treatment of cancer or other cell proliferation diseases and disorders, and for gene therapy of Duchenne Muscular Dystrophy (DMD) and Cystic Fibrosis (CF). Both Ad2 and Ad5 belong to a subclass of adenovirus that are not associated with human malignancies. Adenovirus vectors are capable of providing high levels of transgene delivery to diverse cell types, regardless of the mitotic state of the cell. High titers (10¹³ plaque forming units/ml) of recombinant virus can be easily generated in 293 cells (an adenovirus- transformed, complementation human embryonic kidney cell line: ATCC No. CRLl 573) and cryo-stored for extended periods without appreciable losses. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders. See, e.g., Watanabe, Atherosclerosis 36: 261-268 (1986); Tanzawa et al, FEBS Letters /75(1): 81-84 (1980); Golasten et al, New Engl. J. Med. 309: 288-296 (1983); Ishibashi et al., J. Clin. Invest. 92: 883-893 (1993); Ishibashi et al, J. Clin. Invest. 93: 1889-1893 (1994), all of which are herein incorporated by reference. Recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) gene product has been approved for use in at least two human CF clinical trials. See, e.g., Wilson, Nature 365: 691-692 (1993).

[0060] Some replication-deficient adenoviruses which have been developed for clinical trials contain deletions of the entire EIa region and part of the EIb region. These replication-defective viruses are grown in 293 cells containing a functional adenovirus EIa gene which provides a trans-acting EIa protein. El -deleted viruses are capable of replicating and producing infectious virus in certain cells (e.g., 293 cells), which provide EIa and EIb region gene products in trans. The resulting virus is capable of infecting many cell types and can express the introduced gene (providing it carries its own promoter). However, the virus cannot replicate in a cell that does not carry the El region DNA unless the cell is infected at a very high multiplicity of infection. Other adenoviral vectors developed for clinical trials may be used in the invention. Examples include Ad vectors with recombinant fiber proteins for modified tropism (e.g., van Beusechem et al, Gene Ther. 7: 1940-1946 (2000)), protease pre-treated viral vectors (e.g., Kuriyama et al., Hum. Gene Ther. 11: 2219-2230 (2000)), E2a temperature sensitive mutant Ad vectors (e.g., Engelhardt et al., Hum. Gene Ther. 5: 1217-1229 (1994)), and "gutless" Ad vectors (e.g., Armentano et al, J. Virol. 71: 2408-2416 (1997); Chen et al, Proc. Nat. Acad. Sci. USA 94: 1645-1650 (1997); Schieder et al, Nature Genetics 18: 180-183 (1998)).

[0061] Adenoviruses have a broad host range, can infect quiescent or terminally differentiated cells such as neurons, and appear to be essentially non-oncogenic. Adenoviruses additionally do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. See, e.g., AIi et al 1994, supra, at 373. Recombinant adenoviruses (rAdV) produce very high titers, the viral particles are moderately stable, expression levels are high, and a wide range of cells can be infected.

[0062] Adeno-associated viruses (AAV) have also been used as vectors for somatic gene therapy. AAV is a small, single-stranded (ss) DNA virus with a simple genomic organization (4.7 kb) that makes it an ideal substrate for genetic engineering. Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62, and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VPl, VP2, and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene. See, e.g. , Carter, "The Growth Cycle of Adeno-Associated Virus," in Handbook of Parvoviruses, vol. I, pp. 155-168, Tijssen, ed., CRC Press (1990). It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.

[0063] Administration of the viral particles comprising viral vectors described herein can be via any of the accepted modes of administration for such viral particles well known by a person of ordinary skill in the art. For example, the viral particles may be administered by systemic or local administration, including oral, nasal, parenteral, transdermal, topical, intraocular, intrabronchial, intraperitoneal, intravenous, subcutaneous, and intramuscular administration, or by direct injection into cells, tissues, organs, or tumors. The adenoviral particles/vectors may be formulated in any art-accepted formulation well known to a person of ordinary skill in the art.

Steroids

[0064] This invention provides a method for delivering a viral vector comprising a nucleic acid encoding a therapeutic gene product by administering the vector with a steroid or a fragment, variant, derivative, or analog thereof.

[0065] The steroids encompassed by the invention include corticosteroids that would be understood by one of ordinary skill in the art to fall within classes typified by the following members: (1) hydrocortisone/cortisone; (2) prednisolone/prednisone/methylprednisolone; (3) betamethasone/ dexamethasone; and (4) triamcinolone. [0066] In certain embodiments, the steroid includes, but is not limited to, prednisolone, cortisone, corticosterone, or dexamethasone. In certain embodiments, the steroid is prednisolone.

[0067] Particular steroids encompassed by the present invention include, but are not limited to, the following steroids: alclometasone, alclometasone dipropionate, amcinonide, augmented betamethasone, augmented betamethasone dipropionate, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone benzoate, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, clobetasol, clobetasol propionate, clocortolone, clocortolone pivalate, cortisone, desonide, desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone, diflorasone acetonide, diflorasone diacetate, flucinolone, fludroxycortide, flunisolide, fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasone, fluticasone propionate, halcinonide, halobetasol, halobetasol propionate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone sodium phosphate, hydrocortisone valerate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, mometasone, mometasone furoate, prednicarbate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, tiamcinolone hexacetonide, ulobetasol, a combination of two or more of these steroids, and commercial products of these steroids.

[0068] Steroids of the invention may be administered through any route encompassed by systemic or local administration as defined. For example, steroids of the invention may be applied locally to the skin, applied locally to the eye, ingested orally, inhaled directly into the lungs, injected into a vein or muscle, or injected directly into inflamed joints. Steroids that may be administered by an oral route include, but are not limited to the following steroids: betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, a combination of two or more of these steroids, and commercial products of these steroids. Steroids that may be administered by a parenteral route, such as parenteral injection, include, but are not limited to the following steroids: betamethasone, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, triamcinolone, a combination of two or more of these steroids, and commercial products of these steroids. Steroids that may be administered by inhalation include, but are not limited to the following steroids: beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, a combination of two or more of these steroids, and commercial products of these steroids. Steroids that may be administered by a topical route include, but are not limited to the following steroids: alclometasone, amcinonide, augmented betamethasone, betamethasone, clobetasol, clocortolone, desonide, desoximetasone, dexamethasone, diflorasone, flucinolone, fluocinonide, flurandrenolide, fluticasone, halcinonide, halobetasol, hydrocortisone, methylprednisolone, mometasone, prednicarbate, triamcinolone, a combination of two or more of these steroids, and commercial products of these steroids. One of skill in the art would understand that a particular steroid may be applied by more than one route, e.g. a steroid utilized in a topical formulation may be adapted for intravenous or oral administration.

[0069] One of ordinary skill in the art would understand that steroids have various medical uses, including but not limited to: (1) anti-inflammatory uses, e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone; (2) antiemetic uses, e.g. dexamethasone, hydrocortisone, and prednisone; (3) diagnostic uses, e.g. dexamethasone, as used to detect Cushing's syndrome; and (4) immunosuppressant uses, e.g. betamethasone, cortisone, dexamethasone, hydrocortisone, methylprednicolone, prednisolone, prednisone, and triamcinolone. Moreover, one of ordinary skill in the art would understand that corticosteroid drugs can be used as ingredients contained in eye products (to treat various eye conditions), inhalers (to treat asthma or bronchial disease), nasal drops and sprays (to treat various nasal conditions), and topical products such as ointments and creams (to treat various skin conditions).

[0070] One of ordinary skill in the art would understand that potencies may vary among steroids. For example, as associated with systemic administration, betamethasone and dexamethasone exhibit high overall potencies and high anti-inflammatory potencies; methylprednisolone, triamcinolone, prednisolone, and prednisone exhibit medium overall potencies and medium anti-inflammatory potencies; and hydrocortisone and cortisone exhibit low overall potencies and anti-inflammatory potencies.

[0071] One of ordinary skill in the art would understand that the duration of biological effects elicited by administered steroids may vary among different steroids associated with their respective half-lives. For example, betamethasone and dexamethasone exhibit long half-lives; methylprednisolone, prednisolone, and prednisone exhibit medium half-lives; and cortisone and hydrocortisone exhibit short half-lives. One of skill would understand that the duration of biological effects associated with the half-life of an individual steroid includes the duration of anti-inflammatory effects.

[0072] While not being bound by theory, it is believed that the administered steroid functions by reducing the level of a subject's inflammatory response caused by administration of viral vectors or particles. For example, adenoviral vectors activate host innate immune responses that result in acute inflammation of transduced tissues. The induction of inflammatory cytokines and chemokines is an integral component of the innate immune response to viral vectors. Cytokines and chemokines may have direct antiviral effects but also play a role in recruiting and activating innate effector cells to sites of infection. For example, release of the inflammatory cytokines TNF-α, IL-6, IL-8, and GM-CSF was observed from human peripheral blood mononuclear cells following exposure to adenoviral vectors (Higginbotham et al., Human Gene Therapy, 13:129-141 (2002)). Furthermore, Higginbotham et al. reported that chemokines were also induced upon adenoviral vector administration.

[0073] In certain embodiments, the steroid reduces the levels of an inflammatory response by lowering the production of cytokines. One of skill in the art would understand that an inflammatory response is caused by cytokines. Thus, the ability of a steroid to reduce the inflammatory response is conveniently and routinely determined by measuring cytokine levels as a function of steroid administration. Suitable cytokines to be measured include, but are not limited to, IL-lα, IL-I β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-I l, IL-12, IL-12A, IL-12B, IL-13, IL-15, IL-16, IL-17, IL-18, IL- 20, IL-21, IL-25, IFN-α, IFN-β, TNF-α, and a combination of two or more of these cytokines.

[0074] In other embodiments, the steroid reduces the level of an inflammatory response by lowering the production of chemokines. One of skill in the art would understand that an inflammatory response is caused by chemokines. Thus, the ability of a steroid to reduce the inflammatory response is conveniently and routinely determined by measuring chemokine levels as a function of steroid administration. Inflammatory chemokines fall into two broad groups called cysteine-cysteine (CC) chemokines and cysteine- X-cysteine (CXC) chemokines, classified according to the position of the location of the cysteine residues near the amino terminus. In certain embodiments, the chemokines to be measured are CC chemokines, such as, but not limited to, 1-309, MCP-I, MCP-2, MCP-3, MCP-4, MIP-Ia, MP-Ib, MIP-Id, MIP-3a, MIP-3b, HCC-4, TARC, PARC, SLC, MDC, MPIF-I, MPIF-2, TECK, RANTES, eotaxin, eotaxin-3, CTACK, CCL28, and a combination of two or more of these CC chemokines. In other embodiments, the chemokines to be measured are CXC chemokines, such as, but not limited to, GROl (MBP-2), GRO2, GRO3, ENA-78, GCP-2, MIG, IP-10, I-TAC, SDFl, CXCL4, CXCL7, CXCL13, CXCL14, CXCL16, and a combination of two or more of these CXC chemokines. In certain embodiments, the chemokines to be measured include, but are not limited to, 1-309, MCP-I, MCP-2, MCP-3, MCP-4, MIP-Ia, MIP-Ib, MIP-Id, MIP-3a, MIP-3b, HCC-4, TARC, PARC, SLC, MDC, MPIF-I, MPIF-2, TECK, RANTES, eotaxin, eotaxin-3, CTACK, CCL28, GROl (MJP-2), GRO2, GRO3, ENA-78, GCP-2, MIG, IP-10, I-TAC, SDFl, CXCL4, CXCL7, CXCL13, CXCL14, CXCL16, and a combination of two or more of these chemokines.

[0075] A variety of methods are available to one of ordinary skill in the art for the detection of changes in immune-related status, e.g. changes in an inflammatory response, including: (1) protein assays (e.g. enzyme-linked immunosorbent assay (ELISA), such as the BioSource™ murine IL-6 ELISA kit; Western Blot analysis, Cytometric Bead Array (CBA, such as the BD™ Biosciences CBA Mouse Inflammation Kit); and multiplex assays (including technologies similar to those utilized in the Bio-Plex® or Luminex® multiplex suspension arrays)); (2) DNA assays (e.g. Southern Blot and polymerase chain reaction (PCR, including quantitative-PCR (Q-PCR)); (3) RNA assays {e.g. Northern Blot analysis, and PCR-based assays (including reverse-transcriptase PCR, real time PCR, and Taqman); and (4) other assays directed to immune cell identity, function, or markers {e.g. immunofluorescent staining of cell surface molecules for Flow Cytometric Analysis (FACS); Cytotoxic T Lymphocyte (CTL) assays {e.g. ⁵¹CR release); enzyme-linked immunospot (ELISPOT) assays; and major histocompatibility complex (MHC)-peptide tetramer staining as well as assays directed to numerous other functions {e.g. macrophage activity, antigen-specific T cells, and other cell-based assays for biological response modifiers)).

[0076] hi some embodiments, administration of a steroid suppresses, inhibits, or reduces an inflammatory response against an administered viral vector expressing a therapeutic product.

[0077] In some embodiments, steroid pretreatment suppresses, inhibits, or reduces an undesirable inflammatory response against an administered viral vector expressing a therapeutic product but does not suppress, inhibit, or reduce a desirable inflammatory therapeutic response elicited by a therapeutic product expressed from an administered viral vector. A desirable inflammatory response elicited by a therapeutic product expressed from a viral vector includes, for example, an inflammatory anti-tumor response elicited by a therapeutic product, such as a cytokine, expressed from an administered vector.

[0078] Based on the teachings herein, one of skill in the art would readily understand that the extent and duration of an anti-inflammatory response will be dependent on the type and dosage of the steroid utilized. For example, prednisolone or cortisone may be utilized for transient reduction of inflammation, while dexamethasone or betamethasone may be utilized for comparatively extended reduction of inflammation, hi some embodiments, steroid pretreatment results in transient suppression, inhibition, or reduction of an inflammatory response in a subject, hi some embodiments, steroid pretreatment results in prolonged suppression, inhibition, or reduction of an inflammatory response in a subject.

[0079] While not being bound by theory, it is believed that the steroid may also modulate, e.g. reduce or block, Kupffer cell function. As used herein, the term "modulate" refers to the ability of the steroid to alter the function of Kupffer cells so that they are no longer capable of taking up viral vectors or particles comprising a nucleic acid encoding the therapeutic gene product. An example of a steroid modulating the function of a Kupffer cell is by interfering with the ability of Kupffer cells to phagocytose viral vectors, such as adenoviral vectors. The steroid could also modulate Kupffer cell function by affecting its ability to take up viral vectors via receptor-mediated uptake. As a result of modulating Kupffer cell function, the administered steroid could reduce the ability of Kupffer cells to phagocytose transgene-encoding viral vectors in a subject.

[0080] hi some embodiments, the steroid is administered prior to delivery of a viral vector operably encoding a therapeutic gene product. hi other embodiments, the steroid is administered concurrently with the viral vector. For example, the steroid can be administered 24 hours or less, 10 hours or less, 8 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 10 minutes or less, and even 5 minutes or less prior to administering the viral vector encoding a therapeutic gene product, hi certain embodiments, the steroid is administered 4 hours or less prior to the viral vector.

[0081] Administration of the steroids described herein can be via any of the accepted modes of administration for such steroids well known by those of ordinary skilled in the art. For example, the steroids may be administered by systemic or local administration, including oral, nasal, parenteral, transdermal, topical, intraocular, intrabronchial, intraperitoneal, intravenous, subcutaneous, and intramuscular administration or by direct injection into cells, tissues, organs, or tumors. The steroid is formulated in an art-accepted formulation well-known to a person skilled in the art.

[0082] hi some embodiments, a steroid is administered systemically.

[0083] hi some embodiments, a steroid is administered locally.

[0084] The subject in the above-mentioned methods can be any animal for which introduction of a foreign nucleic acid is desired. Thus, the subject can include, e.g., mammals, reptiles, or birds, hi some embodiments, the subject is a human, mouse, rat, dog, cat, horse, cow, pig, non-human primate, or chicken. Therapeutic Gene Product.

[0085] This invention provides a method for delivering a viral vector comprising a nucleic acid ("therapeutic nucleic acid") encoding a therapeutic gene product by administering the vector with a steroid or a fragment, variant, derivative, or analog thereof. The terms "therapeutic," "therapeutic value," and all variations thereof encompass both therapeutic and prophylactic values. As such, administration can be for therapeutic, including prophylactic, purposes.

[0086] In certain embodiments, the therapeutic gene product is a polypeptide, including a mature form, active form, fragment, variant, or derivative thereof, that has therapeutic value in a subject. In certain embodiments, the therapeutic gene product is a polypeptide that is a membrane protein, such as, but not limited to, CD2, CD4, BAFF, APRIL, CD40, CD 154, and an integrin protein such as the α-1 integrin protein. In certain embodiments, the therapeutic gene product is a polypeptide that is an intracellular protein, such as, but not limited to a caspase, p53, herpes simplex virus thymidine kinase, and retinoblastoma protein. In certain embodiments, the therapeutic gene product is a polypeptide that is a co-stimulatory protein or a polypeptide of the immune system, such as, but not limited to, CD40L, CD27, OX-40, 4-1BB, ICOS, LIGHT, B7.1, B7.2, CD40, CD70, OX-40L, 4- IBBL, ICOS-L, and HVEM. In certain embodiments, the therapeutic gene product is a polypeptide that is a secreted polypeptide such as, but not limited to: an interferon (IFN), such as interferon- beta, interferon-alpha, and interferon-gamma; an interleukin (IL), such as IL- 1, IL-2, IL-4, IL-8, and IL-12; and a growth factor, such as GM-CSF and G- CSF. In certain embodiments, the therapeutic gene product is a polypeptide that includes, but is not limited to, a cytokine, a hormone, an oncogene, and a tumor suppressor gene. In certain embodiments, the therapeutic gene product is a polypeptide that includes, but is not limited to, vascular endothelial factor, TNF-alpha, TNF-beta, TGF-beta, insulin-like growth factor I, insulin, and human growth hormone. In certain embodiments, the therapeutic gene product is a polypeptide that includes, but is not limited to, an antibody, an antigen binding fragment thereof, and an immunoreactive fragment thereof. In certain embodiments, the therapeutic gene product is a polypeptide, including a mature form, active form, fragment, variant, or derivative thereof, having substantial identity, usually at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, or at least about 95% identity with a polypeptide including, but not limited to, a membrane protein, an intracellular protein, a co-stimulatory protein or a polypeptide of the immune system, a secreted polypeptide, a cytokine, a hormone, an oncogene, a tumor suppressor gene, vascular endothelial factor, TNF-alpha, TNF-beta, TGF-beta, insulin-like growth factor I, insulin, and human growth hormone. In certain embodiments, the therapeutic gene product is a fusion or chimeric polypeptide comprising such polypeptides and/or mature forms, active forms, fragments, variants, or derivatives thereof. In certain embodiments, the therapeutic gene product is a combination of two or more gene products including, but not limited to, any of the polypeptides described herein. In a particular embodiment, the therapeutic gene product is selected from LIGHT, interferon-beta, herpes simplex virus thymidine kinase, p53, and a combination of these gene products. In certain embodiments, the therapeutic gene product is used for diagnostic purposes. In certain embodiments, the therapeutic gene product is a polypeptide used for diagnostic purposes including, but not limited to, diagnosis of a disease, disorder, or condition, hi certain embodiments the diagnostic purpose includes, but is not limited to, pre-implantation diagnosis, embryonic diagnosis, prenatal diagnosis, diagnosis of newborns, pre- symptomatic diagnosis, conf rmational diagnosis of a symptomatic individual, monitoring the activity of a target biomolecule in a pre-symptomatic individual, and monitoring the activity associated with delivery of a transgene. hi certain embodiments, the therapeutic gene product is a polypeptide or a fusion or chimeric polypeptide including, but not limited to, a fluorescent polypeptide, a bioluminescent polypeptide, a polypeptide used in imaging processes as a contrast agent, an antibody, an antigen of an antibody, an enzyme, a substrate of an enzymatic activity, and a combination of these gene products. hi certain embodiments, the therapeutic gene product is a polypeptide including, but not limited to, a substrate of a protease. In particular embodiments, the therapeutic gene product is a substrate of a protease for detection of a disease-related protease activity, for monitoring the activity of protease inhibitors, and for monitoring transgene expression of a protease. In certain embodiments, the therapeutic gene product is a polypeptide used for diagnostic purposes through use of in vivo imaging, including but not limited to magnetic resonance imaging and positron emission tomography. In certain embodiments, the therapeutic gene product is a polypeptide, including a mature form, active form, fragment, variant, or derivative thereof, having substantial identity, usually at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, or at least about 95% identity with a product that is used for diagnostic purposes, hi certain embodiments, the therapeutic gene product is a fusion or chimeric polypeptide comprising such polypeptides and/or mature forms, active forms, fragments, variants, or derivatives thereof, hi certain embodiments, the therapeutic gene product is a polynucleotide including, but not limited to, a recombinant polynucleotide, a modified polynucleotide, a labeled polynucleotide, a coding region, a non-coding region, an anti-sense polynucleotide, a fragment of a polynucleotide, and a combination of these gene products. In certain embodiments, the therapeutic gene product is a polynucleotide used for diagnosis including, but not limited to, diagnosis of a disease, disorder, or condition. In certain embodiments the diagnosis includes, but is not limited to, pre-implantation diagnosis, embryonic diagnosis, prenatal diagnosis, diagnosis of newborns, pre-symptomatic diagnosis, confirmational diagnosis of a symptomatic individual, monitoring the activity of a target biomolecule in a pre-symptomatic individual, and monitoring the activity associated with delivery of a transgene. In certain embodiments, the therapeutic gene product is a polynucleotide used for diagnostic purposes through use of in vivo imaging, including but not limited to magnetic resonance imaging and positron emission tomography. hi certain embodiments, the therapeutic gene product is a polynucleotide having substantial homology, usually at least about 70% homology, at least about 75% homology, at least about 80% homology, at least about 85% homology, or at least about 95% homology with a product that may be used for diagnostic purposes. In a particular embodiment of this invention, the therapeutic gene product is interferon-β, including a mature form, active form, fragment, variant, or derivative thereof. In another particular embodiment, the therapeutic gene product is a human interferon-β, including a mature form, active form, fragment, variant, or derivative thereof. In certain embodiments, the therapeutic gene product may be a polypeptide, including a mature form, active form, fragment, variant, or derivative thereof, having substantial identity with interferon-β, usually at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85%, or at least about 95% identity. In certain embodiments, the therapeutic gene product is a fusion or chimeric polypeptide comprising such polypeptides and/or mature forms, active forms, fragments, variants, or derivatives thereof. hi a particular embodiment of this invention, the therapeutic gene product is LIGHT, including a mature form, active form, fragment, variant, or derivative thereof. LIGHT is a molecular entity with a novel function of breaking existing stromal tumor barriers (Mauri et al., Immunity 8:21-30 (1998)("... LIGHT, is homologous to /ymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes." Id. at 22, additional emphasis added); Zhai et al., J. Clin. Investigation 102:1142-1151 (1998)). In addition to its immune- stimulating properties, LIGHT also up-regulates a panel of cytokines and chemokines (including IFN-β, GM-CSF and SLC). In another particular embodiment, the therapeutic gene product is human LIGHT, including a mature form, active form, fragment, variant, or derivative thereof. In another particular embodiment, the therapeutic gene product is a mutant version of LIGHT known as mutant LIGHT, as described in U.S. publication 2005/0025754 and International publication WO 2005/002628. Specifically, mutant LIGHT is generated to prevent protease digestion so that LIGHT can be expressed on tumor cells. The proteolytic site EKLI of the native LIGHT gene is deleted from positions 79-82 of mutant LIGHT and this deletion has been shown to be useful for eliciting high levels of chemokines and adhesion molecules, accompanied by massive infiltration of naive T lymphocytes. The ability of gene delivery of LIGHT or mutant LIGHT to stimulate cellular immunity via its upregulation of cytokines and chemokines can be very powerful in eliminating micrometastatic tumors, suppressing dormant tumor sites, and killing residual tumor cells. Based on the foregoing, treatment with a steroid prior to or concurrently with gene delivery of LIGHT or mutant LIGHT is contemplated as a novel approach for cancer immunotherapy. In certain embodiments, the therapeutic gene product may be a polypeptide, including a mature form, active form, fragment, variant, or derivative thereof, having substantial identity with LIGHT or mutant LIGHT, usually at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85%, or at least about 95% identity. In certain embodiments, the therapeutic gene product is a fusion or chimeric polypeptide comprising such polypeptides and/or mature forms, active forms, fragments, variants, or derivatives thereof.

Method of Enhancing the Expression and Delivery of Virally Encoded Transgenes.

[0090] The invention also provides a method for modulating delivery of a virally encoded transgene to a subject, hi the method, a dosage inflection point is identified for a viral vector that operably encodes a transgene in a subject, wherein the viral vector is administered with a steroid or a fragment, variant, derivative, or analog thereof, hi some embodiments the steroid or fragment, variant, derivative, or analog thereof reduces the level of an inflammatory response and/or modulates Kupffer cell function. As used herein, a "dosage inflection point" is a point at which a small incremental change in the amount of virus delivered to the subject results in a substantial change in the amount of viral gene product. The inflection point is compared to levels of the virally encoded gene product in the subject. The dose of the viral vector containing the transgene and the steroid is then adjusted, if necessary, to deliver an appropriate amount of viral nucleic acid that results in the desired dose of the virally encoded transgene.

Kits.

[0091] The invention also includes kits comprising a viral nucleic acid encoding a therapeutic gene product; a steroid or a fragment, variant, derivative, or analog thereof that reduces the level of an inflammatory response and/or that modulates Kupffer cell function; and an instruction manual for use. The viral nucleic acid can be provided as part of a viral particle, if desired.

Examples

[0092] The invention will be illustrated in the following non-limiting examples.

Example 1

Injection of mice with a steroid prior to administration of adenovirus particles containing human interferon-beta nucleic acid results in enhanced expression of human interferon-beta.

[0093] The effect of pretreating animals with a steroid prior to administration of adenovirus particles operably encoding human interferon-beta nucleic acid on circulating EFN-β levels was examined.

[0094] The El and E3 deleted adenovirus H5.110CMVhIFN-β encodes human IFN-β ("AdhlFN-β"), which is driven by the cytomegalovirus (CMV) early promoter. See, e.g., Qin, et al, Proc. Natl. Acad. Sci. USA 95: 14411-14416 (1998). The virus preparations were highly purified by two rounds of cesium chloride banding and particle titers were determined as previously described. See, e.g., Nyberg-Hoffman, et al, Nat. Med. 3: 808-811 (1997); Chardonnet and Dales, Virology 40: 462-477 (1970).

[0095] Balb/c mice were injected intravenously ("i.v.") via the tail vein with various doses of recombinant adenoviruses in 100 μl phosphate buffered saline ("PBS") as specified below. Doses and virus constructs were as described below. Blood was obtained on day 3 or 4 as specified for hlFN-β assays by tail vein bleeding or cardiac puncture, sera were prepared, and samples were stored at -8O⁰C.

[0096] Interferon-beta levels were measured by an ELISA assay. Ninety-six- well plates were coated overnight at 4⁰C with an anti-human IFN-β antibody, (BO-2®; Summit Pharmaceuticals, Fort Lee, NJ). The antibody was used at 10 μg/ml in the coating buffer containing 50 mM sodium bicarbonate/carbonate, 0.2 raM MgCl₂, and 0.2 mM CaCl₂ (pH 9.6). After the plates were blocked with 0.5% non-fat dry milk in PBS for 1 hr at room temperature, IFN-β samples or IFN-β protein standards (AVONEX™, Biogen Idee), diluted in 10% normal mouse serum/0.5% non-fat dry milk/0.05% Tween-20 in PBS, were then added. After capture for 1.5 hr at room temperature, the plates were washed and successively incubated at room temperature for 1 hr with an anti-IFN-β rabbit sera (available, e.g., from Millipore, 1:2000 dilution), washed again, and then incubated 1 hr with horseradish peroxidase ("HRP")-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch, 1 :5000 dilution). Following a final wash, substrate solution (4.2 mM tetramethylbenzidine, 0.1 M sodium acetate-citric acid, pH 4.9) was then added. The reaction was stopped by the addition of 2 M hydrogen persulfate ("H₂SO₄") and absorbance was measured at 450 nm.

[0097] In one set of experiments, female Balb/c mice (n=5/group) were i.v. injected with increasing doses of prednisolone six hours prior to administration of the adenoviral vector expressing human IFN-β (the AdIFN-β vector described above) via the tail vein. The results are shown in FIG. 1. The concentration of hlFN-β in the sera was determined by ELISA on day 4 post vector dosing. Average serum hlFN-β levels are shown ± SEM.

[0098] As FIG. 1 suggests, increasing doses of prednisolone enhanced hlFN-β expression in a dose-dependent manner. Pretreatment with increasing doses of prednisolone from 1 mg/kg to 25 mg/kg was able to increase the expression levels of hlFN-β upon administration of a low dose of adenoviral vector (2 x 10 particles per mouse) in a dose-dependent manner. As compared to animals in the control group pretreated with PBS, there was an observed increase in the levels of serum hEFN-β detected of up to eight-fold when animals were pretreated with 25 mg/kg of prednisolone.

[0099] Another experiment examined the effect of different steroids and dosages on the expression of hlFN-β from the AdIFN-β vector. Female Balb/c mice (n=5) were injected intravenously with a low dose (2 x 10¹⁰ particles) of the AdhlFN-β vector 4 hours after pretreatment with a subcutaneous administration of PBS or either 5 mg/kg or 25 mg/kg of prednisolone, cortisone, or corticosterone. The serum concentration of hlFN-β was measured by ELISA 3 days after viral dosing. The results are shown in FIG. 2.

[0100] As FIG. 2 suggests, pretreatment for 4 hours with increasing doses of cortisone and corticosterone also enhanced hlFN-β expression upon administration of a low dose of the adenoviral vector (2 x 10 particles per mouse). As compared to animals pretreated with either 5 mg/kg or 25 mg/kg of prednisolone, pretreatment with cortisone and corticosterone was able to enhance serum hlFN-β expression levels more effectively than prednisolone at the lower dose (5 mg/kg).

[0101] In an additional experiment, female Balb/c mice (n=6) were pretreated with PBS or either a low dose (2 mg/kg) or a high dose (10 mg/kg) of dexamethasone either 4 or 24 hours prior to intravenous injection of a low dose (2 x 10¹⁰ particles) of AdhlFN-β vector. The serum concentration of hlFN-β was measured by ELISA 3 days after viral dosing. The results are shown in FIG. 3. Pre-administration of dexamethasone dramatically enhanced the resulting hBFN-β expression levels by as much as three-fold when compared with animals in the control group pretreated with PBS alone. Enhanced IFN-β expression was seen at both dexamethasone doses used.

[0102] These results demonstrate that pretreatment with a steroid can dramatically enhance the resulting levels of hlFN-β expressed from a viral vector. Based on this example, the disclosure herein, and knowledge in the art, one of ordinary skill would be able optimize steroid pretreatment, in terms of the type and dose of steroid, for a particular transgene.

Example 2

Injection of mice with a steroid prior to administration of adenovirus particles containing human LIGHT nucleic acid.

[0103] The effect of pretreating animals with a steroid prior to administration of adenovirus particles operably encoding human LIGHT nucleic acid on circulating LIGHT levels is examined.

[0104] An El and E3 deleted adenovirus encoding human LIGHT

("AdhLIGHT") is highly purified by two rounds of cesium chloride banding and particle titers are determined as previously described. See, e.g., Nyberg- Hoffinan, et al, Nat. Med. 3: 808-811 (1997); Chardonnet and Dales, Virology 40: 462-411 (1970).

[0105] Balb/c mice are injected intravenously ("i.v.") via the tail vein with various doses of recombinant adenoviruses in 100 μl phosphate buffered saline ("PBS") as specified below. Doses and virus constructs are as described below. Blood is obtained on day 3 or 4 as specified for hLIGHT assays by tail vein bleeding or cardiac puncture, sera are prepared, and samples are stored at -8O⁰C. LIGHT levels are measured by an ELISA assay, according to manufacturer's instructions {e.g. Human LIGHT BMS2009, Bender MedSystems GmbH, Vienna, Austria).

[0106] In one set of experiments, female Balb/c mice (n=5/group) are i.v. injected with increasing doses of prednisolone six hours prior to administration of an adenoviral vector expressing human LIGHT (AdhLIGHT) via the tail vein. The concentration of hLIGHT in the sera is determined by ELISA on day 4 post vector dosing. Average serum hLIGHT levels are determined as ± SEM.

[0107] It is expected that increasing doses of prednisolone will enhance hLIGHT expression in a dose-dependent manner. Pretreatment with increasing doses of prednisolone from 1 mg/kg to 25 mg/kg is expected to increase the expression levels of hLIGHT upon administration of a low dose of an AdhLIGHT vector (2 x 10 particles per mouse) in a dose-dependent manner.

[0108] Another experiment examines the effect of different steroids and dosages on the expression of hLIGHT from an AdhLIGHT vector. Female Balb/c mice (n=5) are injected intravenously with a low dose (2 x 10¹⁰ particles) of an AdhLIGHT vector 4 hours after pretreatment with a subcutaneous administration of PBS or either 5 mg/kg or 25 mg/kg of prednisolone, cortisone, or corticosterone. The serum concentration of hLIGHT is measured by ELISA 3 days after viral dosing.

[0109] It is expected that pretreatment for 4 hours with increasing doses of cortisone and corticosterone enhances hLIGHT expression upon administration of a low dose of an AdhLIGHT vector (2 x 10 particles per mouse). As compared to animals pretreated with either 5 mg/kg or 25 mg/kg of prednisolone, pretreatment with cortisone and corticosterone is expected to enhance serum hLIGHT expression levels more effectively than prednisolone at the lower dose (5 mg/kg).

[0110] In an additional experiment, female Balb/c mice (n=6) are pretreated with PBS or either a low dose (2 mg/kg) or a high dose (10 mg/kg) of dexamethasone either 4 or 24 hours prior to intravenous injection of a low dose (2 x 10¹⁰ particles) of an AdhLIGHT vector. The serum concentration of hLIGHT is measured by ELISA 3 days after viral dosing. Pre-administration of dexamethasone is expected to dramatically enhance the resulting hLIGHT expression levels when compared with animals in a control group that are pretreated with PBS alone. It is expected that both dexamethasone doses will result in enhanced hLIGHT expression.

Example 3

Adenoviruses encoding lacZ also enhanced adenoviral EFN-β gene expression when administered nor to the adenovirus encoding IF N-β.

[0111] In order to compare the extent to which pretreatment with steroids can enhance hlFN-β expression, an adenovirus encoding lacZ ("AdLacZ") was administered prior to the adenovirus encoding hlFN-β. See also US 2004- 0086486 Al, herein incorporated by reference in its entirety, describing pretreatment with a viral vector not expressing the therapeutic product prior to administration of the viral vector expressing the therapeutic product.

[0112] Female Balb/c mice (n=5) were injected intravenously with 0.1 x 10¹⁰,

0.3 x 10¹ , or 1 x 10¹⁰ particles of AdhlFN-β vector 4 hours after pretreatment with an intravenous administration of PBS, 10 mg/kg dexamethasone, or AdlacZ vector (5 x 10¹⁰ particles). The serum concentration of hIFN-β (n=5/group, average ± SEM shown) was measured by ELISA 3 days after viral dosing. The results are shown in FIG. 4. When AdlacZ pre-dosing was performed, significant hJFN-β serum levels were observed following administration of very low doses of AdhlFN-β, and the relationship between virus dose and serum hlFN-β was roughly linear. [0113] The time course of administration of the steroid was next examined in vivo. Female Balb/c mice (n=5) were pretreated at various time points ranging from 4 hours to 96 hours as indicated in FIG. 5 with 10 mg/kg of dexamethasone prior to intravenous administration of the AdhlFN-β vector (5 x 10¹ particles), hi parallel, animals were also pretreated with either intravenous administration of the AdlacZ reporter vector (5 x 10¹⁰ particles) or PBS 4 hours prior to intravenous administration of the AdhlFN-β vector. The serum concentration of hlFN-β was determined by ELISA 3 days after viral dosing. The results are shown in FIG. 5.

[0114] Pretreatment with 10 mg/kg of dexamethasone for various time points ranging from 4 hours to 96 hours prior to administration of the adenoviral vector resulted in an enhanced expression of hlFN-β levels as compared with animals in the control group pretreated with PBS, as shown in FIG. 5. There was an approximate 3- to 10-fold increase in the hlFN-β expression level. Consistent with earlier data, animals pretreated with an adenoviral vector encoding the lacZ reporter gene had an even higher hlFN-β expression than the steroid-pretreated mice, resulting in an approximately 300-fold increase in serum hlFN-β levels.

[0115] The effect of pretreatment with steroids or with a control adenovirus was also tested in two different mouse strains. Athymic nude mice (FIG. 6A) or Balb/C mice (FIG. 6B) were intravenously injected with a low dose (1 x 10¹⁰ particles) of AdhlFN-β vector 4 or 24 hours after pretreatment with intravenously administered 10 mg/kg dexamethasone. In parallel, the mice were pretreated with either PBS or intravenously administered AdlacZ vector (5 x 10¹⁰ particles) for 4 hours prior to intravenous administration of the AdhEFN-β vector. The serum concentration of hlFN-β was determined by ELISA 4 days after viral dosing. The results are shown in FIG. 6. Female athymic nude mice (FIG. 6A) or Balb/C mice (FIG. 6B) strains gave essentially similar results in terms of enhancement of hlFN-β levels after pretreatment with dexamethasone or with an adenovirus expressing the lacZ gene. Pretreatment with 10 mg/kg of dexamethasone for 4 or 24 hours prior to administration of the adenoviral vector expressing hlFN-β resulted in a 4-fold and 5-fold increase in serum hDFN-β levels in the athymic nude mice (FIG. 6A) and Balb/c mice (FIG. 6B), respectively. Interestingly, pretreatment with ^lO-

an adenovirus expressing the lacZ reporter gene gave higher EFN-β levels than steroid pretreatment in both the athymic nude and Balb/c mice.

Example 4

Pretreatment with a steroid resulted in a decreased inflammatory response to administered adenovirus.

[0116] The effect of steroid pretreatment on the inflammatory response to administered virus was examined by measuring levels of endogenous cytokines in animals injected with adenovirus particles operably encoding luciferase nucleic acid.

[0117] Female Balb/c mice (n=5) were injected intravenously with either PBS or 10 mg/kg dexamethasone in PBS 4 hours prior to intravenous administration of PBS or 5 x 10¹⁰ particles of AdLUX vector. Small amounts of blood were collected via retro-orbital bleeds at 30 minutes, 60 minutes, 90 minutes, and 120 minutes post-adeno viral treatment, and serum was isolated. The serum was then analyzed for an inflammatory cytokine profile using the BD™ Cytometric Bead Array Mouse Inflammation Kit (BD™ CBA Mouse Inflammation Kit, BD Biosciences), specifically measuring levels of IL-6, MCP-I, and TNF-α. The results are shown in FIG. 7A-7C (DEXPBS = dexamethasone pretreatment followed 4 hours later with injection of PBS; DEXLUX = dexamethasone pretreatment followed 4 hours later with injection of AdLUX; PBSPBS = PBS pretreatment followed 4 hours later with injection of PBS; PBSLUX = PBS pretreatment followed 4 hours later with injection of AdLUX).

[0118] As FIG. 7 suggests, pretreatment with dexamethasone significantly reduced the inflammatory response resulting from administration of AdLUX as seen by expression of the markers IL-6, MCP-I, and TNF-α. As expected, pretreatment with PBS followed by another injection of PBS or pretreatment with dexamethasone followed by an injection of PBS resulted in no inflammatory response. In contrast, pretreatment with PBS followed by injection of AdLUX resulted in a significant increase in IL-6 (FIG. 7A), MCP- 1 (FIG. 7B), and TNF-α (FIG. 7C) shortly after injection with AdLUX. In contrast, pretreatment with dexamethasone followed by injection of AdLUX was able to significantly inhibit increases in the levels of these markers. [0119] These results suggest that pretreatment with a steroid can dramatically reduce the inflammatory response induced by adenoviral vector administration.

Equivalents.

[0120] From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that novel compositions and methods involving nucleic acids, polypeptides, gene therapy, and treatment have been described. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention as a matter of routine for a person of ordinary skill in the art without departing from the spirit and scope of the invention as defined by the claims. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures.

[0121] All documents cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued or foreign patents, or any other documents, are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.

Claims

WHAT IS CLAIMED:

1. A method for enhancing transgene expression of a viral vector- encoded therapeutic gene product comprising administering to a subject:

(a) a viral vector comprising a nucleic acid which operably encodes said therapeutic gene product; and

(b) a steroid or a fragment, variant, derivative, or analog thereof,

wherein said steroid or fragment, variant, derivative, or analog thereof is administered prior to or concurrently with administration of said viral vector.

2. The method according to claim 1, wherein said steroid is selected from the group consisting of prednisolone, cortisone, corticosterone, and dexamethasone.

3. The method according to claim 2, wherein said steroid is prednisolone.

4. The method according to claim 1, wherein said viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a baculoviral vector, an Epstein Ban- viral vector, a papovaviral vector, a vaccinia viral vector, and a herpes simplex viral vector.

5. The method according to claim 1, wherein said steroid is administered prior to administering said viral vector.

6. The method according to claim 5, wherein said steroid is administered 24 hours or less prior to administering said viral vector.

7. The method according to claim 6, wherein said steroid is administered 1 hour or less prior to administering said viral vector.

8. The method according to claim 7, wherein said steroid is administered 5 minutes or less prior to administering said viral vector.

9. The method according to claim 1 , wherein said steroid is administered concurrently with said viral vector.

10. The method according to claim 1, wherein said subject is a rodent.

11. The method according to claim 1 , wherein said subject is a primate.

12. The method according to claim 11, wherein said primate is a human.

13. The method according to claim 1, wherein said viral vector is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of said routes of administration.

14. The method according to claim 1, wherein said steroid is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of said routes of administration.

15. The method according to claim 1, wherein said therapeutic gene product is selected from the group consisting of LIGHT, interferon-β, herpes simplex virus thymidine kinase, p53, and a combination of two or more of said gene products.

16. The method according to claim 15, wherein said therapeutic gene product is LIGHT.

17. The method according to claim 15, wherein said therapeutic gene product is interferon-β.

18. The method according to claim 17, wherein said therapeutic gene product is human interferon-β.

19. The method according to claim 1, wherein said steroid reduces the level of an inflammatory response by lowering said subject's production of cytokines.

20. The method according to claim 19, wherein said cytokine is selected from the group consisting of IL-lα, IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-I l, IL-12, IL-12A, IL-12B, IL-13, IL-15, IL-16, IL- 17, IL-18, IL-20, IL-21, IL-25, IFN-α, IFN-β, TNF-α, and a combination of two or more of said cytokines.

21. The method according to claim 1, wherein said steroid reduces the level of an inflammatory response by lowering said subject's production of chemokines.

22. The method according to claim 21, wherein said chemokine is selected from the group consisting of 1-309, MCP-I, MCP-2, MCP-3, MCP-4, MIP-Ia, MIP-Ib, MIP-Id, MIP-3a, MIP-3b, HCC-4, TARC, PARC, SLC, MDC, MPIF-I, MPIF-2, TECK, RANTES, eotaxin, eotaxin-3, CTACK, CCL28, GROl (MIP-2), GRO2, GRO3, ENA-78, GCP-2, MIG, IP-IO, I-TAC, SDFl, CXCL4, CXCL7, CXCL13, CXCL14, CXCLl 6, and a combination of two or more of said chemokines.

23. The method according to any one of the preceding claims 1-22, wherein said viral vector is an adenovirus vector.

24. The method according to any one of preceding claims 1-22, wherein said viral vector is a replication-defective viral vector.

25. A method for modulating delivery of a viral vector operably encoding a transgene to a subject, the method comprising:

(a) identifying a dosage inflection point of said viral vector, wherein said viral vector is administered with a steroid or a fragment, variant, derivative, or analog thereof;

(b) comparing said inflection point to levels of a therapeutic product of said transgene expressed in said subject; and

(c) adjusting if necessary the doses of said viral vector and said steroid administered to said subject, thereby modulating the dosage of said transgene.

26. The method according to claim 25, wherein said steroid is selected from the group consisting of prednisolone, cortisone, corticosterone, and dexamethasone.

27. The method according to claim 26, wherein said steroid is prednisolone.

28. The method according to claim 25, wherein said viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, and a herpes simplex viral vector.

29. The method according to claim 25, wherein said steroid is administered prior to administering said viral vector.

30. The method according to claim 29, wherein said steroid is administered 24 hours or less prior to administering said viral vector.

31. The method according to claim 30, wherein said steroid is administered 1 hour or less prior to administering said viral vector.

32. The method according to claim 31, wherein said steroid is administered 5 minutes or less prior to administering said viral vector.

33. The method according to claim 25, wherein said steroid is administered concurrently with said viral vector.

34. The method according to claim 25, wherein said subject is a rodent.

35. The method according to claim 25, wherein said subject is a primate.

36. The method according to claim 35, wherein said primate is a human.

37. The method according to claim 25, wherein said viral vector is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of said routes of administration.

38. The method according to claim 25, wherein said steroid is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of said routes of administration.

39. The method according to claim 25, wherein said therapeutic gene product is selected from the group consisting of LIGHT, interferon-β, herpes simplex virus thymidine kinase, p53, and a combination of two or more of said gene products.

40. The method according to claim 39, wherein said therapeutic gene product is LIGHT.

41. The method according to claim 39, wherein said therapeutic gene product is interferon-β.

42. The method according to claim 40, wherein said therapeutic gene product is human interferon-β.

43. The method according to claim 25, wherein said steroid reduces the level of an inflammatory response by lowering said subject's production of cytokines.

44. The method according to claim 43, wherein said cytokine is selected from the group consisting of IL-lα, IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-I l, IL-12, IL-12A, IL-12B, IL-13, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-25, IFN-α, IFN-β, TNF-α, and a combination of two or more of said cytokines.

45. The method according to claim 25, wherein said steroid reduces the level of an inflammatory response by lowering said subject's production of chemokines.

46. The method according to claim 45, wherein said chemokine is selected from the group consisting of 1-309, MCP-I, MCP-2, MCP-3, MCP-4, MIP-Ia, MIP-Ib, MIP-Id, MIP-3a, MIP-3b, HCC-4, TARC, PARC, SLC, MDC, MPIF-I, MPIF-2, TECK, RANTES, eotaxin, eotaxin-3, CTACK, CCL28, GROl (MIP-2), GRO2, GRO3, ENA-78, GCP-2, MIG, IP-10, I-TAC, SDFl, CXCL4, CXCL7, CXCL13, CXCL14, CXCLl 6, and a combination of two or more of said chemokines.

47. The method according to any one of preceding claims 25-46, wherein said viral vector is an adenovirus vector.

48. The method according to any one of preceding claims 25-47, wherein said viral vector is a replication-defective viral vector.

49. A kit for delivering a viral vector encoding a therapeutic gene product, the kit comprising:

(a) a viral vector comprising a nucleic acid which operably encodes said therapeutic gene product,

(b) a steroid, or a fragment, variant, derivative, or analog thereof, and

(c) an instruction manual for use.

50. The kit according to claim 49, wherein said steroid is selected from the group consisting of prednisolone, cortisone, corticosterone, dexamethasone, and a combination of two or more of said steroids.

51. The kit according to claim 50, wherein said steroid is prednisolone.

52. The kit according to claim 49, wherein said viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, and a herpes simplex viral vector.

53. The kit according to claim 49, wherein said therapeutic gene product is selected from the group consisting of LIGHT, interferon-β, herpes simplex virus thymidine kinase, p53, and a combination of two or more of said gene products.

54. The kit according to claim 53, wherein said therapeutic gene product is LIGHT.

55. The kit according to claim 53, wherein said therapeutic gene product is interferon-β.

56. The kit according to claim 55, wherein said therapeutic gene product is human interferon-β.

57. The kit according to claim 49, wherein said steroid reduces the level of an inflammatory response by lowering said subject's production of cytokines.

58. The kit according to claim 57, wherein said cytokine is selected from the group consisting of IL-lα, IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL-12A, IL-12B, IL-13, IL-15, IL-16, IL-17, IL- 18, IL-20, IL-21, IL-25, EFN-α, IFN-β, TNF-α, and a combination of two or more of said cytokines.

59. The kit according to claim 49, wherein said steroid reduces the level of an inflammatory response by lowering said subject's production of chemokines.

60. The kit according to claim 59, wherein said chemokine is selected from the group consisting of 1-309, MCP-I, MCP-2, MCP-3, MCP-4, MIP- Ia, MIP-Ib, MIP-Id, MIP-3a, MIP-3b, HCC-4, TARC, PARC, SLC, MDC, MPIF-I, MPIF-2, TECK, RANTES, eotaxin, eotaxin-3, CTACK, CCL28, GROl (MIP-2), GR02, GRO3, ENA-78, GCP-2, MIG, IP-IO, I-TAC, SDFl, CXCL4, CXCL7, CXCL13, CXCL14, CXCLl 6, and a combination of two or more of said chemokines.

61. The kit according to any one of preceding claims 49-60, wherein said viral vector is an adenovirus vector.

62. The kit according to any one of preceding claims 49-61, wherein said viral vector is a replication-defective viral vector