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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • A61K39/46—Cellular immunotherapy
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  • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
  • A61K39/464416—Receptors for cytokines
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  • A61K39/4643—Vertebrate antigens
  • A61K39/4644—Cancer antigens
  • A61K39/46443—Growth factors
  • A61K39/464434—Transforming growth factor [TGF]
• • A—HUMAN NECESSITIES
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  • A61K39/46—Cellular immunotherapy
  • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
  • A61K39/4643—Vertebrate antigens
  • A61K39/4644—Cancer antigens
  • A61K39/464436—Cytokines
  • A61K39/46444—Interleukins [IL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• the present invention relates to the induction of tolerance to an adenoviral vector (and its adenovirus antigens) comprising a transgene and/or its transgene product in a host, thereby allowing persistent transgene expression in the host, and allowing repeated vector administration to the host while minimizing or eliminating the host immune response.
• the invention provides for pretreatment of the host with an intravenous administration of dendritic cells or other antigen presenting cells infected with an adenoviral vector comprising heterologous nucleic acids encoding a transgene and a heterologous nucleic acid encoding an immunomodulatory molecule.
• the adenoviral vector-infected dendritic cells of the invention migrate predominantly to the lymphoid organs, such as the spleen, where they interact with T cells resulting in high levels of expression of the transgene and immunomodulatory molecule therein.
• the present invention also provides for the infection of dendritic cells in vivo by intradermal injection of an adenoviral vector comprising a transgene and a heterologous nucleic acid encoding an immunomodulatory molecule.
• the dendritic cells thus infected will migrate to the regional lymph nodes where they would interact with T cells.
• the present invention provides for the clonal deletion of T cells through fas/fasL mediated apoptosis of T cells.
• the method further provides for subsequent administration to the host of an adenoviral vector comprising said transgene wherein the host immune response is reduced or eliminated to said adenoviral vector and said transgene, even upon repeated administration of said adenoviral vector.
• transgene The ability to deliver a transgene to a target cell or tissue and have it expressed therein to produce a desired phenotypic effect depends on the development of gene transfer vehicles that can safely and efficiently deliver an exogenous nucleic acid (transgene) to the recipient cell. To this end, most efforts have focused on the use of virus-derived vectors in order to exploit the natural ability of a virus to deliver its genetic content to a target cell.
• retroviral vectors which have been the vectors of choice to deliver transgene coding for a biologically active molecules for gene therapy because of their ability to integrate into the cellular genome.
• retroviral vectors have become evident, including their tropism for dividing cells only, the possibility of insertional mutagenesis in the host genome upon integration of the vector nucleic acid into the host cell genome, decreased expression of the transgene over time, rapid inactivation of retroviruses by the serum complement system, and the possibility of generating replication-competent retroviruses (Jolly, D., Cancer Gene Therapy 1:51-64, 1994; Hodgson, C.P., Bio/Technology 13:222-225, 1995).
• Adenovirus a nuclear double-stranded DNA virus with a genome of about 36 kb, has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M.S., "Adenoviridae and Their Replication,” in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990).
• the adenovirus genome is classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication.
• the cloning capacity of an adenoviral vector is proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, and the deletion of a genomic region such as El whose function may be restored in trans from 293 cells (Graham, F.L., J. Gen. Virol. 36:59-72, 1977) or A549 cells (Imler et al., Gene Therapy 3:75-84, 1996). Such El-deleted vectors are rendered replication-defective.
• the upper limit of vector DNA capacity for optimal carrying capacity is about 105 -108% of the length of the wild-type genome.
• Further adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g., complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Caravokyri et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6: 1575-1586, 1995).
• Adenovirus-derived vectors have several advantages, including tropism for both dividing and non-dividing cells, lessened pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large DNA inserts (Berkner, K.L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
• the cloning capacity of an adenoviral vector is a factor of the deletion of certain regions of the virus genome dispensable for virus growth (e.g., E3) or deletions of regions whose function is restored in trans from a packaging cell line (e.g., El, with complementation of El functions by 293 cells (Graham, F.L., J. Gen. Virol. 36:59-72, 1977).
• the upper limit for optimal packaging may be extended to about 105%-108 of wild-type adenoviral genome length for increased carrying capacity in the vector.
• Genes that have been expressed to date by adenoviral vectors include p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130- 134, 1993); erythropoietin (Descamps et al., Human Gene Therapy 5:979-985, 1994); oraithine transcarbamylase (Stratford-Perricaudet et al., Human Gene Therapy 1:241-256, 1990; We et al., J. Biol. Chem.
• PCT/US98/22886 human beta- galactosidase (Arthur et al., Cancer Gene Therapy 4:17-25, 1997); interleukin-7 (Arthur et al, Cancer Gene Therapy 4:17-25, 1997); a Herpes Simplex Virus thymidine kinase gene (U.S. Patent No. 5,763,415) and cystic fibrosis transmembrane conductance regulator (CFTR) (U.S. Patent Nos. 5,670,488 and 5,882,877; Zabner et al., J. Clin. Invest. 97:1504-1511, 1996).
• CFTR cystic fibrosis transmembrane conductance regulator
• adenoviral vectors which carry the CFTR gene have been developed (Rich, D.
• These foreign molecules can be viral or other parasite proteins; these proteins can be present in the circulatory system of the body or present only within certain cells in the body.
• This form of immunity is based on the ability of the body to recognize foreign agents and to respond with the production of antibodies, so-called humoral immunity, or lymphocytes with killing activity, so-called cellular immunity.
• the effectors of humoral and cellular immunity have a high degree of specificity and immunological memory.
• innate immunity The ability of macrophages to engulf particles, cytokine responses as part of the inflammatory response and the recognition of bacterial DNA methylation patterns are all examples of innate immunity.
• Viruses are examples of infectious agents to which the body responds with both innate and specific immunity. Viral structural proteins can stimulate antibody responses which can neutralize virus infectivity, and viral proteins synthesized within the cell can stimulate a cellular immune response which can target virus-infected cells for destruction. Moreover, viruses can provoke a non-specific inflammatory response as well. Vectors based on viruses can, in theory, be affected by specific and innate immune responses, depending on whether viral genes are retained within the vector and whether the viral genes are expressed at levels sufficient to provoke an immune response. The level at which viral genes are expressed is potentially very significant, since inflammatory and immune responses are proportional to dose. A low dose of antigen can escape detection by the body while a large dose is far more likely to be detected and to provoke a response.
• viral-based vectors are believed to present less of an immunological problem.
• AAV and retroviral vectors for example, in most instances have had all viral genes deleted, thus eliminating the possibility that a cellular immune response will be generated against viral gene products.
• These vectors can, nonetheless, stimulate antibody responses to the virion coat proteins if sufficient amounts of vector are introduced into the body.
• El -deleted replication-defective adenoviral (Ad) vectors are attractive vehicles for gene transfer to host cells because of their ability to transduce a wide variety of dividing and non-dividing cells in vivo (Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256 (1990); Rosenfeld et al., CeU 68:143-155 (1992); Zabner et al., CeU 75:207- 216 (1993); Crystal et al.. Nat. Genetics 8:42-51 (1994); Zabner et al., Nat. Genetics 6:75-83 (1994)).
• Such vectors have been used for transfer of the gene encoding normal human cystic fibrosis transmembrane conductance regulator (CFTR) into airway epithelial cells of experimental animals (e.g. mice, cotton rats, monkeys) and to airway epithelium of individuals with cystic fibrosis (CF) (Rosenfeld et al., CeU 68:143-155 (1992); Zabner et al., CeU 75:207- 216 (1993); Crystal et al.. Nat. Genetics 8:42-51 (1994); Zabner et al.. Nat. Genetics 6:75-83 (1994)).
• CFTR cystic fibrosis transmembrane conductance regulator
• Ad vectors The treatment of chronic diseases such as CF with Ad vectors will likely require repeated administrations of Ad vectors containing the CFTR gene throughout the lifetime of the patient.
• effectiveness of current Ad vectors is limited by the diffi- culty in obtaining successful readministration to an individual using a vector of the same Ad serotype, because of adverse immunologic responses.
• Various groups have demonstrated that a strong dose-dependent humoral immune response is induced by Ad vectors leading to the development of Ad-specific neutralizing antibodies, which leads to the inactivation by the host of readministered vector. (Yang et al., J. Virol. 69:2004-2015 (1995); Kaplan et al., Gene Ther. 3: 117-127 (1996); Smith et al., Gene Ther. 5:397-402 (1993); Yei et al., Gene Ther.
• PAV pseudoadenoviral vectors
• PAV vectors which are adenoviral vectors derived from the genome of an adenovirus containing minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes
• PAV vectors which can accommodate up to 36 kb of foreign nucleic acid, are advantageous because the carrying capacity of the vector is optimized, while the potential for host immune responses to the vector or the generation of replication- competent viruses is reduced.
• PAV vectors contain the 5' inverted terminal repeat (ITR) and the 3' ITR nucleotide sequences that contain the origin of replication, and the s-acting nucleotide sequence required for packaging of the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements.
• ITR inverted terminal repeat
• CTLA4-Ig blocks the B7-CD28 pathway of T cell co-stimulation, which is required for optional activation of T cells.
• Ad-specific antibody levels were reduced in CTLA4-Ig treated mice, the inhibition was not sufficient to allow secondary gene transfer via repeat administration of the vector under the conditions tested (Kay et al., Nat. Genetics 11:191-197 (1995)).
• IL-12 interferon- ⁇
• IL-12 interleukin-12
• INF- ⁇ is known to upregulate MHC class I on antigen presenting cells (Yang et al., Proc. Natl. Acad. Sci. USA 22:7257- 7261 (1995)).
• both INF- ⁇ and IL-12 while capable of inhibiting humoral immunity, might enhance the elimination of Ad vector transduced cells by CTLS (enhanced T hl response).
• interleukin-10 interleukin-10
• IL-10 interleukin-10
• IL-10 Viral IL-10 (vIL-10), a product of the Epstein Barr virus is homologous to both murine and human IL-10 and while vIL-10 shares many biological properties with murine IL-10 and human IL-10, it does not possess some of the immuno- stimulatory activities of cellular IL-10 (de Waal Malefyt et al, J. Exp. Med. 174:915).
• TGF ⁇ transforming growth-factor- ⁇
• TGF ⁇ promotes a reversible phenotypic transformation of non-neoplastic fibroblasts and was originally described to inhibit generation of allogeneic CTL and T cell growth induced by interleukin 2 (IL2) (Fontana et al., J. Immunol. 132:1837, 1984). More recently, TGF ⁇ has been shown to inhibit the generation of cytotoxic T cells in virus-infected mice (Fontana et al., J. Immunol. 143:3230, 1995).
• IL2 interleukin 2
• Still another strategy used to overcome the immune response to adenoviral vector proteins as well as transgene product is the induction of peripheral T-cell tolerance to both the adenoviral vector and the transgene products which, in many cases, may be a neoantigen in the patient.
• activation-induced cell death in T cells in which apoptosis of the T cells is mediated by upregulation of fas and fas ligand (fasL) is responsible for down regulation of the T-cell response and T cell homeostasis (Watanabe-Fukunaga et al., Nature 314-317, 1992; Zhou et al., J. Exp. Med.
• fasL mammalian fas/CD95 ligand
• the p35 gene of baculovirus encodes a protein which blocks apoptosis of baculovirus-infected cells (Clem et al., Science 254:1388-1389, 1991; Hershberger et al., J. Virol. 68:3467-3477, 1994; Bump et al., Science 269:1885-1888, 1995; Xue et al., Nature 377: 248-251, 1995).
• One mechanism of tolerance is via the natural occurrence of immune privileged sites.
• the first is that the method used for expression of the fasL gene in the antigen-presenting cell line involves coinfection with two different adenoviral vectors, AdLoxpfasL (Zhang et al., J. Virol. 72:2483- 2490, 1998) and AxCANCre (Kanegae et al, Nucl. Acids Res. 23:3816-3821, 1995).
• AdLoxpfasL Zahang et al., J. Virol. 72:2483- 2490, 1998)
• AxCANCre Kanegae et al, Nucl. Acids Res. 23:3816-3821, 1995.
• AxCANCre expresses the Cre recombinase and is required to turn on expression of the fasL gene from the AdLoxpfasL vector.
• an extra component besides the vector which contains the fas gene is required for its effective delivery.
• Ad vectors constitutively expressing fasL cannot be grown to high titers, because fasL induces apoptosis of the 293 cells used to propagate the Ad vectors (Larregina et al., Gene Ther. 5:563-568, 1998).
• the antigen-presenting cell line used was derived from a fas-mutant B6-lpr lpr mouse, since fasL expression can kill normal cells expressing the fas receptor (Muruve et al., Hum. Gene Ther. 8:955-963, 1997; Kang et al., Nature. Med. 3:738-743, 1997 and Larregina et al., Gene Ther. 5:563-568, 1998). Therefore, the expression of fasL would be extremely difficult to accomplish in cells derived from normal individuals.
• APCs antigen presenting cells
• DCs dendritic cells
• macrophages macrophages
• B cells are involved in the generation of an optimal immune response to pathogens, alloantigens and tumors.
• Dendritic cells are highly specialized APCs of the immune system (Steinman, Annu. Rev. Immunol. 2:271, 1991).
• the strategic positioning of DCs in nonlymphoid tissue and their ability to circulate via blood and lymph to lymphoid organs after antigen stimulation demonstrate their important role in the induction of immune responses against invading pathogens (Hoefsmit et al., Immunobiology 161:255. 1982; Austyn et al., J. Exp. Med. 166:646, 1988).
• DCs such as Langerhans Cells (LCs) are thought to undergo characteristic modulations of function and phenotype (Larsen et al., J. Exp. Med. 172:1483, 1990; Schuler et al., . Exp. Med. 161:526, 1985).
• LCs Langerhans Cells
• Locally produced inflammatory cytokines and the encounter with an antigen promote the maturation and migration of DCs to regional lymph nodes.
• DCs undergo a differentiation from a processing to a presenting functional cell type, characterized by the expression of co-stimulatory molecules, cytokine production, and a typical morphology ( Schuler et al., . Exp. Med.
• IL-10 treated human dendritic cells can induce tumor- antigen specific anergy in CD8+ T cells (Suzuki et al., J. Exp. Med. 182:477-486, 1995; Steinbringk et al., Blood 23: 1634-1642). It has also been recently shown that IL-10-treated human DCs, generated from peripheral blood, induce a state of antigen specific anergy in various populations of CD4+ cells (Steinbrink, et al., J. Immunol. 159:4772. 1997).
• the present invention is directed to reducing the immune response of a host to administered adenoviral (Ad) vectors carrying a transgene coding for a biologically active molecule, thereby allowing for repeat administration of such vectors to the host without (or with minimized) adverse immune responses.
• Ad adenoviral
• the present invention is also directed to reducing the immune response of a host to immunogenic polypeptides encoded by a transgene without (or with minimized) adverse immune responses to promote the efficacy of the therapeutic effect of the transgene within the host.
• the immune response to Ad vectors appears to be mediated through activation of CD4+ cells by viral antigens leading to CD8+ cytotoxic T lymphocyte (CTL) and humoral immune responses to the Ad vector.
• CTL cytotoxic T lymphocyte
• IL-10 treated human dendritic cells can induce tumor- antigen specific anergy in CD8+ T cells and in CD4+ T cells.
• TGF ⁇ is also a potent immunosuppressant.
• the present invention provides for the pretreatment of a host with an intravenous administration of DCs or other antigen presenting cells which have been infected with an Ad vector comprising a transgene coding for a biologically active molecule and a heterologous nucleic acid encoding an immunosuppressant molecule such as IL-10, vIL-10 and/or TGF ⁇ .
• an Ad vector comprising a transgene coding for a biologically active molecule and a heterologous nucleic acid encoding an immunosuppressant molecule such as IL-10, vIL-10 and/or TGF ⁇ .
• the dendritic cells migrate predominantly to the lymphoid organs like the spleen where they interact with T cells resulting in the expression of IL-10, vIL-10 and/or TGF ⁇ therein.
• IL-10 acts on the DCs making them tolerogenic and TGF ⁇ acts on the splenic T cells, B cells and macrophages.
• a further method for inducing tolerance to an adenoviral vector and/or transgene product in a host entails clonal deletion of antigen specific T cells mediated by apoptosis of T cells via the fas-fasL pathway.
• the present invention also provides for the pretreatment of a host with an intravenous administration of DCs or other antigen presenting cells which have been infected with an Ad vector comprising a nucleic acid encoding fasL, as well as an inhibitor of apoptosis, such as p35.
• an Ad vector comprising a nucleic acid encoding fasL, as well as an inhibitor of apoptosis, such as p35.
• the present invention further provides for the infection of DCs in vivo by intradermal injection of an Ad vector comprising a transgene coding for a biologically active molecule and a heterologous nucleic acid encoding an immunosuppressant molecule such as IL-10, vIL-10 and/or TGF ⁇ or alternatively encoding fasL as well as an inhibitor of apoptosis, such as p35.
• DCs thus infected will migrate to the regional lymph nodes where they would interact with T cells.
• Figure 1 shows an Ad2 vector encoding TGF ⁇ .
• Figure 2 shows an Ad2 vector encoding TGF ⁇ and CFTR.
• Figure 3 shows an Ad2 vector encoding TGF ⁇ and vIL-10.
• Figure 4 shows an Ad2 vector encoding vIL-10.
• Figure 5 shows an Ad2 vector encoding vIL-10 and CFTR.
• Figure 6 shows an Ad2 vector encoding vIL-10 and ⁇ gal.
• Figure 7 shows an Ad2 vector encoding fasL and p35.
• Figure 8 shows the results of the LDH assay.
• Figure 9 shows a DeAd vector genome.
• Figure 10 shows a map of the plasmid pCMV.
• Figure 11 shows a map of the plasmid pAd/E4+/E3 ⁇ 2.9.
• Figure 12 shows an Ad2 vector encoding a transgene, fasL and p35.
• the present invention is directed to diminishing or inhibiting adverse immunological responses in a host individual to an administered Ad vector comprising a transgene coding for a biologically active molecule.
• the invention involves pretreatment of the host with an intravenous administration of dendritic cells or other antigen presenting cells infected with and Ad vector comprising heterologous nucleic acids encoding an immunosuppressant molecule or a molecule capable of mediating T cell apoptosis, and a transgene coding for a biologically active molecule.
• Ad vector-infected dendritic cells allows for repeat administration of the Ad vector comprising the same transgene to the host with minimized adverse immunological consequences and persistent transgene expression in treated host cells.
• the nucleic acid encoding an immunosuppressant molecule or a molecule capable of mediating T cell apoptosis and a transgene may be cloned into any recombinant adenoviral vector suitable for the delivery of a desired nucleic acid to a recipient cell.
• the adenoviral (Ad) vector is derived from adenovirus serotype 2 (Ad 2) and has a substantially deleted El and E3 region.
• Ad 2 adenovirus serotype 2
• Ad 39 Ad 39. From these enumerated adenovirus serotypes, Ad 2, Ad 5, Ad 6 and Ad 17 are preferred.
• the adenoviral vector comprises an adenovirus genome in which the El region and a 1.6 kb portion in the E3 region of the adenovirus genome from nucleotides 29292-30840 are deleted as disclosed in U.S. Application Ser. No. 08/839,553, incorporated herein by reference.
• the adenoviral vector is Ad2/CMV/E3 ⁇ 1.6.
• the vector is a partially-deleted adenoviral (DeAd) vector in which a majority of the adenovirus early genes (E1-E4) required for virus replication have been deleted from the vector.
• the DeAd vector is described in co-pending provisional application Ser. Nos. 60/083,841 filed May 1, 1998 and 60/118,118 filed February 1, 1999 and their corresponding international application No. PCT/US99/09590 filed April 30, 1999, incorporated herein by reference.
• the deleted adenovirus genes with the possible exception of E3, which is not required for replication, are inserted into a producer cell chromosome under the control of a conditional promoter in order to facilitate vector production.
• a preferred producer cell is the human 293 cell line which was established by transfecting human embryonic kidney cells with fragments of the Ad5 genome and selecting for a transformed phenotype (Graham, F.L. et al., J. Gen Virol. 36:59-72, 1997).
• the A549 cell line and the KB cell line may also be used (all of which are available from ATCC).
• the adenoviral vectors are pseudoadenoviral vectors (PAV) which are derived from the genome of an adenovirus which contain minimal c/s-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (See, U.S. Patent No. 5,882,877 which covers PAV vectors and methods for producing PAV, incorporated herein by reference).
• PAV vectors can accommodate up to 36 kb of foreign nucleic acid.
• heterologous nucleic acid encoding immunosuppressant molecules e.g., IL-10, vIL-10 and TGF ⁇
• the transgene coding for a biologically active molecules and the nucleic acids encoding fas, fasL, and p35 may be operably linked to expression control sequences, e.g., a promoter that directs expression of the transgene or the immunomodulatory molecule.
• expression control sequences e.g., a promoter that directs expression of the transgene or the immunomodulatory molecule.
• operably linked refers to the functional relationship of a polynucleotide/transgene with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
• operative linkage of a nucleic acid to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter, such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter, and wherein the promoter directs the transcription of RNA from the polynucleotide.
• Promoter regions include specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. Additionally, promoter regions include sequences that modulate the recognition, binding and transcription initiation activity of RNA polymerase. Such sequences may be cis acting or may be responsive to trans acting factors. Depending upon the nature of the regulation, promoters may be constitutive or regulated.
• promoters examples include SP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter and CMV-derived promoters, mouse mammary tumor virus (MMTV) steroid- inducible promoter, Moloney murine leukemia virus (MMLV) promoter, Rous sarcoma virus long terminal repeat (RSV-LTR) viral promoters, phosphoglycerate kinase (PGK) promoter and the like.
• the promoter may be an endogenous adenovirus promoter, for example the El a promoter or the Ad2 major late promoter (MLP).
• the promoter may also be capable of driving persistent transgene expression such as the CMV derived promoter element described by Armentano et al. in international patent application PCT/US99/00915 incorporated herein by reference.
• CMV derived promoter element described by Armentano et al. in international patent application PCT/US99/00915 incorporated herein by reference.
• adenoviral vectors utilizing endogenous or heterologous polyA adenylation signals, e.g., the SV40, BGH, adenoviral or native polyA signal.
• the nucleic acid molecules may be expressed from different promoters in order to optimize specific expression of each type of gene.
• the transgene bay be placed under the control of a constitutive promoter, and the immunosuppressant molecule may be placed under an inducible promoter.
• the multiple copies of the same promoter may be used for the expression of multiple nucleic acids or a single promoter may be operatively linked to heterologous nucleic acids placed adjacently to achieve simultaneous expression.
• Transgenes are inserted into Ad vectors using methods well known in the art.
• Transgenes are defined herein as nucleic acid molecules or structural genes that encode a particular polypeptide or protein or a ribozyme or an antisense RNA or the like.
• Transgenes encoding polypeptides or proteins include, inter alia, those coding for enzymes, e.g., human lysosomal enzymes, such as -galactosidase A and ⁇ -glucocerebrosidase, hormones, growth factors, cytokines, antigens, and such specific proteins such as CFTR, ⁇ l- antitrypsin, soluble CD4, adenosine deaminase, Herpes Simplex Virus thymidine kinase, the tumor antigens gplOO, MART-1 and TRP-2; and clotting factors, such as factor VIII, factor IX, factor VII and Von Willebrand factor.
• enzymes e.g., human lysosomal enzymes, such as -galactosidase A and ⁇ -glucocerebrosidase, hormones, growth factors, cytokines, antigens, and such specific proteins such as CFTR, ⁇
• lysosomal enzymes in accordance with the present invention are provided in Table I. References relating to isolation and characterization of the lysosomal enzymes and nucleic acid molecules (transgenes) encoding said enzymes in Table I may be found in Scriver et al., The Metabolic Basis of Inherited Disease, 7 th Ed., vol. II, pp. 2427-2879, McGraw Hill, 1995, incorporated herein by reference.
• Polynucleotides encoding immunosuppressant molecules include, inter alia, those coding for IL-10, vIL-10 and TGF ⁇ .
• Other polynucleotides of the present invention include the p35 gene of baculovirus which encodes a protein that blocks apoptosis of baculovirus- infected cells (Clem et al., Science 254:1388-1389, 1991; Hershberger et al., J. Virol.
• fasL mammalian fas/CD95 ligand
• the polynucleotides/transgene and vector nucleic can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
• synthetic nucleic acid linkers can be ligated to the termini of the restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector nucleic acid.
• an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
• a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
• enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
• transcription termination and RNA processing signals from SV40 for mRNA stability transcription termination and RNA processing
• a nucleic acid molecule encoding an immunosuppressant molecule is inserted into the E3- deleted region of the adenoviral vector (Fig. 1 and Fig 4).
• a nucleic acid encoding an immunosuppressant molecule is inserted into the E3-deleted region of the adenoviral vector and a nucleic acid encoding a second molecule (either another immunosuppressant molecule or a transgene) is inserted into the El -deleted region of the adenoviral vector (Fig. 2, Fig. 3, Fig. 5 and Fig. 6).
• a nucleic acid encoding p35 is inserted into the E3-deleted region of the adenoviral vector and a nucleic acid encoding a fasL is inserted into the El -deleted region of the adenoviral vector (Fig. 7) as described in a co-pending U.S. provisional patent application Ser. No. 60/130,415, filed April 21, 1999, incorporated herein by reference.
• the present invention includes a method of using the vectors of the present invention for inducing tolerance to adenovirus antigens and/or transgenes included in an adenoviral vector in antigen-presenting cells (APCs) of an individual to whom the vectors are administered comprising introducing into said APCs an amount of a vector comprising an adenovirus genome comprising nucleic acids encoding said adenoviral antigens, said adenovirus genome also comprising a deletion of the El region and a deletion in a second region, wherein a first nucleic acid molecule is inserted into one deleted region and a second nucleic acid molecule is inserted into the other deleted region and wherein the vector is taken up by the cells and wherein the first and second nucleic acids encode a protein which is expressed in the APCs.
• APCs antigen-presenting cells
• the first and second nucleic acids which encode a protein include, ter alia, vIL-10, TGF ⁇ , fasL, p35 and a transgene.
• the first nucleic acid comprises a transgene and the second nucleic acid encodes TGF ⁇ or IL-10 or vIL- 10.
• a first nucleic acid encodes a transgene
• a second nucleic acid encodes fasL
• a third nucleic acid encodes p35.
• Suitable sources of APCs include but are not limited to whole cells, such as dendritic cells or macrophages and foster antigen-presenting cells.
• the APCs preferably dendritic cells (DCs) may be infected either in vitro or in vivo.
• DCs which are the principle initiators of antigen-specific immune responses have several molecules on their surface that are critical for T-cell activation (reviewed in van Schooten et al., Mol. Medicine Today, pp 254-260, 1997).
• DCs may be purified from bone marrow by isolating bone marrow precursor cells (CD34+) from blood and stimulating them to differentiate into DCs.
• the individual must be treated with cytokines such as GM-CSF to boost the number of circulating CD34+ stem cells in the peripheral blood.
• cytokines such as GM-CSF
• a second approach for isolating DCs is to collect the relatively large numbers of precommitted DCs already circulating in the blood.
• Known procedures for preparing mature DCs from human peripheral blood have involved combinations of physical procedures such as metrisamide gradients and adherence/nonadherence steps (Freudenthal et al., Proc. Natl. Acad. Sci.
• Harvested DCs may then be infected in vitro with an Ad vector comprising either a nucleic acid encoding an immunosuppressant molecule (IL-10, vIL-10 and/or TGF ⁇ ) and a transgene, such as those described above, or the fasL gene and the p35 gene.
• Infected DCs may then used to pretreat a host by the multiple administration of the infected DCs into the host by intravenous injection. After administration of the infected DCs to the host, the DCs migrate to lymphoid organs, such as the spleen, and interact with T cells.
• Such interaction would either suppress the immunogenic response of the T cells where IL-10, vIL-10 and/or TGF ⁇ are expressed, or induce apoptosis of the T cells where the fasL is expressed.
• the p35 will act to protect the DCs from being lysed through the fas/fasL mediated pathway.
• an Ad vector comprising the same transgene as that introduced by the adenoviral vector-infected DCs, may be introduced to the host by any route of administration, e.g., intravenous, intranasal, intramuscular, etc.
• the transgene coding for a biologically active molecule can then be expressed in the absence of an immune response or with a minimized immune response.
• the absent or reduced immune response allows for repeated administration of the Ad vector and persistent expression of the transgene coding for a biologically active molecule with a minimized or eliminated host immune response.
• the Ad vector comprising either a nucleic acid encoding an immunosuppressant molecule (IL-10, vIL-10 and/or TGF ⁇ ) and a transgene, such as those disclosed above, or the fasL gene and the p35 gene may be used to infect DCs in vivo by intradermal injection of the Ad vector or any other suitable route of administration known in the art. DCs thus transduced will migrate to the regional lymph nodes where they can interact with T cells.
• an immunosuppressant molecule IL-10, vIL-10 and/or TGF ⁇
• a transgene such as those disclosed above
• the fasL gene and the p35 gene may be used to infect DCs in vivo by intradermal injection of the Ad vector or any other suitable route of administration known in the art. DCs thus transduced will migrate to the regional lymph nodes where they can interact with T cells.
• an Ad vector comprising the same transgene coding for a biologically active molecule as that introduced by the adenoviral vector-infected DCs, may be introduced by any route of administration, e.g., intravenous, intranasal, intramuscular, etc.
• the transgene coding for a biologically active molecule may then be expressed in the absence of an immune response or with a minimized immune response and may be repeatedly administered.
• Ad vectors comprising the transgene coding for a biologically active molecule
• routes of administration for the Ad vectors comprising the transgene coding for a biologically active molecule include conventional and physiologically acceptable routes such as direct delivery to target cells, organs or tissues, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parenteral routes of administration.
• the Ad vectors may also be administered via inhalation of liquid or dry powder aerosols (e.g. as disclosed in U.S. provisional patent application Ser. No. 60/110,899, filed December 4, 1998, incorporated herein by reference).
• Dosage of an Ad vector which is to be administered to an individual is determined with reference to various parameters, including the condition to be treated, the age, weight and clinical status of the individual and particular molecular defect requiring the provision of a biologically active protein.
• the dosage is preferably chosen so that administration causes a specific phenotypic result, as measured by molecular assays or clinical markers described above.
• Dosages of an Ad vector of the invention which can be used for example in providing a transgene contained in a vector to an individual for persistent expression of a biologically active protein encoded by the transgene and to achieve a specific phenotypic result range from approximately 10 infectious units (I.U.) to 10 u I.U. for humans.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active ingredient calculated to produce the specific phenotypic result in association with the required physiological carrier.
• the specification for the novel dosage unit forms of the invention are dictated by and directly depend on the unique characteristics of the adenoviral vector used in the formulation and the limitations inherent in the art of compounding. The principle active ingredient (the adenoviral vector) is compounded for convenient and effective administration with the physiologically acceptable carrier in dosage unit form as discussed above.
• the Ad vectors administered after pretreatment with Ad infected DCs to induce tolerance may be targeted to specific cells by linking a targeting molecule to the vector, e.g. as disclosed in international application No. PCT/US99/02680 filed February 8, 1999, incorporated herein by reference.
• a targeting molecule is any agent that is specific for a cell or tissue type of interest, including for example, a ligand, antibody, sugar, receptor, or other binding molecule.
• Targeted vectors are particularly useful in the treatment of lysosomal diseases.
• a targeting molecule such as VEGF or an antibody to a VEGF receptor, can provide targeting to vascular endothelial cells in individuals with Fabry's disease.
• Ad vectors administered after pretreatment with infected DCs can be complexed with a cationic amphiphile, such as a cationic lipid, polyL-lysine
• PLL diethylaminoethyldextran
• DEAE-dextran diethylaminoethyldextran
• Representative cationic lipids include those disclosed, for example, in U.S. Patent Nos. 5,238,185 and 5,767,099, with preferred lipids being GL-67 (N 4 -spermine cholesteryl carbamate), GL-53 (N 4 -spermine cholesteryl carbamate), and GL-89 (1-(N 4 - spermine)-2,3-dilaurylglycerol carbamate).
• Ad vectors complexed with DEAE dextran are particularly preferred.
• adenovirus and other viral vectors may be polymer-modified, e.g., complexed with polyethylene glycol (PEG), to reduce viral immunogenicity and allow for repeat administration of the vector (See, e.g., WO/98/44143, incorporated herein by reference).
• PEG polyethylene glycol
• Ad vectors complexed with a cationic molecule, preferably DEAE, and a polyalkylene glycol polymer, e.g. PEG, as disclosed in U.S. provisional patent application Serial NO. 60/097,653, filed August 24, 1998, are contemplated herein.
• Transfer of the transgene to the target cells by the Ad vectors of the invention can be evaluated by measuring the level of the transgene product in the target cell and correlating a phenotypic alteration associated with transgene expression .
• expression of a CFTR transgene in target cells from an individual with cystic fibrosis is correlated with production of a functional chloride ion channel in such cells that may be measured by techniques known in the art.
• the level of transgene product in the target cell directly correlates with the efficiency of transfer of the transgene by the Ad vectors. Any method known in the art can be used to measure transgene product levels, such as ELISA, radioimmunoassay, assays using an fluorescent and chemiluminescent enzyme substrates.
• Immunological procedures useful for in vitro detection of the transgene product in a sample include immunoassays that employ a detectable antibody.
• immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art.
• An antibody can be made detectable by various means well known in the art.
• a detectable marker can be directly or indirectly attached to the antibody.
• Useful markers include, for example, radionuclides, enzymes, fluorogens, chromogens and chemiluminescent labels.
• a detectable antibody can be administered to a subject and the binding of the antibody to the transgene product can be detected by imaging techniques well known in the art.
• Suitable imaging agents include, for example, gamma- emitting radionuclides such as ⁇ u ln, 99m Tc, 51 Cr and the like, as well as paramagnetic metal ions, which are described in U.S. Patent No. 4,647,447.
• the radionuclides permit the imaging of tissues by gamma scintillation photometry, positron emission tomography, single photon emission computed tomography and gamma camera whole body imaging, while paramagnetic metal ions permit visualization by magnetic resonance imaging.
• the method of present invention which induces tolerance to Ad vector gene products and transgene products, can be analyzed for the ability of the method to provide persistence of transgene expression in vivo.
• the method may be assessed in an animal model system where the DCs of the animal are harvested by methods known in the art, infected in vitro with an Ad vector comprising an immunosuppressant and a transgene and used to pretreat the animal to induce tolerance therein or DCs may alternatively be infected with adenoviral vectors in vivo via intradermal injection as described above. After pretreatment, an Ad vector encoding the same transgene may be repeatedly administered to the animal by acceptable routes.
• Such a model may be chosen with reference to such parameters as ease of delivery, identity of transgene, relevant molecular assays and assessment of clinical status.
• an animal model which is representative of the disease state may optimally be used in order to assess a specific phenotypic result correlated with the presence of biologically active transgene product e.g., Fabry knockout mice (as disclosed in international No. PCT/US98/22886, filed October 29, 1998), may be used to assay the ability of Ad vectors comprising an ⁇ -galactosidase A transgene to reduce the levels of GL-3 in such mice.
• the immune response may also be analyzed.
• the ability to achieve persistent gene expression and to repeatedly administer the Ad vector comprising transgene coding for a biologically active molecule with a reduced or eliminated immune response may also be analyzed.
• transgene expression system may be assayed.
• animals in which the transgene expression system may be assayed include, but are not limited to, mice, rats, monkeys and rabbits.
• Suitable mouse strains in which the transgene expression system may be tested include, but are not limited to, C3H, C57B1/6 (wild-type and nude) and Balb/c (available from Taconic Farms, Germantown, New York).
• testing in immune-competent and immune-deficient animals may be compared in order to define specific adverse responses generated by the immune system.
• the use of immune- deficient animals, e.g., nude mice may be used to characterize vector performance and persistence of transgene expression, independent of an acquired host response and to identify other determinants of transgene persistence.
• Ad vectors In order to determine the persistence of Ad vectors in the host, one skilled in the art can assay for the presence of these vectors by any means which identifies the transgene (and its expression), for example, by assaying for transgene or nucleic acid encoding the immunomodulatory molecule mRNA level by RT-PCR, Northern blot or SI analysis, or by assaying for transgene protein expression by Western blot, immunoprecipitation, or radioimmunoassay.
• the presence of the Ad vector or the desired transgene DNA sequences per se in a host can be determined by any technique that identifies DNA sequences, including Southern blot or slot blot analysis, or other methods 'known to those skilled in the art.
• the presence of the vector may be determined by these same assays or a specific functional assay that screens for the marker protein (e.g., X-gal).
• the persistence of a vector of the invention in the host can also be determined from the continued observation of a phenotypic alteration conferred by the administration of the Ad vector containing the transgene, e.g., the improvement or stabilization of pulmonary function following administration of a vector containing the CFTR gene to an individual with cystic fibrosis or the reduction in stored GL3 lipid in an individual with Fabry's disease.
• Spirometry can be used for pulmonary function tests (PFT) in CF individuals. Demonstration of the restoration of chloride ion channel function in vector-treated cells of a CF patient can also be used to assess the persistence of the transgene CFTR (Zabner et al., J. Clin. Invest. 97:1504-1511, 1996).
• PFT pulmonary function tests
• the Ad vectors are all El deleted (Ad2 nucleotides 358-3328 deleted) and E3 deleted (Ad2 nucleotides 27971-30937 deleted).
• the transgene (CFTR, ⁇ -galactosidase ( ⁇ gal), ⁇ - galactosidase, etc.) is inserted in the El-deleted region and IL-10, vIL-10 and TGF ⁇ are inserted in the E3-deleted region. Both genes are driven by the CMV promoter. See Fig. 1 through Fig. 6. All vectors contain wild- type E2 and E4 regions but may also have an E4 deletion.
• the transgene expression cassette replaces the El region of the Ad2 vector.
• This vector is termed Ad2/transgene. See e.g. Ad2/CFTR-2 and Ad2/CFTR-5 of Scaria et al, 1998, J. Virol. 72:7302-7309 and U.S. provisional patent application Ser. No. 60/130,415, incorporated herein by reference.
• the immunomodulatory gene (either IL-10, vIL-10 or TGF ⁇ ) is cloned by PCR and is inserted into an expression cassette plasmid pCMV (see Figure 10) using Xhol and Notl restriction sites, so that the immunomodulatory gene is cloned behind the CMV promoter and uses the SV40 polyA sequence.
• the entire expression cassette is then cut out using the unique Rsrll sites at either end of the cassette and cloned into the E3 ⁇ 2.9 deletion in an adenoviral plasmid, pAd/ ⁇ 4+/E3 ⁇ 2.9 (see Figure 11) which contains the right end of Ad2.
• This plasmid is then co-transfected with digested DNA from Ad2/transgene so that homologous recombination occurs between the plasmid and the vector to yield
• Ad/transgene/CMVimmunomodulatory gene which can express the transgene and also the immunomodulatory molecule from their respective promoters.
• Example 2 Ad vector containing FasL and p35
• mouse fas ligand (fasL) cDNA is cloned by PCR from a mouse testis quick-clone cDNA library (Clonetech Laboratories) and is inserted into the expression plasmid, pCMV using the Xhol and Notl restriction sites ( Figure 7), so that the cDNA is cloned behind the CMV promoter and uses the SV40 polyA site.
• the entire expression cassette is then cut out using the unique Rsrll sites at either end of the cassette and cloned into the Rsrll site in the El deleted (Ad2 nucleotides 358 to 3328 deleted) pre-adenoviral plasmid called pAdEl-R which contains the left end of Ad2 including the protein IX gene and E2B sequences.
• This plasmid is then cotransfected with restricted DNA from an Ad2/CFTR p35 (such as the vector described above) vector so that homologous recombination occurs between the plasmid and the Ad vector to yield Ad/fasL/p35 which expresses fasL from the El region and p35 from the E3 region, both genes being driven by the CMV promoter (Fig. 7).
• the transgene is inserted, as described above in Example 1, into the El- deleted region and nucleic acids encoding fasL and p35 are inserted into the E3-deleted region as a single expression cassette (as described above in Example 1 for a single immunomodulatory molecule) such that all three, the transgene, fasL and p35, are contained within the same Ad vector (see Figure 12).
• the Ad/fasL/p35 or Ad/transgene/fasL/p35 vector is produced in high titers of 10"
• Ad/fasL/p35 or Ad/transgene/fasL/p35 vector kills infected cells
• a cell killing assay is performed on CV-1 cells using an LDH kit purchased from Promega. The results are shown in Figure 8 and indicate that the Ad/fasL/p35 vector does not kill infected cells like the control Ad/fasL vector.
• DCs may be purified from bone marrow by isolating bone marrow precursor cells (CD34+) from blood and stimulating them to differentiate into DCs.
• DCs are isolated from an individual and cultured as described in Bender et al., J. Immunol. Meth. 126:121-135, 1996 and Romani et al, J. Immunol Meth. 126:137-151, 1996.
• Harvested DCs may then be infected in vitro with an Ad vector comprising either a nucleic acid encoding an immunosuppressant molecule (IL-10, vIL-10 and/or TGF ⁇ ) and a transgene, such as those described above in Example 1 (Fig. 1 through Fig. 6), or the fasL gene and the p35 gene as described in Example 2 (Fig. 7).
• an Ad vector comprising either a nucleic acid encoding an immunosuppressant molecule (IL-10, vIL-10 and/or TGF ⁇ ) and a transgene, such as those described above in Example 1 (Fig. 1 through Fig. 6), or the fasL gene and the p35 gene as described in Example 2 (Fig. 7).
• a multiplicity of infection m.o.i.
• Infected DCs may then used to pretreat a host by the multiple administration of the infected DCs into the host accomplished via intravenous injection.
• the DCs migrate to lymphoid organs like the spleen and interact with T cells. Such interaction will either suppress the immunogenic response of the T cells where IL-10, vIL-10 and/or TGF ⁇ are expressed, or induce apoptosis of the T cells where the fasL is expressed.
• the p35 will act to protect the DCs from being lysed through the fas/fasL mediated pathway.
• an Ad vector comprising the same transgene coding for a biologically active molecule is introduced to the host by any route of administration, e.g., intravenous, intranasal, intramuscular, etc.
• the transgene coding for a biologically active molecule is then expressed in the absence of an immune response or with a minimized immune response.
• the absent or reduced immune response allows for repeated administration of the Ad vector and persistent expression of the transgene coding for a biologically active molecule.
• the Ad vector comprising either a nucleic acid encoding an immunosuppressant molecule (IL-10, vIL-10 and/or TGF ⁇ ) and a transgene coding for a biologically active molecule, such as those described above, or the fasL gene and the p35 gene are used to infect DCs in vivo by intradermal injection of the Ad vector or any other suitable route of administration known in the art. DCs thus transduced migrate to the regional lymph nodes where they interact with T cells. Subsequent administration of an Ad vector encoding the same transgene is accomplished as described above.
• an immunosuppressant molecule IL-10, vIL-10 and/or TGF ⁇
• Example 4 DeAd vector
• the present invention also encompasses the use of partially deleted adenoviral (DeAd) vectors into which the transgene and nucleic acid encoding the immunosuppressant molecule (IL-10, vIL-10/TGF ⁇ ) is inserted and is explained more fully in regard to construction of a DeAd vector from an adenovirus of serotype 2 (Ad2).
• the Ad 2 DeAd genome is modified using conventional molecular cloning methods (See, e.g. Ausubel, F.M. et al., eds., 1987-1996, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
• the E2A coding region is deleted by removal of nucleotides 22666 to 23960; this removes the first ATG of the E2A protein encoding sequence, as well as a large portion of the protein coding region, without affecting genes encoded by the opposite strand of the virus.
• the E3 region is deleted by removal of nucleotides 27971 to 30937; this removes all E3 coding regions.
• the E4 region is deleted by removal of nucleotides 32815 to 35977; this removes all E4 coding regions. Through these deletions, the transgene packaging size of the vector is increased to approximately 12kb.
• the DeAd vector genome is further modified by positioning the dimerizer controlled promoter, the ecdysone controlled promoter, or the tetracycline doxycycline controlled promoter (Fontana et al., J. Immunol. 143:3230. 1995) in place of the MLP, i.e., just upstream from position 6038 (Fig. 9).
• Example 5 Construction of a DeAd Vector Containing a nucleic acid molecule(s) encoding an immunomodulatory molecule(s and a CFTR Transgene. a human ⁇ -galactosidase A Transgene. an EPO Transgene. a Factor IX Transgene. a Factor VIII Transgene. or lacZ Reporter Gene and Transfer of the Gene to Recipient Cells
• a DeAd vector comprising the a nucleic acid(s) encoding for an immunomodulatory molecule(s) such as IL-10, vIL-10, TGF ⁇ , and baculo virus p35 with fasL, and a functional CFTR encoding transgene for transferring a functional CFTR coding sequence (Riordan et al., Science 245:1066-1073, 1989; U.S. Patent No. 5,876,974) to cells of an individual with cystic fibrosis, nucleic acid encoding human ⁇ -galactosidase A (e.g. U.S. Patent No.
• Patent 4,994,371), or the factor VIII coding sequence with the B chain present or deleted is constructed by cloning the relevant nucleic acid (transgene encoding CFTR, ⁇ - galactosidase A EPO, factor IX or factor VIII) operably linked to a promoter, such as the CMV promoter, CMV-derived promoter, the PGK promoter, ⁇ -1 antitrypsin promoter, the K19 promoter, or other promoter suitable for expression of the nucleic acid (transgene or immunomodulatory molecule), preferably the CMV promoter, into the region downstream of position 358 in the DeAd vector or any other suitable cloning site, as disclosed in the international application PCT/US99/09590 filed April 30, 1999, incorporated herein by reference, using conventional cloning techniques (Ausubel, F.M.
• DeAd vectors comprising a nucleic acid encoding an immunomodulatory molecule, such as IL-10, vIL-10, TGF ⁇ and p35 with fasL, and the CFTR transgene are propagated in any of the producer cell lines, as disclosed in the international application PCT/US99/09590 filed April 30, 1999, incorporated herein by reference, released from the cells by suitable techniques, such as cell lysis and purified by CsCl gradient centrifugation as described in Zabner et al., Nature Genetics 6:75-83, 1994, incorporated herein by reference.
• an immunomodulatory molecule such as IL-10, vIL-10, TGF ⁇ and p35 with fasL
• the CFTR transgene are propagated in any of the producer cell lines, as disclosed in the international application PCT/US99/09590 filed April 30, 1999, incorporated herein by reference, released from the cells by suitable techniques, such as cell lysis and purified by CsCl gradient centrifug
• DeAd/CFTR vector is administered via DCs to pretreat a host and also administered to a host subsequent to pretreatment as described above in Example 3.
• the DeAd/CFTR vector is administered by aerosol or other topical administration method to airway epithelia cells of a suitable animal (e.g. cotton rats, primates) or to individuals with cystic fibrosis (see, e.g. U.S. Patent No. 5,670,488 and Zabner et al., J. Clin. Invst. 97: 1504- 1511, 1996, incorporated herein by reference).
• CFTR transgene expression in treated cells, animals and individuals is detected by measurement of vector specific CFTR mRNA transcript (see, e.g. Kaplan et al., Hum. Gene Ther. 8:45-56, 1997, incorporated herein by reference) or phenotypic alteration (monitored by the presence of a functional chloride ion channel produced by the functional CFTR) in recipient cells (U.S. Patent No. 5,670,488 and Zabner et al, 1996, supra, Jiang et al., Hum. Gene Ther. 8: 671-680, 1997, incorporated herein by reference).
• Expression of the EPO, factor IX and factor VIII transgenes in host cells transfected with DeAd vectors comprising said transgenes and an immunomodulatory molecule, such as IL-10, vIL-10, TGF ⁇ and p35 with fasL, may be detected by any means known to those of skill in the art, including detection of RNA transcripts and protein production. Phenotypic alterations correlating with expression of the relevant transgene may also be assessed. For example, expression of EPO is measured by increased RBC production in an individual; expression of factors IX and VIII are monitored by measuring clotting in the individual.
• a DeAd vector comprising a nucleic acid encoding an immunomodulatory molecule and a lacZ reporter gene encoding ⁇ -galactosidase is made.
• Effective gene transfer expression of the lacZ gene from the DeAd/lacZ vector to target cells or tissues is detected using an X-gal assay as disclosed in (Armentano et al., J. Virol. 71:2408-2416, 1997; Rich et al., Hum. Gene Ther. 4: 461-476, 1993; U.S. Patent No. 5,670,488, incorporated herein by reference).
• a DeAd/ ⁇ galA vector is constructed, as disclosed in the international application PCT/US99/09590 filed April 30, 1999, incorporated herein by reference, by inserting a transgene encoding human ⁇ -galactosidase A operably linked to a promoter into the DeAd vector and also inserting a heterologous nucleic acid encoding an immunomodulatory molecule(s), such as IL-10, vIL-10, TGF ⁇ , and baculovirus p35 with fasL operably linked to a promoter (any promoter described herein or other suitable promoter may be used to drive the expression of any of the nucleic acids).
• an immunomodulatory molecule(s) such as IL-10, vIL-10, TGF ⁇ , and baculovirus p35 with fasL operably linked to a promoter
• Fibrolasts from normal and Fabry individuals, infected with DeAd/ ⁇ galA vectors produce enzymatically active ⁇ -galactosidase A may be assayed in cell lysates and spent culture medium using the fluorescent substrate 4-methylumbnelliferyl- ⁇ - D-galactopyranoside (4-mu- ⁇ -gal).

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