Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

• DNA-mediated gene transfer involves direct administration of DNA to the patient in various formulations. These methods use genes as medicines in a manner much like conventional organic or protein compounds. DNA-mediated gene transfer however has proven quite difficult. Methodology such as micro- injection, lipofection, and receptor mediated endocytosis have usually resulted in lower gene transfer, and have usually established only transient residence of the novel gene in the targeted cell.
• DNA- mediated gene transfer may be inherently limited to the use of genes as medicines that are administered by conventional parenteral routes to provide a therapeutic effect over predictable period of time.
• Studies of a therapeutic gene product may be constituted by repetitively dosing the patient with degenerate material much like conventional pharmaceutical medicines.
• Viral gene transfer on the other hand involves construction of synthetic virus particles (vectors) that lack pathogenic functions. The virus particles are incapable of replication and contain a therapeutic or diagnostic gene within the viral genome which is delivered to cells by the process of infection. To date the viral vector which has achieved the most success is the retroviral vector.
• Retroviral vectors have l several properties that make them useful for gene therapy. First is the ability to construct a "defective" virus particle that contains the therapeutic gene and is capable of infecting cells but lacks viral genes and expresses no viral gene products which helps to minimize host response to potential viral epitopes. Retroviral vectors are capable of permanently integrating the genes they carry into the chromosomes of the target cell. Considerable experience in animal models and initial experience in clinical trials suggest that these vectors have a high margin of safety.
• Adenoviral vectors are constructed using a deleted adenoviral genome that lacks either the e-3 gene region and/or the e-1 gene region that is required for producing a replicating adenovirus particle. Recombinant genes are inserted into the site of the deleted gene region(s). Adenoviral particles are then produced in a cell line that is able to express e-1 or e-3 genes and thus capable of assembling a viral particle which contains only the recombinant viral genome with the therapeutic gene.
• Adenoviral vectors differ from retroviral vectors in that they do not integrate their genes into the target cell chromosome. Adenoviral vectors will infect a wide variety of both dividing and non-dividing cells in vitro and in vivo with a high level of efficiency providing expression of their recombinant gene for a period of several weeks to months.
• adenoviral vectors that are incapable of proliferating however they are not completely "defective" and will express a series of viral gene products which can generate host immune response to the viral epitopes presented causing quick elimination of the already transient vector.
• Adenoviral vectors remain capable of inducing cell lysis and an inflammatory response. Severe inflammation has been noticed during the experimental clinical trial for the treatment of cystic fibrosis.
• Other viruses exhibit properties that may be useful as potential vectors for gene therapy.
• One such virus is the adeno-associated virus vector. It, like the retrovirus can provide a completely defective vector that permanently integrates in the chromosome of the target cell. This adenoviral vector integrates at a predictable location within the affected cell and could make this type of vector safer than those that integrate randomly into the genome.
• Another promising viral vector is based on the Herpes Simplex virus.
• Herpes virus vectors are capable of infecting cells and persisting indefinitely in a latent state.
• the herpes simplex virus vector involves genetic engineering of the viral genome to render it useful for serial propagation and for sustained expression of foreign genes in a suitable host. Additional components may also be added such as the Epstein Barr virus nuclear antigen gene and latent origin of replication to maintain the vector in episomal state, as described in United States Patent Number 5,830,727.
• nucleotide expression systems for therapeutic or for diagnostic purposes which have decreased immuno ge nicity . It is yet another object of the invention to provide nucleotide expression systems which include immunosuppressive agents isolated from several viral genomes which allow the vehicle to evade the recipient cell immune response.
• the present invention seeks to utilize genetic engineering techniques to provide a recombinant nucleotide expression system which provides for the continued persistence of introduced genes through immune suppression.
• the invention provides a recombinant expression system adapted to express immunosuppression genes isolated from virus genomes or functional equivalents or parts thereof, so that host immune response to the introduced therapeutic gene is minimized.
• immunosuppression gene shall mean any nucleotide sequence, the expression of which provides a protein product which enables a virus to evade, diminish or suppress the immune system reaction of the recipient host to that virus.
• immune responses can include but are not limited to cytotoxic T lymphocyte response, MHC-1, MHC-2, T helper cell, cytokines, Interleukins, natural killer cells, neutrophils, macrophages, ⁇ -cell, plasma cells, tissue macrophages (e.g. Kuffer cell in liver) and dendritic cells.
• Examples of these genes include but are not limited to Epstein-Barr virus BHRF1, LMP-1 and LMP-2A, adenovirus ElB/19k and ElB/55k, cowpox virus crmA and CHOhr, baculovirus p35 and IAP, molluscum contagiosum virus MC159 and MCO66L, equine herpesvirus-2 E8, rabbitpox virus SPI-1, simian virus 40 T-Ag, papilloma virus E7, cy tome galo virus IE2, UL18, and US6, myxoma virus M-T5, MT-2, and M-T4, vaccinia virus E3L and K3L, and herpesvirus samurai Tip.
• expression system is used herein to refer to a genetic sequence which includes a protein encoding region which is operably linked to all of the genetic signals necessary to achieve expression of the protein encoding region.
• the expression system will include a regulatory element such as a promoter or enhancer, to increase transcription and/or translation of the protein encoding region, or to provide control over expression.
• the regulatory element may be located upstream or downstream of the protein encoding region, or may be located at an intron (noncoding portion) interrupting the protein encoding region.
• sequence of the protein encoding region itself to comprise regulatory ability.
• the term “functional equivalent” refers to any derivative which is functionally substantially similar to the referenced sequence or protein.
• the term “functional equivalent” includes derivatives in which nucleotide base(s) and/or amino acid(s) have been added, deleted or replaced without a significantly adverse effect on biological function and which will hybridize under high conditions of stringency according to protocols known in the art and disclosed in Maniantis et. al., "Molecular Cloning” cold Spring Harbor Press, (1989).
• therapeutic gene shall be interpreted in include any nucleotide sequence, the expression of which is desired in a host cell. This can include any genetic engineering protocol for introduction of such sequence which would benefit from reduced immunogenicity to the introduced gene and includes antisense type strategies, diagnostic protocols, or gene therapy.
• the invention in one embodiment includes a recombinant expression system which comprises a therapeutic nucleotide sequence, the expression of which is desired in a cell and a recombinant nucleotide sequence which encodes an immunosuppression agent.
• the immunosuppression agent and nucleotide sequence may be operably linked to a single regulatory system to promote expression of both or may be separately incorporated into unique expression systems.
• the expression system of the invention is included within a gene delivery vehicle or vector for transformation of recipient cells.
• the immune suppression gene is one which is non-native to the vector of choice and preferably is maintained within a vector which has minimal viral encoded proteins.
• the invention is especially useful for transformation protocols which seek to achieve permanent integration and/or expression of gene products.
• the invention further encompasses vectors containing the recombinant expression systems defined above, cells transformed with such vectors, and genetic engineering protocols using these components.
• Figure 1 is a schematic representation of the LXSN retroviral gene therapy vector.
• Figures 2A and 2B are schematic representations of the cloning method and resulting vectors used.
• Figure 2A is the HSV ICP47 encoding gene was amplified from purified HSV DNA and ligated into a shuttle vector. The gene was sequenced to confirm its identity. The gene was then cloned into the
• Figure 2B is the hCMV US 11 gene was cloned using a similar method from a hCMV infected cell line to obtain the LUSN vector.
• Figures 3A, 3B and 3C are Northern blot analysis to confirm that the cell lines constructed were translating the expected RNA.
• Figure 3A is a 1.25% agarose gel electrophoresis separating 10 ⁇ g total RNA preparations from the four indicated cell lines.
• Figure 3B is the RNA was transferred to nylon membrane, probed with radiolabeled ICP 47 DNA, and detected by auto radiography. The band observed is the predicted size for the LISN RNA.
• Figure 3C is the same nylon membrane probed with radiolabeled US11 DNA. The band observed is the predicted size of the LUSN RNA.
• Figure 5 depicts the specific lysis of four target cell lines by TALL-104 CTL cells was determined over five effector/target cell ratios. The percent specific lysis was determined as described in methods.
• Figures 6A-F depict cell surface MHC I inhibition.
• Transduced mixed populations of cells were labeled with anti-MHC I Ab (VMRD Inc.) at 0.1 ⁇ g/.5 x 10 6 cells at 37 degrees for 1 hour.
• Cells were washed with PBS 2x and a 1:150 dilution of goat anti-mouse FITC labeled secondary Ab (Sigma) was added for 1 hour at 37 degrees.
• Cells were again washed 2x and suspended in Phenol red free/serum free Opti-MEM® media for FACS analysis. 3 samples of each were averaged and the standard deviations determined. All cell lines were tested with no Ab, no primary, nontransduced controls, and LXSN transduced controls.
• 6A Transduced 293 Cell Lines
• 6B Transduced VA13 Cell Lines
• 6C Transduced A549 Cell Lines
• 6D Transduced A375 Cell Lines, and this is a repeat of the experiment described in Fig. 4
• 6E Transduced Ovcar Cell Lines
• 6F Transduced IGROV Cell Lines
• Error bars represent the standard error of the mean from three trials.
• Figures 7A and 7B depict specific Lysis of transduced target cells by TALL-104 effector cells.
• 5xl0 3 target cells/well are aliquoted on a 96 well tissue culture plate (4 wells/data point). Effector cells are added at the indicated ratios and incubated for 6 hours. 100% lysis is determined by completely lysing a set of control wells. Cell lysis is determined by measuring the amount of a stable cytosolic enzyme (lactate dehydrogenase, LDH) released into the well and using a color reaction (Promega Corp. Cytotox 96® assay kit). (Sinensky, M.C., A.L. Leiser, and H.
• Figure 8 depicts a schematic diagram of the LISH vector.
• the structure of LISH where the main difference from LISN is the substitution of the hygromycin resistance gene in place of the neomycin resistance gene.
• Figure 9 depicts a schematic diagram of the LUISN vector.
• Figures 10A and 10B depict ICP 47 and USll function in dog and rat cells.
• FIG. 11 depicts Lymphocyte expression vectors.
• the IRES immune suppression cassettes will be constructed and inserted into LGSN vector.
• the LGSN vector was constructed by subcloning the humanized, red shifted eGFP (Clonetech Inc.) into the LXSN vector.
• Figure 12 depicts a schematic representation of L ⁇ SI/N and L ⁇ SU/N vectors. Both vectors will be tested for immune suppression as previously described and ⁇ -ID expression by western analysis.
• Figure 13 is a map of the pLUSN vector which was designed according to the present invention.
• Figure 14 is the nucleotide sequence of the pLUSN vector (SEQ ID NO: 2).
• Figure 15 is a map of the pLXSU-IRES-N vector which was designed according to the present invention.
• Figure 16 is the nucleotide sequence of the pLXSU-IRES-N vector (SEQ ID NO: 5).
• Figure 17 is a map of the pLISN vector which was designed according to the present invention.
• Figure 18 is the nucleotide sequence of the pLISN vector (SEQ ID NO: 1).
• Figure 19 is a map of the pLXSI-IRES-N vector which was designed according to the present invention.
• Figure 20 is the nucleotide sequence of the pLXSI-IRES-N vector (SEQ ID NO: 3).
• Figure 21 is the nucleotide sequence of the pLXSN vector (SEQ ID NO:4).
• HSV Herpes Simplex Virus
• the expression system of the invention in its simplest context comprises an expression system including a therapeutic gene or nucleotide sequence the expression of which is desired in a recipient cell and a recombinant immune suppression gene or expression of recombinant immune suppression gene alone. Any nucleotide sequence as defined supra can be used in the expression system of the invention. Similarly, any of several known and yet to be discovered immunosuppression genes may also be used.
• genes include Epstein-Barr virus BHRFl, LMP-1 and LMP-2A, adenovirus ElB/19k and ElB/55k, cowpox virus crmA and CHOhr, baculovirus 35 and IAP, molluscum contagiosum virus MC159 and MCO66L, equine herpesvirus-2 E8, rabbitpox virus SPI-1, simian virus 40 T-Ag, papilloma virus E7, cytomegalovirus IE2, UL18, and US6, myxoma virus M-T5, MT-2, and M-T4, vaccinia virus E3L and K3L, and herpesvirus samurai Tip.
• Epstein-Barr virus BHRFl Epstein-Barr virus BHRFl, LMP-1 and LMP-2A, adenovirus ElB/19k and ElB/55k, cowpox virus crmA and CHOhr, baculovirus 35 and IAP,
• nucleotide sequences of these genes are generally known and accessible to those of skill in the art from information sources such as GenBank. Further, methods for identification, isolation, and sequencing of other immunosuppression genes are disclosed in the articles incorporated by reference herein and may be used to identify and sequence yet other immunosuppression genes to be used in accordance with the present invention.
• the use of multiple immune suppression genes, either transcriptionally linked by internal ribosome entry sites (IRES) or individually controlled by separate promoters, in a single gene therapy vector promises to increase the effectiveness of this approach (Parks, G.D., G.M. Duke, and A.C. Palmenberg, "Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region", J Virol, 1986. 60(2):376-84.
• nucleotide expression system of the invention as well as methods of the invention employing genetic engineering techniques using this system have the benefit of reducing, inhibiting or evading the immune response generated to the therapeutic gene thus increasing survival time.
• Virtually all conventional gene therapy protocols can benefit from increased survival time of the therapeutic gene and would therefore benefit by inclusion of these immune suppression genes in transformation vehicles.
• expression of recombinant immune suppression genes would be expressed to reduce tissue or cell allograft rejection.
• the nucleotide expression system of the invention is included within an appropriate gene transfer vehicle which is then used to transduce cells to express the gene of interest and to reduce any immune response to the gene by the recipient host cells.
• the gene delivery vehicle can be any delivery vehicle known in the art and can include simply naked DNA which is facilitated by a receptor mediated transfection as well as any of a number of vectors.
• vectors include but are not limited to eukaryotic vectors, prokaryotic vectors (such as for example bacterial vectors) and viral vectors including but not limited to retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentivirus vectors (human and other including porcine), Herpes virus vectors, Epstein-Barr virus vectors, SV40 virus vectors, pox virus vectors, pseudotype virus vectors.
• This invention is particularly useful for viral vector transformation strategies which seek to accomplish permanent as opposed to transient expression of the therapeutic gene.
• the invention is particularly suited to use of vectors such as retroviral, adeno-associated, or lenti viral vectors which integrate into host DNA.
• the invention is also particularly suited to vector systems which persist indefinitely in transformed cells such as HSV vectors or those which are maintained in episomal state such as by use of Epstein Barr genetic elements.
• the invention is also particularly useful with vectors which are defective and thus contain no or minimal amounts of viral epitopes thus minimizing the immune reaction generated to the vector itself or vector components. These vectors may be further optimized by the present invention to minimize any immune response to the therapeutic gene itself.
• Any of a number of standard gene delivery transformation methods can be used such as lipid mediated transfection, receptor mediated transfection, calcium phosphate transfection, electroporation particle bombardment, naked- direct DNA injection, diethylaminoethyl (DEAE-dextran transfection).
• the expression vehicles (vectors) of the invention can be engineered by any of a number of techniques known to those of skill in the art. The following is a summary of techniques for construction and transformation of the vectors of the invention. GENETIC ENGINEERING TECHNIQUES FOR CONSTRUCTION AND DELIVERY OF VECTORS
• Any of a number of standard gene delivery transformation methods can be used for the invention including lipid mediated transfection, receptor mediated transfection, calcium phosphate transfection, electroporation particle bombardment, naked-direct DNA injection, diethylaminoethyl (DEAE-dextran transfection).
• the expression vehicles or vectors of the invention comprising the expression system also comprise a selectable marker gene to select for transformants as well as a method for selecting those transformants for propagation of the construct in bacteria.
• selectable marker may contain an antibiotic resistance gene, such as those that confer resistance to ampicillin, kanamycin, tetracycline, or streptomycin and the like.
• antibiotic resistance gene such as those that confer resistance to ampicillin, kanamycin, tetracycline, or streptomycin and the like.
• These can include genes from prokaryotic or eukaryotic cells such as dihydrofolate reductase or multi-drug resistance I gene, hygromycin B resistance that provide for positive selection.
• Any type of positive selector marker can be used such as neomycin or Zeosyn and these types of selectors are generally known in the art.
• a therapeutic gene to be expressed can then be introduced into the vector of the invention.
• the foreign DNA can comprise an entire transcription unit, promoter-gene-poly A or the vector can be engineered to contain promoter/transcription termination sequences such that only the gene of interest need be inserted.
• These types of control sequences are known in the art and include promoters for transcription initiation, optionally with an operator along with ribosome binding site sequences.
• beta-lactase penicillinase
• lactose promoter systems (Chang et al., Nature, 1977, 198:1056)
• trp Tryptophan
• trp Tryptophan
• Other promoters such as cytomegalovirus promoter or Rous Sarcoma Virus can be used in combination with various ribosome elements such as SV40 poly A.
• the promoter can be any promoter known in the art including constitutive, (supra) inducible, (tetracycline-controlled transactivator (tTA)-responsive promoter (tet system, Paulus, W. et al., “Self-Contained, Tetracycline-Regulated Retroviral Vector System for Gene Delivery to Mammalian Cells", J of Virology, Jan. 1996, Vol. 70, No. 1, pp. 62-67)),or tissue specific, (such as those cited in Costa, et. A , European journal of Biochemistry, 258 "Transcriptional Regulation Of The Tissue-Type
• the vector comprises a specifically engineered multi-cloning site within which several unique restriction sites are created. Restriction enzymes and their cleavage sites are well known to those of skill in the art.
• a packaging cell line is transduced with a viral vector containing the therapeutic nucleotide sequence to form a producer cell line including the viral vector.
• the producer cells may then be directly administered, whereby the producer cells generate viral particles capable of transducing the recipient cells.
• the viral vector is a retroviral vector.
• retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
• Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
• Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the therapeutic gene(s) of interest.
• the structural genes i.e., gag, pol, and env
• the structural genes are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
• These new genes may be incorporated into the proviral backbone in several general ways.
• Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
• a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
• the retroviral vector may be one of a series of vectors described in Bender, et al., J. Virol. 61:1639-1649 (1987), based on the N2 vector (Armentano, et al., J. Virol, 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point.
• MoMuLV Moloney murine leukemia virus
• pPr80 ⁇ a & another glycosylated protein
• MoMuSV Moloney murine sarcoma virus
• the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV.
• the 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
• Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miller, et al., Biotechniques, 7:980-990, 1989).
• Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
• Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).
• the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989).
• Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
• the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragment or truncations thereof, are not expressed.
• the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
• Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol.
• the retroviral vector includes each of these cloning sites.
• a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector.
• the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
• the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
• the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
• the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
• the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
• the vector includes one or more promoters.
• Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, 7:(9):980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
• Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
• the vector then is employed to transduce a packaging cell line to form a producer cell line.
• packaging cells which may be transfected include, but are not limited to the PE501, PA317, ⁇ 2, ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAM12, and DAN cell lines.
• the vector containing the therapeutic nucleic acid sequence may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation.
• the producer cells then are administered directly to or adjacent to desired recipient cells.
• the invention comprises a viral vector which commonly infects humans and packaging cell line which is human based.
• viral vectors derived from viruses which commonly infect humans such as Herpes Virus, Epstein Barr Virus, may be used which do not express an active ⁇ -galactosyl envelope.
• the vector comprises a Herpes Simplex Virus plasmid vector.
• Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potential useful gene delivery vector system for gene therapy, Glorioso, J.C., "Development of Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System. Gene Therapeutics: Methods and Applications of Direct Gene Transfer", Jon A. Wolff, Editor, 1994 Birkhauser Boston, 281-302; Kennedy, P.G., "The Use of Herpes Simplex Virus Vectors for Gene Therapy in Neurological Diseases", Q J Med. Nov. 1993, 86(ll):697-702; Latchman, D.S., "Herpes Simplex Virus Vectors for Gene Therapy", Mol Biotechnol, Oct. 1994, 2(2): 179-95.
• HSV-1 Herpes Simplex virus type-1
• HSV-1 vectors have been used for transfer of genes to muscle.
• Huard, J. "Herpes Simplex Virus Type 1 Vector Mediated Gene Transfer to Muscle", Gene Therapy, 1995, 2, 385-392; and brain, Kaplitt, M.G., "Preproenkephalin Promoter Yields Region-Specific and Long-Term Expression in Adult Brain After Direct In Vivo Gene Transfer Via a Defective Herpes Simplex Viral
• Helper virus dependent mini-viral vectors have been developed for easier operation and their capacity for larger insertion (up to 140 kb), Geller, Al, "An Efficient Deletion Mutant Packaging System for Defective Herpes Simplex Virus Vectors: Potential Applications to Human Gene Therapy and Neuronal Physiology", Proc Natl Acad Sci USA, Nov 1990, 87(22):8950-4; Frenkel, N., "The Herpes Simplex Virus Amplicon: A Versatile Defective Virus Vector", Gene Therapy. 1. Supplement 1, 1994.
• Replication incompetent HSV amplicons have been constructed in the art, one example is the pHSVlac vector by Geller et al, Science, 241, Sept. 1988, incorporated herein by reference.
• HSV amplicons contain large deletions of the HSV genome to provide space for insertion of exogenous DNA. Typically they comprise the HSV-1 packaging site, the HSV-1 "ori S" replication site and the IE 4/5 promoter sequence. These virions are dependent on a helper virus for propagation. Primarily two types of mutant helper viruses have been developed to minimize recombination. Other complementary HSV helper virus systems are contemplated herein and are within the scope of those of skill in the art. One such system which has been developed is a temperature-sensitive mutant. An HSV temperature-sensitive (TS) mutant has been developed with a TS mutation in the IE3 gene. Davison et al, 1984, J. Gen. Virol, 65:859-863.
• TS temperature-sensitive
• helper virus system is a deletion mutant with the majority of the IE3 gene simply deleted. These do not revert to wild type. Therefore HSV-1 vectors packaged using a deletion mutant as helper virus is the most preferred helper virus of the invention. See for example Patterson et al., 1990, J. Gen. Virol, 71:1775-1783.
• Other replication incompetent helper viruses can be used and one of skill in the art will appreciate that other mutations in the IE genes or other genes which result in a replication incompetent helper virus which will provide the appropriate replication and expression functions and which are coordinated with the helper cell line and vector are contemplated within this invention.
• Any cell line can be used for this step so long as it is capable of expressing the IE3 or replication dependent gene, or obtaining a helper cell line which has already been transformed and is commercially available.
• Any cell line can be used by introducing pHE and the plasmid containing the IE3 gene simultaneously.
• the vector is delivered to the helper cell line by electroporation, calcium phosphate DNA transfection or any other suitable method.
• Any cell line can be used by introducing pHE and the plasmid containing the IE3 gene simultaneously.
• the cells are next infected with a helper virus IE3 deletion mutant or other corresponding deletion mutant which is replication incompetent.
• the IE3 gene or other such gene in the helper cell line complements the helper virus resulting in a productive HSV-1 infection and the resulting virus stock consists of HSV-1 particles containing either vector DNA or helper virus DNA, all of which are replication incompetent. Further information about helper cell lines and the methodology is disclosed in Geller et al., PNAS, 87:8950-8954, November 1990, "An Efficient Deletion Mutant Packaging System for Defective Herpes Simplex Virus Vectors: Potential Applications to Human Gene Therapy and Neuronal Physiology".
• the invention comprises a HSV mini vector which combines a replication incompetent HSV amplicon with other viral sequences such as those from Epstein-Barr virus, human papillomavirus, or bovine papillomavirus type 1 which allow the vector to be maintained in the cell in episomal form achieving a 10 times greater titer, and a very large DNA insert capacity.
• helper virus- dependent mini- viral vector comprising: (a) the HSV-1 "a" sequence for the package/cleavage signal and an "ori S" replication origin for the replication packaging of the plasmid (in response to signals to replicate and package from the helper virus); (b) an Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) gene and an EBV latent origin of replication (ori P) which allow the vector to be maintained in episomal form within the nucleus for replication without integration to the host genome and for even replication into each of two dividing cells; preferably (c) genes from prokaryotic cells for propagation of the vector in E.
• EBV Epstein-Barr virus
• EBNA-1 Epstein-Barr virus
• ori P EBV latent origin of replication
• the vector may also comprise prokaryotic genes that provide for a second selectable marker such as the genes for positive Hygromycin selection.
• packaging function of mini-vector a selectable marker gene such as the ampicillin resistance or tetracycline resistance gene and the col. El ori
• a sequence encoding a immune suppression protein such as USll.
• the vector may also comprise prokaryotic genes that provide for a second selectable marker such as the genes for positive Hygromycin selection.
• DNA into Herpes simplex viral capsids is provided by a helper virus and a helper cell line.
• the HSV vector can be engineered to produce a helper free viral vector as in Mann et al., "Construction of a Retro- Virus Packaging Mutant and its Use to Produce Helper-Free Defective
• Retrovirus 33 Sal., p. 153-159, May 1983, Journal of Virology, September 1989, pp. 3822-3829, September 1989; Samulski "Helper Free Stocks of Recombinant Adeno-Associated Viruses: Normal Integration Does Not Require Viral Gene Expression”; and Kohn et al., "High Efficiency Gene Transfer Into Mammalian Cells: Generation of Helper-Free Recombinant Retrovirus With Broad Mammalian Host Range", PNAS, 81:6349-6353, October 1984. See also Okasinki, U.S. Patent No. 4,970,155 "HSV HELPER VIRUS INDEPENDENT VECTOR”, incorporated herein by reference.
• the expression system and transformation vehicle of the present invention can be used for any diagnostic or therapeutic genetic engineering protocol including in vitro, ex vivo, or in vivo expression of a desired nucleotide sequence.
• the expression vehicles of the invention can be used in any of a number of therapeutic treatment protocols in the treatment of cancer such as by the Herpes simplex virus, thymidine kinase gene transfer system Martuza RL et al., "Experimental therapy of human glioma by means of a genetically engineered virus mutant", Science, 1991; 252:854-856).
• the present invention provides nucleotide expression vehicles (pLXSI-IRES-N, pLISN, pLXSU-IRES-N, pLGSN, pLISH, pLUISN, and pLUSN) constructed in accordance with the teachings herein and comprising a polynucleotide having a sequence substantially as set out in SEQ ID NO:l, 2, 3, or 4 or a portion or functional equivalent thereof.
• the immune suppression gene may itself by the therapeutic gene to be introduced to cells. Delivered on their own, these immunosuppression expression systems can be used as therapeutic agents in a number of protocols.
• viruses have evolved immunosuppression gene whose protein products specifically suppress the CTL response.
• McFadden, G. and K. Kane "How DNA viruses perturb functional MHC expression to alter immune recognition", Adv Cancer Res 63:117-209 (2994); Fruh, K., et al., "A viral inhibitor of peptide transporters for antigen presentation", Nature 375:6530):415-8 (1995); Wold, W.S. and L.R. Gooding, "Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions", Virology, 184(l):l-8 (1991); Beersma, M.F., M.J.
• Herpes Simplex Virus (HSV) type 1 ICP-47 encoding gene York, LA., et al, A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell, 1994. 77(4):525-35; Rosenthal, K.L., et al, Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes. J Virol, 1987.
• the CTL response has been identified as a contributing factor in many autoimmune diseases including; Crohn's, diabetes, and allergic reactions (Harris, S.J., et al, Prediction of murine MHC class I epitopes in a major house dust mite allergen and induction of Tl-type CD8+ T cell responses. Int Immunol, 1997. 9(2):273-80; Nagata, M., et al, Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice. J Immunol, 1989. 143(4):1155-62).
• the immune suppression expression system of the invention may be administered as therapeutic agents to inhibit the CTL immune response associated with these diseases.
• the expression system again either alone or in an expression vehicle is administered to the patient in an amount effective for treatment of or reduction in symptoms of the diseases determined by one of skill in the art.
• autoimmune diseases that are potential targets for this therapy including; autoimmune anemias or cytopenias, ulcerative colitis, vascular heart disease, pericardial disease, vasculitis, hypersensitivity pneumonitis, interstitial lung diseases, cardiomyopathies, glomerulopathies, hepatitis/cirrhosis, acute/chronic pancreatitis, foreign body reactions, pulmonary inflammatory syndrome, osteoarthritis, psoriasis, sarcoidosis, vasculitis, Beech's syndrome, siogronssya, polymyositis, dermatomyositis, systemic sclerosis, disease of immediate type hypersensitivity, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, eosinophilic pneumonias, myasthenia gravis, neuromuscular junction diseases, demyelinating diseases, polychondritis, infectious arthritis, arthritis due to deposition of calcium
• the immune suppression system of the invention may be used as a therapeutic agent in a cancer treatment protocol.
• the immune suppression system of the invention includes multiple immune suppression agents which are expressed at high levels. At high levels these agents are become targeted by the immune system and the immune system targets and destroys transduced cells. As such the system may be used to transduce tumor cells to generate a immune response to these cells and to kill the cells.
• the immune suppression system of the invention includes multiple immune suppression agents which are expressed at high levels. At high levels these agents are become targeted by the immune system and the immune system targets and destroys transduced cells.
• the system may be used to transduce tumor cells to generate a immune response to these cells and to kill the cells.
• the expression system will include an inducible or preferably a tumor specific promoter to direct expression of the suicide genes to a tumor cell.
• a tumor specific promoter to direct expression of the suicide genes to a tumor cell.
• Such promoters are known and accessible to those of skill in the art and include those listed in the following articles: Gotoh, et.al., The Journal of Urology, Vol. 160 "Development of Prostate-specific antigen promoter-based Gene Therapy for Androgen-Independent Human Prostate Cancer” pgs. 220- 229 1998; Pang, Shen et. al, Cancer Research Vol 57 "Identification of A Positive Regulatory Element responsible for Tissue-specific Expression of
• CTL cytotoxic T lymphocyte
• MHC I molecules are synthesized in the lumen of the endoplasmic reticulum (ER) and become fully mature. Mature MHC I molecules are transported through the golgi apparatus and presented on the cell surface. Unassembled MHC I molecules are degraded in the golgi and are not presented on the cell surface. (McFadden, G. and K. Kane, "How DNA viruses perturb functional MHC expression to alter immune recognition", Adv Cancer Res 63:117-209 (1993)). The 8-10 amino acid peptides which determine the antigenic epitopes are generated from foreign proteins in the cytosol mainly by proteosomes and are transported into the ER by the transporter associated with antigen processing (TAP) peptide transporter.
• TRIP antigen processing
• the immune suppression agent ICP 47 binds to the peptide loading site on the cytosolic side of the TAP transporter. (Tomazin, R., et al., "Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP", EMBO J, 15(13):3256-66 (1996). This ICP 47/TAP association prevents the 8-10 amino acid viral peptides from entering the ER and thus reduces the number of MHC I molecules present on the cell surface.
• the hCMV USll gene product affects a different step in the antigen processing and presentation pathway.
• the USll gene encodes a transmembrane glycoprotein which translocates mature MHC I molecules from the ER back to the cytosol where they are rapidly degraded.
• newly synthesized MHC I has a half-life of less than 1 minute and is not presented on the cell surface.
• LXSN murine derived retroviral vector LXSN was used to deliver and express the viral immune suppression genes.
• LXSN consists of the necessary bacterial shuttle genes and a retroviral insert ( Figure 1).
• the retroviral insert has 5' and 3' long terminal repeat (LTR) sequences for retroviral integration and a SV40 promoter controls neomycin phosphotransferase expression for use as a selectable marker with the neomycin homologue G418.
• LTR long terminal repeat
• LXSN has a extended packaging signal which allows the vector to produce viral particles in an appropriate packaging cell line.
• the packaging cell line is stably transfected with the viral gag pol and env genes. This system allows for the production of replication incompetent retrovirus particles from an LXSN transfected packaging cell line.
• the LXSN vector can be modified by inserting a gene in the multicloning site located downstream from the 5' LTR promoter.
• HSV Herpes Simplex Virus
• LA. Herpes Simplex Virus
• et al A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes.
• Cell, 1994. 77(4):525-35 Rosenthal, K.L., et al, Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes. J Virol, 1987.
• hCMV human Cytomegalovirus USll gene Jones, T.R., et al., Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains. J Virol, 1995. 69(8):4830-41; Wiertz, E.J., et al, The human cytomegalovirus USll gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell, 1996. 84(5):769-79) to down-regulate the cytotoxic T lymphocyte (CTL) immune response to gene therapy vectors.
• CTL cytotoxic T lymphocyte
• the HSV ICP-47 gene codes for a 87 amino acid immediate early HSV protein.
• the ICP-47 protein binds to the TAP protein of the MHC I processing and presentation pathway and inhibits its ability to transport the viral peptides into the ER (Hill, A., et al., Herpes simplex virus turns off the TAP to evade host immunity. Nature, 1995. 375(6530):411-5; Ahn, K, et al, Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47. Embo J, 1996. 15(13):3247-55).
• MHC-I molecules are rapidly degraded and not transported to the cell surface (York, LA., et al, A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell, 1994. 77(4):525-35; Fruh, K, et al, A viral inhibitor of peptide transporters for antigen presentation. Nature, 1995. 375(6530):415-8).
• MHC-I molecules consist of a heavy chain anchored into the membrane and an associated ⁇ 2 microglobulin chain.
• the hCMV USll gene product has been shown to translocate the MHC-I heavy chain from the ER lumen to the cytosol where it is degraded by proteosomes (Machold, R.P., et al,
• the HCMV gene products USll and US2 differ in their ability to attack allelic forms of murine major histocompatibility complex (MHC) class I heavy chains. J Exp Med, 1997. 185(2):363-6; Jones, T.R. and L.
• the LUSN vector was constructed by cloning the hCMV USll gene from a hCMV infected cell line and subsequently subcloning it into the LXSN vector ( Figure 2B).
• a third vector has been constructed in a similar fashion incorporating the adenovirus GP19k encoding gene (data not shown), LGSN.( Figure 12) GP19k has also been implicated in suppression of the MHC I processing and presentation pathway and the LGSN vector was also be used in these studies.
• LISN and the LUSN vectors were used to construct cell lines expressing the proteins.
• Two murine packaging cell lines were used to generate viral supernatants.
• GPE86 cells were transfected with the vectors using a liposomal transfection method. Supernatants from the GPE86 lines were used to transduce PA317 cells and successful transfectants were identified by selection in G418 containing media.
• A375 is a human melanoma cell line and the above procedure resulted in 2 mixed population cell lines; A375/LISN and A375/LUSN.
• the same 3 step method was used to generate a A375 control cell line with an LXSN vector incorporated A375/LXSN.
• Northern blot analysis confirmed the presence of LISN and LUSN RNA in the appropriate cell lines. ( Figure 3).
• a anti MHC-I monoclonal Ab, H58A and a FITC labeled ant mouse secondary Ab were used. The results were analyzed using a fluorescent activated cell sorter.
• the cells were not permeabolized during the Ab labeling process and thus a decrease in fluorescence indicates a decrease in cell surface MHC-I.
• the autofluorescence of A375 cells and the background binding of the FITC labeled secondary Ab were assessed and found to be negligible (Figure 4).
• the A375/LXSN cell line often showed a marked increase of cell surface MHC-I expression which has been observed as a general response to viral infection.
• the A375/LXSN is the more appropriate control for these experiments since the majority of gene therapy delivery methods are viral based.
• Both the LISN and the LUSN vectors significantly reduced the MHC-I expression from either the A375/LXSN level or the A375 level.
• a cytotoxicity assay was performed (Figure 5). This assay measures the ability of CTL's to lyse target cells by measuring the release of a stable cytosolic enzyme, lactate dehydrogenase (LDH). After incubating the vector transduced target cells with the CTL effector cells, the amount of LDH released is measured using a substrate color reaction read at O.D. 492. Five effector to target ratios were used and both spontaneous release and media background LDH were subtracted from the readings. A clonally selected T acute lymphoblastic leukemia (TALL) cell line (TALL-104, ATCC) was used as effector cells.
• TALL T acute lymphoblastic leukemia
• TALL- 104 cells are known to have a high specific lysis of tumor cells and thus are well suited for this assay.
• A375/LXSN shows the highest specific lysis by TALL cells, followed by A375, A375/LISN, and A375/LUSN respectively.
• the A375 cell lines show up in Figs. 4 and 6D. 6D was a repeat of 4 and thus the numbers are slightly different.
• A375/LXSN (0.93 to 17.5 respectively) resulted in approximately a 43% reduction in TALL lysis at the 10:1 ratio.
• Primers to the Herpes virus gene encoding ICP 47 were designed with the following characteristics: Forward primer: 24 bp complementary to the start of the gene sequence, Kosak sequence to promote ribosome binding, Hind III endonuclease restriction site, and a guanine and a cytosine nucleotide added to help stabilize the primer end and the Hind III site.
• Reverse primer 24 bp complementary to the antisense sequence the end of the gene, Hind III site, and a GC clamp. PCR amplification of the gene was performed and confirmed by agarose gel electrophoresis (data not shown).
• MHC I Expression assay Cell surface MHC I inhibition. Transduced mixed populations of A375 cells were labeled with anti-MHC I Ab (VMRD Inc.) at 0.1 ⁇ g/.5 ⁇ l0 6 cells at 37 degrees for 1 hour. Cells were washed with PBS 2x and a 1:150 dilution of goat anti mouse FITC labeled secondary Ab (Sigma) was added for 1 hour at 37 degrees. Cells were again washed 2x and resuspended in Phenol red free/serum free Opti-MEM ® (Gibco BRL) media for FACS analysis. Three samples of each were averaged and the standard deviations determined. A375 no Ab, A375 no primary, and A375 are nontransduced controls.
• VMRD Inc. anti-MHC I Ab
• CTL response suppression assay Specific Lysis of A375 transduced target cells by TALL- 104 effector cells. 5xl0 3 target cells/well are aliquoted on a 96 well tissue culture plate (4 wells/data point). Effector cells are added at 10:1, 7.5:1, 5:1, 2.5:1, and 1.25:1 ratios and incubated for 7 hours. 100% lysis is determined by completely lysing a set of control wells. Cell lysis is determined by measuring the amount of a stable cytosolic enzyme (lactate dehydrogenase, LDH) released into the well and using a color reaction (Promega Corp. Cytotox 96 ® assay kit). Baseline LDH levels are established and spontaneous LDH release by effector and target cells is subtracted. Extent of color reaction is determined using a 96 well plate spectrophotometer at 492 nm.
• LDH lactate dehydrogenase
• TALL- 104 T acute lymphocytic leukemia
• TALL- 104 is a CD8+ CTL line that shows enhanced cytotoxic effects against tumor cells in general.
• TALL cells were tested to determine their ability to lyse the A375 and VA13 cell lines in a specific manner (Fig. 7A and B).
• the reduced levels of cell surface MHC I observed earlier are mimicked by the reduced levels of specific lysis by TALL cells. From this data we conclude that reduction of MHC I cell surface expression leads to a reduction in the lysis of LISN or LUSN transduced cells by CTL's in vitro. All viral and cell culture materials used in these experiments are treated as biosafety level two agents in accordance with institute policy.
• ICP 47 and USll each reduce the expression of MHC I restricted epitopes.
• two different approaches may be used.
• a single construct expressing two genes eliminates differences in RNA levels as a complicating factor.
• Both of the above mentioned approaches would be pursued concurrently as to avoid unforeseen problems with the relatively new IRES technology. See Figures 15, 16, 19, and 20.
• a vector similar to LISN would be constructed that uses hygromycin resistance as a selection method (Fig. 8). This LISH vector would then be transfected into packaging cells and the supernatant would be used to transduce target cells already containing the LUSN vector. The double transduced cells were be selected for using both G418 and hygromycin.
• IRES internal ribosome entry sites
• HSV ICP 47 and hCMV USll significantly lower the amount of cell surface MHC I and CTL specific lysis of the cell lines tested. Potential application of these genes in a gene therapy protocol would likely involve targeted vector delivery to a variety of organs and tissues. Additionally, it has been demonstrated that there are significant differences in gene expression when comparing established cell lines to primary cell cultures. To assess the function of LISN and LUSN in primary cell cultures, the MHC I transduction, expression, and cytotoxicity experiments will be repeated using selected primary cell cultures. Primary cultures from human liver, lung, kidney, and bone marrow are now commercially available and will be transduced and tested for MHC I cell surface reduction. Primary cell cultures from other tissues may also be tested.
• Packaging cell lines have been established for the LISN and LUSN vectors. Supernatants from these lines will be used to transduce the target cells listed above. The transduced target cells will be selected for 2-4 weeks in G418, a neomycin homologue.
• MHC I cell surface reduction will be assessed using the method described (Fig. 6). Specific lysis by TALL-104 cells will be measured as described (Fig. 7).
• ICP 47 does not function in rat cell lines, such as the 9L cell line shown here, and other rodent lines as reported elsewhere. (Ahn, K., et al., "Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47", EMBO J, 15(13):3247-55 (1996)) This is due to the absence of the ICP 47 binding site on the rodent version of TAP.
• in vivo rodent models will only be used to test USll function.
• ICP 47 in vivo function will be assessed in models where it is known to work, such as dogs as shown by the dog bone marrow cell line D17. Additionally, the complexities of in vivo models make it prudent to pursue several avenues in the hopes of success.
• the first model will express the fluorescent marker gene GFP in bone marrow cells. (Heim, R., A.B. Cubitt, and R.Y. Tsien, "Improved green fluorescence [letter]", Nature, 373(6516):663-4 (1995) This approach will be useful in assessing the incorporation of ICP 47/US11 immune down-regulation in the treatment of many different genetic diseases originating in blood cells.
• a second model will look at using the immune suppression itself as the therapeutic treatment of organ transplant and/or autoimmune disease.
• Recent improvements in whole organ transduction techniques makes it possible to conduct organ transplant experiments after transducing the organ with gene therapy vectors.
• a third model will be pursued in studying ⁇ -L-iduronidase deficiency in dogs.
• Shull, et. Al., Human Gene Therapy, 7:1595-1603 Myoblast gene Therapy in Canine Mucopolysaccharidosis I: Abrogation by an Immune Response to ⁇ -L- Iduronidase” (1996).
• Transduced lymphocytes are rapidly removed from the general lymphocyte population in vivo by both cellular and humoral immune responses.
• Tripathy, S.K., et al. "Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication- defective adenovirus vectors", Nat Med, 2(5):545-50 (1996); Riddell, S.R., et al., "T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients", Nat Med 2(2):216-23 (1996)
• the cellular immune response peaks at 7 days whereas the Ab response peaks at approximately 14 days. Removing the CTL response should prolong the survival of transduced lymphocytes in vivo.
• LGSN LGISN
• LGUSN LGUSN
• Vector producer cells will be established and supernatants used to transduce lymphocytes.
• Murine lymphocytes will be isolated, transduced, selected, and transfused back into syngeneic animals. Persistence of the fluorescent marker will be assessed by FACS analysis of periodic blood samples.
• This protocol requires the construction of a vector expressing three genes.
• the L ⁇ SI/N and L ⁇ SU/N constructs shown in Figure 12 would satisfy the requirements.
• the vector will be used to transduce bone marrow cells and ⁇ -ID production will be monitored long term.
• Previous experiments without immune suppression genes demonstrated that about 5% of bone marrow cells express ⁇ -ID at sub-therapeutic levels long term and that the low expression levels are due in part to an immune response to the ⁇ -ID protein.

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