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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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  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
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  • 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
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
• • A—HUMAN NECESSITIES
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  • 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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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• This invention relates to the treatment of adverse conditions of the nervous system, including, but not limited to tumors of the nervous system such as, for example, meningeal carcinomatosis. More particularly, this invention relates to the treatment of adverse conditions of the nervous system, preferably the central nervous system by administering, to the cerebrospinal fluid of a host, an expression vehicle such as a viral vector contained in a viral producer cell line which produces modified viral particles which include a nucleic acid sequence encoding a therapeutic agent for treatment of adverse conditions of the central nervous system, whereby the modified viruses deliver the nucleic acid sequence encoding the therapeutic agent to cells in the central nervous system.
• an expression vehicle such as a viral vector contained in a viral producer cell line which produces modified viral particles which include a nucleic acid sequence encoding a therapeutic agent for treatment of adverse conditions of the central nervous system, whereby the modified viruses deliver the nucleic acid sequence encoding the therapeutic agent to cells in the central nervous system.
• Meningeal carcinomatosis also known as leptomeningeal carcinomatosis, occurs in from about 5% to about 20% of all cancer patients and results most commonly from the metastatic spread of cancers such as lung cancer, breast cancer, and melanoma to the leptomeningeal coverings of the brain and spinal cord.
• cancers such as lung cancer, breast cancer, and melanoma
• Standard therapy for meningeal carcinomatosis is comprised of radiation therapy and/or intrathecal administration of chemotherapeutic agents.
• the need to irradiate the entire neuroaxis may cause profound bone marrow suppression,which limits the amount of systemic or intrathecal chemotherapy that the patient can tolerate. (Shapiro, 1991).
• Gene transfer is achieved by infection of tumor cells with murine retroviral vectors carrying the Herpes Simplex thymidine kinase gene and integration of this gene into the genome of the host cell. These vectors are produced continuously by murine vector producer cells that are injected into the tumor mass. Because retroviruses can infect only cells that are synthesizing DNA actively (i.e., replicating cells), a preferential transduction of tumor cells is achieved. This approach now is being evaluated in a clinical trial. (Oldfield, et al., Human Gene Therapy, Vol. 4, pgs. 39-69 (1993)).
• Meningeal carcinomatosis and other adverse conditions of the nervous system may provide another application of this approach.
• Vector producer cells which are injected into the cerebrospinal fluid, such as, for example, by injection into the ventricular system or lumbar or subarachnoid space, will circulate in the cerebrospinal fluid, and release viral vectors (such as, for example, retroviral vector particles) continuously.
• viral vectors such as, for example, retroviral vector particles
• a method of treating an adverse condition of the nervous system in a chordate host comprises administering to the cerebrospinal fluid of the chordate host an expression vehicle capable of transducing a cell in order to express a therapeutic agent in the central nervous system.
• the expression vehicle includes a nucleic acid sequence encoding a therapeutic agent for treating the adverse condition of the central nervous system.
• the expression vehicle is administered in an amount effective in treating the adverse condition of the central nervous system in the host.
• the cells which may be transduced with the expression vehicle include tumor cells and normal (i.e, non-tumor) cells, such as epithelial or endothelial cells of the choroid plexus.
• chordate means any animal of the phylum Chordata; i.e., any animal which includes a notochord or analogous structure, such as a spinal cord. Such animals include, but are not limited to, mammals (including human and non-human mammals), reptiles, amphibians, birds, and fish.
• neural system means any part of the animal which has a neurological and/or neuromotor function. Such parts include, but are not limited to, the brain, spinal cord, cranial nerves, and other nerves which are essential for neuromotor function.
• nucleic acid sequence means a DNA or RNA molecule, and includes complete and partial gene sequences, and includes polynucleotides as well. Such term also includes a linear series of deoxyribonucleotides or ribonucleotides connected one to the other by phosphodiester bonds between the 3 ' and 5' carbons of the adjacent pentoses.
• the expression vehicle upon administration to the cerebrospinal fluid to a host, travels throughout the cerebrospinal fluid to cells in the central nervous system, whereby a nucleic acid sequence encoding a therapeutic agent is delivered to cells in the central nervous system, and whereby the therapeutic agent is expressed by such cells.
• Cells which may be transduced include, but are not limited to, tumor cells of the central nervous system, brain cells, cells of the cranial nerves and other nerves essential for neuromotor function, and spinal cord cells.
• the type of cell to be transduced by the expression vehicle and the therapeutic agent is dependent upon the type of adverse condition of the nervous system to be treated.
• abnormal condition of the nervous system includes any disease of the nervous system which may be treated by gene therapy in which an expression vehicle including a nucleic acid sequence is transduced into a cell of the nervous system.
• diseases include, but are not limited to, tumors of the central nervous system, Alzheimer's disease, Parkinson's disease, Huntington's disease, degenerative disorders, mental disorders, and a variety of disorders that can be affected by introducing a new compound or modifying the levels of existing proteins in the nervous system.
• the expression vehicle may be any expression vehicle which is capable of transfecting cells and expressing the therapeutic agent for treating an adverse condition of the central nervous system in vivo.
• Suitable expression vehicles which may be employed include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial plasmids), and viral vectors, DNA- protein complexes, such as DNA-monoclonal antibody complexes, and receptor-mediated vectors.
• the vector may be contained within a liposome.
• the expression vehicle is a viral vector.
• Viral vectors which may be employed include, but are not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Herpes virus vectors.
• the viral vector is a retroviral vector.
• a packaging cell line is transduced with a viral vector containing the nucleic acid sequence encoding the therapeutic agent for treating an adverse condition of the nervous system to form a producer cell line including the viral vector.
• the producer cells then are administered to the cerebrospinal fluid of the host, whereby the producer cells generate viral particles which circulate throughout the cranio-spinal subarachnoid space in the cerebrospinal fluid and are capable of transducing cells, whereby such viral vectors express the therapeutic agent in such cells.
• the adverse condition of the nervous system is a tumor of the nervous system, such as, for example a tumor which infiltrates the leptomeningeal coverings of the central nervous system.
• a tumor of the central nervous system is treated by administering to the cerebrospinal fluid of the host producer cells which are transformed with a viral vector, and which produce a virus including a nucleic acid sequence encoding a therapeutic agent capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells.
• the viruses produced by the producer cells Upon administration of the producer cells to the cerebrospinal fluid, the viruses produced by the producer cells circulate through the cerebrospinal fluid and transduce the tumor cells, whereby the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells is expressed by the tumor cells.
• Tumors of the nervous system which may be treated include, but are not limited to, meningeal carcinomatosis, resulting from a peripheral or a primary brain tumor such as a medulloblastoma; ependynoma; and tumors of the lumbosacral region of the spinal column. It is to be understood, however, that the scope of the present invention is not to be limited to the treatment of tumors of the nervous system.
• 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.
• the retroviral vector is an infectious but non-replication competent retrovirus.
• replication competent retroviruses may also be used.
• 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 gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), 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.
• 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 (Ar entano, 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 sa 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 fragments 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. Such vectors are further described in U.S. Patent Application Serial No. 919,062, filed July 23, 1992, and incorporated herein by reference.
• 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, Vol. 7, No.
• CMV human cytomegalovirus
• 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 promoter also may be a tissue- specific or tumor-specific promoter. 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, ⁇ -2, ⁇ _--AM, PA12, T19-14X, VT-19- 17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines, as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990).
• the vector containing the nucleic acid sequence encoding the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence encoding the agent 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 CaP0 4 precipitation.
• the producer cells then are administered to the cerebrospinal fluid in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
• the producer cells may be administered in an amount of from about 1x10° to about ⁇ x ⁇ o 10cclls P erdose , preferably from about 6xl0 9 to about lxl0 10ceUsperdos .
• the exact amount of producer cells to be administered is dependent upon various factors, including. but not limited to, the type of the tumor and the size of the tumor. In some cases, repeat administration of the producer cells may be required.
• the producer cells are administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
• the carrier may be a liquid carrier such as, for example, a saline solution or a buffer solution or other isomolar aqueous solution.
• the producer cells may be administered to the cerebrospinal fluid intraventricularly, intrathecally, such as through the spinal subarachnoid space, or the producer cells may be administered to the choroid plexus, which may provide for continuous generation of viral vector particles into the cerebrospinal fluid.
• the producer cells Upon administration of the producer cells to the cerebrospinal fluid, the producer cells generate viral vector particles.
• the viral particles circulate throughout the cerebrospinal fluid, and transduce the tumor cells. Because tumor cells, and in particular cancerous tumor cells, in general are actively replicating cells, the retroviral particles would be integrated into and expressed preferentially or exclusively in the tumor cells as opposed to normal cells.
• Tumors of the nervous system which may be treated include malignant and non-malignant tumors.
• the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of such agent is a negative selective marker; i.e., a material which in combination with a chemotherapeutic or interaction agent inhibits, prevents or destroys the growth of the tumor cells.
• an interaction agent is administered to the human host.
• the interaction agent interacts with the negative selective marker in order to prevent, inhibit, or destroy the growth of the tumor cells.
• Negative selective markers which may be employed include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase; and cytosine deaminase.
• thymidine kinase such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase
• cytosine deaminase include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
• the negative selective marker is a viral thymidine kinase selected from the group consisting of Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
• the interaction or chemotherapeutic agent preferably is a nucleoside analogue, for example, one selected from the group consisting of ganciclovir, acyclovir, l-2-deoxy-2- fluoro-3-D-arabinofuranosil-5-iodouracil (FIAU), and 6- methoxypurine-arabinonucleoside (araM).
• FIAU l-2-deoxy-2- fluoro-3-D-arabinofuranosil-5-iodouracil
• arabM 6- methoxypurine-arabinonucleoside
• the negative selective marker is cytosine deaminase.
• cytosine deaminase is the negative selective marker
• a preferred interaction agent is 5-fluorocytosine. Cytosine deaminase converts 5- fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, the tumor cells which express the cytosine deaminase gene convert the 5-fluorocytosine to 5- fluorouracil and are killed.
• the interaction agent is administered in an amount effective to inhibit, prevent, or destroy the growth of the transduced tumor cells.
• the interaction agent may be administered in an amount from about 5 mg/kg to about 10 mg/kg of host weight, depending on overall toxicity to a patient. In one embodiment, such dose may be administered twice per day.
• the interaction agent preferably is administered systemically, such as, for example, by intravenous administration, by parenteral administration, by intraperitoneal administration, or by intramuscular administration.
• a "bystander effect" may result, i.e., tumor cells which were not originally transduced with the nucleic acid sequence encoding the negative selective marker may be killed upon administration of the interaction agent.
• the transformed tumor cells may be producing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent, or is taken up by adjacent, non-transformed tumor cells, which then become susceptible to the action of the interaction agent. It also is possible that one or both of the negative selective marker and the interaction agent are communicated between tumor cells.
• Such bystander effect is described further in U.S. patent application Serial No. 07/877,519 filed May 1, 1992, which is incorporated herein by reference.
• a packaging cell line is transduced with a retroviral vector, such as those hereinabove described, which includes the Herpes Simplex Virus thymidine kinase gene.
• the transduced packaging cells are administered in vivo to the cerebrospinal fluid in an acceptable pharmaceutical carrier and in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
• the producer cells Upon administration of the producer cells to the cerebrospinal fluid, the producer cells generate viral vector particles including a gene encoding the negative selective marker. Such viral particles circulate throughout the cerebrospinal fluid and transduce the tumor cells.
• the human host then is given an agent such as ganciclovir, acyclovir, or l-2-deoxy-2- fluoro-3- D-arabinofuranosil-5-iodouracil (FIAU), which interacts with the Herpes Simplex Virus thymidine kinase to kill the transduced tumor cells.
• agent such as ganciclovir, acyclovir, or l-2-deoxy-2- fluoro-3- D-arabinofuranosil-5-iodouracil (FIAU)
• FIAU Herpes Simplex Virus thymidine kinase
• the expression vehicle is an adenoviral vector.
• the adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome.
• the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.
• the adenoviral vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR'; an adenoviral encapsidation signal; at least one DNA sequence encoding a therapeutic agent; and a promoter controlling the at least one DNA sequence encoding the therapeutic agent.
• the vector is free of the adenoviral El, E2, E3 and E4 DNA sequences, and the vector is free of DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter; i.e., the vector is free of DNA encoding adenoviral structural proteins.
• Such vectors may be constructed by removing the adenoviral 5' ITR, the adenoviral 3' ITR, and the adenoviral encapsidation signal, from an adenoviral genome by standard techniques.
• Such components, as well as a promoter (which may be an adenoviral promoter or a non- adenoviral promoter), tripartite leader sequence, poly A signal, and selectable marker, may, by standard techniques, be ligated into a base plasmid or "starter" plasmid such as, for example, pBluescript II KS-(Strategene) , to form an appropriate cloning vector.
• the cloning vector may include a multiple cloning site to facilitate the insertion of the at least one DNA sequence encoding a therapeutic agent into the cloning vector.
• the multiple cloning site includes "rare" restriction enzyme sites; i.e., sites which are found in eukaryotic genes at a frequency of from about one in every 10,000 to about one in every 100,000 base pairs.
• An appropriate vector in accordance with the present invention is thus formed by cutting the cloning vector by standard techniques at appropriate restriction sites in the multiple cloning site, and then ligating the at least one DNA sequence encoding a therapeutic agent into the cloning vector.
• the vector may then be packaged into infectious viral particles using a helper adenovirus which provides the necessary encapsidation materials.
• a helper adenovirus which provides the necessary encapsidation materials.
• the helper virus has a defective encapsidation signal in order that the helper virus will not encapsidate itself.
• An example of an encapsidation defective helper virus which may be employed is described in Grable, et al., J. Virol. , Vol. 66, pgs. 723-731 (1992).
• the vector and the encapsidation defective helper virus are transfected into an appropriate cell line for the generation of infectious viral particles. Transfection may take place by electroporation, calcium phosphate precipitation, microinjection, or through proteoliposomes. Examples of appropriate cell lines include, but are not limited to, HeLa cells or 293 (embryonic kidney epithelial) cells.
• the infectious viral vector particles may then be transduced into cells in the central nervous system, whereby the at least one DNA sequence encoding a therapeutic agent is expressed by the cells in a host.
• the vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; at least one DNA sequence encoding a therapeutic agent; and a promoter controlling the at least one DNA sequence encoding the therapeutic agent.
• the vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.
• the vector is also free of at least a portion of at least one DNA sequence selected from the group consisting of the E2 and E4 DNA sequences.
• the vector is free of at least the majority of the adenoviral El and E3 DNA sequences, and is free of one of the E2 and E4 DNA sequences, and is free of a portion of the other of the E2 and E4 DNA sequences.
• the vector is free of at least the majority of the El and E3 DNA sequences, is free of at least a portion of at least one DNA sequence selected from the group consisting of the E2 and E4 DNA sequences, and is free of DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.
• Such a vector in a preferred embodiment, is constructed first by constructing, according to standard techniques, a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence, a multiple cloning site (which may be as hereinabove described); a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome.
• a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence,
• Such DNA segment serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such sequence may encompass, for example, a segment of the adenovirus 5 genome no longer than from base 3329 to base 6246 of the genome.
• the plasmid may also include a selectable marker and an origin of replication.
• the origin of replication may be a bacterial origin of replication.
• Representative examples of such shuttle plasmids include pAVS6, shown in Figure 14. A desired DNA sequence encoding a therapeutic agent may then be inserted into the multiple cloning site.
• Homologous recombination then is effected with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequences have been deleted.
• Such homologous recombination may be effected through co-transfection of the shuttle plasmid and the modified adenovirus into a helper cell line, such as 293 cells, by CaP0 4 precipitation.
• a recombinant adenoviral vector which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.
• the homologous recombination fragment overlaps with nucleotides 3329 to 6246 of the adenovirus 5 genome.
• a vector which includes an adenoviral 5' ITR, an adenoviral encapsidation signal; an Ela enhancer sequence; a promoter; a tripartite leader sequence; at least one DNA sequence encoding the therapeutic agent; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences; and an adenoviral 3' ITR.
• This vector may then be transfected into a helper cell line, such as the 293 helper cell line, which will include the Ela and Elb DNA sequences, which are necessary for viral replication, and to generate infectious viral particles.
• the vector consisting of infectious, but replication- defective, viral particles, which contain at least one DNA sequence encoding a therapeutic agent is administered in an amount effective to treat the adverse condition of the central nervous system in a host.
• the vector particles may be administered in an amount of from 1 plaque forming unit to about 10 14 plaque forming units, preferably from about lxlO 6 plaque forming units to about lxlO 13 plaque forming units.
• the host may be a human or non-human animal host.
• the vector particles may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
• the carrier may be a liquid carrier (for example, a saline solution), or a solid carrier, such as, for example, micro- carrier beads.
• the method of the present invention may be employed to deliver other therapeutic gene products to the nervous system by circulation through the cerebrospinal fluid after intrathecal, intraventricular, intraspinal injection, injection into the subarachnoid space, or injection into the choroid plexus.
• gene products include proteins and peptides that are neurotransmitters, neuromodulators, neurohormones, and neurotrophic factors.
• the method of the present invention may be employed to treat other adverse conditions of the nervous system, including, but not limited to, traumatic injury, stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, degenerative disorders, mental diseases, and a variety of disorders that can be affected by introducing a new compound or modifying the levels of existing proteins in the cerebrospinal fluid.
• the method of the present invention may be employed to transduce normal cells or tissue in the nervous system in order to treat non-cancerous adverse conditions of the nervous system.
• Example 1 Construction of pGlTkSvNa
• This vector contains the Thymidine Kinase (hTK) gene from Herpes Simplex virus I regulated by the retroviral promoter and the bacterial gene, neomycin phosphotransferase (Neo R ) driven by an SV40 promoter.
• hTK Thymidine Kinase
• the HTK gene confers sensitivity to the DNA analogs acyclovir and ganciclovir, while the Neo R gene product confer resistance to the neomycin analogue, G418.
• pGlTkSvNa To make pGlTkSvNa, a three step cloning strategy was used. First, the herpes simplex thymidine kinase gene (Tk) was cloned into the Gl plasmid backbone to produce pGlTk. Second, the Neo R gene (Na) was cloned into the plasmid pSvBg to make pSvNa. Finally, SvNa was excised from Psvna and ligated into pGlTk to produce pGlTkSvNa.
• Tk herpes simplex thymidine kinase gene
• Na Neo R gene
• SvNa was excised from Psvna and ligated into pGlTk to produce pGlTkSvNa.
• Plasmid pGlTkSvNa was derived from plasmid PG1 ( Figure 3). Plasmid pGl was constructed from pLNSX (Palmer, et al., Blood, Vol. 73, pgs. 438-445). The construction strategy for plasmid pGl is shown in Figure 1. The 1.6kb EcoRI fragment, containing the 5' Moloney Murine Sarcoma Virus (MoMuSV) LTR, and the 3.0kb EcoRl/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately.
• MoMuSV Moloney Murine Sarcoma Virus
• pGl Figure 3
• pGl Figure 3
• MCS 54 base pair multiple cloning site
• the MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pGl plasmid, the neo r gene, the ⁇ -galactosidase gene, the hygromycin r gene, and the SV40 promoter.
• the structure of the 5' linker was as follows: 5' - 1/2 Ndel - SphI - NotI - SnaBI - Sail - SacII - AccI - Nrul - Bglll - III 27 bp ribosomal binding signal - Kozak consensus sequence/Ncol - first 21 bp of the lacZ open reading frame - 1/2 BamHI - 3' .
• the structure of the 3' linker was as follows: 5' - 1/2 mutated EcoRI - last 55 bp of the lacZ open reading frame - Xhol
• the Kozak consensus sequence (5'-GCCGCCACCATGG-3 ' ) has been shown to signal initiation of mRNA translation (Kozak, Nucl.Acids Res. 12:857-872, 1984).
• the Kozak consensus sequence includes the Ncol site that marks the ATG translation initiation codon.
• pBR322 (Bolivar et al. , Gene 2:95, 1977) was digested with Ndel and EcoRI and the 2.1 kb fragment that contains the ampicillin resistance gene and the bacterial origin of replication was isolated.
• the ligated 5' linker - lacZ - 3' linker DNA described above was ligated to the pBR322 Ndel/EcoRI vector to generate pBg.
• pBg has utility as a shuttle plasmid because the lacZ gene can be excised and another gene inserted into any of the restriction sites that are present at the 5' and 3' ends of the lacZ gene. Because these restriction sites are reiterated in the pGl plasmid, the lacZ gene or genes that replace it in the shuttle plasmid construct can easily be moved into pGl.
• a 1.74 Kb Bglll/PvuII fragment containing the Herpes Simplex Virus Type I thymidise kinase gene was excised from the pXl plasmid (Huberman, et al. , Exptl. Cell Res. Vol. 153, pgs 347-362 (1984) incorporated herein by reference), blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into the unique SnaBI site in the pGl multiple cloning site, to form plasmid pGlTK. ( Figure 5) .
• a producer cell line was made from vector plasmid and packaging cells.
• the PA317/GlTkSvNa producer cell was made by the same general techniques used to make previous clinically relevant retroviral vector producer cell lines.
• the vector plasmid pGlTkSvNa DNA was transfected into a ecotropic packaging cell line, PE501. Supernatant from the PE501 transfected cells was then used to transinfect the amphotropic packaging cell line (PA317).
• Clones of transinfected producer cells were then grown in G418 containing medium to select clones that contain the Neo R gene. The clones were then titered for retroviral vector production. Several clones were then selected for further testing and finally a clone was selected for clinical use.
• 5 x 10 5 PE501 cells (Miller, et al., Biotechniques, Vol. 7, pgs. 980-990 (1989), incorporated herein by reference) were plated in 100 mm dishes with 10 ml high glucose Dulbecco's Modified Essential Medium (DMEM) growth medium supplemented with 10% fetal bovine serum (HGD10) per dish. The cells were incubated at 37°C, in 5% C0 2 /air overnight.
• DMEM Dulbecco's Modified Essential Medium
• the plasmid pGlTKSvNa then was transfected into PE501 cells by CaP0 4 precipitation using 50 ⁇ g of DNA by the following procedure.
• a culture dish(es) with optimum precipitate following the overnight incubation then was (were) selected.
• the dish(es) then was (were) washed again with PBS to remove the salt and the salt solution.
• 10 ml of HGD10 medium then was added to the dish(es), and the dish(es) incubated at 37°C in a 5% C0 2 atmosphere for about 48 hrs.
• PE501 ecotropic containing supernatants from such colonies of PE501 cells were collected in volumes of from about 5 to 10 ml, placed in cryotubes, and frozen in liquid nitrogen at -70°C.
• PA317 cells (Miller et al. Mol. Cell. Biol. 6:2895- 2902 (1986)) then were plated at a density of 5 x 10 4 cells per 100 mm plate on Dulbecco's Modified Essential Medium (DMEM) including 4.5 g/1 glucose, glutamine supplement, and 10% fetal bovine serum (FBS).
• DMEM Dulbecco's Modified Essential Medium
• FBS fetal bovine serum
• the PE501 supernatant then was thawed, and 8 g/ml of polybrene was added to the supernatant.
• the medium was aspirated from the plates of PA317 cells, and 7 to 8 ml of viral supernatant was added and incubated overnight.
• the PE501 supernatant then was removed and the cells refed approximately 18-20 hours with fresh 10% FBS.
• the medium was changed to 10% FBS and G418 (800 g/ml).
• the plate then was monitored, and the medium was changed to fresh 10% FBS and G418 to eliminate dying or dead cells as necessary.
• the plate was monitored for at least 10 to 14 days for the appearance of G418 resistant colonies.
• Cloning rings were placed around all selected colonies.
• the cells were tyrpsinized and incubated into wells in a six well dish in 5 ml of HGD10 plus lx hypoxanthine aminopterin thymidine (HAT) .
• HGD10 lx hypoxanthine aminopterin thymidine
• clones grew to confluency, they were trypsidized and incubated in a 100 ml dish. As a clone in the 100 ml dish approached confluency, its amphotropic vector-containing supernatant was removed and centrifuged at 1,200 to 1,500 rpm for 5 minutes to pellet out cells.
• the plasmid pGlTKlSvNa ( Figure 8), was prepared according to the schematic representation shown in Figure 7. It was prepared to remove the partial open reading frame from pGlTKSvNa ( Figure 6).
• Generation of pSPTK5' DNA from the plasmid pGlNaSvTk was digested with restriction enzymes Bglll and Smal and the 1163 base pair (bp) Herpes thymidine kinase (TK) fragment was fractionated by agarose gel electrophoresis and isolated. This fragment contains 56 bp of the TK 5'-untranslated region and 1107 bp of the TK translation open reading frame.
• the 1163 bp TK fragment was ligated to the plasmid vector pSP73 (Pro ega Corporation, Madison, WI) that had been digested with restriction enzymes Bglll and Smal.
• the resulting ligated plasmid construct was named PSPTK5' because it contains the 5' portion of the TK open reading frame but lacks the last 21 bp of the open reading frame and the translation termination codon.
• PCR of the TK open reading frame: pGlNaSvTK plasmid DNA was linearized by digesting it with Bglll.
• the linearized pGlNaSvTK was used as a template for polymerase chain reaction (PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence for an Xhol site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'-PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence for an Xhol site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'-PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence for an Xhol site, the TK translation termination codon, and the last 19 bp of the
• PCR products were fractionated on an agarose gel and the expected 1215 bp fragment that includes the full-length TK open reading frame was isolated.
• the isolated fragment was digested with restriction enzymes PstI and Xhol, digestion products were fractionated on an agarose gel, and the 420 bp fragment was isolated. This fragment extends from the PstI site at the nucleotides encoding amino acids 249-250 of the TK open reading frame through the Xhol site immediately downstream of the TGA translation termination codon.
• PSPTK5' was digested with PstI and the 3993 bp fragment that contains the PSP73 vector and the 5' portion of the TK open reading frame was isolated following agarose gel electophoresis. This 3993 bp fragment was ligated to the PCR-generated 420 bp Pstl/Xhol fragment that contains the 3' end of the TK open reading frame (above).
• Ligated plasmid DNA was transformed into E. coli DK5 ⁇ competent cells (Gibco/BRL, Gaithersburg, MD) and DNA from ampicillin-resistant colonies was screened by restriction enzyme digestion. Plasmids that appeared to contain the full-length TK open reading frame were termed PSPTKl.
• PSPTKl clone #4 was found to match the expected TK sequence in this region and was used for construction of pGlTKlSvNa.
• PSPTKl DNA was digested with Bglll and the 5' overhanging ends were repaired by incubation of the digested DNA with deoxy nucleotides and Klenow fragment of E. coli DNA polymerase I.
• the DNA was then digested with Xhol to generate a 1225 bp fragment that contains 56 bp of TK 5'-untranslated region and the full-length TK open reading frame. This blunt/XhoI fragment was ligated to pGlXSvNa DNA that had been digested with SnaBI and Sail.
• pGlXSvNa To construct pGlXSvNa, the 1.2 kb SvNa fragment was excised from Psvna (Part A above) with Sail and Hindlll. This fragment was ligated to pGl that had been digested with Sail and Hindlll. The ligated plasmid was termed pGlXSvNa where the "X" denotes a multiple cloning region.
• pGlTKlSvNa Plasmids that appeared to contain the TK fragment by diagnostic restriction enzyme digestion were termed pGlTKlSvNa. ( Figure 8). Clone #2 was dideoxy sequenced from the beginning of the 5'-LTR through the end of the 3'-LTR and was found to contain the intact TK open reading frame. pGlTKlSvNa was used to produce a producer cell by combination with PA 317 by the hereinabove described method (Part B above). Such producer cell line was designated as producer cell line PA317/GlTKlSvNa.7. D. Effect of cerebrospinal fluid on Herpes Simplex thymidine kinase producer cells and vectors; assessment of retroviral vector titer.
• Each of the producer cell clones PA317/GlTkSvNa.53 and PA317/GlTklSvNa.7 was inoculated into two 6-well dishes (12 wells/clone) at 4-5xl0 5 cells/well (37°C, 5%C0 2 , for 24 hours or until nearly confluent) .
• CSF cerebrospinal fluid
• growth medium 0%, 5%, 10%, 25%, 50%, and 75%).
• CPE cytopathic effect
• Supernatant samples then were collected for titer assay.
• supernatant samples of known titer GlTkSvNa.53 and GlTklSvNa.7 were mixed with CSF and titered.
• the cells were grown in this medium until individual colonies (originating from cells that were transduced with the neomycin-resistant gene and thus protected from the toxic effect of G418) were visible microscopically. The cells were then stained with methylene blue and the colonies counted. The titer per ml at each dilution was determined by calculating the average number of colonies in the wells, multiplying by the dilution factor, and dividing by 3 ml. The average titer was determined by calculating the average of the titers for all dilutions.
• the supernatants used as positive control had a neomycin- resistant gene titer between 1 x 10 5 and 4 x 10 6 particles/ml (PA317/GlTklSvNa.7) and 10 3 to 10 4 (PA317/GlTkSvNa.53) .
• Herpes Simplex thymidine kinase vector titer from supernatants of known titer was not affected by cerebrospinal fluid during exposure for 60 minutes prior to overnight incubation over NIH 3T3 cells.
• PA317/GlTkSvNa.53 producer cells were injected (10 6 cells/10 ⁇ l ) into each of four Spraque-Dawley rats (wt. from 230 to 350 g) via cisternal catheter under brief inhalation anesthesia. The rats were sacrificed on days 5 and 10 after cell injection for histologic examination of the brain and spinal cord with hematoxylin and eosin staining. None of the rats showed any signs of neurologic or systemic toxicity, and there was no histologic evidence of meningeal reaction, or parenchymal brain or spinal cord injury.
• PA317/GlTkSvNa.53 producer cells were injected (10 6 cells/10 ⁇ l ) via cistermal intrathecal catheter into each of four Sprague-Dawley rats (each weighing from 230 to 350 g) , and ganciclovir was administered 7 days later (30 mg/kg/day intraperitoneally) .
• the animals were sacrificed on days 3, 6, 10, and 14 of ganciclovir treatment for histologic examination of the brain and spinal cord with hematoxylin and eosin stain. There was no evidence of inflammation of the leptomeninges or parenchymal injury throughout the brain and spinal cord, and the rats showed no signs of neurologic or systemic toxicity throughout the study.
• ganciclovir was given intravenously to two monkeys (10 mg 1 kg/day for 14 days), and was given intrathecally to two monkeys (200 ⁇ g/day for 14 days).
• Two monkeys received only the producer cell injections.
• the monkeys receiving the intravenous ganciclovir treatment underwent placement of an intracardiac catheter via the right external jugular vein with a subcutaneous access port placed in the interscapular region.
• the animals were placed in a stereotactic frame and a midline incision was made to expose sagittal suture and bregma.
• stereotactic coordinates a ventricular catheter was placed in the right lateral ventricle and its location confirmed by pressure tracing.
• the producer cells were injected over a period of time of one minute.
• the ventricular catheter was left in place and connected to a subcutaneous access port in the interscapular region.
• ganciclovir At the completion of the ganciclovir treatment, three monkeys, one from each group, were sacrificed for histologic evaluation of the brains, spinal cords, and peripheral organs. The three remaining monkeys were kept for long-term observation and subsequent repeat intrathecal cell injections. Blood and cerebrospinal fluid samples were collected before cell injection, 7 days after cell injection, after 7 days of ganciclovir treatment, and at the completion of the ganciclovir treatment. Cerebrospinal fluid was analyzed for cytology, protein and glucose content. Blood samples were analyzed for routine chemistry and hematologic profiles.
• Gadolinium-enhanced MRI scans of the brain were obtained before cell injection, 7 days after cell injection, after 7 days of ganciclovir treatment, and at the completion of the ganciclovir treatment.
• the monkeys which were not sacrificed received a repeat intraventricular injection of a higher dose of producer cells (2xl0 8 PA317/GlTKSvNa 53 producer cells mixed with lxlO 7 ⁇ -galactosidase producer cells in a total volume of 2 ml Pbs) .
• seven days after cell injection two of the monkeys received intravenous ganciclovir (10 mg/kg/day) for 14 days. Toxicity was assessed by daily clinical examination, analysis of blood and cerebrospinal fluid samples, and gadolinium-enhanced MRI scans of the brain before cell injection, at the completion of the ganciclovir therapy, and at 9 weeks after cell injection.
• cisternal and lumbar cerebrospinal fluid samples were obtained by percutaneous aspiration on days 1, 3, and 5 after intraventricular cell injection and evaluated for vector titer and cytology to assess the dynamics of producer cell and vector particle distribution in the subarachnoid space.
• Retroviral vector titer was assessed as follows:
• Cultured NIH 3T3 cells were grown in DMEM medium containing 10% fetal bovine serum at a density of lxlO 5 cells/well in 6-well dishes. Serial dilutions of the tested cerebrospinal fluid (0 through 2xl0" 2 ) were made in growth medium containing 8 ⁇ g/ml polybrene. 2 ml of the diluted solution were added to each well. Dilutions of a retroviral supernatant of known titer were used as a positive control. The dishes were incubated at 32°C and 5% C0 2 . After 16-20 hours, the medium was changed to growth medium containing 0.8 mg/ml G418 (a neomycin analogue) and incubated at 37°C.
• G418 a neomycin analogue
• the cells were grown in this medium until individual colonies (originating from cells that were transduced with the neomycin resistance gene and thus protected from the toxic effect of G418) were visible microscopically. The cells then were stained with methylene blue and the colonies counted. The titer per ml at each dilution was determined by calculating the average number of colonies in the wells, multiplying by the dilution factor, and dividing by 2 ml. The average titer was determined by calculating the average of the titers for all dilutions. The supernatant used as positive control has a neomycin-resistant gene titer between lxlO 5 and 4x10° particles/ml.
• the rats were sacrificed by intracardiac perfusion with 4% formaldehyde, and the brains were removed for histologic examination.
• the choroid plexus was dissected from the lateral and 4th ventricles and stained with X-gal after pre-incubation with EGTA to block endogenous ⁇ -galactosidase activity.
• the choroid plexus from both non-ganciclovir and ganciclovir-treated rats showed significant X-gal staining of both the lateral and 4th ventricle choroid plexus compared to control animals that received no cell injections. There was minimal difference in X-gal staining with same disruption of the choroid plexus in ganciclovir-treated rats and the control rats.
• each of 6 rats (Sprague- Dawley, 230g to 350g) were injected intrathecally with a low concentration of ganciclovir (5 ⁇ g/10 ⁇ l PBS daily for 14 days) or a high concentration of ganciclovir (200 ⁇ g/10 ⁇ l PBS daily for 14 days) via an indwelling cisternal catheter.
• the rats were examined daily for evidence of neurological toxicity, and were sacrificed after the 14 days of ganciclovir treatment for histologic examination of the brain and spinal cord with hematoxylin and eosin staining.
• the producer cell line PA317/GlTkSvNa.53 was maintained in culture in Dulbecco Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, Utah), 2 Mm L-glutamine (Gibco BRL, Gaithersburg, MD), 50 units/ml penicillin (Gibco), 50 ⁇ g/ml streptomycin (Gibco), and 2.5 ⁇ g/ml Fungizone (ICN Biomedicals Inc., Costa Mesa, California). The producer cells were grown in T-175 flasks.
• DMEM Dulbecco Modified Eagle's Medium
• Producer cells were harvested prior to intrathecal injection by incubation in 0.05% Trypsin-EDTA (Gibco) for 5 to 10 minutes at 37°C. The cells were collected in Hanks Balanced Salt Solution (HBSS) (Biofluids, Inc., Rockville, MD), washed twice, and resuspended at 8xl0 6 cells/ml for injection.
• HBSS Hanks Balanced Salt Solution
• Some of the producer cells also were infected with the replication-competent retrovirus 4070A.
• 4070A virus-containing supernatant was filtered through a 0.22 ⁇ m filter onto a onolayer of the producer cells. Two passages of the culture were allowed to achieve uniform infection of the producer cells with the 4070A retrovirus.
• Fischer 344 rats weighing 230-300 grams were anesthetized using intraperitoneal (i.p.) Ketamine (90 mg/Kg, Fort Dodge Laboratories, Inc., Fort Dodge, Iowa) and Xylazine (10 mg/Kg, Mobay Corporation, Shawnee, Kansas).
• a sterile PE-10 tube was inserted into the upper thoracic subarachnoid space via the cisterna magna, secured in the subcutaneous soft tissue, and pierced through the skin on the back of the neck. The tube then was obliterated with a sterile removable steel rod. The rats were then observed for 5 to 7 days during which any rat that developed neurological deficits was excluded from the study.
• Rats were housed one per cage to protect the catheters and received oral Amoxicillin (5 mg/kg in water, calculated for an average water consumption of 20 ml/rat/day) and dexamethasone (TechAmerica, Kansas City, MO) in an amount of 0.5 mg/Kg/20 ml for the duration of the study. 159 rats then were reanesthetized, using inhalation anesthesia (N 2 0:0 2 :Halothane mixture), and 8x10 4 syngeneic 9L gliosarcoma cells in 10 ⁇ L Hamilton syringe.
• mice 100 rats were injected with 8xl0 4 PA317/GlTkSvNa.53 producer cells (not co-infected with replication competent virus); 28 rats were injected with 8xl0 4 PA317/GlTkSvNa.53 producer cells which were co-infected with the 4070A replication competent retrovirus; 25 rats were injected with 8xl0 4 PA317/G1TK1SW.7 producer cells; and 6 rats were injected with 8xl0 4 GlBgSvN.29 producer cells, which generate vector particles including a ⁇ -galactosidase gene.
• the low intrathecal dose was chosen to achieve a cerebrospinal fluid concentration of 5 to 10 ⁇ g/ml, which had been shown to be the effective antitumor concentration in vitro studies. (Culver, et al., 1992; Ram, et al., 1993). The higher concentration was chosen to deliver a great excess of the drug into the subarachnoid space.
• the rats were observed daily for development of neurological deficits, which invariably almost manifested as rapidly progressing paraparesis and paraplegia leading to death within 12 to 24 hours.
• the Mantel-Haenzel test Mantel, Cancer Chemother. Rep. , Vol. 50. pgs.
• PA317/GlTkSvNa.53 25 ⁇ g/kg 20.2 0.434 producer cells intrathecal PA317/GlTkSvNa.53 Saline- 19.1 0.434 producer cells intrathecal
• PA317/GlTKSvNa.53 30 mg/kg 17.9 0.017 producer cells intraperitoneally
• Tumor infiltration of the leptomeningeal coverings of the brain and spinal cord presents a unique therapeutic challenge.
• the diffuse narture of this disease requires an aggressive approach, including irradiation and chemotherapy, which results, however, in only a limited tumor response, marginal extension of survival, and significant morbidity.
• Retroviral-mediated gene therapy provides an attractive treatment option in the setting of meningeal carcinomatosis. Since the retroviral vectors are replication-incompetent, and thus unable to propagate infection and gene transfer from one tumor cell to another, efficient distribution of the vector is crucial to maximize gene transfer into as many tumor cells as possible. Efficient delivery of the vector is achieved by injection of the vector-producer cells into the cerebrospinal fluid.
• the retroviral vector particles containg the Herpes Simplex thymidine kinase gene are released continuously from the circulating vector producer cells and thereby reach the whole surface of the tumor-infiltrated meninge ⁇ . Selective transfer of the suicide gene into the proliferating tumor then sensitizes it to the effects of ganciclovir.
• Phase A 20 human patients suffering from meningeal carcinomatosis are grouped into four groups, termed Phase A, Phase B, Phase C, and Phase D.
• Phase A includes 3 patients;
• Phase B includes 3 patients;
• Phase C includes 4 patients; and
• Phase D includes 10 patients.
• Each patient receives an Ommaya reservoir connected to an intraventricular catheter for access to the cerebrospinal fluid.
• the Ommaya reservoir consists of a small bubble-type reservoir and a ventricular catheter.
• the placement of the Ommaya reservoir requires shaving the hair over the right frontal portion of the head, just in front of the coronal suture. The area then is prepped and draped in a sterile fashion.
• the skin is infiltrated with local anesthesia and a small incision is made just in front of the coronal suture and 3 cm to the right of the midline.
• a burr hole opening in the skull is made and a catheter then is placed into the lateral ventricle on that side. The catheter then is attached to the reservoir and the skin then is closed.
• a cerebrospinal fluid sample is taken from the reservoir and tested for the presence of malignant cells and various tumor markers.
• the patients in Phase A receive an intraventricular injection of 2xl0 9 PA317/GlTklSvN.7 producer cells via the Ommaya reservoir. 7 days after the injection of the producer cells, the patients are given ganciclovir by intravenous infusion over one hour at a dose of 5 mg/kg of body weight twice daily for 14 days.
• the patients of Phase B receive an injection of 2xl0 9 PA317/GlTklSvN.7 producer cells into the right lateral ventricle via the Ommaya reservoir, and an injection of 2xl0 9
• the patients of Phase C are given two injections of 2xl0 9 PA317/GlTklSvN.7 producer cells into the right lateral ventricle via the Ommaya reservoir for a total of 4xl0 9 cells injected into the right lateral ventricle, and two injections of 2xl0 9 PA317/GlTklSvN.7 producer cells into the lumbar subarachnoid space via a spinal catheter, for a total of 4xl0 9 cells injected into the lumbar subarachnoid space. 7 days after the patients are injected with the producer cells, the patients are given ganciclovir by intravenous infusion over one hour at a dose of 5 mg/kg of body weight twice daily for 14 days.
• Cerebrospinal fluid samples of the patients in Phases A, B, and C are tested for vector titers twice a day for the first 7 days after the patients are given the producer cells, and at 14, 21, 35, and 48 days after the patients are given the producer cells, followed by monthly checks of vector titer. Cerebrospinal fluid samples also are analyzed for tumor markers. Upon evaluation of the cerebrospinal fluid, samples and the finding that no toxicity in the patients is encountered, the 10 patients of Phase D are treated according to the same protocol as the patients of Phase C.
• Example 3 Adenoviral Gene Transfer into Meningeal Carcinomatosis A. Construction of pAvS6.
• the adenoviral construction shuttle plasmid pAvS6 was constructed in several steps using standard cloning techniques including polymerase chain reaction based cloning techniques.
• Ad-dl327 (Thimmappaya, et al., Cell, Vol. 31, pg.
• the ITR, encapsidation signal, Rous Sarcoma Virus promoter, the adenoviral tripartite leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification ( Figure 12).
• the ITR and encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences from Ad5, Genbank accession #M73260]) were amplified (amplication 1) together from Ad-dl327 using primers containing NotI or Ascl restriction sites.
• the Rous Sarcoma Virus LTR promoter was amplified (amplification 2) from the plasmid pRC/RSV (sequences 209 to 605; Invitrogen, San Diego, CA) using primers containing an Ascl site and an Sfil site. DNA products from amplifications 1 and 2 were joined using the "overlap" PCR method (amplification 3) with only the NotI primer and the Sfil primer. Complementarity between the Ascl containing end of each initial DNA amplication product from reactions 1 and 2 allowed joining of these two pieces during amplification.
• TPL was amplified (amplification 4) (sequences 6049 to 9730 of Ad- dl327 [identical to similar sequences from Ad5, Genbank accession #M73260]) from cDNA made from MRNA isolated from 293 cells infected for 16 hrs. with Ad-dl327 using primers containing Sfil and Xbal sites respectively. DNA fragments from amplification reactions 3 and 4 were then joined using PCR (amplification 5) with the NotI and Xbal primers, thus creating the complete gene block.
• amplification 4 sequences 6049 to 9730 of Ad- dl327 [identical to similar sequences from Ad5, Genbank accession #M73260]
• Ad-dl327 using primers containing Sfil and Xbal sites respectively.
• DNA fragments from amplification reactions 3 and 4 were then joined using PCR (amplification 5) with the NotI and Xbal primers, thus creating the complete gene block.
• the iTR-encapsidation signal-TPL fragment was then purified, cleaved with NotI and Xbal and inserted into the NotI, Xbal cleaved pHR plasmid.
• This plasmid was designated pAvS6A and the orientation was such that the NotI site of the fragment was next to the T7 RNA polymerase site ( Figure 13).
• the SV40 early polyA signal was removed from SV40 DNA as an Hpal-BamHI fragment, treated with T4 DNA polymerase and inserted into the Sail site of the plasmid pAvS6A-( Figure 13) to create pAvS6 ( Figures 13 and 14.)
• the recombinant, replication-deficient adenoviral vector AvlLac Z4, which expresses a nuclear-targetable B-galactosidase enzyme was constructed in two steps. First, a transcriptional unit consisting of DNA encoding amino acids 1 through 4 of the SV40 T- antigen followed by DNA encoding amino acids 127 through 147 of the SV40 T-antigen (containing the nuclear targeting peptide Pro-Lys- Lys-Lys-Arg-Lys-Val) , followed by DNA encoding amino acids 6 through 1021 of E. coli B-galactosidase, was constructed using routine cloning and PCR techniques and placed into the EcoRV site of pAvS6 to yield pAvS6-nlacZ ( Figure 15).
• AvlLacZ4 The infectious, replication-deficient, AvlLacZ4 was assembled in 293 cells by homologous recombination. To accomplish this, plasmid pAvS6-nLacZ was linearized by cleavage with Kpnl. Genomic adenoviral DNA was isolated from purified Ad-dl327 viruses by Hirt extraction, cleaved with Clal, and the large (approximately 35 kb) fragment was isolated by agarose gel electrophoresis and purified. The Clal fragment was used as the backbone for all first generation adenoviral vectors, and the vectors derived from it are known as Avl.
• IMEM-2 IMEM plus 2% FBS, 2mM glutamine (Bio Whittaker 046764)
• IMEM-10 Improved minimal essential medium (Eagle's) with 2x glutamine plus 10% vol./vol. fetal bovine serum plus 2mM supplemental glutamine (Bio Whittaker 08063A) and incubated at 37°C for approximately three days until the cytopathic effect, a rounded appearance and "grapelike" clusters, was observed. Cells and supernatant were collected and designated as CVL-A.
• AvlLacZ4 vector (a schematic of the construction of which is shown in Figure 16) was released by three cycles of freezing and thawing of the CVL-A. Then, a 60 mm dish of 293 cells was infected with 0.5 ml of the CVL-A plus 3 ml of IMEM- 10 and incubated for approximately three days as above. Cells and supernatant from this infection then were processed by three freeze/thaw cycles in the same manner and termed CVL-B. A 2.25ml aliquot of a mixture of 3 ml of CVL-B plus 42 ml of IMEM-2 was used to infect each of 20 15 cm dishes of 293 cells.
• IMEM-10 was added and the incubations were continued for approximately three days until the cytopathic effect was observed.
• the cells and supernatant from CVL-B were harvested, collected into a 50 ml conical tube, and centrifuges at 1,500 rgm for 10 minutes. The cell pellet was resuspended in a reserved portion of the supernatant (5 ml) and stored in a 20°C freezer.
• AvlLacZ4 virus was produced by infecting 293 cells (a human kidney epithelial cell line containing the left 11% of the Adenovirus 5 genome) with AvlLacZ4 at a multiplicity of infection of 10 plaque forming units (pfu)/cell. Growth of the replication- deficient vector in this cell line is possible because the E1A- consituitively active region, which is necessary for viral particle production, is available as a trans activating element in these cells. Purification of the AvlLacZ4 virus yielded concentrations of 2-3x10" pfu/ml.
• Syngeneic Fischer rat 9L gliosarcoma cells were propagated in T-175 tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, Utah), 2mM L-glutamine (Gibco BRL, Gaithersburg, Md.), 50 units/ml penicillin (Gibco), 50 ⁇ g/ml streptomycin (Gibco), and 2.5 ⁇ g/ml Fungizone (ICN Biomedicals, Inc., Costa Mesa, California).
• DMEM Dulbecco's Modified Eagle's Medium
• the 9L syngeneic glioma model of meningeal carcinomatosis was used. (Kooistra, 1986). 12 Fischer 344 rats weighing 230 to 350g were anesthetized and a sterile PE-10 tube was inserted into the upper thoracic subarachnoid space via the cisterna magna, secured in the subcutaneous soft tissue, and pierced through the skin on the back of the neck. The tube then was obliterated with a sterile removable steel rod. The rats were observed for 5 to 7 days during which any rat that demonstrated neurological deficits was excluded from the study.
• Rats were housed one per cage to protect the catheters and received oral amoxicillin (5 mg/kg/day, Beecham Laboratories, Bristol, Tennessee, calculated for an average water consumption of 20 ml/rat/day) and dexamethasone (Tech America, Kansas City, Missouri) 0.5 mg/kg/20 ml for the duration of the study.
• the rats then were reanesthetized, using inhalational anesthesia (N 2 0: 0 2 : halothane mixture), and 8xl0 4 syngeneic 9L gliosarcoma cells in lO ⁇ l Hank's balanced salt solution were injected into the subarachnoid space using a 10 ⁇ l Hamilton syringe.
• AvlLacZ4 viral particles (lxlO 9 or 2xl0 8 in 10 ⁇ l PBS) then were injected into 6 rats on the day of tumor inoculation, and to 6 rats 7 days after inoculation.
• the catheter then was flushed with additional 10 ⁇ l PBS and sealed with a steel rod.
• the rats were sacrificed on days 3, 7, and 10 after vector injection.
• the brains and spinal cords were removed, sectioned, and stained with X-Gal to identify cells expressing jS-galactosidase.
• ⁇ -galactosidase expression was detected using the X-Gal histochemical stain. (Bondi, et al., Histochemistry, Vol. 76, pgs.
• the choroid plexus a specialized intraventricular organ that develops during embryogenesis from infolding of the ependymal cell layer, is comprised of ciliated epithelial cells surrounding a mesh of capillaries. It actively secretes cerebrospinal fluid (CSF) into the cerebral ventricles.
• CSF cerebrospinal fluid
• the CSF circulates in the subarachnoid space to bathe the surface of the brain and spinal cord and to penetrate deeply into the Vircho-Robin spaces of the brain and spinal cord.
• choroid plexus epithelium and endothelial cells are the most mitotically-active cells in the normal adult brain (Johnson, et al . , Cancer, Vol. 13, pgs. 336-342 (1960); Kaplan, The Anatomical Record, Vol. 197, pgs. 496-502 (1980)), and, thus, are preferentially susceptible to transduction by retroviral vectors.
• Targeted transduction of these choroid plexus cells may allow continuous secretion of therapeutic proteins, such as nerve growth factors, neurotransmitters, enzymes, and hormones into the CSF for therapy or research.
• hGM-CSF human granulocyte macrophage colony stimulating factor
• the vector contains the human GM-CSF gene downstream of the 5' long terminal repeat (LTR) promoter and retroviral packaging signal.
• the vector also contains the neomycin resistance gene internally promoted by the SV40 promoter.
• the GIGmSvNa vector is packaged by the amphotropic retroviral vector producer cell line PA317 which is derived from NIH 3T3 cells. Cloned, G418-selected, human GM-CSF vector producer cells (PA317/GlGmSvNa.9) were maintained in culture and harvested by trypsinization just prior to intraventricular injection.
• Fifteen Fischer 344 rats received bilateral injections of 2 x 10 6 PA317/GlGmSvNa.
• producer cells suspended in 20 ⁇ l PBS into the lateral ventricles using a stereotactic frame (coordinates for injection: AP -1.0 mm, L 1.5 mm, and DV 3.5 mm, from the bregma, and dura, respectively).
• the rats were sacrificed on days 1, 2, 3, 4, and 7 after cell injections to obtain cerebrospinal fluid and choroid plexus specimens (3 animals were sacrificed each day to provide triplicate specimens for each time point).
• the brains were removed and choroid plexus of the lateral and 4th ventricle was dissected using an operating microscope.
• DNA was extracted from the choroid plexus specimens and amplified using a polymerase chain reaction (PCR) with: 1. primers that anneal specifically to the envelope gene in the pPAM3 helper plasmid in the vector-producer cells (Miller, et al . , Somat. Cell. Mol. Genet., Vol. 12, pgs. 175-183 (1985)) and 2. primers that anneal specifically to the hGM-CSF open reading frame.
• PCR polymerase chain reaction
• Transduction of the choroid plexus was documented by Southern analysis of the PCR products in one rat 3 days after vector-producer cell injection and in three rats 7 days after vector-producer cell injection.
• the remaining specimens of the choroid plexus showed evidence of pPAM3 sequence amplification in addition to human GM-CSF sequence amplification, indicating that these specimens contained surviving vector-producer cells that were adherent to the choroid plexus and could not allow estimation of transduction efficiency.
• Quantitative evaluation of the PCR bands from the choroid plexus specimens indicated that at 7 days approximately 0.4% of the choroid plexus cells were transduced with the hGM-CSF gene.
• Cerebrospinal fluid specimens were collected by exposing the cisterna magna surgically and siphoning the CSF-using a capillary tube. Samples were evaluated by enzyme-linked immunoassay for human GM-CSF. Levels of human GM-CSF greater than the lowest ELISA standard curve value (10 pg/ml) were detected in all CSF samples. A significant proportion of the human GM-CSF found in the CSF was contributed by the human GM-CSF vector-producer cells themselves. However, human GM-CSF levels as high as 110 pg/ml were detectable at late time points when no vector-producer cells were present in the choroid plexus samples.
• the measured levels of the cytokine could have resulted from a combination of secretion from transduced choroid plexus epithelium and residual, decaying human GM-CSF that had been previously secreted by the producer cells.

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