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WO1995009654A9 - Therapie genique concernant le systeme nerveux - Google Patents

Therapie genique concernant le systeme nerveux

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Publication number
WO1995009654A9
WO1995009654A9 PCT/US1994/010977 US9410977W WO9509654A9 WO 1995009654 A9 WO1995009654 A9 WO 1995009654A9 US 9410977 W US9410977 W US 9410977W WO 9509654 A9 WO9509654 A9 WO 9509654A9
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cells
tumor
nervous system
vector
agent
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WO1995009654A1 (fr
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Priority to JP7510881A priority patent/JPH09505561A/ja
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Publication of WO1995009654A9 publication Critical patent/WO1995009654A9/fr
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  • 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
  • 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.
  • 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)).
  • Vector producer cells which are injected into the cerebrospinal fluid, such as, for example, by injection into the ventricular system or lumbar or
  • viral vectors such as, for example, retroviral vector particles
  • Such viral vectors will contact tumor-infiltrated leptomeninges, transduce the replicating tumor cells, and enable the selective eradication of the tumor cells with the systemic administration of ganciclovir.
  • 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
  • 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
  • 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.
  • tumors of the central nervous system 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
  • 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
  • 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 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
  • 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.
  • LTR long terminal repeat
  • 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.
  • 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
  • Moloney murine sarcoma virus (MoMuSV) has alterations in this 5' region , including a frameshift and loss of glycosylation sites, which obviate potential expression of the amino terminus of pPr80 gag . Therefore, 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
  • Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miller, et al.,
  • 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 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
  • 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 XhoI.
  • 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 XhoI 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
  • 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 ß-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 , ⁇ - ⁇ M, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAm12, 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 CaPO 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 1 ⁇ 10 9 to about 1 ⁇ 10 10cells per dose , preferably from about 6 ⁇ 10 9 to about 1 ⁇ 10 10cells per dose .
  • 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 is selected from the group consisting of:
  • 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.
  • 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, 1-2-deoxy-2-fluoro- ⁇ -D-arabinofuranosil-5-iodouracil (FIAU), and 6-methoxypurine-arabinonucleoside (araM).
  • FIAU 1-2-deoxy-2-fluoro- ⁇ -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
  • 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.
  • 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
  • a packaging cell line is transduced with a retroviral vector, such as those
  • 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 1-2-deoxy-2-fluoro- ⁇ - 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 1-2-deoxy-2-fluoro- ⁇ - 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
  • 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 E1, 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
  • 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
  • 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
  • the vector is free of at least the majority of adenoviral E1 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. In one embodiment, 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 E1 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
  • 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 E1a 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 E1a 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);
  • 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
  • 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 E1 and E3 adenoviral DNA sequences have been deleted.
  • homologous recombination may be
  • a recombinant adenoviral vector is formed which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous
  • 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 E1a 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 may then be transfected into a helper cell line, such as the 293 helper cell line, which will include the E1a and E1b DNA sequences, which are necessary for viral replication, and to generate infectious viral particles.
  • helper cell line such as the 293 helper cell line, which will include the E1a and E1b 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 1 ⁇ 10 6 plaque forming units to about 1 ⁇ 10 13 plaque forming units.
  • the host may be a human or non-human animal host.
  • the vector particles may be administered in any order.
  • 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
  • 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.
  • 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.
  • the HTK gene confers
  • Neo R gene product confer resistance to the neomycin analogue, G418.
  • pGITkSvNa To make pGITkSvNa, a three step cloning strategy was used. First, the herpes simplex thymidine kinase gene (Tk) was cloned into the G1 plasmid backbone to produce pG1Tk. Second, the Neo R gene (Na) was cloned into the plasmid pSvBg to make pSvNa. Finally, SvNa was excised from Psvna and ligated into pG1Tk to produce pG1TkSvNa.
  • Tk herpes simplex thymidine kinase gene
  • Na Neo R gene
  • SvNa was excised from Psvna and ligated into pG1Tk to produce pG1TkSvNa.
  • Plasmid pG1TkSvNa was derived from plasmid PG1 ( Figure 3). Plasmid pG1 was constructed from pLNSX (Palmer, et al., Blood, Vol. 73, pgs. 438-445). The construction strategy for plasmid pG1 is shown in Figure 1. The 1.6kb EcoRI fragment, containing the 5' Moloney Murine Sarcoma Virus (MoMuSV) LTR, and the 3.0kb EcoRl/ClaI fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated
  • pG1 ( Figure 3) consists of a retroviral vector backbone composed of a 5' portion derived from MoMuSV, a short portion of gag in which the authentic ATG start codon has been mutated to TAG (Bender, et al.
  • MCS 54 base pair multiple cloning site
  • the structure of the 5' linker was as follows: 5' - 1/2 NdeI - SphI - NotI - SnaBI - SalI - SacII - AccI - NruI - BglII - III 27 bp ribosomal binding signal - Kozak consensus sequence/NcoI - 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 - XhoI
  • the restriction sites in the linkers were chosen because they are not present in the neomycin resistance gene, the ⁇ -galactosidase gene, the hygromycin resistance gene, or the SV40 promoter.
  • the 27 bp ribosomal binding signal was included in the 5' linker because it is believed to enhance mRNA stability
  • 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 NdeI 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
  • the lacZ gene or genes that replace it in the shuttle plasmid construct can easily be moved into pG1.
  • accession no. V00467 accession no. V00467, incorporated herein by reference
  • pX1 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 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 pG1TK.
  • EcoRI/AsuII fragment containing the coding sequence of the neomycin resistance gene was removed from pN2 (Armentano, et al., J. Virol., Vol. 61, pgs. 1647-1650 (1987)), blunted with Klenow fragment and ligated into the 2.5 kb blunted Bg1II/XhoI fragment generated hereinabove, resulting in pSvNa.
  • the SV40 promoter/neomycin resistance gene cassette was then removed from pSvNa as a 1191bp SalI/HindIII fragment.
  • the pG1Tk plasmid was then digested with
  • 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 pG1TkSvNa 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).
  • 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.
  • DMEM Dulbecco's Modified Essential Medium
  • HFD10 fetal bovine serum
  • the plasmid pG1TKSvNa then was transfected into PE501 cells by CaPO 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% CO 2 atmosphere for about 48 hrs.
  • the six plates of cells were examined daily. The medium was changed as needed to remove dead cells. Live cells or colonies were allowed to grow to a size such that the colonies are large enough to clone (i.e., the colonies are visible to the naked eye). PE501 ecotropic containing supernatants from such colonies of PE501 cells were
  • PA317 cells (Miller et al. Mol. Cell. Biol. 6:2895-2902 (1986)) then were plated at a density of 5 ⁇ 10 4 cells per 100 mm plate on Dulbecco's Modified Essential Medium (DMEM) including 4.5 g/l 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.
  • the cells were tyrpsinized and incubated into wells in a six well dish in 5 ml of HGD10 plus 1x
  • hypoxanthine aminopterin thymidine HAT
  • the plasmid pG1TK1SvNa ( 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).
  • telomere sequence DNA from the plasmid pG1NaSvTk was digested with restriction enzymes BgIII and SmaI 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 (Promega Corporation, Madison, WI) that had been digested with restriction enzymes BgIII and SmaI. 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
  • pG1NaSvTK plasmid DNA was linearized by digesting it with BgIII.
  • the linearized pG1NaSvTK was used as a
  • PCR polymerase chain reaction
  • 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 XhoI site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'- CCTGCATCGATTCTCGAGTCAGTTAGCCTCCCCCATCTCC-3').
  • 30 cycles of PCR were performed as follows: 1 minute at 94°C and 2 minutes at 60°C with a final 7 minute extension cycle at 72°C.
  • 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 XhoI, 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 XhoI site immediately downstream of the TGA translation termination codon.
  • PSPTK5' was digested with PstI and the 3993 bp
  • PSPTK1 Plasmids that appeared to contain the full-length TK open reading frame were termed PSPTK1.
  • the DNA from several PSPTK1 clones was dideoxy sequenced in the region from the PstI site through the XhoI site (the region that was generated by PCR). PSPTK1 clone #4 was found to match the expected TK sequence in this region and was used for construction of pG1TK1SvNa.
  • PSPTK1 DNA was digested with BgIII and the 5'
  • pG1XSvNa To construct pG1XSvNa, the 1.2 kb SvNa fragment was excised from Psvna (Part A above) with Sail and HindIII. This fragment was ligated to pG1 that had been digested with Sail and HindIII. The ligated plasmid was termed pG1XSvNa where the "X" denotes a multiple cloning region.
  • Plasmids that appeared to contain the TK fragment by diagnostic restriction enzyme digestion were termed pG1TK1SvNa.
  • pG1TK1SvNa 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/G1TKl1vNa.7.
  • thymidine kinase producer cells and vectors assessment of retroviral vector titer.
  • Each of the producer cell clones PA317/G1TkSvNa.53 and PA317/GlTk1SvNa.7 was inoculated into two 6-well dishes (12 wells/clone) at 4-5 ⁇ 10 5 cells/well (37°C, 5%CO 2 , for 24 hours or until nearly confluent).
  • CSF cerebrospinal fluid
  • concentrations of cerebrospinal fluid (CSF) in growth medium 0%, 5%, 10%, 25%, 50%, and 75%).
  • CPE cytopathic effect
  • Supernatant samples then were collected for titer assay. Independently, supernatant samples of known titer (G1TkSvNa.53 and G1Tk1SvNa.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 (undiluted with CSF) had a neomycin-resistant gene titer between 1 ⁇ 10 5 and 4 ⁇ 10 6
  • Herpes Simplex thymidine kinase vector titer was not affected by cerebrospinal fluid during exposure for 60 minutes prior to overnight incubation over NIH 3T3 cells.
  • thymidine kinase vector producer cells and gar, iclovir.
  • PA317/G1TkSvNa.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/G1TkSvNa.53 producer cells were injected (10 6 cells/10 ⁇ l) via cistermal
  • mice 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.
  • the B-galactosidase producer cell line known as G1BgSvN.29, was formed by transfecting the PA317 cell line with pG1BgSvNa, a vector in which the lacZ gene replaces the Herpes Simplex thymidine kinase gene.
  • G1BgSvN.29 The B-galactosidase producer cell line, known as G1BgSvN.29, was formed by transfecting the PA317 cell line with pG1BgSvNa, a vector in which the lacZ gene replaces the Herpes Simplex thymidine kinase gene.
  • 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
  • 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.
  • 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 intrathecal ganciclovir the ventricular catheter was left in place and connected to a subcutaneous access port in the interscapular region.
  • Gadolinium-enhanced MRI scans of the brain were obtained before cell injection, 7 days after cell
  • Cerebrospinal fluid protein and glucose levels remained normal, although the two monkeys treated with ganciclovir intraventricularly developed mild cerebrospinal fluid pleocytosis which was asymptomatic and did not progress to meningitis.
  • the choroid plexus from the control animal appeared normal.
  • X-Gal staining showed diffuse staining of choroid plexus cells, but no evidence of transduction with the ⁇ -galactosidase vector was detected in the brain parenchymal or spinal cord.
  • the monkeys which were not sacrificed received a repeat intraventricular injection of a higher dose of producer cells (2 ⁇ 10 8 PA317/G1TKSvNa 53 producer cells mixed with 1 ⁇ 10 7 ⁇ -galactosidase producer cells in a total volume of 2 ml Pbs).
  • a repeat intraventricular injection of a higher dose of producer cells (2 ⁇ 10 8 PA317/G1TKSvNa 53 producer cells mixed with 1 ⁇ 10 7 ⁇ -galactosidase producer cells in a total volume of 2 ml Pbs).
  • 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 1 ⁇ 10 5 cells/well in 6-well dishes. Serial dilutions of the tested cerebrospinal fluid (0 through 2 ⁇ 10 -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% CO 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. 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
  • the titer per ml at each dilution was determined by calculating the average number of colonies in the wells, multiplying by the
  • 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 1 ⁇ 10 5 and 4 ⁇ 10 6 particles/ml.
  • Vector titers were detected in the cerebrospinal fluid samples 24 hours after repeat cell injections. Comparable titers were found in the cisternal and lumbar cerebrospinal fluid samples.
  • 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
  • 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. There was no evidence of neurologic sequelae in any of the treatment animals, and histologic appearance of the brain and spinal cord in both the high and low-concentration intrathecal ganciclovir treated rats showed no indication of meningeal reaction or parenchymal injury.
  • F Administration of producer cells to rats having
  • the producer cell line PA317/G1TkSvNa.53 was
  • DMEM Dulbecco Modified Eagle's Medium
  • fetal bovine serum Hyclone Laboratories, Inc., Logan, Utah
  • 2 Mm L-glutamine Gibco BRL
  • the producer cells were grown in T-175 flasks. 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 8 ⁇ 10 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 monolayer of the producer cells. Two passages of the culture were allowed to achieve uniform infection of the producer cells with the 4070A retrovirus.
  • Ketamine 90 mg/Kg, Fort Dodge Laboratories, Inc., Fort Dodge, Iowa
  • 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
  • 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
  • gliosarcoma cells in 10 ⁇ L Hamilton syringe. 100 rats were injected with 8xl0 4 PA317/G1TkSvNa.53 producer cells (not co-infected with replication competent virus); 28 rats were injected with 8 ⁇ 10 4 PA317/G1TkSvNa.53 producer cells which were co-infected with the 4070A replication competent retrovirus; 25 rats were injected with 8 ⁇ 10 4
  • PA317/G1TK1SVN.7 producer cells; and 6 rats were injected with 8xl0 4 G1BgSvN.29 producer cells, which generate vector particles including a ß-galactosidase gene.
  • Spo producer cells are formed by transfecting the PA317 cell line with pG1BgSvNa, a vector in which the lac Z (ß-galactosidase) gene replaces the Herpes Simplex thymidine kinase gene.
  • the catheter then was flushed with 10 ⁇ l of phosphate buffered saline (PBS), and sealed with a steel rod.
  • Rats injected with the ß-galactosidase producer cell line were sacrificed on days 3, 6, and 10 after cell injection. The brain and spinal cord were removed and histological
  • the rats which received ganciclovir received the drug daily by intraperitoneal injections (30 mg/kg/ml PBS) or intrathecal injections (25 ⁇ g/kg or 600 ⁇ g/kg; 10 ⁇ l PBS) for 14 days.
  • 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. 163-170 (1966)) was used to compare survival between ganciclovir and saline-treated rats in the survival experiments.
  • PA317/G1TkSvNa.53 producer cells as compared with the saline-treated rats.
  • a survival curve for the rats of Group 1 is shown in Figure 9.
  • 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
  • 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 meninges. Selective transfer of the suicide gene into the
  • tumoricidal effect can be achieved with systemic (intraperitoneal) ganciclovir therapy. Histological examination of spinal cords from rats with meningeal carcinomatosis that had received intrathecal ganiclovir demonstrated almost complete eradication of the thin layers of tumor cells, which characteristically infiltrate the circumference of the spinal cord in this model of meningeal
  • ganciclovir has been previously described.
  • One of the major components of such bystander effect was linked to transfer of phosphorylated ganciclovir from transduced to non-transduced cells via gap junctions.
  • topical ganciclovir interacts with the superficial layers of tumor cells in the leptomeninges, such bystander effect may be generated and account for the efficient tumor eradication of the infiltrating tumor in the circumference of the spinal cord.
  • 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 2 ⁇ 10 9 PA317/G1Tk1SvN.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 2 ⁇ 10 9
  • the patients of Phase C are given two injections of 2 ⁇ 10 9 PA317/G1Tk1SvN.7 producer cells into the right lateral ventricle via the Ommaya reservoir for a total of 4 ⁇ 10 9 cells injected into the right lateral ventricle, and two injections of 2 ⁇ 10 9
  • 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.
  • the adenoviral construction shuttle plasmid pAvS6 was
  • Ad-d1327 (Thimmappaya, et al., Cell, Vol. 31, pg. 543 (1983)) is identical to adenovirus 5 except that an XbaI fragment including bases 28591 to 30474 (or map units 78.5 to 84.7) of the adenovirus 5 genome, and which is located in the E3 region, has been deleted.
  • the ITR and encapsidation signal (sequences 1-392 of Ad-d1327 [identical to sequences from Ad5, Genbank accession
  • 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 AscI site and an SfiI site. DNA products from amplifications 1 and 2 were joined using the
  • the TPL was amplified (amplification 4) (sequences 6049 to 9730 of Ad-d1327 [identical to similar sequences from Ad5, Genbank accession #M73260]) from cDNA made from MRNA isolated from 293 cells infected for 16 hrs. with Ad-d1327 using primers containing SfiI and XbaI sites respectively.
  • DNA fragments from amplification reactions 3 and 4 were then joined using PCR (amplification 5) with the NotI and XbaI primers, thus creating the complete gene block.
  • the ITR-encapsidation signal-TPL fragment was then purified, cleaved with NotI and XbaI and inserted into the NotI, XbaI 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 KpnI. Genomic adenoviral DNA was isolated from purified Ad-d1327 viruses by Hirt extraction, cleaved with ClaI, and the large (approximately 35 kb) fragment was isolated by agarose gel electrophoresis and purified. The ClaI fragment was used as the backbone for all first generation adenoviral vectors, and the vectors derived from it are known as Av1.
  • IMEM-2 IMEM plus 2% FBS, 2mM glutamine (Bio Whittaker 046764)
  • IMEM-10 Improved minimal
  • AvlLacZ4 vector (a schematic of the
  • 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 2-3 ⁇ 10" 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
  • AvlLacZ4 viral particles (1 ⁇ 10 9 or 2 ⁇ 10 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 ⁇ -galactosidase.
  • ⁇ -galactosidase expression was detected using the X-Gal histochemical stain. (Bondi, et al., Histochemistry, Vol. 76, pgs.
  • ⁇ -galactosidase activity was detected in the superficial layers of the extensively infiltrating tumor, both around the catheter and along the surface of the tumor mass, including tumor infiltrates surrounding nerve roots.
  • the depth of transduction was limited to several cell layers from the subarachnoid surface of the tumor while the center of the tumor mass showed no X-Gal-positive cells.
  • Seven days after vector injection, transduced cells were limited to the area of the
  • 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
  • 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.
  • hGM-CSF human granulocyte macrophage colony stimulating factor
  • GlGmSvNa hGM-CSF retroviral vector
  • McLachlin Moloney Murine Leukemia Virus
  • LTR long terminal repeat
  • the vector also contains the neomycin resistance gene internally promoted by the SV40 promoter.
  • the GlGmSvNa vector is packaged by the amphotropic retroviral vector producer cell line PA317 which is derived from NIH 3T3 cells.
  • Fifteen Fischer 344 rats received bilateral injections of 2 x 10 6 PA317/GlGmSvNa.9 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:
  • primers that anneal specifically to the envelope gene in the pPAM3 helper plasmid in the vector-producer cells (Miller, et al. , Somat. Cell. MoI. Genet., Vol. 12, pgs. 175-183 (1985)) and 2.
  • amplification of human GM-CSF sequences in the absence of pPAM3 sequence amplification provides evidence of choroid plexus transduction with the human GM-CSF gene.
  • 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
  • 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.
  • the duration and levels of expression of gene products secreted into the CSF after choroid plexus transduction may vary, but can be modulated by repeat injection and through the use of vectors that contain tissue-specific promoters. This approach may be applied for therapy as a drug delivery system for proteins into the CSF or used as a research tool to study the effects of continuous delivery of specific gene products in to the CSF.

Abstract

Un procédé permet de traiter un trouble du système nerveux chez un sujet chordé (mammifère de préférence). Il consiste à administrer dans le liquide céphalo-rachidien du sujet un vecteur d'expression pouvant transduire une cellule pour exprimer un agent thérapeutique dans le système nerveux. Ce vecteur d'expression comporte une séquence d'acide nucléique codant un agent thérapeutique destiné à traiter un trouble du système nerveux et on l'administre en quantité efficace à cette fin. Le procédé inventé s'applique particulièrement au traitement de la carcinomatose méningée et les cellules productrices, qui contiennent un vecteur rétroviral comportant un gène qui code un marqueur sélectif négatif (tel que la thymidine kinase de l'Herpes simplex), sont administrées dans le liquide céphalo-rachidien du patient. Ces cellules productrices donnent des particules virales qui transduisent les cellules de la carcinomatose méningée, qui, une fois transduites, sont alors tuées par l'administration d'un agent d'interaction (le ganciclovir par exemple) qui interagit avec le marqueur sélectif négatif.
PCT/US1994/010977 1993-10-01 1994-09-28 Therapie genique concernant le systeme nerveux Ceased WO1995009654A1 (fr)

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Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204052B1 (en) 1994-08-16 2001-03-20 Introgene B.V. Adenoviral vectors with reduced TNF response and partial E3 region deletion
US6544523B1 (en) 1996-11-13 2003-04-08 Chiron Corporation Mutant forms of Fas ligand and uses thereof
WO1998032869A1 (fr) * 1997-01-29 1998-07-30 Neurosearch A/S Vecteurs d'expression et procedes d'expression in vivo de polypeptides therapeutiques
ATE476508T1 (de) 1997-11-06 2010-08-15 Novartis Vaccines & Diagnostic Neisseriale antigene
SG152917A1 (en) 1998-01-14 2009-06-29 Chiron Srl Neisseria meningitidis antigens
CA2323067A1 (fr) * 1998-03-12 1999-09-16 The Trustees Of The University Of Pennsylvania Cellules productrices pour virus aptes a la replication utilisees dans le traitement de la malignite
EP2261341A3 (fr) 1998-05-01 2012-01-04 Novartis Vaccines and Diagnostics, Inc. Antigènes de Neisseria meningitidis et compositions
EP1228217B1 (fr) 1999-04-30 2012-11-21 Novartis Vaccines and Diagnostics S.r.l. Antigenes de neisseria conserves
GB9911683D0 (en) 1999-05-19 1999-07-21 Chiron Spa Antigenic peptides
GB9916529D0 (en) 1999-07-14 1999-09-15 Chiron Spa Antigenic peptides
GB2397063A (en) * 1999-10-21 2004-07-14 Cedars Sinai Medical Center A medicament comprising a cytotoxic pro drug activated by HSV TK
GB9924981D0 (en) 1999-10-21 1999-12-22 Univ Manchester Gene therapy
EP2275552B1 (fr) 1999-10-29 2015-09-09 GlaxoSmithKline Biologicals SA Peptides antigéniques de Neisseria
EP2275129A3 (fr) 2000-01-17 2013-11-06 Novartis Vaccines and Diagnostics S.r.l. Vaccin omv complèté contre la méningocoque
WO2002034771A2 (fr) 2000-10-27 2002-05-02 Chiron Srl Acides nucleiques et proteines derives des groupes de streptocoques a et b
GB0107661D0 (en) 2001-03-27 2001-05-16 Chiron Spa Staphylococcus aureus
GB0107658D0 (en) 2001-03-27 2001-05-16 Chiron Spa Streptococcus pneumoniae
ES2312649T3 (es) 2001-12-12 2009-03-01 Novartis Vaccines And Diagnostics S.R.L. Inmunizacion frente a chlamydia trachomatis.
GB0308198D0 (en) 2003-04-09 2003-05-14 Chiron Srl ADP-ribosylating bacterial toxin
EP1736541B1 (fr) 2004-03-29 2013-01-23 Galpharma Co., Ltd. GALECTINE-9 PROTEINE Modifiée ET UTILISATION DE CELLE-CI
EP2338524B1 (fr) 2004-08-12 2013-05-22 Cedars-Sinai Medical Center Thérapie génique combinée destinée au traitement de gliomes macroscopiques
WO2008020335A2 (fr) 2006-06-09 2008-02-21 Novartis Ag Compositions immunogènes pour streptococcus agalactiae
US8883493B2 (en) 2007-01-30 2014-11-11 Cedars-Sinai Medical Center Adenoviral vector comprising herpes simplex virus type 1 thymidine kinase and a transgene for increasing the expression of the transgene
MX373332B (es) * 2013-07-26 2020-05-21 Univ Iowa Res Found UNA PARTÍCULA rAAV Y UN AGENTE DE INMUNOSUPRESIÓN PARA USARSE EN EL TRATAMIENTO DE ENFERMEDADES.
CA3187131A1 (fr) 2020-09-02 2022-03-10 Fredrik JOHANSSON SWARTLING Prediction de rechute de cancer et traitement de maladies cancereuses

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2051288C (fr) * 1990-09-14 2002-02-05 Robert L. Martuza Destruction ciblee de cellules neoplasiques par un virus
US5529774A (en) * 1991-08-13 1996-06-25 The Regents Of The University Of California In vivo transfer of the HSV-TK gene implanted retroviral producer cells

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