WO2009039390A2 - Développement de procédés thérapeutiques à base de glycobiologie pour le traitement de tumeurs cérébrales - Google Patents

Développement de procédés thérapeutiques à base de glycobiologie pour le traitement de tumeurs cérébrales Download PDF

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WO2009039390A2
WO2009039390A2 PCT/US2008/077045 US2008077045W WO2009039390A2 WO 2009039390 A2 WO2009039390 A2 WO 2009039390A2 US 2008077045 W US2008077045 W US 2008077045W WO 2009039390 A2 WO2009039390 A2 WO 2009039390A2
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seq
gene
seqidno
glioma
genes
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WO2009039390A3 (fr
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Joseph R. Moskal
Roger A. Kroes
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Naurex Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Brain tumors are presently the leading cause of cancer death in children under the age of 20, only recently surpassing acute lymphoblastic leukemia (ALL). In addition, they are the third leading cause of cancer death in young adults ages 20-39 [4, 5]. Although not the most common form of cancer, brain tumors are clearly among the most devastating. Brain tumors are phenotypically and genotypically diverse, with over 120 different types of brain tumors currently classified [7]. As with most tumors, the classification of primary brain tumors is based on the precise tumor site, the anatomical structures involved, as well as tumorigenic tendencies.
  • GBM glioblastoma multiforme
  • meningiomas which are the most common primary brain tumor and represent 26% of all primary brain tumors [I].
  • Glioblastoma multiforme is nearly uniformly fatal, with median survival between 9 and 12 months from initial diagnosis.
  • GBMs are highly invasive tumors, making complete surgical resection unachievable.
  • Current multimodality therapy for GBMs including surgery, radiation therapy (RT), and chemotherapy is still the cornerstone of treatment but is largely ineffective, as the majority of patients experience progression or recurrence.
  • the present invention in a general and overall sense, presents a broad spectrum of glycobiology focused therapeutic agents and molecular biology based materials that, among other applications, possess accurate and specific clinical diagnostic and therapeutic uses in the field of neuro-oncology.
  • the invention provides a focused microarray of synthesized 45-mer oligonucleotides. These oligonucleotides may also include a 5 '-amino linker.
  • the invention provides for a solid substrate, such as a glass microscope slide, nylon membrane, nitrocellulose support material, glass or silica support, or other appropriate solid or semisolid medium, to provide a microchip that includes placed thereon a microarray of the oligonucleotides described herein, or a subset of selected of these oligonucleotides.
  • Robotic systems which pipette nano to picomolar amounts of gene products onto the support are commercially available or can be built using commercially available materials.
  • the oligonucleotides may be described as having been arranged using robotic placement as covalently linked oligonucleotides onto a solid or semisolid substrate of choice.
  • the oligonucleotides are to be provided as an array spotted in quadruplicate onto an aldehyde-coated glass microscope slide.
  • the focused microarray oligonucleotides panel comprises two (2) or more oligonucleotide sequences of Table 1.
  • the panel comprises 2 or more specific probes having a sequence of SEQ ID No: 2, SEQ ID No: 6, SEQ ID No: 11, SEQ ID No: 20, SEQ ID No: 23, SEQ ID No: 27, SEQ ID No. 31, SEQ ID No: 39 or any combination of 2 or more of these.
  • the Focused Microarray Panel comprises forty-two (42) glioma and non- glioma gene specific probes. These are listed in Table 1 :
  • a method for screening a candidate library of compounds suitable for identifying a selected candidate compound for treating a glioma is provided.
  • the devised 45-mer oligonucleotides that were created with information disclosed in the present paper that compose the transcriptome signature comprising the genes encoding MAN2A2, ST6GALNAC5, ST6GAL1, OGT, B3GNT6, POFUTl, CHI3L1, ST3GAL3, alone or in any combination with each other as well as one or more of the other genes described here in the glioma associated gene panel or non-glioma associated gene panel, will be included as part of a screening panel to be used in selecting therapeutic agent(s). These panels may also be used to identify target genes for inhibiting cancer (glioma) in a particular patient.
  • a method for identifying glycobiology based therapeutics for treating glioblastoma through use of preparations that modify the expression of a specific set of genes.
  • the screening method comprises exposing a candidate substance from a library of potential therapeutic compounds to a cell culture of human glioma brain cells, such as a culture of cells from cell line SNB 19, D54MG, U87MG, U373MG, Ul 18MG, U251 or A172, and measuring expression levels of a selected target gene or genes from a human glioma-associated gene panel and optionally for expression levels of a selected target gene from a non-glioma associated gene panel.
  • a candidate substance will be selected that is capable of reducing and/or inhibiting expression levels of 2 or more genes of the glioma-associated gene panel, increasing or reducing inhibition of expression of 2 or more selected target nonglioma associated genes of a non-glioma associated gene panel, or 1 selected target glioma associated gene and 1 selected target non-glioma associated gene.
  • the level of expression of a selected target gene from a culture having been exposed to a candidate substance may be assessed using any 1, 2 or more of the 45-mer oligonucleotides provided in Table 1.
  • each of the 45-mer oligonucleotides specifically hybridizes to a uniquely identifiable transcript of a specific glioma-associated or non-glioma associated gene
  • determining which specific gene product is being inhibited and/or enhanced by a candidate substance may also be specifically identified using this method.
  • the present methods provide for specifically identifiable glioma-associated gene targeted therapeutics and specifically targeted non-glioma associated gene targeted therapeutics.
  • the glioblastoma-associated genes include those listed in Table 2.
  • NM 001276 Chitinase 3-like 1 (cartilage glycoprotein-39, YKL-40) (CHI3L1)
  • NM 002103 Glycogen synthase 1 (GYS 1 )
  • GYS 1 Glycogen synthase 1
  • NM 003032 Sialyltransferase l( ⁇ -galactoside ⁇ 2, 6-sialyltransferase) (SIATl), transcript variant 2
  • NM 005228 Epidermal growth factor receptor (EGFR) NM 015352 O-fucosyltransferase 1 (POFUTl), transcript variant 1 NM 031302 Glycogene 8 domain containing 2 (GLT8D2) NM 032528 ⁇ -galactoside ⁇ 2, 6-sialyltransferase II (ST6GaIII) NM 033167 UDP-GaI : ⁇ -GlcNAc ⁇ l, 3-galactosyltransferase, polypeptide 3
  • NM 153286 Hyaluronoglucosaminidase 1 (HYALl), transcript variant 6 NM 174963 ST3 ⁇ -galactoside ⁇ 2,3-sialyltransferase 3 (ST3GAL3), transcript variant 1 NM_006278 Sialyltransferase 4C ( ⁇ -galactoside ⁇ 2,3-sialyltransferase) (SIAT4C) NM 000521 Hexosaminidase B ( ⁇ -polypeptide) (HEXB) NM 002409 Mannosyl ( ⁇ 1 ,4-)-glycoprotein ⁇ 1 ,4-N-acetylglucosaminyltransferase
  • the non-glioblastoma-associated gene panel includes genes listed in Table 3.
  • Non-Glioma Associated Gene Panel AB 032956 UDP-N-acetyl- ⁇ -d-galactosamine polypeptide N- acetylgalactosaminyltransferase-like 1 (GALNTLl) NM 000153 Galactosylceramidase (Krabbe disease) (GALC) NM 000188 Hexokinase 1 (HKl), transcript variant 1 NM 002372 ⁇ -mannosidase, class 2 A, member 1 (MAN2A1) NM 003360 UDP glycogene 8 (UDP galactose ceramide galactosyltransferase) (UGT8) NM 003605 O-linked N-acetylglucosamine (GIcNAc) transferase (UDP-N- acetylglucosamine : polypeptide-N-acetylglucosaminyltransferase) (OGT), transcript variant 3
  • NM 004455 Exostoses (multiple)-like 1 (EXTLl) NM 004737 Glycogene-like, LARGE (MDCID), transcript variant 1 NM 006122 ⁇ -mannosidase, class 2 A, member 2 (MAN2A2)
  • NM 006876 UDP-GIcNAc ⁇ -Gal, ⁇ l ,3-N-acetylglucosaminyltransferase 6 (B3GNT6) NM 012215 Meningioma expressed antigen 5 (hyaluronidase) (MGEA5) NM 013443 ST6 ( ⁇ -N-acetyl-neuraminyl-2,3 ⁇ galactosyl- l,3)-N-acetylgalactosaminide ⁇ 2,6-sialyltransferase 6 (ST6GALNAC6) NM 018644 ⁇ l, 3 -glucurony ⁇ transferase 1 (glucuronosyltransferase P) (B3GAT1), transcript variant 1
  • transcript variant 1 NM 019109 ⁇ 1 ,4 mannosyltransferase (HMT- 1 ) NM 020742 Neuroligin 4 (NLGN4) transcript variant 1 NM 024344 Calpain 3 (p94) (CAPN3), transcript variant 2 NM 030965 ST6 ( ⁇ -N-acetyl-neuraminyl-2,3- ⁇ -galactosyl- 1 ,3)-N- acetylgalactosaminide ⁇ 2,6-sialyltransferase 5 (ST6GALNAC5) NM 033158 Hyaluronoglucosaminidase 2 (HYAL2), transcript variant 2 NM 033309 UDP-GIcNAc : ⁇ Gal ⁇ l,3-N-acetylglucosaminyltransferase 9 (B3GNT9) NM 054025 ⁇ 1,3 -glucurony ⁇ transferase 1 (glucuronosyltransferas
  • the present invention provides a method for treating glioblastoma in a patient.
  • the method comprises treating a patient having or at risk of having a glioma whose tumor demonstrates an identifiable glioma gene expression profile comprising a decreased expression level of a MAN2A2 gene, a ST6GALNAC5 gene, a ST6GAL1 gene, a OGT gene, a B3GNT6 gene, or any combination of these, an increased (elevated) expression level of a POFUTl gene, a CHI3L1 gene, a ST3GAL3 gene, or any combination of these, with a viral vector having a sequence that comprises a therapeutic gene sequence appropriate for the patients identifiable glioma gene expression profile.
  • the adenoviral vector will comprise a gene sequence that corresponds to the protein coding region of the gene exhibiting decreased expression.
  • the adenoviral vector will comprise a gene sequence that corresponds to an antisense molecule specific for the gene that is increased (elevated) relative to a non-glioma expression level of one or more of these genes.
  • a method for inhibiting a glioma brain tumor comprising altering expression pattern levels of a glioma-associated gene or genes in a patient, such as a POFUTl gene, a CHI3L1 gene, a ST3GAL3 gene, or any combination of these, in a patient having being diagnosed as having glioma, particularly, a grade 4 glioma.
  • glioma-associated genes have been characterized in the present disclosure as being more highly expressed in the tumor of a patient having been diagnosed as having a glioma, compared to expression levels of the gene in normal, non-glioma brain tissue.
  • a treatment method would comprise inhibiting the level of expression of the POFUTl gene, a CHI3L1 gene, a ST3GAL3 gene,, alone or in conjunction with other of the glioma-associated gene panel described herein, in a patient having a glioma so as to achieve a relative level of the expression of these genes that is about the same as, or at least a reduced level of expression that is not more than about 20% to 30% higher, than the expression levels of these genes in a normal (non-glioma) human brain tissue.
  • the method may comprise administering a recombinant adenoviral vector particle having a sequence encoding an antisense molecule (e.g., antisense oligonucleotide, ribozyme, siRNA, or shRNA) specific for a selected glioma-associated target gene or genes, such as POFUTl gene, a CHBLl gene, a ST3GAL3 gene, or any combination of these, to a patient having been diagnosed to have a glioma, and inhibiting and/or reducing the elevated expression of the glioma-associated target gene or genes in the patient to a non-glioma associated gene expression level of the selected glioma-associated target gene or genes, such as POFUTl, CHBLl, or ST3GAL3.
  • an antisense molecule e.g., antisense oligonucleotide, ribozyme, siRNA, or shRNA
  • a method for inhibiting a glioma comprising altering expression pattern levels of a non-glioma associated gene or genes in a patient, such as a MAN2A2 gene, a ST6GALNAC5 gene, a ST6GAL1 gene, a OGT gene, a B3GNT6 gene, or any combination of these, within the tissue of a patient having been diagnosed as having a glioma.
  • a method for inhibiting a glioma comprising altering expression pattern levels of a non-glioma associated gene or genes in a patient, such as a MAN2A2 gene, a ST6GALNAC5 gene, a ST6GAL1 gene, a OGT gene, a B3GNT6 gene, or any combination of these, within the tissue of a patient having been diagnosed as having a glioma.
  • These genes have been characterized as being expressed in relatively lower levels in the tumor of a glioma-afflicted patient,
  • a patient having been diagnosed or suspected to have a glioblastoma would be treated so as to achieve an elevated level of expression of a selected non-glioma associated gene or genes, such as those listed in Table 3, or in particular embodiments, the MAN2A2 gene, the ST6GALNAC5 gene, the ST6GAL1 gene, the OGT gene, the B3GNT6 gene, or any combination of these.
  • a selected non-glioma associated gene or genes such as those listed in Table 3, or in particular embodiments, the MAN2A2 gene, the ST6GALNAC5 gene, the ST6GAL1 gene, the OGT gene, the B3GNT6 gene, or any combination of these.
  • a selected non-glioma associated target gene or genes such as the MAN2A2 gene, the ST6GALNAC5 gene, the ST6GAL1 gene, the OGT gene, the B3GNT6 gene, or any combination of these
  • the treatment would provide the patient with a non-glioma associated gene expression level of the selected non-glioma associated target genes, MAN2A2, ST6GALNAC5, ST6GAL1, OGT, B3GNT6, or any combination of these.
  • a sufficient expression level of these genes would comprise an expression level that is characteristic of a normal, non-glioblastoma, brain tissue culture.
  • the present invention further provides a method for screening a tissue for glioblastoma, comprising measuring a brain tissue specimen for levels of expression of a gene selected from a group of genes comprising 2 or more of the genes: MAN2A2 gene, ST6GALNAC5 gene, ST6GAL1 gene, OGT gene, B3GNT6 gene, POFUTl gene, CHBLl gene, or ST3GAL3 gene. All or any combination of these genes may be examined for expression level within the brain tissue specimen.
  • any method for detection of the expression level of the identified gene product may be utilized, including but not limited to assays for the presence or activity of the glycogene protein within a cell or assays for detecting nucleic acids encoding or involved in the expression of a glycogene. Detection of a nucleic acid encoding a glycogene may be accomplished by detection of glycogene mRNA using any of several techniques available to one skilled in the art such as Northern blot (Alwine, et al. Proc. Natl. Acad. Sci. 74:5350), RNase protection (Melton, et al. Nuc. Acids Res. 12:7035), or RT-PCR (Berchtold, et al. Nuc. Acids. Res. 17:453).
  • the gene comprises MAN2A2, ST6GALNAC5, ST6GAL1, OGT, B3GNT6, POFUTl, CHI3L1, ST3GAL3 or any one or combination thereof.
  • the present invention comprises a kit for determining the tumorigenicity or malignancy of a brain tumor.
  • the kit may comprise a panel of independent or paired nucleic acid molecules (such as any 1, 2 or more of the 45-mer oligonucleotides provided in Table 1, such as the 45 mer that corresponds to detection of POFUTl gene activity, CHI3L1 gene activity, ST3GAL3 gene activity, or all of these) specific for the detection of the expression of specific nucleic acid sequences corresponding to specific glioma and/or non-glioma associated genes.
  • kits utilizes enzyme-mediated nucleic acid amplification such as the polymerase chain reaction (PCR) in which a pair of nucleic acid molecules (i.e., primers) that allow for amplification of a nucleic acid sequence, are included.
  • PCR polymerase chain reaction
  • a kit allowing for determining levels of expression of these genes may be utilized to predict or determine tumorigenicity of a patient tumor biopsy sample.
  • nucleic acid samples used in the methods and assays of the invention may be prepared by any available method or process. Methods of isolating total mRNA are well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24, Hybridization With Nucleic Acid Probes: Theory and Nucleic Acid Probes, P. Tijssen, Ed., Elsevier Press, New York, 1993. Such samples include RNA samples, but also include cDNA synthesized from a mRNA sample isolated from a cell or tissue of interest. Such samples also include DNA amplified from the cDNA, and RNA transcribed from the amplified DNA. One of skill in the art would appreciate that it is desirable to inhibit or destroy RNase present in homogenates before homogenates are used.
  • glioma associated gene expression profile is understood to mean an elevated level of expression of 2 or more glioma associated genes.
  • an elevated level of glioma-associated gene expression would include an expression level of these genes that is 1.5 fold greater than the gene expression level of the same genes by a normal (non-glioma) human brain cell culture.
  • 3 glioma-associated genes are POFUTl, CHBLl, and ST3GAL3.
  • non-glioma associated gene expression profile is understood to mean a gene expression level of 2 or more non-glioma-associated genes that is not less than about 80% the expression level of the same 2 or more non-glioma associated genes by a non-glioma human brain cell culture, a gene expression level of 2 or more glioma associated genes that is not more than about 20% greater than the expression level of the same 2 or more glioma associated genes by a non-glioma human brain cell culture, or both.
  • 5 non-glioma associated genes are MAN2A2, ST6GALNAC5, ST6GAL1, OGT, and B3GNT6.
  • 3 glioma-associated genes are POFUTl, CH13L1, and ST3GAL3.
  • glioma associated gene is a gene that encodes one or more of the glioma-associated genes POFUTl, CHI3L1, ST3GAL3, or any combination of the glioma associated genes in Table 2.
  • non-glioma associated gene is a gene that encodes one or more of the non-glioma-associated genes selected from the group consisting of 2 or more of MAN2A2, ST6GALNAC5, ST6GAL1, OGT, and B3GNT6, or any combination of the non- glioma associated genes in Table 3.
  • a panel of glioma associated genes is understood to mean a panel of differentially expressed genes comprising one or more glioma associated genes, one or more non-glioma associated genes, or a combination thereof, that present a differentially identifiable expression profile compared to a non-glioma brain tissue culture of the same genes.
  • the panel of glioma associated genes comprises a MAN2A2 gene, a ST6GALNAC5 gene, a ST6GAL1 gene, a OGT gene, a B3GNT6 gene, or any combination of two or more of these genes.
  • the term “bind(s) to” or “hybridizes to” refers to complementary hybridization between a probe nucleic acid (or oligonucleotide) and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence
  • Figure 2 qRT-PCR corroboration of selected gly co-targets identified by microarray analysis. For each mRNA, transcript abundance, normalized to GAPDH, was calculated by qRT-PCR, as described in Materials and methods. Data are presented for MAN2A2 (panel 2a), ST6GALNAC5 (panel 2b), POFUTl (panel 2c), and CHI3L1 (panel 2d) and represent mean ( ⁇ SD). Significant differences between GBM and normal brain were observed for all genes (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001; two-tailed, unpaired student's t-test). n/s, not significant (p > 0.05).
  • nucleic acid construct directing expression of a protein or nucleic acid product having the ability to alter expression of a glycogene.
  • a nucleic acid construct directing expression of a protein or nucleic acid product having the ability to alter expression of a glycogene.
  • multiple viral and non- viral methods suitable for introduction of a nucleic acid molecule into a target cell. Genetic manipulation of primary tumor cells has been described previously (Patel et al., 1994). Genetic modification of a cell may be accomplished using one or more techniques well known in the gene therapy field (Human Gene Therapy April 1994, Vol. 5, p. 543-563; Mulligan, R. C. 1993).
  • Viral transduction methods may comprise the use of a recombinant DNA or RNA virus comprising nucleic acid sequence that drives or inhibits the expression of a protein having glyco-related protein, while retaining sufficient activity to infect a target cell.
  • a suitable DNA virus for use in the present invention includes but is not limited to an adenovirus (Ad), adeno-associated virus (AAV), herpes virus, vaccinia virus or a polio virus.
  • a suitable RNA virus for use in the present invention includes but is not limited to a retrovirus or Sindbis virus. It is to be understood by those skilled in the art that several such DNA and RNA viruses exist that may be suitable for use in the present invention.
  • Adenoviral vectors have proven especially useful for gene transfer into eukaryotic cells (Stratford-Perricaudet and Perricaudet. 1991). Adenoviral vectors have been successfully utilized to study eukaryotic gene expression (Levrero, M., et al. 1991), vaccine development (Graham and Prevec, 1992), and in animal models (Stratford-Perricaudet, et al. 1992.; Rich, et al. 1993). The first trial of Ad-mediated gene therapy in human was the transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to lung (Crystal, et al., 1994).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Adeno-associated virus has recently been introduced as a gene transfer system with potential applications in gene therapy. Wild-type AAV demonstrates high-level infectivity, broad host range and specificity in integrating into the host cell genome (Hermonat and Muzyczka 1984). Herpes simplex virus type-1 (HSV-I) is attractive as a vector system for use in the nervous system because of its neurotropic property (Geller and Federoff. 1991; Glorioso, et al. 1995). Vaccinia virus, of the poxvirus family, has also been developed as an expression vector (Smith and Moss, 1983; Moss, 1992). Each of the above- described vectors is widely available to one skilled in the art and would be suitable for use in the present invention.
  • Retroviral vectors are capable of infecting a large percentage of the target cells and integrating into the cell genome (Miller and Rosman. 1989). Retroviruses were developed as gene transfer vectors relatively earlier than other viruses, and were first used successfully for gene marking and transducing the cDNA of adenosine deaminase (ADA) into human lymphocytes.
  • ADA adenosine deaminase
  • a viral vector in vivo by implantation of a "producer cell line" in proximity to the target cell population.
  • a producer cell line infiltiation of a brain tumor with cells engineered to produce a viral vector carrying an effector gene results in the continuous release of the viral vector in the vacinity of the tumor cells for an extended period of time (i.e, several days).
  • the vector is retroviral vector which preferentially infects proliferating cells, which, in the brain, would include mainly tumor cells.
  • the present invention provides a methodology with which a viral vector supplies a nucleic acid sequence encoding a protein having glyco-related activity to cells involved in a neurological disorder such as brain cancer.
  • Non-viral delivery techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO 4 precipitation, gene gun techniques, electroporation, and lipofection (Mulligan, 1993). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success (Mulligan, R. 1993).
  • Lipofection may be accomplished by encapsulating an isolated DNA molecule within a liposomal particle and contacting the liposomal particle with the cell membrane of the target cell.
  • Liposomes are self-assembling, colloidal particles in which a lipid bilayer, composed of amphiphilic molecules such as phosphatidyl seine or phosphatidyl choline, encapsulates a portion of the surrounding media such that the lipid bilayer surrounds a hydrophilic interior.
  • Unilammellar or multilammellar liposomes can be constructed such that the interior contains a desired chemical, drug, or, as in the instant invention, an isolated DNA molecule.
  • the cells may be transfected in vivo (preferably at the tumor site), ex vivo (following removal from a primary or metastatic tumor site), or in vitro.
  • the cells may be transfected as primary cells isolated from a patient or as a cell line derived from primary cells, and are not necessarily autologous to the patient to whom the cells are ultimately administered.
  • the cells may be implanted into a host, preferably a patient having a neurological disorder and even more preferably a patient having a brain tumor. Genetic manipulation of primary tumor cells has been described previously (Patel et al. 1994). Genetic modification of the cells may be accomplished using one or more techniques well known in the gene therapy field (Human Gene Therapy. April 1994. Vol. 5, p. 543-563; Mulligan, R. C. 1993).
  • a transcriptional regulatory region capable of driving gene expression in the target cell.
  • the transcriptional regulatory region may comprise a promoter, enhancer, silencer or repressor elements and is functionally associated with a nucleic acid of the present invention.
  • the transcriptional regulatory region drives high level gene expression in the target cell. It is further preferred that the transcriptional regulatory region drives transcription in a cell involved in a neurological disorder such as brain cancer.
  • Transcriptional regulatory regions suitable for use in the present invention include but are not limited to the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter and the chicken beta-actin promoter coupled to the CMV enhancer (Doll, et al. 1996).
  • CMV human cytomegalovirus
  • SV40 early enhancer/promoter the SV40 early enhancer/promoter
  • JC polyomavirus promoter the chicken beta-actin promoter coupled to the CMV enhancer
  • the vectors of the present invention may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif).
  • nucleic acid constructs useful for practicing the present invention comprise a transcriptional regulatory region such as the CMV immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter, or the chicken beta-actin promoter coupled to the CMV enhancer operably linked to a nucleic acid encoding a glyco-related enzyme that is preferably MAN2A2, ST6GALNAC5, ST6GAL1, OGT, B3GNT6.
  • a nucleic acid sequence encoding the enzyme may be processed using one or more restriction enzymes such that certain sequences flank the nucleic acid. Processing of the nucleic acid may include the addition of linker or adapter sequences.
  • a nucleic acid sequence comprising a preferred transcriptional regulatory region may be similarly processed such that the sequence has flanking sequences compatible with the nucleic acid sequence encoding the enzyme. These nucleic acid sequences may then be joined into a single construct by processing of the fragments with an enzyme such as DNA ligase. The joined fragment, comprising a transcriptional regulatory region operably linked to a nucleic acid encoding a glyco-related enzyme, may then be inserted into a plasmid capable of being replicated in a host cell by further processing using one or more restriction enzymes.
  • vectors containing nucleic acid sequences encoding antisense molecules designed to inhibit POFUTl, CH 13Ll, or ST3GAL3 are envisioned and may be similarly constructed.
  • a nucleic acid of the present invention may be administered using any of a variety of techniques well known to those skilled in the art.
  • Such reagents may be administered by intravenous injection or using a technique such as stereotactic injection to administer the reagent into the target cell or the surrounding areas (Badie, et al. 1994; Perez-Cruet, et al. 1994; Chen, et al. 1994; Oldf ⁇ eld, et al. 1993; Okada, et al. 1996).
  • the vectors of the present invention may be administered orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • the dosage regimen for treating a neurological disorder disease with the vectors of this invention and/or compositions of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.
  • the pharmaceutically active compounds (i.e., vectors, selected candidate compounds) of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.
  • the pharmaceutical composition may be in the form of, for example, a capsule, a tablet, a suspension, or liquid.
  • the pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of DNA or viral vector particles (collectively referred to as "vector").
  • vector may contain an amount of vector from about 10 3 -10 15 viral particles, preferably from about 10 6 -10 12 viral particles.
  • a suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.
  • the vector may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water.
  • Injectable preparations such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known are using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • a suitable topical dose of active ingredient of a vector of the present invention is administered one to four, preferably two or three times daily.
  • the vector may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
  • the pharmaceutical compositions may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsif ⁇ ers, buffers etc.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch
  • inert diluent such as sucrose, lactose, or starch
  • additional substances other than inert diluents e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.
  • Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, and perfuming agents.
  • nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • the present invention may comprise elevation or depression of enzyme levels in cells expressing various amounts of enzyme.
  • Introduction of a glycogene expression vector into a cell already expressing a high level of that enzyme may alter glycosylation patterns within that cell.
  • introduction of a nucleic acid construct that inhibits expression of such an enzyme in a cell expressing low levels of that enzyme may also serve to alter glycosylation patterns in that cell. Either of these methodologies may decrease the tumorigenicity of the cell by any of multiple mechanisms.
  • Brain cancer is defined herein as any cancer involving a cell of neural origin, as well as cells of non-neural origin that have metastasized to the brain.
  • brain cancers include but are not limited to intracranial neoplasms such as those of the skull (i.e., osteoma, hemangioma, granuloma, xanthona, osteitis deformans), the meninges (i.e., meningioma, sarcoma, gliomatosis), the cranial nerves (i.e., glioma of the optic nerve, schwannoma), the neuroglia (i.e., gliomas) and ependyma (i.e., ependymomas), the pituitary or pineal body (i.e., pituitary adenoma, pinealoma), and those of congenital origin (i.e., craniopharygioma, chordoma, germinoma, teratoma, dermoid cyst, angioma, hemangioblastoma), as well as those intracranial
  • Example 1 Materials and Methods
  • Human Tissue Samples For the microarray analysis, six surgical specimens and six normal human brain specimens were used. For the qRT-PCR analyses, four additional glioblastoma and four additional normal brain specimens were used.
  • Brain tumor tissue (GBM, WHO Grade IV) from patients was acquired from Field Neurosciences Institute, Saginaw, MI and from the tumor bank maintained by the FaIk Center. None of the patients had been subjected to chemotherapy or radiotherapy prior to resection. Protocols for tissue accrual and use were approved by the appropriate Institutional Review Boards. Immediately upon resection, tissue was stored in KNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA, USA) and subsequently snap frozen and maintained in liquid nitrogen. All tissue samples used in these analyses were evaluated by a neuropathologist; all specimens selected were characterized by dense tumor cellularity and the presence of >90% tumor tissue. The average patient age at time of resection was 56.1 ( ⁇ 13.1) years. There were six males and four females represented, and the mean survival was 8.7 ( ⁇ 3.0) months from time of diagnosis.
  • Normal brain tissue was obtained from representative tissue punches of gray matter from 10 appropriately age-matched hemicoronal brain tissue sections obtained from autopsy specimens provided by the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, Maryland. All tissue sections were maintained in liquid nitrogen. The average age at time of death was 58.0 ( ⁇ 7.6) years. There were seven males and three females represented, and the mean postmortem interval for sample preservation was 17.2 ( ⁇ 5.4) h.
  • Human Glioma Cell Culture The following cell lines were used for qRT-PCR analysis: human glioblastomas, SNB19 and D54MG, U87MG, U373MG, Ul 18MG, U251, and A172 (obtained from ATCC, Rockville, MD, USA). All established human brain tumor cell lines were maintained using Dulbecco's modified Eagle's medium (containing 4.5 g/L glucose) supplemented with 10% heat-inactivated fetal bovine serum (Whittaker BioProducts, Walkersville, MD, USA).
  • Microarray fabrication, validation, and quality control The 359 genes comprising the present focused Human Glycobiology microarray are compiled from NCBI/EMBL/TIGR human sequence databases and the Consortium for Functional Glycomics-CAZY databases (available at http://www.cazy.org/CAZY/) and represented all of the cloned human glycogenes, glycosylhydrolases, polysaccharide lyases, and carbohydrate esterases. Only those genes with fully curated Reference Sequence (RefSeq) ID's were used.
  • RefSeq Reference Sequence
  • oligonucleotide complementary to sequences within these human mRNAs were designed and prioritized using stringent selection criteria, including minimal secondary structure, minimal homology to other genes in the available human genomic databases, no low complexity or repeat regions, and all with a similar, yet well-defined T m [ArrayDesigner v2.03, Premier Biosoft, Palo Alto, CA, USA] to provide optimal hybridization efficiency across all oligos on the array.
  • Control oligonucleotides representing the most traditionally accepted and commonly utilized housekeeping genes (Lee et al. 2002) were similarly designed and prioritized.
  • oligonucleotides were individually synthesized with the addition of a 5'-amino linker (C6-TFA, Glen Research, Sterling, VA, USA) onto each oligonucleotide, as described (Kroes et al. 2006). The oligonucleotides were then robotically arrayed, covalently linked in quadruplicate to aldehyde-coated glass microscope slides, and quality controlled prior to use.
  • C6-TFA 5'-amino linker
  • the dynamic range, discrimination power, accuracy, reproducibility, and specificity of the focused oligonucleotide microarrays used in these studies were evaluated by exogenous mRNA spiking studies. Exogenous mRNA spiking studies were conducted as described in Kroes et al, 2006), which reference is specifically incorporated herein by reference for this purpose.
  • the dynamic range defined as the range of transcript abundance over which hybridization intensity was linearly correlated, was identified in six independent experiments and was found to be between two and three orders of magnitude. The data presented here fell within this dynamic range. Discrimination power, or the ability to discriminate authentic signal from background at the low end of the dynamic range, was also used here to set appropriate cutoffs prior to statistical analysis of the data.
  • the accuracy of the microarray results was determined by direct comparison to individual mRNA abundance determined by qRT-PCR analysis of the spiked mRNA samples. Conformity between the two datasets (i.e., qRT-PCR and the spiked microarray samples) was measured, with a Pearson correlation coefficient of +0.96, which is in good agreement with reported values (Baum et al. 2003).
  • Hybridization specificity was evaluated using a range of 1-6 sequence mismatches synthesized within the gene-specific 45-mer oligonucleotide immobilized on the array.
  • the oligonucleotides used on the microarrays were gene specific since adverse effects on hybridization efficiency were not found with less than three mismatches.
  • Example 2 Target Preparation; RNA Extraction and Labeling, and Microarray Hybridization
  • a universal human reference RNA (Stratagene, La Jolla, CA, USA) was used in the present analyses. Identical aliquots were treated concurrently with the tissue samples.
  • Equivalent amounts of Cy5 -labeled (experimental) and purified Cy3 -labeled (reference) amplified RNA (aRNA) targets were combined, denatured and hybridized at 46°C for 16 h. Following sequential high-stringency washes, individual Cy3 and Cy5 fluorescence hybridization to each spot on the microarray was quantitated by a high resolution confocal laser scanner.
  • the data from each channel were normalized using the LOWESS curve-fitting equation on a print-tip specific basis (GeneTraffic v2.8, Iobion Informatics, La Jolla, CA, USA).
  • Statistical analyses were performed using the permutation-based SAM (significance analysis of microarrays) algorithm (v2.20, Stanford University, see Tusher et al., 2001), that reports the median false discovery rate (FDR) as the percentage of genes in the identified gene list (rather than in the entire cohort of genes present on the microarray) that are falsely reported as showing statistically significant differential expression.
  • FDR median false discovery rate
  • Quantitative real-time PCR analysis The expression levels of selected genes were analyzed by real-time PCR using Brilliant SYBR Green qRT-PCR Master Mix (Strata-gene) on an Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA). Reverse transcription of 1 ⁇ g of DNAsed, total RNA was primed with oligo(dT) and random hexamers and was performed exactly as described (Kroes et al. 2006). All primer sets were designed across intron:exon boundaries to derive -100 bp amplicons (Table 1), with individual primer concentrations and final amplification conditions optimized for each gene. Dissociation curves were performed on all reactions to assure product purity.
  • RNA amounts were calculated by comparison to standard curves using purified PCR product as a template for the mRNAs of interest and were normalized to amount of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH). Studies were performed in triplicate for each data point.
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • a SAM analysis was used to determine statistically significant differences in the measured expression levels for each gene on the array and demonstrated that a number of genes can be identified with high confidence as differentially expressed between the two cell types (Fig. 1).
  • Table 6 shows the identities, functional annotations, and relative expression ratios of these differentially expressed genes.
  • 34 glyco-genes genes differed in their expression between GBM and normal brain by at least 1.5-fold (at 0% FDR). Importantly, at this stringent FDR, none of these changes was expected to be a false- positive.
  • 34 genes 10 had increased and 24 had decreased measured expression levels in the grade IV tumors relative to normal brain.
  • ⁇ -mannosidase 2A2 MAN2A2
  • POFUTl protein O-fucosyltransferase 1
  • ST6 ⁇ -N-acetyl-neuraminyl-2,3- ⁇ -galactosyl 1,3 N-acetylgalactosaminide ⁇ 2,6-sialyltransferase 5 (ST6GALNAC5)
  • CHI3L1 chitinase 3-like 1
  • mRNA levels were measured by real-time quantitative RT-PCR.
  • the core approach to transcriptome profiling was to use the focused microarrays as a screening tool to identify statistically significant differentially expressed genes followed by corroboration of a subset of these in larger sample panels using higher throughput methodology, including quantitative qRT-PCR.
  • the number of samples was expanded for the qRT-PCR corroboration to analyze total RNA from 10 GBM samples, 10 age-matched normal brain controls, and seven glioma cell lines and the expression of MAN2A2, ST6GALNAC5, POFUTl and CHI3L1 was analyzed relative to the level of GAPDH mRNA in each sample.
  • GAPDH was chosen for a reference (control) because its mRNA levels, measured by microarray analysis, were comparable between GBM and normal brain.
  • oligonucleotide microarrays were utilized to even further define the intrinsically complex regulation and activity of glyco-related genes.
  • genes encoding key glyco-related mRNAs are drastically underrepresented on most commercial arrays, a microarray core facility was created to provide a comprehensive platform of glycoconjugate metabolism-associated oligonucleotides assuring the most up-to- date coverage of these gene families.
  • This technology has not been systematically applied to the global analysis of glyco-related gene expression in brain tumors. Production, hybridization and data analysis of the present oligonucleotide arrays have been optimized in order to measure subtle, phenotypically relevant changes in glyco-gene expression associated with brain tumorigenesis both in vitro and in vivo.
  • the 359 genes comprising the present Human Glycobiology microarray are compiled from NCBI/EMBL/TIGR human sequence databases and the Consortium for Functional Glycomics-CAZY databases. Unique sense 45-mer oligonucleotides corresponding to mRNAs of each gene used as probes are individually synthesized, purified and immobilized via a 5 '-amino linker onto aldehyde-coated microarrays. Total RNA is reverse transcribed and used as the substrate for RNA amplification and labeling using the indirect aminoallyl methodology based on the Eberwine protocol [89].
  • Cell lines that have been transfected to include the glioblastoma associated gene or genes as identified here also provide an in vitro model systems for characterizing the regulation of a given gene or associated gene family, (3) confirm and extend the present microarray data with in situ hybridization studies and quantitative RT-PCR analyses, and (4) Continuing to evaluate therapeutic candidates using viral vectors in animal models such as the SCID mouse.
  • Group I These unified 45 mers (SEQ ID Nos. 1-14) were designed, synthesized, and used to identify fourteen (14) glioma associated genes that are over expressed by at least 1.5- fold in human glioma tissue as compared to non-glioma, normal human brain tissue.
  • SEQ ID No.1 identifies FUT3 5' CAAATATTCTGGGGTTGAGGGAAATTGCTGCTGTCTACAAAATGC 3'
  • Group II These 45-mers (SEQ ID Nos. 15-42) were designed, synthesized and used to identify a selection of 28 genes that were more highly expressed in non-glioma human brain tissue by at least 1.5 fold as compared to non-glioma (normal) human brain tissue.
  • SEQ ID No. 15 (identifies GALNTLl) 5' GGACCTCGTCTTTATGAAACACACACCTGGAATAAAACCACTTCT 3' (P) SEQ ID No. 16 (identifies GALC)
  • T SEQ ID No. 20 (identifies OGT, transcript variant 3)
  • JJ SEQ ID No. 36 (identifies CAPN3, transcript variant 4)
  • KK SEQ ID No. 37 (identifies CAPN3, transcript variant 5)

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Abstract

L'invention concerne l'identification d'une liste unique de gènes associés au gliome, exprimés de manière différentielle, ainsi qu'une liste unique et exprimée de manière différentielle de gènes non associés au gliome. Des procédés de sélection d'agents thérapeutiques ciblant un gène pour le traitement et l'inhibition du glioblastome, ainsi que les procédés thérapeutiques antiglioblastome eux-mêmes, sont décrits. Des constructions moléculaires telles que des vecteurs adénoviraux, qui comprennent une sélection des gènes associés au gliome exprimés de manière différentielle et/ou des gènes non associés au gliome identifiés sont également fournies. Un microréseau oligonucléotidique focalisé de séquences oligonucléotidiques hautement spécifiques au gliome est également fourni. Des micropuces et/ou d'autres matrices de support solides et semi-solides sur lesquelles les oligonucléotides sont fixés sont également fournies. Des procédés pour diagnostiquer le glioblastome chez un patient sont également proposés, qui utilisent un microréseau génétique comprenant les séquences oligonucléotidiques.
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US11376250B2 (en) 2016-08-01 2022-07-05 Aptinyx Inc. Spiro-lactam NMDA receptor modulators and uses thereof
US11427585B2 (en) 2016-08-01 2022-08-30 Aptinyx Inc. Spiro-lactam NMDA modulators and methods of using same
US11512051B2 (en) 2016-08-01 2022-11-29 Aptinyx Inc. Spiro-lactam NMDA receptor modulators and uses thereof
US11530223B2 (en) 2016-08-01 2022-12-20 Aptinyx Inc. Spiro-lactam NMDA receptor modulators and uses thereof
CN107937527A (zh) * 2017-12-28 2018-04-20 中南大学湘雅医院 胶质瘤诊断标志物circ1:43920404|43920928及应用
US11578072B2 (en) 2018-01-31 2023-02-14 Aptinyx Inc. Spiro-lactam NMDA receptor modulators and uses thereof
CN113736878A (zh) * 2021-08-24 2021-12-03 复旦大学附属肿瘤医院 一种神经***肿瘤检测的基因panel、试剂盒及其应用

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