Manipulation of meningeal lymphatic vasculature for brain and CNS tumor therapy

A technology of lymphatic and immunotherapy, applied in antineoplastic drugs, cytokines/lymphokines/interferon, angiogenin, etc.

Pending Publication Date: 2021-09-10
YALE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Notably, less than 2% of drugs typically cross the blood-brain barrier, presenting further challenges in the treatment of brain cancer patients

Method used

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  • Manipulation of meningeal lymphatic vasculature for brain and CNS tumor therapy
  • Manipulation of meningeal lymphatic vasculature for brain and CNS tumor therapy
  • Manipulation of meningeal lymphatic vasculature for brain and CNS tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1: Determination of VEGF-C Induced Lymphatic Proliferation in Dural Lymphatic Vessels

[0143] To investigate the question of how immune privilege affects tumor growth, the inventors used a model to examine tumor growth with increased meningeal lymphatic vessels.

[0144] GL261 (C57BL / 6 syngeneic GBM cell line) was transduced to express the luciferase gene (GL261-Luc) [58]. GL261 parental cells were obtained from the NIH Cancer Cell Bank. GL 261-luciferase cells were obtained from Yale Universit. GL261 and GL261-Luc cells were cultured in RPMI (10% FBS, 1% penicillin / streptomycin).

[0145] All experiments used 8-week-old in-house bred wild-type C57BL / 6 mice. C57BL / 6 mice were purchased from the National Cancer Institute and Jackson Laboratory, and subsequently bred and maintained at Yale University. All procedures were performed according to the animal experiment protocol. For tumor inoculation, intraperitoneal injections of ketamine (50 mg kg -1 ) and xy...

Embodiment 2

[0152] Example 2: Assay of VEGF-C Induction of Lymphatic Vascular Proliferation in Dural Lymphatic Vessels Manipulation of lymphatic vessels can affect the clearance of macromolecules from the brain parenchyma [34-36,50].

[0153] Mice were treated with EGFC-AAV or mock AAV as described in Example 1. A third group of wild-type mice was treated with PBS only. Go through two months.

[0154] Syngeneic mouse GBM cells (GL261-Luc) were implanted into the striatum of mice that had received AAV encoding VEGF-C, a scrambled sequence (mock AAV), or PBS (WT ). Specifically, 5,000 or 50,000 GL261-Luc cells were intracranially implanted into the striatum (the coordinates are 3 mm lateral to the bregma and 1 mm posterior). Mice were observed for signs of significant weight loss, lethargy, dorsal arch, or periorbital hemorrhage as survival endpoints. All brains were collected after death or endpoint to confirm tumor growth. 500,000 GL261 cells suspended in Matrigel (Corning) at a volu...

Embodiment 3

[0161] Example 3: Production and validation of VEGFC-mRNA

[0162] VEGFC-mRNA was custom-made from TriLink Biotechnologies, the sequence is as follows Figure 12 shown. The mRNA was produced using TriLink Biotechnologies Cap1 with full 5-methylcytosine and pseudouridine substitutions for stability. Specifically, 5-methylcytosine enhances stability and reduces deamination, while pseudouridine substitution reduces nuclease activity, reduces innate recognition and can increase translation. Cap 1 does not activate PR receptors. The mRNA is also polyadenylated (120A).

[0163] 2 μg of VEGFC-mRNA was transfected into HEK293T cells using lipofectamine. HEK293T cells were purchased from ATCC. HEK293T cells were cultured in complete DMEM (4.5 g / L glucose, 10% FBS, 1% penicillin / streptomycin). Cy5-GFP mRNA was used as a control. Cell lysates were collected 6 hours and 24 hours after transfection. Dissolve samples in RIPA buffer and boil with sample buffer for 5 min. Culture med...

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Abstract

A method of inducing lymphangiogenesis in the brain or central nervous system of a subject is provided in which an effective amount of a lymphangiogenesis inducer is administered. A method of inducing lymphangiogenesis in a tumor in the brain or central nervous system of a subject is provided in which an effective amount of a lymphangiogenesis inducer is administered. A method of treating a cancer of the brain or central nervous system is also provided in which an effective amount of a lymphangiogenesis inducer is administered. An example of a lymphangiogenesis inducer is VEGFC. The lymphangiogenesis inducer can be in the form of a protein or a polynucleotide encoding the protein, such as an mRNA or AAV. The lymphangiogenesis inducer can be administered to the cistema magna or directly into the lymphatic system. An immunotherapeutic agent, such as a checkpoint inhibitor, may also be administered.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 768,390, filed November 16, 2018, and U.S. Provisional Application No. 62 / 929,527, filed November 1, 2019, which are incorporated by reference in their entirety Incorporated into this article. [0003] sequence listing [0004] This application contains a Sequence Listing filed in ASCII format via the EFS Web and is hereby incorporated by reference in its entirety. Created on November 14, 2019, the ASCII text file is named Yale23sequence_ST25.txt and is 24,080 bytes in size. [0005] field of invention [0006] The present invention relates to methods and compositions for inducing lymphangiogenesis in tumors of the brain or central nervous system. [0007] Background of the invention [0008] There are more than 15,000 newly diagnosed patients with glioblastoma multiforme (also referred to herein as glioblastoma or GBM) in the United States each y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61P35/04A61P37/04C07K14/515C07K14/52
CPCC07K14/52A61P37/04A61P35/04Y02A50/30A61K2039/505C07K16/2818C07K16/2878A61K2039/507A61K39/39541A61K48/005A61K48/0075C12N2750/14143A01K2227/105A01K2267/0331A01K2207/12A61K38/1866A61K2300/00A61K9/513A61K39/395A61K45/06A61P35/00C07K14/485C07K14/515C12N15/86
Inventor A·伊瓦萨基J-L·托马斯E·宋
Owner YALE UNIV
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