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Macrophage transfection method

A macrophage, monocyte technology

Inactive Publication Date: 2010-08-11
托马斯・E・瓦格纳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the '612 patent does not describe the use of yeast cell wall particles such as zymosan for direct entry of nucleic acids into cells of monocyte origin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] This example demonstrates the transfection of macrophages with an adenovirus-vector coupled to a particle carrier.

[0079] 1. Coupling of Adenoviral Vectors to Streptavidin-Magnetic Beads

[0080] Adenovirus (Ad) particles (suspended in PBS) were bioacylated with Sulfo-NHS-LC-biotin and added to streptavidin-conjugated magnetic Beads (MB) for 2 hours. Ad-MB conjugates were washed thoroughly with PBS and stored at 4°C until use.

[0081] 2. Conjugation of Adenoviral Vectors to Zymosan

[0082] 2.1. Derivatization of Zymosan for Conjugation

[0083] The sugar group of zymosan was slightly oxidized by sodium metaperiodate, and then adipic acid dihydrazide (ADH) was added to introduce the amino group. The resulting conjugate was stabilized by the addition of sodium cyanoborohydride. The ADH-modified zymosan was further reacted with SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate), each particle Introduce about 106 active protection sulfhydryl groups, wash with PB...

Embodiment 2

[0091] This example demonstrates that adenovirus-mediated gene transfer stimulates the secretion of macrophage anti-tumor activity.

[0092] Thioglycollate (salt)-induced mouse peritoneal macrophages were transfected with Ad-Z (zymosan)-vector for 48 hours at a ratio of about 4 zymosan particles per macrophage (approximately equivalent to 40 Ad-particles). Thereafter, the medium was collected, clarified by filtration (0.22 μm), concentrated 50-fold by ultrafiltration (10 kDa cut-off), and dialyzed against HEPES buffered saline solution (HBS). Serial dilutions of concentrated macrophage culture supernatants were incubated with YAC-1 mouse lymphoma cells for 40 hours. Thereafter, viable tumor cells were stained with MTT and the relative cytotoxicity of the samples (relative to controls incubated with HBS) was determined photometrically. Cytotoxicity was expressed in units per milliliter (U / ml), and 1 U / ml was defined as the concentration that caused 50% cytotoxicity. Culture ...

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Abstract

Described are a method and a composition for transfecting monocytes, as well as use of the same for therapeutic purposes. The composition is composed of a nucleic acid component, a lysosome evading component and a digestible particle that can be phagocytized. Preferably, the monocyte is a macrophage and the digestible particle is from a natural source, such as from a microbial source. More preferably, the digestible particle is a yeast cell wall particle such as zymosan. The composition itself, or cells pretreated with the composition, are useful in all gene medicine applications, such as gene therapy, gene vaccination, cancer treatment as well as immunomodulation and tissue repair.

Description

[0001] Cross references to related patent applications [0002] This application claims priority to U.S. Provisional Application No. 60 / 907,977, filed April 25, 2007, and U.S. Provisional Application No. 60 / 924,868, filed June 4, 2007, both Both U.S. provisional applications are hereby incorporated by reference in their entirety. field of invention [0003] The present invention relates to a monocyte transfection method and its therapeutic use. Background of the invention [0004] Monocytes play a central role in the immune response. After maturation, monocytes become macrophages and dendritic cells, which are the main antigen-presenting cells of the body. Furthermore, as tumors grow, they produce macrophage-attracting chemokines that attract monocytes to the tumor as part of the angiogenic process. Thus, once monocytes are specifically targeted, they may be immediately used to deliver therapeutic gene products to tumor cells or generate therapeutic or preventive immune r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K39/12A61K31/713
CPCA61K47/48776C12N2810/10C12N2799/022A61K47/4823A61K47/48876A61K47/61A61K47/6901A61K47/6927A61P19/02A61P29/00A61P31/04A61P31/12A61P33/00A61P35/00A61P37/02A61P37/04A61P37/06A61P37/08
Inventor T·E·沃纳G·施瓦姆伯格X·于
Owner 托马斯・E・瓦格纳
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