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Hepatocyte targeting polyethylene glyco-grafted poly-lysine polymeric gene carrier

A technology of polyethylene glycol and gene carrier, applied in gene therapy, genetic engineering, plant gene improvement, etc., can solve the problem of low transfection rate

Inactive Publication Date: 2002-01-30
EXPRESSION GENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using PLL alone as a gene delivery vehicle has several disadvantages
First, since PLL has no functional groups other than the amino group used in charge neutralization, its transfection efficiency is very low

Method used

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  • Hepatocyte targeting polyethylene glyco-grafted poly-lysine polymeric gene carrier
  • Hepatocyte targeting polyethylene glyco-grafted poly-lysine polymeric gene carrier
  • Hepatocyte targeting polyethylene glyco-grafted poly-lysine polymeric gene carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1L

[0048] The synthesis of embodiment 1Lac-PEG diacid

[0049] The synthesis of lactose-PEG diacid was carried out as follows. Under a nitrogen atmosphere in an ice-water bath, 600 mg (1.0 mM) of PEG diacid (molecular weight = 600; Fluka, Ronkonkoma, NY) was dissolved in 1.5 mL of anhydrous N,N-Dimethylformamide (DMF; Aldrich). 1 mM isobutyl chloroformate (IBCF; Aldrich) dissolved in 1.0 mL of dry DMF was added dropwise in an ice-water bath, and the mixture was stirred for 15 minutes. IBCF acts as a carboxylic acid activator. Then p-aminophenyl-α-D-lactopyranoside (1.0 mM; Sigma) dissolved in 1.0 mL of DMF was added, and the mixture was stirred in an ice-water bath for 30 minutes, then at room temperature for a further 3 hours. The product was precipitated in excess dry diethyl ether (25 mL). The precipitate was then dissolved in 20 mL of distilled water, dialyzed against distilled water (MWCO of the dialysis bag was 1,000; Spectrum, Houston, TX) and lyo...

Embodiment 2

[0050] The grafting of embodiment 2 lactose-PEG diacid and PLL

[0051] In this example the synthesis of Lac-PEG-PLL with 30% (mol / mol%) lactose-PEG) is described. Lactose-PEG and lactose-PEG-lactose mixture (51.5 mg; containing 0.042M lactose-PEG) prepared according to the procedure of Example 1 were dissolved in 0.5 mL of anhydrous DMF under a nitrogen atmosphere in an ice-water bath. IBCF (0.036M) dissolved in 0.5 mL DMF was added dropwise to the above solution. The mixture was stirred in an ice-water bath for 30 minutes, then added dropwise to 1 mL of anhydrous dimethylsulfoxide (DMSO; Aldrich) containing 25 mg of PLL-HBr (120 repeating units; molecular weight = 25,000; Sigma) and 10 μLTEA (which had been purged with nitrogen in an ice-water bath for 10 minutes), then stirred at room temperature for an additional 3 hours. The product was precipitated in 100 mL of anhydrous ether, and the precipitate was dissolved in 10 mL of distilled water. Disso...

Embodiment 3

[0052] The 1H-NMR analysis of embodiment 3 lactose-PEG-PLL

[0053] 1H-NMR analysis of Lac-PEG-PLL prepared by the procedures of Examples 1 and 2 was carried out in water. exist figure 2 The 1H-NMR spectrum of 30 mol% Lac-PEG-PLL is shown in . A peak at approximately 3.5 ppm indicates the presence of PEG in the composition. The PEG content was calculated from the NMR spectrum by correlating the PEG (-CH2CH2-.s.3.4-3.7ppm) peak with the PLL side chain (-CH2CH2CH2-.m.1.1-1.8ppm) peak. The following ratios of lactose-PEG content in four illustrative carrier compositions made by the present invention were confirmed by this method: 6, 12, 20 and 30 mole %, respectively.

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Abstract

A compound for polymeric targeted gene delivery carrier consisting of polyethylene glycol (PEG) grafted poly(L-lysine) (PLL) and a targeting moiety (TM), wherein at least one free amino function of the PLL is substituted with said PEG, at least one free amino function of the PLL is substituted with the TM, and the grafted PLL contains at least 50 % unsubstituted free amino function groups. TM is preferably lactose or galactose which are capable of specifically targeting a hepatoma cell or a liver cell. The new synthetic carriers with various substitution ratios of TM-PEG were characterized using NMR spectroscopy. The new polymeric gene carriers of this invention are capable of forming stable and soluble complexes with nucleic acids, which in turn are able to efficiently transform cells. PEG attached to the PLL gives better solubility properties to the gene / carrier complex and improved transfection efficiency without considerable cytotoxicity. Methods of preparing and using the TM-PEG-PLL as polymeric gene carriers to efficiently transfect cells are disclosed.

Description

[0001] Cross-references with related applications [0002] This application claims priority to US Provisional Application 60 / 086,072, filed May 20,1998. Background of the invention [0003] The present invention relates to gene therapy. More specifically, the present invention relates to a composition of polyethylene glycol-grafted poly-L-lysine and hepatocyte-directed molecule-polyethylene glycol-grafted poly-L-lysine, the combination for gene delivery into hepatocytes. [0004] Twenty-five years ago, Friedmann pointed out the prospect of human gene therapy (T. Friedmann and R. Roblin (1972) "Gene therapy for human genetic diseases?" 175 "Science" 949-955 (1972)). Since then, gene therapy has represented a new way of treating human disease and drug delivery. The implicit focus of existing studies has been to determine the safety of the gene transfer step, often with efficacy as a secondary goal. The main technical obstacle to gene transfer is the...

Claims

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Application Information

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IPC IPC(8): C12N15/00A61K9/127A61K47/26A61K47/34A61K47/48A61K48/00C12N15/87
CPCA61K47/48315C12N15/87A61K47/645A61K47/32
Inventor J·S·帕克Y-H·乔F·刘
Owner EXPRESSION GENETICS INC
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