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Method for inducing in vitro the generation of erythrocyte medicine containing l-aspase II

A technology of erythrocytes and drugs, applied in the field of regenerative medicine, can solve the problems of reduced deformability of carrier erythrocytes, increased fragility of carrier erythrocytes, damage of carrier erythrocyte membranes, etc., and achieve the effect of reducing toxic and side effects

Active Publication Date: 2017-08-04
厦门三一造血技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are several problems in changing the permeability of red blood cell membrane to embed drugs: 1) There are different degrees of deformability reduction in carrier red blood cells; 2) There are different degrees of membrane damage in carrier red blood cells; 3) There are different degrees of morphological abnormalities in carrier red blood cells; 4) Increased fragility of carrier erythrocytes; these changes will directly affect the clearance time of drug-loaded erythrocytes in the body and drug release effect

Method used

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  • Method for inducing in vitro the generation of erythrocyte medicine containing l-aspase II
  • Method for inducing in vitro the generation of erythrocyte medicine containing l-aspase II
  • Method for inducing in vitro the generation of erythrocyte medicine containing l-aspase II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction and packaging of lentiviral vector

[0032] The Escherichia coli L-asparaginase II gene sequence (981bp, the last group is terminator) and amino acid sequence (326aa) were obtained from NCBI, as shown in 1 and 2 in the sequence table.

[0033] The experimental process of lentiviral vector construction is as follows (the process is as follows: figure 2 shown):

[0034] 1) Construction of cloning vector: the target gene L-asparaginase II gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the target gene was connected to the pUC57 vector through BamH I and EcoR I restriction sites to construct a cloning vector pUC57-L-ASPase II.

[0035] 2) Construction of expression vector: plasmid pUC57-L-ASPase II and vector plasmid pLenti6.3 / V5-GW / Em-GFPVerA (Invitrogen) were simultaneously digested with BamH I and EcoR I enzymes, enzyme digestion system: 10x buffer 2 μl , 0.4 μl each of BamH I and EcoR I, 4 μl of plasmid solution (plasmi...

Embodiment 2

[0050] Example 2: Genetic modification of IPS cells using lentiviral vectors

[0051] 1) Plating: Digest and resuspend the IPS in the logarithmic growth phase with 0.05% trypsin in PBS, press 1*10 5 / ml density inoculated in 12-well plate, grown overnight (12h).

[0052] 2) Infection: Aspirate 70-80% of the culture solution in the 12-well plate, replace with fresh culture solution, and add virus solution diluted in PBS concentration gradients at the same time (5 gradients in total, prepare 5 1.5ml EP tubes, Add 90 μl PBS to each tube, add 10 μl virus stock solution to the first tube, mix well, pipette 10 μl into the second tube and mix well, and so on, make 5 dilutions (10~10 -4 )), mix well and put in 37℃, 5%CO 2 cultured in an incubator. 3) Change the medium after 24 hours of culture, and you can see the fluorescence after 48 hours. Place the cells on the MEF trophoblast cells for low-density culture (1*10 4 / ml), 12-14 days later, the clones were picked for the next ste...

Embodiment 3

[0053] Example 3: Inducing IPS cells in vitro to generate erythrocyte drugs containing L-ASPase II

[0054] 1) In vitro differentiation of IPS cells into EBs (Erythroid Body)

[0055] Human IPS cells (hIPS) (prepared in Example 2) passaged for 12 to 30 passages were digested with collagenase IV (collagenase IV), and then transferred to a culture medium prepared with IMDM (Biochrome) as the base culture medium. In the culture medium Contains SCF (Sigma, 100ng / mL), TPO (Sigma, 100ng / mL), FLT3 ligand (Sigma, 100ng / mL), BMP4 (Peprotech, 10ng / mL), VEGF-A165 (Sigma, 5ng / mL), IL -3 (Sigma, 5ng / mL), IL-6 (Peprotech, 5ng / mL) and erythropoietin EPO (Sigma, 3U / mL); cells were placed at 37°C, 5% CO 2 EBs were cultured for 20 days under conditions; after the formation of EBs, the cells were digested with collagenase B (Sigma, 0.4 U / mL) at 37°C for 30 minutes and placed in buffer (invitrogen) for 10 minutes.

[0056] 2) EB induces differentiation into mature red blood cells

[0057] ① Af...

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Abstract

The invention discloses a method for producing a red blood cell drug containing L-ASPase II (L-asparaginase) by in vitro induction. The method comprises the steps of: 1) using a lentiviral vector system to transform induced pluripotent stem (IPS) cells: I. constructing a pLenti6.3 / V5-GW / Em-GFP VerA-L-ASPase II plasmid; II. using 293T cells to perform packaging so as to generate high titer virus particles containing L-ASPase II; III. using the virus particles to transfect induced pluripotent stem cells; and 2) inducing the induced pluripotent stem cells in vitro to generate L-ASPase II-containing red blood cells: I. subjecting IPS cells to in vitro differentiation induction to form an erythroid body EB; and II. performing induced differentiation on EB to obtain mature red cells. The method provided in the invention performs genetic modification on IPS cells by the lentiviral vector system, and induces them in vitro to generate red blood cells containing L-ASPase II. The generated red blood cells can be used as a sustained release carrier of L-ASPase II, and also can maintain the original shapes and functions of red blood cells. The toxic and side effects of L-ASPase II are further reduced, so that the method lays a solid foundation for future clinical application.

Description

technical field [0001] The invention relates to a method for transforming induced pluripotent stem cells (IPS cells) by using a lentiviral vector system and inducing them to generate erythrocytes containing L-asparaginase II drug in vitro, which belongs to the field of regenerative medicine. Background technique [0002] L-asparagine is an essential amino acid for the growth of certain tumor cells. For normal human cells, they have the ability to synthesize L-asparagine, while tumor cells lack asparagine synthetase and cannot synthesize asparagine. Its cells require the uptake of exogenous L-asparagine to survive. L-asparaginase II (L-asparaginase, L-ASPase II) can catalyze the hydrolysis of L-asparagine to generate L-aspartic acid and ammonia, so it can effectively inhibit the growth of cancer cells and eventually make cancer cells die . Therefore, L-ASPase II is an important drug for the treatment of tumors, especially for acute lymphoblastic leukemia (Actute lymphoblast...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10A61K38/50A61K47/46A61P35/00A61P35/02
Inventor 叶永清
Owner 厦门三一造血技术有限公司
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