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A kind of gene medicine and its application for treating the dysfunction of Leydig cells

A Leydig cell and gene drug technology, applied in the field of gene drugs for the treatment of Leydig cell dysfunction, can solve problems such as inability to solve the problem of male infertility, and achieve the effect of high safety, specificity and safety

Active Publication Date: 2022-02-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In existing clinical practice, testosterone replacement therapy can restore serum testosterone levels and promote gonadal development (such as "Testosterone replacement therapy should be carried out correctly and reasonably, Zhang Guiyuan, National Family Planning Commission Research Institute, Beijing 100081", "Testosterone replacement therapy and its impact Pharmacogenetics of Body Function and Metabolism, Michael Zitzmann, ASIAN JOURNAL OFANDROLOGY, 2008"), however, existing therapies can hardly solve the problem of male infertility caused by genetic LCD

Method used

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  • A kind of gene medicine and its application for treating the dysfunction of Leydig cells
  • A kind of gene medicine and its application for treating the dysfunction of Leydig cells
  • A kind of gene medicine and its application for treating the dysfunction of Leydig cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Construction and separation and purification of AAV vector

[0039] Plasmid AAV-Lhcgr was constructed as figure 1 As shown, the main elements include CAG promoter and Lhcgr sequence. Viral vectors are obtained by plasmid co-transfection. Different types of AAV Rep / Cap plasmids, AAV-Helper plasmids and AAV-Lhcgr plasmids are co-transfected with HEK 293 cells to form AAV vectors. After purification by iodixanol density gradient ultracentrifugation, the titer of the virus was determined by real-time fluorescent quantitative PCR, and finally the silver staining method was used to confirm that the virus vector particles were not contaminated and did not contain endotoxin, and were divided into -80 Store at ℃.

Embodiment 2

[0040] Example 2: Carrier Screening

[0041] To construct an AAV vector carrying mCherry, we screened 11 AAV serotypes AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV7m8, AAVnc80, AAVDJ. Dilute the constructed viral vector composition to 1×10 with saline 13 vg / mL, after the mouse was anesthetized with 0.2% avertin (0.2 mL / 10 g), 8 μL of the diluted carrier composition was injected into each testis on both sides with a microinjector. One week later, the mouse testis tissue was taken for tissue immunofluorescence staining, and Leydig cell precursor cell marker Nestin was stained; finally, a confocal microscope was used to observe and take pictures.

[0042] figure 2 It shows that AAV5, AAV6, AAV8, AAV9, AAVrh10, AAVnc80, AAVDJ and Nestin have a higher co-expression ratio, suggesting that the above AAV types can better infect Leydig cell precursor cells.

Embodiment 3

[0043] Example 3: gene drug expresses Lhcgr, restores testosterone level, and promotes the maturation of Leydig cells

[0044]AAV8 in Example 2 was selected as a gene delivery tool, and an AAV8-CAG-Lhcgr viral vector (gene drug) was constructed. In order to detect whether the gene carrier is successfully expressed and functioning in the testis; 3w-age Lhcgr - / - Mice were injected with AAV8-Lhcgr or PBS, Lhcgr from littermate + / + or Lhcgr + / - as comparison.

[0045] The results of immunofluorescence staining showed that Lhcgr in the AAV8-Lhcgr treatment group - / - There is a strong expression of Lhcgr in the interstitium of mouse testis, while Lhcgr injected in PBS - / - There was no expression of Lhcgr in mouse testis interstitium ( image 3 ).

[0046] Furthermore, compared with the PBS-injected group, Lhcgr treated with AAV8-Lhcgr - / - Serum testosterone levels were also significantly increased in mice. Its serum testosterone level can reach Lhcgr + / + or Lhcgr + / - Mous...

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Abstract

The present invention relates to a gene medicine for treating Leydig cell dysfunction and its application. The gene medicine is a recombinant adeno-associated virus that can express the luteinizing hormone / chorionic gonadotropin receptor by utilizing host cells. The drug can not only restore the level of testosterone, promote the development of gonads, but also can significantly promote the production of sperm and rebuild fertility; at the same time, the gene drug has no obvious side effects and is highly safe. In addition, the gene medicine can be applied to the preparation of the gene medicine composition or the bioreactor for the production of luteinizing hormone / chorionic gonadotropin receptor, which has high application value.

Description

technical field [0001] The invention belongs to the field of gene medicine, and in particular relates to a gene medicine for treating testicular interstitial cell dysfunction. Background technique [0002] Testosterone is essential for the development and maintenance of male reproductive function and secondary sex characteristics. More than 95% of testosterone in men is synthesized and secreted by Leydig cells under the joint action of various key genes for testosterone synthesis. When gene mutations lead to inactivation of key genes for testosterone synthesis, testosterone synthesis decreases, which in turn causes gonadal dysplasia, spermatogenesis block, and male infertility, which is called hereditary Leydig cell dysfunction (LCD). [0003] In existing clinical practice, testosterone replacement therapy can restore serum testosterone levels and promote gonadal development (such as "Testosterone replacement therapy should be performed correctly and rationally, Zhang Guiyu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P15/08
CPCA61K48/005A61K48/0008A61P15/08
Inventor 项鹏邓春华夏凯汪富林
Owner SUN YAT SEN UNIV
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