CN113018459A

CN113018459A - Biological agent for losing weight through gene therapy and preparation method thereof

Info

Publication number: CN113018459A
Application number: CN202110256123.1A
Authority: CN
Inventors: 李生斌; 李小明; 刘晓唤
Original assignee: Baima Technology Shenzhen Co ltd
Current assignee: Baima Technology Shenzhen Co ltd
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-06-25

Abstract

The invention relates to a biological medicament for losing weight by gene therapy, which comprises AAV2, TBG and APOA4, wherein the AAV2, TBG and APOA4 are subjected to gene recombination in vitro to form a gene vector biological medicament for intravenous injection, and a preparation method of the biological medicament is also disclosed, and the biological medicament is prepared by gene acquisition, cloning, transfection, amplification and purification. The invention uses AAV2 as a carrier for transferring therapeutic gene to target cell of target organ of body; TBG is a liver-specific expression promoter, ensuring targeted delivery of the gene to the liver, where it produces the gene product; the therapeutic gene is APOA4, and can code apolipoprotein A4 synthesized as 46 kDa; the weight-reducing medicament does not need to be repeatedly used every day, has no toxic or side effect, and can continuously play the weight-reducing effect for a long time by being used once.

Description

Biological agent for losing weight through gene therapy and preparation method thereof

Technical Field

The invention relates to a weight-losing medicament, in particular to a biological medicament for gene therapy weight loss and a preparation method thereof.

Background

Obesity has become a serious global medical and social problem. According to the statistics in 2013, obesity affects the health of more than 4 hundred million Chinese people, and the obesity people in China are expected to rise rapidly in the coming decades. Obesity is a high risk factor for hyperlipidemia, type 2 diabetes, fatty liver, hypertension, cardiovascular disease and cancer, and the number of deaths worldwide is about 300 million each year.

The current drugs used for weight loss did not make a significant improvement over 10 years ago because of the discovery of serious adverse reactions. The existing weight-reducing drugs comprise two categories, pancreatic lipase inhibitors and appetite inhibitors acting on the central nervous system, including orlistat (orlistat) capsules, lorcaserin (lorcaserin), phentermine hydrochloride/topiramate, naltrexone/bupropion compound sustained-release tablets (conjugate) and liraglutide (liraglutide) injection, and have safety uncertainty in the aspects of the brain center, the cardiovascular system and the like; appetite suppressants are limited in use because they cause adverse reactions in the nervous system, and the pancreatic lipase inhibitor orlistat is used for weight loss by inhibiting pancreatic lipase activity and thus inhibiting the decomposition and absorption of fat in food, but it causes steatorrhea, may cause fat-soluble vitamin deficiency, and may also cause liver function damage.

The existing weight-reducing medicine has the defects of curative effect, side effects and adverse reactions: long-term administration is required, and the weight is rebounded. Long-term taking of the weight-reducing medicine can cause gastrointestinal dysfunction and dyspepsia of a human body, a series of gastrointestinal diseases are caused, and some weight-reducing medicines can cause the function of the kidney to be degraded; female eating weight loss drugs can also lead to infertility; in addition, some people have symptoms of mental diseases such as hyperexcitability, insomnia, emotional instability, delusion, hallucination and the like after taking the weight-reducing medicine. Furthermore, amphetamines also cause addiction like drugs, and once addicted, withdrawal is difficult, further causing symptoms such as anxiety, depression, fatigue, somnolence, overeating and the like.

Disclosure of Invention

Aiming at the existing defects, the invention provides a biological agent for gene therapy weight reduction and a preparation method thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows: a biological agent for reducing weight in gene therapy comprises AAV2, TBG and APOA4, wherein the AAV2, TBG and APOA4 are subjected to gene recombination in vitro to form a gene vector biological agent for intravenous injection.

Preferably, the APOA4 is 46kDa apolipoprotein a 4.

Preferably, the AAV2 is an adeno-associated virus type 2.

Preferably, the TBG is a liver-specific expression promoter.

Preferably, the viral vector of AAV2 is a PFD-rAAV-TBG-GFP empty plasmid.

A method for preparing a biological agent for reducing weight by gene therapy comprises the following steps of S1, obtaining therapeutic genes, amplifying an APOA4 full-length cDNA fragment from a normal organism tissue cDNA library, and adding SpeI and Sa1I enzyme cutting sites on two sides of the fragment; s2, cloning APOA4 cDNA into the AAV2 virus vector PFD-rAAV-TBG-GFP empty plasmid through SpeI and Sa1I enzyme cutting sites to prepare PFD-rAAV-TBG-APOA4-GFP therapeutic plasmid; s3, the PFD-rAAV-TBG-APOA4-GFP plasmid is made into PFD-rAAV-TBG-mAPPOA 4-GFP gene carrier biological agent through transfection, amplification and purification.

Preferably, the method further comprises a step S4 of diluting the PFD-rAAV-TBG-mAPOA4-GFP gene vector biological agent by physiological saline to form a biological agent capable of intravenous injection.

Preferably, the biopharmaceutical is diluted to a concentration of 2 x 10¹⁰vg/ml。

The invention has the beneficial effects that: the invention provides a safe and convenient biological medicament for losing weight by using gene therapy, which is a biological medicament AAV2-TBG-APOA4 based on gene therapy, is recombinant DNA constructed by gene separation and cloning in vitro, and takes AAV2 as a carrier for transferring therapeutic genes to target cells of target organs of organisms; TBG is a liver-specific expression promoter, ensuring targeted delivery of the gene to the liver, where it produces the gene product; the therapeutic gene is APOA4, and can code apolipoprotein A4 synthesized as 46 kDa; the weight-reducing medicament does not need to be repeatedly used every day, has no toxic or side effect, and can continuously play the weight-reducing effect for a long time by being used once.

Drawings

FIG. 1 is a schematic illustration of weight loss in a treatment group according to an embodiment of the present invention;

FIG. 2 is a sequence of the therapeutic APOA4 gene according to an embodiment of the present invention;

FIG. 3 is the plasmid structure of AAV-TBG-APOA4 as an exemplary therapeutic gene vector according to the present invention;

FIG. 4 is a schematic diagram of the knocking-out of mouse liver without APOA4 by APOA4 in accordance with the embodiment of the present invention;

FIG. 5 is a schematic diagram of the weight change of mice with high fat-induced obesity caused by APOA4 gene knockout and gene overexpression in an embodiment of the present invention;

FIG. 6 is a schematic diagram of APOA4 protein expressed in liver of a treatment group according to an embodiment of the present invention;

FIG. 7 is a schematic diagram of apoA4 protein detected in skeletal muscle of a treated group according to an embodiment of the present invention.

Detailed Description

To more clearly illustrate the objects, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described with reference to the following embodiments, which are clearly and completely described, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, are within the scope of the present invention.

A gene therapy slimming biological agent, including AAV2, TBG, APOA4, said AAV2, TBG, APOA4 through gene recombination in vitro to form gene carrier biological agent that can be used for intravenous injection, the biological agent formed is biological agent AAV2-TBG-APOA4 based on gene therapy, recombinant DNA constructed through gene separation and cloning in vitro, wherein AAV2 is adeno-associated virus type 2, as the carrier of the therapeutic gene to the target cell of the body target organ, the viral vector is PFD-rAAV-TBG-GFP empty plasmid, as shown in figure 3, about 80% carries this virus, it will produce the influence to the human body, but will not cause disease; wherein TBG is a liver-specific expression promoter, so that the gene is directionally delivered to the liver, and the liver is taken as a target organ to produce a gene product in the liver; the APOA4 is apolipoprotein A4 of 46kDa, is induced after human and animal diets, is an endogenous apolipoprotein synthesized and secreted in small intestine and liver, can reach all organs of the whole body to play a role through blood, is used as a therapeutic gene, and has no toxic or side effect; in addition, the APOA4 protein can be continuously generated in the liver for a long time by one-time intravenous injection administration, and the APOA4 protein can be generated locally in the liver and secreted and transported to other organs of the body to generate curative effect, reduce the weight and maintain the reduced weight without rebound; selectively fat-reducing without protein consumption; improving metabolic disorders in obese patients; safe, has little adverse reaction and no possibility of addiction and abuse. The efficacy of the anti-obesity agent was evaluated by (1) the difference in weight loss between the subjects in group 2 after 1 year of administration of the anti-obesity agent and placebo should be no less than 5%; (2) the proportion of subjects with weight loss of more than 5% in the drug-treated group is not less than 35%, and the proportion is 2 times that of the placebo group; as shown in fig. 1, is a graph of body weight change of obese model mice after injection of the weight-reducing biopharmaceutical.

A biological preparation for gene therapy for reducing weight is prepared by the following steps,

s1, acquiring a therapeutic gene, amplifying an APOA4 full-length cDNA fragment from a normal organism tissue cDNA library, and adding SpeI and Sa1I enzyme cutting sites on two sides of the fragment; therapeutic gene selection mouse APOA4(mAPOA4) cDNA: firstly, amplifying an APOA4 full-length cDNA fragment from a normal mouse liver tissue cDNA library as shown in figure 2, wherein SpeI and SalI enzyme cutting sites are added on two sides of the fragment during amplification;

s2, cloning APOA4 cDNA into the AAV2 virus vector PFD-rAAV-TBG-GFP empty plasmid (also a negative control plasmid) through SpeI and Sa1I enzyme cutting sites to prepare PFD-rAAV-TBG-APOA4-GFP therapeutic plasmid, the structure of which is shown in figure 3, and the therapeutic gene vector AAV-TBG-APOA4 plasmid structure;

s3, preparing the PFD-rAAV-TBG-APOA4-GFP plasmid into the PFD-rAAV-TBG-mAPPOA 4-GFP gene carrier biological agent through transfection, amplification and purification,at this time, the plasmid carries a liver tissue-specific promoter TBG, the target gene carries fluorescent protein GFP, and the titer of the prepared biological preparation is 5X 10¹²vg/ml, as gene vector biopharmaceutical to be used.

When in use, the method also comprises a step S4, the PFD-rAAV-TBG-mAPOA4-GFP gene carrier biological medicament is diluted by physiological saline to form a biological medicament capable of being injected intravenously, and the biological medicament is diluted to a corresponding concentration according to different use requirements, wherein the concentration of the biological medicament is 2 x 10¹⁰vg/ml can meet the conventional use requirement, and 100 mu L (2X 10) of each fundus is injected into the fundus vein of a male mouse aged 5 weeks⁹vg) is carried out.

The specific embodiment is as follows:

1 obesity model mouse APOA4 gene deletion, weight gain and overexpression weight loss,

1.1 knocking out APOA4 gene and building an obesity model of an overexpression genetic engineering mouse: male APOA4 knockout (KO group), APOA4 transgenic (TG group) and C57BL/6J wild type (WT negative control group) mice 6 weeks old were randomly selected, fed with high fat diet for 16 weeks, weighed weekly, and sacrificed at 16 weeks to obtain material.

1.2 results: immunofluorescence experiments showed that the APOA4 knockout liver did not express APOA4, as shown in fig. 4, and the KO group body weight was significantly increased compared to the WT group, as shown in fig. 5, by 5%, 10.7%, and 8.3% at weeks 12, 14, and 16, respectively. And the body weight of the APOA4 transgenic overexpression group is reduced from 4 weeks, and the reduction at 4, 6, 8, 10, 12 and 14 weeks is 6.6%, 13.0%, 14.8%, 13.6%, 11.2% and 5.5%, respectively.

The APOA4 gene overexpression has obvious curative effect on obese mice, and the weight loss reaches 12% in 4 weeks and 12.9% in 16 weeks after high-fat feeding.

2 the weight-losing biological medicament AAV2-TBG-APOA44 can treat weight gain caused by the gene knockout of APOA4,

2.1 injecting fat-reducing biological agent into APOA4 gene knock-out fat model mouse: the mice of APOA4 gene knockout high fat induced obesity model described in 1.1 are injected with the weight-reducing biological agent AAV2-TBG-APOA44 once (KO-A4 group), and the mice injected with AAV2-TBG-GFP (KO-GFP group) are used as negative control. The injection method was performed by injecting 100. mu.L (2X 109vg) per fundus intravenously into 5-week-old male mice, weighing the body weight weekly after injection, and obtaining the material from the mice sacrificed at 16 weeks.

2.2 results: immunofluorescence experiments show that the liver of the biological slimming agent treatment group KO-A4 expresses APOA4, the liver of the negative control group KO-GFP group does not express APOA4, as shown in figure 6, meanwhile, the skeletal muscle tissue of the treatment group KO-A4 group detects APOA4 protein, the skeletal muscle tissue of the negative control group KO-GFP group does not detect APOA4 protein, as shown in figure 7, the result shows that the liver expresses APOA4 protein and can reach other peripheral tissue organs such as skeletal muscle through blood, the weight change is shown in figure 1, compared with a negative control group mouse, the weight of the treatment group is remarkably reduced at the 4 th week, and the weight reductions of the 4 th week, the 8 th week, the 12 th week and the 16 th week are respectively 12.0%, 9.6%, 10.1% and 12.9%. The weight-losing biological medicament is far beyond the accepted treatment standard of weight-losing medicaments, and the weight-losing biological medicament has obvious weight-losing treatment effect.

It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Claims

1. A biological agent for losing weight by gene therapy is characterized in that: the AAV2, TBG and APOA4 are included, and the AAV2, the TBG and the APOA4 are subjected to in vitro gene recombination to form a gene vector biological agent for intravenous injection.

2. The biological agent for reducing weight by gene therapy according to claim 1, wherein the APOA4 is apolipoprotein A4 of 46 kDa.

3. The biological agent for gene therapy to reduce weight according to claim 1, wherein AAV2 is adeno-associated virus type 2.

4. The biological agent for weight loss through gene therapy according to claim 1, wherein the TBG is a promoter for liver-specific expression.

5. The biological agent for gene therapy to reduce weight according to claim 1, wherein the viral vector of AAV2 is PFD-rAAV-TBG-GFP empty plasmid.

6. A method for preparing a biological medicament for losing weight by gene therapy is characterized by comprising the following steps,

s1, acquiring a therapeutic gene, amplifying an APOA4 full-length cDNA fragment from a normal organism tissue cDNA library, and adding SpeI and Sa1I enzyme cutting sites on two sides of the fragment;

s2, cloning APOA4 cDNA into the AAV2 virus vector PFD-rAAV-TBG-GFP empty plasmid through SpeI and Sa1I enzyme cutting sites to prepare PFD-rAAV-TBG-APOA4-GFP therapeutic plasmid;

s3, the PFD-rAAV-TBG-APOA4-GFP plasmid is made into PFD-rAAV-TBG-mAPPOA 4-GFP gene carrier biological agent through transfection, amplification and purification.

7. The method for preparing a biological agent for gene therapy to reduce weight according to claim 6, further comprising a step S4 of diluting the PFD-rAAV-TBG-mAPOA4-GFP gene vector biological agent with physiological saline to form a biological agent capable of intravenous injection.

8. The method for preparing biological agent for gene therapy to lose weight as claimed in claim 7, wherein the concentration of the diluted biological agent is 2X 10¹⁰vg/ml。