RU2815935C1 - Method for controlling insulin secretion by pancreatic β-cells using thermogenetics - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method for increasing insulin secretion β-cells of pancreatic islets of Langerhans by means of thermogenetics. In β-cells deliver a genetic construct containing a promoter specific for them and a gene of human thermally activated channel TRPV1. Then β-cells are heated in a PCR machine with a certain mode of heating and cooling cycles.

EFFECT: invention enables non-invasive stimulation β-cells of pancreatic islets of Langerhans and stimulate insulin secretion.

3 cl, 2 dwg, 1 ex

Description

Field of technology to which the invention relates

The presented invention relates to the field of medicine and biotechnology, namely to technologies for controlling the activity of excitable tissues, and can be used to control insulin secretion by pancreatic β-cells.

State of the art

More than three hundred million people in the world suffer from diabetes mellitus, a complex disease associated with impaired glucose absorption. The consequence of the disease is hyperglycemia, leading to disorganization of all main types of metabolism: carbohydrate, protein, lipid and water-salt.

The pathogenetic mechanism for the development of type 1 diabetes is the insufficiency of insulin synthesis and secretion by endocrine cells of the pancreas (pancreatic β-cells), caused by their destruction as a result of exposure to certain factors (viral infection, stress, autoimmune aggression, and others). The prevalence of type 1 diabetes reaches 10-15% of all cases of diabetes. This disease is characterized by the manifestation of the main symptoms in childhood or adolescence, the rapid development of complications against the background of decompensation of carbohydrate metabolism. The main treatment method is insulin injections, which normalize the body's metabolism. If left untreated, type 1 diabetes progresses rapidly and leads to severe complications such as ketoacidosis and diabetic coma.

The basis of the pathogenesis of type 2 diabetes is a decrease in the sensitivity of insulin-dependent tissues to the action of insulin (insulin resistance). In the initial stage of the disease, insulin is synthesized in normal or even increased quantities. Diet and weight loss of the patient in the initial stages of the disease help normalize carbohydrate metabolism, restore tissue sensitivity to the action of insulin and reduce glucose synthesis at the liver level. However, as the disease progresses, insulin biosynthesis by pancreatic β-cells decreases, which makes it necessary to prescribe hormone replacement therapy with insulin preparations. Type 2 diabetes accounts for 85-90% of all cases of diabetes mellitus in adults and is most common among people over 40 years of age, usually accompanied by obesity. The disease develops slowly and is mild. The clinical picture is dominated by accompanying symptoms; ketoacidosis develops rarely. Persistent hyperglycemia over the years leads to the development of micro- and macroangiopathy, nephro- and neuropathy, retinopathy and other complications.

In addition, there are several other types of diabetes, such as MODY diabetes and gestational diabetes, but all of them are clinically manifested by hyperglycemia and diabetes.

Currently, treatment of diabetes mellitus in the vast majority of cases is symptomatic and is aimed at eliminating existing symptoms without eliminating the cause of the disease, since an effective treatment for diabetes has not yet been developed. The main objectives in the treatment of diabetes mellitus are: compensation of carbohydrate metabolism, prevention and treatment of complications, normalization of body weight. Compensation of carbohydrate metabolism is achieved in two ways: by providing cells with insulin, in different ways depending on the type of diabetes, and by ensuring a uniform, equal supply of carbohydrates, which is achieved by following a diet.

In the case of drug therapy to stimulate the secretion of additional insulin by pancreatic β-cells and restore normal blood glucose concentrations, various oral hypoglycemic drugs are used:

- Sulfonylurea derivatives (Tolbutamide, Carbutamide, Chlorpropamide, Glibenclamide, Glipizide, Gliclazide, Gliquidone, Glimepiride) increase insulin secretion by beta cells of the pancreas.

- Prandial glycemic regulators (Repaglinide, Nateglinide) are secretagogues with rapid absorption and a short period of hypoglycemic action.

- Biguanides (metformin) reduce the absorption of glucose in the intestine and its production in the liver, as well as increase the sensitivity of tissues to the action of insulin.

- Thiazolidinediones (rosiglitazone, pioglitazone) stimulate genetic mechanisms involved in glucose metabolism, as well as increase tissue sensitivity to glucose.

- α-glycosidase inhibitors (acarbose) inhibit intestinal enzymes that break down complex carbohydrates into glucose, thereby reducing the absorption of glucose at the intestinal level.

If these drugs are ineffective, insulin replacement therapy is prescribed.

The production of the hormone insulin is carried out by β-cells of the endocrine part of the pancreas, and the presence of a tool for controlling their activity is of utmost importance for the treatment of diabetes mellitus. Cell activation is regulated by membrane depolarization followed by the entry of calcium into the cytoplasm, where calcium is a second messenger in the regulation of their metabolism. There is a need for methods to control calcium concentration at the intracellular level. However, at the moment, such technologies are practically absent, and the existing approaches have disadvantages that limit their widespread use.

Solutions are known from the prior art that make it possible to control insulin secretion by β-cells using optogenetics methods. In the article by Reinbothe and co-authors [Reinbothe TM, Safi F, Axelsson AS, Mollet IG, Rosengren AH. Optogenetic control of insulin secretion in intact pancreatic islets with β-cell-specific expression of Channelrhodopsin-2. Islets. 2014; 6(1):e28095. doi: 10.4161/isl.28095. PMID: 25483880; PMCID: PMC4593566] describes an optogenetics-based approach that allows specific study of β-cells in the islets of Langerhans. The authors used transgenic mice expressing the light-sensitive cation channel channelhodapsin-2 (ChR2) under the control of the insulin promoter. Light stimulation of the islets of ChR2 transgenic mice caused a rapid increase in intracellular calcium and enhanced insulin secretion in the islets. β-cells from diabetic mice on a high-fat diet showed a 3.5-fold increase in light-induced calcium influx compared to mice on a control diet. In addition, light induced an increase in insulin secretion.

In a study by Kushibiki et al [Kushibiki T, Okawa S, Hirasawa T, Ishihara M. Optogenetic control of insulin secretion by pancreatic β-cells in vitro and in vivo. Gene Ther. July 2015; 22(7):553-9. doi: 10.1038/gt.2015.23. Epub 2015 Mar 26. PMID: 25809465.] assessed the ability of optogenetic methods to control insulin secretion and blood glucose homeostasis by regulating intracellular calcium ion concentrations in a mouse pancreatic β-cell line (MIN6) transfected with the optogenetic channel protein ChR2. MIN6 cells transfected with ChR2 (ChR2-MIN6) secreted insulin after laser irradiation (470 nm). The increase in calcium was accompanied by increased levels of messenger RNAs that encode calcium/calmodulin-dependent protein kinase II delta and adenylate cyclase 1. ChR2-MIN6 cells suspended in Matrigel were transplanted into streptozotocin-induced diabetic mice. which were then subjected to a glucose tolerance test. Laser irradiation of these mice caused a significant decrease in blood glucose levels, and the irradiated implanted cells expressed insulin.

The results demonstrate the utility and versatility of optogenetics for studying mechanisms of glucose homeostasis and for developing treatments for metabolic diseases such as diabetes. A significant drawback of these approaches is the use of channelrhodopsins, which are not found in mammals and cause an immune reaction, which in a relatively short time leads to the death of cells expressing channelrhodopsins. A promising approach to solve this problem may be the use of thermogenetic calcium switches.

Thermogenetics is an approach for regulating cell activity using thermally activated channels of the TRP (Transient Receptor Potential) family, used both in model cell cultures and in animals in vivo. TRPs are nonselective cation channels that, when activated, allow calcium, sodium, potassium, and magnesium ions to pass through. The use of TRP channels, which allow control of intracellular calcium concentration, is a promising approach to control cell activity, in particular, insulin secretion by β-cells.

Approaches are known from the prior art that allow controlling cells using temperature-sensitive ion channels, but these solutions are not used to control the activity of pancreatic β-cells. For example, methods have been patented that allow stimulating immune cells by expressing mechano- and temperature-sensitive ion channels in them, followed by activation using ultrasound [patent WO2018098315A1].

One of the approaches presented in the scientific literature, closest to what we have stated, is non-invasive neuromodulation of the deep parts of the brain [Yang Y, Pacia CP, Ye D, Zhu L, Baek H, Yue Y, Yuan J, Miller MJ, Cui J, Culver JP , Bruchas MR, Chen H. Sonothermogenetics for noninvasive and cell-type specific deep brain neuromodulation. Brain Stimul. 2021 Jul-Aug; 14(4):790-800. doi: 10.1016/j.brs.2021.04.021]. This approach uses the temperature-sensitive TRPV1 ion channel and heats brain cells using high-intensity focused ultrasound. However, this approach has not been applied to control pancreatic β-cell activity.

Controlling cellular activity using temperature-activated channels of the TRP superfamily is a promising approach because these channels are present in the human body and their expression does not induce an immune response. The radiation required to activate the thermosensitive channels is invisible to living organisms, which reduces the animal’s stress level during the stimulation process. Due to the low absorption by its own pigments, infrared and microwave radiation does not cause phototoxic effects in the upper layers of tissues, as is the case when blue light is used to activate ChRs.

Disclosure of the Invention

Technical problem,solved by the present invention is the absence of the possibility of non-invasive control of insulin secretion by pancreatic β-cells.

When solving the stated technical problem, the following technical results are achieved: the possibility of non-invasive control of insulin secretion by β-cells of individual islets of Langerhans of the pancreas using heating.

To solve the stated technical problem and achieve the stated technical result, a method is proposed for controlling insulin secretion by β-cells of the islets of Langerhans of the pancreas using thermogenetics approaches, including the following actions:

- creation of genetic constructs containing a β-cell-specific promoter and genes for thermally activated channels of the TRP superfamily;

- delivery of these constructs using adeno-associated viruses into β-cells of the islets of Langerhans for subsequent expression of the corresponding channel;

- heating of β-cells of the isolated islets of Langerhans of the pancreas using pulses of a given frequency or in a constant mode, using an infrared (IR) laser or focused ultrasound.

Brief description of drawings

The invention is illustrated by figures, which show:

Fig. 1. Map of the genetic construct based on the AAV plasmid containing the β-cell-specific Ins2 promoter, as well as the sequence of the human thermally activated TRPV1 channel with the FLAG-tag signal peptide.

Fig. 2. Results of heating individual islets of Langerhans transduced with the AAV-Ins2-hTRPV1-FLAG construct. Heating is carried out in the mode cycle 1 (20 minutes 37C, (1 minute 43C, 1 minute 37C) x10), cycle 2 (20 minutes 37C, (10 seconds 43C, 50 seconds 37C) x20.

Carrying out the invention

In general, the method of controlling insulin secretion by β-cells of the islets of Langerhans of the pancreas using thermogenetics approaches includes the following steps:

- creation of a genetic construct by cloning into a commercially available vector a β-cell specific promoter, a thermally activated channel gene, as well as the FLAG signal peptide sequence necessary for identifying TRPV1 using immunohistochemistry;

- development of the resulting genetic construct using the transformation of E. coli bacteria with their subsequent expansion and isolation of plasmid DNA;

- generation of a viral vector suitable for delivery of the genetic construct into β-cells;

- delivery of the genetic construct through transduction of β-cells of the islets of Langerhans;

- non-contact thermal impact on the isolated islets of Langerhans using an IR laser or high-intensity focused ultrasound with control of insulin concentration.

Examples

Example 1. Control of insulin secretion by β-cells of the islets of Langerhans using thermogenetics.

Creation and development of a genetic construct, generation of a viral vector

To create genetic constructs, we used the AQUA cloning method (Beyer et al., 2015). The basis for cloning was the AAV plasmid, into which the β-cell-specific Ins2 promoter, the sequence of the human heat-activated channel TRPV1, and the sequence of the FLAG signal peptide necessary for identifying TRPV1 using immunohistochemistry were cloned. A map of the resulting AAV-Ins2-hTRPV1-FLAG construct is shown in Fig. 1. Adeno-associated viruses of the PHP.S serotype carrying the AAV-Ins2-hTRPV1-FLAG construct were generated using a commercially available service.

Isolation and transduction of islets of Langerhans

Isolations of the islets of Langerhans were performed in mice aged 2 to 3 months using a modified version of the method published by Stull et al [Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG. Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease. J Vis Exp. 2012 Sep 7; (67):4137. doi: 10.3791/4137. PMID: 22987198; PMCID: PMC3490250].

Isolation was carried out according to the following protocol:

In the first stage, the mouse was euthanized by placing it in a chamber with a high carbon dioxide content. To gain access to the pancreas, a V-shaped incision in the skin and abdominal wall was made under an operating microscope. To avoid excessive blood entering the removed pancreas, an incision was made in the inferior vena cava and blood was collected. Next, the common hepatic duct was closed with a clamp. In the area where the pancreatic duct flows into the duodenum, under the duct, the connective tissues were ruptured using tweezers and a thread was threaded into the resulting hole and a loop was made. Using small, very sharp scissors, the junction of the pancreatic duct and the duodenum was cut. A 31G needle was inserted into the resulting hole 0.5-1 cm and the loop was tightened. A tube with a syringe filled with a cold solution of collagenase XI (Sigma, C7657, USA) with a volume of 2 ml and a concentration of 1.5 mg/ml was attached to the needle. Next, the pancreas was removed and placed in 4 ml of collagenase solution, incubated in a water bath at +37C for 15 minutes. After incubation, Krebs-Ringer bicarbonate solution supplemented with HEPES (KRBH) with 0.3% bovine serum albumin (BSA) was added to the pancreas and vigorously shaken 15 times to dissociate the pancreas. The resulting suspension was centrifuged for 1 minute at 400 G, the supernatant was drained, and 20 ml of KRBH with 0.3% BSA was added to the sediment. The sediment was intensively broken up two or three times by pipetting with a serological pipette. Then the cell suspension was passed through a mesh with a mesh size of 0.5 mm, centrifuged for 1 minute at 400 G. Next, separation was carried out using a density gradient, the resulting sediment was resuspended in 10 ml of a solution for isolating lymphocytes with a density of 1.077 g/ml (Capricorn scientific, Germany) . KRBH was layered on top and centrifuged for 20 minutes at 900 G. Langerhans islets were collected at the solution interface using a serological pipette and passed through a 40-μm filter. The islets of Langerhans were washed using RPMI 1640 medium with the addition of an antibiotic, glutamine and 10% fetal bovine serum in a petri dish with a diameter of 60 mm. At the final stage, islets of Langerhans with normal physiology were selected manually and transferred 25-30 pieces into one well of a 12-well plate without surface coating for further cultivation.

Transduction with an adeno-associated virus at a concentration of 5.2*10^13 viral genomes/ml, 1.5-2 µl of a virus solution per 0.5 ml of medium and 25-30 islets of Langerhans carrying the AAV-Ins2-hTRPV1-FLAG construct was carried out immediately after selection. Isolated islets were cultured at 37°C, 5% CO2 for 3-5 days.

Non-contact thermal effect on β-cells of individual islets of Langerhans using heat.

To control insulin secretion by β-cells, transduced islets of Langerhans with a virus carrying the AAV-Ins2-hTRPV1-FLAG construct were transferred to PCR tubes for heating in a PCR machine. Heating is carried out in cycle 1 (20 minutes 37C, (1 minute 43C, 1 minute 37C) x10), cycle 2 (20 minutes 37C, (10 seconds 43C, 50 seconds 37C) x20. Measurements of the concentration of secreted insulin were carried out using an ELISA kit (Mercodia, Sweden).

The experiment demonstrated that heating individual islets of Langerhans leads to an increase in insulin secretion by at least 1.8 times.

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Claims (3)

1. A method of increasing insulin secretion by β-cells of the islets of Langerhans of the pancreas using thermogenetics, characterized by the fact that a genetic construct is delivered to the β-cells containing a promoter specific to them and the gene for the human thermally activated channel TRPV1 (transient receptor potential vanilloid 1 - vanilloid receptor ), followed by heating of β-cells in the PCR machine, which is carried out in cycle 1 mode: the temperature is maintained at 37°C for 20 minutes, followed by 10 heating-cooling cycles: 1 minute at 43°C and 1 minute at 37 °C; cycle 2: the temperature is maintained at 37°C for 20 minutes, followed by 20 heating-cooling cycles: 10 seconds at 43°C and 50 seconds at 37°C. 2. The method according to claim 1, characterized by the use of vectors based on adeno-associated viruses as a method for delivering genetic constructs. 3. The method according to claim 1, characterized by the use of a thermal cycler for periodic heating of the islets of Langerhans.