Advancements in Gene Therapy for Type 2 Diabetes: Insights from CRISPR Cas9 Mediated Gene Editing and Insulin Production

Abstract

This literature presents a comprehensive overview of the application of CRISPR-based gene editing technology in the treatment of diabetes mellitus (DM). The introduction highlights the significance of DM as one of the oldest human diseases and the need for effective control to prevent potential consequences. It emphasizes the limitations of conventional medications for hyperglycemia and the challenges in achieving optimal glucose concentrations and minimizing long-term consequences. The abstract then delves into the advancements in CRISPR technology, discussing various delivery methods for the CRISPR-Cas complex, including non-viral vectors, viral vectors, and nanocarriers. The use of CRISPR-Cpf1 as an alternative to Cas9 is explored, highlighting its advantages and functionality. The abstract further explores the potential of CRISPR gene therapy and nanocarriers in treating DM, particularly targeting the NLRP3 inflammasome and downregulating the DPP-4 enzyme. Liposomal particles and lecithin nano-liposomal particles are discussed as efficient delivery systems for CRISPR/Cas9, with potential applications in T2DM treatment. The role of islet amyloid polypeptide (IAPP) in T2DM and its study using CRISPR Cas9-based gene editing technology is also presented. Overall, this abstract provides a comprehensive overview of the current advancements and potential applications of CRISPR technology in the treatment of DM.

1. Introduction

According to some sources, diabetes mellitus (DM) is among the oldest human diseases. Diabetes mellitus (DM) is a metabolic condition that is frequently characterized by elevated blood glucose levels that necessitate frequent monitoring and effective control. The hormone insulin is produced by pancreatic beta cells (−cells), which also play a number of other roles in the body. Insulin helps the body’s cells absorb glucose for energy (Figure 1). Diabetes mellitus (DM) is brought on by a deficiency in insulin synthesis or sensitivity. It is primarily divided into numerous forms, however type 1 and type 2 DM are the most prevalent types. Type 1 diabetes (T1DM) is characterized by a failure in the pancreatic -cells to produce insulin as a result of T-cell-mediated autoimmunity [1]. Contrarily, type 2 diabetes (T2DM) is characterized by reduced insulin production and insulin resistance. Due to the considerably higher incidence of cardiovascular illnesses and acute metabolic abnormalities in the former group, T1DM was reported to have a shorter life expectancy than T2DM [2]. In order to prevent or delay the development of potential consequences involving other organs such as diabetic nephropathy, retinopathy, neuropathy, cardiovascular illnesses, and diabetic foot ulcers, it is crucial that all kinds of diabetes be recognized and handled at an early stage (Figure 2a and b) [3, 4, 5].

Figure 1.

Insulin receptor gets activated by the binding of ligand, this triggers the islets of Langerhans cells, activating the beta cells (one among four cells types in islets of Langerhans), these produce the hormone insulin and secret them into blood stream. Insulin regulates the activity i.e. the uptake of glucose by the different body organs, tissues and muscles by the action of GLUT4 factor. Any alteration or inhibition in this pathway of glucose production would lead to lack of/or no production of insulin, thus leading to a state of insulin deficiency and further leading to diabetes. If diabetes I s caused by auto immune disfunctioning then its termed as type 1 diabetes and if its caused as a result of insufficient insulin production then it is termed as type 2 diabetes or insulin dependent diabetes.

Figure 2.

(a) First image shows a healthy body condition in which pancreatic cells produce enough insulin and thus helping in the adequate glucose intake by organ and other body tissues, this also regulates the blood glucose level, keeping them at normal level. The image shows the glucose being taken up by the organs and also being stored up for further usage, and because of this we see adequate amount of glucose in blood flow and normal blood glucose levels in the body; (b) This figure represents the lack of insulin production and also imbalanced uptake of glucose by the body tissues and organs thus leading to higher concentration of glucose in blood and this condition is referred to as insulin dependent diabetes mellitus. In this the glucose is not adequately absorbed by the cells and organs thus leading to high glucose inflow in blood, eventually leading to increased blood glucose level and also a pathogenic state leading to type II DM.

This metabolic condition develops into chronic, life-threatening microvascular, macrovascular, and neuropathic consequences over time. Diabetes mellitus (DM) is brought on by a lack of insulin production, pancreatic cell injury, or insulin resistance brought on by inadequate insulin use. The trend toward sedentary living may be the main cause of the rising number of diabetic patients worldwide, which is predicted to reach 366 million in the older population (>65 years) by 2030 [6]. Nephropathy, neuropathy, cardiovascular and renal issues, retinopathy, food-related diseases, and more are among the many consequences linked to DM.

Type 2 diabetes (T2D) is a prevalent metabolic disorder characterized by insulin resistance and impaired glucose metabolism. Traditional treatments for T2D have limitations in achieving optimal glucose control and minimizing long-term complications. In recent years, there have been significant advancements in gene therapy that utilize CRISPR-Cas9-mediated gene editing and insulin production, offering potential solutions for the challenges in T2D treatment. The CRISPR-Cas9 technology allows precise modification of specific genes associated with T2D pathogenesis. To ensure effective delivery of the CRISPR-Cas9 complex to target tissues and cells, researchers have explored different methods such as non-viral vectors, viral vectors, and nanocarriers. Moreover, the use of liposomal particles and lecithin nano-liposomal particles as delivery systems enhances the stability and effectiveness of CRISPR-based therapies. These scientific developments hold significant promise in advancing the field of gene therapy for T2D, opening new avenues for personalized and efficacious treatment approaches.

Sulfonylureas, biguiands, peroxisome proliferator activated receptor-, agonists (boosts the action of insulin), and -glucosidase inhibitors (interferes with absorption of glucose in the stomach) are the main conventional types of medications for treating hyperglycemia [7]. These pharmacological types are either used alone or in conjunction with other hypoglycemic medications. The main drawbacks of using the aforementioned conventional drugs include severe hypoglycemia, weight gain, lower therapeutic efficacy due to improper or ineffective dosage regimen, low potency and altered side effects due to drug metabolism and lack of target specificity, solubility and permeability issues [8]. Despite the development of potential anti-hyperglycemic drugs, refining the current therapies to provide optimal and balanced glucose concentrations and lowering long-term consequences from diabetes are the main obstacles to effective diabetes therapy [9]. Type 2 diabetes mellitus (T2DM) is one of the most common metabolic illnesses due to the growth in sedentary behaviors, obesity, and genetic predisposition.

90% of cases of diabetes globally are of type 2, making it the most prevalent type. T2DM is characterized by insulin resistance in peripheral tissues, insulin insufficiency, and poor glucose homeostasis. These factors combine to start a debilitating process that then encourages -cell dysfunction, which further raises morbidity and mortality while decreasing the effectiveness of treatment. Sulphonyurea, meglitinide, biguanide, thiazolidinediones (TZD), and -glucosidase inhibitors are antidiabetic medications. Unfortunately, these therapy approaches are typically limited by gastrointestinal discomfort, weight gain, safety, and tolerability, even when used in combination. The rapid development of genome editing in recent years has boosted human genome research and given researchers a better understanding of the role of single-gene products in the regulation of various disorders [10].

2. CRISPR

With several applications in both scientific research and clinical trials, CRISPR has emerged as the most advantageous and effective genome editing technology [11]. There is still potential for improvement even though the use of CRISPR technology for DNA editing has advanced dramatically in recent years. It is challenging and time-consuming to deliver the CRISPR-Cas complex to particular tissues or cells because its components must be delivered into the nucleus for their effect on the nuclear genome to overcome tissue and cell membrane barriers [12]. For the delivery of CRISPR-Cas9, non-viral vectors, viral vectors, and physical delivery are frequently used [13, 14]. Electroporation and single-cell microinjection, which are frequently used in embryonic gene editing and the creation of transgenic animals, are the most widely used physical techniques for introducing the CRISPR-Cas complex into cells [15]. Transfer of the Cas DNA or protein components has been done well, with minimal to no cytotoxicity. Microinjection is a laborious and drawn-out procedure that can only be used to deliver the CRISPR-Cas complex to a small number of species [16]. Because they are efficient and effective, particularly for in vivo investigations, viral vectors are frequently utilized as CRISPR-Cas 9 delivery vectors. Given their better cellular uptake and editing potency, viral vectors are currently thought to be the undisputed masters of in vivo CRISPR delivery. Examples of viral delivery vectors include full-sized adenoviruses, lentiviruses, and genetically altered adeno-associated viruses (AAVs). Adeno-associated viral vectors (AAVVs), which have low immunogenicity, cytotoxicity, and limited integration into the host cell, have become the dominant in vivo delivery method for CRISPR components [17]. The lentiviral vector (LV) is another viral vehicle for delivering CRISPR components. It is more effective at cloning than the AAV vector. It can package two copies of an RNA genome (about 10 kilobases), making it a platform for delivering the most common CRISPR/Cas protein (Cas 9) and sgRNA cassette in a single viral transfection event [18]. Due to insertional mutagenesis and the ongoing generation of site-specific nucleases, both of which can result in off-target mutations, LVs present a safe method in therapeutic applications [19]. Scientists have recently created non-integrating lentivirus vectors (NILVs) to deliver CRISPR components in an effort to reduce the risk of integration through either a change in the viral integrase gene or a change in the attachment sequence of lengthy terminal repeats (LTRs) [20]. It is hard to suggest a single viral vector as the best method of delivering CRISPR components because each viral vector has a unique set of advantages and disadvantages. Despite the fact that viral vectors have a high in vivo transfection efficiency, there are substantial problems with their clinical application, such as immunogenicity, integration, and off-target effects. New methods are continually being developed to address these challenges.

Recent studies have shown that using nanocarriers to carry the CRISPR-Cas complex’s genes has specific advantages [21]. demonstrates the CRISPR-Cas system’s timeframe for use in medicines. The CRISPR-Cas complex has been generated and delivered via a variety of nano-delivery strategies, including cationic liposomes, lipid nanoparticles (LNPs), cationic polymers, vesicles, and gold nanoparticles [14]. Non-viral vectors have emerged as a promising technology in preclinical investigations for the delivery of CRISPR-Cas 9 systems as unique vehicles for extending the application of this technology, hence triggering strong gene-editing in the life sciences and therapeutic settings [22]. As an illustration, cationic arginine gold nanoparticles to transport the Cas 9 protein for causing tumor regression in CC cells by targeting the human AAVS1 gene [23]. Black phosphorus nanosheets (BPs) with Cas 9-RNPs targeting EGFP via cytosolic delivery in a mouse model are used as a biodegradable 2-D delivery platform to elicit tumor regression (Table 1) [30].

Types of species	In vitro/in vivo	Method of gene delivery	Target gene	Genetic alteration	Types of disease created	Reference
Pig	In vitro	Somatic cell nuclear tRansfer	HTT	Knock in	Huntington	Yan et al. [24]
Mouse	In vivo	Stereotactic injection	Ptch1	Deletion	Medulloblastoma and glioblastoma	Zuckermann et al. [25]
New born mice	In vivo	Intravenous infection	OTC	Gene correction	Metabolic liver disease	Yang et al. [26]
Rat	In vivo	Subretinal injection	Rho	INDEL mutation	Retinal dystrophy	Bakondi et al. [27]
Zebra fish	In vitro	Microinjection	Abcc9,kcnj8 or pln	INDEL mutation	Cardiovascular disease	Tessadori et al. [28]
Human	In vitro	Adenoviral transduction	SMAD3	Gene inactivation	Fibrosis	Voets et al. [29]

Table 1.

Cellular and animal model of human diseases generated by CRISPR/Cas9.

3. CRISPR in diabetes mellitus

There are 463 million instances of diabetes mellitus (DM), a chronic endocrine and metabolic condition with a significant mortality rate, globally [31]. Patients with DM have been treated with insulin, insulin analogs, and non-insulin oral hypoglycemic medications. However, due to inherent pharmacological deficits and restrictions on delivery methods (subcutaneous administration or oral distribution), which led to enzymatic hydrolysis, chemical instability, and subpar gastrointestinal absorption, they were rendered ineffective [32]. A promising therapeutic approach for treating DM is CRISPR gene therapy and nanocarriers, which carry CRISPR to the target locations utilizing liposomes, polymer-based nanoparticles, and inorganic nanoparticles. The CRIPSR can be protected from an enzymatic breakdown in the stomach chambers, improved in vivo stability, and improved bioavailability using nano-carriers. The use of a nanocarrier and the CRISPR-Cas complex reduces the risk of hypoglycemia and boosts patient compliance while replicating endogenous insulin delivery through external stimulation. This treatment can be more focused on specific areas and released gradually over an extended period of time, avoiding adverse effects and maximizing therapeutic effect for the treatment of diabetes [33].

4. Treatment of T2DM by lecithin nano-liposomal particle as a CRISPR/Cas9 delivery system

Liposomal particles are great candidate materials given their simple preparation method, easy surface modification, and high biocompatibility. Skyler [34] have recently reported a bio-reducible lipid nanocarrier complex for protein-based Cas9 genome editing. It was produced through electrostatic interaction of cationic lipids and super-negatively charged complexes via protein–protein fusion. Type 2 diabetes mellitus (T2DM) was used as a target disease given its suitability for genetic therapy concerning liver. T2DM is a complex disease characterized by high glucose levels in the bloodstream, reduced glucose processing capacity in adipocytes, and insulin resistance in the body [35]. Elevating glucagon-like peptide-1 (GLP-1), an important target hormone that stimulates insulin secretion, is one of recent therapeutic approaches [36]. However, this hormone has a short half-life due to its extremely rapid degradation by enzyme dipeptidyl peptidase-4 (DPP-4) [37]. To prevent GLP-1 degradation, various drugs inhibiting DPP-4 such as sitagliptin, vildagliptin, saxagliptin, and linagliptin have been developed for insulin-mediated glucose control of T2DM [38]. Moreover, since DPP-4 inhibitors are widely used in clinical practice, they are also investigated as potential new therapeutics against the development of hepatic fibrosis and steatosis [39]. However, small-molecule antidiabetic drugs must be administered daily. In addition, they are associated with adverse effects such as hepatic impairment. T2DM could be treated using a low-risk therapeutic approach such a CRISPR/Cas9-based approach that can efficiently downregulate the DPP-4 enzyme. Cas9-RNP, a ribonucleoprotein made of recombinant Cas9 nuclease complexes and a sgRNA, is intended to modify the DPP-4 gene. A lecithin-based liposomal nanocarrier particle (NL) was created to convey the Cas9-RNP complex. A cationic polymer was included with the Cas9-RNP complex to make up for the negatively charged lipid structure of the NL and boost encapsulation effectiveness. This is so because electrostatic interactions are a major determinant of loading efficiency [40]. Due to the liver’s natural metabolism of lecithin, NL are also ideal for addressing liver illnesses from the perspective of biodistribution.

The use of a positively charged polymer improved loading efficiency, which improved Cas9-RNP complex encapsulation. Negatively charged lipids and charge-compensated complexes spontaneously interacted electrostatically, causing NL spheres with a uniform size distribution to self-assemble. The genome platform is ideal for treating genetic and chronic human diseases because it has excellent biocompatibility, low cytotoxicity, and high solution stability, in contrast to unprotected protein therapy techniques that have low delivery efficacy due to enzymatic degradation.

According to the findings, the NL@Cas9-RNP system has a number of benefits, including very effective Cas9-RNP complex encapsulation, efficient and stable in vivo administration, and efficient treatment for liver disease. It is necessary to conduct additional research to characterize and improve the pharmacokinetics, effectiveness, and safety of this DPP-4 gene editing method in animals.

5. CRISPR based new tool Cpf1

Recently, a dual-RNA construct known as single guide RNA (sgRNA), which consists of the two components of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), has been designed by genetic engineering in line with the CRISPR/Cas9 system type II and to ease the usage of this system. When Cas9 binds to tracrRNA, crRNA directs it to the target sequence [41]. This approach relies on RNA rather than protein to assess target specificity, making the technology far more straightforward and accessible. Furthermore, CRISPR/Cas9 is one of the best nuclease-mediated genome editing approaches due to its benefits over meganucleases, ZFNs, and TALENs, including the simplicity of target design, efficiency, and numerous mutations. The Cpf1 family proteins are a group of Cas9 orthologues that were found [42]. These proteins’ HNH endonuclease domains are structurally distinct from Cas9’s [43]. Technically, the discovery of Cpf1 provided a more straightforward and limited alternative to the CRISPR toolbox by opening up the prospect of more precise genome editing. In terms of its functionality, it belongs to the class of class 2 CRISPR systems. The CRISPR system uses Cpf1 in a different way than Cas9. One RNA molecule would be sufficient to continue the process because, in this case, Cpf1 leaves behind a sticky end rather than a flat one following cleavage (Figure 3). Therefore, genes of interest can be consciously put into vectors utilizing CRISPR/Cpf1 technology. Because of this, Cpf1 is more effective than Cas9 [44]. Cpf1 circumvents HDR’s efficiency restriction for genome editing in non-diverging cells [45]. The CRISPR/Cas9 system can only target sites with PAMs with NGG sequences, which restricts gene editing at target sites with G-rich sequences. Cpf1 fixes this issue in the interim by identifying the T-rich target locations. The other benefit of Cpf1 over Cas9 is the ability to target a greater variety of places in the genome thanks to the addition of this option to the gene editing toolbox. Researchers recently created Cpf1 proteins that include uridine-rich 3′ ends in addition to a complementary 20-bp target site to increase the efficacy of Cpf1 in inducing INDEL mutations in target locations [46].

Figure 3.

The CRISPR/Cpf1 technology’s molecular processes. Although it differs from CRISPR/Cas9 technically, CRISPR/Cpf1 technology expands the toolkit thanks to its enzyme. Component. A class 2 nuclease, the Cpf1 enzyme exclusively recognizes the target site using one strand of RNA. Based on the location of a T-rich PAM with a TTTN sequence at the 5′ end of crRNA, this enzyme can identify the target sequence. As can be observed, one strand is exactly split at 19 base pairs (bp) after the PAM sequence, and the opposing strand is cleaved at 23 base pairs (bp). When compared to CRISPR/Cas9, the CRISPR/Cpf1 sticky ends boost the specificity and functional efficiency.

6. CRISPR Cas9 treatment using CLAN nano-particle-

Although the NLRP3 inflammasome is a well-researched target for the therapy of a number of auto-inflammatory illnesses, including diabetes, there is still much to be done. Using a cationic lipid-Nano-carrier to deliver CRISPR-Cas9 into macrophages for the downregulation of NLRP3 via CLAN encapsulated mCas9 and gRNA-targeting NLRP3 in macrophages is a potential method for treating NLRP3-dependent DM. This increases insulin vulnerability and decreases inflammation in the adipose tissue of type 2 diabetes caused by high-fat diets (HFDs) by inhibiting the activation of the NLRP3 inflammasome to minimize acute inflammation post-intravenous injection [47].

Another study used a library of cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN) with varying polymer compositions (PEG5K-b-PLGA11K, PLGA8K, and cationic lipid BHEM-Chol), surface density, and surface charges to deliver the CRISPR-Cas9 system to neutrophils in the epididymal white adipose tissue and liver [47]. Similar to this, macrophage-specific CRISPR-Cas9 plasmids targeting Ntn1 gene encased in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN) via intravenous injection have been shown to decrease Ntn1 gene expression and improve Type 2 diabetes (T2D) symptoms both in vitro and in vivo [48]. Based on the aforementioned research, it can be deduced that the precision delivery of the CRISPR-Cas9 system to the Ntn1, elastase, NLRP3, and DPP4 genes to produce Type 2 DM regression is aided by cationic and lipid nanoparticles. To learn more about their molecular process, however, additional research in the animal model is required.

7. Islet amyloid polypeptide (IAPP) and T2DM

Islet amyloid polypeptide (IAPP, amylin), a polypeptide hormone of [49] amino acid residues that is released by islet beta cells [50], was identified, isolated, and given the name from islet tumor cells. Under the influence of glucose and other secretagogues, it is mostly deposited in the halo of the secretory granule and secreted in pulses [51]. IAPP creates oligomers that lead to beta cell death and so promote islet amyloidosis. In short, IAPP produces oligomers that lead to beta cell death and promote islet amyloidosis, which in turn cause insulin secretion to gradually fail [52]. The main function of IAPP in the process of glucose metabolism is to block the manufacture of glycogen, the transit of glucose, the uptake of glucose by muscle tissue, and the use of glucose by hepatocytes [53]. The single subunit state of the soluble IAPP protein is unfolded. In order to more accurately control human blood sugar, it can work in synergy with hormones that control blood glucose, such as insulin [54]. The mature human IAPP protein (hIAPP) can form amyloid aggregates and has a strong propensity to misfold. Of the 20 amyloid aggregated peptides that have been found so far, hIAPP is one of the most heavily aggregated polypeptides [55]. According to several investigations, islet amyloid deposition was discovered in the islets of people with type 2 diabetes. Amyloidosis of the hIAPP is seen as a potentially.

Since pigs are biologically the most similar to humans than any other species, they were chosen to serve as the model for this HiAPP protein study. This protein’s expression and analysis were studied to determine its impact on T2DM using a CRISPR Cas9-based gene editing technology.

8. Conclusion

In conclusion, this literature provides a comprehensive overview of the advancements in gene therapy for type 2 diabetes using CRISPR-Cas9-mediated gene editing and insulin production. The introduction highlights the significance of diabetes mellitus (DM) as a chronic metabolic condition and the limitations of conventional medications in achieving optimal glucose control and minimizing long-term consequences. The abstract delves into the advancements in CRISPR technology, discussing various delivery methods for the CRISPR-Cas complex, including non-viral vectors, viral vectors, and nanocarriers. It also explores the potential of CRISPR gene therapy and nanocarriers in treating DM, targeting specific factors such as the NLRP3 inflammasome and the DPP-4 enzyme. Liposomal particles and lecithin nano-liposomal particles are discussed as efficient delivery systems for CRISPR/Cas9, with potential applications in treating type 2 diabetes. The role of islet amyloid polypeptide (IAPP) in type 2 diabetes and its study using CRISPR-Cas9-based gene editing technology is also presented. The treatment of type 2 diabetes using CRISPR technology holds great promise for overcoming the limitations of current therapies. The use of CRISPR-Cas9 and nanocarriers provides advantages such as improved stability, targeted delivery, and prolonged release of therapeutic agents. Non-viral vectors, viral vectors, and physical delivery methods have been explored for delivering the CRISPR-Cas complex to specific tissues or cells. Each method has its advantages and limitations, and ongoing research aims to address the challenges associated with clinical applications. The application of CRISPR technology in type 2 diabetes involves targeting specific genes or factors involved in the disease process. The downregulation of the NLRP3 inflammasome and DPP-4 enzyme using CRISPR gene therapy and nanocarriers shows promise in improving insulin sensitivity, reducing inflammation, and enhancing glucose control. The use of liposomal particles and lecithin nano-liposomal particles as CRISPR/Cas9 delivery systems has demonstrated efficient encapsulation and targeted delivery to the liver, a relevant organ in type 2 diabetes management. Furthermore, the study of islet amyloid polypeptide (IAPP) using CRISPR-Cas9-based gene editing technology provides insights into the role of this peptide in glucose metabolism and its potential contribution to beta cell dysfunction. By understanding the mechanisms underlying IAPP aggregation and its impact on insulin secretion, researchers can develop innovative strategies for managing type 2 diabetes. Overall, the advancements in CRISPR technology offer exciting possibilities for the treatment of type 2 diabetes. The use of gene editing and nano-carriers can improve the effectiveness and specificity of therapeutic interventions, potentially leading to better glucose control, reduced complications, and improved quality of life for individuals with type 2 diabetes. Further research and development in this field are needed to optimize delivery methods, ensure safety and efficacy, and translate these advancements into clinical practice. The discovery and development of novel therapies, including those derived from natural products, in combination with CRISPR technology, hold great promise for the future of diabetes treatment.