shRNA in Metabolic Disorders-Long-Term Gene Silencing for Chronic Conditions

Introduction of shRNA in Metabolic Disorders

20–25 nt short hairpin RNAs (shRNA) are artificially synthesized and can be used to knock down gene expression. They must be delivered to the nucleus for transcription in an appropriate plasmid or viral vector, where the primary transcript (pri-shRNA) is cleaved by the Drosha complex to form precursor shRNAs (pre-shRNAs). These are exported from the nucleus via Exportin-5 and loaded into the Dicer complex, where the shRNA hairpin loop is cleaved and the resulting double-stranded siRNA product has 2nt 3' overhangs and is loaded into the Ago/RISC complex. Once loaded into the Ago/RISC complex, the action mechanism that leads to cleavage or translation repression. Metabolic disorders associated with obesity, type 2 diabetes, and dyslipemiaid, are related to energy homeostasis and metabolism. shRNA technology may hold the potential to treat metabolic disorders through targeting genes associated with metabolism. Targeting genes for cholesterol and lipid metabolism was shown to be promising in preclinical studies. shRNA targeting PCSK9 showed a potential to lower plasma cholesterol level through inhibition of LDLR degradation in liver. This would potentially be useful for treatment of hypercholesterolemia and prevention of cardiovascular disease.

What are the Metabolic Disorders

Abnormal chemical reactions in the body can produce metabolic disorders by altering body metabolism. It can also be described as a type of inherited single gene anomaly, most of which are autosomal recessive. Some symptoms that could be present with metabolic disorders are lethargy, weight loss, jaundice and seizures. The symptoms that would be expressed would depend on the type of metabolic disorder being expressed. There are four types of symptoms: acute symptoms, late onset of acute symptoms, progressive general symptoms, and permanent symptoms. Obesity, Diabetes Type 2 (T2DM), and Inflammatory Bowel Diseases are the most common metabolic disorders around the world. A defective gene responsible for enzyme deficiency leads to inherited metabolic disorders which contribute to metabolic disease development. The many different types of this disease are referred to as inborn errors of metabolism. The malfunction of liver and pancreatic organs can lead to metabolic diseases.

Design and the Delivery systems for shRNA applications in Metabolic Disorders

Target Sequence Selection

The target sequence must be located in the coding region of the mRNA and have low homology to other genes to reduce off-target effects. It must not be located in regions that fold into complex secondary structures, as this can interfere with binding of the shRNA. GC content should be between 30% and 50%. The stem of the shRNA should be 19-29 nucleotides long, with the target sequence on one strand and the complementary target sequence on the other. The loop connecting the two arms of the hairpin should be 4-10 nucleotides long. Common loop sequences include UCAAGAGA and UUCG, which aid in the folding and processing of the shRNA by Dicer. Include a termination signal such as a polyA tract for expression by RNA polymerase III. RNA Polymerase III Promoters: Commonly used promoters include the human U6 and H1 promoters. Different promoters impact shRNA expression and U6 demonstrates improved performance in specific tissue types. Use bioinformatics tools such as BLAST to find homologous sequences and to avoid regions of high similarity. Avoid sequences with long stretches of identical nucleotides, which can change the structure and function of the shRNA.

Delivery Systems

• Viral Vectors

Lentiviral vectors integrate within the host genome to sustain long-term shRNA expression. They are useful for hard-to-transfect cells and in vivo applications. Adenoviral vectors provide high transduction efficiency and are suitable for transient expression. They are less likely to integrate into the host genome, thus avoiding insertional mutagenesis. Adeno-Associated Virus (AAV) vectors are known for their low immunogenicity and their ability to target specific cell types, making them a good choice for targeted therapy.

• Non-Viral Delivery Methods

Today lipid-based nanoparticles stand out as leading delivery systems in nanomedicine due to simple production processes and safe therapeutic properties combined with strong biocompatibility. Solid lipid nanoparticle (SLN) stands out as an effective delivery vehicle for shRNA macromolecules. A study was conducted on the new approach of treating chronic infection of Hepatitis C virus (HCV). The research used SLNs to deliver shRNA74 to the stem loop II in the 5' UTR to target and suppress the IRES function of the HepG2 cell line which produces viral protein. Solid lipid nanoparticle was also used for the delivery of shRNA-encoding plasmid against 5-α reductase (p5α-Red). In vitro study demonstrated significant reduction in the specific protein level of human prostate cancer cell line (DU-145 cell line). Another study was conducted on in vivo efficacy of lipid nanoparticle (LNP) conjugated with sshRNAs (short synthetic shRNAs) targeting the HCV IRES. ShRNA SG220 was formulated into the LNP and injected in to a mouse, which was expressing HCV IRES-luciferase. The formulation exhibited substantial uptake during in vivo imaging which led to dose-dependent suppression of luciferase expression.

shRNA Applications in Metabolic Disorders

• Diabetes

Diabetes mellitus (DM) is a metabolic disorder of elevated blood glucose and a series of complications. The intravenous injection of NORAD lentivirus shRNA for 4 weeks can reduce the body weight and serum biochemical indexes, improve cardiac function, and reduce the inflammation and fibrosis in DCM mice. NORAD acts as a sponge to absorb miR-125a-3p, and miR-125a-3p binds to Fyn. Intravenous injection of miR-125a-3p adenovirus can improve cardiac function and fibrosis, and reduce the inflammatory response in DCM mice. Co-overexpression of miR-125-3p and Fyn partly restored the beneficial effect of miR-125-3p overexpression on cardiac fibrosis in DCM mice. Knockdown of NLRP3 by shRNA inhibited the activation of NLRP3 inflammasome, reduced the expression of intimal adhesion molecules ICAM-1 and VCAM-1, suppressed atherosclerosis formation and plaque stabilization in diabetic atherosclerosis mouse model.

• Hypercholesterolemia

As an E3 ubiquitin ligase, IDOL could mediate the ubiquitination and degradation of liver LDLR and this would make IDOL a potentially therapeutic target for treating dyslipidemia, hypercholesterolemia, and atherosclerotic cardiovascular disease (ASCVD). We designed pseudotyped recombinant lentivirus IDOL-shRNA with VSV-G envelope mediated by different liver-targeting ligands. CS8-LV-shIDOL exhibited a robust therapeutic effect in reducing serum low-density lipoprotein cholesterol (LDL-C) and atherosclerotic lesions in mice. Moreover, CS8-LV-shIDOL showed better therapeutic effect than wild-type LV-shIDOL in mice, including reducing hepatic lipid accumulation and attenuating liver injury. Specific knockdown of BCHE in hepatocytes by shRNA with targeting BChE reduced LDLR transcription and led to markedly decreased plasma cholesterol and improved hypercholesterolemia. Hyperlipidemia and increased PCSK9 expression were detected in HFD mice. Cerebral histological injury and neuronal apoptosis, as well as PCSK9 and ApoER2 levels increased upon ischemia in hyperlipidemic mice, were attenuated by PCSK9 shRNA. The anti-apoptotic effect of PCSK9 shRNA interference was closely associated with decreased neuronal apoptosis and reduced level of ApoER2 expression in the hippocampus and cortex. In conclusion, the PCSK9 shRNA-mediated anti-apoptotic effect induced by MCAO in hyperlipidemic mice was closely related to ApoER2 downregulation and it might be a novel therapeutic strategy for stroke treatment in hyperlipidemic patients.

Fig. 1 Schematic diagram illustrating the mechanism of liver-targeted lentiviral vector CS8-LV-shIDOL treating atherosclerosis in C57/BL6 mice ^1,6.

• Obesity

Obesity is a metabolic disease. Targeting IL-1β by blocking IL-1 receptor antagonist (IL-1Ra) could improve blood glucose and β-cell function in obese mice. Non-virus mediated IL-1β shRNA interference vectors and non-pathogenic Saccharomyces cerevisiae (S. cerevisiae) were constructed to synthesize oral shRNA/yeast microcapsules. Body weight and fat mass of obese mice were reduced by IL-1β shRNA/yeast treatment. Mice in the IL-1β shRNA/yeast group had a higher average food intake and lower energy conversion than control group. In addition, cytokines associated with lipid metabolism and blood glucose concentration were improved in circulating blood of mice treated with IL-1β shRNA/yeast. Yeast microcapsule-mediated IL-1β shRNA delivery could efficiently alleviate obesity. Notably, this non-dietary weight loss strategy did not need to control diet and showed good biocompatibility, which was very beneficial for obese patients who could not control their diet. Local silencing of ACSL1 by shRNA electroporation in the gastrocnemius muscle of HFD-fed C57BL/6J mice induced upregulation of ACSL1 in non-silenced tissues. Silencing of Acsl1 by shRNA caused decrease in the levels of LCACoA, Cer (C18: 1-Cer and C24:1-Cer) and DAG (C16:0/18:0-DAG, C16:0/18:2-DAG, C18:0/18:0-DAG) in the muscle. Knockdown of Acsl1 enhanced insulin sensitivity and glucose uptake. Silencing Acsl1 through shRNA reduced mitochondrial β-oxidase expression as well as SCA-Car and SCACoA levels in muscle tissue.

Fig. 2 Schematic illustration of anti-obesity therapy by yeast microcapsules mediated oral delivery of shRNA^2,6.

Clinical Status and Future Directions

• Current Status

ShRNA holds great promise as a potential treatment for metabolic diseases, but there are no shRNA-based drugs on the market yet. Several shRNA-based preclinical candidates are currently being investigated. Some of these have shown promising results in animal models and in early clinical trials. One of the most promising candidates is shRNA against the glucagon receptor (GCGR) gene. ShRNA against the GCGR gene has been shown to significantly lower blood glucose levels in an animal model of DM. shRNA against GCGR may be a novel therapeutic for type 2 diabetes(T2DM), increasing insulin sensitivity and maintaining normal glucose levels. ShRNA targets apolipoprotein CIII (apoCIII) has been shown to reduce liver fat content, increase insulin sensitivity and maintain normal glucose homeostasis in an animal model, this strategy may be a novel treatment for NAFLD, which is commonly associated with obesity and T2DM.

• Future Directions

The main drawback of shRNA-based therapies is the effective and targeted delivery of the molecules to the desired tissue of interest. Improvements in delivery systems (i.e. lipid nanoparticles and viral vectors) are promising to improve the delivery of shRNA. However, there is still room for optimization to improve the delivery and reduce off-target effects. Apart from their use in therapy, shRNAs are promising biomarkers for metabolic disorders. ShRNAs have been identified in the circulation in human biofluids, including blood, urine, and serum. shRNAs targeting the metabolic pathways could be used as biomarkers for obesity, type 2 diabetes and dyslipidemia. Researchers will keep developing the delivery system of shRNA to further increase the therapeutic effect and reduce the off-target effect. The possibility of shRNAs as biomarkers for early diagnosis and follow-up of metabolic disorders will also be a new hot spot. As the knowledge of shRNA biology and delivery technology progresses, shRNA-based therapies are expected to play a more important role in the management of metabolic disorders.

References

1. Wang, Wei, et al. "Engineering lentivirus envelope VSV-G for liver targeted delivery of IDOL-shRNA to ameliorate hypercholesterolemia and atherosclerosis." Molecular Therapy-Nucleic Acids 35.1 (2024). https://doi.org/10.1016/j.omtn.2024.102115.
2. Zhang, Li, et al. "Oral gene therapy of HFD-obesity via nonpathogenic yeast microcapsules mediated shRNA delivery." Pharmaceutics 13.10 (2021): 1536. https://doi.org/10.3390/pharmaceutics13101536.
3. Zhao, Yongli, et al. "Knockdown of Tlr4 in the arcuate nucleus improves obesity related metabolic disorders." Scientific reports 7.1 (2017): 7441. https://doi.org/10.1038/s41598-017-07858-6.
4. Lin, Yen-Kuang, et al. "Pterostilbene increases LDL metabolism in HL-1 cardiomyocytes by modulating the PCSK9/HNF1α/SREBP2/LDLR signaling cascade, upregulating epigenetic hsa-miR-335 and hsa-miR-6825, and LDL receptor expression." Antioxidants 10.8 (2021): 1280. https://doi.org/10.3390/antiox10081280.
5. Roszczyc-Owsiejczuk, Kamila, et al. "shRNA-mediated down-regulation of Acsl1 reverses skeletal muscle insulin resistance in obese C57BL6/J mice." Plos one 19.8 (2024): e0307802. https://doi.org/10.1371/journal.pone.0307802.
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