Adeno-associated virus (AAV) vectors have become indispensable tools in gene therapy, owing to their safety, tissue specificity, and ability to enable long-term transgene expression. By tailoring serotypes, promoters, and injection strategies, AAV can target specific organs to treat diseases effectively. This article explores the applications of AAV in skeletal muscle, liver, and pancreas gene therapy, highlighting key methodologies and case studies.

 

Key words：Adeno-associated virus (AAV)，gene therapy，serotypes，promoter

 

  1. AAV Applications in Muscle Gene Therapy

 

1.1 Importance of Skeletal Muscle Targeting

 

Skeletal muscle is one of the largest tissues in the human body, constituting 40–50% of total body mass. This tissue plays a vital role in locomotion and metabolism, making it a key target for treating disorders such as Duchenne muscular dystrophy (DMD) and metabolic syndromes.

 

1.2 AAV Serotypes for Skeletal Muscle

 

AAV serotypes 1, 6, 8, and 9 have demonstrated high transduction efficiency in skeletal muscle. Among them, AAV9 is particularly effective due to its ability to transduce both skeletal and cardiac muscle when delivered systemically.

 

1.3 Delivery Methods

 

Two primary delivery methods are commonly employed for muscle-targeted AAV delivery:

  1. Intramuscular Injection: Provides localized expression but is limited in spread, often necessitating multiple injection sites for broader coverage.
  2. Systemic Administration: Through intravenous routes such as tail vein injection, vectors distribute widely, enabling systemic transduction. However, non-muscle tissues may also absorb a significant portion of the vector.

 

1.4 Case Studies

  • Case 1: Duchenne Muscular Dystrophy (DMD) Gene Editing

Using AAV9, researchers delivered CRISPR-Cas9 and guide RNAs targeting exon 23 of the DMD gene in dystrophic mice.

  • Promoter: CAG.
  • Protocol: Tibialis anterior muscle injection; dose of 7.5 × 10¹¹ vg/mouse.
  • Results: Exon skipping successfully restored dystrophin expression, leading to significant improvement in muscle structure and function.

 

Figure 1. Comparison of AAV Transduction Efficiency in Muscle Tissue of C57BL/6 Mice

 

  • Case 2: Enhanced Muscle Targeting with Capsid Optimization

MyoAAV1A, a capsid variant engineered to enhance muscle tropism, was compared with standard AAV9.

  • Protocol: Intravenous injection; dose of 1.0 × 10¹² vg/mouse.
  • Results: MyoAAV1A demonstrated superior transduction efficiency in both skeletal and cardiac muscle, achieving higher gene expression at a lower dose.

 

Figure 2. MyoAAV1A Efficiently Transduces Mouse Skeletal Muscle

 

  1. AAV Applications in Liver Gene Therapy

 

2.1 Why Target the Liver?

 

The liver is a central hub for metabolic regulation and protein synthesis, making it a primary target for addressing diseases like hemophilia, hypercholesterolemia, and liver fibrosis.

 

2.2 AAV Serotypes for Liver Targeting

 

AAV8 is the preferred serotype for liver-targeted gene therapy, exhibiting transduction efficiency 10 to 100 times higher than AAV7, AAV9, and other serotypes. Its ability to cross the liver’s endothelial barrier makes it uniquely suited for hepatocyte targeting.

 

2.3 Delivery Methods

 

  1. Tail Vein Injection: Common in small animal models, providing efficient and widespread hepatic transduction.
  2. Portal Vein Injection: Directly delivers vectors to the liver, reducing off-target effects and increasing localized expression.

 

2.4 Case Studies in Liver Gene Therapy

 

  • Case 1: CRISPR-Mediated Gene Editing for Hypercholesterolemia

In this study, AAV8 was used to deliver the SaCas9 nuclease and guide RNAs targeting PCSK9, a gene associated with cholesterol metabolism. The goal was to reduce serum cholesterol levels, a crucial aspect in treating cardiovascular diseases.

  • Vector: AAV8-TBG-SaCas9.
  • Promoter: TBG (liver-specific).
  • Injection Protocol: Tail vein injection at a dose of 1.0 × 10¹¹ vg/mouse.
  • Results: The treatment led to a 95% decrease in serum PCSK9levels, accompanied by a 40% reduction in total cholesterol. This reduction was sustained for over four weeks, demonstrating the efficacy of AAV-mediated CRISPR-based gene editing in the liver.

 

Figure 3: AAV-Mediated SaCas9 for Liver Genome Editing

 

  • Case 2: Let-7 Therapy for Liver Fibrosis

Let-7, a microRNA with a known anti-fibrotic role, was delivered via AAV2/8 to treat liver fibrosis induced by CCl4 in a mouse model.

  • Vector: AAV2/8-let-7a.
  • Promoter: CMV.
  • Injection Protocol: Tail vein injection, once a week, with a dose of 2 × 10¹⁰ vg/mouse for 3 weeks.
  • Results: Let-7 expression effectively alleviated liver fibrosis by targeting TGF-β signaling and promoting hepatocyte apoptosis. This demonstrates AAV’s potential to treat fibrotic diseases through gene modulation.

 

Figure 4: Effects of Let-7 on CCl4-Induced Liver Fibrosis Model

 

 

  1. AAV Applications in Pancreas Gene Therapy

 

3.1 Targeting the Pancreas

 

The pancreas is a vital organ responsible for glucose metabolism and the secretion of digestive enzymes. Gene therapies targeting pancreatic β-cells and ductal cells are especially important for the study and treatment of diabetes and pancreatic cancer. AAV’s ability to transduce these cells with high efficiency is key for advancing research in these areas.

 

3.2 AAV Serotypes for Pancreas Targeting

 

Among the various AAV serotypes, AAV8 and AAV9 have shown strong transduction efficiency in both endocrine and exocrine regions of the pancreas. Capsid modifications, such as Y447F+Y733F-AAV8, have further enhanced the specificity and efficiency of transduction in pancreatic tissues.

 

3.3 Promoter Selection for Pancreas Targeting

 

Promoter selection is crucial for achieving tissue-specific expression in the pancreas. Different promoters can direct gene expression to specific pancreatic cells:

 

Promoter Names Infection Characteristics
Sox9   Duct cell-specific promoter Ductal Cells
RIP/mlP Insulin promoter Islet β-Cells
CMV Cytomegalovirus promoter Broad Infection

Table 1: Promoter Selection for AAV Pancreatic Infection

 

3.4 Delivery Methods for Pancreas Gene Therapy

 

  1. Bile Duct Injection: This approach provides targeted delivery directly into the pancreas, enabling efficient infection of both the exocrine and endocrine cells.
  2. Intraperitoneal Injection: A more convenient method, though it lacks the same level of tissue specificity as bile duct injection.

 

3.5 Case Studies in Pancreas Gene Therapy

 

  • Case 1: β-Cell-Specific Gene Expression Using AAV8

AAV8 was used to deliver the mIP (mouse insulin promoter)-driven GFP expression construct to target pancreatic β-cells in diabetic mice. The goal was to achieve specific expression in β-cells to facilitate diabetes research.

  • Promoter: mIP (mouse insulin promoter).
  • Injection Protocol: Intraperitoneal injection; dose of 4.0 × 10¹² vg/mouse.
  • Results: The GFP expression was restricted to β-cells, confirming the high specificity of the mIP promoter. This approach enables precise investigation of β-cell function and insulin production in the context of diabetes.

 

Figure 5: Enhanced Green Fluorescent Protein (eGFP) Expression in Pancreatic Islets

  • Case 2: Enhanced Pancreatic Transduction Using Capsid-Optimized AAV8

In this case, a capsid-optimized variant of AAV8 (Y447F+Y733F-AAV8) was tested for its ability to enhance transduction efficiency in the pancreas.

  • Promoter: CBA (chicken β-actin promoter).
  • Injection Protocol: Intraperitoneal injection, dose range from 1.0 × 10¹¹ to 3.0 × 10¹¹ vg/mouse.
  • Results: The optimized AAV8 vector achieved higher transduction efficiency at a lower dose compared to standard AAV8, demonstrating improved pancreatic gene delivery. This case underlines the importance of capsid modifications for improving vector efficiency in organ-specific gene therapy.

 

Figure 6: Intraperitoneal Injection of Y447F+Y733F-AAV8 Achieves Higher Transduction Efficiency and Gene Expression Compared to Tail Vein Injection

 

AAV-based gene therapies have demonstrated significant potential across a range of organs, including skeletal muscle, liver, and pancreas. By optimizing serotype selection, promoter design, and delivery methods, AAV vectors can target specific tissues with high efficiency and precision. The continued development of novel capsids and therapeutic strategies will pave the way for more effective treatments for genetic and acquired diseases.

 

As research advances, AAV’s versatility in treating a broad spectrum of diseases will undoubtedly expand, bringing gene therapies closer to becoming a standard treatment modality. Brain Case is at the forefront of these innovations, offering cutting-edge solutions in viral packaging and vector optimization. With expertise in engineering AAV vectors tailored for specific organ targeting, Brain Case is playing a pivotal role in advancing the clinical applications of AAV-based gene therapies, helping bring these transformative treatments closer to widespread use.

 

References

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  2. Rehman KK, Trucco M, Wang Z, Xiao X, Robbins PD. AAV8-mediated gene transfer of interleukin-4 to endogenous beta-cells prevents the onset of diabetes in NOD mice. Mol Ther. 2008;16(8):1409-1416.
  3. Chen M, Maeng K, Nawab A, et al. Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors. Hum Gene Ther Methods. 2017;28(1):49-59.