Pancreas Series | Application of AAV in Gene Therapy: Serotype, Promoter Selection, and Injection Strategies

The pancreas is a critical endocrine organ in the body, playing a role in digestion, glucose metabolism, and lipid metabolism. Its dysfunction can lead to diseases such as diabetes and pancreatic cancer. Therefore, identifying virus vectors that can specifically target the pancreas is crucial for metabolic and cancer research. Conducting in vivo genetic engineering on the pancreas, particularly gene transfer to β-cells, could help us study and elucidate the physiological and pathological roles of key genes in the onset and progression of diabetes.

Studies have shown that AAV vectors demonstrate higher transduction efficiency in the pancreas compared to adenoviral and lentiviral vectors. Among single-stranded AAV vectors, serotypes 8 (AAV8) and 9 (AAV9) exhibit strong transduction efficiency in both the exocrine and endocrine regions of the pancreas when administered systemically, intrapancreatically, intraductally, or via pancreatic vasculature.

1. Pancreatic Tropism of Different AAV Serotypes

AAV8 and Y447F+Y733F-AAV8 are currently the most commonly used serotypes for pancreatic infection.

2. Promoter Selection for AAV Pancreatic Infection

When AAV carrying the Sox9 promoter (ductal cell-specific) to mediate Cre recombinase expression is injected into R26R-Tomato reporter mice, ductal cells are specifically and efficiently labeled. When AAV carrying the RIP promoter (insulin promoter) to mediate recombinant SMAD7 expression is injected, SMAD7 is found to be specifically overexpressed in pancreatic β-cells. When AAV carrying the CMV promoter (cytomegalovirus promoter) to mediate GFP expression is injected, GFP expression is observed throughout the entire pancreas. ?

3. Injection Methods for AAV Pancreatic Infection

Currently, there are two main methods for AAV infection of the pancreas:

(1) Injection via the bile duct:

This method involves using microclamps to block the proximal hepatic and duodenal ends of the bile-pancreatic duct. The virus is then delivered into the bile duct using a pump to infect the pancreas.

(2) Intraperitoneal injection:

This method is more convenient. However, it exhibits weaker tissue specificity compared to the bile duct injection method.

4. Example Application

Case 1: Comparison of the efficiency of AAV6, AAV8, and AAV9 in transducing the endocrine pancreas via pancreatic duct injection.

Serotypes: AAV6, AAV8, and AAV9

Promoter: CAG

Experimental Animals: Mice

Injection Protocol:?Pancreatic duct injection, Dose: 3×1010?to 3×1012?vg?per mouse, Expression duration-4 weeks

Experimental Results:?Compared to AAV9, AAV6 and AAV8 transduced more pancreatic cells at all tested doses (3E+10/3E+11/3E+12).?At the highest dose (3.0×10^12 vg/per mouse), transduction was achieved across all islets in the pancreas.

Case 2: AAV8-mediated expression of the interleukin-4 gene in β-cells

Serotype: AAV2/8

Promoter: mIP (mouse insulin promoter)

Experimental Animals: NOD (Non-Obese Diabetic) mice

Injection Protocol:?Intraperitoneal injection, 4.0×1012vg per mouse, Expression duration?- 2 weeks

Experimental Results: The mouse insulin promoter (mIP) in the dsAAV vector demonstrated β-cell specificity, effectively driving AAV-mediated expression of enhanced green fluorescent protein (eGFP) specifically in pancreatic β-cells.?In young female NOD mice, the infection efficiency of dsAAV8-mIP-eGFP in pancreatic β-cells and its ability to mediate eGFP expression were comparable to those observed in normal BALB/C mice, achieving β-cell-specific expression.

Case 3: Enhanced Expression Efficiency of Capsid-Optimized AAV8 Vector in the Pancreas

Serotype: AAV2/8

Promoter: CBA (chicken β-actin promoter)

Experimental Animals: Mice

Injection Protocol:Intraperitoneal injection, 1.0×1011?to?3.0×1011?vg per mouse, Expression duration?- 2 weeks

Experimental Results:?Compared to intravenous injection, intraperitoneal injection of AAV8 significantly increased transduction efficiency in the pancreas.?Using the Y447F+Y733F mutant AAV8 vector (AAV2/8 pan-CBA-mCherry), a dose three times lower (1.0×1011vg per mouse) achieved higher transduction efficiency in the pancreas than previously reported doses (3.0×1011?to 5.0×1011?vg per mouse) with standard AAV8 vectors.

Case 4: AAV-Mediated Pancreatic Overexpression of Igf1 Inhibits the Progression of Autoimmune Diabetes in Mice

Serotype: AAV2/8

Promoter: mIP (mouse insulin promoter)

Experimental Animals: Mice

Injection Protocol:?Pancreatic duct injection, Titer: 1.0×1013?vg/ml, Expression duration: 2 weeks

Experimental Results:?To investigate the specific effects of mPGES-2 on β-cell function and glucose metabolism, AAV8-MIP-mPGES-2-GFP was delivered via pancreatic duct injection to achieve β-cell-specific overexpression of mPGES-2.?The findings indicated that mPGES-2 plays a significant role in regulating glucose metabolism and β-cell senescence.

Reference

1、Jimenez V, Ayuso E, Mallol C, et al. In vivo genetic engineering of murine pancreatic beta cells mediated by single-stranded adeno-associated viral vectors of serotypes 6, 8 and 9. Diabetologia. 2011;54(5):1075-1086.?

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.

4、Zhong D, Wan Z, Cai J, et al. mPGES-2 blockade antagonizes β-cell senescence to ameliorate diabetes by acting on NR4A1. Nat Metab. 2022;4(2):269-283.

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