Evaluating the transduction efficiency of systemically delivered AAV vectors in the rat nervous system

Abstract

Gene delivery or manipulation with viral vectors is a frequently used tool in basic neuroscience studies. Adeno-associated viruses (AAV) are the most widely used vectors due to their relative safety and long-term efficacy without causing overt immunological complications. Many AAV serotypes have been discovered and engineered that preferentially transduce different populations of neurons. However, efficient targeting of peripheral neurons remains challenging for many researchers, and evaluation of peripheral neuron transduction with AAVs in rats is limited. Here, we aimed to test the efficiency of systemic AAVs to transduce peripheral neurons in rats. We administered AAV9-tdTomato, AAV-PHP.S-tdTomato, or AAV-retro-GFP systemically to neonatal rats via intraperitoneal injection. After 5 weeks, we evaluated expression patterns in peripheral sensory, motor, and autonomic neurons. No significant difference between the serotypes in the transduction of sensory neurons was noted, and all serotypes were more efficient in transducing NF200 + neurons compared to smaller CGRP + neurons. AAV-retro was more efficient at transducing motor neurons compared to other serotypes. Moreover, PHP.S was more efficient at transducing sympathetic neurons, and AAV-retro was more efficient at transducing parasympathetic neurons. These results indicate that specific AAV serotypes target peripheral neuron populations more efficiently than others in the neonatal rat.

Keywords: AAV-retro; AAV9; Adeno Associated Virus (AAV); PHP.S; autonomic nervous system (ANS); dorsal root ganglion (DRG); peripheral nervous system (PNS); sensory nervous system.

FIGURE 1

Systemic PHP.S, AAV9, and adeno-associated viruses (AAV)-retro transduce dorsal root ganglion neurons at similar rates. (A) Representative image of tdTomato expression in dorsal root ganglion of PHP.S-injected rats. Neurons marked with Nissl (scale bar, 50 μm). (B) Representative image of tdTomato expression in dorsal root ganglion of AAV9-injected rats. Neurons marked with Nissl (scale bar, 50 μm). (C) Representative image of Green florescent protein (GFP) expression in dorsal root ganglion of AAV-retro-injected rats. Neurons marked with β-tubulin (scale bar, 50 μm). (D) Quantification of transduced neurons in dorsal root ganglia using Welch’s ANOVA indicated no significant difference between PHP.S (n = 11), AAV9 (n = 8), and AAV-retro (n = 9) (p = 0.4196). (E,F) Images of dorsal root ganglia from vehicle-injected rats with the corresponding fluorescent channels for GFP (E) and tdTomato (F) (scale bar, 50 μm).

FIGURE 2

Systemic PHP.S, AAV9, and adeno-associated viruses (AAV)-retro transduce pelvic ganglion neurons at similar rates. (A) Representative image of tdTomato expression in the pelvic ganglion of PHP.S-injected rats. Neurons marked with Nissl (scale bar, 50 μm). (B) Representative image of tdTomato expression in the pelvic ganglion of AAV9-injected rats. Neurons were marked with Nissl (scale bar, 50 μm). (C) Representative image of GFP expression in the pelvic ganglion of AAV-retro-injected rats. Neurons marked with Nissl (scale bar, 50 μm). (D) Quantification of transduced neurons in pelvic ganglia using one-way ANOVA indicated no significant difference between PHP.S (n = 9 animals), AAV9 (n = 6), and AAV-retro (n = 6) (p = 0.3176). (E,F) Images of pelvic ganglia from vehicle-injected rats with the corresponding fluorescent channels for GFP (E) and tdTomato (F) (scale bar, 50 μm).

FIGURE 3

PHP.S, AAV9, and adeno-associated viruses (AAV)-retro transduce sensory neurons innervating the dorsal horn of the spinal cord, while only AAV-retro transduced neurons in the ventral horn. (A) Representative images of tdTomato expression from three different spinal cord segments in PHP.S injected animals (scale bar, 500 μm). (B) Representative images of tdTomato expression from three different spinal cord segments in AAV9 injected animals (scale bar, 500 μm). (C) Representative images of GFP expression from three different spinal cord segments in AAV-retro injected animals (scale bar, 500 μm). The insets magnify cell body transduction in the ventral horn at a lower exposure (scale bar, 50 μm).

FIGURE 4

Example of urinary bladder neuronal transduction. (A) Representative images from the PHP.S group showing colocalization of β-tubulin with tdTomato (scale bar, 50 μm). (B) Representative images from the AAV9 group showing colocalization of β-tubulin with tdTomato (scale bar, 50 μm). The white triangle points to a non-neuronal cell expressing tdTomato. (C) Representative images from the AAV-retro group showing colocalization of β-tubulin with GFP (scale bar, 50 μm). White arrows indicate transduced nerve fibers.

FIGURE 5

Nerve fibers and non-neuronal cells are transduced by PHP.S, AAV9, and adeno-associated viruses (AAV)-retro in the external urethral sphincter. Representative images of tdTomato (A,B) or GFP (C) expression in the external urethral sphincter (EUS) (scale bar, 500 μm). Skeletal (S) and endothelium (E) squares are magnified to display expression in the skeletal muscle layers and nerve fibers in the endothelium (scale bar, 50 μm). (D) Images of EUS from vehicle-injected rats showing the corresponding fluorophore channels for GFP and tdTomato as well as skeletal muscle and endothelial magnifications (scale bar, 50 μm). White arrows indicate transduced nerve fibers.

FIGURE 6

PHP.S and adeno-associated viruses (AAV)-retro transduce large, myelinated neurons at similar rates. (A) Representative images of tdTomato expression colocalizing with NF200 + neurons in dorsal root ganglia from PHP.S-injected rats (scale bar, 50 μm). (B) Representative images of GFP expression colocalizing with NF200 + neurons in dorsal root ganglia from AAV-retro-injected rats (scale bar, 50 μm). (C) Percent of transduced neurons that were NF200 + (p = 0.9373). (D) Percent of NF200 + neurons transduced by virus (^*⁣*⁣**p < 0.0001). (E) Images of dorsal root ganglia from rats injected with PHP.S (top) or AAV-retro (bottom) stained with secondary antibody only for the corresponding fluorophore used for NF200 in experimental tissue. Statistics were performed using the student’s t-test to compare PHP.S (n = 7) to AAV-retro (n = 9). White arrows indicate neurons with colocalization; black arrows with a white outline indicate viral transduced without colocalization.

FIGURE 7

PHP.S and adeno-associated viruses (AAV)-retro transduce small, non-peptidergic neurons at similar rates. (A) Representative images of tdTomato expression colocalizing with IB4 + neurons in dorsal root ganglia from PHP.S-injected rats (scale bar, 50 μm). (B) Representative images of GFP expression colocalizing with IB4 + neurons in dorsal root ganglia from AAV-retro-injected rats (scale bar, 50 μm). (C) Percent of transduced neurons that were IB4 + (p = 0.7330). (D) Percent of IB4 + neurons that were transduced (p = 0.8585). (E) Images of dorsal root ganglia tissue from rats injected with PHP.S (top) or AAV-retro (bottom) with no antibody staining in the corresponding fluorophore used for IB4 in experimental tissue. Statistics were performed using the student’s t-test to compare PHP.S (n = 7) to AAV-retro (n = 9). White arrows indicate neurons with colocalization.

FIGURE 8

PHP.S and adeno-associated viruses (AAV)-retro transduces small, peptidergic neurons at similar rates. (A) Representative images of tdTomato expression colocalizing with calcitonin gene-related peptide (CGRP) + neurons in dorsal root ganglia from PHP.S-injected rats (scale bar, 50 μm). (B) Representative images of GFP expression colocalizing with CGRP + neurons in dorsal root ganglia from AAV-retro-injected rats (scale bar, 50 μm). (C) Percent of transduced neurons that were CGRP + (p = 0.4728). (D) Percent of CGRP + neurons that were transduced (^**p = 0.0066). (E) Images of dorsal root ganglia from rats injected with PHP.S (top) or AAV-retro (bottom) stained with secondary antibody only for the corresponding fluorophore used for CGRP in experimental tissue. Statistics were performed using the student’s t-test to compare PHP.S (n = 7) and AAV-retro (n = 9). White arrows indicate neurons with colocalization; black arrows with a white outline indicate viral transduced without colocalization.

FIGURE 9

Adeno-associated viruses (AAV)-retro shows tropism for sympathetic neurons over PHP.S in pelvic ganglia. (A) Representative images of tdTomato expression colocalizing with TH + neurons in pelvic ganglia from PHP.S-injected rats (scale bar, 50 μm). (B) Representative images of GFP expression colocalizing with TH + neurons in pelvic ganglia from AAV-retro-injected rats (scale bar, 50 μm). (C) Percent of transduced neurons that were TH + (^***p = 0.0002). (D) Percent of TH + neurons that were transduced (p = 0.0536). (E) Images of pelvic ganglia from rats injected with PHP.S (top) or AAV-retro (bottom) with secondary antibody staining only in the corresponding fluorophore used for TH in experimental tissue. Statistics were performed using the student’s t-test to compare PHP.S (n = 5) and AAV-retro (n = 6). White arrows indicate neurons with colocalization; black arrow with a white outline indicates viral transduced without colocalization.

FIGURE 10

PHP.S shows tropism for parasympathetic neurons over adeno-associated viruses (AAV)-retro in pelvic ganglia. (A) Representative images of tdTomato expression colocalizing with neuronal nitric oxide synthase (nNOS) + neurons in pelvic ganglia from PHP.S-injected rats (scale bar, 50 μm). (B) Representative images of GFP expression colocalizing with nNOS + neurons in pelvic ganglia from AAV-retro-injected rats (scale bar, 50 μm). (C) The percent of transduced neurons that were nNOS +, Welch’s t-test was used to compare PHP.S (n = 5) and AAV-retro (n = 6) because the data set had unequal variances (*p = 0.0394). (D) Percent of nNOS + neurons that were transduced. Mann–Whitney test was used to compare PHP.S (n = 5) and AAV-retro (n = 6) because the data had unequal variances and did not reach normalcy (*p = 0.0043 and ^**p = 0.0043). (E) Images of pelvic ganglia tissue from rats injected with PHP.S (top) or AAV-retro (bottom) with secondary antibody staining only in the corresponding fluorophore used for nNOS in experimental tissue. White arrows indicate neurons with colocalization; black arrow with a white outline indicates viral transduced without colocalization.