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In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.

Xu Q, Chou B, Fitzsimmons B, Miyanohara A, Shubayev V, Santucci C, Hefferan M, Marsala M, Hua XY - PLoS ONE (2012)

Bottom Line: We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection.In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons.This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of California San Diego, San Diego, California, United States of America. q1xu@ucsd.edu

ABSTRACT
We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

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Expression of GFP in different populations of neurons in the lumbar DRG.A,B C and D) GFP (green) was co-labeled with A) NF200, B) TRPV1, C) CGRP and D) IB4 (red) at two week following vector administration. Arrowheads indicate co-localization of GFP with the respective cell marker. E) Bar graph represents percentage of GFP-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3 rats. Scale bar, 100 µm.
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pone-0032581-g002: Expression of GFP in different populations of neurons in the lumbar DRG.A,B C and D) GFP (green) was co-labeled with A) NF200, B) TRPV1, C) CGRP and D) IB4 (red) at two week following vector administration. Arrowheads indicate co-localization of GFP with the respective cell marker. E) Bar graph represents percentage of GFP-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3 rats. Scale bar, 100 µm.

Mentions: To study the transduction efficacy and tropism of AAV5 in rats, we first constructed a vector encoding GFP driven by a cytomegalovirus (CMV) promoter. Ten microliters of virus (1011 viral particles) was administered in adult rats through an intrathecal catheter, with the tip ending at the spinal level of L3–L4. The GFP expression began to be visualized in the DRG and spinal cord at 1 week following the vector administration, however the intensity of GFP immunoreactivity at this time point was very weak (not shown), and the distribution pattern was not analyzed. A profound GFP expression was observed after 2 weeks in the lumbar DRG neurons (Figure 1a), while in the thoracic and cervical DRGs GFP was not detectable (figure 1b and c). We have analyzed a total of 3781 neurons in the L4, L5 and L6 DRGs from three rats, 44% (1672) of those neurons were GFP-positive. Interestingly, the GFP-labeled neurons were predominantly of large to medium size (figure 1d). Out of all the GFP-positive profiles in the DRG, 38% were large (>1200 µm2), 41% were medium (>600 µm2and <1200 µm2) and only 21% were small (<600 µm2). Conversely, out of all the neuronal profiles, GFP was observed in 100%, 52% and 20% in large, medium and small profiles respectively. Double immunofluorescence study revealed that over 80% of the GFP-positive neurons were co-labeled with NF200, a marker for myelinated fibers (figure 2a and e). A small portion of transduced neurons are co-labeled with nociceptor markers such as TRPV1, CGRP and IB4 (figure 2b–e).


In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.

Xu Q, Chou B, Fitzsimmons B, Miyanohara A, Shubayev V, Santucci C, Hefferan M, Marsala M, Hua XY - PLoS ONE (2012)

Expression of GFP in different populations of neurons in the lumbar DRG.A,B C and D) GFP (green) was co-labeled with A) NF200, B) TRPV1, C) CGRP and D) IB4 (red) at two week following vector administration. Arrowheads indicate co-localization of GFP with the respective cell marker. E) Bar graph represents percentage of GFP-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3 rats. Scale bar, 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293818&req=5

pone-0032581-g002: Expression of GFP in different populations of neurons in the lumbar DRG.A,B C and D) GFP (green) was co-labeled with A) NF200, B) TRPV1, C) CGRP and D) IB4 (red) at two week following vector administration. Arrowheads indicate co-localization of GFP with the respective cell marker. E) Bar graph represents percentage of GFP-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3 rats. Scale bar, 100 µm.
Mentions: To study the transduction efficacy and tropism of AAV5 in rats, we first constructed a vector encoding GFP driven by a cytomegalovirus (CMV) promoter. Ten microliters of virus (1011 viral particles) was administered in adult rats through an intrathecal catheter, with the tip ending at the spinal level of L3–L4. The GFP expression began to be visualized in the DRG and spinal cord at 1 week following the vector administration, however the intensity of GFP immunoreactivity at this time point was very weak (not shown), and the distribution pattern was not analyzed. A profound GFP expression was observed after 2 weeks in the lumbar DRG neurons (Figure 1a), while in the thoracic and cervical DRGs GFP was not detectable (figure 1b and c). We have analyzed a total of 3781 neurons in the L4, L5 and L6 DRGs from three rats, 44% (1672) of those neurons were GFP-positive. Interestingly, the GFP-labeled neurons were predominantly of large to medium size (figure 1d). Out of all the GFP-positive profiles in the DRG, 38% were large (>1200 µm2), 41% were medium (>600 µm2and <1200 µm2) and only 21% were small (<600 µm2). Conversely, out of all the neuronal profiles, GFP was observed in 100%, 52% and 20% in large, medium and small profiles respectively. Double immunofluorescence study revealed that over 80% of the GFP-positive neurons were co-labeled with NF200, a marker for myelinated fibers (figure 2a and e). A small portion of transduced neurons are co-labeled with nociceptor markers such as TRPV1, CGRP and IB4 (figure 2b–e).

Bottom Line: We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection.In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons.This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of California San Diego, San Diego, California, United States of America. q1xu@ucsd.edu

ABSTRACT
We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

Show MeSH
Related in: MedlinePlus