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Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

Towne C, Pertin M, Beggah AT, Aebischer P, Decosterd I - Mol Pain (2009)

Bottom Line: Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression.Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG.Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, EPFL, Lausanne, Switzerland. chris.towne@epfl.ch

ABSTRACT

Background: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice.

Results: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types.

Conclusion: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

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Neuronal transduction pattern following sciatic nerve injection. Representative confocal images demonstrating colocalization of eGFP-positive neurons with markers against neuronal subpopulations following injection of 2.6 × 105 tu rAAV2/6 into the sciatic nerve. TRPV1, Substance P, CGRP and TrkA for small peptidergic neurons, IB4 for non-peptidergic neurons and NF200 for large neurons. Scale bar: 100 μm.
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Figure 3: Neuronal transduction pattern following sciatic nerve injection. Representative confocal images demonstrating colocalization of eGFP-positive neurons with markers against neuronal subpopulations following injection of 2.6 × 105 tu rAAV2/6 into the sciatic nerve. TRPV1, Substance P, CGRP and TrkA for small peptidergic neurons, IB4 for non-peptidergic neurons and NF200 for large neurons. Scale bar: 100 μm.

Mentions: Immunohistochemistry was next performed to further characterize the vector tropism for putative nociceptive neurons. L4 DRG sections were labeled with antibodies against markers of various neuronal populations and assessed for colocalization with eGFP (Fig. 3, 4). More eGFP-positive cells colocalized with transient receptor potential vanilloid 1 (TRPV1) than with neurofilament 200 (NF200) for both modes of delivery. Indeed NF200 colocalization with eGFP was rare, although, interestingly, was more frequent for the intrathecal than the sciatic nerve delivery. There was also colabeling with markers of the small peptidergic neurons, Substance P, α-calcitonin gene-related peptide (CGRP) and neurotrophic tyrosine kinase receptor type 1 (TrkA) and the non-peptidergic neuronal population, isolectin B4 (IB4) for both modes of delivery.


Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

Towne C, Pertin M, Beggah AT, Aebischer P, Decosterd I - Mol Pain (2009)

Neuronal transduction pattern following sciatic nerve injection. Representative confocal images demonstrating colocalization of eGFP-positive neurons with markers against neuronal subpopulations following injection of 2.6 × 105 tu rAAV2/6 into the sciatic nerve. TRPV1, Substance P, CGRP and TrkA for small peptidergic neurons, IB4 for non-peptidergic neurons and NF200 for large neurons. Scale bar: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2747840&req=5

Figure 3: Neuronal transduction pattern following sciatic nerve injection. Representative confocal images demonstrating colocalization of eGFP-positive neurons with markers against neuronal subpopulations following injection of 2.6 × 105 tu rAAV2/6 into the sciatic nerve. TRPV1, Substance P, CGRP and TrkA for small peptidergic neurons, IB4 for non-peptidergic neurons and NF200 for large neurons. Scale bar: 100 μm.
Mentions: Immunohistochemistry was next performed to further characterize the vector tropism for putative nociceptive neurons. L4 DRG sections were labeled with antibodies against markers of various neuronal populations and assessed for colocalization with eGFP (Fig. 3, 4). More eGFP-positive cells colocalized with transient receptor potential vanilloid 1 (TRPV1) than with neurofilament 200 (NF200) for both modes of delivery. Indeed NF200 colocalization with eGFP was rare, although, interestingly, was more frequent for the intrathecal than the sciatic nerve delivery. There was also colabeling with markers of the small peptidergic neurons, Substance P, α-calcitonin gene-related peptide (CGRP) and neurotrophic tyrosine kinase receptor type 1 (TrkA) and the non-peptidergic neuronal population, isolectin B4 (IB4) for both modes of delivery.

Bottom Line: Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression.Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG.Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, EPFL, Lausanne, Switzerland. chris.towne@epfl.ch

ABSTRACT

Background: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice.

Results: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types.

Conclusion: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Show MeSH
Related in: MedlinePlus