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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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eGFP expression in DRG and spinal cord following intravenous delivery of rAAV2/6. (A) eGFP expression in whole L4 DRG following intravenous injection of 1.2 × 107 tu rAAV2/6. Colocalization against NF200 (B) and eGFP expression in the deep lamina of the dorsal horn (C) demonstrate predominantly non-nociceptive transduction. Scale bar: 100 μm.
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Figure 8: eGFP expression in DRG and spinal cord following intravenous delivery of rAAV2/6. (A) eGFP expression in whole L4 DRG following intravenous injection of 1.2 × 107 tu rAAV2/6. Colocalization against NF200 (B) and eGFP expression in the deep lamina of the dorsal horn (C) demonstrate predominantly non-nociceptive transduction. Scale bar: 100 μm.

Mentions: Two reports have recently described transduction of fibers within the dorsal spinal cord following intravenous administration of rAAV [18,22]. To determine whether this mode of delivery results in transduction of cells within the DRG, we injected 1.2 × 107 tu rAAV2/6 into the tail vein of C57BL/6 mice. Three weeks following intravenous delivery we observed relatively low levels of eGFP expression in the DRG at all levels of the spine (L4 DRG shown in Fig. 8A). These cells colocalized predominantly with NF200 (Fig. 8B) and not with peripherin (data not shown). Similarly, only transduced fibers within the deeper lamina of the spinal cord were observed (Fig. 8C).


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)

eGFP expression in DRG and spinal cord following intravenous delivery of rAAV2/6. (A) eGFP expression in whole L4 DRG following intravenous injection of 1.2 × 107 tu rAAV2/6. Colocalization against NF200 (B) and eGFP expression in the deep lamina of the dorsal horn (C) demonstrate predominantly non-nociceptive transduction. Scale bar: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: eGFP expression in DRG and spinal cord following intravenous delivery of rAAV2/6. (A) eGFP expression in whole L4 DRG following intravenous injection of 1.2 × 107 tu rAAV2/6. Colocalization against NF200 (B) and eGFP expression in the deep lamina of the dorsal horn (C) demonstrate predominantly non-nociceptive transduction. Scale bar: 100 μm.
Mentions: Two reports have recently described transduction of fibers within the dorsal spinal cord following intravenous administration of rAAV [18,22]. To determine whether this mode of delivery results in transduction of cells within the DRG, we injected 1.2 × 107 tu rAAV2/6 into the tail vein of C57BL/6 mice. Three weeks following intravenous delivery we observed relatively low levels of eGFP expression in the DRG at all levels of the spine (L4 DRG shown in Fig. 8A). These cells colocalized predominantly with NF200 (Fig. 8B) and not with peripherin (data not shown). Similarly, only transduced fibers within the deeper lamina of the spinal cord were observed (Fig. 8C).

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