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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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rAAV2/6 transduction of the dorsal root ganglia following subcutaneous, intramuscular, sciatic nerve and intrathecal delivery. (A) Macroscopic view of L4 DRG of mice injected with rAAV2/6 expressing eGFP. From left to right: uninjected, dose response of sciatic nerve injected, subcutaneous (high dose), intramuscular (high dose), intrathecal (high dose). (B) Native eGFP expression and corresponding bright field image in a typical 12 μm L4 DRG section following sciatic nerve delivery. Scale bar, 100 μm. (C) Transduction rates within L4 DRG following subcutaneous, intramuscular, sciatic nerve delivery at three doses (n = 3 per group and dose) and intrathecal delivery at the highest dose (n = 5) expressed as a percentage of eGFP expressing profiles per total number of DRG cells observed. LD, low dose (2.6 × 104 tu); MD, medium dose (8.0 × 104 tu); HD, high dose (2.6 × 105 tu). (D) qPCR against the vector genome (beta globin intron of rAAV2/6) in DRG, spinal cord and liver for the high dose of the four administration routes. (E) Reverse transcription qPCR against eGFP mRNA transcripts normalized with GAPDH mRNA in the DRG for the various protocols.
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Figure 1: rAAV2/6 transduction of the dorsal root ganglia following subcutaneous, intramuscular, sciatic nerve and intrathecal delivery. (A) Macroscopic view of L4 DRG of mice injected with rAAV2/6 expressing eGFP. From left to right: uninjected, dose response of sciatic nerve injected, subcutaneous (high dose), intramuscular (high dose), intrathecal (high dose). (B) Native eGFP expression and corresponding bright field image in a typical 12 μm L4 DRG section following sciatic nerve delivery. Scale bar, 100 μm. (C) Transduction rates within L4 DRG following subcutaneous, intramuscular, sciatic nerve delivery at three doses (n = 3 per group and dose) and intrathecal delivery at the highest dose (n = 5) expressed as a percentage of eGFP expressing profiles per total number of DRG cells observed. LD, low dose (2.6 × 104 tu); MD, medium dose (8.0 × 104 tu); HD, high dose (2.6 × 105 tu). (D) qPCR against the vector genome (beta globin intron of rAAV2/6) in DRG, spinal cord and liver for the high dose of the four administration routes. (E) Reverse transcription qPCR against eGFP mRNA transcripts normalized with GAPDH mRNA in the DRG for the various protocols.

Mentions: rAAV2/6 (rAAV serotype 2 genome packaged in the serotype 6 capsid) was delivered to C57BL/6 mice through four routes; subcutaneously at the middle and lateral plantar surface of the hind foot; intramuscularly in the triceps surae muscle of the hind limb; in the sciatic nerve; and intrathecally at the lumbar level. Three different doses of vector were examined (except for the intrathecal administration which received only the high dose) and the transduction was assessed three weeks later. All modes of delivery resulted in eGFP epifluorescence within the whole dissected L4 DRG demonstrating that rAAV2/6 is capable of transducing sensory neurons (Fig. 1A, 1B). The levels of transduction were dependant upon the mode of delivery (from highest to lowest) with intrathecal, sciatic nerve, subcutaneous and intramuscular delivery resulting in 57.5 ± 0.1, 28.1 ± 3.1, 3.5 ± 1.7 and 1.6 ± 0.6 percent of eGFP expressing profiles per total number of DRG cells observed, respectively. Furthermore, the transduction rate was dose dependent in instances where examined (Fig. 1C).


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)

rAAV2/6 transduction of the dorsal root ganglia following subcutaneous, intramuscular, sciatic nerve and intrathecal delivery. (A) Macroscopic view of L4 DRG of mice injected with rAAV2/6 expressing eGFP. From left to right: uninjected, dose response of sciatic nerve injected, subcutaneous (high dose), intramuscular (high dose), intrathecal (high dose). (B) Native eGFP expression and corresponding bright field image in a typical 12 μm L4 DRG section following sciatic nerve delivery. Scale bar, 100 μm. (C) Transduction rates within L4 DRG following subcutaneous, intramuscular, sciatic nerve delivery at three doses (n = 3 per group and dose) and intrathecal delivery at the highest dose (n = 5) expressed as a percentage of eGFP expressing profiles per total number of DRG cells observed. LD, low dose (2.6 × 104 tu); MD, medium dose (8.0 × 104 tu); HD, high dose (2.6 × 105 tu). (D) qPCR against the vector genome (beta globin intron of rAAV2/6) in DRG, spinal cord and liver for the high dose of the four administration routes. (E) Reverse transcription qPCR against eGFP mRNA transcripts normalized with GAPDH mRNA in the DRG for the various protocols.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: rAAV2/6 transduction of the dorsal root ganglia following subcutaneous, intramuscular, sciatic nerve and intrathecal delivery. (A) Macroscopic view of L4 DRG of mice injected with rAAV2/6 expressing eGFP. From left to right: uninjected, dose response of sciatic nerve injected, subcutaneous (high dose), intramuscular (high dose), intrathecal (high dose). (B) Native eGFP expression and corresponding bright field image in a typical 12 μm L4 DRG section following sciatic nerve delivery. Scale bar, 100 μm. (C) Transduction rates within L4 DRG following subcutaneous, intramuscular, sciatic nerve delivery at three doses (n = 3 per group and dose) and intrathecal delivery at the highest dose (n = 5) expressed as a percentage of eGFP expressing profiles per total number of DRG cells observed. LD, low dose (2.6 × 104 tu); MD, medium dose (8.0 × 104 tu); HD, high dose (2.6 × 105 tu). (D) qPCR against the vector genome (beta globin intron of rAAV2/6) in DRG, spinal cord and liver for the high dose of the four administration routes. (E) Reverse transcription qPCR against eGFP mRNA transcripts normalized with GAPDH mRNA in the DRG for the various protocols.
Mentions: rAAV2/6 (rAAV serotype 2 genome packaged in the serotype 6 capsid) was delivered to C57BL/6 mice through four routes; subcutaneously at the middle and lateral plantar surface of the hind foot; intramuscularly in the triceps surae muscle of the hind limb; in the sciatic nerve; and intrathecally at the lumbar level. Three different doses of vector were examined (except for the intrathecal administration which received only the high dose) and the transduction was assessed three weeks later. All modes of delivery resulted in eGFP epifluorescence within the whole dissected L4 DRG demonstrating that rAAV2/6 is capable of transducing sensory neurons (Fig. 1A, 1B). The levels of transduction were dependant upon the mode of delivery (from highest to lowest) with intrathecal, sciatic nerve, subcutaneous and intramuscular delivery resulting in 57.5 ± 0.1, 28.1 ± 3.1, 3.5 ± 1.7 and 1.6 ± 0.6 percent of eGFP expressing profiles per total number of DRG cells observed, respectively. Furthermore, the transduction rate was dose dependent in instances where examined (Fig. 1C).

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