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Intrathecal administration of IGF-I by AAVrh10 improves sensory and motor deficits in a mouse model of diabetic neuropathy.

Homs J, Pagès G, Ariza L, Casas C, Chillón M, Navarro X, Bosch A - Mol Ther Methods Clin Dev (2014)

Bottom Line: Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy.As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons.Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona , Bellaterra, Barcelona, Spain.

ABSTRACT
Different adeno-associated virus (AAV) serotypes efficiently transduce neurons from central and peripheral nervous systems through various administration routes. Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy. Here, we show that lumbar puncture of a nonhuman AAV leads to wide and stable distribution of the vector along the spinal cord in adult mice. AAVrh10 efficiently and specifically infects neurons, both in dorsal root ganglia (60% total sensory neurons) and in the spinal cord (up to one-third of α-motor neurons). As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons. IGF-I-treated diabetic animals showed increased vascular endothelial growth factor expression, activation of Akt/PI3K pathway, and stimulated nerve regeneration and myelination in injured limbs. Moreover, we achieved restoration of nerve conduction velocities in both sensory and motor nerves by AAVrh10, whereas we reached only sensory nerve improvement with AAV1. Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.

No MeSH data available.


Related in: MedlinePlus

AAVrh10 coding for IGF-I promotes regeneration and myelin proteins expression in injured sciatic nerves of diabetic mice. Injured sciatic nerves and DRG from diabetic mice treated with AAVrh10 coding for GFP or IGF-I were dissected 4 weeks after treatment. Graphs represent means ± SEM (n = 3–5 animals). mRNA and protein of GAP-43, a marker for nerve regeneration, are significantly increased in IGF-I–treated animals as assessed by (a) quantitative PCR from DRG and (b) western blot from sciatic nerve protein extracts, where levels of GAP-43 were quantified and compared to actin. (c) Quantitative PCR from sciatic nerve shows increased mRNA for myelin proteins P0, PMP22, MAG, and MBP in animals that received IGF-I compared to GFP-treated mice. (d) Protein levels of myelin protein P0 analyzed by western blot corroborated quantitative PCR results. Student’s t-tests show statistically significant results (***P < 0.005; **P < 0.01; *P < 0.05). AAV, adeno-associated virus; DRG, dorsal root ganglia; GAP, growth associated protein; GFP, green fluorescent protein; IGF, insulin-like growth factor; MBP, myelin basic protein.
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fig6: AAVrh10 coding for IGF-I promotes regeneration and myelin proteins expression in injured sciatic nerves of diabetic mice. Injured sciatic nerves and DRG from diabetic mice treated with AAVrh10 coding for GFP or IGF-I were dissected 4 weeks after treatment. Graphs represent means ± SEM (n = 3–5 animals). mRNA and protein of GAP-43, a marker for nerve regeneration, are significantly increased in IGF-I–treated animals as assessed by (a) quantitative PCR from DRG and (b) western blot from sciatic nerve protein extracts, where levels of GAP-43 were quantified and compared to actin. (c) Quantitative PCR from sciatic nerve shows increased mRNA for myelin proteins P0, PMP22, MAG, and MBP in animals that received IGF-I compared to GFP-treated mice. (d) Protein levels of myelin protein P0 analyzed by western blot corroborated quantitative PCR results. Student’s t-tests show statistically significant results (***P < 0.005; **P < 0.01; *P < 0.05). AAV, adeno-associated virus; DRG, dorsal root ganglia; GAP, growth associated protein; GFP, green fluorescent protein; IGF, insulin-like growth factor; MBP, myelin basic protein.

Mentions: Four weeks after AAV injection and 8 weeks after induction of diabetes, animals were euthanized. IGF-I expression was demonstrated by immunohistochemistry in lumbar DRG and spinal cord in both AAV1- and AAVrh10-injected animals (Figure 4a,d, respectively). IGF-I immunohistochemistry in DRG differs from GFP expression shown in Figure 1 because IGF-I is a secreted protein, and as IGF-I receptor is also expressed in DRG cells,23 it can be uptaken by other nontransduced neighboring cells, showing the global image of IGF-I signal in the DRG. Quantitative real-time PCR from lumbar DRG and ventral horn spinal cord also showed significant increase of IGF-I mRNA with both vectors compared to GFP (Figure 4b; n = 5–7/group; **P < 0.01 and ***P < 0.005 by one-way analysis of variance and Tukey’s multiple comparison test), correlating with immunohistochemistry images. Moreover, levels of IGF-I were also detected by enzyme-linked immunosorbent assay in the CSF of treated animals at the time of sacrifice, particularly for those injected with AAVrh10 (Figure 4c; n = 3/group; *P < 0.05 by Student’s t-test). AAV1-injected animals also showed increased IGF-I in CSF but not statistically significant from GFP-treated animals (Figure 4c). Increased IGF-I in sensory neurons probably activated Akt signaling pathway, as shown by phosphorylation of Akt compared to total Akt protein levels in AAVrh10 IGF-I and but not in GFP-injected mice (Figure 5a). IGF-I is capable to activate different signaling pathways and to promote cell survival as well as to regulate the expression of different trophic factors, VEGF being one among them. VEGF has been shown to be essential for neuronal survival, and its secretion and mRNA expression in many tissues is being induced by IGF-I. In Figure 5b, we show a significant increase in VEGF protein in lumbar DRG of IGF-I–injected animals, compared to GFP-injected mice. On the other hand, growth associated protein 43 (GAP-43) is overexpressed by neurons in the process of regeneration, and it is located in the growth cones of regenerating axons. We analyzed GAP-43 in regenerating sciatic nerves from animals injected with vectors expressing IGF-I or GFP at the time of injury. We detected a significant increase in GAP-43 mRNA in DRG and GAP-43 protein in sciatic nerve, 4 weeks after AAVrh10 IGF-I treatment (Figure 6a,b, respectively), correlating with the electrophysiological results and confirming a positive effect of IGF-I in the regeneration of injured nerves in diabetes. Levels of GAP-43 mRNA were also found increased in sciatic nerves of AAV1-injected animals but at lower levels than that in AAVrh10-injected animals. AAV1-mediated GAP-43 protein was not significantly increased in these animals (Supplementary Figure S6a,b).


Intrathecal administration of IGF-I by AAVrh10 improves sensory and motor deficits in a mouse model of diabetic neuropathy.

Homs J, Pagès G, Ariza L, Casas C, Chillón M, Navarro X, Bosch A - Mol Ther Methods Clin Dev (2014)

AAVrh10 coding for IGF-I promotes regeneration and myelin proteins expression in injured sciatic nerves of diabetic mice. Injured sciatic nerves and DRG from diabetic mice treated with AAVrh10 coding for GFP or IGF-I were dissected 4 weeks after treatment. Graphs represent means ± SEM (n = 3–5 animals). mRNA and protein of GAP-43, a marker for nerve regeneration, are significantly increased in IGF-I–treated animals as assessed by (a) quantitative PCR from DRG and (b) western blot from sciatic nerve protein extracts, where levels of GAP-43 were quantified and compared to actin. (c) Quantitative PCR from sciatic nerve shows increased mRNA for myelin proteins P0, PMP22, MAG, and MBP in animals that received IGF-I compared to GFP-treated mice. (d) Protein levels of myelin protein P0 analyzed by western blot corroborated quantitative PCR results. Student’s t-tests show statistically significant results (***P < 0.005; **P < 0.01; *P < 0.05). AAV, adeno-associated virus; DRG, dorsal root ganglia; GAP, growth associated protein; GFP, green fluorescent protein; IGF, insulin-like growth factor; MBP, myelin basic protein.
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Related In: Results  -  Collection

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fig6: AAVrh10 coding for IGF-I promotes regeneration and myelin proteins expression in injured sciatic nerves of diabetic mice. Injured sciatic nerves and DRG from diabetic mice treated with AAVrh10 coding for GFP or IGF-I were dissected 4 weeks after treatment. Graphs represent means ± SEM (n = 3–5 animals). mRNA and protein of GAP-43, a marker for nerve regeneration, are significantly increased in IGF-I–treated animals as assessed by (a) quantitative PCR from DRG and (b) western blot from sciatic nerve protein extracts, where levels of GAP-43 were quantified and compared to actin. (c) Quantitative PCR from sciatic nerve shows increased mRNA for myelin proteins P0, PMP22, MAG, and MBP in animals that received IGF-I compared to GFP-treated mice. (d) Protein levels of myelin protein P0 analyzed by western blot corroborated quantitative PCR results. Student’s t-tests show statistically significant results (***P < 0.005; **P < 0.01; *P < 0.05). AAV, adeno-associated virus; DRG, dorsal root ganglia; GAP, growth associated protein; GFP, green fluorescent protein; IGF, insulin-like growth factor; MBP, myelin basic protein.
Mentions: Four weeks after AAV injection and 8 weeks after induction of diabetes, animals were euthanized. IGF-I expression was demonstrated by immunohistochemistry in lumbar DRG and spinal cord in both AAV1- and AAVrh10-injected animals (Figure 4a,d, respectively). IGF-I immunohistochemistry in DRG differs from GFP expression shown in Figure 1 because IGF-I is a secreted protein, and as IGF-I receptor is also expressed in DRG cells,23 it can be uptaken by other nontransduced neighboring cells, showing the global image of IGF-I signal in the DRG. Quantitative real-time PCR from lumbar DRG and ventral horn spinal cord also showed significant increase of IGF-I mRNA with both vectors compared to GFP (Figure 4b; n = 5–7/group; **P < 0.01 and ***P < 0.005 by one-way analysis of variance and Tukey’s multiple comparison test), correlating with immunohistochemistry images. Moreover, levels of IGF-I were also detected by enzyme-linked immunosorbent assay in the CSF of treated animals at the time of sacrifice, particularly for those injected with AAVrh10 (Figure 4c; n = 3/group; *P < 0.05 by Student’s t-test). AAV1-injected animals also showed increased IGF-I in CSF but not statistically significant from GFP-treated animals (Figure 4c). Increased IGF-I in sensory neurons probably activated Akt signaling pathway, as shown by phosphorylation of Akt compared to total Akt protein levels in AAVrh10 IGF-I and but not in GFP-injected mice (Figure 5a). IGF-I is capable to activate different signaling pathways and to promote cell survival as well as to regulate the expression of different trophic factors, VEGF being one among them. VEGF has been shown to be essential for neuronal survival, and its secretion and mRNA expression in many tissues is being induced by IGF-I. In Figure 5b, we show a significant increase in VEGF protein in lumbar DRG of IGF-I–injected animals, compared to GFP-injected mice. On the other hand, growth associated protein 43 (GAP-43) is overexpressed by neurons in the process of regeneration, and it is located in the growth cones of regenerating axons. We analyzed GAP-43 in regenerating sciatic nerves from animals injected with vectors expressing IGF-I or GFP at the time of injury. We detected a significant increase in GAP-43 mRNA in DRG and GAP-43 protein in sciatic nerve, 4 weeks after AAVrh10 IGF-I treatment (Figure 6a,b, respectively), correlating with the electrophysiological results and confirming a positive effect of IGF-I in the regeneration of injured nerves in diabetes. Levels of GAP-43 mRNA were also found increased in sciatic nerves of AAV1-injected animals but at lower levels than that in AAVrh10-injected animals. AAV1-mediated GAP-43 protein was not significantly increased in these animals (Supplementary Figure S6a,b).

Bottom Line: Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy.As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons.Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona , Bellaterra, Barcelona, Spain.

ABSTRACT
Different adeno-associated virus (AAV) serotypes efficiently transduce neurons from central and peripheral nervous systems through various administration routes. Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy. Here, we show that lumbar puncture of a nonhuman AAV leads to wide and stable distribution of the vector along the spinal cord in adult mice. AAVrh10 efficiently and specifically infects neurons, both in dorsal root ganglia (60% total sensory neurons) and in the spinal cord (up to one-third of α-motor neurons). As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons. IGF-I-treated diabetic animals showed increased vascular endothelial growth factor expression, activation of Akt/PI3K pathway, and stimulated nerve regeneration and myelination in injured limbs. Moreover, we achieved restoration of nerve conduction velocities in both sensory and motor nerves by AAVrh10, whereas we reached only sensory nerve improvement with AAV1. Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.

No MeSH data available.


Related in: MedlinePlus