dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm

I., H.; Han, Jun-Ming; Tao, Pao‐Luh; Law, Ping Yee; Loh, Horace H.

doi:10.1073/pnas.0703409104

Cited by 22 publications

(25 citation statements)

References 22 publications

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“…Expression of the transgene was monitored by the EGFP fluorescence. Similar to previous studies (Chen et al, 2007), the intrathecal injection of the dsAAV2 resulted in the expression of the MORS196ACSTA at the L3-L5 spinal cord starting from 2 weeks after the virus injection. A representative picture of EGFP fluorescence in mice 8 weeks after the intrathecal injection is shown in Fig.…”

Section: Expression Of the Mutated Mor In Mice After Intrathecal Injesupporting

confidence: 70%

“…Mice injected with dsAAV2 exhibited a naloxone-mediated antinociceptive response that was not accompanied by tolerance or dependence development during subchronic antagonist treatment (Chen et al, 2007). These results suggest that opioid antagonist is activating the mutant but not the endogenous MOR.…”

Section: Introductionsupporting

confidence: 48%

“…Experiment 3. To determine the rewarding effects induced by morphine or naloxone, the CPP test was carried out as reported previously (Chen et al, 2007).…”

Section: Detection Of the Expression Of Mutated -Opioid Receptorsmentioning

confidence: 99%

“…The cloned MOR or the mutant MORS4.45AT7.44AC7.47A (MORS196ACSTA) with EGFP as reporter was cloned into the dsAAV2 expression cassette pV4.1c containing the cytomegalovirus promoter as described previously (Chen et al, 2007). The dsAAV2 was produced in human embryonic kidney 293 cells by using the adenovirus-free, triple-plasmid cotransfection method (Wang et al, 2003) and purified by heparin affinity column chromatography following previously published methods (Clark et al, 1999;Wang et al, 2003).…”

Section: Construction Of the Dsaav2 Virusmentioning

confidence: 99%

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Intrathecal Delivery of a Mutant μ-Opioid Receptor Activated by Naloxone as a Possible Antinociceptive Paradigm

Kao

Chen

et al. 2010

J Pharmacol Exp Ther

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Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a -opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.

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Section: Expression Of the Mutated Mor In Mice After Intrathecal Injesupporting

confidence: 70%

Section: Introductionsupporting

confidence: 48%

“…Experiment 3. To determine the rewarding effects induced by morphine or naloxone, the CPP test was carried out as reported previously (Chen et al, 2007).…”

Section: Detection Of the Expression Of Mutated -Opioid Receptorsmentioning

confidence: 99%

Section: Construction Of the Dsaav2 Virusmentioning

confidence: 99%

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Intrathecal Delivery of a Mutant μ-Opioid Receptor Activated by Naloxone as a Possible Antinociceptive Paradigm

Kao

Chen

et al. 2010

J Pharmacol Exp Ther

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“…This conserved residue (Ser196 in the MOR) is located at the junction of the second intracellular loop and transmembrane domain 4, in the same vicinity as Asn190. In mice genetically engineered to express a S196A MOR construct in the spinal cord, the putative "antagonist" naloxone induced antinociceptive responses without signs of tolerance or dependence (Chen et al, 2007). On the basis of this finding and our observations, it is possible that naltrexone will paradoxically induce antinociceptive actions in individuals harboring the N190K polymorphism.…”

Section: Discussionmentioning

confidence: 44%

The μ-Opioid Receptor Variant N190K Is Unresponsive to Peptide Agonists yet Can be Rescued by Small-Molecule Drugs

Fortin

Ci²,

Schroeder³

et al. 2010

Mol Pharmacol

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The -opioid receptor (MOR) plays an important role in modulating analgesia, feeding behavior, and a range of autonomic functions. In the current study, we investigated the degree to which 13 naturally occurring missense mutations affect the pharmacological properties of the human MOR. After expression of each receptor in human embryonic kidney 293 cells, signaling (G␣ i/o -mediated) induced by peptide agonists was assessed using luciferase reporter gene assays. Multiple mutants (S66F, S147C, R260H, R265C, R265H, and S268P) show a significant reduction in agonist potency. At the N190K variant, agonist-mediated signaling was essentially absent. Enzymelinked immunosorbent assay, microscopic analysis, and radioligand binding assays revealed that this mutant shows markedly reduced cell-surface expression, whereas all other receptor variants were expressed at normal levels. Surface expression of the N190K variant could be increased by incubation with the alkaloid agonist buprenorphine or with either naltrexone or naloxone, structurally related MOR antagonists. We were surprised to find that both putative antagonists, despite being inactive at the wild-type MOR, triggered a concentration-dependent increase in N190K receptor-mediated signaling. In contrast, peptidic ligands failed to promote expression or rescue function of the N190K mutant. Subsequent analysis of the N190K variant in an ethnically diverse cohort identified this isoform in a subgroup of African Americans. Taken together, our studies reveal that the N190K mutation leads to severe functional alterations and, in parallel, changes the response to established MOR ligands. The extent to which this mutation results in physiological abnormalities or affects drug sensitivity in selected populations (e.g., those with chronic pain or addiction) remains to be investigated.

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Rapid, long‐term labeling of cells in the developing and adult rodent visual cortex using double‐stranded adeno‐associated viral vectors

Lowery

Zhang

Kelly

et al. 2009

Developmental Neurobiology

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Chronic in-vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, non-toxic and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV’s main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation allowing rapid transgene expression. Here we have injected different serotypes (1,2,6,7,8,9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days post-injection and as long as a month, surpassing conventional AAV’s onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to non-labeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex.

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dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm

Cited by 22 publications

References 22 publications

Intrathecal Delivery of a Mutant μ-Opioid Receptor Activated by Naloxone as a Possible Antinociceptive Paradigm

Intrathecal Delivery of a Mutant μ-Opioid Receptor Activated by Naloxone as a Possible Antinociceptive Paradigm

The μ-Opioid Receptor Variant N190K Is Unresponsive to Peptide Agonists yet Can be Rescued by Small-Molecule Drugs

Rapid, long‐term labeling of cells in the developing and adult rodent visual cortex using double‐stranded adeno‐associated viral vectors

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