Current gene therapy using viral vectors for chronic pain

The complexity of chronic pain and the challenges of pharmacotherapy highlight the importance of development of new approaches to pain management. Gene therapy approaches may be complementary to pharmacotherapy for several advantages. Gene therapy strategies may target specific chronic pain mechanisms in a tissue-specific manner. The present collection of articles features distinct gene therapy approaches targeting specific mechanisms identified as important in the specific pain conditions. Dr. Fairbanks group describes commonly used gene therapeutics (herpes simplex viral vector (HSV) and adeno-associated viral vector (AAV)), and addresses biodistribution and potential neurotoxicity in pre-clinical models of vector delivery. Dr. Tao group addresses that downregulation of a voltage-gated potassium channel (Kv1.2) contributes to the maintenance of neuropathic pain. Alleviation of chronic pain through restoring Kv1.2 expression in sensory neurons is presented in this review. Drs Goins and Kinchington group describes a strategy to use the replication defective HSV vector to deliver two different gene products (enkephalin and TNF soluble receptor) for the treatment of post-herpetic neuralgia. Dr. Hao group addresses the observation that the pro-inflammatory cytokines are an important shared mechanism underlying both neuropathic pain and the development of opioid analgesic tolerance and withdrawal. The use of gene therapy strategies to enhance expression of the anti-pro-inflammatory cytokines is summarized. Development of multiple gene therapy strategies may have the benefit of targeting specific pathologies associated with distinct chronic pain conditions (by Guest Editors, Drs. C. Fairbanks and S. Hao)

Fgr contributes to hemorrhage-induced thalamic pain by activating NF-κB/ERK1/2 pathways

Thalamic pain, a type of central poststroke pain, frequently occurs following ischemia/hemorrhage in the thalamus. Current treatment of this disorder is often ineffective, at least in part due to largely unknown mechanisms that underlie thalamic pain genesis. Here, we report that hemorrhage caused by microinjection of type IV collagenase or autologous whole blood into unilateral ventral posterior lateral nucleus and ventral posterior medial nucleus of the thalamus increased the expression of Fgr, a member of the Src family nonreceptor tyrosine kinases, at both mRNA and protein levels in thalamic microglia. Pharmacological inhibition or genetic knockdown of thalamic Fgr attenuated the hemorrhage-induced thalamic injury on the ipsilateral side and the development and maintenance of mechanical, heat, and cold pain hypersensitivities on the contralateral side. Mechanistically, the increased Fgr participated in hemorrhage-induced microglial activation and subsequent production of TNF-α likely through activation of both NF-κB and ERK1/2 pathways in thalamic microglia. Our findings suggest that Fgr is a key player in thalamic pain and a potential target for the therapeutic management of this disorder

MicroRNA-181a Suppresses Mouse Granulosa Cell Proliferation by Targeting Activin Receptor IIA

<div><p>Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.</p> </div

Effect of miR-181a inhibitor on mouse granulosa cell (mGC) proliferation.

<p>mGC was transfected with indicated miR-181a inhibitor (anti-sense oligonucleotide of miR-181a) or miRNA inhibitor negative control (miRNA inhibitor control) for 48 h. (A) MiR-181a expression was measured by qRT-PCR. (B) The proliferation of mGC was examined by CCK-8 after transfection of miR-181a inhibitor. Cyclin D2 mRNA (C) and protein (D) levels measured by qRT-PCR and Western blotting. (E) qRT-PCR and (F) Western blot analysis showed acvr2a mRNA and protein levels in mGC treated with miR-181a inhibitor. Relative protein levels were measured by densitometry using Quantity One Software and normalized to β-actin, the control group; the ratios were presented above the Western blot bands. *p<0.05, **p<0.01, compared with controls.</p

Effect of activin A on miR-181a expression and mouse granulosa cell (mGC) proliferation.

<p>mGC was isolated from 21-day-old mouse ovaries. (A) mGC was treated with indicated concentrations of activin A for 24 h. MiR-181a level was determined by qRT-PCR. (B) qRT-PCR analysis was performed to measure miR-181a level in mGC treated with activin A (50 ng/ml) for up to 48 h. (C) The proliferation of mGC was measured by CCK-8 after treated with activin A for 48 h. Cyclin D2 mRNA (D) and protein (E) levels were examined in mGC after treated with activin A (50 ng/ml) for 48 h by qRT-PCR and Western blotting, respectively. Relative protein levels were measured by densitometry using Quantity One Software and normalized to β-actin, the control group; the ratios were presented above the Western blot bands. All experiments were performed three times. *p<0.05, **p<0.01, compared with untreated controls.</p

Variation of miR-181a and acvr2a expression in development of ovaries and during ovarian follicle maturation.

<p>Expression of miR-181a (A) and acvr2a (B) assessed by qRT-PCR in day 3, 8, 12, and 21 mouse ovaries. *p<0.05, **p<0.01, compared with the day 3 group. qRT-PCR analysis of miR-181a (C) and acvr2a (D) in primary (pri), preantral (pre), and antral follicles of 21-day-old mouse ovaries. *p<0.05, compared with primary follicles.</p

Inactivation of the activin signaling pathway by miR-181a.

<p>Western blot analysis of the levels of Smad2 and phosphorylated Smad2 (Ser465/467) in mouse granulosa cells (mGC) treated with 50 ng/ml activin A (A) or infected with 50 MOI Ad-miR-181a (B) for 24 h. (C) mGC was infected with 50 MOI Ad-miR-181a for 24 h, and cells were then treated with 50 ng/ml activin A for another 24 h. The protein level of Smad2 and phosphorylated Smad2 were measured by Western blotting. (D) qRT-PCR analysis of Smad2 and cyclin D2 expression in mGC transfected with 50 nM siRNA duplexes targeting mouse Smad2 (siSmad2) or siRNA negative control for 48 h. *p<0.05, compared with controls. (E, F, and G) mGC was infected with 50 MOI Ad-miR-181a or Ad-LacZ for 24 h, and cells were then treated with 50 ng/ml activin A for another 24 h. (E) mGC proliferation was examined by CCK-8. Cyclin D2 mRNA (F) and protein (G) levels were measured by qRT-PCR and Western blotting. Relative protein levels of phosphorylated Smad2 and cyclin D2 were measured by densitometry using Quantity One Software and normalized to β-actin, the control group; the ratios were presented above the Western blot bands. Bars labeled with different letters indicate statistically significant differences (p<0.05).</p