MicroRNAs (miRNAs) are non-coding endogenous RNAs that direct post-transcriptional regulation of gene expression by several mechanisms. Activity is primarily through binding to the 3’ untranslated regions (UTRs) of messenger RNAs (mRNA) resulting in degradation and translation repression. Unlike other small-RNAs, miRNAs do not require perfect base pairing, and thus, can regulate a network of broad, yet specific, genes. Although we have only just begun to gain insights into the full range of biologic functions of miRNA, their involvement in the onset and progression of disease has generated significant interest for therapeutic development. Mounting evidence suggests that miRNA-based therapies, either restoring or repressing miRNAs expression and activity, hold great promise. However, despite the early promise and exciting potential, critical hurdles often involving delivery of miRNA-targeting agents remain to be overcome before transition to clinical applications. Limitations that may be overcome by delivery include, but are not limited to, poor in vivo stability, inappropriate biodistribution, disruption and saturation of endogenous RNA machinery, and untoward side effects. Both viral vectors and nonviral delivery systems can be developed to circumvent these challenges. Viral vectors are efficient delivery agents but toxicity and immunogenicity limit their clinical usage. Herein, we review the recent advances in the mechanisms and strategies of nonviral miRNA delivery systems and provide a perspective on the future of miRNA-based therapeutics.

Whereas tissue injury increases spinal dynorphin expression, the functional relevance of this upregulation to persistent pain is unknown. Here, mice lacking the prodynorphin gene were studied for sensitivity to non-noxious and noxious stimuli, before and after induction of experimental neuropathic pain. Prodynorphin knock-out (KO) mice had normal responses to acute nonnoxious stimuli and a mild increased sensitivity to some noxious stimuli. After spinal nerve ligation (SNL), both wild-type (WT) and KO mice demonstrated decreased thresholds to innocuous mechanical and to noxious thermal stimuli, indicating that dynorphin is not required for initiation of neuropathic pain. However, whereas neuropathic pain was sustained in WT mice, KO mice showed a return to baselines by post-SNL day 10. In WT mice, SNL upregulated lumbar dynorphin content on day 10, but not day 2, after injury. Intrathecal dynorphin antiserum reversed neuropathic pain in WT mice at post-SNL day 10 (when dynorphin was upregulated) but not on post-SNL day 2; intrathecal MK-801 reversed SNL-pain at both times. Opioid (, ␦, and ) receptor density and G-protein activation were not different between WT and KO mice and were unchanged by SNL injury. The observations suggest (1) an early, dynorphinindependent phase of neuropathic pain and a later dynorphindependent stage, (2) that upregulated spinal dynorphin is pronociceptive and required for the maintenance of persistent neuropathic pain, and (3) that processes required for the initiation and the maintenance of the neuropathic pain state are distinct. Identification of mechanisms that maintain neuropathic pain appears important for strategies to treat neuropathic pain.

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We expressed the cloned μ‐opioid receptor (μR) in high abundance (5.5 × 106 sites/cell) with an amino‐terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope‐tagged receptor (EE‐μR) was similar to the untagged μR in ligand binding and agonist‐dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the μ‐specific peptide agonist [d‐Ala2,MePhe4,Glyol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist‐induced cellular redistribution of EE‐μR. Tracer binding studies suggested partial net internalization and a small degree of down‐regulation caused by DAMGO. EE‐μR‐containing membranes were solubilized in detergent [3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate] and immunoprecipitated by an anti‐epitope monoclonal antibody. Immunoblotting revealed a prominent band at ∼70 kDa with weaker bands at ∼65 kDa. EE‐μR was labeled with [γ‐32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65–70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced ∼1.8‐fold with the addition of morphine. In conclusion, intracellular trafficking of the μR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein‐coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.

There are differences between human individuals and between mouse strains in levels of opiate receptor (OR) expression, responses to painful stimuli, and responses to opiate drugs. One of the best candidates for contributing to these differences is variation at the OR gene locus. Support for this idea comes from analyses of the human and murine OR genes. Assessments of individual differences in human OR expression add further support. Studies with mice, including knockout-transgenic, quantitative trait locus, and strain-comparison studies, also strongly support the possibility that OR gene alleles would be strong candidates for contributing to individual differences in human nociception and opiate drug responses. This paper reviews current analyses of the murine and human OR genes, their important variants, and correlations between these variants and opiate inf luences on pain.Opiates remain major weapons in pain therapy, but individual differences in the effectiveness of these drugs and in their side effects can be a major limitation for effective pain treatment for many patients. A number of lines of evidence now indicate convincingly that the morphine-preferring opiate receptor (OR) is the major site for the analgesic action of most clinically important opiate drugs.The powerful analgesic effects of morphine and related drugs focus attention on morphine-preferring ORs and their endogenous and exogenous agonists. A number of laboratories, including our own, have had success in cloning OR cDNAs and genomic sequences from several species (1-4), thereby opening new avenues from which to approach this receptor's neurobiology and its relationships with nociceptive responses. This work has laid substantial groundwork for genetic analyses, although it remains incomplete (see below).Data from animal models provide powerful motivation to search out and understand possible genetic bases for individual differences in levels of human OR gene expression. Recent data from transgenic mice provide important information about the role of OR expression levels in mouse models of human pain (4, 5). The data indicate strongly that the OR gene product is the principal route for opiate effects on nociception. Morphine is not analgesic without ORs. Prototypical ␦ and agonists can also function poorly without ORs (refs. 6 and 7; I.S. and G.R.U., unpublished observations).Several studies of the mice that lack OR provide evidence that ORs are important for baseline nociception (ref. 4; see also ref. 5). Nociceptive thresholds vary in gene dosedependent fashions in such mice. Mice with no ORs have lower nociceptive thresholds than heterozygous knockouts that have 50% of wild-type receptor densities. These heterozygous mice, in turn, have lower nociceptive thresholds than wild-type mice with intact ORs.Mouse-strain comparisons and studies in recombinant inbred mouse lines also provide powerful models for possible sources and consequences of genetic variation in humans.