MicroRNA let-7b enhances spinal cord nociceptive synaptic transmission and induces acute and persistent pain through neuronal and microglial signaling.

ABSTRACT: Secreted microRNAs (miRNAs) have been detected in various body fluids including the cerebrospinal fluid, yet their direct role in regulating synaptic transmission remains uncertain. We found that intrathecal injection of low dose of let-7b (1 µg) induced short-term (<24 hours) mechanical allodynia and heat hyperalgesia, a response that is compromised in Tlr7-/- or Trpa1-/- mice. Ex vivo and in vivo calcium imaging in GCaMP6-report mice revealed increased calcium signal in spinal cord afferent terminals and doral root ganglion/dorsal root ganglia neurons following spinal perfusion and intraplantar injection of let-7b. Patch-clamp recordings also demonstrated enhanced excitatory synaptic transmission (miniature excitatory postsynaptic currents [EPSCs]) in spinal nociceptive neurons following let-7b perfusion or optogenetic activation of axonal terminals. The elevation in spinal calcium signaling and EPSCs was dependent on the presence of toll-like receptor-7 (TLR7) and transient receptor potential ion channel subtype A1 (TRPA1). In addition, endogenous let-7b is enriched in spinal cord synaptosome, and peripheral inflammation increased let-7b in doral root ganglion/dorsal root ganglia neurons, spinal cord tissue, and the cerebrospinal fluid. Notably, let-7b antagomir inhibited inflammatory pain and inflammation-induced synaptic plasticity (EPSC increase), suggesting an endogenous role of let-7b in regulating pain and synaptic transmission. Furthermore, intrathecal injection of let-7b, at a higher dose (10 µg), induced persistent mechanical allodynia for >2 weeks, which was abolished in Tlr7-/- mice. The high dose of let-7b also induced microgliosis in the spinal cord. Of interest, intrathecal minocycline only inhibited let-7b-induced mechanical allodynia in male but not female mice. Our findings indicate that the secreted microRNA let-7b has the capacity to provoke pain through both neuronal and glial signaling, thereby establishing miRNA as an emerging neuromodulator.

Aptamer-programmable adeno-associated viral vectors as a novel platform for cell-specific gene transfer.

Adeno-associated viruses (AAVs) are commonly used for in vivo gene therapy. Nevertheless, the wide tropism that characterizes these vectors limits specific targeting to a particular cell type or tissue. Here, we developed new chemically modified AAV vectors (Nε-AAVs) displaying a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. We were able to identify AAV vectors that would tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging efficiency. We functionalized the Nε-AAVs through conjugation with DNA (AS1411) or RNA (E3) aptamers or with a folic acid moiety (FA). E3-, AS1411-, and FA-AAVs showed on average a 3- to 9-fold increase in transduction compared with their non-conjugated counterparts in different cancer cell lines. Using specific competitors, we established ligand-specific transduction. In vivo studies confirmed the selective uptake of FA-AAV and AS1411-AAV without off-target transduction in peripheral organs. Overall, the high versatility of these novel Nε-AAVs might pave the way to tailoring gene therapy vectors toward specific types of cells both for ex vivo and in vivo applications.

An Aptamer for Broad Cancer Targeting and Therapy.

Recent advances in chemotherapy treatments are increasingly targeted therapies, with the drug conjugated to an antibody able to deliver it directly to the tumor. As high-affinity chemical ligands that are much smaller in size, aptamers are ideal for this type of drug targeting. Aptamer-highly toxic drug conjugates (ApTDCs) based on the E3 aptamer, selected on prostate cancer cells, target and inhibit prostate tumor growth in vivo. Here, we observe that E3 also broadly targets numerous other cancer types, apparently representing a universal aptamer for cancer targeting. Accordingly, ApTDCs formed by conjugation of E3 to the drugs monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) efficiently target and kill a range of different cancer cells. Notably, this targeting extends to both patient-derived explant (PDX) cancer cell lines and tumors, with the E3 MMAE and MMAF conjugates inhibiting PDX cell growth in vitro and with the E3 aptamer targeting PDX colorectal tumors in vivo.

Tunable cytotoxic aptamer-drug conjugates for the treatment of prostate cancer.

Therapies that can eliminate both local and metastatic prostate tumor lesions while sparing normal organ tissue are desperately needed. With the goal of developing an improved drug-targeting strategy, we turned to a new class of targeted anticancer therapeutics: aptamers conjugated to highly toxic chemotherapeutics. Cell selection for aptamers with prostate cancer specificity yielded the E3 aptamer, which internalizes into prostate cancer cells without targeting normal prostate cells. Chemical conjugation of E3 to the drugs monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) yields a potent cytotoxic agent that efficiently kills prostate cancer cells in vitro but does not affect normal prostate epithelial cells. Importantly, the E3 aptamer targets tumors in vivo and treatment with the MMAF-E3 conjugate significantly inhibits prostate cancer growth in mice, demonstrating the in vivo utility of aptamer-drug conjugates. Additionally, we report the use of antidotes to block E3 aptamer-drug conjugate cytotoxicity, providing a safety switch in the unexpected event of normal cell killing in vivo.