Gene Therapy for Neuropathic Pain through siRNA-IRF5 Gene Delivery with Homing Peptides to Microglia

Abstract

Astrocyte- and microglia-targeting peptides were identified and isolated using phage display technology. A series of procedures, including three cycles of both in vivo and in vitro biopanning, was performed separately in astrocytes and in M1 or M2 microglia, yielding 50–58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocytes (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely, astrocytes, M1 microglia, or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between peptide MG1 and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells.

Stem cell factor induces polarization of microglia to the neuroprotective phenotype in vitro

“…After decapitation of six C57BL/6 pups 1–2 days postpartum at the same time, mixed glial cells from brain cortex were dissociated, and primary mixed glial cell-culture was established ( Fig. 1 ) [ 24 , 25 ]. After incubation of these mixed glial cells with Dulbecco's Modified Eagle Medium (DMEM)/F12 with 10% FBS (foetal bovine serum) for 14 days at 37 °C, many number of microglia were appeared on layered astrocytes, were isolated by mechanical tapping and transferred to another culture dish.…”

Advanced Technology for Gene Delivery with Homing Peptides to Spinal Cord through Systemic Circulation in Mice

“…35 In previous reports, we demonstrated DRG-targeting gene therapy or microglia-specific gene delivery in the spinal cord by intrathecal injection. 6,10 Lumbar puncture and intrathecal injection are usually performed clinically. Side effects include headache and intra- cranial hypotension.…”

“…6 In another study, homing peptides to microglia and astrocytes were also identified and applied for the delivery of small interfering RNA (siRNA) to treat neuropathic pain in a mouse model. 10 Although homing peptides have great potential applicability and could become powerful tools for drug and gene delivery, these experiments showed successful results of gene therapy only by the intrathecal route of administration. 6,10 Intrathecal injection after lumbar puncture is routinely performed clinically.…”

“…10 Although homing peptides have great potential applicability and could become powerful tools for drug and gene delivery, these experiments showed successful results of gene therapy only by the intrathecal route of administration. 6,10 Intrathecal injection after lumbar puncture is routinely performed clinically. However, a less-invasive route, such as intravenous injection, is desirable for patients, especially if repeated injection is required for therapy.…”

The involvement of microglia in Alzheimer's disease: a new dog in the fight

“…In addition, novel microglial targets have been identified. Homing peptides directed towards microglia that contained siRNA for IRF5 resulted in a decrease in neuropathic pain (Terashima et al, ). Injection of microglial precursor cells transduced with TREM2 ex vivo in an EAE model of multiple sclerosis facilitated the clearance of cellular debris and ameliorated the disease (Takahashi et al, ).…”