Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients

Survival of syngeneic and allogeneic iPSC–derived neural precursors after spinal grafting in minipigs

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)–mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis

オリゴヌクレオチドを用いた長期的脊髄グリシントランスポーター2 (GlyT2)発現抑制による反射亢進及び痙縮に対する抑制効果は経時的・ニ相性に変化する

博士(医学)琉球大

Thoracic 9 Spinal Transection-Induced Model of Muscle Spasticity in the Rat: A Systematic Electrophysiological and Histopathological Characterization.

The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABAB receptor agonist), tizanidine (α2-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity

Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats.

Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury

Development of tactile hypersensitivity in rats at chronic stages after spinal transection.

<p><b>(A)</b>—Application of tactile stimuli (von Fray filaments; 1-15g) on the plantar surface of hind paw led to a clear EMG response measured by surface EMG electrodes from gastrocnemius muscle in animals at 3 months post-spinal-transection (TSCT 1, 2, 3). No response was seen in naive non-injured animals. Application of 5 repetitive stimuli at the same pressure (15g) and delivered every 5 seconds led to a consistent responses after each stimulus (A-right panels). <b>(B, C)</b>- Statistical analysis of repetitive (5x stimuli) tactile stimulus-evoked EMG responses separated by 10–15 seconds intervals showed a significant increase in transected animals (compared to naïve controls) at paw pressures between 2–15 grams (one-way ANOVA; Bonferroni post hoc; *-p< 0.05; **-p< 0.01; ***-p< 0.001). <b>(D, E)</b>- Statistical analysis of repetitive tactile stimulus-evoked (5x stimuli) EMG responses separated by 5 seconds intervals showed a comparable significant increase in animals with transection as seen after application of stimuli separated by 10–15 sec intervals (one-way ANOVA; Bonferroni post hoc; *-p< 0.05; **-p< 0.01; ***-p< 0.001).</p

Potentiation of spinal hyper-reflexia by increased angle and velocity of ankle rotation.

<p><b>(A, B, C, D)</b>—Comparing the effect of 40° to 80°of ankle rotation if ankle is rotated at 40, 200 or 400°/sec showed the most potent EMG response and corresponding increase in peripheral muscle resistance (PMR) at 80°of ankle rotation delivered at 400°/sec.</p

Increase in Hoffmann reflex and loss of rate-dependent depression (RDD) in spinally transected rats at 3 months after transection.

<p><b>(A, B)</b>—Measurement of H-reflex and statistical analysis of the H/M ratio showed a significant increase in responses at 3 months after transection if compared to wild-type non-injured animals (unpaired two-tailed t-test; ***P< 0.001). <b>(C)</b>—Testing of RDD showed a significant loss of RDD in spinally-transected animals at stimulation frequencies of 1, 5 and 10 Hz (one-way ANOVA; Bonferroni post hoc; ***P< 0.001).</p