Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

• Klíčová slova
• GAD65 and VGAT, long-lasting pain reversal, neuropathic pain, precision spinal vector delivery, subpial injection technique, subpial non-human primates, subpial pig, targeted AAV9 delivery,
• MeSH
• buňky zadních rohů míšních MeSH
• mícha MeSH
• myši MeSH
• neuralgie * etiologie   terapie   MeSH
• nociceptory * MeSH
• prasata MeSH
• technika přenosu genů MeSH
• zadní rohy míšní MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

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.

• Klíčová slova
• glia limitans formation from grafted neural precursors, human-specific mRNA sequencing, immunodeficient rat, neuraxial neural precursor migration, subpial stem cell injection,
• MeSH
• krysa rodu Rattus MeSH
• nervové kmenové buňky metabolismus   MeSH
• parenchymatická tkáň cytologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

• MeSH
• amyotrofická laterální skleróza genetika   patofyziologie   terapie   MeSH
• atrofie MeSH
• degenerace nervu genetika   patofyziologie   terapie   MeSH
• Dependovirus metabolismus   MeSH
• interneurony patologie   MeSH
• lidé MeSH
• malá interferující RNA aplikace a dávkování   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mícha diagnostické zobrazování   patologie   patofyziologie   MeSH
• motorické evokované potenciály MeSH
• motorické neurony patologie   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• pia mater patologie   patofyziologie   MeSH
• prasata MeSH
• primáti MeSH
• progrese nemoci MeSH
• regulace genové exprese MeSH
• sbalování proteinů MeSH
• superoxiddismutasa 1 genetika   metabolismus   MeSH
• technika přenosu genů * MeSH
• umlčování genů * MeSH
• vývoj svalů MeSH
• zánět patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• malá interferující RNA MeSH
• messenger RNA MeSH
• superoxiddismutasa 1 MeSH

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

• MeSH
• audiovizuální záznam MeSH
• Dependovirus genetika   MeSH
• fluorescenční mikroskopie MeSH
• genetické vektory genetika   metabolismus   MeSH
• mícha metabolismus   MeSH
• mozek metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH