subpial-adeno-associated-virus-9-aav9-vector-delivery-in-adult-mice

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

University of California, San Diego

In the present study, we have characterized an atraumatic grafting technique which permits multiple, segmental, and lamina-specific injections into cervical or lumbar spinal cord. Cell injections were performed in spinally mounted rats of different ages and spinal cord size, using a micromanipulator and glass microcapillary connected to a digital microinjector. For grafting, we used human neuroteratoma (hNT) cells, BrdU-labeled rat spinal precursors or primary embryonic spinal cord neurons isolated from E14 spinal cord of the eGFP+ rat. Systematic quantification of grafted cells was performed using stereological principles of systematic random sampling and semi-automated optical Disector software. Volume reconstruction was performed using serial sections from grafted areas and custom-developed software (Ellipse) which permits "two reference points" semi-automated alignment of images, as well as volume reconstruction and calculation. By coupling these techniques, it is possible to achieve a relatively precise and atraumatic cell delivery into multiple spinal cord segments and specific spinal laminae. Consistency of the multiple grafts position in the targeted laminar areas was verified by a systematic volume reconstruction. Good survival of implanted cells for the three different cell lines used indicate that this grafting technique coupled with a systematic analysis of the individual grafting sites can represent a valuable implantation-analytical system.

Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study.

Transient spinal ischemia, a complication associated with aortic cross-clamp may lead to spastic paraplegia. Once fully developed this deficit is permanent. Quantitative histopathological assessments and pharmacological studies show that the ischemic spasticity is secondary to the loss of lumbar GABA and glycinergic inhibitory interneurons. In the present study, we investigated whether human hNT neurons or committed Sprague-Dawley rat spinal neuronal precursors (SNPs) when grafted into previously ischemic spinal segments depleted of inhibitory neurons would restore local inhibitory tone and ameliorate spasticity. Rats with functionally and electrophysiologically defined spasticity that received spinal graft of hNT neurons or neuronal precursors and immunosuppressive treatment displayed a progressive recovery of motor function that correlated with the improvement of otherwise exacerbated peripheral motor response evoked by stimulation of motor cortex. In contrast, in control, medium-injected or oligodendrocyte-grafted animals no significant therapeutic effect was seen. Stereological quantification of grafted neurons revealed 1-2% survival at three months after transplantation. These surviving neurons displayed a robust axo-dendritic sprouting and expression of markers typical of mature neurons including NSE, NeuN and synaptophysin. In both treatment groups a subpopulation of grafted neurons developed GABA immunoreactivity. These data provide evidence that a region specific grafting of hNT neurons or other neuronally committed cells, which have a potential to develop inhibitory neurotransmitter phenotype, represent an effective treatment modality to modulate ischemia-induced spastic paraplegia.

Spinal implantation of hNT neurons and neuronal precursors: graft survival and functional effects in rats with ischemic spastic paraplegia.

In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague-Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1-4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3-4 weeks after plating. A sub-population of plated neurons (35-40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10 mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 +/- 950%) and glutamate release (130 +/- 42%) during first 10 min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.

Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.

In recent studies using a rat aortic balloon occlusion model, we have demonstrated that spinal grafting of rat or human neuronal precursors or human postmitotic hNT neurons leads to progressive amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In the present study, we characterized the optimal dosing regimen and safety profile of human spinal stem cells (HSSC) when grafted into the lumbar spinal cord segments of naive immunosuppressed minipigs. Gottingen-Minnesota minipigs (18-23 kg) were anesthetized with halothane, mounted into a spine-immobilization apparatus, and received five bilateral injections of HSSC delivered in 2, 4, 6, 8, or 10 μl of media targeted into L2-L5 central gray matter (lamina VII). The total number of delivered cells ranged between 2,500 and 100,000 per injection. Animals were immunosuppressed with Prograf® for the duration of study. After cell grafting, ambulatory function was monitored daily using a Tarlov's score. Sensory functions were assessed by mechanically evoked skin twitch test. Animals survived for 6-7 weeks. Three days before sacrifice animals received daily injections of bromodeoxyuridine (100 mg/kg; IV) and were then transcardially perfused with 4% paraformaldehyde. Th12-L6 spinal column was then dissected; the spinal cord was removed and scanned with MRI. Lumbar transverse spinal cord sections were then cut and stained with a combination of human-specific (hNUMA, hMOC, hNSE, hSYN) or nonspecific (DCX, MAP2, GABA, CHAT) antibodies. The total number of surviving cells was estimated using stereological quantification. During the first 12-24 h after cell grafting, a modest motor weakness was observed in three of eight animals but was no longer present at 4 days to 7 weeks. No sensory dysfunction was seen at any time point. Postmortem MRI scans revealed the presence of the individual grafts in the targeted spinal cord areas. Histological examination of spinal cord sections revealed the presence of hNUMA-immunoreactive grafted cells distributed between the base of the dorsal horn and the ventral horn. In all grafts intense hMOC, DCX, and hSYN immunoreactivity in grafted cells was seen. In addition, a rich axodendritic network of DCX-positive processes was identified extending 300-700 μm from the grafts. On average, 45% of hNUMA-positive neurons were GABA immunoreactive. Stereological analysis of hNUMA-positive cells showed an average of 2.5- to 3-fold increase in number of surviving cells compared with the number of injected cells. Analysis of spinal structural morphology showed that in animals injected with more than 50,000 cells/injection or volumes of injectate higher than 6 μl/injection there was tissue expansion and disruption of the local axodendritic network. Based on these data the safe total number of injected cells and volume of injectate were determined to be 30,000 cells delivered in ≤6 μl of media. These data demonstrate that highly reproducible delivery of a potential cell therapeutic candidate into spinal parenchyma can be achieved across a wide range of cell doses by direct intraspinal injections. The resulting grafts uniformly showed robust cell survival and progressive neuronal maturation.

Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs.

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.

The goal of the present study was to develop a porcine spinal cord injury (SCI) model, and to describe the neurological outcome and characterize the corresponding quantitative and qualitative histological changes at 4-9 months after injury. Adult Gottingen-Minnesota minipigs were anesthetized and placed in a spine immobilization frame. The exposed T12 spinal segment was compressed in a dorso-ventral direction using a 5-mm-diameter circular bar with a progressively increasing peak force (1.5, 2.0, or 2.5 kg) at a velocity of 3 cm/sec. During recovery, motor and sensory function were periodically monitored. After survival, the animals were perfusion fixed and the extent of local SCI was analyzed by (1) post-mortem MRI analysis of dissected spinal cords, (2) qualitative and quantitative analysis of axonal survival at the epicenter of injury, and (3) defining the presence of local inflammatory changes, astrocytosis, and schwannosis. Following 2.5-kg spinal cord compression the animals demonstrated a near complete loss of motor and sensory function with no recovery over the next 4-9 months. Those that underwent spinal cord compression with 2 kg force developed an incomplete injury with progressive partial neurological recovery characterized by a restricted ability to stand and walk. Animals injured with a spinal compression force of 1.5 kg showed near normal ambulation 10 days after injury. In fully paralyzed animals (2.5 kg), MRI analysis demonstrated a loss of spinal white matter integrity and extensive septal cavitations. A significant correlation between the magnitude of loss of small and medium-sized myelinated axons in the ventral funiculus and neurological deficits was identified. These data, demonstrating stable neurological deficits in severely injured animals, similarities of spinal pathology to humans, and relatively good post-injury tolerance of this strain of minipigs to spinal trauma, suggest that this model can successfully be used to study therapeutic interventions targeting both acute and chronic stages of SCI.

Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study.

Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B) receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments.

Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1(G93A) rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1(G93A) rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1(G93A) animals.

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation.

Intraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury.

Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation.

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague-Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6 weeks to 3 months after cell grafting. In animals implanted with TAC pellets (3.4 mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1 ± 4.9 ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4-5.1 mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats.

Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival.

Development of progressive muscle spasticity resulting from spinal traumatic injury can be mediated by loss of local segmental inhibition and/or by an increased sensory afferent drive with resulting exacerbated α-motoneuron activity. To identify potential contributions of neuroactive substances in the development of such spasticity state, we employed a well-defined spinal injury-evoked spasticity rat model. Signaling molecules were analyzed in the spinal parenchyma below the level of spinal injury and in the corresponding dorsal root ganglion cells using Kinex™ antibody microarrays. The results uncovered the involvement of angiogenesis and neurodegeneration pathways together with direct cross-talk mediated by several hub proteins with SH-2 domains. At 2 and 5weeks after transection, up-regulation of several proteins including CaMKIV, RONα and PKCδ as well as MAPK3/ERK1 phosphorylation was observed in the spinal ventral horns. Our results indicate that these signaling molecules and their neuronal effector systems cannot only play an important role in the initiation but also in the maintenance of spasticity states after spinal trauma. The exclusivity of specific protein changes observed in lumbar spinal parenchyma but not in dorsal root ganglia indicates that new treatment strategies should primarily target specific spinal segments to prevent or attenuate spasticity states.

Signaling proteins in spinal parenchyma and dorsal root ganglion in rat with spinal injury-induced spasticity.

Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan.

A transgenic minipig model of Huntington's Disease.

Pluripotent stem cell-derived committed neural precursors are an important source of cells to treat neurodegenerative diseases including spinal cord injury. There remains an urgency to identify markers for monitoring of neural progenitor specificity, estimation of neural fate and follow-up correlation with therapeutic effect in preclinical studies using animal disease models. Cell surface capture technology was used to uncover the cell surface exposed N-glycoproteome of neural precursor cells upon neuronal differentiation as well as post-mitotic mature hNT neurons. The data presented depict an extensive study of surfaceome during neuronal differentiation, confirming glycosylation at a particular predicted site of many of the identified proteins. Quantitative changes detected in cell surface protein levels reveal a set of proteins that highlight the complexity of the neuronal differentiation process. Several of these proteins including the cell adhesion molecules ICAM1, CHL1, and astrotactin1 as well as LAMP1 were validated by SRM. Combination of immunofluorescence staining of ICAM1 and flow cytometry indicated a possible direction for future scrutiny of such proteins as targets for enrichment of the neuronal subpopulation from mixed cultures after differentiation of neural precursor cells. These surface proteins hold an important key for development of safe strategies in cell-replacement therapies of neuronal disorders.

Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation.

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.

Article	Year
Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study. Experimental neurology\| PubMed ID: 15473986	2004
Spinal implantation of hNT neurons and neuronal precursors: graft survival and functional effects in rats with ischemic spastic paraplegia. The European journal of neuroscience\| PubMed ID: 15525281	2004
Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts. Journal of neuroscience methods\| PubMed ID: 15585285	2005
Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs. Cell transplantation\| PubMed ID: 20412634	2010
Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PloS one\| PubMed ID: 21407814	2011
Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study. Journal of neurotrauma\| PubMed ID: 22029501	2012
Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity. PloS one\| PubMed ID: 22291989	2012
Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation. PloS one\| PubMed ID: 22916141	2012
Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation. Stem cell research & therapy\| PubMed ID: 23710605	2013
Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival. Experimental neurology\| PubMed ID: 23748136	2013
Signaling proteins in spinal parenchyma and dorsal root ganglion in rat with spinal injury-induced spasticity. Journal of proteomics\| PubMed ID: 23835433	2013
A transgenic minipig model of Huntington's Disease. Journal of Huntington's disease\| PubMed ID: 25063429	2013
Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation. Journal of proteomics\| PubMed ID: 26581640	2016
Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs. Molecular therapy. Methods & clinical development\| PubMed ID: 27462649	2016

Silvia Marsala

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