Tumor microenvironment in a minipig model of spinal cord glioma.

BACKGROUND: Spinal cord glioma (SCG) is considered an orphan disease that lacks effective treatment options with margins that are surgically inaccessible and an overall paucity of literature on the topic. The tumor microenvironment is a critical factor to consider in treatment and modeling design, especially with respect to the unresectable tumor edge. Recently, our group developed a high-grade spinal cord glioma (SCG) model in Göttingen minipigs. METHODS: Immunofluorescence and ELISA were performed to explore the microenvironmental features and inflammation cytokines in this minipig SCG model. Protein carbonyl assay and GSH/GSSG assay were analyzed in the core and edge lesions in the minipig SCG model. The primary core and edge cells proliferation rate were shown in vitro, and the xenograft model in vivo. RESULTS: We identified an elevated Ki-67 proliferative index, vascular and pericyte markers, CD31 and desmin in the tumor edge as compared to the tumor core. In addition, we found that the tumor edge demonstrated increased pro-inflammatory and gliomagenic cytokines including TNF-α, IL-1ß, and IL-6. Furthermore, the mediation of oxidative stress is upregulated in the tumor edge. Hypoxic markers had statistically significant increased staining in the tumor core, but were notably still present in the tumor edge. The edge cells cultures derived from SCG biopsy also demonstrated an increased proliferative rate compared to core cell cultures in a xenotransplantation model. CONCLUSIONS: Our study demonstrates heterogeneity in microenvironmental features in our minipig model of high-grade SCG, with a phenotype at the edge showing increased oxidative stress, proliferation, inflammatory cytokines, neovascularization, and decreased but present staining for hypoxic markers. These findings support the utility of this model as a means for investigating therapeutic approaches targeting the more aggressive and surgically unresectable tumor border.

Lentiviral-Induced Spinal Cord Gliomas in Rat Model.

Intramedullary spinal cord tumors are a rare and understudied cancer with poor treatment options and prognosis. Our prior study used a combination of PDGF-B, HRAS, and p53 knockdown to induce the development of high-grade glioma in the spinal cords of minipigs. In this study, we evaluate the ability of each vector alone and combinations of vectors to produce high-grade spinal cord gliomas. Eight groups of rats (n = 8/group) underwent thoracolumbar laminectomy and injection of lentiviral vector in the lateral white matter of the spinal cord. Each group received a different combination of lentiviral vectors expressing PDGF-B, a constitutively active HRAS mutant, or shRNA targeting p53, or a control vector. All animals were monitored once per week for clinical deficits for 98 days. Tissues were harvested and analyzed using hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Rats injected with PDGF-B+HRAS+sh-p53 (triple cocktail) exhibited statistically significant declines in all behavioral measures (Basso Beattie Bresnahan scoring, Tarlov scoring, weight, and survival rate) over time when compared to the control. Histologically, all groups except the control and those injected with sh-p53 displayed the development of tumors at the injection site, although there were differences in the rate of tumor growth and the histopathological features of the lesions between groups. Examination of immunohistochemistry revealed rats receiving triple cocktail displayed the largest and most significant increase in the Ki67 proliferation index and GFAP positivity than any other group. PDGF-B+HRAS also displayed a significant increase in the Ki67 proliferation index. Rats receiving PDGF-B alone and PDGF-B+ sh-p53 displayed more a significant increase in SOX2-positive staining than in any other group. We found that different vector combinations produced differing high-grade glioma models in rodents. The combination of all three vectors produced a model of high-grade glioma more efficiently and aggressively with respect to behavioral, physiological, and histological characteristics than the rest of the vector combinations. Thus, the present rat model of spinal cord glioma may potentially be used to evaluate therapeutic strategies in the future.

A Stereotactic Device for Intraparenchymal Spinal Cord Injections: Latest Developments for Practical Clinical Use.

This manuscript introduces the latest generation of a patient-mounted platform designed for segmental injections of therapeutics direct into the spinal cord parenchyma. It emphasizes its importance and it presents the rationale for developing this delivery methodology. It compares the newest with the previous generations, detailing how the modifications can streamline transportation, assembly, sterilization, and utilization of the platform by different surgeons. Finally, the illustrations depict the main alterations, as well as a cadaveric assessment of the device prototype in the cervical and thoracolumbar regions.

Thoracoscopic delivery of therapeutics in the swine sympathetic chain: Implications for future neuromodulation.

The thoracic sympathetic chain is implicated in several disease processes including palmar hyperhidrosis and complex regional pain syndrome. These diseases are often medically refractory and require surgical treatments including sympathectomies and ganglion blocks. The use of chemogenetic or optogenetic technologies to modulate sympathetic chain activity may be a potential treatment for these diseases. However, there is no established thoracoscopic surgical approach to deliver viral vectors into the thoracic sympathetic chain and ganglia. Our objective was to evaluate the feasibility of thoracoscopic injection of the swine sympathetic chain. Two Landrace farm pigs underwent a novel procedure for thoracoscopic sympathetic chain injections. One was non-survival and one was a five-day survival surgery. Both procedures successfully delivered methylene blue in the thoracic sympathetic chain. Over the five-day postoperative period, the animal displayed stable vital signs. Thoracoscopic targeted injections of the sympathetic chain is a feasible approach to deliver therapeutics in swine. Future studies should investigate the use of transgene expression as a potential means to control sympathetic output for the development of novel therapies for palmar or axillary hyperhidrosis, thoracic neuropathic pain syndromes and select peripheral vascular diseases.

Lentiviral Vector Induced Modeling of High-Grade Spinal Cord Glioma in Minipigs.

BACKGROUND: Prior studies have applied driver mutations targeting the RTK/RAS/PI3K and p53 pathways to induce the formation of high-grade gliomas in rodent models. In the present study, we report the production of a high-grade spinal cord glioma model in pigs using lentiviral gene transfer. METHODS: Six Gottingen Minipigs received thoracolumbar (T14-L1) lateral white matter injections of a combination of lentiviral vectors, expressing platelet-derived growth factor beta (PDGF-B), constitutive HRAS, and shRNA-p53 respectively. All animals received injection of control vectors into the contralateral cord. Animals underwent baseline and endpoint magnetic resonance imaging (MRI) and were evaluated daily for clinical deficits. Hematoxylin and eosin (H&E) and immunohistochemical analysis was conducted. Data are presented using descriptive statistics including relative frequencies, mean, standard deviation, and range. RESULTS: 100% of animals (n = 6/6) developed clinical motor deficits ipsilateral to the oncogenic lentiviral injections by a three-week endpoint. MRI scans at endpoint demonstrated contrast enhancing mass lesions at the site of oncogenic lentiviral injection and not at the site of control injections. Immunohistochemistry demonstrated positive staining for GFAP, Olig2, and a high Ki-67 proliferative index. Histopathologic features demonstrate consistent and reproducible growth of a high-grade glioma in all animals. CONCLUSIONS: Lentiviral gene transfer represents a feasible pathway to glioma modeling in higher order species. The present model is the first lentiviral vector induced pig model of high-grade spinal cord glioma and may potentially be used in preclinical therapeutic development programs.

Minimally Invasive Injection to the Phrenic Nerve in a Porcine Hemidiaphragmatic Paralysis Model: A Pilot Study.

BACKGROUND: Neurodegenerative diseases and spinal cord injury can affect respiratory function often through motor neuron loss innervating the diaphragm. To reinnervate this muscle, new motor neurons could be transplanted into the phrenic nerve (PN), allowing them to extend axons to the diaphragm. These neurons could then be driven by an optogenetics approach to regulate breathing. This type of approach has already been demonstrated in the peripheral nerves of mice. However, there is no established thoracoscopic approach to PN injection. Also, there is currently a lack of preclinical large animal models of diaphragmatic dysfunction in order to evaluate the efficacy of potential treatments. OBJECTIVE: To evaluate the feasibility of thoracoscopic drug delivery into the PN and to assess the viability of hemidiaphragmatic paralysis in a porcine model. METHODS: Two Landrace farm pigs underwent a novel procedure for thoracoscopic PN injections, including 1 nonsurvival and 1 survival surgery. Nonsurvival surgery involved bilateral PN injections and ligation. Survival surgery included a right PN injection and transection proximal to the injection site to induce hemidiaphragmatic paralysis. RESULTS: PN injections were successfully performed in both procedures. The animal that underwent survival surgery recovered postoperatively with an established right hemidiaphragmatic paralysis. Over the 5-d postoperative period, the animal displayed stable vital signs and oxygenation saturation on room air with voluntary breathing. CONCLUSION: Thoracoscopic targeting of the porcine PN is a feasible approach to administer therapeutic agents. A swine model of hemidiaphragmatic paralysis induced by unilateral PN ligation or transection may be potentially used to study diaphragmatic reinnervation following delivery of therapeutics.

Frameless Stereotactic Targeting of the Cerebellar Dentate Nucleus in Nonhuman Primates: Translatable Model for the Surgical Delivery of Gene Therapy.

BACKGROUND: Stereotactic targeting techniques in nonhuman primate (NHP) models are often utilized in the preclinical investigation of new drug therapies with the goal of demonstrating accurate and reliable delivery of a therapy to the target tissue. However, targeting certain neuroanatomical structures can be challenging. The deep cerebellar nuclei, specifically the dentate nucleus, are potential stereotactic targets for the treatment of certain ataxias. Currently, there are no detailed techniques describing frameless targeting of these structures in a NHP model. A well-defined, accurate, and reliable stereotactic surgical approach to the dentate in these animal models is critical to prove the feasibility and safety of drug delivery in order to develop clinical protocols. METHODS: Frameless stereotactic neuronavigation was employed to target the bilateral dentate nuclei of the cerebellum in four healthy juvenile Cynomolgus monkeys via a suboccipital, transcerebellar approach. The precision and accuracy of the targeting were evaluated radiologically and histologically. RESULTS: Using the described surgical methodology, we were successful in hitting the target deep cerebellar nuclei seven out of eight times. CONCLUSION: Frameless stereotactic targeting of the cerebellar dentate nuclei in NHPs for future investigational drug delivery is feasible, safe, and accurate as described by this report. Potential areas for improving the technique are discussed.

Peripheral blood detection of systemic graft-specific xeno-antibodies following transplantation of human neural progenitor cells into the porcine spinal cord.

Extensive pre-clinical and clinical studies have searched for therapeutic efficacy of cell-based therapeutics in diseases of the Central Nervous System (CNS) with no other viable options. Allogeneic cells represent the primary source of these therapies and immunosuppressive regimens have been empirically employed based on experience with solid organ transplantation, attempting to avoid immune mediated graft rejection. In this study, we aimed to 1) characterize the host immune response to stem cells transplanted into the CNS and 2) develop a non-invasive method for detecting immune response to transplanted cell grafts. Human neural progenitor cells were transplanted into the spinal cord of 10 Göttingen minipigs, of which 5 received no immunosuppression and 5 received Tacrolimus. Peripheral blood samples were collected longitudinally for flow cytometry cross match studies. Necropsy was performed at day 21 and spinal cord tissue analysis. We observed a transient increase in xeno-reactive antibodies was detected on post-operative day 7 and 14 in pigs that did not receive immunosuppression. This response was not detected in pigs that received Tacrolimus immunosuppression. No difference in graft survival was observed between the groups. Infiltration of numerous immune mediators including granulocytes, T lymphocytes, and activated microglia, and complement deposition were detected. In summary, a systemic immunologic response to stem cell grafts was detected for two weeks after transplantation using peripheral blood. This could be used as a non-invasive biomarker by investigators for detection of immunologic rejection. However, the absence of a detectable response in peripheral blood does not rule out a parenchymal immune response.

AAV gene delivery to the spinal cord: serotypes, methods, candidate diseases, and clinical trials.

INTRODUCTION: Adeno-associated viral (AAV) vector-mediated gene delivery to the spinal cord has finally entered the pathway towards regulatory approval. Phase 1 clinical trials using AAV gene therapy for pediatric disorders - spinal muscular atrophy (SMA) and giant axonal neuropathy (GAN) - are now underway. AREAS COVERED: This review addresses the latest progress in the field of AAV gene delivery to the spinal cord, particularly focusing on the most prominent AAV serotypes and delivery methodologies to the spinal cord. Candidate diseases and scaling up experiments in large animals are also discussed. EXPERT OPINION: Intravenous (IV) and intrathecal (IT) deliveries seem to undoubtedly be the preferred routes of administration for diffuse spinal cord delivery of therapeutic AAV vectors that can cross the blood-brain barrier (BBB) and correct inherited genetic disorders. Conversely, intraparenchymal delivery is still an undervalued but very viable approach for segmental therapy in afflictions such as ALS or Pompe Disease as a means to prevent respiratory dysfunction.

Ferumoxytol Labeling of Human Neural Progenitor Cells for Diagnostic Cellular Tracking in the Porcine Spinal Cord with Magnetic Resonance Imaging.

We report on the diagnostic capability of magnetic resonance imaging (MRI)-based tracking of ferumoxytol-labeled human neural progenitor cells (hNPCs) transplanted into the porcine spinal cord. hNPCs prelabeled with two doses of ferumoxytol nanoparticles (hNPC-FLow and hNPC-FHigh ) were injected into the ventral horn of the spinal cord in healthy minipigs. Ferumoxytol-labeled grafts were tracked in vivo up to 105 days after transplantation with MRI. Injection accuracy was assessed in vivo at day 14 and was predictive of "on" or "off" target cell graft location assessed by histology. No difference in long-term cell survival, assessed by quantitative stereology, was observed among hNPC-FLow , hNPC-FHigh , or control grafts. Histological iron colocalized with MRI signal and engrafted human nuclei. Furthermore, the ferumoxytol-labeled cells retained nanoparticles and function in vivo. This approach represents an important leap forward toward facilitating translation of cell-tracking technologies to clinical trials by providing a method of assessing transplantation accuracy, delivered dose, and potentially cell survival. Stem Cells Translational Medicine 2017;6:139-150.

Magnetic Resonance Imaging-Guided Transplantation of Neural Stem Cells into the Porcine Spinal Cord.

BACKGROUND: Cell-based therapies are a promising treatment option for traumatic, tumorigenic and degenerative diseases of the spinal cord. Transplantation into the spinal cord is achieved with intravascular, intrathecal, or direct intraparenchymal injection. The current standard for direct injection is limited by surgical invasiveness, difficulty in reinjection, and the inability to directly target anatomical or pathological landmarks. The objective of this study was to present the proof of principle for minimally invasive, percutaneous transplantation of stem cells into the spinal cord parenchyma of live minipigs under MR guidance. METHODS: An MR-compatible spine injection platform was developed to work with the ClearPoint SmartFrame system (MRI Interventions Inc.). The system was attached to the spine of 2 live minipigs, a percutaneous injection cannula was advanced into the spinal cord under MR guidance, and cells were delivered to the cord. RESULTS: A graft of 2.5 × 106 human (n = 1) or porcine (n = 1) neural stem cells labeled with ferumoxytol nanoparticles was transplanted into the ventral horn of the spinal cord with MR guidance in 2 animals. Graft delivery was visualized with postprocedure MRI, and characteristic iron precipitates were identified in the spinal cord by Prussian blue histochemistry. Grafted stem cells were observed in the spinal cord of the pig injected with porcine neural stem cells. No postoperative morbidity was observed in either animal. CONCLUSION: This report supports the proof of principle for transplantation and visualization of pharmacological or biological agents into the spinal cord of a large animal under the guidance of MRI.

Transplantation of spinal cord-derived neural stem cells for ALS: Analysis of phase 1 and 2 trials.

OBJECTIVE: To test the safety of spinal cord transplantation of human stem cells in patients with amyotrophic lateral sclerosis (ALS) with escalating doses and expansion of the trial to multiple clinical centers. METHODS: This open-label trial included 15 participants at 3 academic centers divided into 5 treatment groups receiving increasing doses of stem cells by increasing numbers of cells/injection and increasing numbers of injections. All participants received bilateral injections into the cervical spinal cord (C3-C5). The final group received injections into both the lumbar (L2-L4) and cervical cord through 2 separate surgical procedures. Participants were assessed for adverse events and progression of disease, as measured by the ALS Functional Rating Scale-Revised, forced vital capacity, and quantitative measures of strength. Statistical analysis focused on the slopes of decline of these phase 2 trial participants alone or in combination with the phase 1 participants (previously reported), comparing these groups to 3 separate historical control groups. RESULTS: Adverse events were mostly related to transient pain associated with surgery and to side effects of immunosuppressant medications. There was one incident of acute postoperative deterioration in neurologic function and another incident of a central pain syndrome. We could not discern differences in surgical outcomes between surgeons. Comparisons of the slopes of decline with the 3 separate historical control groups showed no differences in mean rates of progression. CONCLUSIONS: Intraspinal transplantation of human spinal cord-derived neural stem cells can be safely accomplished at high doses, including successive lumbar and cervical procedures. The procedure can be expanded safely to multiple surgical centers. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that for patients with ALS, spinal cord transplantation of human stem cells can be safely accomplished and does not accelerate the progression of the disease. This study lacks the precision to exclude important benefit or safety issues.

Preclinical Validation of Multilevel Intraparenchymal Stem Cell Therapy in the Porcine Spinal Cord.

BACKGROUND: Although multiple clinical trials are currently testing different stem cell therapies as treatment alternatives for many neurodegenerative diseases and spinal cord injury, the optimal injection parameters have not yet been defined. OBJECTIVE: To test the spinal cord's tolerance to increasing volumes and numbers of stem cell injections in the pig. METHODS: Twenty-seven female Göttingen minipigs received human neural progenitor cell injections using a stereotactic platform device. Cell transplantation in groups 1 to 5 (5-7 pigs in each) was undertaken with the intent of assessing the safety of an injection volume escalation (10, 25, and 50 µL) and an injection number escalation (20, 30, and 40 injections). Motor function and general morbidity were assessed for 21 days. Full necropsy was performed; spinal cords were analyzed for graft survival and microscopic tissue damage. RESULTS: No mortality or permanent surgical complications were observed during the 21-day study period. All animals returned to preoperative baseline within 14 days, showing complete motor function recovery. The histological analysis showed that there was no significant decrease in neuronal density between groups, and cell engraftment ranged from 12% to 31% depending on the injection paradigm. However, tissue damage was identified when injecting large volumes into the spinal cord (50 µL). CONCLUSION: This series supports the functional safety of various injection volumes and numbers in the spinal cord and gives critical insight into important safety thresholds. These results are relevant to all translational programs delivering cell therapeutics to the spinal cord.

Rational design and engineering of a modified adeno-associated virus (AAV1)-based vector system for enhanced retrograde gene delivery.

BACKGROUND: After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. OBJECTIVE: To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. METHODS: Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. RESULTS: Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. CONCLUSION: This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.

Analysis of graft survival in a trial of stem cell transplant in ALS.

OBJECTIVE: The first US Food and Drug Administration-approved clinical trial to treat amyotrophic lateral sclerosis (ALS) with neural stem cell-based therapy is in progress. The goal of the current study was to identify and assess the survival of human spinal cord-derived neural stem cells (HSSCs) transplanted into the spinal cord in patients with ALS. METHODS: Spinal cords transplanted with HSSCs were examined from six autopsy cases. Homogenized tissues were interrogated for the presence of donor versus recipient DNA using real-time PCR methods (qPCR). Fluorescence in situ hybridization (FISH) was performed using DNA probes for XY chromosomes to identify male donor HSSCs in one female case, and immunohistochemistry (IHC) was used to characterize the identified donor cells. RESULTS: Genomic DNA from donor HSSCs was identified in all cases, comprising 0.67-5.4% of total tissue DNA in patients surviving 196 to 921 days after transplantation. In the one female patient a "nest" of cells identified on H&E staining were XY-positive by FISH, confirming donor origin. A subset of XY-positive cells labeled for the neuronal marker NeuN and stem cell marker SOX2. INTERPRETATION: This is the first study to identify human neural stem cells transplanted into a human spinal cord. Transplanted HSSCs survived up to 2.5 years posttransplant. Some cells differentiated into neurons, while others maintained their stem cell phenotype. This work is a proof of concept of the survival and differentiation of human stems cell transplanted into the spinal cord of ALS patients.

Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). Previously, we showed that central nervous system (CNS) delivery of an adeno-associated viral (AAV) vector encoding SMN1 produced significant improvements in survival in a mouse model of SMA. Here, we performed a dose-response study in SMA mice to determine the levels of SMN in the spinal cord necessary for efficacy, and measured the efficiency of motor neuron transduction in the spinal cord after intrathecal delivery in pigs and nonhuman primates (NHPs). CNS injections of 5e10, 1e10, and 1e9 genome copies (gc) of self-complementary AAV9 (scAAV9)-hSMN1 into SMA mice extended their survival from 17 to 153, 70, and 18 days, respectively. Spinal cords treated with 5e10, 1e10, and 1e9 gc showed that 70-170%, 30-100%, and 10-20% of wild-type levels of SMN were attained, respectively. Furthermore, detectable SMN expression in a minimum of 30% motor neurons correlated with efficacy. A comprehensive analysis showed that intrathecal delivery of 2.5e13 gc of scAAV9-GFP transduced 25-75% of the spinal cord motor neurons in NHPs. Thus, the extent of gene expression in motor neurons necessary to confer efficacy in SMA mice could be obtained in large-animal models, justifying the continual development of gene therapy for SMA.

Intraspinal neural stem cell transplantation in amyotrophic lateral sclerosis: phase 1 trial outcomes.

OBJECTIVE: The US Food and Drug Administration-approved trial, "A Phase 1, Open-Label, First-in-Human, Feasibility and Safety Study of Human Spinal Cord-Derived Neural Stem Cell Transplantation for the Treatment of Amyotrophic Lateral Sclerosis, Protocol Number: NS2008-1," is complete. Our overall objective was to assess the safety and feasibility of stem cell transplantation into lumbar and/or cervical spinal cord regions in amyotrophic lateral sclerosis (ALS) subjects. METHODS: Preliminary results have been reported on the initial trial cohort of 12 ALS subjects. Here, we describe the safety and functional outcome monitoring results for the final trial cohort, consisting of 6 ALS subjects receiving 5 unilateral cervical intraspinal neural stem cell injections. Three of these subjects previously received 10 total bilateral lumbar injections as part of the earlier trial cohort. All injections utilized a novel spinal-mounted stabilization and injection device to deliver 100,000 neural stem cells per injection, for a dosing range up to 1.5 million cells. Subject assessments included detailed pre- and postsurgical neurological outcome measures. RESULTS: The cervical injection procedure was well tolerated and disease progression did not accelerate in any subject, verifying the safety and feasibility of cervical and dual-targeting approaches. Analyses on outcome data revealed preliminary insight into potential windows of stem cell biological activity and identified clinical assessment measures that closely correlate with ALS Functional Rating Scale-Revised scores, a standard assessment for ALS clinical trials. INTERPRETATION: This is the first report of cervical and dual-targeted intraspinal transplantation of neural stem cells in ALS subjects. This approach is feasible and well-tolerated, supporting future trial phases examining therapeutic dosing and efficacy.

Intraspinal stem cell transplantation in amyotrophic lateral sclerosis: a phase I trial, cervical microinjection, and final surgical safety outcomes.

BACKGROUND: The first US Food and Drug Administration approved clinical trial for a stem cell-based treatment of amyotrophic lateral sclerosis has now been completed. OBJECTIVE: Primary aims assessed the safety of a direct microinjection-based technique and the toxicity of neural stem cell transplantation to the ventral horn of the cervical and thoracolumbar spinal cord. Results from thoracolumbar-only microinjection groups have been previously published. Cervical and cervical plus thoracolumbar microinjection group perioperative morbidity results are presented. METHODS: Eighteen microinjection procedures (n = 12 thoracolumbar [T10/11], n = 6 cervical [C3-5]) delivered NSI-566RSC (Neuralstem, Inc), a human neural stem cell, to 15 patients in 5 cohorts. Each injection series comprised 5 injections of 10 µL at 4-mm intervals. The patients in group A (n = 6) were nonambulatory and received unilateral (n = 3) or bilateral (n = 3) thoracolumbar microinjections. The patients in groups B to E were ambulatory and received either unilateral (group B, n = 3) or bilateral (group C, n = 3) thoracolumbar microinjection. Group D and E patients received unilateral cervical (group D, n = 3) or cervical plus bilateral thoracolumbar microinjection (group E, n = 3). RESULTS: Unilateral cervical (group D, n = 3) and cervical plus thoracolumbar (group E, n = 3) microinjections to the ventral horn have been completed in ambulatory patients. One patient developed a postoperative kyphotic deformity prompting completion of a laminoplasty in subsequent patients. Another required reoperation for wound dehiscence and infection. The solitary patient with bulbar amyotrophic lateral sclerosis required perioperative reintubation. CONCLUSION: Delivery of a cellular payload to the cervical or thoracolumbar spinal cord was well tolerated by the spinal cord in this vulnerable population. This encouraging finding supports consideration of this delivery approach for neurodegenerative, oncologic, and traumatic spinal cord afflictions.

Regulated neuronal neuromodulation via spinal cord expression of the gene for the inwardly rectifying potassium channel 2.1 (Kir2.1).

BACKGROUND: Neuromodulation is used to restore neural function in disorders that stem from an imbalance in the activity of specific neural networks when they prove refractory to pharmacological therapy. The Kir2.1 gene contributes to stabilizing the resting potential below the threshold of activation of voltage-gated sodium channels and action potentials. Therefore, the delivery of the Kir2.1 gene to neuronal cells could reduce the probability of action potential generation, inhibiting excessive neural activity. OBJECTIVE: To address the hypothesis that overexpression of the inwardly rectifying potassium channel 2.1 (Kir2.1) gene could inhibit motor neuron activity and therefore be therapeutically used in gene-based neuromodulation. METHODS: To induce expression of Kir2.1, the inducible RheoSwitch promoter was used and controlled by ligand. In vivo gene expression was accomplished by an adenoviral vector to deliver unilaterally into the lumbar spinal cord of rats. RESULTS: Behavioral assays demonstrated that neuromuscular inhibition was exclusive to rats that received the ligand. Histological analysis also showed evidence of some motor neuron loss in these animals. Behavioral effects of Kir2.1 expression were completely reversible, arguing that the behavioral effect did not result from motor neuron death. CONCLUSION: Delivery of the gene for Kir2.1 inhibits neurons by resisting depolarization to the action potential threshold. Regulated neuronal expression of Kir2.1 may provide an elegant means for neuromodulation in a selected neuronal population.

Surgical technique for spinal cord delivery of therapies: demonstration of procedure in gottingen minipigs.

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.