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. ...

Czech Academy of Sciences

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.

This study describes the results achieved using a combination of allogeneic mesenchymal stem cells (MSCs) with chondrocytes (CHC) and a new scaffold consisting of type-I collagen and chitosan nanofibers in the prevention of partial growth plate arrest after iatrogenic injury in pigs.

Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs.

A safe closure technique of transluminal access is essential for the widespread application of natural orifice transluminal endoscopic surgery (NOTES).

Single loop-and-clips technique (KING closure) for gastrotomy closure after transgastric ovariectomy: a survival experiment.

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.

An important component for successful translation of cell replacement-based therapies into clinical practice is the utilization of large animal models to conduct efficacy and/or safety cell dosing studies. Over the past few decades, several large animal models (dog, cat, nonhuman primate) were developed and employed in cell replacement studies; however, none of these models appears to provide a readily available platform to conduct effective and large-scale preclinical studies. In recent years, numerous pig models of neurodegenerative disorders were developed using both a transgenic approach as well as invasive surgical techniques. The pig model (naïve noninjured animals) was recently used successfully to define the safety and optimal dosing of human spinal stem cells after grafting into the central nervous system (CNS) in immunosuppressed animals. The data from these studies were used in the design of a human clinical protocol used in amyotrophic lateral sclerosis (ALS) patients in a Phase I clinical trial. In addition, a highly inbred (complete major histocompatibility complex [MHC] match) strain of miniature pigs is available which permits the design of comparable MHC combinations between the donor cells and the graft recipient as used in human patients. Jointly, these studies show that the pig model can represent an effective large animal model to be used in preclinical cell replacement modeling. This review summarizes the available pig models of neurodegenerative disorders and the use of some of these models in cell replacement studies. The challenges and potential future directions in more effective use of the pig neurodegenerative models are also discussed.

Pig models of neurodegenerative disorders: Utilization in cell replacement-based preclinical safety and efficacy studies.

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.

Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes.

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars.

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.

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

Novel, less invasive approaches such as single-incision laparoscopic cholecystectomy or natural orifice transluminal endoscopic surgery require preclinical evaluation and training. Therefore, there is a need for an experimental model closely mimicking the clinical situation. The aim of our study was to create an experimental model of calculous cholecystitis in a large laboratory animal and test its feasibility for the evaluation of different techniques of cholecystectomy.

Article	Year
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
Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs. Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia\| PubMed ID: 22837133	2012
Single loop-and-clips technique (KING closure) for gastrotomy closure after transgastric ovariectomy: a survival experiment. Wideochirurgia i inne techniki maloinwazyjne = Videosurgery and other miniinvasive techniques\| PubMed ID: 23362421	2012
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
Pig models of neurodegenerative disorders: Utilization in cell replacement-based preclinical safety and efficacy studies. The Journal of comparative neurology\| PubMed ID: 24610493	2014
A transgenic minipig model of Huntington's Disease. Journal of Huntington's disease\| PubMed ID: 25063429	2013
Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars. Neuro-degenerative diseases\| PubMed ID: 26959244	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
A new experimental model of calculous cholecystitis suitable for the evaluation and training of minimally invasive approaches to cholecystectomy. Surgical endoscopy\| PubMed ID: 27495340	2017

Jana Juhasova

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