Gene therapy is the delivery of a gene designed to correct or lessen a disease state. This therapeutic approach has shown promise for many neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), and Parkinson’s disease (PD). However, in order to introduce a functional, therapeutic gene into the cells of an animal, a biological carrier termed a “vector” must be used.
Recently, the adeno-associated virus serotype 9 (AAV9) has been investigated as a potential vector for the delivery of therapeutic genes. Although, AAV9 has been shown to transduce both neuronal and non-neuronal cells, the distribution of transduction is dependent on the method of vector delivery. In this study we are investigating the biodistribution of AAV9 delivered through the ventricles of a rodent. Unlike other delivery approaches that may be more systemic or direct, such as intravenous or intraparenchymal delivery, respectively, delivery into the CSF-filled ventricles limits distribution of the vector to the central nervous system. Moreover, AAV9 has shown to transduce regions of the CNS that are commonly impaired in neurodegenerative disease.
Understanding the biodistribution of AAV9 injected intraventricularly may eventually allow us to exploit this vector to deliver therapeutic genes to treat neurodegenerative diseases.
Aberrant neuronal activity is the underlying mechanism of numerous neurological conditions such as spasticity, pain and epilepsy. The current standard of care for this aberrant activity includes both neurosurgical and pharmacological interventions. The surgical approaches of ablation or implanted devices though effective, have the pitfalls of irreversibility and device failure or infection, respectively. Pharmacological approaches to treat pain and spasticity can be subject to tolerance, addiction and other negative side effects. There is a clear need for a more elegant and reversible way to control aberrant neuronal activity to treat this wide array of neurological conditions. This type of therapy would have far reaching applications. Gene based neuro-modulation, (the use of gene therapy to control neuronal activity by delivering genes, which ultimately have an effect on synaptic activity) may be just such an approach.
The human cranial ventricular system is located deep within the substance of the brain and is comprised of interconnected fluid spaces that serve as a reservoir for cerebrospinal fluid (CSF). The ventricular system serves as both a site of CSF production and the conduit by which CSF gains access to bathe the entirety of the brain and spinal cord. Because of this unique access, CSF has been used as a conduit for delivery of some medications such as antibiotics and chemotherapeutics. Additionally, intraventricular cannulation and infusion represents an attractive methodology to achieve delivery of gene- or peptide-based therapeutics to the central nervous system. Other researchers have used non-human primates to effectively deliver viral vectors to the CSF while our laboratory has achieved a similar result when delivery is focused to the spinal cord in swine with minimal off-target delivery to peripheral tissues.
Current efforts intend to extend our demonstrated capability to deliver vectors to the spinal intrathecal space to cranial intraventricular delivery approaches in swine. Early efforts have used a freehand approach (top left) with accurate placement confirmed by spontaneous CSF flow (top right) and fluoroscopic visualization of contrast dye within the ventricle (bottom left). Catheter position has also been confirmed post-necropsy (bottom right). Results from these studies may be expected to validate swine as an alternative to non-human primates for application to a broad variety of vector-and peptide-based therapeutics delivered through an intraventricular route.
The treatment of patients with chronic craniofacial pain (pain in the head and face) presents a challenge to clinicians, as they are often refractory to traditional medical or surgical treatment interventions. The Boulis lab is working on the novel application of gene-based neuromodulation for the control of craniofacial pain, using established rodent models of Temporomandibular joint disorder (TMJD) and Trigeminal Inflammatory Compression (TIC) to assess efficacy. The TMJD model is a model for chronic nociceptive pain (pain caused by stimulation of pain receptors), while the TIC model is a model for neuropathic pain (pain caused by injury to the nerve).
Transgenes designed to affect the process of neural transmission will be delivered to the pain-processing pathway of the face – the trigeminal pain pathway. Special interest will be directed towards components of the trigeminal pain pathway such as the Trigeminal Ganglion (TG) and Spinal Nucleus of V (SNV). During this process, the utility of different viral vector-based delivery systems and routes will be studied in the two rodent models and compared. As a translational laboratory, the ultimate intention is to develop a gene-based neuromodulatory therapy that can be validated through human clinical trials.
Accurate targeting and graft tracking challenges limit the current methods available for cellular therapeutic delivery to the spinal cord. Improving how we visualize spinal cord anatomy in the operating room and how we locate cell grafts post-transplantation is essential to widespread clinical translation of stem cell therapy in the central nervous system.
In the lab, we are studying how using MR-guided delivery methods could improve the future of stem cell therapies. The cells are labeled with contrast agent which allows them to be visualized using MRI. MR-guided techniques permit real time visualization of the grey and white matter of the spinal cord, thus improving targeting accuracy and making it possible to confirm the delivery of cells immediately after injection. Furthermore, MR-guidance allows the possibility of a minimally invasive approach to the spinal cord. Employing an MRI compatible injection platform and adapted cannula, we intend to determine the feasibility of MR-guided intraparenchymal injections in the porcine model.
While neurotrophic factors have shown promise in animal studies, delivery of therapeutic amounts of proteins to patients remains a significant hurdle to effective therapy. One way to overcome these delivery problems is to provide the patient’s body with a way to produce these molecules on its own. Gene therapy allows us to genetically modify a patient’s own cells and turn them into neurotrophic factor factories. Using this approach, the patient’s motor neurons are being supported 24 hours a day, 7 days a week. Recent studies using invasive techniques have shown that this approach has merit. In order to find a safer alternative, we are studying the use of intrathecal injection of gene therapy vectors expressing the neurotrophic factor IGF-1. This approach involves the delivery of vector into the space between the spinal cord and its protective sheath (the dura), which causes fewer traumas than other delivery methods.
Stem cell transplantation represents a promising approach for the treatment of ALS. Although, multiple clinical trials are currently underway using this approach to treat ALS, there are still many gaps in our knowledge. These gaps include: understanding the maximum volume and number of stem cell injections tolerated by the spinal cord, understanding the immune response to transplantation in the spinal cord and optimizing immunosuppression treatment to minimize transplant rejection. Our Department of Defense funded study attempts to answer most of these questions. The study is divided into two Aims:
Aim 1: This aim focuses on providing critical data on the safety and accuracy of our cell transplantation technique. It is critical to understand the time course of transient surgical complications (morbidity). As surgeons, we must have a threshold for determining when to re-explore these patients in search of reversible causes of unexpected morbidity such as epidural hematomas. Additionally, it will help us to refine our technique to understand dangerous thresholds for number and volume of multiple injections. Finally, it will establish expectations to guide pre- and post-operative care.
Aim 2: Little is understood about the appropriate immunosuppressive therapy for spinal cord stem cell transplant recipients. Aggressive immunosuppressant therapy has formed the single biggest source for adverse events in current stem cell trials. Aim 2 will help us to minimize immunosuppression, preventing needless complications.
Graft localization, survival, and migration are crucial for the outcome and efficacy of spinal cord cell therapies. ALS patients whose cell therapy fails to penetrate the ventral horn are unlikely to achieve a substantial benefit from these cells. Survival, migration, and differentiation of neural cell grafts have been characterized in the rodent brain and spinal cord. Clinical trials have demonstrated variable results regarding survival of neural grafts. Cell transplantation, independent of the cell type, donor, or site, seems to be associated with long-lasting immunological rejection around the grafts.
While a considerable amount of literature exists for solid organ transplantation, little is known about long-term survival/rejection of cell grafts. Such disparities reinforce the challenges of translating cell graft strategies from large animal models into humans. Our understanding of the factors that predict survival of cell grafts in the CNS remains inadequate and requires detailed investigation.
Our aims are to elucidate the mechanisms of immunological rejection and to optimize immunosuppressive strategies. To achieve these aims, we employ both xenograft (human to pig) and allograft (pig to pig) models of intraspinal cell transplantation in our pig model.
Multiple clinical investigations of cell-based therapies transplanted in to the spinal cord are underway for a range of neurological diseases. Accurate assessment of clinical outcomes and therapeutic efficacy is complicated by the unknown fate of transplanted cell grafts, secondary to limited evidence confirming graft delivery and survival.
We aim to develop a clinically relevant method to pre-label transplanted cell grafts with MRI contrast agents, such as Super-Paramagnetic Iron Oxide Nanoparticles (SPION) and Gadolinium-conjugated nanoparticles. These approaches will allow us to visualize transplanted intraspinal cell grafts in vivo with MRI. With this ability, we will better understand the post-transplant fate of cell grafts.
To achieve these aims, we will transplant labeled cells in to the porcine spinal cord. The pigs will be serially scanned using a clinical MR scanner to monitor the fate of the transplanted cell grafts in vivo. Lastly, we will use this labeling and tracking approach to enable MR-guided intraspinal transplantation.