MRI-guided, intrathecal delivery of hydrogel-embedded glial progenitors for treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disorder without healing. Patients suffering from ALS will usually die within 2-5 years of diagnosis. Recent advances in stem cells and nanotechnology have raised hopes of breakthroughs in treatment. It has recently been reported that glia cells have a significant role for the proper functioning of motor nerve cells, and effective methods of isolating glia cells (GPR) are established. Models with rodents have shown that GPR has great therapeutic potential. A cell-based therapy relies on the effective delivery of cells to areas with degenerated neurons. By using new nanomedicine, to characterize and monitor cell delivery systems and progression in treatment, we wanted to administer GRP to the spinal cord with the goal of saving lung function, which is a primary problem in ALS. To improve the survival and differentiation of the transplanted cells, we will utilize growth factors on nano-carriers that will be integrated with cells into an alginate gel for slow release. Both cells and gel were marked with MRI visible markers for monitoring, distributing, stability and degradation of alginate gel as well as cell migration. A hydrogel formulation that supports the survival of incorporated cells has been established and intrathecal injection into cerebrospinal fluid (CSF) has been performed in a svine model. The focus during these injections was feasibility and safety. Injection into CSF is associated with high uncertainty around the distribution of the hydrogel, and this was an important motivation for developing a technique that facilitates visualization of this procedure in real time. An alginate hydrogel with Mn2+ as a contrast agent for MRI was developed. Optimization of Mn concentration has been carried out in the alginate hydrogel and studies of Mn concentration have been carried out to determine in vitro toxicity with stem cells of svine (pMSCs). The MRI signal at both clinical field strength of 3 Tesla and high-field scanner of 7 Tesla has been assessed. The optimal concentration of MnCl2 to achieve the highest possible MRI signal with low toxicity was defined and used in intrathecal injections in svine. Good visualization of the location of the hydrogel was achieved. Our data with hydrogel visualization provided the basis for a patent application. To streamline these studies and reduce the number of animals, we developed a model system of anatomical conditions in the spinal cord. We used these screening formulations that also support long-term visualization. We identified three formulations with satisfactory injectability and stability of the MRI signal for at least a week. We then developed two formulations; one for quick assessment of biodistribution and signal lapse within 24 hours and another formulation where the hydrogel is visible with NMR for several days after injection. We have optimized alginate hydrogels with manganese for Mr-guided transplantation of cells in large animals. The tested hydrogels retain their structure after transplantation, which was an important goal of the project. Mn simplifies MRI monitoring of real-time distribution during transplantation. This is very important as it enables immediate intervention and relocation of the catheter to adjust the biodistribution of the hydrogel. Fast clearance within 24 hours is also a desired feature when diagnostic imaging after gel delivery is important in some cases. On the other hand, another formulation of the hydrogel is needed for long-term observation of the hydrogel. To achieve this, we have optimized hydrogels where the implementation of manganese oxide nanoparticles is used to extend the visualization time in magnetic resonance after administration in addition to new formulations of Mn alginate particles. Furthermore, in the project there are optimized formulations of injectable hydrogels (both alginate and HA) for intrathecal delivery in rodents. We have shifted the composition of the hydrogel and the timing of injection to facilitate injection into the Cisterna magna of ALS mice. Since one of the main goals of the project is to develop Imaging tools for monitoring injection and degradation of hydrogels in vivo, the hydrogel is as previously described, marked with Mn2 +. Practically all elements of this task have one achieved good detectability with T1 weighted MRI. As a final remark a start up company has been established in Poland based on the finings and achievements in the NanoTech4ALS project.

NovaMatrix tasks for the project was to development of injectable hydrogel formulations, rheological verification of formulation functionality and gel characterization. The development of intrathecally injectable and MRI-traceable hydrogel/nanocarrier composites may have a wide array of applications beyond the current application for treatment of ALS. The cells embedded within a hydrogels and supported by growth factor-laden nanocarriers could also be applied for treatment of other diseases with pathology in the spinal cord such as multiple sclerosis, transverse myelitis, ischemia or trauma. Hydrogels with highly tunable biodegradation and precise method of delivery in concert with the nanocarrier features could be used for slow release drug delivery to the spinal cord. NovaMatrix has successfully developed injectable hydrogels with real time MRI visibility and with functionality to serve cell theraphy applications.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with no cure. Patients who suffer from ALS pypically die within 2-5 years of diagnosis. Recent progress in the field of stem cells and nanotechnology has raised hope for a treatment breakthrough. The significant role of glia for the proper function of motor neurons has been recently reported, and efficient methods to isolate glial restricted progenitors (GRPs) have been established. Rodent models have shown that GRPs display high therapeutic potential; however, due to disseminated pathology in ALS, efficacy of cell-based therapy is contingent upon effective delivery of cells to sites of motor neuron degeneration. Thus we propose to use novel nanomedicine and imaging tools, to characterize cell delivery systems and monitor cell treatment progression. We will use human fetal GRPs and deliver them intrathecally targeting the cells primarily to the cervical spinal cord with the goal of rescuing respiratory function, which is a primary problem in ALS. To improve survival and differentiation of transplanted cells we will utilize growth factor-laden nanocarriers that will be embedded with cells into the hydrogel for slow release. Both cells and the gel will be labeled with MRI tracker for monitoring distribution, stability/degradation of the hydrogel and cell migration. We will first evaluate and optimize properties of the hydrogel in vitro focusing on biomechanical properties and supporting function for GRPs. We will then assess applicability of MRI for non-invasive monitoring of 19F-labeled hydrogel and SPIO-labeled GRPs. Hydrogel/nanocarrier/GRP composites will be delivered via the intrathecal route and evaluated in shiverer and SOD1 mice, followed by studies in pigs. For pig experiments we will use porcine GRPs (allografts) to maintain full relevance to human allotransplantation studies.Finally we will perform pre-clinical study in dogs suffering from degenerative myelopathy.