SBIR-STTR Award

Innovative drug/therapy combinations directed at multiple and proven therapeutic targets have the potential to dramatically improve outcomes after SCI. Studies on axon regeneration and recovery in rodents have revealed that the spinal cord is not hard wired and "learning" can occur in spinal cord circuits. Compounds that elicit axonal regeneration and sprouting help plasticity in the spinal cord and dramatically improve recovery from traumatic injury, at least in rodents. Many different compounds/therapies tested in rodents promote regeneration. Almost without exception, compounds that promote regeneration also improve functional recovery after spinal cord injury. Recently, very impressive axon regeneration was obtained from deletion of PTEN. The goal of this application is to simultaneously evaluate PTEN as target for therapeutic treatment of spinal cord injury and a novel RNAi delivery technology to provide prolonged in vivo gene knockdown effect. We will create "self-delivering" small interfering RNAs (sdRNA) targeting PTEN. The chemical modification introduced into sdRNA molecules makes them cell- permeable. This technology has been demonstrated to work in vivo in several applications, including delivery to the retina. As the first step we will synthetize a panel of sdRNA sequences and evaluate them in mammalian cell cultures to select lead candidate(s) efficient in PTEN knockdown. Next, we will test the compounds in neuron cell cultures, then with primary neurons. We will also examine effects on morphology and proliferation of primary glial cells and human glioblastoma cells. Once we determine lead candidate(s) that promote robust neurite growth, we will test the compounds for efficacy of knockdown in vivo. We will confirm ability to promote regeneration using an optic nerve model because retinal ganglion cells are known to respond to PTEN knockdown. Safety will be assessed by following clinical signs and clinical chemistry.

Public Health Relevance Statement:


Public Health Relevance:
A novel target to promote robust regeneration of axons in the injured central nervous system (CNS) has been identified as a protein called PTEN. The goal of this application is to create a novel RNA interference therapeutic that will easily penetrate neurons to silence PTEN and promote axon regeneration after injury. Modulation of PTEN expression has the potential to improve recovery after CNS injury by helping neurons create new adaptive circuitry to overcome deficits caused by lost connections after injury.

Project Terms:
Aftercare; Animals; Axon; axon regeneration; axonal sprouting; Biochemical; Cell Culture Techniques; Cell Survival; Cells; central nervous system injury; Chemicals; Clinical; Clinical Chemistry; Combination Drug Therapy; Dermal; design; Development; Dose; Double-Stranded RNA; drug candidate; drug development; efficacy testing; EGF gene; Ensure; Environment; experience; Gene Silencing; Genes; Glioblastoma; Goals; Growth; Hela Cells; Human; improved; In Vitro; in vitro testing; in vivo; Inflammation; injured; Injury; innovation; knock-down; Label; Lead; Learning; Length; Lysine; Mammalian Cell; Measurement; Measures; Mediating; Methods; Modeling; Modification; Molecular; Morphology; Natural regeneration; Nerve Crush; Neuraxis; neurite growth; Neurites; Neuroglia; Neuronal Differentiation; Neurons; novel; Operative Surgical Procedures; Optic Nerve; Outcome; PC12 Cells; Pharmaceutical Preparations; programs; Proteins; PTEN gene; public health relevance; Recombinants; Recovery; Recovery of Function; regenerative; repaired; Reporter; Research; research study; response; Retina; Retinal; Retinal Ganglion Cells; Risk; RNA; RNA Interference; RNA Sequences; Rodent; Safety; Signal Transduction; Silicon Dioxide; Small Interfering RNA; Spinal Cord; Spinal cord injury; System; Technology; Testing; Therapeutic; therapeutic target; Therapeutic Uses; Toxic effect; Transgenic Mice; Translating; tumor; Viral Vector; Western Blotting; Work

Studies on axon regeneration and recovery in rodents have revealed that the spinal cord is not hard wired and “learning” can occur in spinal cord circuits. Compounds that elicit axonal regeneration and sprouting help plasticity in the spinal cord and dramatically improve recovery from traumatic injury, at least in rodents. Intrinsic signals that progress with normal neuronal differentiation play a significant role in preventing axon regeneration. Several intrinsic negative regulators of regeneration have been identified through studies of knock-out mice. Suppression of synthesis of PTEN protein can promote axon regeneration and recovery of motor function in adult rodents. There has been a concerning lack of translation in SCI research for the most promising targets, and therefore we decided to create a compound with potential for therapeutic development. We chose the technology of self-deliverable RNA interference (sd-rxRNAi) because of the simplicity and proven efficacy of in vivo delivery to the central nervous system. BA- 434 as a novel sd-rxRNA that targets mRNA encoding PTEN and suppress the expression of PTEN protein. It has sequence homology to rat, pig and human, and was selected from 20 that we screened. We propose to further development of BA-434 and carry out IND-enabling safety and efficacy experiments to translate these findings to clinical study. We will optimize stability of BA-434, and dose and efficacy in spinal cord injury models. We will also confirm appropriate drug delivery in pig spinal cord because the size of pig spinal cord better approximates the size of human spinal cord. The proposed research will form the basis of non-GLP safety and efficacy studies in support of an IND application.

Public Health Relevance Statement:
After neurotrauma, neurons in the central nervous system (CNS) do not regenerate their injured axons. A protein called PTEN, known to be an intrinsic barrier to regeneration, blocks regeneration in the adult CNS. Through Phase I SBIR funding, we have created a novel therapeutic compound, BA-434, that acts by RNA interference to suppress PTEN mRNA expression. BA-434 easily penetrates cells because of specialized chemistry, and when applied to the injured CNS it promotes robust axon regeneration. The goal of this application is to examine the safety and feasibility to use BA-434 to promote recovery of motor function after spinal cord injury.

Project Terms:
Adult; Aftercare; Animals; Astrocytes; Axon; axon regeneration; axonal sprouting; base; Biochemical; Biological Assay; Biology; Blood specimen; Cell Line; Cell Survival; cell type; Cells; Chemistry; Clinical; Clinical Chemistry; Clinical Research; Coagulation Process; Control Groups; Contusions; Corticospinal Tracts; Data; Databases; Development; Distal; Dose; Drug Delivery Systems; Drug Kinetics; efficacy study; Enzyme-Linked Immunosorbent Assay; experience; experimental study; Family suidae; Female; foot; Formulation; Funding; Future; Goals; Growth; Hematology; Histologic; Human; Hybrids; immunocytochemistry; improved; In Vitro; in vivo; in vivo regeneration; injured; Injury; Interferons; knock-down; Knockout Mice; Label; Lead; Learning; Longevity; male; Measurable; Measures; Mediating; Messenger RNA; Methods; Modeling; Modification; Molecular; motor function recovery; Motor Neurons; mRNA Expression; Natural regeneration; Nervous System Trauma; Neuraxis; Neuroglia; Neuronal Differentiation; Neurons; novel; novel therapeutics; nuclease; Nucleic Acids; Operative Surgical Procedures; Outcome; Pathology; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Pharmacology; Phase; phosphorothioate; Play; pre-clinical research; prevent; programs; protein expression; Protein phosphatase; Proteins; PTEN gene; PTEN protein; Rattus; Recovery; Recovery of Function; Regulatory Pathway; Research; response; RNA Interference; Rodent; Role; Route; Safety; safety and feasibility; safety study; Sequence Homology; Serotonin; Serum; Signal Transduction; Site; Small Business Innovation Research Grant; Spinal Cord; Spinal cord injury; Technology; Testing; Therapeutic; therapeutic development; Time; Tissue Sample; Toxic effect; Translating; Translations; Traumatic injury; Walking; Western Blotting