Development of a Translational Strategy to Overcome Muscle Paralysis Using Stem Cell Derived Neural Grafts and Optogenetics

The ability to control muscle contraction is critical to all motor behaviour in humans, including respiration. However, as a result of disease or traumatic injury, this ability is often lost, causing muscle paralysis that can be permanent, and even life-threatening. The extent of paralysis can vary greatly between affected individuals, ranging at one end of the spectrum from tetraplegia following high-level spinal cord injury and locked-in syndrome in late-stage amyotrophic lateral sclerosis (ALS), to more localized paralysis of individual limbs or muscle groups, following peripheral nerve injury. In addition to the devastating personal effects of paralysis, the socio-economic implications are also profound, for example following severe spinal cord injury. Importantly, no effective therapy currently exists that can restore lost motor function. There is therefore an urgent need to develop effective therapies to overcome paralysis.

In this project, we will build upon a novel strategy we have recently developed to overcome muscle paralysis in which specialised nerve cells produced from stem cells are transplanted into injured nerves in order to replace lost or damaged motor nerves. Since these nerve cells are grafted outside the central nervous system, away from their normal source of stimulation, we have genetically modified them so they can be stimulated with high specificity, using pulses of light. Thus, in our recent high-impact study, we showed that grafts of these photosensitive nerve cells, produced from mouse stem cells, survive when implanted in injured nerves in the hindlimb of mice, grow fibres to connect with paralysed muscles, and following stimulation with pulses of light, can induce contraction of the previously paralysed muscles.

This strategy has several key advantages over existing, conventional approaches to artificially restore muscle function, which only work when the motor nerve is intact and depend on delivering electrical stimuli directly to the nerve. Electrical stimulation is not only non-specific, activating both motor nerves that control muscle contraction as well as sensory fibres that convey pain, but can also directly damage muscle fibres. In contrast, optical stimulation is highly specific and painless, only activating the grafted photosensitive nerve cells, and critically, unlike electrical stimulation, activates muscles in "normal" fashion, thereby avoiding the rapid muscle fatigue associated with electrical stimulation.

In this project, we have assembled a team of leading experts including specialists in stem cells, electronics and muscle physiology, that will enable the development of this strategy from the current proof of concept stage, using mouse nerve cells in mouse models of muscle paralysis, towards a more clinically relevant stage applicable for use in humans. Specifically, we will develop human motor nerve cells, produced from adult human stem cells that have been safely modified to enable them to be controlled using pulses of light. Moreover, we will develop implantable light stimulators that can be used to control the function of specific muscles in awake, freely moving animals. Finally, we will demonstrate the ability to finely control a pair of paralysed opposable muscles in the rat forepaw to emulate hand-grasping, as an example of a functionally useful human movement that could be restored using this strategy.

In principle, the complexity of the motor function that can be artificially controlled using this approach is limited only by the sophistication of the optical stimulation device. In addition, by coupling this strategy with emerging technology that can decipher the brain's intention to carry out a specific movement (brain-computer interface technology), it will pave the way for restoring autonomous control over the body's own muscle in a diverse range of patients affected by paralysis.

This project aims to develop our novel strategy to restore lost motor function, using a combination of stem cell-derived neural replacement of lost or damaged motor neurons and optogenetic stimulation to restore control of muscle function. We have previously demonstrated the feasibility of using peripherally grafted mouse embryonic stem cell-derived (mESC) motor neurons that have been optogenetically modified, to restore function to paralysed muscles, using optical stimulation. This approach has significant advantages over conventional approaches to overcome paralysis that typically involve electrical stimulation: i) the neural graft can be specifically and physiologically controlled using light, thus avoiding painful activation of sensory afferents and rapid muscle fatigue; ii) this approach does not depend on the survival of endogenous axons, as the grafted neurons grow to reinnervate target muscles following degeneration or damage to motor neuron axons.
A key objective of this study is to pave the way for the essential translational transition from proof-of-concept stage, using mESCs in murine injury and disease models, towards pre-clinical development that will provide an efficient method for safely modifying human induced pluripotent stem cells (iPSCs) and demonstrate the ability of iPSC-derived motor neurons to reinnervate flexor and extensor muscles in the forelimb of adult rats.
In principle, the complexity of the motor function that can be artificially controlled using this strategy is limited only by the sophistication of the optical stimulation device. Moreover, coupling of this strategy with brain-computer interface technology would enable the restoration of autonomous control of complex motor functions in a diverse range of patients affected by paralysis. In summary, this strategy enables the bypassing of motor signal blockade that causes paralysis, irrespective of the cause, thus providing a novel therapeutic route to restore lost motor function.

Exploitation and Application (Year 3): The impact of this multidisciplinary, highly-translational application is potentially enormous, as this strategy is applicable to all forms of paralysis caused by injury such as spinal cord injury or disease, such as ALS. Moreover, the proposed strategy to restore function to paralysed muscles could in the future be coupled with brain computer interface technology to provide a means of restoration of autonomous motor control in paralysed patients. Given the diversity of causes of paralysis it is difficult to accurately estimate the number of potential beneficiaries, however, in the case of SCI alone, the WHO estimates an annual incidence of 250,000 to 500,000 new cases worldwide. Any treatment capable of improving quality of life for people with paralysis, and reducing the high socio-economic costs associated with caring with these patients, would thus have enormous benefits, even if it were to only restore basic motor functions.

In addition, the development of efficient and safe methods to carry out gene-targeting of human adult stem cells would have broad utility in an array of cell-therapy strategies for different diseases.

This research promises to create valuable intellectual property (IP), as an optical stimulator device would be highly desirable for the treatment of patients suffering from a variety of disorders. The PIs will engage with UCL Business to identify new IP should it arise. IP arising shall be owned by UCL. Commercial Partners will be sought at an early stage following the filing of any patent applications.

Advancing Training (Years 1-3): The proposed grouping (PIs and collaborators) have considerable expertise in the molecular, cellular and morphological analyses of neural tissue, as well as the development of implantable stimulator devices, which will provide excellent training opportunities for the postdoctoral researchers employed on this grant, in a variety of skill sets. Our postdoctoral researchers will be actively involved in the supervision of PhD and MSc student projects, which will facilitate the development of skills required to manage and supervise lab projects.

Communication and Dissemination of Data (Years 2-3): We support the RCUK's policy to make the research data generated via their funding to be available to the community in as timely a manner as possible. Data will be archived in the collaborating Departments and centrally collected in Prof. Greensmith's group. Our data will be made available through peer-reviewed publications with an Open-Access policy of not more than 12 months ('Green' option). Whenever possible, we will choose immediate unrestricted access ('Gold' option). Any reagents produced will be made freely available to the scientific community immediately on publication. We plan to publish and/or present our data at meetings.

Public Understanding of Science (Years 1-3): We will engage with school students for example by working with the educational charity In2ScienceUK, which places school students from low-income backgrounds for work experience in laboratories to encourage them to pursue a career in science. We have a strong track record in both conventional and innovative public engagement. Our overarching vision of engagement is to inform, listen to and collaborate with diverse, clearly defined, audiences to help shape our research and public engagement priorities. Our proposal lends itself to innovative engagement through dance and/or theatre (particularly concepts such as stem cells, optogenetics, paralysis). We have established collaborations with a dance company and a talented set of actors and directors with a proven track record of high quality engagement. We will also generate video blogs or 'vlogs' to promote widely disseminated engagement, encouraging feedback through a jargon-free dialogue.