Biotech startup puts new spin on regenerative medicine for nervous system repair
An early stage, Midwest biotech startup with a unique approach to treating damaged nervous system tissue is looking for its first round of capital. Axonia Medical has raised $1 million of the $3 million it hopes to secure in a series A financing that would carry the Kalamazoo, Michigan-based biotech through large animal trials of its regenerative nervous system repair technology. The nervous system has a limited ability to repair itself after injury, and there is a lack of effective treatment options to restore function, especially in brain and spinal cord injuries, according to Axonia cofounder, President and CEO Harry Ledebur. “There’s not a well-established market or standard of care for peripheral nervous system injuries, and a number of injuries with large lesions or that are close to the spinal cord can’t be treated right now,” he said. The startup company’s technology aims to surgically rebuild and restore damaged nervous systems using living functional tissue-engineered nerve grafts, which can bridge lost nervous tissue and jump-start regenerative mechanisms in the body to restore functionality. These grafts are essentially implantable nervous tissue — not cells — made of two neuron populations connected by long stretches of functioning axons that are grown and stretched out in a lab.
Approximately 400,000 individuals in the U.S. suffer from injuries to the peripheral nervous system, with less a quarter of them achieving good restorative function. Although it’s still in the preclinical phase, Axonia’s technology has applications to repair damage that is currently untreatable in a market Ledebur believes to be worth more than $1 billion. He said trials have produced positive preclinical results in small rodents with peripheral and spinal injuries, and the series A funding will help transition the company to 18 to 24 months of large animals trials. If those trials produce positive results, Ledebur thinks he’ll be able to secure more funding from coastal venture groups and partners. The company anticipates a $25 million series B round. Founded in 2011, Axonia leverages technology developed by Dr. Douglas Smith at the University of Pennsylvania. CEO Ledebur is a two-time entrepreneur and an executive-in-residence at the Southwest Michigan First Life Science Fund. For now, the company will focus on the peripheral nervous system application but will continue researching the use of its technology in follow-on products for spinal cord and brain injuries.
Smith Neurotrauma Laboratory
The Smith Neurotrauma Laboratory focuses on the mechanical events at the time of brain injury and how they relate to long-term outcome. It specifically concentrates on the fate of nerve fibers in the brain, called “axons,” that appear to be exquisitely vulnerable to trauma. Brain trauma is a devastating disease that affects over 2 million people in the United States each year. However, the mechanisms of brain trauma have only begun to be elucidated. Scientists know that some unique features of the physical damage induced by brain trauma can trigger progressive degenerative damage. This startling finding is the basis of the research efforts at Penn.
Researchers have found that the key features of the human brain that allow humans to design and drive automobiles are also their greatest liability in the event of a crash. With its massive size and high organization, the human brain can literally pull itself apart under the physical forces associated with traumatic brain injury. In particular, axons in the white matter appear poorly prepared to withstand damage from rapid mechanical deformation of the brain during trauma. Accordingly, axonal injury is one of the most common pathologies resulting from brain trauma, where the extent of axonal damage is thought to play a major role in functional outcome.
While a primary strategy to repair spinal cord and other nerve injuries is to bridge the damage with axons, producing axons of sufficient length and number has posed a significant challenge. In the Smith Laboratory, scientists explore the ability of integrated central and peripheral nervous system (CNS and PNS) axons to grow long distances in response to continuous mechanical tension. Using a microstepper motor system, they have physically split an integrated neuron culture in two and progressively separated the two populations of neurons. The tension on the axons spanning the expanding gap induced “stretch-induced growth.” Using this novel mechanism of axon growth, they are able to produce remarkably thick bundles of thousands of axons that can be grown to several centimeters in length. They propose that this technique may be exploited to produce transplant materials to bridge extensive nerve damage.
Engineering Nerve Constructs for Clinical Application
B.J. Pfister1, J. Huang1, E.L. Zager1, A. Iwata1, D.F.Meaney2, A.S. Cohen3,4, D.H.Smith1
Departments of (1)Neurosurgery, (2)Bioengineering, and (3)Pediatrics, University of Pennsylvania and Division of Neurology; (4)Children’s Hospital of Philadelphia
In the United States, tens of thousands of peripheral nerve injuries occur each year, many resulting in the loss of bodily functions and even permanent disability. The gold standard of peripheral nerve repair traditionally relies on a surgical procedure that involves the removal and transplantation of an autograft, a separate, less important length of nerve from the patient.
In some patients the damaged nerve will regenerate, using the autograft as a guide, leading to restoration of lost bodily functions. However, some major peripheral nerve deficits that can typically be repaired are not due to the limited availability of nerves that the patient can spare (for example, an insufficient supply of donor nerves for the reconstruction of a major brachial plexus injury).
There are a number of disadvantages associated with this technique including the additional loss of neurological function associated with the harvesting other nerves for grafting (including scarring and painful neuroma formation), increased post-operative pain due to additional incisions, increased risk of infection, and increased operating room and anesthesia time. For this approach, the extent of functional recovery is largely dependent on the distance of regeneration required to bridge the damaged nerve. Typically only about 50 percent of all patients will recover some useful functions and full recovery is rare [1,2].
In the case of spinal cord injuries, there is presently no effective treatment that can restore lost function. In contrast to peripheral nerve injuries, spinal cord injuries occur in an environment that is non-permissive to regeneration. While active research is focusing on the obstacles to regeneration within the spinal cord, an effective repair strategy has yet to be uncovered [3,4].
Currently, repair of spinal cord and peripheral nerve injuries relies on the ability of axon fibers (a nerve cell process that conducts nervous signals) to regenerate across the damaged area to restore nervous system communication. Accordingly, primary repair strategies have aimed to enhance and guide axon outgrowth by bridging the damaged area with materials of biologic or synthetic origin. Several approaches have been vigorously studied including: biomaterials to act as physical guides, transplantation of various cell types to support axon growth, administration of drugs to counteract elements that inhibit axon growth, and agents that enhance axon growth [1,5]. For peripheral nerve damage, these approaches have only been successful for injuries spanning a short distance, much smaller than what autogenous grafting can repair 6. For spinal cord injury, some approaches have been able to enhance the outgrowth of a few axons, but fall far short of number that would be necessary to restore lost function .
Here a distinct approach to engineering an effective man-made nerve construct for nerve repair is described. This construct consists of numerous bundles of axons, which are embedded in a collagen gel and packaged in a biocompatible conduit. Sized to the length of the damaged nerve, this construct can be directly transplanted to provide a living and functional connection. Researchers hypothesize that nerve constructs spanned by axons may establish or promote functional pathways necessary for nervous system repair that have not been achieved by any other approach. In order for this to be feasible, axons must be grown in a short period of time to lengths that can bridge any size lesion and consist of enough axons to adequately restore function.
Supporting a long held hypothesis , research has shown that tracts containing up to a million axons can be mechanically elongated in the laboratory at rates and to lengths that greatly exceeds what an axon can grow on its own. This process is similar to one of two distinct forms of axonal growth that occur in succession during development. First, axons grow out from the neural cell body and find their way to their final destination. After the axon integrates with its target, the growth of an animal induces the continued growth of axons as a result of mechanical stretching [7,8]. Anecdotal evidence of this form of growth can be found throughout nature. For example, the blue whale can grow an estimated 4cm per day and the giraffe’s neck increases by about 2cm per day at peak growth. It has also been shown that sensory axons in the deer antler are forced to grow at a rate of over 1cm per day 9. This process is referred to as axon stretch-growth that likely represents the primary mechanism that drives the formation of long nerves in animals.
Research has found that axons from embryonic dorsal root ganglion neurons (DRG, a type of neuron that resides in the peripheral nervous system) can sustain amazingly high stretch growth rates of up to 10mm/day and potentially much faster. Furthermore, axon tracts consisting of 105 to 106 axons can be stretch grown to 1cm in length within 4 days and up to 10cm in length in only 28 days while remaining healthy in culture and maintaining a normal structure 10. By exploiting this stretch growth process, ideal axonal tissue for transplant can be created in a matter of days for even extensive lesions.
Nonetheless, for clinical application, an embryonic source tissue for human transplantation is controversial and largely unattainable. A more ideal source of neurons would be from adult sources such as organ donors or from the patients themselves. The research team therefore examined the stretch-growth potential of axons from adult rat DRG neurons and subsequently from adult human donors for use in transplantable nerve constructs. Human DRG neurons were harvested from 18 patients, 14 who had undergone a pain management surgery, and 4 who were organ donors. It was found that human and rat DRG neurons survive in culture for more than three months and their axons can be successfully stretch-grown to a length of at least 1cm. The incorporation of elongated axonal tissue into a nerve construct offers an unexplored and potentially important new direction in bridging nerve lesions. Combining living axons derived from adult humans with other promising nerve construct designs may lead to a clinically applicable strategy for the repair of spinal cord and other nerve injuries.
Supported by Sharpe Trust and NIH grants AG21527, NS38104, NS45975.
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Peripheral Nerve Transplant
Traditional methods for repairing peripheral nerve deficits include bridging the nerve gap with an autologous graft or a bioabsorbable tube. The autologous graft is considered the gold standard for repair of significant lesions within the PNS, but its challenges include the limited supply of donor nerves and donor site morbidity. Researchers sought to test a new concept in which living dorsal root ganglion neurons and mechanically stretch-grown axons are transplanted into a 12mm sciatic nerve gap in the rat.
Thirty rats were divided into four groups
- Transplant Group: Nine rats were transplanted with the living nerve construct.
- Reverse Autologous Graft Group: Nine rats were repaired with a reverse autologous graft.
- Transection/No Repair Group: Six rats were injured without repair.
- Sham Group: Six rats were subjected to sham surgery in which the sciatic nerve was exposed, but not transected.
All rats were survived for four months.
Behavior: Rats were tested using an angle board paradigm in which they were required to stand without slipping on increasingly greater angles of incline.
The repaired sciatic nerve segment was recorded extracellularly immediately before sacrifice. Nerve conduction velocities were calculated as a measure of regeneration.
Spinal Cord Transplant
There are an estimated 10,000 patients who suffer spinal cord injury (SCI) each year in the United States and approximately 250,000 chronic SCI patients. Accordingly, there are extensive research efforts to develop techniques that enhance axon growth in the injured spinal cord. A primary goal of these efforts is to promote axon growth across the lesion to integrate with viable tissue on either side and create functional relays.
There have been numerous notable attempts at promoting axon bridges across spinal cord lesions. Some of the previous techniques have been successful in promoting axon sprouting into or around spinal cord lesions in animal models. While promising, this sprouting typically includes only a small number of spinal axons growing a limited distance. It has been proposed that the improvements in functional recovery often found after SCI in these transplanted animals is primarily due to physical and biochemical support for the host tissue surrounding the lesion, rather than the formation of new intraspinal circuits across the lesion. Considering that human SCI lesions typically extend several centimeters, bridging these lesions with axons of sufficient number and length to form functional relays remains an enormous challenge.
In contrast to inducing axon growth by promoting sprouting of axons, researchers have recently developed technology that induces rapid axon growth through continuous mechanical elongation of integrated axon tracts in vitro (Smith et al., 2001; Pfister et al., 2004). This axon stretch-growth technology was recently adapted to create nerve constructs consisting of living and functional axon tracts that are capable of bridging even extensive SCI lesions (Figure: Illustration of the SCI transplant). Most recently, it was found that transplanted elongated cultures from GFP transgenic rats survived four weeks in the injured spinal cord (Figure: Survival of transplanted tissues). These results demonstrate the promise of the lab’s nerve constructs consisting of stretch-grown axons to bridge even extensive spinal cord lesions. The research team is now investigating the functional recovery after SCI in the transplanted animals.
Smith Neurotrauma Laboratory
Cultured Axonal Injury Diffuse axonal injury (DAI) is thought to be the most common and important pathology in mild, moderate, and severe traumatic brain injury. In severe cases of DAI, shearing forces can cause primary disconnection of axons.
However, the vast majority of posttraumatic axonal pathologies evolve over time due to a series of deleterious cascades that include activation of proteases, second messengers, and mitochondrial failure. It has been previously demonstrated that dynamic mechanical stretch injury of cultured axons (Figure: Illustration of the CAI device) replicates many of the morphological and ultrastructural changes found in DAI in vivo (Smith et al., 1999).
With this model, it was found that the first evidence that rapid stretch of axons induces an immediate increase in intra-axonal calcium levels ([Ca2+]i) and that this response could be completely reversed with the voltage-gated sodium channel (NaCh) blocker tetrodotoxin (TTX) (Wolf et al., 2001).
Thus, while sustained elevated [Ca2+]i may be an important mediator of secondary damage to axons after trauma, as has been previously proposed ( Wolf et al., 2001), this increase in [Ca2+]i is dependent on trauma-induced Na+ influx through NaChs. However, the disposition of NaChs following dynamic stretch injury has not previously been examined.
Non-inactivation of NaChs has been shown to cause pathological Na+ influx and membrane depolarization, a state that could potentiate Ca2+ influx through voltage-gated Ca2+ channels and reversal of the Na+/Ca2+ exchanger (Wolf et al., 2001). Most recently, researchers have used the model of dynamic stretch injury of axons from primary cortical neurons to find that Na+ influx through NaChs due to axonal deformation triggers initial increases in [Ca2+]i and subsequent proteolysis of the III-IV intra-axonal loop of the NaCh alpha-subunit, suggesting a unique “feed forward” deleterious process initiated by mechanical trauma of axons (Iwata et al., 2004).