After a spinal cord injury (SCI), complex pathological interactions lead to a cascade of secondary damage that results in the blockade of axonal growth. Our work and that of other labs establishes that regeneration can be induced by targeting intrinsic growth capacity but regeneration is still impeded by the growth-hostile environment of the damaged spinal cord. Several critical molecules that impede axon regeneration in the injury territory have been identified, but one of the most important is a molecule called chondroitin sulfate proteoglycan (CSPG) produced by reactive astrocytes at the injury site, which creates an especially powerful stop signal for growing axons. In addition to blocking regeneration, CSPG also forms an extracellular structure called the peri-neuronal net, which stabilizes synaptic connections in the mature brain and spinal cord. The peri-neuronal net forms a scaffold around synapses sort of cementing them in place; this can impede formation of new connections by regenerating axons. Based on these properties of CSPGs, scientists believe that blocking or degrading CSPGs may be a good strategy for addressing the extrinsic factors that impede regeneration at the injury site and impede formation of replacement connections once axons get beyond the injury.
Previous studies injected the bacterial enzyme chondroitinase ABC (ChASE) to break down CSPGs and have shown enhanced axonal regeneration and neuroplasticity in vivo, and functional recovery following spinal cord injury in rats. Some studies have injected the enzyme directly into the nervous system, but this strategy has the disadvantage that the enzyme doesn’t remain active for very long and is degraded over the course of days.
Another approach has been to inject a viral vector that carries a gene cassette that expresses ChASE. For example, Dr. Elizabeth Bradbury and colleagues at Kings College London have used a lentiviral vector (derived from the virus that causes HIV) to deliver a mammalian-compatible ChASE gene to the spinal cord. When this vector is taken up by cells at the injury site, the cells express and release ChASE over a long time period. One disadvantage is that lentiviruses induce innate and adaptive immune responses that could limit the effectiveness of the transgene.
Adeno-associated virus (AAV) vectors are derived from a non-pathogenic replication deficient parvovirus that have the ability to transduce nerve cells and present low innate immunity (see Anatomy 101, Spinal Connections 31 for the basic biology of AAV vectors). Our studies using AAV/shPTEN in rats and retro-AAV/Cre in mice show that AAVs have robust transduction efficiency in the brain that persists for months without any apparent negative consequences. This vector system is an ideal platform to express ChASE in the spinal cord as a combinatorial approach with retrogradely transported-AAV against shPTEN (retro-AAV/shPTEN).
We’ve previously addressed the importance of targeting neuron-intrinsic mechanisms by knocking down PTEN, showing regeneration and enhanced recovery of skilled motor function. However, all studies involving interventions to enhance intrinsic growth capacity indicate that the injury continues to be a huge barrier to regenerating axons. Accordingly, a combinatorial strategy that activates intrinsic mechanisms and also targets the growth inhibitory molecules at the injury site is an obvious next step. This combinatorial approach is possible because our technique to deleting PTEN (and thus activating growth) uses retrogradely transported AAVs (retro-AAVs), which are injected into the spinal cord near the injury site. This is of course exactly the same area to be targeted by interventions to reduce growth inhibitory molecules.
Building on this extensive prior literature, the funded project will test an innovative combinatorial approach to simultaneously target intrinsic and extrinsic factors that impede axonal growth after spinal cord injury. To assess this, we will test two sets of AAVs injected above and below the injury. One of the AAVs will knock-down PTEN (retro-AAVshPTEN), that targets intrinsic factors promoting regeneration of cut axons; and the other AAV will express ChASE (AAV-ChASE), that targets CSPGs that contribute to the growth-hostile environment present after SCI and promote sprouting of spared axons. A gene therapy method of transgene delivery with injections performed under the same procedure, where host cells themselves express the transgene, bypasses the need for repeated, invasive administration of treatment.
People often ask whether we collaborate with other scientists. The answer is “yes” and this is a perfect example. Dr. Liz Muir from the University of Cambridge, who created a version of ChASE that could be expressed by human cells, generously provided us with the DNA that expresses ChASE that we used to engineer a new AAV/ChASE vector. Dr. Muir and Dr. Bradbury have also been very generous with technical advice to help us get the approach going. Thank you to both Dr. Muir and Bradbury!