Anatomy 101: Gene Targeting to Enable Regeneration After Spinal Cord Injury
Over the past decade, researchers have begun to see some real successes with approaches that target genes to enhance intrinsic growth capacity and modify some of the chemical barriers at the lesion scar.
It’s well accepted by scientists that the best hope for functional recovery following spinal cord injury is to regenerate the connections that were damaged. It’s also well accepted that there are 2 major reasons that regeneration doesn’t occur: 1) Adult neurons have very limited “intrinsic capacity” for growth; 2)The lesion site is a hostile environment for growing nerve connections (axons) with chemical and physical impediments that block growth, many of which are expressed by inflammatory cells and glial cells (astrocytes). Over the past decade, researchers have begun to see some real successes with approaches that target genes to enhance intrinsic growth capacity and modify some of the chemical barriers at the lesion scar.
What do we mean by “gene targeting”. This isn’t an intervention to alter a person’s genes (DNA sequence) as might be done to correct a defective gene. Instead, scientists are using new approaches that target the molecules that carry information from the gene in DNA to the cytoplasm of the cell where proteins are made called “messenger RNA”, abbreviated “mRNA”. Remember the “central dogma” of molecular biology, genes are DNA, DNA encodes for mRNA, mRNA encodes for different proteins. So, if you have a protein (gene product) that is doing something you don’t want, target the mRNA that makes the protein.
In the context of SCI, the idea is to deplete (knock down) proteins that halt regeneration. For example, O.Steward’s work follows up on the discovery that intrinsic growth capacity of adult neurons is shut down by proteins that are expressed as neurons mature (growth suppressors like the molecule PTEN).Intrinsic growth capacity is what enables neuron growth during development. So, to enable regeneration of the corticospinal tract, which controls our ability to move voluntarily, the strategy is to knock down PTEN. Similar approaches are being used to knock down molecules that inhibit growth at the lesion site, like the inhibitory molecule chondroitin sulphate proteoglycan, which is expressed by astrocytes at the lesion scar.
Viral-mediated delivery routes for therapeutic genes.
So, how do we target mRNA in cells like neurons to knock down expression? There are two parts to the story; the first is to harness viruses as delivery vehicles; the second is to engineer the viruses to express what might be called “anti-mRNAs”.
Viruses are very simple organisms that have a protein coat (called a “capsid”) surrounding genetic material (genome) which is in the form of a circle. Viruses work by binding to cells and injecting their genetic material into the “host” cell. The viral genes take over the machinery of the host cell so that the host starts making viral proteins, which cause disease, often by killing the infected cell.
Scientists have been able to take advantage of the ability of viruses to bind to host cells and inject theirDNA. The trick is to engineer the viral genome, removing harmful genes and genes that enable viral replication and inserting genes that do something else (in our case, synthesize an “anti-RNA”). The result is a “viral vector” that is a therapeutic candidate.
Anti-RNAs are short RNA molecules that have a sequence that is “complimentary” to a short stretch of the target mRNA. Depending on their physical form, these “anti-RNAs” are called short hairpin RNA(shRNA) or short interfering RNA(siRNA). When these anti-RNAs bind to the target mRNA, this causes the cell to mount a response that rapidly degrades (knocks down) the target mRNA. As a result, the targeted protein is not synthesized.
Using AAV to knock down PTEN expression.
This is the scientific background for the candidate therapy being developed by Steward’s research group at RIRC. The approach was initially developed by Dr. Gail Lewandowski in Steward’s research team and uses an AAV vector that targets neurons.
The AAV vector expresses shRNA against PTEN, so when the AAV/shPTEN is injected into the motor cortex, the vector is taken up by the cells of origin of the CST, and PTEN expression is knocked down.This activates a growth program in adult CST neurons that enables regeneration.
Our published studies have shown that decreasing PTEN promotes unprecedented regeneration of the injured tracts and improves motor function of the upper extremities. So far, we haven’t seen any treatment-related adverse events, but one potential issue for translation is that we used an AAV virus that once it is injected, it is always “on” meaning that it will continue to knock down PTEN even after regeneration has been achieved. A desirable characteristic for a therapeutic candidate is to be able to regulate expression of the shRNA in a defined time frame after the injury. For this, Steward’s research team is now testing AAVs with an “on-switch”.
AAVs with regulated expression
A huge advance in AAV vector biology has been the development of new AAV vectors that allow “regulated expression”. Synthesis of theAAV cargo (in our case shRNA)can be turned on by giving a drug that is actually already approved for human use (the antibiotic tetracycline ordoxycycline). What is exciting about this approach is that we can precisely control how long the therapy is delivered by using the 'on-switch', and turnoff the AAV by discontinuing the drug when regeneration and recovery are complete.
Steward’s research team is launching studies to test out this new approach, which will be an important step toward developing the final “therapeutic candidate” to advance to the FDA as an investigational new drug (IND).There are already several ongoing trials involving AAV based therapies and just this year, a clinical trial of an AAV based therapy for the treatment of spinal muscular atrophy(SMA) reported remarkable efficacy. These are exciting times for sure in terms of developing new therapies forSCI.
