Tuesday 15 November 2016

Getting a grip on gene therapy delivery of chondroitinase

One inhibitory aspect of the glial scar, an enrichment of chondroitin sulphate proteoglycans or CSPGs,  is problematic. CSPGs can either cause regenerating and sprouting axons to retract or become stupified and trapped by it - the latter being a bit like a kid in a candy store. The bacterial enzyme chondroitinase degrades CSPGs, and experimentally, it has been shown to have a number of reparative effects making it a very promising treatment option for SCI.

Nevertheless, there are concerns relating to how we should deliver this bacterial protein in the clinic. For one, ChABC activity is short-lived and therefore repeated injections or delivery by intrathecal catheter is needed, which is unattractive.

Gene therapy approaches may provide the answer. Modified viruses have long been used as a tool to carry inserted genes into cells, “infecting” them with a copy of the gene which in turn causes the cell to produce (express) the protein. Obviously, concerns surround gene therapy as well, but the prospect of only needing a single administration of a therapeutic viral vector which gives rise to long-term expression of the therapeutic gene is compelling.

A few years back the Bradbury lab (Kings College London, UK) presented a poster on the successful application of a gene therapy approach. I posted something on this at the time and the work was eventually published a little while after.

Sustained and widespread degradation of CSPGs was seen after a single injection of a lentiviral vector carrying a gene of ChABC. This led to a dramatic reduction of lesion pathology: sparing of neuronal tissue and dramatic reduction in cavity volume. It significantly improved signal conduction across the injury site coupled with improved performance on a horizontal ladder test

The viral vector used in this earlier work was based on a lentiviral carrier and the expression of chondroitinase was permanently on.

Yesterday two posters from the Guest lab at the Miami Project showed data from a non-human primate study using either gene therapy chondroitinase alone or in combination with autologous Schwann cell transplants [A. Y. Flores  #158.26 & R. De Negri #158.25]. The gene vector system was the same as that used by the Bradbury group, i.e. always on.

Chondroitinase gene therapy in an upper cervical hemi-contusion injury resulted in improved hand function. Interestingly, the group receiving the chondroitinase + cell graft combination lost significant upper limb function. It is unfair to speculate too much as to why this combination fared so badly as the group haven't had an opportunity to look at the histology, but the take home is that gene therapy alone looked very promising in a non-human primate study which is what I'm interested in here.

In all of the above studies the gene vector used permanently produced the gene product, chondroitinase.

Today, we were treated to three posters that reported on the first controllable chondroitinase gene therapy system. Controlled (inducible) gene expression can be achieved by engineering into the vector a antibiotic responsive transactivator which only switches on the production of the gene of interest (in our case chondroitinase) when exposed to the antibiotic doxycycline. Such a system has been around for years but it can't be used in the clinic because the transactivator causes an immune reaction that damages and kills the cells containing the vector. Not good.

The Verhaagen group have produced a "stealthy" version that evades the immune system and the results were impressive and from a translational point of view, very significant.

F. De Winter [#323.09] was able to show that this stealthy, inducible system could be repeatedly switched on and off many, many times over the course of 47 weeks without losing any efficacy. This markedly contrasted with the results from a non-stealthy version which could only be switched on over three cycles before losing "switchability". In addition, histological examination of the tissue at the end of the experiment revealed a lot of cell debris and very unhealthy-looking cells in the spinal cord tissue of animals injected with the non-stealthy version; the tissue from the stealthy group look just fine!

In the above study De Winter used a reporter gene (luciferase) to characterise the system. R. Eggers on the other hand looked at a therapeutic option [#323.10]. Eggers treated an avulsion injury at L3, L4 & L5 with an inducible, stealthy GDNF gene-carrying vector. The importance of being able to induce the production GDNF in an controlled way became evident when it was shown that 12 week exposure to doxycycline (12 weeks on) resulted in exuberant regeneration of motor neuron axons in and around the site of the injection but these axons remained there and went no further. The axons became trapped by their thirst for GDNF. When doxycycline was given for only 4 weeks (4 weeks on) the axons made their way onwards towards the denervated target.

Finally, E. R. Burnside from the Bradbury lab showed further evidence of the value of being able to control the delivery of chondroitinase using this vector system [#323.11]. Using a cervical contusion injury model and treatment with the inducible, stealthy chondroitinase vector two treatment groups were studied ; (i) treatment with short-term expression (2.5 weeks exposure to doxycyline) and (ii) treatment with 8 weeks exposure. Both groups showed improved locomotor function over controls. What was most impressive was the finding that fine motor control in the forelimb improved only in the 8 week treated group. This was the first time this type of skilled motor function has been shown to recover in this injury model.

The Verhaagen and Bradbury labs are part of the CHASE-IT consortium.

Monday 14 November 2016

SCI and the bowel ..... does that sound like a band?

OK, maybe not.

A couple of papers caught my attention today on bowel function.

More than 90% of acute and between 30-50% of chronic patients have bowel incontinence and/or diarrhoea. Even long after injury most individuals require at least one method to facilitate defecation when constipation and faecal retention are prevalent.

Andrew Gaudet, University of Colorado Boulder [#142.16] presented a nice piece of work being carried out in his lab on a rodent model/study of bowel function after SCI. SCI rats show characteristic alterations in body mass - losing weight initially before slowly regaining much of it - coupled with a compensatory increase in food intake during the weight recovery phase. Interestingly, transit time (bit of blue dye in the diet and time how long it takes for the poo to come out blue is their simple and effective way to measure this) is reduced which, Gaudet suggests could be seen as a correlate of incontinence.

With the increased food intake there is a corresponding increase in faecal output. His group also found a disruption to the normal circadian rhythms. As with humans, rats poo less at night. After SCI this distinction is abolished, they found. Other circadian rhythms are also disturbed. Temperature and general activity no longer follow the typical day/night cycle.

It's a small but important start towards developing models and understanding of bowel function in the SCI rodent, which is needed. Hopefully more will follow and we can start looking at developing treatments that address recovery of bowel function rather than merely manage it.

Which brings me to the poster of April Herrity, University of Louisville [#158.11]. Herrity is part of the Harkema team, famed for the work on epidural stimulation (ES) on patients that results in voluntary inducible stand/stepping outcomes in ASIA A patients, for example. In the papers about this work, Harlema et al., reported non-locomoter benefits to ES. These are being more systematically examined now.

Study of exercise and locomotor training (LT) on the bladder can be traced back as far as the 1930's. As a precursor to a poster presented tomorrow, Herrity's presented findings on the effect of LT in patients with SCI on not only bladder but also bowel and sexual function and the results were pretty interesting.

By LT they mean 80 treadmill sessions of 1 hr duration, 5 times per week. Eight patients (a mix of AIS A to D) where studied. If you're interested in your urodynamics, bladder capacity increased (105 ml to 205 ml), contraction duration  improved, there was a reduction in leak point pressure, improvements in bladder muscle responsiveness and contractability and voiding efficiency improved from 40% to 80%. Bladder compliance - that's the ability of the bladder to maintain safe pressure - also improved.

Other benefits included reduction in bladder medication, less time needed for bowel management and increased sexual desire. The poster reporting results from combining LT with epidural stimulation will be presented tomorrow.

Sunday 13 November 2016

Incontinence doesn't kill you.....

…. it just takes away your life".

Is not a bad way to start a talk. It is a quote from a woman who had an overactive bladder. The session was on “Autonomic Nerves as Targets for Precision Bioelectronic Medicines”, the talk [#6.02] was by John Hokanson from Duke University, Durham, NC.

For the purposes of this discussion let's say the lady in question was otherwise healthy but we know the underlying cause was not SCI. Nevertheless, it does resonate with those with SCI as it reflect just one of the many secondary consequences of paralysis. Arguably, incontinence per se might not kill you in the context of SCI, but bladder dysfunction can and does cause serious complications in SCI patients and resolving bladder function consistently rates highly as a major priority for people with SCI.

Treatment for overactive bladder (OAB) is varied and includes physical therapy, drugs and as a third tier, Botox and sacral nerve stimulation (e.g. Medtronic InterStim(R) System). For those that are offered neurostim, approximately 32% are treated successfully, says Hokanson. So that means 68% aren't well treated by neuromodulation.

Hokanson described current work being done to optimise the sacral nerve stimulation parameters. In animals models, he showed that increasing the stimulation frequency (1 Hz to 20 Hz) led to broadly doubling the bladder capacity (how they did this without busting the bladder is another story but involves filling the bladder with a solution containing prostaglandins for those who are interested). Increasing stimulation strength led to further increases in bladder capacity.

The take home here appeared to be that increased stimulation frequencies help sustain bladder filling but stimulation strength ultimately proves the deciding parameter where capacity is concerned.

But for SCI, high bladder pressure causes the real problems as it backs up to the kidneys causing renal damage. Neuromodulation to increase bladder volume might not be a good idea. Whilst Hokanson could increase bladder capacity he also stated this was at the expense of voiding efficiency - the bladder retained too much urine. Not good either. Hokanson demonstrated that a switch in stimulation parameters at time of voiding took good care of that problem. As a final aside, he also showed data to suggest neuromodulation led to (beneficial) plasticity within the system. Little is currently know about the physiology of this and further work was promised.

So, neuromodulation of the bladder may be getting some helpful refinement in the future.

But we could do with more helpful in vivo outcome models of bladder function to help translate the above studies to SCI and speed up assessment of potential therapies. That's why I liked Faiza Qureshi's poster [#59.08]. Qureshi set out to determine whether there was any benefit to be had from treadmill training on recovery of bladder function after SCI. The experiment was simple in concept - test baseline cystometric parameters (bladder volume, thresholds, peak pressures etc.) - perform SCI and record changes in the above parameters in two groups, one receiving treadmill training the other not.

What was particularly nice about the study was the development of an effective method of taking these measurements in individual animal over many time points without using terminal studies or chronically indwelling catheters and electrodes that can result inflammation, infection etc.

 Each time they wanted to take a measurement they simply anaesthetised the animal, placed a catheter into the bladder, placed a recording electrode on the urethral sphincter. They could do this multiple times on each animal throughout the experiment, gathering data. A little more validation is needed but it could lead to an effective method for repeated evaluation of therapeutic interventions for bladder function.

And what of the effect of treadmill training? Well, a full control is needed but treadmill training might result in some benefit a number of key bladder and sphincter measures. But we need that control.

Spinal Research is committed to developing clinical therapies for this unmet medical need by setting up a Special Emphasis Network of researchers and clinical experts. Learn more about our Special Emphasis Networks on our website www.spinal-research.org.

Saturday 12 November 2016

It's SfN 2016 - how time flies



They've had record breaking temperatures in San Diego by all accounts, but now I've arrived it's about to "break" to a comfortable 80 degrees C - at least for a few days. All will be missed in the bowels of the poster arena (Hall B) in the convention centre. Actually, it can get pretty chilly down there with industrial air con hard at work, which is why it is advisable to seek out some "hot air" emanating from a poster presentation from time to time.

SfN can be daunting if you're new to it, unwieldy and pretty much the worst place to learn the basics. It's great for networking, getting things off your chest and exploring areas not core to your particular interest.

Keep it quite, but last year disappointed a little. Reviewing this year's sessions gives me hope.

This year there is a helpful curated itinerary on Brain and Spinal Cord Injury, but I'm sure there's stuff worth seeing beyond that.

We can be found at Booth Number 3924. If you're here, come by.





Sunday 18 October 2015

Healthy body, healthy bladder

It's established that epidural stimulation in SCI patients leads to changes in motor output. Basically, electrical stimulation (ES) primes the circuitry responsible for standing/walking to an extent that it becomes capable of responding appropriately to feedback from the sensory system; weight bearing leads to standing whereas introduction of locomotor activity (via a treadmill, for example) leads to the generation of patterned sensory signals coming from the legs that the cord responds to by trying to fire muscles in a coordinated extensor-flexor, left-right fashion. The work of Harkema and colleagues - a few years ago now - was published with much fanfare and interest remains high in the potential of electrical stimulation, particularly as it is so clinically feasible.

Anyway, one of the more intriguing observations from this seminal work was the anecdotal reports of improvements in bladder function in some of those earliest patients receiving epidural ES. What of this? The mechanism by which ES and locomotor training might couple to bladder function and other non-locomotor systems is less intuitive. Ward et al., recently showed that quadrupedal locomotor training in rats with SCI (contusion; T10) led to improved bladder function and that training led to changes in neurotrophin levels.

One question then is, does ES specifically engage with circuitry that contributes to improvements in bladder function or might it be the training "allowed" by ES that it key? L.R. Montgomery [#46.09] has added to the story. Montgomery presented data from a study that tested bladder function in three rodent groups; (i) injured, (ii) injured receiving quadrupedal training and, (iii) injured receiving forelimb locomotor training (basically, treadmill training with hind limbs hoisted off the ground, wheelbarrow fashion).

What she showed was that it didn't matter what training was received as both groups showed equivalent improvements in bladder function measures over injury alone, suggesting there are global and systemic mechanisms at play. There's a couple of more papers being presented this week relevant to this which might be worth looking at.

Elsewhere, a paper caught my eye on neuropathic pain. Tan presented [#66.01] a nice little poster on the correlation of dendritic spine (post-synaptic micron sized structures) distribution and morphology with the presentation of neuropathic pain. The correlation is tight such that neuropathic pain is always associated with remodelling of the dendritic spines. The group had already shown that if you acutely inhibited remodelling of the spines with Rac-1 inhibitor, neuropathic pain states did not develop. This poster looked at what happened if you inhibited remodelling after pain had been established. They found the pain state diminished and furthermore, when the drug is withdrawn remodelling is re-established and pain comes back.




Saturday 17 October 2015

Neuroscience 2015 - Chicago

It's been a few years since Neuroscience landed in Chicago and it's pretty nice to be back. Sessions, proper, start today and for those interested in research of spinal cord injury we have a dedicated session in the afternoon. Entitled "Restorative Therapeutic Strategies" it will be interested to see what they've decided to put into this one. 

Tuesday 18 November 2014

Take time to draw breath

Application of the bacterial enzyme, chondroitinase, led to restoration of breathing even after long term paralysis of the diaphragm, it was revealed at a press event at the annual Society for Neuroscience meeting. Philippa Warren, a Case Western Reserve scientists and lead on this work, described how the paralysed diaphragm recovered function after a remarkable 1.5 years post injury. "This treatment holds great promise for those with respiratory dysfunction", said Warren. Case Western's own press release can be read here.

A subsequent poster [#523.10] expanding on her work revealed that some of the animals treated responded perhaps too well to the treatment resulting in a less than rhythmic pattern of muscle contraction. Investigation showed that there was particularly strong reactivation of excitatory nerve fibres and that recovery and function in these was likely to be responsible for the recovery. Indeed, treating with a drug that could interfere with signals from these fibres brought things back in line.

Elsewhere, Kevin Hoy was reporting similar (respiratory) recovery after treatment with a licensed anti-cancer drug[#523.08]. Taxol is used in chemotherapy and stabilises the cellular scaffolding in cells. At low doses, however, the stabilisation appears to make for a more robust growth response in nerve fibres. Taxol is by its nature toxic but the dose used in these studies was 1/100 that used clinically. Nevertheless, Hoy looked at whether a less toxic drug with similar properties also led to recovery of breathing function. Alas, no but further work does need to be done, he said.

I was also impressed with Caitlin Hill's study [#523.26]. We know that cellular grafts often survive poorly and efficacy is disappointing. The question Hill set out to answer was; is the lack of effect due to poor choice of cells or simply due to so much cell death which in and of itself has a negative impact on the outcome? It's a subtle question but potentially an important one to answer given the shear amount of effort going into cell transplantations these days.