AAV-PHP.B: The future is apparently now

In addition to looking at current Parkinson’s disease research on this website, I like to look at where technological advances are taking us with regards to future therapies.

In July of this year, I wrote about a new class of engineered viruses that could potentially allow us to treat conditions like Parkinson’s disease using a non-invasive, gene therapy approach (Click here to read that post). At the time I considered this technology way off at some point in the distant future. Blue sky research. “Let’s wait and see” – sort of thing.

So imagine my surprise when an Italian research group last weekend published a new research report in which they used this futurist technology to correct a mouse model of Parkinson’s disease. Suddenly the distant future is feeling not so ‘distant’.

In today’s post we will review and discuss the results, and look at what happens next.

Technological progress – looking inside the brain. Source: Digitial Trends

I have said several times in the past that the pace of Parkinson’s disease research at the moment is overwhelming.

So much is happening so quickly that it is quite simply difficult to keep up. Not just here on the blog, but also with regards to the ever increasing number of research articles in the “need to read” pile on my desk. It’s mad. It’s crazy. Just as I manage to digest something new from one area of research, two or three other publications pop up in different areas.

But it is the shear speed with which things are moving now in the field of Parkinson’s research that is really mind boggling!

Source: Pinterest

Take for example the case of Squalamine.

In February of this year, researchers published an article outlining how a drug derived from the spiny dogfish could completely suppress the toxic effect of the Parkinson’s associated protein Alpha Synuclein (Click here to read that post).

The humble dogfish. Source: Discovery

And then in May (JUST 3 MONTHS LATER!!!), a biotech company called Enterin Inc. announced that they had just enrolled their first patient in the RASMET study: a Phase 1/2a randomised, controlled, multi-center clinical study evaluating a synthetic version of squalamine (called MSI-1436) in people with Parkinson’s disease. The study will enrol 50 patients over a 9-to-12-month period (Click here for the press release).

Source: Onemednews

Wow! That is fast.

Yeah, I thought so too, but then this last weekend a group in Italy published new research that completely changed my ideas on the meaning of the word ‘fast’. Regular readers will recall that in July I discussed amazing new technology that may one day allow us to inject a virus into a person’s arm and then that virus will make it’s way up to the brain and only infect the cells that we want to have a treatment delivered to. This represents non-invasive (as no surgery is required), gene therapy (correcting a medical condition with the delivery of DNA rather than medication). This new study used the same virus we discussed in July.

Here is the study:

Title: AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy.
Authors: Morabito G, Giannelli SG, Ordazzo G, Bido S, Castoldi V, Indrigo M, Cabassi T, Cattaneo S, Luoni M, Cancellieri C, Sessa A, Bacigaluppi M, Taverna S, Leocani L, Lanciego JL, Broccoli V.
Journal: Molecular Therapy. 2017 Aug 10. pii: S1525-0016(17)30363-5.
PMID: 28882452

In this study, the investigators conducted a characterisation of the amazing virus (called AAV-PHP.B) that I discussed in a previous post. The virus is amazing because it preferentially infects brains cells after being injected in the tail vein of adult mice. In this study, the scientists injected mice and 3-5 weeks later looked at where the virus had infected cells.

How did they do that?

In order to do this, the investigators needed a marker that would only be present in infected cells. They inserted a piece of DNA inside the AAV-PHP.B virus that would provide cells with the instructions to make a protein that glows when in the presence of ultraviolet light. That protein is called green fluorescent protein (or GFP).

Since it was discovered in Jellyfish in 1962, GFP has come to play an extremely significant role in research, allowing researchers to determine which cells are producing a particular protein (by replacing the DNA of that protein with the GFP DNA) or where in a cell a protein can be found (again by replacing the DNA of that protein with the GFP DNA). Its uses are many – so much so, that in 2008, the researchers who contributed to its discovery/utillity were awarded the Nobel prize in Chemistry (Click here to read more about this).

Organisms that produce GFP. Source: PNAS

By inserting GFP into the AAV-PHP.B virus, the researchers were able to determine which cells in each organ of the body was infected after the virus was injected into the tail of the mice. The virus would simply infect cells and release their DNA cargo, which the cell would incorporate and start to produce the protein according to the instructions.

When they looked at the brains of the injected mice, the investigators that approximately 65% of the neurons were producing GFP – meaning that they were infected by the AAV-PHP.B virus. This is a rather remarkable result, given that only one injection was made and that injected volume of viral particles passed through the entire body before infecting just the cells in the brain. Some cells in other parts of the body were also GFP, but only the heart exhibited a high (approximately 60%) level of infection, and the investigators found that they could reduce this by more 10-times in the heart by injecting the AAV-PHP.B virus into the carotid artery – which takes blood up to the brain.

The carotid artery (red). Source: ReadersDigest

After establishing that the virus was working very well, the investigators next decided to determine its utility in a disease model. They chose a genetically engineered mouse that produces too much of the Parkinson’s disease-associated protein, Alpha Synuclein. The particular type of Alpha Synuclein that these mice produce is a faulty version of the protein that is produced in people with a genetic mutation in the gene that provides the instructions for making the protein. At 6 months of age, these mice (called A53T-SCNA mice) gradually start accumulating Alpha Synuclein deposits throughout their brain, and by 10–12 months of age they start developing a loss of voluntary movements.

The investigators decided to try and rescue these mice by injecting them with an AAV-PHP.B virus that contained a piece of DNA that contains the GBA1 gene.

What is the GBA1 gene?

The GBA1 gene provides the instructions for making an enzyme, called Glucocerebrosidase. Approximately 5%–8% of people with Parkinson’s have a genetic mutation in the GBA1 gene (Click here and here to read more about this). This mutation causes a reduction in the activity of the Glucocerebrosidase enzyme.

What does Glucocerebrosidase do?

Glucocerebrosidase helps with the digestion and recycling of certain waste inside cells. The enzyme is located and active inside ‘lysosomes‘.

What are Lysosomes?

Lysosomes are small bags of digestive enzymes that can be found inside cells. They help to break down proteins that have either been brought into the cell or that have served their function and need to be digested and disposed of (or recycled).


How lysosomes work. Source: Prezi

Inside the lysosomes there are enzymes like glucocerebrosidase which help to break material down into useful basic parts. The lysosome will fuse with other small bags (called vacuole) that act as storage vessels of surplus material/waste inside a cell. The enzymes from the lysosome will mix with the material in the vacuole and digest it (or it break down into more manageable components).

Now people with a genetic mutation in their GBA1 gene will often have an abnormally short, non-functioning version of the glucocerebrosidase enzyme. In those cases the breaking down of waste inside the lysosome becomes inhibited. And if waste can not be disposed of or recycled properly, things start to go wrong in the cell. Generally, people with Parkinson’s disease and a GBA1 mutation will exhibit more severe symptoms than people without the GBA1 mutation (Click here to read more about this), but this is not always the case. In fact, there are cases of identical twins who both have a GBA1 mutation, but only one of them has Parkinson’s disease (Click here to read more about this). Regardless, increasing Glucocerebrosidase activity in individuals affected by this mutation represents a reasonable therapeutic approach to Parkinson’s disease.

And that is why the Italian researchers decided to have a look at whether an AAV-PHP.B virus that contained the GBA1 gene could rescue the alpha synuclein over-producing A53T-SCNA mice. So the question was: by increasing the level of waste recycling, could the investigator help these mice?

And the answer was: Yes.

Five-month-old A53T-SCNA mice were injected with with either the AAV-PHP.B GFP (control) virus or the AAV-PHP.B GBA1 GFP virus. Interestingly, glucocerebrosidase activity was strongly reduced in control A53TSCNA mice, suggesting that the build-up of alpha synuclein protein affects normal glucocerebrosidase protein processing.

The researchers found that the AAV-PHP.B GBA1 GFP virus significantly rescued of glucocerebrosidase enzyme levels and activity, which elicited a strong reduction of alpha synuclein in all of the brain regions examined (including the dopamine neurons – which are severely affected by Parkinson’s disease). The AAV-PHP.B GBA1 GFP virus injected mice also exhibited a strong recovery in learning and cognitive performance AND survived longer compared with the control treated mice.

Thus, a single injection of AAV-PHP.B was sufficient to rescue these mice, by infecting the majority of cells in the brain and only a fraction of cells in all the other organs. A rather remarkable achievement with an amazing new piece of technology.

What does it all mean?

Ok, so summing up: Delivery of therapies has always been problem #1 for medicine. Many drugs and treatments often have side effects because they are doing stuff in other parts of the body where they are not supposed to go. To improve this situation, researchers have recently been engineering viruses that only target cells in specific organs (eg. the heart or liver or brain). Italian researchers have used one of these viruses and published data suggesting that non-invasive gene therapy for Parkinson’s disease is possible. By injecting an engineered virus into a mouse model of Parkinson’s disease, the researchers were able to rescue the mice.

An important part of what we look at here on the SoPD website is not just current state of Parkinson’s research, but also where things are heading in the future. It is my reckoning (and others in the research community agree) that this new viral technology is sure to be an important part of that future as it provides a near perfect delivery system for therapeutic agents. Having said that, it must be acknowledged that there will be limited clinical applications for these advances in the near future – this technology is at least five years away from the clinic. The Italian researchers indicate that they are now investigating other mouse models of Parkinson’s disease as well as planning for preclinical testing in larger animals, but there will be significant hurdles to cross when taking this technology to the clinic (such as, regulatory – regulators are ok with localised injections of gene therapy viruses into the brain (see the Voyager clinical trial as an example), but the idea of a systemic injection (that is, exposing the whole body) of viruses will raise concerns at the FDA and EMA). 

All of that said, I still believe that it is important to bring this new information/technology to the attention of readers. I meet a lot of people in the clinic who selflessly want to take part in research so that their children and grand children do not have to worry about inheriting this disease. Posts like these are for those individuals, to reassure them that impressive new technologies are coming down the pipe – 

Hang on a minute Simon. That’s really nice, but I have a GBA mutation and Parkinson’s disease. How is this future technology going to help me? What can I do right now?

Well, luckily for you there is currently the clinical testing of a drug called Ambroxol in Parkinson’s disease.

What is Ambroxol?

Ambroxol is a commonly used treatment for respiratory diseases. It promotes mucus clearance and eases coughing. Ambroxol is also anti-inflammatory, reducing redness in a sore throat.


Ambroxol. Source: Skinflint

Ok, but why is it being clinically trialled in Parkinson’s disease?

In May of 2014, this study was published:


Title: Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells.
Authors: McNeill A, Magalhaes J, Shen C, Chau KY, Hughes D, Mehta A, Foltynie T, Cooper JM, Abramov AY, Gegg M, Schapira AH.
Journal: Brain. 2014 May;137(Pt 5):1481-95.
PMID: 24574503    (This report is OPEN ACCESS if you want to read it)

In this study the researchers collected skin cells (called fibroblasts) from 11 people with GBA1 mutations (some had been diagnosed with Parkinson’s disease). They measured the amount of glucocerebrosidase protein and enzyme activity in these cells, and they found that glucocerebrosidase enzyme activity was significantly reduced in fibroblasts from people with GBA mutations (on average just the enzyme was acting at just 5% of normal levels). They found that Ambroxol increased glucosylceramidase activity in fibroblasts from people with GBA1 mutations AND in fibroblasts from healthy controls. Ambroxol treatment also reduced markers of oxidative stress in GBA1 mutant cells.

Given the increase in glucocerebrosidase activity after Ambroxol treatment, the researchers wondered whether the drug would reduce levels of the Parkinson’s disease-associated protein Alpha Synuclein in cells. To test this idea, the researchers grew cells that were over producing this protein, and amazingly, after 5 days of Ambroxol treatment, levels of alpha synuclein had decreased significantly (on average 15%), and this was accompanied by a 40% increase in levels of glucocerebrosidase activity.

Additional studies have subsequently confirmed these initial results (Click here and here to read more about those studies).

And there’s a clinical trial?

Not one, but two!

The first study (named AiM-PD) is a phase II trial funded by the Cure Parkinson’s Trust and the Van Andel Research Institute (USA). Conducted at the Royal Free Hospital in London, the trial will test the safety of slowly increasing doses of Ambroxol in Parkinson’s disease. The researchers will also look to see if Ambroxol can increase levels of glucocerebrosidase activity and whether this has any beneficial effects in the subjects. The study will be conducted on 20 people with Parkinson’s disease who also have GBA1 mutations. They will be given the drug and followed over the next 24 months (Click here to read more about it).

The second study is being conducted by the Lawson Health Research Institute (and the Weston Foundation) in Canada, and it will be a phase II, 52 week trial of Ambroxol in 75 people with Parkinson’s Disease Dementia. In this study, two doses of Ambroxol will be tested – a high dose (1050 mg) and a low dose (525 mg) – as well as a placebo treated group (Click here to read more about this study).

If these trials are successful, there will be a clinically available drug that we can repurpose for Parkinson’s disease. Longer term it will be interesting to see if Ambroxol can also help people with Parkinson’s disease who do not have a GBA1 mutation (note that the mice in the virus study described above did not have the GBA1 mutation).

What does it all mean? (Part 2)

So, briefly summing up again: Amazing new technology, lots of interesting clinical trials. The future looks bright and exciting, except of course if you are a scientist in the field of Parkinson’s disease research then the story unfortunately very different: the pace of discovery is only going to increase and ultimately it will put you out of a job.

But hey, it’ll be worth it and it’s going to be an amazing ride.

The banner for today’s post was sourced from Blenderartist


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