Thread by @SimonWad

Thrilled to see our mouse gene therapy for Dopamine Transporter Deficiency Syndrome. DTDS is a rare inherited genetic disease, caused by mutated SLC6A3 encoding dopamine transporter. stm.sciencemag.org/content/13/594…

Dopamine transporter reuptakes dopamine from the synaptic cleft after neurotransmission. Current treatments are palliative, not very effective, and prognosis is very poor.

For me, the story started 21/9/2011. @profahadrahim, @drsuzybuckley +me were extending our #genetherapy to harder targets such as neuronopathic #GaucherDisease @gauchersA. Paediatricians Paul Gissen & Manju Kurian (

https://twitter.com/SaraEMole/status/1154690452194582528

) (

https://twitter.com/UCLchildhealth/status/907906073591517184

) emailed.

They wanted to start doing gene therapy for genetic disease of children. 2 years before this, Manju published SLC6A3 as the gene underlying DTDS (jci.org/articles/view/…). @DrJo_Ng, a paediatric neurologist, joined us for her PhD to develop gene therapy for DTDS.

Plan was: treat neonatal hDAT knockout mice by vector injection into lateral ventricles. We chose AAV9 because it spread well throughout the brain. Jo screened for dopaminergic neuron-specific promoters to avoid ectopic/overexpression of DAT. A year later, no specificity ☹

We proceeded with a pan-neuronal synapsin promoter, figured was better to see anything – even toxicity, than screen forever. The first commercially-synthesised vector gave GFP in lots of non-neuronal cells! Turns out was the wrong promoter- “eSyn” had a chunk of CMV enhancer.

When we finally tested AAV9-Synapsin-GFP (without CMV enhancer) it gave abundant brain expression – all in cells that looked like neurons so onto the DAT knockout mice.

Untreated knockouts are hyperkinetic and survive on a high-calorie peanut butter diet. 2/3rds decline to terminal parkinsonian akinesia and tremors while the remainder keep zipping around, doing badly on the grid-walk and pole-descent tests

The highest dose of gene therapy with AAV9-Syn-DAT didn’t improve this survival at all though it did prevent the hyperactivity and other motor problems in the remainder.

Worse, there was a LOT of GFAP expression in the treated brains, indicative of neuroinflammation, possibly caused by the very high, widespread hDAT expression. This was what we feared, but at least we now had something to improve upon.

Dropping the dose 10x yielded a beautiful outcome - so good it didn’t even get dumped in the supps (they are Fig 6). All 6 treated KOs were healthy and indistinguishable from wild type littermates for 1 yr of follow-up – grid walk, pole descent, open field all normal.

However, there was cortical GFAP elevation, cell loss and vacuolation, probably from hDAT ectopic and over-expression.

Clinically we would want to target expression to those regions by MRI-guided stereotactic injection so we switched to AAV2, a vector already being used in trials to treat a similar movement disorder, Aromatic Acid Decarboxylase Deficiency (researchsquare.com/article/rs-738…).

@The_MRC provided funding to validate stereotactic AAV2-Syn-hDAT delivery to adult DAT knockout mice. (🙏🏻 to @jowinx of the @UCLTRO and @CatrionaCrombie).

@DrJo_Ng’s results were really quite nice – dose-dependent therapeutic effect looking great at the highest dose

and much more localised expression and no collateral cortical inflammation or cell loss.

I haven’t given enough credit to the tremendous efforts of Serena Barral and her studies on patient iPSC-derived dopaminergic neurons, which you can read about in the paper

Alongside the prodigious efforts of Manju, Jo & Serena, are the great contributions from many colleagues including @LignaniLab_UCL @gabrilignani @Dr_RKarda @RickPrivolizzi @WadeMartinsLab @SuffNatalie @SimonHeales @jantinaod

And of course, tremendous support from funders @RosetreesT, @wellcometrust, @The_MRC, @GOSHCharity, Robert Luff Foundation, John Black Foundation, @NIHRresearch, @UCLHresearch @actionmedres, @lifearc1 and many more