Oscar Wilkins1, Maria Pisliakova1, Jernej Ule2, Pietro Fratta1
1UCL Queen Square Motor Neuron Disease Centre, Department of Neuromuscular Diseases, University College London (UCL), 2RNA Networks Laboratory, Francis Crick Institute
Objective:
Leverage the regulatory role of TDP-43 in splicing to design a transgene vector for gene therapy applications in ALS
Background:
Amyotrophic Lateral Sclerosis (ALS) is a fatal and incurable neurological disorder characterized by the prion-like aggregation of TAR DNA-binding protein 43 (TDP-43). TDP-43 is also implicated in other neurodegenerative conditions, such as frontotemporal dementia (FTD) and Alzheimer's disease, making it a promising candidate for clinical intervention. Challengingly, only a small proportion of neurons exhibit TDP-43 pathology, and thus broad administration of therapeutics could drive significant off-target toxicity. Loss of TDP-43 function during ALS pathophysiology leads to expression of CEs, introducing premature termination codons into transcripts and hindering production of essential neurological proteins. We questioned whether this cryptic splicing mechanism could be exploited to control protein expression.
Design/Methods:
To exploit cryptic splicing to regulate protein expression, we developed a vector that contains a CE which promotes protein expression. We selected a CE within the AARS1 gene and designed a modified transgene construct in which an upstream regulatory CE controls downstream transgene expression. To enable direct visualisation of transgene expression, we used the fluorescent reporter protein mCherry as our transgene in these pilot experiments.
Results:
We created doxycycline-inducible TDP-43 knockdown cell lines and transfected them with our vector. Upon inducing TDP-43 knockdown in cell lines, expression of mCherry was increased >16-fold. Additionally, we conducted Nanopore sequencing analysis to quantify CE inclusion, which revealed a >43-fold increase in cryptic exon inclusion upon TDP-43 knockdown. We therefore demonstrate that protein expression is activated by splicing changes induced by TDP-43 knockdown.
Conclusions:
These proof-of-concept experiments demonstrate the potential to produce therapeutic transgenes regulated by TDP-43's splicing function. Given the critical role of TDP-43 loss of function in ALS and other neurodegenerative diseases, this approach could be applied in treatments and further explored for precise gene therapies in affected neurons.