AVV5 mediated microRNA delivery for treatment of ALS

AAV5 mediated delivery of micro-RNA for treatment of ALS and FTD with C9orf72 G4C2 expansion

Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons, leading to muscle atrophy and paralysis. A significant number of ALS patients also develop frontal temporal dementia (FTD) caused by progressive degeneration of frontal and temporal lobes. There are no disease-modifying drug and most patients die within 3-5 years after the onset of the disease. The most common genetic cause of ALS and FTD is an expansion of G4C2 repeats (> one hundred repeats) in the first intronic site of the c9orf72 gene present on chromosome 9.  

The two underlying pathogenic mechanisms have been proposed for c9orf72 related neurotoxicity. First, the loss of function of c9orf72 caused by G4C2 expansion disrupts mRNA splicing and decreased protein expression resulting in neurotoxicity. But the lack of motor deficit symptoms in mice without c9orf72 expression and non-pathogenic loss of function mutations suggest that the loss-of-function is not sufficient to cause the disease pathology. Second, the gain of toxicity caused by the accumulation of abnormal sense and antisense RNA and the recruitment of RNA binding proteins resulting in the formation of the RNA foci. The RNA foci sequester various RNA binding proteins, dipeptide repeat proteins, TDP-43 protein that hinder cellular function and result in neurotoxicity.

Based on this evidence, the approach to reduce the expression of aberrant mRNA and RNA foci formation is a formidable target for drug discovery. The gene silencing approach is promising but it comes with challenges that including
a) accessibility to the target tissue,
b) requirement of repeated administration and
c) toxicity due to large bolus injection.
Theoretically, a single injection of AAV packaged with a microRNA gene may allow sustained efficacious concentration in the target tissues with a single injection. Indeed, UniQure is developing gene therapy and result from their studies reveal a feasible expression of miRNA in brain tissue, decreased RNA foci in cytoplasm and nuclei cell and non-clinical disease model.

The studies highlight the following key points

a) Characterization of c9orf72 RNA transcripts from normal and disease populations shows a higher level of the first intronic region in ALS patients with variable sequences in each patient. Exonic regions are conserved in both ALS patients and healthy population. 

b) The design and optimization of candidate miRNAs result in specific binding to the intronic and conserved exonic regions of c9orf72 mRNA. 

c) Validation of c90rf72 gene silencing and AAV5 mediated gene delivery in HEK cells and iPSC neurons show successful transduction and expression of microRNA and decrease in c9orf72 RNA and RNA foci

 d) Proof-of-concept of AVV5 mediated gene delivery shows transduction of AAV5 vector on various brain tissue, expression of microRNA transgene and decreased nuclear and cytoplasmic RNA foci in c9orf72_3 transgenic mice.

Figure 1: Decreased RNA foci after treatment with microRNA specific for c90rf72. (Martier et al)

Figure 2: Expression of  GFP proteins in brain tissues after administration of AVV5 via striatum (Martier et al)

These proof of concept studies are performed in cell culture model and transgenic mice. Although the mouse model possesses some disease features such as RNA foci formation, the typical motor neuron-related inactivity and neurodegeneration are not observed. Therefore, it is still a pending question on whether this candidate miRNA mediated suppression is sufficient to prevent neurotoxicity and progressive neurodegeneration.

Path forward and challenges
a) The non-clinical and clinical studies to explore safety and tolerability of AAV 5 mediated gene delivery of microRNA.
b) Exploring the immunogenicity against the microRNA and the viral vectors with the administration of the miRNA package viral genome in the striatum.
c) Optimal efficacious dosage/copy of the vector genome with minimal tolerable toxicity.
d) The durability of the transgene expression in non-human primates and clinical subjects