Gene Therapy for Neurodegenerative Diseases: Slowing Down the Ticking Clock

Abstract

Gene therapy is an emerging and powerful therapeutic tool to deliver functional genetic material to cells in order to correct a defective gene. During the past decades, several studies have demonstrated the potential of AAV-based gene therapies for the treatment of neurodegenerative diseases. While some clinical studies have failed to demonstrate therapeutic efficacy, the use of AAV as a delivery tool has demonstrated to be safe. Here, we discuss the past, current and future perspectives of gene therapies for neurodegenerative diseases. We also discuss the current advances on the newly emerging RNAi-based gene therapies which has been widely studied in preclinical model and recently also made it to the clinic.

Keywords: AAV (adeno-associated virus); ALS; RNA interference (RNAi); gene therapy; neurodegenarative disease.

FIGURE 1

(A) Schematic of wild-type AAV. Its genome consists of the viral Rep and Cap genes flanked by two inverted terminal repeats (ITRs). (B) Schematic of recombinant AAV (rAAV) generated by replacing the viral genes for a therapeutic gene. The therapeutic transgene expression cassette consists of a promoter of choice, a therapeutic gene and a polyadenylation signal (not shown) and is flanked by the AAV ITRs. The Rep and Cap genes are expressed from a different plasmid or viral vector. The rAAV is generated by co-transfecting cells with the transgene cassette flanked by AAV ITRs, the Rep and Cap genes of a specific AAV serotype, and an adenovirus helper plasmid.

FIGURE 2

Mechanisms of toxicity associated with C9orf72 G₄C₂ repeat. (A) RNA-mediated toxicity. Repeat-containing sense and antisense RNA transcripts accumulate and sequester RNA binding proteins (B) RAN translation. The sense and antisense repeat-containing transcripts undergo RAN translation into five, potentially toxic DPRs (C) Haploinsufficiency. Hypermethylation of the expansion leads to reduced transcription of C9orf72.

FIGURE 3

Ataxin-3 mediated mechanisms of toxicity. (A) RNA toxicity. RNA containing the CUG repeats can sequester function of transcription factor (B) Mutant ataxin-3 toxicity. The CAG repeat-containing ATXN3 transcript is translated into a protein with a polyQ expansion. Proteolytic cleavage of mutant ataxin-3 can generate C-terminal protein fragments containing the polyQ repeat. The mutant ataxin-3 and C-terminal protein fragments can both cause several cellular disturbances such as transcriptional deregulation, impaired autophagy, mitochondrial dysfunction, proteasomal impairment, compromised axonal transport, and DNA damage.

FIGURE 4

Schematic of miRNA processing pathway. Most miRNAs are processed in the Dicer dependent (canonical) pathway shown in the left part of the figure. After being transcribed, the pri-miRNA transcripts are cropped by Drosha in the nucleus, resulting in a 60–70 nt pre-miRNA. The pre-miRNA is exported to the cytoplasm by Exportin 5 and the hairpin is then diced by Dicer into ∼22-nt miRNA duplex, after which it is separated into a guide and passenger strand. The guide strand is usually loaded into Ago proteins to form RISC.

FIGURE 5

Artificial miRNA design. Artificial miRNAs can be made by replacing the mature miRNA sequence of a natural pri-miRNA for a complementary sequence of a target mRNA of interest. The artificial pri-miRNA sequence can be cloned in an expression construct. Upon transfection with the artificial miRNA construct, the artificial miRNA is processed in the cell into a mature artificial miRNA which can bind and knockdown expression of an mRNA of interest.