Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting over 2500 people in Poland and nearly half a million people worldwide. It is sporadic in approximately 90% of cases (sporadic ALS, SALS). The remaining 10-15% of patients suffers from a familial condition mostly inherited as an autosomal dominant trait (familial ALS, FALS). From 12 to 23.5% of FALS and over 7% of SALS cases are due to mutations in a gene encoding for Cu, Zn-superoxide dismutase (SOD1), a free radical scavenging enzyme (1). Several new genes were found to be associated with classic ALS in recent years. They encode for transactive response (TAR) DNA-binding protein TDP-43 (TARDP), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), each responsible for 4% of FALS and 1% of SALS, angiogenin (ANG), optineurin (OPTN) and others (2, 3). The greatest number of mutations associated with ALS was found in the SOD1 gene (for complete list please refer to http://www.alsod.org, http://alsod1.iop.kcl.ac.uk/reports/mutations). A vast majority of over 155 causative SOD1 mutations identified to date induce changes in stability of the enzyme. Some also influence its enzymatic activity. Experiments with the use of transgenic animals harboring human mutated SOD1 have shown that mutations in the SOD1 gene lead to a toxic gain of function of the SOD1 protein. In transgenic mice, the disease phenotype directly correlates with the level of transgene expression (4). On the other hand, the animals deprived of wild type SOD1 (wt SOD1) do not develop ALS-like phenotype. Overall, the experimental data leads to conclusion that genetic silencing of mutant SOD1 may potentially decrease the mutation-induced toxicity and ameliorate the clinical phenotype (5).

The mutation in TARDP, FUS/TLS, ANG and OPTN associated with FALS, are also inherited in an autosomal dominant manner (with only single exceptions). Both mutated TDP-43 and FUS/TLS form intraneuronal and glial inclusions, what may suggest a toxic gain of protein function rather than its loss (6). In fact, increased expression of TDP-43 in the nuclei was proved toxic to neurons (7). On the other hand, a depletion of TDP-43 from mouse adult brain with antisense oligonucleotides resulted in numerous abnormal splicing events. It also lead to decrease of mRNA levels of several genes involved in synaptic activity, including FUS/TLS and progranulin (8). Silencing of either TARDBP or FUS/TLS by RNA interference was also shown to reduce the expression of histone deacetylase (HDAC) 6 mRNA in cell culture (9). The impact of such silencing on mammal physiology has not been evaluated. The lack of similar experiments designed for other genes, reduces their consideration as immediate candidates for gene therapy. More information on the physiological and pathological function of the newly discovered genes associated with ALS will arrive along with new in vivo studies (2).

For these reasons, at present, the gene therapy mostly orientates on patients who carry ALS-associated mutations in the SOD1 gene. The autosomal dominant inheritance and toxic gain of protein function makes them prone to profit from therapy based on gene silencing. A specific reduction of expression of a target gene would assure the therapeutic effects. Two molecular mechanisms of potential use is gene silencing have been described to date. They are RNA interference and antisense olignucleotides.

RNAi is a highly conserved cellular mechanism responsible for gene silencing. It is triggered by double stranded RNAs (dsRNAs) capable of degrading the complementary mRNA or blocking its translation. RNAi is a natural cell defense against viruses and excessive transposon mobilization. It also regulates gene expression during development (10, 11). DsRNAs may be synthesized within the nucleus as pre-microRNA (miRNA), or introduced to the cell by viral vectors or plasmids. In either case, dsRNAs are processed to small (21-23 nucleotide)-interfering RNAs (siRNAs) by RNase III-like enzyme, called Dicer (12). siRNAs are effector RNAs, which target cognate cellular mRNAs and recruit them to the cytosolic RNA-induced silencing complex (RISC), a multiprotein complex containing ribonuclease. Attachment to RISC leads to an ATP-dependent mRNA unwinding and binding a single stranded RNA to the target mRNA. The process ends by mRNA rapid degradation catalyzed by cellular exonucleases present in the cytoplasm or, dependent on the degree of complementarity, the inhibition of translation (12, 13). MiRNA are non coding RNAs responsible for gene regulation and not the protein synthesis. They are produced by Dicer from endogenous short hairpin RNAs (shRNA) precursors (12). After binding to protein complex they associate to ribosomes thus inhibiting translation of partially compatible mRNA. If miRNA is thoroughly complementary to the target RNA, it produces target degradation, just like in the case of exogenous siRNA. The possibility to use RNAi in gene therapy arouse from a study that permitted to trigger gene silencing with the use of exogenous siRNAs (14). It’s worth noticing that introducing ds RNAs longer than 23 nt provokes enhanced immune response mediated by interferons and inflammatory cytokines, which leads to global repression of translation and cell death (15). Since RNA interference is quite specific, it may be therapeutically used to repress expression of genes responsible for synthesis of mutated proteins with toxic properties (16). In light of our knowledge considering genetic background of ALS, and more particularly on ALSOD (ALS due to SOD1 mutation), this disorder seems to be a perfect candidate for treatment using RNAi-based approach.

The biggest concern about the use of RNAi in therapy is to assure its efficient transport to target areas. siRNAs do not cross the blood-brain barrier and are highly instable in biological fluids. They need to be introduced to the RISC in the cytoplasm by direct CSF/brain parenchyma infusion (anterograde transport). Since they are rapidly degraded after gene silencing (36-48 h), the potential effectiveness of this therapy implicates introduction of numerous siRNA copies. Unexpectedly, even very short siRNAs induce interferon response in a dose-dependent manner (17). The only solution of this issue seems to be a long-term repetitive siRNA administration, which may encounter important technical problems.