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Neurocalcin delta (NCALD) was identified as a protective SMA modifier in humans. Preliminary work in cellular and non-mammalian models showed that reducing NCALD ameliorated defects mediated by SMN deficiency: particularly, the neuronal outgrowth which is strongly reduced in SMN-deficient cells and zebrafish (McWhorter et al. 2003, Kwon et al. 2011) was restored to control levels when NCALD was concomitantly downregulated. The work presented here attempted to answer the question whether NCALD reduction can rescue the SMA phenotype in mice. Studies of SMA mouse models contributed enormously to the understanding of the disease pathology: by unraveling NMJ defects as one of the earliest SMA symptoms (Kariya et al. 2008), the developmental synaptopathy of the neuromuscular junctions has been identified as the primary pathology both in severe and mild SMA mouse models and, most importantly, in SMA patients (Murray et al. 2008, Bogdanik et al. 2015, Harding et al. 2015). SMA mice serve also as a platform to test potential therapeutics against this devastating disorder which are reviewed in (Kaczmarek et al. 2015). By studying the effect of NCALD reduction in SMA mice we aimed at answering two questions: first, which SMA symptoms are potentially rescued by NCALD reduction, and second, to explore the therapeutic potential of NCALD knock-down. In order to do so, we had to find the optimal method to reduce NCALDin vivo: in the scope of this work, three different approaches have been tested and each has shown certain advantages but also some caveats.

Our first strategy to reduce NCALD involved generating a novel mouse line carrying an shRNA that by RNA interference would likely promote the degradation ofNcald transcript (Kleinhammer et al. 2011). The shRNA would be expressed only upon induction, while the inducer (Doxycycline) can be either added to the medium in vitro or conveniently administered in vivo in food or drinking water. This strategy of inducible and incomplete depletion of a gene product is particularly suitable when a gene knock-out would result in embryonic death (McJunkin et al. 2011). Moreover, by merely reducing the levels of a protein of interest the human phenotype can be recapitulated more faithfully, as is the case in the asymptomatic individuals, where NCALD is not completely absent, but expressed at lower levels. The greatest challenge of this approach lies in the identification of potent shRNA sequences, and indeed, we had difficulties identifying an shRNA sequence that would strongly downregulate endogenous NCALD in a mouse cell line. Only when we overexpressedNcald cDNA in a cell line that does not express NCALD endogenously, we could find two efficient shRNA sequences. As also identification of a strong siRNA for

transient NCALD knock-down in mouse cell lines proved difficult, it is feasible thatNcald mRNA takes on a secondary structure which is not easily targeted by RNAi (Gutschner et al. 2011).

Second, as the completeNcaldko/ko_{mouse line has become available and turned out to be} both viable and fertile, we decided to adapt our strategy accordingly and instead of the shRNA transgene to use the heterozygousNcaldko/wt_{mice as a model of NCALD depletion,} which we crossed with the SMA mice. The great advantage of a genetic knock-out model is a consistent degree of protein reduction between individual animals while an exogenous treatment inevitably introduces additional variability. A genetic model is best suited for initial assessment of candidate modifier genes for a disease, however, it has obviously little translational potential. Moreover, most likely virtually any knock-out mouse line has its own phenotype arising from the disruption of the cellular homeostasis. In the case of theNcaldko/ko_{mice, their phenotypical analysis has revealed numerous changes of mainly} neurological and metabolic nature (Jackson Laboratory 2016). It remains unclear how many of these changes are already present in the Ncaldko/wt_{animals and have an effect} on the SMA-Ncaldko/wt_{mice that is independent of SMN deficiency. NCALD reduction had} a readily noticeable impact on the adipose tissue and body weight (5.2.3), still further analysis of theNcaldko/wt_andNcaldko/ko_{animals is required to fully comprehend the function} of NCALD.

Third, we attempted to deplete NCALD using exogenous compounds with a direct therapeutic potential, antisense oligonucleotides (ASOs). ASOs have the ability to target RNA in a sequence-specific way and depending on the locus they bind to, they can either silence gene expression by a variety of mechanisms or alter the splicing (Bennett and Swayze 2010). The ASOs we used here represent the second generation of antisense compounds with the mixed 2’ MOE chemistry: the center residues are phosphorothioate oligodeoxyribonucleotides, while the several terminal residues carry a methoxyethoxy substitution on the 2’ position of ribose. This combination increased hybridization affinity and potency, as well as resistance to nuclease cleavage; it also improved the tolerability profile by decreasing the proinflammatory effects (Chery and Naar 2016). Due to their large molecular weight and size, ASOs do not cross the blood brain barrier, therefore targeting ASO to neuronal tissue involves intrathecal or intracerebreventricular administration. In our tests of Ncald-ASO we observed that the 2’MOE chemistry (assessed from control ASO) was well tolerated despite the invasive nature of i.c.v. injection. WhenNcald-ASOs were administered i.c.v., they mediated an NCALD knock- down in the CNS even up to 30% of control level (5.4). Unfortunately, an apparent sequence-related toxicity ofNcald-ASOs resulted in increased mortality. One possible way

to overcome this drawback would be to test yet another ASO sequence; this approach is currently in progress. Another way to achieve a milder injection would be the use of a fine glass capillary needle in order to minimize the damage to the sensitive brain tissue (Glascock et al. 2011); also this refinement is under investigation.

Testing modifier genes in SMA mouse models has previously required laborious generation of new mouse lines carrying the modifying variants. Conveniently, a growing number of knock-out lines has become available for the scientific community to study the functional basis of human diseases (Rosen et al. 2015). In the future, functional genomic screens in mammalian animal models, e.g. in mouse, might be facilitated by the break- through gene editing technique using the endonuclease CRISPR/Cas9, especially as the libraries of verified guide RNA sequence are already being generated for many common laboratory species (Hsu et al. 2014). The advantages of the CRISPR/Cas9 mediated gene editing are its specificity, as specific gene variants can be tested instead of disease models only phenocopying a particular disorder, and rapidness, through bypassing the typical ES cell targeting step and directly targeting zygotes to generate transgenic lines (Paquet et al. 2016). This technique has already proven helpful in targeting difficult genes that failed in previous attempts (Schick et al. 2016). It harbors also a therapeutic potential for treating genetic disorders by correcting (even postnatally) the causative mutations while the gene remains expressed in its natural context which is an advantage in comparison to viral gene delivery (Long et al. 2016).