## 2 Mutation Type and Compatible Therapeutic Approaches
### Gene Therapy Approaches  
The type of autosomal dominant mutation associated with a particular disorder dictates the appropriate gene therapy approach. For loss-of-function mutations, higher protein levels are required. Therefore, supplementation of an extra copy of the wild type gene may be sufficient for phenotype restoration. In such cases, adeno-associated viral (AAV) vectors are most commonly used (Figure 3)due to their high transduction efficiency and excellent safety profile [12]. However, in cases where the cell is sensitive to the levels of the encoded protein, supplementation may not be suitable. Gene supplementation alone has also been suggested for some cases of dominant-negative mutations [13; 20; 21]. The reasoning is that by increasing the ratio of wild type versus mutant protein, the mutant protein will be out-competed and some level of function will be restored. However, caution needs to be taken, as this approach may only alleviate symptoms but not necessarily represent a cure. A reason is that even though a mutation may behave primarily as dominant-negative, it may also have toxic side effects for the cell, which will not be lessened by the extra levels of wild type protein [13; 22]. Therefore, generally, in order to treat dominant-negative as well as gain-of-function mutations, it is necessary to interfere with the mutant allele at the DNA or RNA level, prior to, or in lieu of, gene supplementation (for comprehensive review see [23; 24]). Three basic strategies (Figure 3) have been explored in order to fulfil this purpose:  
Invalidation of both alleles and gene supplementationThis is probably the most straightforward approach in terms of gene invalidation. The rationale is to design molecules that target both the mutant and wild type allele thus blocking protein production prior to supplying an exogenous gene copy. Figure 3: Flow chart showing the appropriate gene therapy strategy based on the type of mutation. Loss-of-function mutations (in yellow) could be treated by gene supplementation using, for example, viral vectors or non-viral nanoparticles to vehicle the transgene into cells. A future alternative would be to use CRISPR activation (CRISPRaR) to enhance gene expression (see Section 5). Dominant-negative (in blue) and gain-of-function (in red) mutations can be treated by mutation-independent or -dependent gene invalidation using the CRISPR/Cas system and the error-prone non-homologous end-joining (NHEJ) repair pathway. In the case of mutation-independent invalidation, this would systemically need to be coupled to gene supplementation. The more recent CRISPR interference (CRISPRRi) or base-editor technology, which can be used to suppress gene expression (see Section 5), also hold potential. Alternatively, these mutations can be treated by mutation-dependent correction using the CRISPR/Cas system and homology-directed repair (HDR). The recent base-editor technology also holds promise as a future gene correction strategy. An example at the DNA level is transcriptional repression using engineered zinc finger proteins, which has been shown to successfully silence human rhodopsin [25; 26]. At the RNA level, examples include the use of ribozymes [27] to mediate destruction of the target RNA, or RNA interference (si/shRNA) technologies [28] to down-regulate translation. Invalidation of the mutant alleleIt has been a longstanding goal of researchers to specifically target the mutant allele while sparing the wild type. The strategies can be the same as those for the invalidation of both alleles, but using molecules that specifically recognize the mutant allele. At the DNA level, genome editing by the relatively recent CRISPR/Cas system, which is detailed in a separate section below, has been used to introduce targeted double-strand breaks for specific allele ablation [29; 30; 31]. Examples for RNA destruction or interference include ribozymes [32] and anti-oligosense nucleotides [33], respectively.