**3. Animal models to test precision medicine approaches**

Genetic testing revealed the incredible diversity of mutations in *DMD* gene. However, mutations are not equally presented throughout the gene. As much as 80% of all mutations are concentrated in exons 2–20 and 45–55 representing two hotspots. Mutations can be divided into two groups: frequent (one or more exons deletions and duplications) and rare (point substitutions in exons and introns, small deletions and duplications) [81]. Mutation-specific precision medicine approaches are mostly based on the reading frame rule and convert mutations from Duchenne to Becker type. In the presence of frameshift generating mutations additional removal of one or several exons can restore the reading frame and cause expression of shortened yet functional dystrophin protein. For exons removal during splicing process antisense oligonucleotides (ASO, AON) are used. AON binds specifically to the splice sites of selected exons hiding them from cellular splicing machinery and leading to their exclusion from mRNA. Different chemical structures are used for reduced AON cleavage, prolonged circulation, better cellular and nuclear penetration. The most popular backbones are presented by PMO (phosphorodiamidate morpholino oligomers), 2-OMePS (2'O-methylated phosphorothioate), vivo-morpholino (morpholino oligo covalently linked to octaguanidine dendrimer), LNA (locked nucleic acids), tcDNA (tricyclo-DNA). Indeed, AON can be delivered naked or in the lipid complex, fused with targeting peptides or other molecules enhancing biodistribution. In addition to AON, vectorized drug candidates are tested for exon skipping. Their design is based on U7 snRNA, naturally participating in histone pre-mRNA processing. Deletion of additional exons directly from genomic DNA (gDNA) is also proposed as a mutation-specific therapeutic strategy for DMD. For this purpose viral delivery of one or two single guide RNA (sgRNA) and Cas9 encoding sequences is tested. Targeted Cas9-induced double strand cleavage is also applied for indel generation in affected or neighborhood exons. Indels lead to +1 or − 1 frameshifts with a certain probability. This approach is known as reframing. Exon can be excluded from mRNA due to another DNA modification - base editing in conservative splice site sequence. For this approach Cas9 fused with base editing enzymes is utilized. Both for U7 snRNA (U7 small nuclear RNA) and Cas9 delivery viral vectors such as lentiviruses and AAV are used. Majority of experiments for mutation-specific approach examinations are conducted on patients-derived cell cultures and modified human embryonic stem (ES) cells. However, complexity of the disease and limitations of functional tests applicable *in vitro* force to generate and use genetically modified animal models.

#### *Duchenne Muscular Dystrophy Animal Models DOI: http://dx.doi.org/10.5772/intechopen.96738*

Mice with various mutations in the dystrophin gene, replicating mutations found in individual patients or groups of patients are the most common among the genomeedited models. The timeline of disease progression and traits of new models are usually not well studied. Main DMD symptoms are similar to those found in *mdx* mice. The purpose of these "genetic" models is to test mutation-specific therapies and show not only restoration of dystrophin expression but also improvement in locomotor activity, illustrating the functionality of the shortened protein. The ease of maintenance and reproduction, extensive experience in obtaining and speed of reproduction, together with the high conservativeness of the dystrophin gene, make mice the optimal objects for such work, nevertheless, there are other, larger animal models with mutations often found in patients with DMD.

Deletions of one or more exons are the most common mutations in the DMD gene. They account for 68% of all mutations. Among them, deletions of single exon 44 (3%), 45 (4%), 50 (2%), 51 (3%), 52 (3%) are represented with approximately the same frequency [81]. Directed mutations in the dystrophin gene in laboratory animals were obtained for the purpose of selecting drugs for exon-skipping. Exon structures of popular models with deletions in mutation hotspot are shown on **Figure 1**. The first models were obtained by homologous recombination using embryonic stem cells. In 1997 a mouse model *mdx52* with a deletion of exon 52 was created [82], where this exon was replaced with a neomycin resistance cassette. This mouse model was used to test various drug candidates: PS-modified tcDNA (phosphorothioate-modified Tricyclo-DNA) based ASO for skipping of the exon 51 [83], PMO for exon 51 skipping [84, 85], AAV9-U7snRNA for exon 51 skipping [86],

#### **Figure 1.**

*Animal models representing DMD exon deletions in mutation hotspot. Gene fragment structures around exon with frameshift mutation are shown on the left. Currently tested therapeutic approaches and resulting exon structures are shown on the right.*

mix of vivo-morpholinos for simultaneous skipping of the exons 45–55 [87, 88]. With the advent of effective genome editing techniques, frequent mutations were the first to be reproduced in animals. TALEN were used to create mice with exon 52 deletion resulting in **del52hdmd/***mdx* model [89]. This model is notable for the fact that the mutation was introduced into the sequence of the human gene. Thus, **del52hdmd/***mdx* model can be used to test drugs that are designed to target unique human sequences. The authors showed the effectiveness of AON for skipping exons 51 and 53 to the human sequence during intramuscular delivery [89]. This line was used to test CRISPR/Cas9 genome editing complexes for reframing in exons 51 and 53 during lentiviral delivery [90]. AAV9 double SaCas9 (*Staphylococcus aureus* Cas9 ortholog) and guide mix was tested for deletion with borders within exons 47 and 58 for himeric exon formation [91]. Later, another mouse model with a deletion of exon 52, **∆52**, was obtained using CRISPR/Cas9 genome editing system [92]. This model was used to test CRISPR/Cas9-based drugs for exon 53 removal or reframing [92].

Reframing in exon 51 was also tested in mouse models with deletion of exon 50 **ΔEx50** and **ΔEx50-Dmd-Luc** [93]. In the *Dmd* gene of **ΔEx50-Dmd-Luc** mice, in addition to the deletion of exon 50, the luciferase gene sequence is also introduced at the C-terminus, connected to the protein sequence via an autocatalytic 2A peptide. Thus, luminescence was observed during the restoration of the reading frame, which allowed to assess the effectiveness of drugs *in vivo* without resorting to invasive methods [93]. Bioluminescence was detected both after intramuscular and systemic delivery of Cas9 and sgRNA-51 by AAV9. The presence of bioluminescence was shown to correlate with dystrophin expression as verified by western blotting and immunohistochemistry (IHC) [93].

One of the most frequent deletions, the deletion of exon 44, was reproduced in mice **Δex44 DMD** [94]. Correction of exon 44 deletions by gene editing of surrounding exons could potentially restore the reading frame of dystrophin in ~12% of patients with DMD. Authors created AAV9-Cas9 and AAV9-sgRNA mix targeting 5′-end of exon 45 and tested them *in vivo* during intramuscular injections on this model. The most perspective guide sequence 6 (G6) was used for systemic delivery and selection of a better Cas9 to sgRNA AAV particles ratio. Selected conditions lead to force increase from 59% to 107% in the extensor digitorum longus (EDL) muscle of **ΔEx44 DMD** mice [94]. 20-fold lower dose of self-complementary adenoassociated virus (scAAV) bearing Cas9 + sgRNA was used for exon 45 skipping and reframing on the same model [95]. Weekly injection of (1,2-dioleoyl-3-trimethylammonium-propane) LNPs (lipid nanoparticles) encapsulating Cas9/sgDMD RNPs (ribonucleoproteins) into Tibialis Anterior (TA) muscles was tested on **ΔEx44 DMD** mice. The expression of dystrophin in TA muscles was successfully restored after skipping or reframing of exon 45 induced by treatment, as demonstrated by immunofluorescence and western blot analysis. Quantitative analysis of the western blot result showed that 4.2% of dystrophin protein was restored [96].

CRISPR/Cas9 genome edited **hDMD del45** model represents deletion of exon 45 in human dystrophin gene in the presence of wild type *Dmd* gene while **hDMD del45** *mdx* **D2** has dystrophic phenotype due to *Dmd* gene knock-out [97]. In the same paper exons 45–55 deletion strategy (Cas9 + gRNAs to introns 44 and 55) aiming to help 60–65% of patients was tested [97, 98]. A more realistic approach from the clinical application point of view is multiple exon 45–55 skipping using U7 snRNAs [99]. It was tested on **hDMD/***mdx* model [100]. Similar multiple exonskipping strategy using PMOs cocktail [98] was tested on **hDMD/Dmd Null** mice [85]. The **hDMD/Dmd Null** model compares favorably with the previous models, since it does not have a mouse dystrophin sequence and allows us to quantify the level of exon skipping and compare the effectiveness of different sequences and

#### *Duchenne Muscular Dystrophy Animal Models DOI: http://dx.doi.org/10.5772/intechopen.96738*

drugs with each other, which is demonstrated by the example of exon 51 skipping [85]. Moreover, the presence of normal dystrophin in some models leads to the absence of the necessary symptoms for the delivery of oligonucleotides and viruses, such as inflammation in the muscles and intact cellular membranes. It's necessary to point out the crucial role of **hDMD** mouse model with full-length *DMD* gene integrated into chromosome 5 [100]. It is not very useful for any drug substances by itself due to simultaneous expression of wild type human and murine dystrophin proteins. But when crossing to *mdx* or other *Dmd* knockout mice (**hDMD/***mdx***, hDMD/Dmd Null**) it becomes an extremely important background for creation of new models. Any antisense or guide molecules designed and tested on subsequent animals can be transferred to human cells without sequence adaptation.

Models with single exon deletions **∆43** (exon 43 deletion), **∆45** (exon 45 deletion) were reported together with **∆52** (exon 52 deletion) **DMD** mice [92]. These mouse models were used to test single guide genome editing procedures aiming at exon skipping or reframing. AAV9 and scAAV9 viruses with a guide and Cas9 sequences were used. Intramuscular delivery of guide RNA to exon 44 and Cas9 encoding viruses to TA muscle restored dystrophin expression in both **∆43** and **∆45 DMD** models. Interestingly, the selected guide generated both exon reframed and exon skipped transcripts in **∆45 DMD** muscle, but only exon skipped transcripts in **∆43 DMD** muscle. Restoration of dystrophin in **∆45 DMD** muscle was more efficient than in **∆43 DMD** muscle when using the same sgRNA for gene editing [92].

Deletions of several exons are quite common in patients, but to date only one mouse model with an extended mutation in the hotspot is known. Deletion of exons 52–54 was simulated in **Dmd Δ52–54** in which authors declare severe cardiac dysfunction in addition to common skeletal muscle symptoms. CRISPR-mediated single sgRNA exon skipping of exon 55 was tested on this model. Also another model with deletion of exons 52–55 was created to check potential benefit of the treatment [101]. CRISPR/Cas9 system popularity, easy to use and high efficiency allow to generate such 100% skipping (editing) models to check generated shortened dystrophin protein functionality.

Duplications of one or several exons are also highly widespread mutations, affecting 5–10% of all DMD patients [102]. The most common duplication in the patient population **Dup2** (duplication of exon 2) was recreated in the mouse model created in 2015 [103]. Correction of the mutation was shown on this model after intramuscular injections of AAV1.U7-ACCA [104]. Main attribute of this mutation and corresponding model is that precision skipping of a duplicated exon results in full-length dystrophin expression. Thus, it is the rare case when exon-skipping converts Duchenne type mutation to wild type rather than Becker type. At the same time it is a challenge to accurately skip only copy of exon 2 not affecting the main sequence.

Nonsense mutations are also very common in the human population affecting approximately 25% of the patients [102]. The most popular *mdx* mouse model representing nonsense mutation was found in the natural population [105]. A lot of treatment strategies targeting downstream disease mechanisms were tested on this model. Precision medicine approach on *mdx* model includes stop codon readthrough [106]. Targeted single nucleotide mutation was created in **DMD-KO** (D108) mouse [107] representing C-to-T conversion generating stop codon in exon 20 (Q871Stop). This mouse model was used for adenine base editing using guided SpCas9 (*Streptococcus pyogenes* Cas9 ortholog) nickase [108]. Created by ENU mutagenesis, *mdx*4cv model also has C-to-T conversion in exon 53 generating TAA stop codon [33]. Adenine base editor was also tested on this mouse model with modified Cas9 recognizing relaxed minimal PAM (protospacer adjacent motif) sequence - NG [109]. Both *mdx* and *mdx*4cv models were used for trans-splicing method

efficiency demonstration [110]. Intramuscular delivery of AAV vectors expressing trans-splicing template (PTM) allowed detectable levels of dystrophin in *mdx* and *mdx*4cv, illustrating that a given PTM can be suitable for a variety of mutations.

Rare mutations found in patients were repeated in models to test precision gene editing methods. Those contain big deletion of exons 8–34 in **DmdDel8–34** mouse model (Egorova et al., 2019). Reading frame in this model can be restored by simultaneous skipping of exons 6 and 7. The fact that the N-terminal actin binding domain is partly encoded by these exons, gives the opportunity to better understand structure-functional interplay in dystrophin protein and its shortened forms. Several approaches including vivo-morpholino induced exon-skipping and CRISPR/Cas9 gene editing are tested on this model ([111]; unpublished data). Other variants of rare mutations generated by Koo and colleagues, represent small frameshift mutations [112]. In vivo treatment with AAV vectors encoding CjCas9 (*Campylobacter jejuni* Cas9 ortholog) and single guide to the affected exon restored the reading frame and enhanced muscle strength.

Large mammalian models have more pronounced DMD symptoms in comparison to murine models. But limited availability and less extensive experience in their genome modification led to reduced use in precision medicine approaches testing. Pig model with deletion of exon 52 **DMDΔ52** was created using somatic nuclear transfer from bacterial artificial chromosome (BAC)-edited cells [113]. AAV9 vector with intein splitted Cas9 and two guides around exon 51 was tested on these animals reaching widespread dystrophin expression, prolonged survival and reduced arrhythmogenic vulnerability [114]. Another example of CRISPR/Cas9 Dmd gene targeting in pigs resulted in indels in exon 27 which lead to premature piglet death at day 52 [115]. So this model was not tested yet for any treatment approach.

**DMD KO** rabbits represent different mutations in exon 51 which is within the mutation hotspot in human *DMD* gene [116]. Many of these mutations are small deletions or insertions, disrupting the reading frame of the *DMD* gene, resulting in frameshift and complete absence of dystrophin expression followed by main phenotypic features in skeletal muscles and cardiomyopathy. Animals could benefit from 50–51, 51–52 or 45–55 exons skipping, however this model animals were not used for any precision medicines testing.

Identified in natural population **CKCS-MD** (deltaE50-MD) dogs have a splice site missense mutation in intron 50 of the *DMD* gene, causing out-of-frame skipping of exon 50 and resulting in a lack of dystrophin and a severe dystrophic phenotype resembling DMD [61]. In the same paper authors show that additional skipping of exon 51 could restore dystrophin expression on cultured myoblasts [61]. Single guide genome editing aiming at exon 51 skipping or reframing shows big potential after intramuscular and intravenous delivery [117].

Naturally occurring intron splice site mutation that leads to the loss of exon 7 was identified in Golden retriever dogs leading to generation of **GRMD** (**CXMDJ**) canine model [62]. It is the most widespread canine model which is used in numerous studies including exons 6 and 8 skipping driven by PMO and 2'OMePS [118, 119], AAV1-U7snRNA [120], rAAV6-U7snRNA [121], AAV8-U7snRNA [122].

The next model stands out and its value is more in demonstrating the possibility of creating new models than in itself. The new mice obtained by transgenesis carry randomly embedded copies of the EGFP under the CAG promoter (strong synthetic promoter that consists of regulatory elements from **C**MV, chicken beta-**a**ctin gene and rabbit beta-**g**lobin gene), which are separated by exon 23 with the murine *mdx* mutation. EGFP expression is possible only in case of exon 23 skipping, which was demonstrated using PMO and LNA/2'-OMe based AONs [123]. Thus, the resulting mouse model allows noninvasive assessment of the effectiveness of exon skipping, as well as studying the bio-distribution of drugs. The creation of similar

mouse models for testing exon skipping, more applicable to patients, will allow more intensive studies of future drugs.
