Finding an Appropriate Mouse Model to Study the Impact of a Treatment for Friedreich Ataxia on the Behavioral Phenotype.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by a GAA repeat in the intron 1 of the frataxin gene (FXN) leading to a lower expression of the frataxin protein. The YG8sR mice are Knock-Out (KO) for their murine frataxin gene but contain a human frataxin transgene derived from an FRDA patient with 300 GAA repeats. These mice are used as a FRDA model but even with a low frataxin concentration, their phenotype is mild. We aimed to find an optimized mouse model with a phenotype comparable to the human patients to study the impact of therapy on the phenotype. We compared two mouse models: the YG8sR injected with an AAV. PHP.B coding for a shRNA targeting the human frataxin gene and the YG8-800, a new mouse model with a human transgene containing 800 GAA repeats. Both mouse models were compared to Y47R mice containing nine GAA repeats that were considered healthy mice. Behavior tests (parallel rod floor apparatus, hanging test, inverted T beam, and notched beam test) were carried out from 2 to 11 months and significant differences were noticed for both YG8sR mice injected with an anti-FXN shRNA and the YG8-800 mice compared to healthy mice. In conclusion, YG8sR mice have a slight phenotype, and injecting them with an AAV-PHP.B expressing an shRNA targeting frataxin does increase their phenotype. The YG8-800 mice have a phenotype comparable to the human ataxic phenotype.

Prime editing strategies to mediate exon skipping in DMD gene.

Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.

Removal of the GAA repeat in the heart of a Friedreich's ataxia mouse model using CjCas9.

Most Friedreich ataxia (FRDA) cases are caused by the elongation of the GAA repeat (GAAr) sequence in the first intron of the FXN gene, leading to a decrease of the frataxin protein expression. Deletion of this GAAr with CRISPR/Cas9 technology leads to an increase in frataxin expression in vitro. We are therefore aiming to develop FRDA treatment based on the deletion of GAAr with CRISPR/Cas9 technology using a single AAV expressing a small Cas9 (CjCas9) and two single guide RNAs (sgRNAs) targeting the FXN gene. This AAV was intraperitoneally administrated to YG8sR (250-300 GAAr) and to YG8-800 (800 GAAr) mice. DNA and RNA were extracted from different organs a month later. PCR amplification of part of intron 1 of the FXN gene detected some GAAr deletion in some cells in heart and liver of both mouse models, but the editing rate was not sufficient to cause an increase in frataxin mRNA in the heart. However, the correlation observed between the editing rate and the distribution of AAV suggests a possible therapy based on the removal of the GAAr with a better delivery tool of the CRISPR/Cas9 system.

Small-molecule inhibitors of proteasome increase CjCas9 protein stability.

The small size of CjCas9 can make easier its vectorization for in vivo gene therapy. However, compared to the SpCas9, the CjCas9 is, in general, less efficient to generate indels in target genes. The factors that affect its efficacity are not yet determined. We observed that the CjCas9 protein expressed in HEK293T cells after transfection of this transgene under a CMV promoter was much lower than the SpCas9 protein in the same conditions. We thus evaluated the effect of proteasome inhibitors on CjCas9 protein stability and its efficiency on FXN gene editing. Western blotting showed that the addition of MG132 or bortezomib, significantly increased CjCas9 protein levels in HEK293T and HeLa cells. Moreover, bortezomib increased the level of CjCas9 protein expressed under promoters weaker than CMV such as CBH or EFS but which are specific for certain tissues. Finally, ddPCR quantification showed that bortezomib treatment enhanced CjCas9 efficiency to delete GAA repeat region of FXN gene in HEK293T cells. The improvement of CjCas9 protein stability would facilitate its used in CRISPR/Cas system.

Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy.

The Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (DMD) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the DMD gene in HEK293T cells were obtained with the PRIME Editor 2 (PE2) and PE3, respectively. Three repeated treatments increased the percentage of specific mutations with PE2 to 16%. An additional mutation in the protospacer adjacent motif (PAM) sequence improved the PE3 result to 38% after a single treatment. We also carried out the correction of c.428 G>A point mutation in exon 6 of the DMD gene in a patient myoblast. Myoblast electroporation showed up to 8% and 28% modifications, respectively, for one and three repeated treatments using the PE3 system. The myoblast correction led to dystrophin expression in myotubes detected by Western blot. Thus, prime editing can be used for the correction of point mutations in the DMD gene.

Serum extracellular vesicles for delivery of CRISPR-CAS9 ribonucleoproteins to modify the dystrophin gene.

Extracellular vesicles (EVs) mediate intercellular biomolecule exchanges in the body, making them promising delivery vehicles for therapeutic cargo. Genetic engineering by the CRISPR system is an interesting therapeutic avenue for genetic diseases such as Duchenne muscular dystrophy (DMD). We developed a simple method for loading EVs with CRISPR ribonucleoproteins (RNPs) consisting of SpCas9 proteins and guide RNAs (gRNAs). EVs were first purified from human or mouse serum using ultrafiltration and size-exclusion chromatography. Using protein transfectant to load RNPs into serum EVs, we showed that EVs are good carriers of RNPs in vitro and restored the expression of the tdTomato fluorescent protein in muscle fibers of Ai9 mice. EVs carrying RNPs targeting introns 22 and 24 of the DMD gene were also injected into muscles of mdx mice having a non-sense mutation in exon 23. Up to 19% of the cDNA extracted from treated mdx mice had the intended deletion of exons 23 and 24, allowing dystrophin expression in muscle fibers. RNPs alone, without EVs, were inefficient in generating detectable deletions in mouse muscles. This method opens new opportunities for rapid and safe delivery of CRISPR components to treat DMD.

CRISPR-SCReT (CRISPR-Stop Codon Read Through) method to control Cas9 expression for gene editing.

CRISPR/Cas9 has paved the way for the development of therapies that correct genetic mutations. However, constitutive expression of the Cas9 gene can increase off-target mutations and induce an immune response against the Cas9 protein. To limit the time during which the Cas9 nuclease is expressed, we proposed a simple drug inducible system. The approach consists of introducing a premature termination codon (PTC) in the Cas9 gene and subsequently treating with an aminoglycoside drug, which allows readthrough of the complete protein. To validate that system, HEK293T cells were co-transfected with a PX458 plasmid, which was mutated to introduce a PTC in the SpCas9 gene and two sgRNAs targeting the DMD gene (exons 50 and 54). Cells were treated with different doses of geneticin (G418) for 48 h. Western blot confirmed that the Cas9 protein expression, which was shut down by the PTC mutation, can be induced by the drug. The hybrid exon 50-54 formed by the deletion of part of the DMD gene was detected by PCR only in the cells treated with G418. The approach was also used successfully with CjCas9 to edit the FXN gene. Our results show that it is possible to control SpCas9 and CjCas9 expression by CRISPR-SCReT (CRISPR-Stop Codon Read Through) method.

Rotavirus and norovirus in children with severe diarrhea in Burkina Faso before rotavirus vaccine introduction.

Burkina Faso introduced rotavirus vaccine (RotaTeq) to the national immunization program in November 2013. This study describes the detection rates, clinical profiles, and molecular epidemiology of rotavirus and norovirus (NoV) infections among children <5 years hospitalized (n = 154) because of acute diarrhea in Ouagadougou, Burkina Faso, from December 2012 to November 2013, just before the start of vaccination. Overall, 44% and 23% of fecal samples were positive for rotavirus and NoV, respectively, most of them detected during the cold dry season (December-March). The predominant G/P combinations were G12P[8] (47%) and G6P[6] (30%). G2P[4] (n = 3), G12P[6] (n = 3), and G6P[8] (n = 1) were also detected. Nearly all (94%) successfully genotyped NoV strains belonged to genotype GII.4. The predominance of rotavirus and NoV was noteworthy in the age group ≤6 months, with 67% rotavirus and 22% NoV, respectively. Vomiting was significantly more common among rotavirus-infected children. To conclude, this study shows high detection rates of both rotavirus and NoV in children with severe diarrhea in Burkina Faso just before the introduction of rotavirus group A vaccination. The results can be used for estimating the impact of rotavirus group A vaccination, which started in the end of 2013. Furthermore, this study shows that the G6P[6] rotavirus strains emerging in Burkina Faso in 2010 is now established as a regionally important genotype.

Prevalence, genetic variants and clinical implications of G-6-PD deficiency in Burkina Faso: a systematic review.

BACKGROUND: It is now well-known that some antimalarials such as primaquine may induce severe hemolytic anemia in people with G-6-PD deficiency. Antimalarial drug prescriptions must, therefore take into account the patient's G-6-PD status in malaria endemic areas such as Burkina Faso, where the prevalence of this genetic abnormality is relatively high. Although great clinical heterogeneity is observed depending on the molecular nature of the deficiency and the residual enzyme activity in the red blood cell, there is very poor data on the prevalence of G-6-PD deficiency and the distribution of involved genetic variants in Burkina Faso. In this systematic review, we present a synthesis of the various studies carried out on the G-6-PD deficiency in Burkina Faso in order to determine its prevalence, probable distribution of the genetic variants involved and their clinical implications for a national systematic screening policy among the groups most vulnerable to malaria. METHODS: A systematic review was carried out to analyze available published data on the prevalence, phenotypes and mutations responsible for G-6-PD deficiency in Burkina Faso. The key words used were "G-6-PD deficiency AND Burkina Faso" or "Déficit en G-6-PD AND Burkina Faso" in French. To identify the relevant articles, two independent reviewers reviewed the titles, abstracts and the full text of the selected papers. RESULTS: An average prevalence of 16.6% (183/1100; CI 95%: 0.145-0.190) and 6.5% (69/1066; CI 95%: 0.051-0.081) of G-6-PD deficiency was found respectively in men and women in this systematic review. Although the predominance (99.8% of G-6-PD deficient cases) of 202A/376G G-6-PD A- variant, the Santamaria and Betica Selma variants were identified in Burkina Faso. Independently of the method used, the enzymatic deficiency was significantly higher in males (2.5-20.5%) compared to females (3.3-12.3%). CONCLUSION: This systematic review suggests that despite the ubiquity of the 202A/376G G-6-PD A- variant in Burkina Faso, it will be necessary to consider the Santamaria and Betica Selma variants although their frequencies remain to be specified. A systematic screening of the G-6-PD deficiency is also needed to prevent the occurrence of iatrogenic hemolytic accidents.

Major Polymorphisms of Genes Involved in Homocysteine Metabolism in Malaria Patients in Ouagadougou, Burkina Faso.

This study analyzed the four main polymorphisms of the genes in homocysteine metabolism in malaria patients. Forty-two randomly selected subjects, diagnosed positive for Plasmodium falciparum, were included. The four genotypes were detected by real-time PCR using the MTHFR 677C>T, MTHFR 1298A>C, MTR 2756A>G, and MTRR 66A>G detection kit (Sacace Biotechnologies REF: T01002-96-S). The results revealed frequencies of 90% 677CC, 10% 677CT, and 00% 677TT for MTHFR C677T; 78.6% 1298AA, 19% 1298AC, and 2.4% 1298CC for MTHFR A1298C; 61.9% 2756AA, 33.3% 2756AG, and 4.8% 2756GG for MTR A2756G; and 50% of 66AA, 45% of 66AG, and 5% of 66GG for MTRR A66G. Correlations were found between A2756G MTR genotypes and parasitaemia (P = 0.02), MTRR A66G and hemoglobin genotypes (P = 0.009), and MTHFR A1298C and sex (P = 0.01). This study demonstrated for the first time an association between the A2756G MTR alleles and Plasmodium falciparum malaria in Burkina Faso and gave an overview of the genotypic distribution of the major SNPs influencing the metabolism of homocysteine.

Molecular Heterogeneity of Glucose-6-Phosphate Dehydrogenase Deficiency in Burkina Faso: G-6-PD Betica Selma and Santamaria in People with Symptomatic Malaria in Ouagadougou.

The G-6-PD deficiency has an important polymorphism with genotypic variants such as 202A/376G, 376G/542T and 376G/968T known in West African populations. It would confer protection against severe forms of malaria although there are differences between the various associations in different studies. In this study we genotyped six (06) variants of the G-6-PD gene in people with symptomatic malaria in urban areas in Burkina Faso. One hundred and eighty-two (182) patients who tested positive using rapid detection test and microscopy were included in this study. A regular PCR with the GENESPARK G6PD African kit was run followed by electrophoresis, allowing initially to genotype six SNPs (G202A, A376G, A542T, G680T, C563T and T968C). Women carrying the mutations 202A and/or 376G were further typed by real-time PCR using TaqMan probes rs1050828 and rs1050829. In the study population the G-6-PD deficiency prevalence was 9.9%. In addition of G-6-PD A- (202A/376G) variant, 376G/542T and 376G/968T variants were also detected. Hemoglobin electrophoresis revealed that 22.5% (41/182) of the individuals had HbAC compared with2.2% with HbAS and one individual had double heterozygous HbSC. There was no correlation between the G-6-PD deficiency or haemoglobinopathies and symptomatic malaria infections in this study. Our study confirms that the G-6-PD deficiency does not confer protection against Plasmodium falciparum infections. As opposed to previous genotyping studies carried out in Burkina Faso, this study shows for the first time the presence of the variant A- (376G/968C) and warrants further investigation at the national level and in specific ethnic groups.