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Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

ABSTRACT: Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns^-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H⁺ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS^-/- kidney cells and injection into ctns^-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns^-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns^-/- larvae, and restoration of the zebrafish pronephros function.

SUBMITTER: Bondue T

PROVIDER: S-EPMC10684520 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

Bondue Tjessa T Berlingerio Sante Princiero SP Siegerist Florian F Sendino-Garví Elena E Schindler Maximilian M Baelde Hans Jacobus HJ Cairoli Sara S Goffredo Bianca Maria BM Arcolino Fanny Oliveira FO Dieker Jürgen J Janssen Manoe Jacoba MJ Endlich Nicole N Brock Roland R Gijsbers Rik R van den Heuvel Lambertus L Levtchenko Elena E

Scientific reports 20231128 1

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns<sup>-/-</sup> zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H<sup>+</sup> symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidn ...[more]

PMID: 38016974

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Project description:BACKGROUND:Cystinosis is an autosomal recessive lysosomal storage disorder characterized by accumulation of cystine in lysosomes throughout the body. Cystinosis is caused by mutations in the CTNS gene that encodes the lysosomal cystine carrier protein cystinosin. CTNS mutations result in either complete absence or reduced cystine transporting function of the protein. The diagnosis of nephropathic cystinosis is generally based on measuring leukocyte cystine level, demonstration of corneal cystine crystals by the slit lamp examination and confirmed by genetic analysis of the CTNS gene. CASE PRESENTATION:A boy born to consanguineous Caucasian parents had the characteristic clinical features of the infantile nephropathic cystinosis including renal Fanconi syndrome (polydipsia/polyuria, metabolic acidosis, hypokalemia, hypophosphatemia, low molecular weight proteinuria, glycosuria, cystine crystals in the cornea) and elevated WBC cystine levels. Initially we performed RFLP analysis of the common in the Northern European population 57-kb deletion of proband's DNA, then a direct Sanger sequencing which revealed no mutations in the coding part of the CTNS gene. To confirm the diagnosis we performed RT-PCR analysis of total RNA obtained from patient-derived fibroblasts in combination with cDNA sequencing. This revealed the skipping of exon 4 and exon 5 in the CTNS in our patient. Therefore, we detected a novel 9-kb homozygous deletion in the CTNS gene at genomic DNA level, spanning region from intron 3 to intron 5. In order to identify the inheritance pattern of the deletion we analyzed DNA of proband's mother and father. Both parents were found to be heterozygous carriers of the CTNS mutation. CONCLUSIONS:Analysis of CTNS gene transcript allowed to identify a large homozygous deletion in the patient with infantile nephropathic cystinosis. Mutational detection at RNA level may be an efficient tool to establish the genetic defect in some cystinosis patients.

Project description:BackgroundOcular cystinosis is a rare autosomal recessive disorder characterized by intralysosomal cystine accumulation in renal, ophthalmic (cornea, conjunctiva), and other organ abnormalities. Patients with ocular cystinosis are mostly asymptomatic and typically experience mild photophobia due to cystine crystals in the cornea observed accidently during a routine ocular examination. The ocular cystinosis is associated with different mutations in CTNS gene. Cysteamine therapy mostly corrects the organ abnormalities.MethodsThis study was performed in collaboration with the department of ophthalmology of Farhat Hached Hospital. The Optical Coherence Tomography (OCT) of the cornea and retinal photography were used to search cystine crystals within the corneas and conjunctiva in eight Tunisian patients. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the entire CTNS gene.ResultsThe studied patients were found to have cystine crystal limited anterior corneal stroma and the conjunctiva associated with retinal crystals accumulation. CTNS gene sequencing disclosed 7 mutations: three missense mutations (G308R, p.Q88K, and p.S139Y); one duplication (C.829dup), one framshift mutation (p.G258f), one splice site mutation (c.681 + 7delC) and a large deletion (20,327-bp deletion). Crystallographic structure analysis suggests that the novel mutation p.S139Y is buried in a first transmembrane helix closed to the lipid bilayer polar region, introducing a difference in hydrophobicity which could affect the hydrophobic interactions with the membrane lipids. The second novel mutation p.Q88K which is located in the lysosomal lumen close to the lipid membrane polar head region, introduced a basic amino acid in a region which tolerate only uncharged residue. The third missense mutation introduces a positive change in nonpolar tail region of the phospholipid bilayer membrane affecting the folding and stability of the protein in the lipid bilayer.ConclusionsOur data demonstrate that impaired transport of cystine out of lysosomes is the most common, which is obviously associated with the mutations of transmembrane domains of cystinosine resulting from a total loss of its activity.

Dataset Information

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

Publications

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

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