Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5

Yamamura, Takehira; Horinouchi, Tomoko; Adachi, Tomomi; Terakawa, Maki; Takaoka, Yutaka; Omachi, Kohei; Takasato, Minoru; Takaishi, Kiyosumi; Shoji, Takao; Onishi, Yasunobu; Kanazawa, Yoshito; Koizumi, Makoto; Tomono, Yasuko; Sugano, Aki; Shono, Atsuko; Minamikawa, Shogo; Nagano, China; Sakakibara, Noburu; Ishiko, Shinya; Aoto, Yuya; Kamura, Misato; Harita, Yutaka; Miura, Kenichiro; Kanda, Shoichiro; Morisada, Naoya; Rossanti, Rini; Ye, Ming Juan; Nozu, Yoshimi; Matsuo, Masafumi; Kai, Hirofumi; Iijima, Kazumoto; Nozu, Kandai

doi:10.1038/s41467-020-16605-x

Cited by 59 publications

(49 citation statements)

References 27 publications

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“…Note clear immunostaining of p57-positive cells limited to the podocyte. Average number of p57-positive cells per glomerular profiles showed no significant difference between WT mice and mutant mice in 6 and www.nature.com/scientificreports/ caused exclusive crescents, and exon-skipping therapy enabled trimer formation of collagen IV and rescued glomeruli from crescents in this model 21 , indicating a causal relationship between this mutation and crescents. Sequential analysis of kidney pathology yielded several key findings about the mechanism of crescent formation in this XLAS model.…”

Section: Discussionmentioning

confidence: 79%

Bidirectional, non-necrotizing glomerular crescents are the critical pathology in X-linked Alport syndrome mouse model harboring nonsense mutation of human COL4A5

Song

Saga

Kawanishi

et al. 2020

Sci Rep

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X-linked Alport syndrome (XLAS) is a progressive kidney disease caused by genetic abnormalities of COL4A5. Lack of collagen IV α5 chain staining and “basket-weave” by electron microscopy (EM) in glomerular basement membrane (GBM) are its typical pathology. However, the causal relationship between GBM defects and progressive nephropathy is unknown. We analyzed sequential pathology in a mouse model of XLAS harboring a human nonsense mutation of COL4A5. In mutant mice, nephropathy commenced from focal GBM irregularity by EM at 6 weeks of age, prior to exclusive crescents at 13 weeks of age. Low-vacuum scanning EM demonstrated substantial ragged features in GBM, and crescents were closely associated with fibrinoid exudate, despite lack of GBM break and podocyte depletion at 13 weeks of age. Crescents were derived from two sites by different cellular components. One was CD44 + cells, often with fibrinoid exudate in the urinary space, and the other was accumulation of α-SMA + cells in the thickened Bowman’s capsule. These changes finally coalesced, leading to global obliteration. In conclusion, vulnerability of glomerular and capsular barriers to the structural defect in collagen IV may cause non-necrotizing crescents via activation of PECs and migration of interstitial fibroblasts, promoting kidney disease in this model.

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Section: Discussionmentioning

confidence: 79%

Bidirectional, non-necrotizing glomerular crescents are the critical pathology in X-linked Alport syndrome mouse model harboring nonsense mutation of human COL4A5

Song

Saga

Kawanishi

et al. 2020

Sci Rep

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“…During such study, we have produced a ready-made splicing reporter minigene named H492 minigene [34]. This minigene has been utilized not only for analysis of exon skippable agents [11,19] but also for in vitro determination of splicing outcomes caused by nucleotide changes in genetic diseases [35][36][37][38][39][40][41][42]. However, the minigene is facing two drawbacks of RT-PCR amplification and sequencing step.…”

Section: Discussionmentioning

confidence: 99%

“…Exon skipping is recognized as a promising therapy for many diseases [11]. However, it has been a big issue to establish a mass screening system to identify agents to induce skipping of exons.…”

Section: Discussionmentioning

confidence: 99%

“…There are three main aims of exon skipping therapy: gain of function, loss of function and modulation of function. For the gain of function, exon skipping has been applied to transform out-of-frame mutations into in-frame in the DMD gene [4] and to eliminate disastrous mutations of the DYSF, CEP290, COL4A5 and GNTAB genes [9][10][11][12]. For the loss of function, skipping of an out-of-frame exon of the MSTN gene has been reported [13].…”

Section: Introductionmentioning

confidence: 99%

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Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

Fukushima

Farea

Maeta

et al. 2020

IJMS

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Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.

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“…We conducted an in vivo study using an AS mouse model possessing a nonsense mutation in exon 21 of col4a5 [46] and treated mice with ASO leading to exon 21 (84 bp) skipping. This treatment had a remarkable effect of improving the pathological findings with the recovery of α5(IV) chain expression, reduced urine protein excretion level, delayed development of ESKD, and extension of survival [10]. To date, we have only developed ASO for exon 21, so further work is needed to develop this therwww.krcp-ksn.org apy to target other exons.…”

Section: Exon Skipping Therapymentioning

confidence: 99%

Genetic background, recent advances in molecular biology, and development of novel therapy in Alport syndrome

Nozu

Takaoka

Kai

et al. 2020

Kidney Res Clin Pract

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Alport syndrome (AS) is a progressive inherited kidney disease characterized by hearing loss and ocular abnormalities. There are three forms of AS depending on inheritance mode: X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, which encodes type IV collagen α5 chain, while ADAS and ARAS are caused by variants in COL4A3 or COL4A4, which encode type IV collagen α3 or α4 chain, respectively. In male XLAS or ARAS cases, end-stage kidney disease (ESKD) develops around a median age of 20 to 30 years old, while female XLAS or ADAS cases develop ESKD around a median age of 60 to 70 years old. The diagnosis of AS is dependent on either genetic or pathological findings. However, determining the pathogenicity of the variants detected by gene tests can be difficult. Recently, we applied the following molecular investigation tools to determine pathogenicity: 1) in silico and in vitro trimer formation assay of α345 chains to assess triple helix formation ability, 2) kidney organoids constructed from patients' induced pluripotent stem cells to identify α5 chain expression on the glomerular basement membrane, and 3) in vitro splicing assay to detect aberrant splicing to determine the pathogenicity of variants. In this review article, we discuss the genetic background and novel assays for determining the pathogenicity of variants. We also discuss the current treatment approaches and introduce exon skipping therapy as one potential treatment option.

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Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5

Cited by 59 publications

References 27 publications

Bidirectional, non-necrotizing glomerular crescents are the critical pathology in X-linked Alport syndrome mouse model harboring nonsense mutation of human COL4A5

Bidirectional, non-necrotizing glomerular crescents are the critical pathology in X-linked Alport syndrome mouse model harboring nonsense mutation of human COL4A5

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

Genetic background, recent advances in molecular biology, and development of novel therapy in Alport syndrome

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