Antisense oligonucleotides for the treatment of stargardt disease

An antisense oligonucleotide and nucleotide technology, applied in gene therapy, sensory diseases, medical preparations containing active ingredients, etc., can solve problems such as frameshift loss and instability

Pending Publication Date: 2020-04-10
プロキューアールセラピューティクスツーベスローテンフェンノートシャップ
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Skipping of exon 39 resulted in a frameshift of 124 nucleotides, whereas double skipping of exons 39 and 40 resulted in a frameshift of 254 nucleotides
Notably, this shorter version of the protein was not detected, possibly because they are unstable (Aukrust et al., 2016)

Method used

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  • Antisense oligonucleotides for the treatment of stargardt disease
  • Antisense oligonucleotides for the treatment of stargardt disease
  • Antisense oligonucleotides for the treatment of stargardt disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Generation and testing of antisense oligonucleotides (AONs) for blocking skipping from exon 39 of human ABCA4 pre-mRNA

[0070] Antisense oligonucleotides designed to target exonic or intronic splice sites (ESS and ISS) were tested for their ability to block skipping from exon 39 of the human ABCA4 pre-mRNA, which Dots exist in intron 38 between exon 38 and exon 39, in exon 39, and intron between exon 39 and exon 40 of human ABCA4 precursor-mRNA 39, and at the boundaries of introns and exons. The aim is to design AONs that are not too long (for manufacturing purposes, for drug delivery efficiency, and to limit self-annealing), contain at most one CpG motif, and do not contain too high a percentage of guanosine. All originally designed AONs are provided in Table 1. All AONs were in the range of 20-25 nucleotides in length, and all AONs were fully 2'-O-methyl modified. All internucleoside linkages are phosphorothioate linkages. After preparation, AON was re...

Embodiment 2

[0100] Example 2. Additional testing of AON for blocking exon 39 skipping from human ABCA4 precursor-mRNA in HEK293 cells using ddPCR analysis

[0101] The antisense oligonucleotides described in Example 1 and originally tested were further evaluated in HEK293 cells using quantitative and isotype-specific ddPCR assays. Briefly, the ABCA4 exon 39 minigene (MG3) containing the c.5461-10T>C mutation was transiently expressed in HEK293 cells and treated with different AONs. Samples not treated with AON (mock) were used as reference controls. A ddPCR assay was used to quantify the ability of AON to block exon 39 skipping from human ABCA4 pre-mRNA.

[0102] Cell culture conditions, transfection, RNA isolation

[0103] MG3 is described in Example 1. HEK293 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. cells at 0.2 x 10 6 The density of cells / well was seeded into 12-well plates. Two samples (replicate samples) were used for each treatment ...

Embodiment 3

[0109] Example 3. Testing of AON Blocking Exon 39 Skipping from Human ABCA4 Pre-mRNA Using an Alternative (MG3_2) Minigene Construct

[0110] Various AONs described and tested as shown in Examples 1 and 2 were further evaluated using the new minigene constructs. Briefly, a novel ABCA4 exon 39 minigene containing the c.5461-10 T>C mutation (herein referred to as MG3_2) was transiently expressed in HEK293 cells and treated with different AONs. Samples not treated with AON (mock) were used as reference controls. In addition, a scrambled AON not complementary to ABCA4 pre-mRNA was used as a control. The ability of AON to block exon 39 skipping from human ABCA4 pre-mRNA was quantified using a quantitative isoform-specific ddPCR assay.

[0111] Construction of Microgene MG3_2

[0112] A DNA insert including the ABCA4 exon 39 sequence and its flanking intronic regions (including the c.5461-10T>C mutation) was chemically synthesized as a gBlock with EcoR1 and SalI sites at the 5′ a...

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Abstract

The invention relates to the fields of medicine and biotechnology. In particular, the invention relates to novel antisense oligonucleotides (AONs) that may be used in the treatment, prevention and / ordelay of Stargardt disease and / or ABCA4-associated eye disease. More in particular, the invention relates to AONs that are used in inhibiting or blocking exon 39 skipping in the human ABCA4 pre-mRNA.

Description

[0001] field of invention [0002] The invention relates to the fields of medicine and biotechnology. In particular, the present invention relates to antisense oligonucleotides (AONs) useful for the treatment, prevention and / or delay of ocular diseases, preferably macular dystrophy, more preferably Stargardt disease. Background technique [0003] Stargardt's disease (STGD or STGD1) is the most common inherited macular dystrophy that causes progressive damage to central vision. Onset usually occurs in childhood or early adulthood and is least common in late adulthood, and the later the onset, the better the prognosis. The disease has a prevalence of 1 in 8,000-10,000 and is inherited in an autosomal recessive manner, associated with disease-causing mutations in the gene encoding the photoreceptor-specific ATP-binding cassette transporter ABCA4. The protein consists of 2273 amino acids and is mainly expressed in the retina, where it is located in the limbus and cone outer segm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K31/7088
CPCC12N15/111A61K31/713A61P27/02C12N2310/11C12N15/113C12N2310/315C12N2310/321C12N2310/322C12N2320/33
Inventor A·S·伊尔马兹P·亚当森K·C·杜拉I·A·E·舒尔克斯
Owner プロキューアールセラピューティクスツーベスローテンフェンノートシャップ
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