Trans-splicing ribozyme targeting rhodopsin transcript and uses thereof

Inventors

LEE, SEONG-WOOK • Kim, Ji Hyun • Han, Seung Ryul

Assignees

Rznomics Inc

Abstract

A trans-splicing ribozyme capable of splicing a rhodopsin transcript at a target splicing site and containing a sequence that is capable of complementarily binding to a target binding site of the rhodopsin transcript is disclosed. The trans-splicing ribozyme may further containing a desired rhodopsin transcript at 3′-end. The trans-splicing ribozyme may further contains an antisense sequence that is complementary to a region downstream the target binding site of the rhodopsin transcript. A nucleotide molecule encoding the trans-splicing ribozyme is also disclosed. Delivery systems to delivery the nucleotide molecule and/or the trans-splicing ribozyme to target tissue or cells as well as uses of the trans-splicing ribozyme, the nucleotide molecule, delivery systems, or pharmaceutical compositions containing any of them are also disclosed.

Core Innovation

The invention provides a recombinant nucleic acid molecule that comprises a trans-splicing ribozyme capable of splicing at a target splicing site of a target rhodopsin transcript. The trans-splicing ribozyme includes an internal guide sequence (IGS) that complementarily binds to a binding region of the target rhodopsin transcript containing the target splicing site, and the binding region comprises 5 to 10 consecutive nucleotides. The IGS comprises 5-10 nucleotides and is capable of complementarily binding and forming G/U wobble base pairs at the target splicing site.

The target splicing site is defined by nucleotide positions identified with reference to SEQ ID NO: 1, including positions such as +30, +35, +42, +43, +52, +54, +55, and +59, as well as additional enumerated positions including +661 or +688. In described embodiments, a +59/5′UTR site is highlighted as a target splicing site for performing trans-splicing on the rhodopsin (RHO) transcript. The trans-splicing process cleaves and replaces a downstream fragment with a wild-type (WT) RHO transcript.

The invention further frames this trans-splicing ribozyme system for treating retinitis pigmentosa (RP), particularly autosomal dominant RP arising from mutant RHO (for example, P23H). The nucleic acid constructs and gene constructs encode the ribozyme and, where described, a wild-type RHO payload and optionally include an antisense sequence to increase specificity. The document also describes recombinant expression vectors and delivery systems including non-viral carriers (liposomes and lipid nanoparticles) and recombinant viral vectors including adeno-associated virus (AAV), with an emphasis on avoiding reliance on external protein introduction such as Cas9.

Claims Coverage

The independent claim covers recombinant nucleic acid molecules encoding a trans-splicing ribozyme that targets enumerated rhodopsin transcript splicing sites using an IGS (5–10 nt) forming G/U wobble base pairs. The claim includes the constraint that the binding region comprises 5–10 consecutive nucleotides and that target splicing site nucleotide positions are identified relative to SEQ ID NO: 1, with several example positions explicitly listed, including +59.

Overall claim coverage centers on a recombinant nucleic acid molecule encoding a trans-splicing ribozyme that uses a 5–10 nt IGS to complementarily bind a 5–10 nt binding region and form G/U wobble base pairs at an enumerated rhodopsin transcript target splicing site defined relative to SEQ ID NO: 1.

Stated Advantages

Documented Applications