Permanent gene correction by means of nucleotide-modified messenger rna

Abstract

The present invention relates to a nucleotide-modified messenger RNA for the permanent correction of a genetic alteration on a DNA. The invention further relates to a nucleotide-modified messenger RNA in combination with a repair template. It also relates to a pharmaceutical composition. It finally relates to methods for the correction of a genetic alteration on a DNA.

Therefore, what is claimed, is: 
     
         1 . Nuclease-encoding nucleotide-modified messenger RNA (nec-mRNA) configured for the correction of a genetic alteration on a DNA. 
     
     
         2 . nec-mRNA of  claim 1 , wherein in the nec-mRNA up to including approx. 50% of the uridine nucleotides and up to including approx. 50% of the cytidine nucleotides are modified by exchanging uridine for 2-thiouridine (s2U) or pseudouridine (ψ), and by exchanging cytidine for 5-methylcytidine (m5C). 
     
     
         3 . nec-mRNA of  claim 1 , wherein the genetic alteration exists in a lung protein. 
     
     
         4 . nec-mRNA of  claim 3 , wherein the genetic alteration exists in a surfactant protein. 
     
     
         5 . nec-mRNA of  claim 4 , wherein the genetic alteration exists in a lung protein selected from the group consisting of: surfactant protein B (SP-B), cystic fibrosis transmembrane and conductance regulator (CFTR), Foxp3. 
     
     
         6 . nec-mRNA of  claim 1 , wherein the nec-mRNA encodes a nuclease which is configured in such a way that it can bind upstream or downstream of the genetic alteration on the DNA. 
     
     
         7 . nec-mRNA of  claim 1 , wherein the nuclease is selected from the group consisting of: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), CRISPR/Cas9, and dimeric CRISPR RNA guided FokI nucleases. 
     
     
         8 . nec-mRNA of  claim 1 , wherein the nec-mRNA is coupled to an aptamer. 
     
     
         9 . nec-mRNA of  claim 1 , wherein the nec-mRNA is packed into a nanoparticle. 
     
     
         10 . nec-mRNA of  claim 9 , wherein the nanoparticle is coated with chitosan. 
     
     
         11 . nec-mRNA of  claim 1  associated with a repair template. 
     
     
         12 . nec-mRNA of  claim 11 , wherein the repair template comprises a nucleotide section which is exchangeable by homologous recombination (HR) against a section on the DNA comprising the genetic alteration. 
     
     
         13 . nec-mRNA of  claim 12 , wherein the repair template is one of the following: packed into an adeno-associated viral vector (AAV), encoded by a plasmid DNA, packed into a lentiviral vector, and is packed into a protein-capped adenoviral vector (AdV). 
     
     
         14 . Pharmaceutical composition comprising a nuclease-encoding nucleotide-modified messenger RNA (nec-mRNA). 
     
     
         15 . Pharmaceutical composition of  claim 14 , which further comprises a repair template. 
     
     
         16 . Pharmaceutical composition of  claim 14  configured for the treatment of a lung diseaseselected from the group consisting of: surfactant protein B deficiency, cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD). 
     
     
         17 . Pharmaceutical composition of  claim 14 , which comprises the nec-mRNA of  claims 1  to  8  and/or the combination of any of the  claims 9  to  11 . 
     
     
         18 . Pharmaceutical composition of  claim 14 , which comprises the nec-mRNA of  claim 11 . 
     
     
         19 . Method for the correction of a genetic alteration on a DNA comprising the following steps:
 (1) introducing a repair template into a DNA-containing cell, which comprises the genetic alteration to be corrected,   (2) introducing a nec-mRNA into the cell.   
     
     
         20 . The method of  claim 19 , wherein the cell is a lung cell and the introduction is realized by means of high pressure application of the repair template and the nec-mRNA into the lung. 
     
     
         21 . The method of  claim 20 , wherein the nec-mRNA is the nec-mRNA of  claim 11 . 
     
     
         22 . Method for the correction of a genetic alteration on a DNA comprising the following steps:
 (1) introducing a repair template into a living being having a genetically altered DNA to be corrected,   (2) introducing a nec-mRNA into the living being.   
     
     
         23 . The method of  claim 22 , wherein the introduction is realized by means of high pressure application of the repair template and the nec-mRNA into the lung of the living being. 
     
     
         24 . The method of  claim 23 , wherein the nec-mRNA is the nec-mRNA of  claim 11 .