Method for reducing the expression of nkcc1 in a subject

Abstract

The invention relates to an RNA interference (RNAi) strategy based on the use of artificial microRNA (amiR) to reduce NKCC1 expression. In particular, the invention relates to a vector that achieves neuron-specific expression of specific amiR against NKCC1 by using a human Synapsin promoter to drive transgene expression.

1 . A vector comprising a polynucleotide encoding an artificial microRNA (amiR) capable of reducing the expression of NKCC1 in a subject for use in a therapeutic treatment method of reducing the expression of NKCC1 in a subject in need thereof, characterized in that the amiR comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs:4, 8, 15, 16, 18, 23, 25, 39, 40, 44, 45, 53, 57, 64, 66, 67, 68, 69, 82 and 88. 
     
     
         2 . The vector according to  claim 1 , wherein the subject suffers from a disease selected from the group consisting of Down syndrome, Fragile X syndrome, Rett syndrome, Tuberous sclerosis, traumatic brain injury, epilepsy, autism, Schizophrenia, Parkinson disease and hypertension. 
     
     
         3 . The vector according to  claim 1 , wherein the amiR consists of a 5′ flanking region derived from a natural miRNA, a TGCT nucleotide overhang, a 5′G+the short 21 nucleotide antisense sequence derived from the hNKCC1 selected from the group consisting of SEQ ID NOs:4, 8, 15, 16, 18, 23, 25, 39, 40, 44, 45, 53, 57, 64, 66, 67, 68, 69, 82 and 88, a loop forming sequence, a short 19 nucleotides sense sequence derived from hNKCC1 with 2 nucleotides removed (Δ2) to create an internal loop, a CAGG overhang and a 3′ flanking region derived from a natural miRNA. 
     
     
         4 . The vector according to  claim 1 , wherein the 5′ flanking region derived from a natural miRNA is SEQ ID NO: 90. 
     
     
         5 . The vector according to  claim 1 , wherein the loop forming sequence is SEQ ID NO: 91. 
     
     
         6 . The vector according to  claim 1 , wherein the 3′ flanking region derived from a natural miRNA is SEQ ID NO: 92. 
     
     
         7 . The vector according to  claim 1 , wherein the vector is a non-viral vector or a viral vector. 
     
     
         8 . The vector according to  claim 7 , wherein the viral vector is an AAV vector. 
     
     
         9 . The vector according to  claim 1 , wherein the vector comprises a neuronal specific promoter. 
     
     
         10 . The vector according to  claim 9 , wherein the neuronal specific promoter is selected from the group consisting of synapsin-1 (Syn) promoter, neuron-specific enolase (NSE) promoter, neurofilament light-chain gene promoter, and the neuron-specific vgf gene promoter. 
     
     
         11 . The vector according to  claim 1 , wherein the therapeutic treatment method comprises delivering the vector to the hippocampus of the subject. 
     
     
         12 . The vector according to  claim 1 , wherein the therapeutic treatment method comprises delivering the vector to the subject by intravenous injection. 
     
     
         13 . The vector according to  claim 1 , wherein the therapeutic treatment method comprises delivering the vector to the subject by intrathecal administration. 
     
     
         14 . A pharmaceutical composition for use in a therapeutic treatment method of reducing the expression of NKCC1 in a subject in need thereof, the composition comprising a vector according to  claim 1   
     
     
         15 . A method of reducing the expression of NKCC1 in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a vector according to  claim 1 .