Ldlr variants and their use in compositions for reducing cholesterol levels

Abstract

A recombinant vector having an expression cassette comprising a modified human low density lipoprotein receptor (hLDLR) gene is provided, wherein said hLDLR gene encodes a modified hLDLR comprising (a) one or more of the following amino acid substitutions: L318H, N295D, H306D, V307D, N309A, D310N, L318H, and/or L318D; or (b) an amino acid substitution of any of (a) in combination with one or more of the following amino acid substitutions: K796, K809R and/or C818A. Also provided are pharmaceutical compositions containing this vector and uses therefor in lowering cholesterol and/or treating familial hypercholesterolemia.

1 . A recombinant vector having an expression cassette comprising a modified human low density lipoprotein receptor (hLDLR) gene, wherein said hLDLR gene encodes a modified hLDLR that reduces cholesterol following expression, and wherein said modified hLDLR comprises:
 (a) one or more amino acid substitutions that interfere with the wild-type hLDLR IDOL pathway; and/or   (b) one or more amino acid substitutions which are resistant to degradation of hLDLR by interfering with the PCSK9 pathway.   
     
     
         2 . The recombinant vector of  claim 1 , wherein the modified hLDLR comprises (a) an amino acid substitution at one or more of N295, H306, V307, N309, D310, and/or L318, based on the numbering of SEQ ID NO:1. 
     
     
         3 . The recombinant vector of  claim 2 , wherein the hLDLR comprises one or more amino acid substitutions selected from: N295D, H306D, V307D, N309A, D310N, L318H, and/or L318D, based on the numbering of SEQ ID NO:1. 
     
     
         4 . The recombinant vector of  claim 1 , wherein the modified hLDLR comprises (b) an amino acid substitution of at least one of L769R, K809R and/or C818A, based on the numbering of SEQ ID NO:1, optionally in combination with an amino acid substitution of (a). 
     
     
         5 . The recombinant vector of  claim 1 , wherein the vector is a recombinant adeno-associated virus (rAAV) vector. 
     
     
         6 . The recombinant vector of  claim 1 , wherein the vector is a rAAV comprising a capsid selected from AAV8, rh64R1, AAV9, or rh10. 
     
     
         7 . The recombinant vector of  claim 1 , wherein the expression cassette comprises a promoter which specifically directs expression of the modified hLDLr in liver cells. 
     
     
         8 . A rAAV vector having an expression cassette comprising a modified hLDLR gene, wherein said hLDLR gene encodes a modified hLDLR comprising an L318D amino acid substitution, based on the numbering of SEQ ID NO:1. 
     
     
         9 . The rAAV vector of  claim 8 , wherein the modified hLDLR further comprises a K809R and/or C818A amino acid substitution, based on the numbering of SEQ ID NO:1. 
     
     
         10 . The rAAV vector of  claim 8 , wherein the vector comprises a capsid selected from AAV8, rh64R1, AAV9, or rh10. 
     
     
         11 . The rAAV vector of  claim 8 , wherein the expression cassette comprises a promoter which specifically directs expression of the modified hLDLr in liver cells. 
     
     
         12 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant vector of  claim 1 . 
     
     
         13 . A method for reducing circulating cholesterol levels by administering to a subject in need thereof a recombinant vector of  claim 1 , wherein said expression cassette further comprises regulatory control sequences which direct expression of modified hLDLR in the subject. 
     
     
         14 . A method for reducing circulating cholesterol levels by administering to a subject in need thereof a rAAV vector of  claim 8 , wherein said expression cassette further comprises regulatory control sequences which direct expression of modified hLDLR in the subject. 
     
     
         15 . A synthetic or recombinant hLDLR comprising:
 (a) an amino acid substitution at one or more of N295, H306, V307, N309, D310, L318, and/or L318, based on the numbering of SEQ ID NO:1; or   (b) an amino acid substitution of any of (a) in combination with an amino acid substitution of at least one of K769R, K809R and/or C818A, based on the numbering of SEQ ID NO:1.   
     
     
         16 . The synthetic or recombinant hLDLR of  claim 15 , wherein the one or more amino acid substitutions of (a) are selected from: N295D, H306D, V307D, N309A, D310N, L318H, and/or L318D, based on the numbering of SEQ ID NO:1. 
     
     
         17 . A pharmaceutical composition comprising a synthetic or recombinant hLDLR protein of  claim 15  and a pharmaceutically acceptable carrier.