Recombinant aav-crumbs homologue composition and methods for treating lca-8 and progressive rp

Abstract

The present invention relates to a Crumbs homologue (CRB) therapeutic for use as a medicament or in a method of treatment or prophylaxis, for example in the treatment or prophylaxis of a retinal disorder due to mutations in the Crumbs homologue-1 (CRB1) gene, such as Leber's congenital amaurosis 8 (LCA8) or retinitis pigmentosa 12 (RP12). In particular, the present invention relates to a recombinant viral vector comprising CRB2 or modified non-toxic forms of either CRB1 or CRB3 that resemble CRB2.

1 .- 15 . (canceled) 
     
     
         16 . A gene therapy vector comprising at least one of:
 a) a nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein; and   b) a nucleotide sequence encoding a modified Crumbs homologue-I (CRB1) protein or a modified Crumbs homologue-3 (CRB3) protein, wherein the modified CRB1 protein or the modified CRB3 protein comprises a modification in the C-terminal part of the protein and is less toxic or not toxic in an in vivo assay, which assay comprises the steps of:
 i) intravitreal transduction in one eye of a Crb2 conditional knock-out (cKO) or Crb1Crb2F/+ cKO mouse with recombinant adeno-associated virus (rAAV) comprising the nucleotide sequence encoding the modified CRB1 or modified CRB3 protein; 
 ii) intravitreal transduction in the other eye of the mouse with rAAV comprising a nucleotide sequence encoding a wild-type CRB2 protein, wild-type CRB1 or wild-type CRB3 as a control; and, 
 iii) making an electroretinogram one and three months after transduction, wherein an increased percentage in the maximum a-wave and/or b-wave amplitude in the electroretinogram in the modified CRB1 or modified CRB3 transduced retinas compared to the electroretinogram in the wild-type CRB1 or wild-type CRB3 transduced retinas indicates that the modified CRB1 protein or the modified CRB3 protein is less toxic; or 
   
       wherein a maximum a-wave and/or b-wave amplitude in the electroretinogram of at least 60% of the difference of maximum a-wave and/or b-wave amplitude with wild type CRB2 subtracted with the maximum a-wave and/or b-wave amplitude with modified CRB1 or modified CRB3 protein indicates that the modified CRB1 protein or the modified CRB3 protein is not toxic. 
     
     
         17 . A gene therapy vector according to  claim 16 , wherein at least one of:
 a) the CRB2 protein is a eumetazoan CRB2 protein;   b) the modified CRB1 protein is a modified eumetazoan CRB1 protein; and   c) the modified CRB3 protein is a modified eumetazoan CRB3 protein.   
     
     
         18 . A gene therapy vector according to  claim 16 , wherein the gene therapy vector is a recombinant parvoviral vector or a lentiviral vector. 
     
     
         19 . A gene therapy vector according to  claim 18 , wherein the vector is a recombinant adeno-associated virus (rAAV) vector. 
     
     
         20 . A gene therapy vector according to  claim 19 , wherein the gene therapy vector is a recombinant adeno-associated virus vector selected from the group consisting of recombinant adeno-associated virus serotype 1 (rAAV1), recombinant adeno-associated virus serotype 2 (rAAV2), recombinant adeno-associated virus serotype 3 (rAAV3), recombinant adeno-associated virus serotype 4 (rAAV4), recombinant adeno-associated virus serotype 5 (rAAV5), recombinant adeno-associated virus serotype 6 (rAAV6), recombinant adeno-associated virus serotype 7 (rAAV7), recombinant adeno-associated virus serotype 8 (rAAV8), recombinant adeno-associated virus serotype 9 (rAA9), or a serotype variant of any thereof. 
     
     
         21 . A gene therapy vector according to  claim 16 , wherein the CRB2 protein comprises or consists of an amino acid sequence that has at least 80% sequence identity with the amino acid sequences of any one of SEQ ID NO: 40-63, 65-83 and wherein the CRB2 protein is functionally active as measured by electroretinography. 
     
     
         22 . A gene therapy vector according to  claim 16 , wherein the nucleotide sequence encoding CRB2, modified CRB1 or modified CRB3 is operably linked to expression control elements comprising a promoter that produces sufficient expression of CRB2, modified CRB1 or modified CRB3 protein, respectively, to obtain a therapeutic effect. 
     
     
         23 . A gene therapy vector according to  claim 16 , wherein the modification in the C-terminal part of the amino acid sequence of the modified CRB1 or modified CRB3 protein is selected from the group consisting of:
 i) the PDZ binding domain of CRB1 or CRB3 is replaced by amino acid residues 1282-1285 of SEQ ID NO:40;   ii) the FERM binding domain of CRB1 or CRB3 is replaced by amino acid residues 1251-1264 of SEQ ID NO:40;   iii) the transmembrane domain of CRB1 or CRB3 is replaced by amino acid residues 1225-1247 of SEQ ID NO:40;   iv) the 16 C-terminal amino acid residues of CRB1 or CRB3 are replaced by amino acid residues 1270-1285 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1270-1285 of SEQ ID NO:40;   v) an amino acid sequence of CRB1 or CRB3 consisting of the FERM binding domain of CRB1 or CRB3, two N-terminal amino acid residues and five C-terminal amino acid residues is replaced by amino acid residues 1249-1269 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1249-1269 of SEQ ID NO:40;   vi) an amino acid sequence of CRB1 or CRB3 consisting of the transmembrane domain of CRB1 or CRB3, two N-terminal amino acid residues and one C-terminal amino acid residue is replaced by amino acid residues 1223-1248 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1223-1248 of SEQ ID NO:40;   vii) an amino acid sequence of CRB1 or CRB3 comprising any of a serine, threonine or tyrosine at a position that corresponds to position 1243, 1254, 1257, 1258, 1259, 1261 and/or 1274 of SEQ ID NO:40; and,   viii) combination of one or more i)-vii).   
     
     
         24 . A nucleic acid construct comprising at least one of:
 a) a nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein and at least one parvoviral inverted terminal repeat (ITR) sequence, wherein preferably the nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein is operably linked to expression control elements comprising a promoter that is capable of sufficient CRB2 protein expression to obtain a therapeutic effect; and   b) a nucleotide sequence encoding a Crumbs homologue-1 (modified CRB1) protein or a Crumbs homologue-3 (modified CRB3) protein, and at least one ITR sequence, wherein preferably the nucleotide sequence encoding a modified CRB1 protein or a modified CRB3 protein is operably linked to expression control elements comprising a promoter that is capable of sufficient modified CRB1 or modified CRB3 protein expression to obtain a therapeutic effect and wherein the modified CRB1 protein or the modified CRB3 protein comprises a modification in the C-terminal part of the protein and is less toxic or not toxic in an in vivo assay, which assay comprises the steps of:
 i) intravitreal transduction in the one eye of a Crb2 conditional knock-out (cKO) or Crb1Crb2F/+ cKO mouse with recombinant adeno-associated virus (rAAV) comprising the nucleotide sequence encoding the modified CRB1 or modified CRB3 protein; 
 ii) intravitreal transduction in the other eye of the mouse with rAAV comprising a nucleotide sequence encoding a wild-type CRB2 protein, wild-type CRB1 or wild-type CRB3 as a control; and, 
 iii) making an electroretinogram one and three months after transduction, wherein an increased percentage in the maximum a-wave and/or b-wave amplitude in the electroretinogram in the modified CRB1 or modified CRB3 transduced retinas compared to the electroretinogram in the wild-type CRB1 or wild-type CRB3 transduced retinas indicates that the modified CRB1 protein or the modified CRB3 protein is less toxic; or wherein a maximum a-wave and/or b-wave amplitude in the electroretinogram of at least 60% of the difference of maximum a-wave and/or b-wave amplitude with wild type CRB2 subtracted with the maximum a-wave and/or b-wave amplitude with modified CRB1 or modified CRB3 protein indicates that the modified CRB1 protein or the modified CRB3 protein is not toxic. 
   
     
     
         25 . A pharmaceutical composition comprising a gene therapy vector according to  claim 16  and a pharmaceutically acceptable excipient. 
     
     
         26 . A kit comprising:
 (a) a gene therapy vector in accordance with  claim 16 , or a pharmaceutical composition comprising the gene therapy vector and a pharmaceutically acceptable carrier; and,   (b) instructions for using the gene therapy vector or pharmaceutical composition according to (a) in the prevention, treatment, or amelioration of one or more symptoms of a retinal disorder due to mutations in CRB1 gene.   
     
     
         27 . A method for treatment or prophylaxis of a retinal disorder due to mutations in a CRB1 gene, comprising administering an effective amount gene therapy vector according to  claim 16  to a subject in need thereof. 
     
     
         28 . A method according to  claim 27 , wherein the retinal disorder is Leber's congenital amaurosis or retinitis pigmentosa. 
     
     
         29 . A method according to  claim 28 , wherein the retinal disorder is LCA8 or RP12. 
     
     
         30 . A gene therapy vector according to  claim 17 , wherein:
 a) the CRB protein is a CRB2 protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin;   b) the modified CRB1 protein is a modified CRB1 protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin; or   c) the modified CRB3 protein is a modified CRB3 protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin.   
     
     
         31 . A gene therapy vector according to  claim 30 , wherein:
 a) the CRB2 protein is a human CRB2 protein;   b) the modified CRB1 protein is a modified human CRB1 protein; or   c) the modified CRB3 protein is a modified human CRB3 protein.   
     
     
         32 . A gene therapy vector according to  claim 20 , wherein the serotype variant comprises rAAV6 ShH10, rAAV6 ShH10Y, or a combination thereof. 
     
     
         33 . A gene therapy vector according to  claim 21 , wherein the CRB2 protein comprises or consists of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 40-42. 
     
     
         34 . A gene therapy vector according to  claim 22 , wherein the promoter is selected from the group consisting of: a truncated CMV promoter, a CMV promoter, a truncated human RLBP1 promoter, a human photoreceptor specific rhodopsin kinase promoter, and a human rod photoreceptor specific rhodopsin promoter. 
     
     
         35 . A gene therapy vector according to  claim 34 , wherein the promoter is selected from the group consisting of: a CMV promoter according to SEQ ID NO: 121, a truncated human RLBP1 promoter according to SEQ ID NO:122, a human photoreceptor specific rhodopsin kinase promoter according to SEQ ID NO:123, a human rod photoreceptor specific rhodopsin promoter according to SEQ ID NO:124, and a truncated CMV promoter according to SEQ ID NO:133. 
     
     
         36 . A gene therapy vector according to  claim 23 , wherein the amino acid residues at positions 1243, 1254, 1259, 1261 and 1274 are serine; the amino acid residue at position 1257 is threonine; and the amino acid residue at position 1258 is tyrosine.