Importation of mitochondrial protein by an enhanced allotopic approach

Abstract

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

1 - 5 . (canceled) 
     
     
         6 . An expression vector adapted to the efficient and stable delivery of a protein into the mitochondrion of a mammalian cell, the vector comprising:
 at least one mitochondrion-targeting nucleic acid sequence (nucleic acid sequence);   at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (CDS sequence); and   at least one 3′ nucleic acid sequence, which is located 3′ of said at least one MTS nucleic acid sequence and of said at least one CDS sequence,   wherein,   said at least one MTS nucleic acid sequence is   the cDNA sequence of a MTS of a nuclearly-encoded mitochondrially-targeted mRNA, or   a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function,   said at least one 3′ nucleic acid sequence is   the cDNA sequence of the 3′UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or   a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3′UTR of said naturally-occurring mRNA, still allows for a mitochondrial targeting of the resulting mRNA,   said vector does not comprise any sequence which would be identical to   the UTR of a naturally-occurring mRNA which is a nuclearly-transcribed but not mitochondrially-targeted mRNA, or   the cDNA sequence of such a 3′UTR sequence, or   a DNA sequence coding for such a 3′UTR of a naturally-occurring mRNA in accordance with the universal genetic code, and   said vector does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.   
     
     
         7 . The expression vector of  claim 6 , wherein said at least one MTS nucleic acid sequence is the MTS nucleic acid sequence of SOD2, or of COX10, or of AC02, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2. 
     
     
         8 . The expression vector of  claim 6 , wherein said at least one 3′ nucleic acid sequence is:
 the 3′UTR sequence of SOD2, or of COX10, or of ACO2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of AK2, or 
 the cDNA sequence of such a 3′UTR sequence, or 
 a DNA sequence coding for such a 3′UTR sequence in accordance with the universal genetic code. 
 
     
     
         9 . The expression vector of  claim 8 , wherein said at least one 3′ nucleic acid sequence is SEQ ID NO: 35 or SEQ ID NO: 47. 
     
     
         10 . The expression vector of  claim 6 , wherein said at least one MTS nucleic acid sequence is the MTS nucleic acid sequence of SOD2, and said 3′ UTR sequence is the 3′ UTR sequence of SOD2. 
     
     
         11 . The expression vector of  claim 6 , wherein said at least one MTS nucleic acid sequence is the MTS nucleic acid sequence of COX10, and said 3′ UTR sequence is the 3′ UTR sequence of COX10. 
     
     
         12 . The expression vector of  claim 6 , wherein said at least one CDS sequence is the sequence of a naturally-occurring mitochondrial nucleic acid, recoded in accordance with the universal genetic code. 
     
     
         13 . The expression vector  claim 12 , wherein said at least one CDS sequence is a nucleic acid sequence of ATP6, or of ND1, or of ND4, recoded in accordance with the universal genetic code. 
     
     
         14 . The expression vector of  claim 6 , comprising at least one sequence of SEQ ID NO: 25 (COX10 MTS-re-coded ND1-COX10 3′UTR), SEQ ID NO: 26 (COX10 MTS-re-coded ND4-COX10 3′UTR), SEQ ID NO: 21 (COX10 MTS-re-coded ATP6-COX10 3′UTR), and SEQ ID NO: 22 (SOD2 MTS-re-coded ATP6-SOD2 3′UTR). 
     
     
         15 . The expression vector of  claim 6 , wherein said at least one CDS sequence is the nucleic acid sequence of a naturally-occurring nuclear nucleic acid which encodes a functional mitochondrial protein. 
     
     
         16 . The expression vector of  claim 6 , wherein said mammalian cell is a human cell. 
     
     
         17 . A non-naturally occurring nucleic acid construct comprising:
 at least one mitochondrion-targeting nucleic acid sequence (MTS nucleic acid sequence);   at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (CDS sequence); and   at least one 3′ nucleic acid sequence, which is located 3′ of said MTS nucleic acid sequence and of said CDS sequence,   wherein,   said at least one MTS nucleic acid sequence is   the cDNA sequence of a MTS RNA sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or   a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function,   said at least one 3′ nucleic acid sequence is   the cDNA sequence of the 3′UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or   a conservative variant or fragment of such a cDNA 3′UTR sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3′UTR of said nuclearly-encoded mitochondrially-targeted mRNA, still allows for a mitochondrial targeting of the resulting mRNA,   provided that, when said at least one MTS nucleic acid sequence and said at least one 3′ nucleic acid sequence, respectively, are the MTS nucleic acid sequence and 3′UTR sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA sequences of such a mRNA, or a DNA sequence coding for such a mRNA, then said CDS sequence is not the CDS sequence of said naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA,   said nucleic acid construct does not comprise any sequence which would be identical to   the 3′UTR of a naturally-occurring mRNA which is a nuclearly-transcribed but not-mitochondrially-targeted mRNA, or   the cDNA sequence of such a 3′UTR sequence, or   a DNA sequence coding for such a 3′UTR of a naturally-occurring mRNA in accordance with the universal genetic code, and   said nucleic acid construct does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.   
     
     
         18 . The nucleic acid construct of  claim 17 , wherein said at least one MTS nucleic acid sequence is the MTS nucleic acid sequence of SOD2, or of COX10, or of ACO2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2. 
     
     
         19 . The nucleic acid construct of  claim 17 , wherein said at least one 3′ nucleic acid sequence is:
 the 3′UTR sequence of SOD2, or of COX10, or of ACO2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of AK2, or 
 the cDNA sequence of such a 3′UTR sequence, or 
 a DNA sequence coding for such a 3′UTR sequence in accordance with the universal genetic code. 
 
     
     
         20 . An engineered mammalian cell which has been transduced,
 infected and/or transfected by at least one vector according to  claim 6 .   
     
     
         21 . A pharmaceutical composition comprising at least one vector according to  claim 6 . 
     
     
         22 . A method for the in vivo or ex vivo therapy of a subject or patient in need of a therapeutic, palliative or preventive treatment of a disease, condition, or disorder related to a defect in activity or function of mitochondria, comprising administering to said subject an expression vector adapted to the efficient and stable delivery of a protein into the mitochondrion of a mammalian cell, the vector comprising:
 at least one mitochondrion-targeting nucleic acid sequence (nucleic acid sequence);   at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (CDS sequence); and   at least one 3′ nucleic acid sequence, which is located 3′ of said at least one MTS nucleic acid sequence and of said at least one CDS sequence,   wherein,   said at least one MTS nucleic acid sequence is   the cDNA sequence of a MTS of a nuclearly-encoded mitochondrially-targeted mRNA, or   a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function,   said at least one 3′ nucleic acid sequence is   the cDNA sequence of the 3′UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or   a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3′UTR of said naturally-occurring mRNA, still allows for a mitochondrial targeting of the resulting mRNA,   said vector does not comprise any sequence which would be identical to   the UTR of a naturally-occurring mRNA which is a nuclearly-transcribed but not mitochondrially-targeted mRNA, or   the cDNA sequence of such a 3′UTR sequence, or   a DNA sequence coding for such a 3′UTR of a naturally-occurring mRNA in accordance with the universal genetic code, and   said vector does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.   
     
     
         23 . An engineered mammalian cell which has been transduced, infected and/or transfected by at least one nucleic acid construct according to  claim 17 . 
     
     
         24 . A pharmaceutical composition comprising at least one nucleic acid construct according to  claim 17 . 
     
     
         25 . A method for the in vivo or ex vivo therapy of a subject or patient in need of a therapeutic, palliative or preventive treatment of a disease, condition, or disorder related to a defect in activity or function of mitochondria, comprising administering to said patient a construct according to  claim 17 .