Methods and compositions for treatment of pompe disease

Abstract

In certain embodiments, the present disclosure provides compositions and methods for treating Pompe disease.

We claim: 
     
         1 . A chimeric polypeptide comprising: (i) a GAA polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22 and (ii) an internalizing moiety that binds DNA with a K D  of less than 100 nM and/or promotes transit across cellular membranes via an equilibrative nucleoside transporter 2 (ENT2) transporter;
 wherein the internalizing moiety is an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or a humanized variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10, or a humanized variant thereof.   
     
     
         2 . The chimeric polypeptide of  claim 1 , further comprises a linker sequence comprising the amino acid sequence set forth in SEQ ID NO: 30. 
     
     
         3 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide does not comprise the full length, GAA precursor polypeptide set forth in SEQ ID NO: 1. 
     
     
         4 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide does not comprise the portion of GAA polypeptide set forth in residues 1-57 of SEQ ID NO: 1 or 2. 
     
     
         5 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide has acid alpha-glucosidase activity. 
     
     
         6 . The chimeric polypeptide of  claim 1 , wherein neither the GAA polypeptide nor the chimeric polypeptide comprise a contiguous amino acid sequence corresponding to the amino acids 1-60 of SEQ ID NO: 1 or 2. 
     
     
         7 . The chimeric polypeptide of  claim 1 , wherein neither the GAA polypeptide nor the chimeric polypeptide comprise a contiguous amino acid sequence corresponding to the amino acids 1-66 of SEQ ID NO: 1 or 2. 
     
     
         8 . The chimeric polypeptide of  claim 1 , wherein the mature GAA polypeptide has a glycosylation pattern that differs from that of naturally occurring human GAA. 
     
     
         9 . The chimeric polypeptide of  claim 1 , wherein the internalizing moiety promotes delivery of the chimeric polypeptide into cytoplasm of cells. 
     
     
         10 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide is capable of being taken up by an autophagic vacuole. 
     
     
         11 . The chimeric polypeptide of  claim 1 , wherein the internalizing moiety promotes transport of said chimeric polypeptide into muscle cells or hepatocytes. 
     
     
         12 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide further comprises one or more polypeptide portions that enhance one or more of in vivo stability, in vivo half life, uptake/administration, production, or purification. 
     
     
         13 . The chimeric polypeptide of  claim 1 , wherein the antibody is:
 a) a monoclonal antibody 3E10, or a variant thereof that retains cell penetrating activity, or a variant thereof that binds the same epitope as 3E10, or an antibody that has substantially the same cell penetrating activity as 3E10 and binds the same epitope as 3E10; or   b) a monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10.   
     
     
         14 . The chimeric polypeptide of  claim 1 , wherein the antibody is a chimeric, humanized, or fully human antibody. 
     
     
         15 . The chimeric polypeptide of  claim 1 , wherein the antibody comprises
 a VH CDR1 having the amino acid sequence of SEQ ID NO 24;   a VH CDR2 having the amino acid sequence of SEQ ID NO: 25;   a VH CDR3 having the amino acid sequence of SEQ ID NO: 26;   a VL CDR1 having the amino acid sequence of SEQ ID NO: 27;   a VL CDR2 having the amino acid sequence of SEQ ID NO: 28; and   a VL CDR3 having the amino acid sequence of SEQ ID NO: 29;   which CDRs are according to the IMGT system.   
     
     
         16 . A nucleic acid construct, comprising a nucleotide sequence that encodes the chimeric polypeptide of  claim 1 . 
     
     
         17 . A nucleic acid construct, comprising a nucleotide sequence that encodes a GAA polypeptide, operably linked to a nucleotide sequence that encodes an internalizing moiety, wherein the nucleic acid construct encodes a chimeric polypeptide comprising: (i) the amino acid sequence set forth in SEQ ID NO: 22 and (ii) an internalizing moiety that promotes transit across cellular membranes via an equilibrative nucleoside transporter 2 (ENT2) transporter and/or that binds DNA with a K D  of less than 100 nM;
 wherein the internalizing moiety is an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or a humanized variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10, or a humanized variant thereof.   
     
     
         18 . A vector comprising the nucleic acid construct of claim  1760 . 
     
     
         19 . A host cell comprising the vector of  claim 18 . 
     
     
         20 . A method of producing a chimeric polypeptide comprising culturing the host cell of  claim 19  under appropriate conditions to allow expression of the polypeptide to occur. 
     
     
         21 . A method of treating Pompe disease in a subject in need thereof, comprising administering to the subject an effective amount of the chimeric polypeptide of  claim 1 . 
     
     
         22 . The method of  claim 21 , wherein said subject in need thereof is a subject:
 a) whose disease has been refractory to one or more previous enzyme replacement therapies;   b) having pathologic cytoplasmic glycogen accumulation prior to initiation of treatment with said chimeric polypeptide;   c) diagnosed with Pompe disease greater than six months prior to initiation of treatment with said chimeric polypeptide;   d) diagnosed with Pompe disease at least one year prior to initiation of treatment with said chimeric polypeptide;   e) in whom the onset of symptoms of Pompe disease occurred greater than six months prior to initiation of treatment with said chimeric polypeptide; and/or   f) in whom the onset of symptoms of Pompe disease occurred at least one year prior to initiation of treatment with said chimeric polypeptide.   
     
     
         23 . A method of decreasing glycogen accumulation in cytoplasm, lysosomes, and/or autophagic vacuoles of muscle cells, comprising contacting muscle cells with a chimeric polypeptide, which chimeric polypeptide comprising: (i) the amino acid sequence set forth in SEQ ID NO: 22, and (ii) an internalizing moiety that promotes transit across cellular membranes via an equilibrative nucleoside transporter 2 (ENT2) transporter; wherein the internalizing moiety is an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or a humanized variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10, or a humanized variant thereof.