Dual-aav vector-based systems and methods for delivering oversized genes to mammalian cells

Abstract

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide: and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

1 .- 25 . (canceled) 
     
     
         26 . A hybrid polynucleotide vector system comprising:
 i) a first AAV vector polynucleotide comprising a first sequence, a splice donor site, and a first synthetic intron sequence; and   ii) a second AAV vector polynucleotide comprising a second sequence, a splice acceptor site, and a second synthetic intron sequence;   wherein the first synthetic intron sequence and the second synthetic intron sequence comprise an overlapping polynucleotide sequence.   
     
     
         27 . The hybrid polynucleotide vector system of  claim 26 , wherein the first synthetic intron sequence and the second synthetic intron sequence comprise a synthetic alkaline phosphatase intron. 
     
     
         28 . The hybrid polynucleotide vector system of  claim 26 , wherein the overlapping sequence is about 50 to about 500 nucleotides in length. 
     
     
         29 . The hybrid polynucleotide vector system of  claim 26 , wherein the first synthetic intron sequence and the second synthetic intron sequence comprise part of the human MYO7A gene. 
     
     
         30 . The hybrid polynucleotide vector system of  claim 26 , wherein the first synthetic intron sequence and the second synthetic intron sequence comprise intron 23 of the human MYO7A gene. 
     
     
         31 . The hybrid polynucleotide vector system of  claim 26 , wherein the first synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         32 . The hybrid polynucleotide vector system of  claim 26 , wherein the second synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 4. 
     
     
         33 . The hybrid polynucleotide vector system of  claim 26 , wherein the first AAV vector polynucleotide and the second AAV vector polynucleotide further comprise an inverted terminal repeat sequence. 
     
     
         34 . The hybrid polynucleotide vector system of  claim 26  further comprising an AAV capsid. 
     
     
         35 . The hybrid polynucleotide vector system of  claim 34 , wherein the AAV capsid is selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. 
     
     
         36 . A method of treating a subject in need thereof, the method comprising administering a hybrid polynucleotide vector system comprising:
 i) a first AAV vector polynucleotide comprising a first sequence, a splice donor site, and a first synthetic intron sequence; and   ii) a second AAV vector polynucleotide comprising a second sequence, a splice acceptor site, and a second synthetic intron sequence;   wherein the first synthetic intron sequence and the second synthetic intron sequence comprise an overlapping polynucleotide sequence.   
     
     
         37 . The method of  claim 36 , wherein the hybrid polynucleotide vector system is administered by a method selected from intravenous administration, intramuscular administration, intraocular administration, and intranasal administration. 
     
     
         38 . The method of  claim 36 , wherein the overlapping sequence in the first and second AAV vector polynucleotides is about 50 to about 500 nucleotides in length. 
     
     
         39 . The method of  claim 36 , wherein the first synthetic intron sequence and the second synthetic intron sequence comprise part of the human MYO7A gene. 
     
     
         40 . The method of  claim 36 , wherein the first synthetic intron sequence and the second synthetic intron sequence comprises intron 23 of the human MYO7A gene. 
     
     
         41 . The method of  claim 36 , wherein the first synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         42 . The method of  claim 36 , wherein the second synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 4. 
     
     
         43 . The method of  claim 36 , wherein the hybrid polynucleotide vector system further comprises an AAV capsid. 
     
     
         44 . The method of  claim 43 , wherein the AAV capsid is selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. 
     
     
         45 . A pharmaceutical composition, comprising
 a) a hybrid polynucleotide vector system comprising:
 i) a first AAV vector polynucleotide comprising a first sequence, a splice donor site, and a first synthetic intron sequence; and 
 ii) a second AAV vector polynucleotide comprising a second sequence, a splice acceptor site, and a second synthetic intron sequence; 
   wherein the first synthetic intron sequence and the second synthetic intron sequence comprise an overlapping polynucleotide sequence, and   b) a pharmaceutical excipient.