US2023287452A1PendingUtilityA1

Method of increasing the function of an aav vector

86
Assignee: UNIV PENNSYLVANIAPriority: Apr 7, 2005Filed: Nov 11, 2022Published: Sep 14, 2023
Est. expiryApr 7, 2025(expired)· nominal 20-yr term from priority
Inventors:Luk H. VandenbergheGuangping GaoJames M. Wilson
C12N 15/86A61K 48/0091C12N 2750/14122C12N 2750/14143A61P 31/12A61P 37/00A61P 43/00A61K 48/00C07K 14/005C12N 7/00C12N 2750/14142C12N 2750/14152A61K 48/005
86
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Claims

Abstract

A method of correcting singletons in a selected AAV sequence in order to increasing the packaging yield, transduction efficiency, and/or gene transfer efficiency of the selected AAV is provided. This method involves altering one or more singletons in the parental AAV capsid to conform the singleton to the amino acid in the corresponding position(s) of the aligned functional AAV capsid sequences.

Claims

exact text as granted — not AI-modified
1 . A cultured host cell containing a recombinant nucleic acid molecule encoding an AAV vp1 capsid protein having i) a sequence comprising amino acids 1 to 738 of SEQ ID NO: 4 (AAVrh46), or ii) an amino acid sequence at least 95% identical to the full length of amino acids 1 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 1 to 738 of SEQ ID NO: 4; and wherein the recombinant nucleic acid molecule further comprises a heterologous non-AAV sequence. 
     
     
         2 . The cultured host cell according to  claim 1 , which further comprises a rep gene. 
     
     
         3 . The cultured host cell according to  claim 2 , wherein the rep gene is from AAV2. 
     
     
         4 . The cultured host cell according to  claim 1 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         5 . The cultured host cell according to  claim 1 , wherein the AAV vp1 capsid protein has an amino acid sequence which is at least 97% identical to the full length of amino acids 1 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 1 to 738 of SEQ ID NO: 4. 
     
     
         6 . The cultured host cell according to  claim 1 , wherein the AAV vp1 capsid protein has an amino acid sequence which is at least 99% identical to the full length of amino acids 1 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 1 to 738 of SEQ ID NO: 4. 
     
     
         7 . (canceled) 
     
     
         8 . A cultured host cell containing a recombinant nucleic acid molecule encoding an AAV vp2 capsid protein having i) a sequence comprising amino acids 138 to 738 of SEQ ID NO: 4 (AAVrh46), or ii) an amino acid sequence at least 95% identical to the full length of amino acids 138 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 138 to 738 of SEQ ID NO: 4; and wherein the recombinant nucleic acid molecule further comprises a heterologous non-AAV sequence. 
     
     
         9 . The cultured host cell according to  claim 8 , which further comprises a rep gene. 
     
     
         10 . The cultured host cell according to  claim 9 , wherein the rep gene is from AAV2. 
     
     
         11 . The cultured host cell according to  claim 8 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         12 . The cultured host cell according to  claim 8 , wherein the AAV vp2 capsid protein has an amino acid sequence which is at least 97% identical to the full length of amino acids 138 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 138 to 738 of SEQ ID NO: 4. 
     
     
         13 . The cultured host cell according to  claim 8 , wherein the AAV vp2 capsid protein has an amino acid sequence which is at least 99% identical to the full length of amino acids 138 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 138 to 738 of SEQ ID NO: 4. 
     
     
         14 . (canceled) 
     
     
         15 . A cultured host cell containing a recombinant nucleic acid molecule encoding an AAV vp3 capsid protein having i) a sequence comprising amino acids 204 to 738 of SEQ ID NO: 4 (AAVrh46), or ii) an amino acid sequence at least 95% identical to the full length of amino acids 204 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4; and wherein the recombinant nucleic acid molecule further comprises a heterologous non-AAV sequence. 
     
     
         16 . The cultured host cell according to  claim 15 , which further comprises a rep gene. 
     
     
         17 . The cultured host cell according to  claim 16 , wherein the rep gene is from AAV2. 
     
     
         18 . The cultured host cell according to  claim 15 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         19 . The cultured host cell according to  claim 15 , wherein the AAV vp3 capsid protein has an amino acid sequence which is at least 97% identical to the full length of amino acids 204 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4. 
     
     
         20 . The cultured host cell according to  claim 15 , wherein the AAV vp3 capsid protein has an amino acid sequence which is at least 99% identical to the full length of amino acids 204 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4. 
     
     
         21 . (canceled) 
     
     
         22 . A cultured host cell containing a recombinant nucleic acid molecule comprising nucleotides 1 to 2217 of SEQ ID NO: 22 or a sequence at least 98% identical to nucleotides 1 to 2217 of SEQ ID NO: 22 that encodes an AAV capsid protein, wherein the amino acid residue of the encoded AAV capsid protein corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 1 to 738 of SEQ ID NO: 4;
 wherein the recombinant nucleic acid molecule further comprises a heterologous non-AAV sequence.   
     
     
         23 . The cultured host cell according to  claim 22 , wherein said nucleotide sequence is at least 99% identical to nucleotides 1 to 2217 of SEQ ID NO: 22. 
     
     
         24 . The cultured host cell according to  claim 22 , which further comprises a rep gene. 
     
     
         25 . The cultured host cell according to  claim 24 , wherein the rep gene is from AAV2. 
     
     
         26 . The cultured host cell according to  claim 22 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         27 . (canceled) 
     
     
         28 . A cultured host cell containing a recombinant nucleic acid molecule comprising nucleotides 412 to 2217 of SEQ ID NO: 22 or a sequence at least 98% identical to nucleotides 412 to 2217 of SEQ ID NO: 22 that encodes an AAV capsid protein, wherein the amino acid residue of the encoded AAV capsid protein corresponding to position 665 in SEQ ID NO:4 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4;
 wherein the recombinant nucleic acid molecule further comprises a heterologous non-AAV sequence.   
     
     
         29 . The cultured host cell according to  claim 28 , wherein said nucleotide sequence is at least 99% identical to nucleotides 412 to 2217 of SEQ ID NO: 22. 
     
     
         30 . The cultured host cell according to  claim 28 , which further comprises a rep gene. 
     
     
         31 . The cultured host cell according to  claim 30 , wherein the rep gene is from AAV2. 
     
     
         32 . The cultured host cell according to  claim 28 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         33 . (canceled) 
     
     
         34 . A cultured host cell containing a recombinant nucleic acid molecule comprising nucleotides 610 to 2217 of SEQ ID NO: 22 or a sequence at least 98% identical to nucleotides 610 to 2217 of SEQ ID NO: 22 that encodes an AAV capsid protein wherein the amino acid residue of the encoded AAV capsid protein corresponding to position 665 in SEQ ID NO:4 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4;
 wherein the recombinant nucleic acid molecule further comprises a heterologous non-AAV sequence.   
     
     
         35 . The cultured host cell according to  claim 34 , wherein said nucleotide sequence is at least 99% identical to nucleotides 610 to 2217 of SEQ ID NO: 22. 
     
     
         36 . The cultured host cell according to  claim 34 , which further comprises a rep gene. 
     
     
         37 . The cultured host cell according to  claim 36 , wherein the rep gene is from AAV2. 
     
     
         38 . The cultured host cell according to  claim 34 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         39 . (canceled)

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