Adeno-associated viral vectors for gene therapy

Abstract

Genetically modified compositions, such as adeno-associated viral vectors and primary cells, for treating various conditions and diseases. Disclosed are also modified adeno-associated viruses for the treatment of cancer. Also disclosed are the methods of making and using the genetically modified compositions in treating various diseases, conditions, and cancer.

1 - 232 . (canceled) 
     
     
         233 . An engineered AAV virion, wherein the engineered AAV virion comprises:
 a capsid, wherein the capsid comprises functional AAV capsid viral proteins that comprise a VP1, a VP2, and a VP3 polypeptide, wherein at least two of said VP1, VP2, and VP3 polypeptides are from a first AAV serotype, wherein at least one of said VP1, VP2, and VP3 polypeptides are from a second AAV serotype, and wherein said first serotype or said second serotype is AAV12.   
     
     
         234 . The engineered AAV virion of  claim 233 , wherein said first AAV serotype is AAV12. 
     
     
         235 . The engineered AAV virion of  claim 233 , wherein said VP1 and VP2 sequences are from the first serotype that is AAV12. 
     
     
         236 . The engineered AAV virion of  claim 233 , wherein said engineered AAV virion upon transduction with a virus containing a CMV-promoter driven GFP-coding nucleic acid sequence has increased transduction efficiency as compared to that generated by a wild-type AAV virion comprising a VP1, VP2, and VP3 polypeptide of said first or said second serotype when measured by quantifying fluorescence of mammalian cells after 24 hours. 
     
     
         237 . The engineered AAV virion of  claim 233 , wherein the engineered AAV virion further comprises a transgene, and wherein said engineered AAV virion upon transduction with a virus containing a CMV-promoter driven GFP-coding nucleic acid sequence has increased expression of the transgene as compared to a wild-type AAV virion comprising a VP1, VP2, and VP3 polypeptide of said first or second serotype when measured by quantifying fluorescence of mammalian cells after 24 hours. 
     
     
         238 . The engineered AAV virion of  claim 237 , wherein the transgene codes for at least a portion of an exogenous receptor. 
     
     
         239 . The engineered AAV virion of  claim 238 , wherein the exogenous receptor is a T cell receptor or a chimeric antigen receptor. 
     
     
         240 . An engineered mammalian immune cell infected with an engineered AAV virion, wherein after infection the immune cell comprises:
 a capsid, wherein the capsid comprises functional AAV capsid viral proteins that comprise a VP1, a VP2, and a VP3 polypeptide, wherein at least two of said VP1, VP2, and VP3 polypeptides are from a first AAV serotype, wherein at least one of said VP1, VP2, and VP3 polypeptides are from a second AAV serotype, and wherein said first serotype or said second serotype is AAV12.   
     
     
         241 . The engineered immune cell of  claim 240 , wherein said first AAV serotype is AAV12. 
     
     
         242 . The engineered immune cell of  claim 240 , wherein said VP1 and VP2 sequences are from the first serotype that is AAV12. 
     
     
         243 . The engineered immune cell of  claim 240 , wherein the engineered immune cell is a primary cell or immortalized cell. 
     
     
         244 . The engineered immune cell of  claim 240 , wherein the engineered immune cell is a tumor infiltrating lymphocyte. 
     
     
         245 . The engineered immune cell of  claim 240 , wherein the engineered immune cell is a T cell or natural killer cell. 
     
     
         246 . The engineered immune cell of  claim 240 , wherein the engineered immune cell further comprises a transgene. 
     
     
         247 . The engineered immune cell of  claim 246 , wherein the transgene codes for at least a portion of an exogenous receptor. 
     
     
         248 . The engineered immune cell of  claim 247 , wherein the exogenous receptor is a T cell receptor or a chimeric antigen receptor. 
     
     
         249 . An isolated non-naturally occurring nucleic acid, wherein the isolated non-naturally occurring nucleic acid comprises:
 an adeno-associated virus (AAV) capsid nucleotide sequence that comprises VP1, VP2, and VP3 sequences coding for functional AAV capsid viral proteins, wherein two of said VP1, VP2, and VP3 sequences are from a first AAV serotype, wherein one of said VP1, VP2, and VP3 sequences is from a second AAV serotype, and wherein said first serotype or said second serotype is AAV12.   
     
     
         250 . The isolated non-naturally occurring nucleic acid of  claim 249 , wherein said first AAV serotype is AAV12. 
     
     
         251 . The isolated non-naturally occurring nucleic acid of  claim 249 , wherein said VP1 and VP2 sequences are from the first serotype that is AAV12. 
     
     
         252 . The isolated non-naturally occurring nucleic acid of  claim 249 , wherein said isolated non-naturally occurring nucleic acid upon transduction with a virus containing a CMV-promoter driven GFP-coding nucleic acid sequence has increased efficiency compared to that generated by a wild-type AAV nucleic acid comprising a VP1, VP2, and VP3 sequence of first or second serotype when measured by quantifying fluorescence of mammalian cells after 24 hours. 
     
     
         253 . The isolated non-naturally occurring nucleic acid of  claim 249 , wherein the isolated non-naturally occurring nucleic acid further comprises a transgene, and wherein said isolated non-naturally occurring nucleic acid upon transduction with a virus containing a CMV-promoter driven GFP-coding nucleic acid sequence has an increased expression of the transgene as compared to a wild-type AAV nucleic acid comprising a VP1, VP2, and VP3 sequence of first or second serotype when measured by quantifying fluorescence of mammalian cells after 24 hours. 
     
     
         254 . The isolated non-naturally occurring nucleic acid of  claim 253 , wherein the transgene codes for at least a portion of an exogenous receptor. 
     
     
         255 . The isolated non-naturally occurring nucleic acid of  claim 254 , wherein the exogenous receptor is a T cell receptor or a chimeric antigen receptor. 
     
     
         256 . The isolated non-naturally occurring nucleic acid of  claim 249 , wherein said isolated non-naturally occurring nucleic acid comprises DNA.