University of Washington

Fred Hutchinson Cancer Center

David W Russell

Roli K Hirata

A Dusty Miller

Ian E Alexander

Elizabeth A Rutledge

The invention provides isolated primate cells preferably human cells that comprise a genetically engineered disruption in a beta-2 microglobulin (B2M) gene, which results in deficiency in MHC class I expression and function. Also provided are the method of using the cells for transplantation and treating a disease condition.

Beta-2 microglobulin-deficient cells

This invention provides methods for obtaining targeted gene modification in vertebrate cells using parvoviral vectors, including adeno-associated virus (AAV). The parvoviral vectors used in the methods of the invention are capable of targeting a specific genetic modification to a preselected target locus in a cellular genome by homologous pairing.

Targeted gene modification by parvoviral vectors

The present invention provides methods of generating a mammalian cell that is homozygous at a locus of interest, as well as cells made by the method. The present invention further provides methods of using the cells.

HLA homozygous cells

Parvoviral production of HLA homozygous cells

The present invention provides isolated adenovirus-associated viruses ('AAV'), including AAV isolates designated AAV3B and AAV6. The invention also provides nucleic acid molecules of AAV3B (SEQ ID NO: 1) or AAV6 (SEQ ID NO: 2), including DNA or RNA, and provides substantially purified polypeptides encoded by AAV3B or AAV6, as well as antibodies specific for such polypeptides. The invention also provides infectious AAV3B and AAV6 clones; AAV viral vectors, which can be hybrid AAV viral vectors; AAV vector plasmids; and AAV helper plasmids; each comprising at least a portion of an AAV3B (SEQ ID NO: 1) or AAV6 (SEQ ID NO: 2) nucleic acid molecule. The invention further provides host cells containing at least a portion of an AAV3B or AAV6 nucleic acid molecule and progeny cells derived therefrom, and provides non-human transgenic mammals having an AAV vector genome stably integrated in a chromosome. The invention also provides methods of producing a polypeptide or an RNA by expressing the polypeptide or the RNA from an AAV viral vector in a cell, and methods of treating a pathologic condition in a mammal, comprising transducing cells of the mammal with an AAV viral vector containing a heterologous nucleic acid sequence. The invention also provides AAV3B or AAV6 nucleotide sequences, which can be useful, for example, as probes for identifying the presence of AAV3B or AAV6 nucleic acids in a sample. The present invention further provides a method of identifying an AAV viral vector suitable for administration to an individual.

Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom

Method for increasing transduction of cells by adeno-associated virus

The invention includes methods for increasing the efficiency of transduction of cells, including non-dividing cells, by recombinant AAV vectors. The methods utilize agents that alter certain aspects of DNA metabolism, more specifically, that affect DNA synthesis and/or affect repair, that impact on maintenance of chromosomal integrity, and/or that cause damage to the cellular DNA. Agents and vectors can now also be preselected and screened for transducing ability and/or transducing agents for their effect on DNA metabolism. These agents include tritiated nucleotides such as thymidine, gamma irradiation, UV irradiation, cis-platinum, etoposide, hydroxyurea and aphidicolin.

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Seattle, WA, United States of America

David W Russell