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RECOMBINANT VIRUS AND PREPARATIONS THEREOF

  • US 20140287938A1
  • Filed: 03/14/2014
  • Published: 09/25/2014
  • Est. Priority Date: 03/15/2013
  • Status: Active Grant
First Claim
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1. A method for obtaining and optionally storing a sample containing a set amount of rAAV comprising or consisting essentially of:

  • (a) creating infected or transfected cells by a process comprising or consisting essentially of one or more methods selected from;

    (i) transfecting plasmid(s) containing or consisting essentially of exogenous DNA including DNA for expression into AAV-infected cells along with another helper plasmid that provides AAV rep and/or cap genes which are obligatory for replication and packaging of the rAAV;

    or(ii) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, and helper virus wherein the rAAV lacks functioning cap and/or rep and the helper virus provides the cap and/or rev function that the rAAV lacks;

    or(iii) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, and transfecting said cells with a plasmid supplying cap and/or rep function that the rAAV lacks;

    or(iv) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, wherein said cells supply cap and/or rep function that the recombinant construct lacks;

    or(v) transfecting the susceptible cells with an AAV lacking functioning cap and/or rep and plasmids for inserting exogenous DNA into the recombinant construct so that the exogenous DNA is expressed by the recombinant construct and for supplying rep and/or cap functions whereby transfection results in an rAAV containing or consisting essentially of the exogenous DNA including DNA for expression that lacks functioning cap and/or rep; and

    (b) incubating the infected or transfected cells, whereby there results infected or transfected cells and supernatant containing the rAAV lacking functioning cap and/or rep;

    (c) after incubating, extracting an aliquot from the supernatant;

    (d) filtering the aliquot, whereby the filtered aliquot contains and the method obtains a sample containing set amount of the rAAV relative to the type and amount of susceptible cells infected or transfected; and

    (e) optionally freezing the filtered aliquot,whereby the method optionally includes storing a sample containing set amount of the rAAV relative to the type and amount of susceptible cells infected or transfected.

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