RECOMBINANT VIRUS AND PREPARATIONS THEREOF
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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Abstract
The present invention generally relates to methods and compositions used delivery of gene editing compositions including transcriptional effectors with parvovirus and preferred methods for making same.
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Citations
18 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification