RECOMBINANT VIRUS AND PREPARATIONS THEREOF
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.
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.
2 . A method for screening rAAV comprising or consisting essentially of,
preparing the filtered aliquot or the stored filtered aliquot of claim 1 ,
if necessary, thawing the stored filtered aliquot,
contacting the filtered aliquot with cells, and
determining whether the exogenous DNA is expressed in an amount and/or duration sufficient for an intended use.
3 . The method of claim 2 wherein the contacting of the filtered aliquot with cells comprises or consists essentially of transducing said cells.
4 . The method of claim 3 wherein the contacting is for 5-6 days.
5 . The method of claim 2 wherein the rAAV expresses a TALE and the contacting includes or consists essentially of detecting nuclease, activator or repressor activity.
6 . The method of claim 2 wherein the rAAV expresses a LITE, and the contacting includes or consists essentially of inducing gene expression or subjecting the contacted cells to a suitable stimulus, and detecting whether a transcriptional effector has been induced.
7 . The method of claim 6 wherein detecting whether a transcriptional effector has been induced includes or consists essentially of detecting a color change.
8 . The method of claim 2 wherein the rAAV expresses a CRISPR system, and the contacting includes or consists essentially of detecting gene knockdown or other effects of the CRISPR system.
9 . The method of claim 1 wherein the AAV is AAV1, AAV2, AAV5 or an AAV having a hybrid or mosaic AAV1, AAV2 and/or AAV5 capsid.
10 . The method of claim 1 wherein the susceptible cells are 293FT cells.
11 . The method of claim 10 wherein 2×10 5 cells are transfected or infected.
12 . The method of claim 11 wherein a 250 μL filtered aliquot contains the recombinant AAV at a concentration of about 5.6+/−0.24×10 5 .
13 . The method of claim 1 including freezing the filtered aliquot.
14 . The method of claim 13 wherein the filtered aliquot is frozen at about −80 C.
15 . The method of claim 1 including adding a secretion enhancer to the cells before, during or after and within the incubating.
16 . The method of claim 15 wherein the secretion enhancer is polyethylenimine (PEI).
17 . A method of high-throughput screening of a sample comprising or consisting essentially of contacting the supernatant containing the rAAV lacking functioning cap and/or rep of claim 1 with the sample and determining whether the exogenous DNA of claim 1 is present in the sample.
18 . The method of claim 17 , wherein the supernatant is thawed from the filtered aliquot.