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Methods for the titration of AAV vectors

100%

Rutkowski A. , Jazwa A. , Popowa S. , Jozkowicz A. , Dulak J.

nr 3

157-166

Adeno-associated viral (AAV) vectors are promising tools for gene therapy. However, for trustworthy comparison of the results produced from different clinical trials, the exact amount of the used infectious vector particles must be known. We have produced AAV using a commercially available system and compared three common methods for the quantification of the number of produced vectors: ELISA, dot-blot and quantitavive PCR (qPCR). Although ELISA is a very reproducible and precise method, it is able only to determine the number of viral capsids in the vector preparation, also those which contain no genetic material and are therefore useless. With this method we established that we are able to produce ~ 6.5 x 1011 viral capsids/mL. Dot-blot assay determines the number of genomic particles in the vector preparation in a quite precise manner, but it is a very labor- and time-consuming method. qPCR is also a method determining the number of genomic particles. It is, however, much faster and simpler than the dot-blot assay. Both dot-blot and qPCR gave similar results (~ 4 x 1011 viral genomes/mL), which indicated only about 2/3 of the produced vectors containing genetic material. Our results show that qPCR is the most convenient and reliable method for quantification of AAV vectors and we believe it could be routinely used to titer the vectors prior to their usage in clinical trials.