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. 2019 Nov 13;28(4):997–1015. doi: 10.1016/j.ymthe.2019.11.006

Figure 6.

Figure 6

Gene Editing by AAV-Delivered ZFN and Donor DNA Template

The zinc finger nucleases (ZFNs) are designed to place the normal copy of the clotting factor gene within the albumin intron 1, under control of the endogenous albumin locus promoter. Three adeno-associated virus (AAV) vectors are delivered, each providing one of the three components: a right or left ZFN or clotting factor cDNA donor template.140,141,143 (1) The AAV-expressed ZFN fuses a DNA-cleavage domain (FokI endonuclease) to a zinc finger DNA-binding domain. (2) The ZFN binding domain targets a specific sequence and then the cleavage domain induces a double-strand break. (3) The double-strand break can be repaired by homologous recombination if a DNA donor template is present with flanking arms homologous to the DNA at the ZFN cleavage site. (4) Flanking the clotting factor cDNA is a poly(A) sequence (pA) and a splice acceptor signal (SA), which splices the transcribed clotting factor RNA to the splice donor (SD) of albumin exon 1 RNA to produce an mRNA fusion transcript. (5) The mRNA fusion transcript is translated into secreted clotting factor protein.