Fig. 3: Capsid DNA shuffling and selection in NF1 xenograft mice created AAV vectors for NF1 tumors.
From: Development of an adeno-associated virus vector for gene replacement therapy of NF1-related tumors
A Example of an orthotopic xenograft ST88-14 tumor grown in the mouse sciatic nerve. ST88-14 cells were injected in the sciatic nerve of NSG mice and tumor was formed in the nerve. B Capsid 557-2 is the top candidate selected from a capsid DNA shuffling library. A DNA shuffling library was created from 12 natural serotypes of AAV capsids and packaged in 293T cells. Selection in ST88-14 tumor-bearing mice resulted in 557-2, whose resemblance to the template capsids was shown in the graph generated by Xover 3.0. The solid black lines represent the parts of the 557-2 sequence found in the capsids of natural serotypes. C Improved transduction by 557-2-GFP in ST88-14 tumor compared to AAV9-GFP. AAVs were injected IV to the ST88-14 tumor-bearing mice at the dose of 5 × 1011 vg (viral genome)/mouse and after 2 weeks tumors were harvested for anit-GFP IHC. Scale bar = 10 µm. D AAV-557-2 showed a significantly reduced liver retention with more tumor distribution in a panel of three orthotopic NF1 xenografts. Three xenografts of NF1-related tumor cells, ST88-14, ipNF03.3 and RHT92, were injected in the sciatic nerve of NSG mice. AAV9-GFP or 557-2-GFP were injected IV to the mice at a dose of 5 × 1011 vg. Tissues were harvested after two weeks and quantitative PCR was used to determine titers of AAV genome in different tissues with the linearized AAV plasmid as standard. Three mice were used in each data point. Data are presented as mean values with SD and analyzed by two-tailed t-test. E AAV-557-2-GRDC24 significantly slowed down the ST88-14 tumor growth in mice. ST88-14 cells were implanted in the sciatic nerve in the NSG mice and after 14 days, tumor sizes were assessed by IVIS imaging and AAV 557-2-GRDC24 was injected IV to at a dose of 1 × 1012 vg per mouse. Two weeks later, the tumor growth was imaged and compared to the control. Data are presented as mean values with SD and analyzed by one-tailed t-test. Con: n = 14 mice; 557-2: n = 13 mice.
