1. Engineered Exosomes as Vehicles for Biologically Active Proteins
Ulrich Sterzenbach, Ulrich Putz, Ley-Hian Low, John Silke, Seong-Seng Tan, Jason Howitt 
Molecular Therapy 
Volume 25, Issue 6, Pages (June 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Molecular Therapy 2017 25, 1269-1278DOI: (10.1016/j.ymthe.2017.03.030)
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 1 The Packaging of Targeted Fusion Proteins into Exosomes
(A) Flow cytometric analysis of transfected mT/mG MEFs showed that the WW-tagged Cre protein (WW-Cre) maintained Cre-recombinase activity compared to wild-type Cre protein. Unpaired t test. (B) Cre protein is not exported in exosomes with or without Ndfip1 expression. WW-Cre protein is efficiently exported into exosomes in the presence of Ndfip1 in LN18 cells. The asterisk indicates the WW-Cre band above the Tsg101 band after reprobing the western blot. (C) NanoSight tracking analysis was used to measure the particle size of the purified exosomes. (D) GW4869 inhibited the release of WW-Cre protein into exosomes. Data points are the average ± SEM of three independent experiments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 2
Exosomes Containing Target WW-Cre Proteins Are Functionally Active in Recipient Reporter Cells
(A–D) Exosomes purified from LN18 cells transfected with Cre only (A), Cre and Ndfip1 (B), WW-Cre only (C), or WW-Cre and Ndfip1 (D) were delivered to mT/mG MEFs. (E) Quantification of the number of positive reporter mT/mG MEFs after exosome delivery. Scale bar, 50 μm. Data points are the average ± SEM of three independent experiments. One-way ANOVA tests with Bonferroni post-tests, ****p <
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 3
Ndfip1 Expression Is Required for Functional WW-Cre Exosomes and Mediates the Ubiquitination of WW-Cre
(A) Immunoprecipitation assay of overexpressed WW-Cre and Ndfip1 in HEK293T cells showed an interaction between the two proteins. (B) Ndfip1+/+ and Ndfip1−/− MEFs used in the co-culture assay in (C) expressed similar levels of WW-Cre, as shown by western blotting. (C) Ndfip1+/+ and Ndfip1−/− MEFs infected with lentiviral construct of WW-Cre were co-cultured with mT/mG reporter MEFs. Only Ndfip1+/+ MEFs resulted in a positive recombination of mT/mG MEFs. (D) Denaturing ubiquitin assays show that Cre protein is not ubiquitinated with or without Ndfip1. In contrast, WW-Cre protein is predominantly monoubiquitinated in the presence of Ndfip1. Scale bar, 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 4 The Stability and Function of Stored Exosomes
(A) Proteinase treatment of exosomes did not result in the degradation of WW-Cre unless Triton X-100 was added to permeabilize the exosome membrane. (B) Exosomes were stored for 3 days under different conditions before western blotting was performed to determine the stability of WW-Cre protein compared to a control fresh preparation of WW-Cre exosomes. (C) Stored exosomes were analyzed for the ability to deliver active WW-Cre to mT/mG reporter MEFs. Data points are the average ± SEM of three independent experiments. One-way ANOVA tests with Bonferroni post-tests, *p < 0.05.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 5 In Vivo Activity of Exogenous Exosomes
(A) Quantification of positive reporter cells after nasal delivery of exosomes derived from LN18 cells transfected with WW-Cre and Ndfip1, Cre and Ndfip1, or untransfected to Ai14 mice. Mouse brains were collected 14 days after last nasal delivery, and sections from multiple brain regions were analyzed for red reporter cells indicating Cre-recombination (n = 4 mice per condition). (B) Overview of the coronal brain section analyzed for Cre recombination, with the white box indicating the area imaged in (C). (C) Higher magnification of brain regions showing Cre recombination after delivery of WW-Cre containing exosomes. (D) Immunocytochemistry with anti-NeuN antibody shows colocalization in cells with Cre-recombinase activity (tdTomato) in Ai14 mice after WW-Cre exosome delivery. (E) Immunocytochemistry with anti-Iba-1 antibody shows colocalization with Cre-recombinase activity (tdTomato) in Ai14 mice after WW-Cre exosome delivery. One-way ANOVA tests with Bonferroni post-tests, *p < Scale bars, (B) 2.5 mm, (C) 250 μm, and (D and E) 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions