Figure 5: Gene editing in mouse BM stem cell populations in vivo and human CD34+ cells ex vivo.
(a) Thalassemic mice were treated with either blank NPs or NPs containing γtcPNA4/donor DNA plus SCF (four doses at 2-day intervals as in Fig. 4). Deep sequencing analysis was performed to measure gene editing in the β-globin gene in either total BM (total BM) cells or in BM stem/progenitor cell sub-populations selected based on the indicated markers. BM cells were harvested either on day 36 post-treatment (total BM samples) or on day 65 post-treatment (sorted cell sub-populations). Data represent the combined analysis of BM from n=3 mice in each group. Data are mean±s.e.m., n=3; statistical analysis by Student’s t-test, *P<0.05. (b) Schematic showing experimental design in which human CD34+ cells were treated ex vivo with either blank NPs or with γtcPNA4/donor DNA NPs plus SCF. Treated cells were either harvested 2 days later for deep sequencing analysis of gene editing in the β-globin gene or were transplanted into NOD-scid IL2rγnullmice. Eight weeks after transplantation, BM cells were harvested from the mice and human CD34+ cells were isolated, followed by deep sequencing of the the β-globin gene alleles. (c) Deep sequencing results to quantify β-globin gene editing in either pre-transplanted human CD34+ cells or in human CD34+ cells that were harvested from NOD-scid IL2rγnull mice 8 weeks after transplant, as described in b. Data are mean±s.e.m., n=3 statistical analysis by Student’s t-test, *P<0.05.
