Experimental design of tumorigenesis assay.

<p>(<b>a</b>) Transgenic mice were taken off of doxycycline drinking water at 7–8 weeks of age to induce expression of the human <i>C-MYC</i> transgene specifically in the liver from the LAP promoter, except for one control group (MYC off). Exactly one week after C-MYC induction, all groups except one control group (MYC on, no injection) were given hydrodynamic injections of phosphate-buffered saline alone or DNA plasmids diluted in phosphate-buffered saline. Mice were monitored weekly, imaged every two or three weeks, and sacrificed when tumors were detectable by gross distention of the abdomen as pictured. Inset shows the dissected liver and tumors from the pictured mouse, which was representative of all mice in all groups. (<b>b</b>) The plasmids given by hydrodynamic injection and their features.</p

Treatment group and number of days from initiation of C-MYC overexpression until sacrifice (survival time) for each case of metastasis.

<p>Treatment group and number of days from initiation of C-MYC overexpression until sacrifice (survival time) for each case of metastasis.</p

Luciferase activity and PCR analysis of tumors from mice in the pCSI/pLiLucB group provide no evidence of φC31 integrase activity.

<p>(<b>a</b>) Protein extracts were prepared and the luciferase activity was measured in absolute counts per second (CPS). Controls included HeLa cells given FuGene 6 alone [HeLa (-)] or the CMV-luciferase plasmid pNBL2 via FuGene 6 [HeLa (+)], the normal-appearing part of the tumor-ridden liver taken from either a saline-injected mouse [Liver (-)] and pCSI/pLiLucB-injected mouse [Liver (+)]. Eight tumor samples (Tumor 1 through 8) and one metastasis (Met 1) that were obtained from four animals were also analyzed. The error bars give standard error of the mean for four replicates of each sample. (<b>b</b>) PCR analysis to detect the pLiLucB plasmid by amplification of the luciferase transgene. Plasmid DNA (20 ng pLiLucB) and no DNA controls show specific amplification of luciferase only in the reaction containing plasmid. One mouse each from the saline-only and pCSmI/pLiLucB groups was analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues (none found). Three mice in the pCSI/pLiLucB group were analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues. Luciferase could be detected in 2/3 normal-appearing liver samples and none of the tumors. (<b>c</b>) PCR analysis for integration at the mpsL1 pseudo <i>attP</i> site was done on 18 tumors (lanes 5 through 22) and one metastasis (lane 23) taken from nine mice given pCSI/pLiLucB by hydrodynamic injection. Controls included no DNA (1^st round, lane 1 and 2^nd round, lane 25), and a DNeasy performed on no tissue (lane 2) to show no contamination from the DNA isolation procedure. Normal-appearing liver from a mouse in the pCSI/pLiLucB group (lane 3) served as the positive control. DNA isolated from a tumor in the saline-only group served as the negative control (lane 4). PCR for the <i>GAPDH</i> gene showed that sufficient DNA was added to all reactions. Seven tumors and one metastasis were subjected to the analysis in <i>both </i><b>a</b> and <b>c</b>. Six tumors were analyzed by all assays (<b>a</b>, <b>b</b> and <b>c</b>).</p

Survival curves suggest that hydrodynamic injection may contribute to C-MYC-induced tumor formation in the mouse liver.

<p>(<b>a</b>) Key showing the number of animals (n), median survival time in days (Median), and statistical results for each group. The significance as determined by the Gehan-Breslow-Wilcoxon test comparing each group to the MYC on, no injection (no inj?) or MYC on, saline injection (saline?) control groups is given. (<b>b</b>) A comparison of MYC off, no injection (yellow), MYC on, no injection (orange), and MYC on, saline injection (red) survival curves. (<b>c</b>) A comparison of MYC on, saline injection and MYC on, pCS/pLiLucB (purple) injection survival curves. (<b>d</b>) A comparison of MYC on, pCS/pLiLucB, MYC on, pCSmI/pLiLucB (cyan), and MYC on, pCSI/pLiLucB (blue) survival curves. (<b>e</b>) The survival curves of groups given pCSI with and without (green) donor plasmid. (<b>f</b>) A comparison of pCSI/pLiLucB to the control groups of no injection and saline-only injection. (<b>g</b>) The survival curves of all groups shown on the same plot. All plots and statistics were done using GraphPad Prism software.</p

Luciferase expression and the relationship between initial expression values and long-term survival.

<p>(<b>a</b>) <i>TRE-MYC/LAP-tTA</i> transgenic mice given pCS/pLiLucB (purple diamonds), pCSmI/pLiLucB (cyan squares), or pCSI/pLiLucB (blue triangles) by hydrodynamic injection were imaged at Day 1, Week 2, 4, 6, 8, and 10. The normalized luciferase levels were obtained by dividing the average luciferase expression in reflective light units (RLU) at each time point by the average level at day 1 for that group. Propagation of errors was used to determine the standard error at each time point given the division calculation (error bars). By student's t-test of the values at day 70, the pCSI/pLiLucB group and pCS/pLiLucB group had a p-value of 0.067, while the pCSI/pLiLucB group and pCS/pLiLucB group were significantly higher than the pCSmI/pLiLucB group (p = 0.014 and p = 0.000031, respectively). (<b>b</b>) Each mouse is represented by one point on the scatterplot, using the day 1 luciferase value in reflective light units (RLU) as the x-coordinate and the days of survival as the y-coordinate. The symbols and colors are identical to those used in <b>a</b>. The linear line-of-best-fit was calculated by GraphPad Prism and is plotted for each group (R-squared values of pCS/pLiLucB, 0.1065; pCSmI/pLiLucB, 0.3839; pCSI/pLiLucB, 0.0033). No R-squared values exceeded 0.95, which would have indicated that there was a trend relating transfection efficiency and survival.</p

Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in <i>mdx</i> Mouse Induced Pluripotent Stem Cells

<div><p>A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the <i>mdx</i> mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram <i>mdx</i> fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.</p></div

Myofiber differentiation and engraftment.

<p>(a) Immunofluorescence staining of dystrophin in W9, W987, and ESC. Myosin heavy chain (MHC) identified muscle cells after differentiation. DAPI was used to stain nuclei. (b) Myotube formation in differentiated W987 and W9 iPSC and ESC. Note that W9 iPSC did not form myotubes. (c) Engraftment of corrected <i>mdx</i> iPSC in mouse TA muscle. Approximately 750,000 W987 iPSC that were differentiated for 13 days <i>in vitro</i> and sorted for the SM/C-2.6 antibody were injected into the TA muscle of an irradiated <i>mdx/;SCID</i> mouse. After three weeks, muscle sections were prepared and stained. Staining for laminin delineated individual muscle fibers, while staining for dystrophin revealed engraftment of corrected iPSC (arrows). (d) Numbers of dystrophin-positive fibers per TA muscle, total section, are shown for engrafted muscle versus uninjected contralateral muscle (control).</p

Muscle marker expression in differentiating <i>mdx</i> iPSC.

<p>W9 and W987 iPSC and ESC were differentiated <i>in vitro</i>, and at various time points, cells were stained with the SM/C-2.6 antibody and analyzed by flow cytometry (FC). (a) FC plots of W9, W987, and ESC differentiated for 13 days, indicating that approximately 4.5%, 46%, and 59% of cells were positive for SM/C-2.6, respectively, at this time point. Anti-rat IgG was used as a staining control. (b) Differentiation efficiency at the day 6, 13, 20, 27, and 34 time points for W9, W987, and ESC, as judged by percent positive for SM/C-2.6. (c) qRT-PCR analysis of muscle lineage markers <i>Pax7</i>, <i>Pax3</i>, <i>MyoD</i>, and <i>myogenin</i> during the time course in W9, W987, and ESC. (d) qRT-PCR analysis of <i>MyoD</i> and <i>myogenin</i> expression in W987, W9 and ESC cells at 20 and 27 days of differentiation. (e) qRT-PCR analysis of dystrophin expression at various time points.</p

Pluripotency of <i>mdx</i> iPSC.

<p>(a) Reprogrammed <i>mdx</i> iPSC colony W9; bright field, GFP, and alkaline phosphastase staining. (b) GFP fluorescence and immunofluorescence staining of Oct4, Sox2, Nanog, and SSEA-1 in W9 iPSC before and after (W987) Cre-mediated excision of reprogramming genes and in mESC. Scale bar  = 50 µm. (c) Quantitative RT-PCR data showing expression of Oct4, Sox2, Nanog, and c-Myc in W9 and W987 iPSC, as well as in mESC controls and in the parental <i>mdx</i> adult fibroblasts. (d) Promoter methylation status of Oct4 in W9 and W987 iPSC and in mESC and adult fibroblast controls. Five different CpG islands were analyzed, indicated by their distance from the transcription start site. Open circles reflect low methylation (0–25%), gray circles represent medium (26–75%), and black circles indicate high (76–100%) methylation. (e) Embryoid bodies grown from W987 iPSC and stained for markers of the three germ layers. Day 14 embryoid bodies were stained with antibodies against smooth muscle actin (SMA), α-fetoprotein (AFP), and βIII-tubulin (Tuj1), indicating mesodermal, endodermal and ectodermal differentiation <i>in vitro</i>, respectively. DAPI was used to stain the nuclei. Alexa 594-labeled secondary antibodies were used. (f) Chromosome counts were performed in the parental <i>mdx</i> adult fibroblasts and in iPSC before (W9) and after (W987) Cre excision. The normal murine chromosome number of 40 was observed.</p

Genome engineering.

<p>(a) Southern blot analysis. iPSC clones derived from <i>mdx</i> fibroblasts and reprogrammed using pCOBLW and pVI were probed with a sequence from the EGFP gene. The number of bands indicated the number of copies of the reprogramming plasmid that integrated. W3, W4, W5, W10, and W9 represented a subset of the reprogrammed colonies that were screened. (b) Bxb1-mediated site-specific integration of therapeutic plasmid. Puromycin-resistant colonies were picked. (c) Representative subclones W9D1–W9D10 were analyzed by PCR to detect the expected <i>attR</i> (591 bp) and <i>attL</i> (431 bp) Bxb1 junction bands. (d) Left: Cre-mediated excision of unwanted sequences causes a loss of GFP fluorescence. Cre resolvase was introduced in representative subclones W9D8 and W9D10 to excise the reprogramming cassette and other sequences no longer needed. After excision, GFP expression was extinguished in colonies. Right: Representative subclones W9D8E3, W9D8E4, W9D8E7 (W987), W9D8E8, W9D8E17, and W9D10E1 were analyzed by nested PCR to detect the expected <i>loxP</i> 167 bp junction fragment indicative of successful excision.</p