Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms

Treatment of Terminal Peritoneal Carcinomatosis by a Transducible p53-Activating Peptide

Advanced-stage peritoneal carcinomatosis is resistant to current chemotherapy treatment and, in the case of metastatic ovarian cancer, results in a devastating 15%–20% survival rate. Therapeutics that restore genes inactivated during oncogenesis are predicted to be more potent and specific than current therapies. Experiments with viral vectors have demonstrated the theoretical utility of expressing the p53 tumor suppressor gene in cancer cells. However, clinically useful alternative approaches for introducing p53 activity into cancer cells are clearly needed. It has been hypothesized that direct reactivation of endogenous p53 protein in cancer cells will be therapeutically beneficial, but few tests of this hypothesis have been carried out in vivo. We report that a transducible D-isomer RI-TATp53C′ peptide activates the p53 protein in cancer cells, but not normal cells. RI-TATp53C′ peptide treatment of preclinical terminal peritoneal carcinomatosis and peritoneal lymphoma models results in significant increases in lifespan (greater than 6-fold) and the generation of disease-free animals. These proof-of-concept observations show that specific activation of endogenous p53 activity by a macromolecular agent is therapeutically effective in preclinical models of terminal human malignancy. Our results suggest that TAT-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to malignant cells in vivo

Clumpiness enhancement of charged cosmic rays from dark matter annihilation with Sommerfeld effect

Boost factors of dark matter annihilation into antiprotons and electrons/positrons due to the clumpiness of dark matter distribution are studied in detail in this work, taking the Sommerfeld effect into account. It has been thought that the Sommerfeld effect, if exists, will be more remarkable in substructures because they are colder than the host halo, and may result in a larger boost factor. We give a full calculation of the boost factors based on the recent N-body simulations. Three typical cases of Sommerfeld effects, the non-resonant, moderately resonant and strongly resonant cases are considered. We find that for the non-resonant and moderately resonant cases the enhancement effects of substructures due to the Sommerfeld effect are very small ($\sim \mathcal{O}(1)$) because of the saturation behavior of the Sommerfeld effect. For the strongly resonant case the boost factor is typically smaller than $\sim \mathcal{O}(10)$. However, it is possible in some very extreme cases that DM distribution is adopted to give the maximal annihilation the boost factor can reach up to $\sim 1000$. The variances of the boost factors due to different realizations of substructures distribution are also discussed in the work.Comment: 28 pages, 8 figures, 2 table. The detailed fomula of the propagation and boost factor are moved to the Appendix. Accepted by JCA

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms

Tumor-Reconstituted Cells from Treated Mice Remain Sensitive to RI-TATp53C′ Peptide-Induced G1 Arrest or Apoptosis in Culture

<div><p>(A) TA3/St cells were recovered from an A/J mouse treated with RI-TATp53C′ peptide and grown in DMEM/10% FBS. Recovered cells were treated with increasing concentrations of RI-TATp53C′ peptide and then analyzed for DNA content by flow cytometry 24 h later.</p> <p>(B) Namalwa cells were recovered from a SCID mouse treated with RI-TATp53C′ peptide and grown in RPMI plus 10% FBS. Recovered cells were treated with increasing concentrations of RI-TATp53C′ peptide. After 2 d, the number of viable cells was assessed by Trypan blue exclusion and normalized to the number of viable untreated cells. Mean and standard deviation of multiple experiments are depicted.</p></div

RI-TATp53C′ Treatment Leads to 50% Long-Term Survival of Mice Bearing Terminal Peritoneal Lymphoma

<div><p>(A) Treatment of human Namalwa B-cell lymphoma cells with RI-TATp53C′ peptide induces apoptosis. Cells were treated with wild-type or mutant peptide, and DNA content was analyzed by flow cytometry 24 h after peptide addition.</p> <p>(B) Long-term survival of SCID mice harboring lethal peritoneal Namalwa lymphoma tumor burden after RI-TATp53C′ peptide treatment. Namalwa lymphoma cells were IP injected into SCID mice and allowed to proliferate for 48 h. Mice were then injected 16 times over 20 d with vehicle control (<i>n</i> = 16), 900 μg of wild-type RI-TATp53C′ peptide (<i>n</i> = 12), or 900 μg of mutant peptide (<i>n</i> = 6). Mean survival duration was 35 d for vehicle-treated mice and 33 d for mice receiving mutant peptide, whereas 50% of mice treated with wild-type RI-TATp53C′ peptide remained healthy at 150 d after tumor cell injection.</p></div

RI-TATp53C′ Peptide Activates p53-Dependent Transcription and Inhibits Tumor Cells Expressing p53

<div><p>(A, left) Induction of transcription from a p53-dependent promoter by RI-TATp53C′ only when p53 protein is expressed. H1299 cells (<i>p53^−/−</i>) were cotransfected with p53-responsive reporter (PG13-Luc) and either empty vector or p53 expression vector. Depicted are mean and standard deviation of triplicate results that are representative of multiple experiments.</p> <p>(A, right) RI-TATp53C′ peptide activates p53-dependent transcription in cells expressing DNA contact mutant p53. SW480 cells containing a DNA contact mutant (R273H) p53 were transfected with p53-dependent reporter (PG13-Luc). H1299 cells (<i>p53</i>^−/−) were co-transfected with PG13-Luc and either R248Q or R273H mutant p53 expression vector. RI-TATp53C′ was added to cells, and promoter activity was assessed 24 h later.</p> <p>(B) Inhibition of tumor cell proliferation in a p53-dependent manner by RI-TATp53C′. Increasing concentrations of peptide were added to HCT 116 cells (<i>p53^+/+</i>) and their <i>p53^−/−</i> isogenic derivatives. After 2 d, the number of viable cells was assessed by Trypan blue exclusion and normalized to the number of viable untreated cells. Mean and standard deviation of multiple experiments are depicted.</p> <p>(C) Inhibition of the proliferation of tumor cells expressing wild-type or mutant p53, but not <i>p53^−/−</i> tumor cells or nontransformed human fibroblasts. Cell viability was assessed as in (B). Mean and standard deviation of multiple experiments are depicted.</p></div

RI-TATp53C′ Treatment Extends Survival of Mice Harboring Terminal Peritoneal Carcinomatosis

<div><p>(A) A 6-fold increase in survival of A/J immune-competent mice harboring lethal TA3/St mammary peritoneal carcinomatosis burden after RI-TATp53C′ peptide treatment. A/J mice were given IP injections of TA3/St cells, and cells were allowed to double in number (approximately 24 h). Peritoneal tumor-bearing mice were then treated once a day for 12 consecutive days with vehicle (<i>n</i> = 15), 600 μg of wild-type RI-TATp53C′ (<i>n</i> = 10), or 600 μg of mutant peptide (<i>n</i> = 10). Mean survival duration was 11 d for vehicle-treated mice, 10 d for mice receiving mutant peptide, and greater than 70 d for the group receiving wild-type RI-TATp53C′ peptide.</p> <p>(B) Reduction of tumor cell number in vivo by RI-TATp53C′ treatment. Mice were injected with TA3/St tumor cells and treated with wild-type peptide as in (A). Three days after tumor cell injection, cells were flushed from the peritoneal cavity and serially diluted in 6-well plates. Growth of colonies was then assessed by methylene blue staining and used to measure the number of viable tumor cells present in the peritoneum after treatment with vehicle or wild-type peptide.</p></div

RI-TATp53C′ Induces the Hallmarks of p53 Activity in TA3/St Mammary Carcinoma Cells

<div><p>(A) Sequence of p53C′TAT peptide (L-amino acids) and its retro-inverso analogue (D-amino acids). To generate a negative control peptide, three essential lysine residues (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020036#pbio-0020036-Selivanova1" target="_blank">Selivanova et al. 1997</a>) were mutated while leaving the remaining peptide sequence intact.</p> <p>(B) Induction of G1 arrest in TA3/St cells by wild-type RI-TATp53C′, but not mutant peptide, 24 h after peptide addition.</p> <p>(C) Dose-dependent induction of G1 arrest by RI-TATp53C′ (open square) (D-amino acids) and the less potent p53C′TAT (open circle) (L-amino acids) but not mutant (open triangle) peptide at 24 h (left) and 48 h (right) after single treatment.</p> <p>(D) Induction of a permanent growth arrest in TA3/St cells by RI-TATp53C′. Cells were treated with RI-TATp53C′ peptide or vehicle for 2 d, replated, and allowed to proliferate in the presence of serum for 10 d. Colonies were then stained with methylene blue.</p> <p>(E) Induction of a senescence-like phenotype in TA3/St cells by RI-TATp53C′. Cells were treated with RI-TATp53C′ peptide and stained for acidic β-galactosidase activity.</p></div