CRISPR-ERA for Switching Off (Onco) Genes

Genome-editing nucleases like the popular CRISPR/Cas9 enable the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks (DSBs) within a genome. In most cases, when a DNA template is not present, the DSB is repaired by nonhomologous end joining (NHEJ), resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, for several reasons, these mutations do not produce the desired null result in all cases, instead generating a similar protein with functional activity. This undesirable effect could limit the therapeutic efficiency of gene therapy strategies focused on abrogating oncogene expression by CRISPR/Cas9 and should be taken into account. This chapter reviews the irruption of CRISPR technology for gene silencing and its application in gene therapy

Influence of IFN-gamma and its receptors in human breast cancer

<p>Abstract</p> <p>Background</p> <p>Interferons are a group of proteins that trigger multiple responses including prevention of viral replication, inhibition of cell growth, and modulation of cell differentiation. In different mammary carcinoma cell lines IFNγ induces growth arrest at mid-G1. At the present there are no <it>in vivo </it>studies in human breast. The aim of this study was to investigate the expression patterns of IFNγ and its two receptors (IFNγ-Rα and IFNγ-Rβ) by Western blot and immunohistochemistry, in order to elucidate its role in the different types of human breast cancer (<it>in situ </it>and infiltrative).</p> <p>Methods</p> <p>Immunohistochemical and semiquantitative study of IFNγ, its receptors types (IFNγ-Rα and IFNγ-Rβ), cell proliferation (proliferating cell nuclear antigen, also named PCNA), and apoptosis (TUNEL method) was carried between the three breast groups (fibrocystic lesions, <it>in situ</it> tumors and infiltrating tumors).</p> <p>Results</p> <p>In the three groups of patients, IFNγ and IFNγ-Rα immunoreactions appeared in the cytoplasm while IFNγ-Rβ also was found in the nucleus. The optical density to IFNγ was higher in <it>in situ </it>carcinoma than in benign and infiltrating tumors. When we observed IFNγ-Rα, the optical density was lower in infiltrating carcinoma than in benign and <it>in situ </it>tumors (the higher density). To IFNγ-Rβ, the optical density was similar in the three group samples. In tumor samples PCNA and TUNEL index was significantly higher; than in benign diseases. PCNA index increased with the malignance. No significant differences were found between cancer types to TUNEL. IFNγ could be a potential therapeutic tool in breast cancer. However, tumor cells are able to escape from the control of this cytokine in the early tumor stages; this is probably due to a decreased expression of IFNγ, or also to an alteration of either its receptors or some transduction elements.</p> <p>Conclusion</p> <p>We conclude that the decrease in the % positive samples that expressed IFNγ and IFNγ-Rα together with the nuclear localization of IFNγ-Rβ, could be a tumoral cell response, although perhaps insufficient to inhibit the uncontrolled cell proliferation. Perhaps, IFNγ might be unable to activate p21 to stop the cell cycle, suggesting a possible participation in breast cancer development.</p

Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice

A: Survival curve of normal-risk corneal grafts.

<p>Graft survival was significantly shortened by stromal injection of any of the cell types, compared with vehicle injection, and graft survival was similar among the different cell-treated groups (p = .008): the sham group (rabbits treated with vehicle), the hAD-MSCs (rabbits treated with stromal injection of human adipose-derived MSCs), the activated hAD-MSCs (rabbits treated with stromal injection of previously IFN-γ and TNF-α activated human adipose derived-MSCs) and the rAD-MSCs (rabbits treated with stromal injection of rabbit adipose derived-MSCs). B-E: Rejection index score (haze, haze, edema, and neovascularization) comparison between groups. Different colors show different rejection index scores in the surviving grafts at any time point.</p

AD-MSC characterization.

<p>A-D: Fluorescence-activated cell sorting of a sample of the stromal vascular fraction of human adipose tissue for markers CD45, CD34, CD90 and CD105 (open lines) with respect to autofluorescence (shadow lines). Percentages of each population are given within the graph for each marker. E: Phase contrast of cultured living AD-MSC cells. Observe the normal fusiform morphology. F: In vitro osteogenesis of the human AD-MSC, revealed by alkaline phosphatase staining with Fast Red. G: In vitro chondrogenesis of the human AD-MSC cultured using the micromass technique and revealed by Alcian blue. H: In vitro adipogenesis of the human AD-MSC, revealed by Oil red staining in red.</p

Representative photographs of transplanted corneas at the time of rejection.

<p>A) sham group; B) human AD-MSCs; C) activated human AD-MSCs; D) rabbit AD-MSCs after transplantation; E) unrejected cornea of one of the sham group rabbits; F) unrejected cornea of one of the MSC treated rabbits. Magnification 2x.</p

Representative photographs of histological sections of the transplanted corneas of the high-risk model of corneal transplant.

<p>A) sham group; B) rabbit AD-MSCs. Note the intense edema causing thickening of the corneal stroma (Str), and leukocyte infiltration in the stroma and blood vessels (arrows). Epi: corneal epithelium; end: corneal endothelium. Hematoxylin and eosin staining. C) Corneal thickness measurements in the transplanted corneas. Stars indicate statistical significance at the p≤0.05 level. D) CD45 leukocyte infiltration measurements. No statistical difference. E) CD45 immunohistochemistry in a transplanted cornea with sham treatment. F) CD45 immunohistochemistry in an AD-MSC-treated transplanted cornea. Positive cells are labeled in black (arrows).</p

Fluorescently CM-DiI labeled MSCs remain in the corneal stroma even after graft rejection.

<p>A) human AD-MSCs; B) activated human AD-MSCs; and C) rabbit AD-MSCs are localized in different layers in the corneal stroma around the site of injection.</p

AD-MSC immunophenotypical markers and in vitro immunosuppressive ability characterization.

<p>A), B), C), G:) Expression of costimulatory molecules in naive AD-MSCs (Filled: control isotype; line: sample). D), E), F), H): Expression of costimulatory molecules after stimulation with INF-γ and TNF-α (Filled: naive; line: upon stimulation). I): AD-MSCs effect on T cell proliferation (expressed in %) at ratios MSC:T cells 1:1, 1:10 and 1:100. NS: non stimulated cocultures; S-MSCs: stimulated AD-MSCs, S-T cells: stimulated T cells; DS: double stimulation (T cells and AD-MSCs). Asterisks: statistically significant p≤0,05.</p

Representative photographs of the corneas of the high-risk model of corneal transplantation.

<p>A) prevascularization of the corneas, showing profusion of blood vessels invading the cornea; B) sham group of intravenous injection of vehicle, showing recovering of prevascularization and transparent cornea; and C) rabbit AD-MSC intravenous injection showing opacity of the cornea and rejection. Hematoxylin and eosin staining.</p