Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.

Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome.

Various methods have been used in targeted gene knock-in applications. CRISPR-based knock-in strategies based on homology-independent repair pathways such as CRISPR HITI have been shown to possess the best efficiency for gene knock-in in mammalian cells. However, these methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. In this study, we introduce a new homology-independent knock-in strategy, Transposase-CRISPR mediated Targeted Integration (TransCRISTI), that exploits a fusion of Cas9 nuclease and a double mutant piggyBac transposase. In isogenic mammalian cell lines, we show that the TransCRISTI method demonstrates higher efficiency (72%) for site-specific insertions than the CRISPR HITI (44%) strategy. Application of the TransCRISTI method resulted in site-directed integration in 4.13% and 3.69% of the initially transfected population in the human AAVS1and PML loci, respectively, while the CRISPR HITI strategy resulted in site-directed integration in the PML locus in only 0.6% of cells. We also observed lower off-target and random insertions in the TransCRISTI group than the CRISPR HITI group. The TransCRISTI technology represents a great potential for the accurate and high-efficiency knock-in of the desired transposable elements into the predetermined genomic locations.

CRISPR/dCas9-mediated transposition with specificity and efficiency of site-directed genomic insertions.

The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboring-specific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them.

CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells.

BACKGROUND: With the increasing discovery of long noncoding RNAs (lncRNAs), the application of functional techniques that could have very specific, efficient, and robust effects and readouts is necessary. Here, we have applied and analyzed three gene knockout (KO) strategies to ablate the CCAT1 gene in different colorectal adenocarcinoma cell lines. We refer to these strategies as "CRISPR excision", "CRISPR HDR", and "CRISPR du-HITI". RESULTS: In order to obstruct the transcription of lncRNA or to alter its structure, in these strategies either a significant segment of the gene is removed, or a transcription termination signal is inserted in the target gene. We use RT-qPCR, RNA-seq, MTT, and colony formation assay to confirm the functional effects of CCAT1 gene ablation in knockout colorectal adenocarcinoma cell lines. We applied three different CRISPR/Cas9 mediated knockout strategies to abolish the transcription of CCAT1 lncRNA. CCAT1 knockout cells displayed dysregulation of genes involved in several biological processes, and a significant reduction for anchorage-independent growth. The du-HITI strategy introduced in this study removes a gene segment and inserts a reporter and a transcription termination signal in each of the two target alleles. The preparation of donor vector for this strategy is much easier than that in "CRISPR HDR", and the selection of cells in this strategy is also much more practical than that in "CRISPR excision". In addition, use of this technique in the first attempt of transfection, generates single cell knockouts for both alleles. CONCLUSIONS: The strategies applied and introduced in this study can be used for the generation of CCAT1 knockout cell lines and in principle can be applied to the deletion of other lncRNAs for the study of their function.

Neuregulin 1-alpha regulates phosphorylation, acetylation, and alternative splicing in lymphoblastoid cells.

Neuregulins (NRGs) are signaling molecules involved in various cellular and developmental processes. Abnormal expression and (or) genomic variations of some of these molecules, such as NRG1, have been associated with disease conditions such as cancer and schizophrenia. To gain a comprehensive molecular insight into possible pathways/networks regulated by NRG1-alpha, we performed a global expression profiling analysis on lymphoblastoid cell lines exposed to NRG1-alpha. Our data show that this signaling molecule mainly regulates coordinated expression of genes involved in three processes: phosphorylation, acetylation, and alternative splicing. These processes have fundamental roles in proper development and function of various tissues including the central nervous system (CNS)--a fact that may explain conditions associated with NRG1 dysregulations such as schizophrenia. The data also suggest NRG1-alpha regulates genes (FBXO41) and miRNAs (miR-33) involved in cholesterol metabolism. Moreover, RPN2, a gene already shown to be dysregulated in breast cancer cells, is also differentially regulated by NRG1-alpha treatment.

Human PTCHD3 nulls: rare copy number and sequence variants suggest a non-essential gene.

BACKGROUND: Copy number variations (CNVs) can contribute to variable degrees of fitness and/or disease predisposition. Recent studies show that at least 1% of any given genome is copy number variable when compared to the human reference sequence assembly. Homozygous deletions (or CNV nulls) that are found in the normal population are of particular interest because they may serve to define non-essential genes in human biology. RESULTS: In a genomic screen investigating CNV in Autism Spectrum Disorders (ASDs) we detected a heterozygous deletion on chromosome 10p12.1, spanning the Patched-domain containing 3 (PTCHD3) gene, at a frequency of ~1.4% (6/427). This finding seemed interesting, given recent discoveries on the role of another Patched-domain containing gene (PTCHD1) in ASD. Screening of another 177 ASD probands yielded two additional heterozygous deletions bringing the frequency to 1.3% (8/604). The deletion was found at a frequency of ~0.73% (27/3,695) in combined control population from North America and Northern Europe predominately of European ancestry. Screening of the human genome diversity panel (HGDP-CEPH) covering worldwide populations yielded deletions in 7/1,043 unrelated individuals and those detected were confined to individuals of European/Mediterranean/Middle Eastern ancestry. Breakpoint mapping yielded an identical 102,624 bp deletion in all cases and controls tested, suggesting a common ancestral event. Interestingly, this CNV occurs at a break of synteny between humans and mouse. Considering all data, however, no significant association of these rare PTCHD3 deletions with ASD was observed. Notwithstanding, our RNA expression studies detected PTCHD3 in several tissues, and a novel shorter isoform for PTCHD3 was characterized. Expression in transfected COS-7 cells showed PTCHD3 isoforms colocalize with calnexin in the endoplasmic reticulum. The presence of a patched (Ptc) domain suggested a role for PTCHD3 in various biological processes mediated through the Hedgehog (Hh) signaling pathway. However, further investigation yielded one individual harboring a homozygous deletion (PTCHD3 null) without ASD or any other overt abnormal phenotype. Exon sequencing of PTCHD3 in other individuals with deletions revealed compound point mutations also resulting in a null state. CONCLUSION: Our data suggests that PTCHD3 may be a non-essential gene in some humans and characterization of this novel CNV at 10p12.1 will facilitate population and disease studies.

Gene and miRNA expression profiles in autism spectrum disorders.

Accumulating data indicate that there is significant genetic heterogeneity underlying the etiology in individuals diagnosed with autism spectrum disorder (ASD). Some rare and highly-penetrant gene variants and copy number variation (CNV) regions including NLGN3, NLGN4, NRXN1, SHANK2, SHANK3, PTCHD1, 1q21.1, maternally-inherited duplication of 15q11-q13, 16p11.2, amongst others, have been identified to be involved in ASD. Genome-wide association studies have identified other apparently low risk loci and in some other cases, ASD arises as a co-morbid phenotype with other medical genetic conditions (e.g. fragile X). The progress studying the genetics of ASD has largely been accomplished using genomic analyses of germline-derived DNA. Here, we used gene and miRNA expression profiling using cell-line derived total RNA to evaluate possible transcripts and networks of molecules involved in ASD. Our analysis identified several novel dysregulated genes and miRNAs in ASD compared with controls, including HEY1, SOX9, miR-486 and miR-181b. All of these are involved in nervous system development and function and some others, for example, are involved in NOTCH signaling networks (e.g. HEY1). Further, we found significant enrichment in molecules associated with neurological disorders such as Rett syndrome and those associated with nervous system development and function including long-term potentiation. Our data will provide a valuable resource for discovery purposes and for comparison to other gene expression-based, genome-wide DNA studies and other functional data.

Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology. RESULTS: Using RNA interference (RNAi) to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (Pvalb and Casq1), or cardiomyopathy (Obscurin, Tcap). In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels in vivo and in vitro. CONCLUSION: Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage. Therefore, it becomes apparent that any gene therapy approaches for DMD should target early stages in muscle development to attain a maximum clinical benefit. With a clear and specific definition of the transcriptome of dystrophin deficiency, manipulation of identified dysregulated molecules downstream of dystrophin may lead to novel ameliorative approaches for DMD.

RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice, the animal model of DMD. Accordingly, we hypothesized that the pathology observed with dystrophin deficiency may be developmentally regulated. Initially, we demonstrated that profound small interfering RNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus vectors to express short-hairpin RNAs targeting dystrophin in skeletal muscle in vivo yielded a potent and specific dystrophin knockdown, but only after approximately 5 months, indicating the very long half-life of dystrophin. Interestingly, and in contrast to what is observed in congenital dystrophin deficiency, long-term ( approximately 1 year) dystrophin knockdown in adult mice did not result, per se, in overt dystrophic pathology or upregulation of utrophin. This supports our hypothesis and suggests new pathophysiology of the disease. Furthermore, taking into account the rather long half-life of dystrophin, and the notion that the development of pathology is age-dependent, it indicates that a single gene therapy approach before the onset of pathology might convey a long-term cure for DMD.