Posts Tagged ‘Microbiology’

CRISPR/Cas9 and HIV1

Posted in Clinical & Translational, Clinical Genomics, CRISPR/Cas9 & Gene Editing, Infectious Disease & New Antibiotic Targets, Viral diseases, tagged CRISPR/Cas9, gene activation, gene editing, gene excision, HIV infections, HIV-1, latency, Microbiology, RNA therapy on April 16, 2016| Leave a Comment »

CRISPR/Cas9 and HIV1

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus

Hirotaka Ebina, Naoko Misawa, Yuka Kanemura & Yoshio Koyanagi

Scientific Reports 2013; 2510(3) http://dx.doi.org:/10.1038/srep02510

Even though highly active anti-retroviral therapy is able to keep HIV-1 replication under control, the virus can lie in a dormant state within the host genome, known as a latent reservoir, and poses a threat to re-emerge at any time. However, novel technologies aimed at disrupting HIV-1 provirus may be capable of eradicating viral genomes from infected individuals. In this study, we showed the potential of the CRISPR/Cas9 system to edit the HIV-1 genome and block its expression. When LTR-targeting CRISPR/Cas9 components were transfected into HIV-1 LTR expression-dormant and -inducible T cells, a significant loss of LTR-driven expression was observed after stimulation. Sequence analysis confirmed that this CRISPR/Cas9 system efficiently cleaved and mutated LTR target sites. More importantly, this system was also able to remove internal viral genes from the host cell chromosome. Our results suggest that the CRISPR/Cas9 system may be a useful tool for curing HIV-1 infection.

Integration of reverse transcribed viral DNA into the host cell genome is an essential step during the HIV-1 life cycle¹. The integrated retroviral DNA is termed a provirus, which serves as the fundamental source of viral protein production. HIV-1 gene expression is regulated by LTR promoter and enhancer activities, where cellular transcription factors such as NF-κB, SP-1 and TBP bind to promote RNA polymerase II processivity. Subsequently, Tat protein is expressed from early double-spliced transcripts and binds to the trans activation response (TAR) region of HIV-1 RNA for its efficient elongation².

Latent infection occurs when the HIV-1 provirus becomes transcriptionally inactive, resulting in a latent reservoir that has become the main obstacle in preventing viral eradication from HIV-1 infected individuals. However, the mechanisms of viral silencing and reactivation remain incompletely understood³. Previous studies have suggested that the position of the integration site strongly influences viral gene expression and may be one of the determinants of HIV-1 latency⁴. While highly active anti-retroviral therapy (HARRT) has dramatically decreased mortality from HIV-1 infection, there is currently no effective strategy to target the latent form of HIV-1 proviruses⁵.

Over the last decade, novel genome-editing methods that utilize artificial nucleases such as zinc finger nucleases (ZFNs)⁶ and transcription activator like-effector nucleases (TALENs)⁷ have been developed. These molecularly engineered nucleases recognize and cleave specific nucleotide sequences in target genomes for digestion, resulting in various mutations such as substitutions, deletions and insertions induced by host DNA repair machinery. These technologies have enabled the production of genome-manipulated animals in a wide range of species such as Drosophila⁸, Zebrafish⁹ and Rat¹⁰. However, ZFNs or TALENs remain somewhat difficult and time-consuming to design, develop, and empirically test in a cellular context¹¹. Recently, a third genome-editing method was developed based on clustered regularly interspaced short palindromic repeat (CRISPR) systems. CRISPR systems were originally identified in bacteria and archaea¹² as part of an adaptive immune system, dependent on a complex consisting of CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) proteins to degrade complimentary sequences of invading viral and plasmid DNA. Mali et al. created a novel version of the genome-editing tool applicable to mammalian cells, termed the CRISPR/Cas9 system, which is based on modifications of the Streptococcus pyogenes type II CRISPR system in crRNA fused to trans-encoded tracrRNA¹³. This CRISPR/Cas9 system is composed of guide RNA (gRNA) and a human codon-optimized Cas9 nuclease that forms an RNA-protein complex to digest unique target sequences matching those of gRNA. The CRISPR/Cas9 system can be utilized by simple transfection of designed gRNA and a humanized Cas9 (hCas9) expression plasmid into target mammalian cells, making it a promising tool for various applications.

In this study, we tested the ability of the CRISPR/Cas9 system to suppress HIV-1 expression by editing HIV-1 integrated proviral DNA. Cas9 and gRNA, designed to target HIV-1 LTR, were transfected and significantly inhibited LTR-driven expression under the control of Tat. This LTR-targeted CRISPR/Cas9 system can also excise provirus from the cellular genome.

http://www.nature.com/article-assets/npg/srep/2013/130826/srep02510/images_hires/w926/srep02510-f2.jpg

CRISPR/Cas9 system can target the latent form of HIV-1 provirus in Jurkat cell

Because the putative latently infected cells are CD4⁺ T cells, we next tested the genome editing potential of the CRISPR/Cas9 system in these cells.

…..

In this study, we successfully disrupted the expression of HIV-1 provirus utilizing the CRISPR/Cas9 system (Fig. 1). Importantly, this disruption not only restricted transcriptionally active provirus, it also blocked the expression of latently integrated provirus (Fig. 3). Cas9 proteins are predicted to contain RuvC and HNH motifs¹⁵, which possess autonomous ssDNA cleavage activity. Interestingly, mutants lacking one of the motifs become nicking endonucleases¹⁶. It is plausible that the independent nicking activity of each domain may enhance efficient access to the heterochromatin state of latently integrated provirus. Another possibility is that Cas9 has a highly efficient target surveillance system similar to what has been previously reported for the Cas3 system¹⁷.

T6 gRNA that targeted the NF-κB binding site, also strongly suppressed the LTR promoter activity (Fig. 1). However, the effect was weaker than that of T5 gRNA. In this study we used an LTIG vector modified from the LTR of HIV-1 strain NL4-3 that possesses two adjacent NF-κB binding sites¹⁸. The T6 target site is at the end of the 5′ NF-κB binding site, meaning that mutations may not completely render transcription inactive since the 3′ NF-κB binding site may remain functional. On the other hand, T5 gRNA that targeted TAR, is profoundly effective in disrupting HIV-1 gene expression. The putative cleavage site was positioned at the neck of the stem loop region of TAR, which is critical for Cyclin T1-Tat-TAR ternary complex formation¹⁹. Therefore, the TAR sequence may be one of the best targets for blocking HIV-1 provirus expression. Target specificity of the CRISPR/Cas system is very high and a single mutation can disrupt targeting²⁰, meaning that some provirus may escape from this genome-editing machinery if mutations arise in target sequences. However, given that the TAR region is relatively conserved and there is little variation among HIV-1 subtypes²¹, it could still be an appropriate target for the elimination of latently infected provirus.

Perhaps the most important finding in this study is that we could excise provirus from the host genome of HIV-1 infected cells, which may provide a ray of hope to eradicate HIV-1 from infected individuals. However, there are numerous hurdles that must be cleared before utilizing genome editing for HIV-1 eradication therapies such as gene therapy. First, the efficiency of genome-editing and/or proviral excision should be quantified in HIV infected primary cells, including latently infected CD4⁺ quiescent T cells. Second, an efficient delivery system must be developed. Fortunately, the CRISPR/Cas9 system has the advantage in size compared with TALENs²². Thus, the CRISPR system has the potential to be delivered by lentivirus vectors, whereas TALENs do not because of their large size and repeat sequences²³. The final hurdle concerns possible off-target effects, which are pertinent concerns for all genome-editing strategies that may lead to nonspecific gene modification events. If Cas9 has off-target effects, then removal of the off-target activity may be the best approach before utilizing CRISPR/Cas system for anti-HIV treatment.

Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

Rafal Kaminski, Yilan Chen, Tracy Fischer, Ellen Tedaldi, Alessandro Napoli,Yonggang Zhang, Jonathan Karn, Wenhui Hu & Kamel Khalili

Scientific Reports 6, Article number: 22555 (2016) http://dx.doi.org:/10.1038/srep22555

We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.

AIDS remains a major public health problem, as over 35 million people worldwide are HIV-1-infected and new infections continue at steady rate of greater than two million per year. Antiretroviral therapy (ART) effectively controls viremia in virtually all HIV-1 patients and partially restores the primary host cell (CD4+ T-cells), but fails to eliminate HIV-1 from latently-infected T-cells^1,2. In latently-infected CD4+ T cells, integrated proviral DNA copies persist in a dormant state, but can be reactivated to produce replication-competent virus when T-cells are activated, resulting in rapid viral rebound upon interruption of antiretroviral treatment^3,4,5,6,7,8. Therefore, most HIV-1-infected individuals, even those who respond very well to ART, must maintain life-long ART due to persistence of HIV-1-infected reservoir cells. During latency HIV infected cells produce little or no viral protein, thereby avoiding viral cytopathic effects and evading clearance by the host immune system. Because the resting CD4+ memory T-cell compartment⁹is thought to be the most prominent latently-infected cell pool, it is a key focus of research aimed at eradicating latent HIV-1 infection.

Recent efforts to eradicate HIV-1 from this cell population have used primarily a “shock and kill” approach, with the rationale that inducing HIV reactivation in CD4+ memory T-cells may trigger elimination of virus-producing cells by cytolysis or host immune responses. For example, epigenetic modification of chromatin structure is critical for establishing viral reactivation. Consequently, inhibition of histone deacetylase (HDAC) by Trichostatin A (TSA) and vorinostat (SAHA) led to reactivation of latent virus in cell lines^10,11,12. Accordingly, other HDACi, including vorinostat, valproic acid, panobinostat and rombidepsin have been tested ex vivo and have led, in the best cases, to transient increases in viremia^13,14. Similarly, protein kinase C agonists, can potently reactivate HIV either singly or in combination with HDACi^15,16. However, there are multiple limitations of this approach: (i) since a large fraction of HIV genomes in this reservoir are non-functional, not all integrated provirus can produce replication-competent virus¹⁷; (ii) total numbers of CD4+ T cells reactivated from resting CD4+ T cell HIV-1 reservoirs, has been found by viral outgrowth assays to be much smaller than the numbers of cells infected, as detected by PCR-based assays, suggesting that not all cells within this reservoir are reactivated¹⁸; (iii) the cytotoxic T lymphocyte (CTL) immune response is not sufficiently robust to eliminate the reactivated infected cells¹⁹ and (iv) uninfected T-cells are not protected from HIV infection and can therefore sustain viral rebound.

These observations suggest that a cure strategy for HIV-1 infection should include methods that directly eliminate the proviral genome from the majority of HIV-1-positive cells, including CD4+ T-cells, and protect cells from future infection, with little or no harm to the host. The clustered, regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) nuclease has wide utility for genome editing in a broad range of organisms including yeast, Drosophila, zebrafish, C. elegans, and mice, and has been applied in a broad range of in vivo and in vitro studies toward human diseases^{20,21,22,23,24}. Recently we modified the CRISPR/Cas9 system to enable recognition of specific DNA sequences positioned within the HIV-1 promoter spanning the 5′ long terminal sequence (LTR)^25,26. Using this modified system, we now demonstrate excision of integrated copies of the proviral DNA fragment from a latently HIV-1-infected human T-lymphoid cell line, completely eliminating HDAC inhibition-elicited viral production. Results of whole-genome sequencing and comprehensive bioinformatic analysis ruled out any genotoxicity to host cell DNA. Further, we found that lentivirally-delivered CRISPR/Cas9 reduces viral replication upon HIV-1 infection of primary cultured CD4+ T-cells. The results point toward this approach as a promising potential therapeutic avenue to eradicating HIV-1 from T reservoir cells of host patients, to prevent AIDS re-emergence.

Despite its remarkable therapeutic success and efficacy, ART treatment is unable to eradicate HIV-1 from infected patients who must therefore undergo life-long treatment. A new therapeutic strategy is thus needed in order to achieve permanent remission allowing patients to stop ART and reduce it’s attendant costs and potential long-term side effects. Our findings address key barriers to this goal, as we developed CRISPR/Cas9 techniques that eradicated integrated copies of HIV-1 from human CD4+ T-cells, inhibited HIV-1 infection in primary cultured human CD4+ T-cells, and suppressed viral replication ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells of HIV-1+ patients. They also address a further key issue, providing evidence that such gene editing effectively impedes viral replication without causing genotoxicity to host DNA or eliciting destructive effects via host cell pathways. Prior studies using gene editing based on zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems prompted much interest in their potential abilities to suppress viral infection, either by altering virus receptors or introducing mutations in the viral genome (for review see^26,30). All these studies suggest that gene editing strategies can be engineered for targeting specific regions of the viral genome and once efficiently delivered to infected cells, their robust antiviral activity effectively suppresses viral replication. However, there are several important issues that require close attention including the careful design of the targeting strategy that achieves the highest levels of specificity and safety with optimum efficiency of editing.

In this study, due to the complexity associated with determination of the sites and numbers of randomly integrated proviral HIV-1 DNA in in vitro infected primary cell culture and the difficulty in full scale characterization of the InDel/Excision by Cas9/gRNAs in these cells, as a first step, we chose to use the clonal 2D10 cell line as a human T-cell latency model to establish: (i) the ability of Cas9/gRNA in removing the entire coding sequence of the integrated copies of the HIV-1 DNA using ultradeep whole genome sequencing and (ii) investigate its safely related to off-target effects and cell viability. Once these goals were accomplished, we shifted our attention to primary cell culture as well as patient samples to examine the efficiency of the CRISPR/Cas9 in affecting viral DNA load in a laboratory setting.

We found that CRISPR/Cas9 edited multiple copies of viral DNA scattered among the chromosomes. Combined treatment of latently-infected T cells with Cas9 plus gRNAs A and B that recognize specific DNA motifs within the LTR U3 region efficiently eliminated the entire viral DNA fragment spanning between the two LTRs. The remaining 5′ LTR and 3′ LTR cleavage sites by Cas9 and gRNA B in chromosome 1, and by Cas9 and gRNAs A and B in chromosome 16, were joined by host DNA repair at sites located precisely three nucleotides upstream of the PAM. Genome-wide assessment of CRISPR/Cas9-treated HIV-1-infected 2D10 cells clearly verified complete excision of the integrated copies of viral DNA from the second intron of RSBN1 and exon 2 of MSRB1 genes. To address the critical issue related to its specificity and potential off-target and adverse effects, we analyzed this comprehensively and at an unprecedented level of detail, by whole-genome sequencing and bioinformatic analyses. These revealed many naturally-occurring mutations in the genomes of control cells and gRNAs A- and B-mediated HIV-1 DNA eradication. The mutations discovered included naturally-occurring InDels, base excisions, and base substitutions, all of which are, more or less, expected in rapidly growing cells in culture, including Jurkat 2D10 cells. The critical issue is our discovery that none of these mutations resulted from our gene-editing system, as we identified no sequence identities with either gRNA A or B within 1200 nucleotides of any such mutation site. Further, our method of HIV-1 DNA excision had no adverse effects on proximal or distal cellular genes and showed no impact on cell viability, cell cycle progression or proliferation, and did not induce apoptosis, thus strongly supporting its safety at this translational phase, by all in vitromeasures assessed in cultured cells. We found that the expression levels of Cas9 and the gRNAs diminished after several passages and eventually disappeared, but as long as Cas9 and single or multiplex gRNAs were present, cells remained protected against new HIV-1infection.

Another key translational feasibility question we addressed is whether CRISPR/Cas9-mediated HIV-1 eradication can prevent or suppress HIV-1 infection in the most relevant human and patient target cell populations. We provide a critical new advance, by observing in PBMCs and CD4+ T-cells from HIV-1 infected patients that lentivirally-delivered Cas9/gRNAs A/B significantly decreased viral copy numbers and protein levels. Using primer sets directed within the LTR, we amplified and detected residual viral DNA fragments that were not completely deleted in these cells, yet were affected by Cas9/gRNAs and contained InDel mutants near the PAM sequence. These findings verified that CRISPR/Cas9 exerted efficacious antiviral activity in the PBMCs of HIV-1 patients. We also found that introducing Cas9/gRNAs A/B via lentiviral delivery into primary cultured human CD4+ HIV-1_JRFL– or HIV-1_NL4-3-infected T-cells significantly reduced viral copy numbers, corroborating earlier findings by us and others that stably-integrated HIV-1-directed Cas9 and gRNAs (distinct from our gRNAs A and B used presently) conferred resistance to HIV-1 infection in cell lines^31,32. With the notion that CRISPR/Cas9 can target both integrated, as well as episomal DNA sequences, as evidenced by its editing ability of various human viruses as well as plasmid DNAs in either configuration^{31,32,33,34,35,36}, it is likely that both the integrated as well as pre-integrated, free-floating intracellular HIV-1 DNA are edited by Cas9/gRNA.

As noted, during the course of our studies no ART was included prior to the treatment with CRISPR/Cas9 as our goal in this study was to determine the extent of viral suppression during the productive stage of viral infection. We observed a significant level of suppression suggesting that CRISPR/Cas9 may effectively disable expression of the functionally active integrated copies of HIV-1 DNA in the host chromosome. This notion is supported by our observations using 2D10 CD4+ T-cells where the latent copies of HIV-1 that are integrated in chromosomes 1 and 16 were effectively eliminated by CRISPR/Cas9. Our future studies are aimed to address the impact of CRISPR/Cas9 in in vitro infected CD4+ T-cells where the virus is controlled by ART and a cohort of naïve and ART-treated patient CD4+ T-cells. Results from these studies should determine whether or not, in the context of ART, the virus enters into the latent stage and remains responsive to CRISPR/Cas9. Of note, results from these ex vivo studies using ART treated patient PBMCs and CD4+ T-cells show that CRISPR/Cas9 effectively suppresses viral replication by introducing InDel mutations.

Our findings show comprehensively and conclusively that the entire coding sequence of host-integrated HIV-1 was eradicated in human 2D10 T cells, providing a strong first step of support for potential translatability of such a system to T-cell-directed HIV-1 therapies in patients. The complete absence of genomic and off-target functional effects in all assays also provides critical support for the promise of developing this approach for future therapeutic applications.

When evaluating a therapeutic strategy based on CRISPR/Cas9, it is critical to understand that not only will HIV-1 be eliminated from latently infected cells, but the majority of uninfected cells will become resistant to HIV infection. Thus, there is a high likelihood that rebounding viral infections will be contained by the resistant cells. Still, some formidable challenges remain before this type of strategy can be implemented. First, it will be important to maximize elimination of viral sequences from patients. This will require analysis of the HIV-1 quasi-species harbored by patients’ CD4+ T-cells and design of suitable, i.e. personalized CRISPRs. Second, improved delivery of CRISPR/Cas9 will be required to target the majority of circulating T-cells. In summary, our novel ex vivo findings that our lentiviral delivery-based approach reduced HIV-1 DNA copy numbers and protein levels in PBMCs of HIV-1 infected patients provides strong proof-of-concept evidence that CRISPR/Cas9 can be effectively utilized as part of HIV Cure strategies.

The therapeutic application of CRISPR/Cas9 technologies for HIV Preview Full text HTML PDF

Sheena Saayman^ab, Stuart A Ali^b, Kevin V Morris^ac& Marc S Weinberg^*abd

Expert Opinion on Biological Therapy 2015; 15(6): 819-830 http://dx.doi.org:/10.1517/14712598.2015.1036736

Introduction: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system.

Areas covered: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus.

Expert opinion: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.

Genetic therapy for HIV/AIDS

Ananthalakshmi Poluri, Marc van Maanen & Richard E Sutton

Expert Opinion on Biological Therapy Sept 2003; 3(6):951-963

Towards a durable RNAi gene therapy for HIV-AIDS

Ben Berkhout & Olivier ter Brake

Expert Opinion on Biological Therapy Feb 2009; 9(2): 161-170

Read Full Post »

Microbe meets cancer

Posted in Bacterial Resistance, Biochemical pathways, Biological Networks, Gene Regulation and Evolution, Cancer - General, Cancer Informatics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Genomics, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gastroenterology, Gene Regulation, Genome Biology, Inflammasome, Liver & Digestive Diseases Research, Metabolism, Methylation, Microbiologial genetics, Microbiology, Molecular Genetics & Pharmaceutical, Oxidative phosphorylation, Warburg effect, tagged Cancer immunology, Cancer immunotherapy, gut microbiome, gut microbiota, Immunotherapy, Microbiology, microbiome on April 10, 2016| Leave a Comment »

Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Microbes Meet Cancer

Understanding cancer’s relationship with the human microbiome could transform immune-modulating therapies.

By Kate Yandell | April 1, 2016 http://www.the-scientist.com/?articles.view/articleNo/45616/title/Microbes-Meet-Cancer

In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types and improved survival to a far greater extent in mice with intact microbiomes.¹ The French group, led by INSERM’s Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to develop tumors of the lung.²

The findings incited a flurry of research and speculation about how gut microbes contribute to cancer cell death, even in tumors far from the gastrointestinal tract. The most logical link between the microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or tamp it down, and can modulate immune cells’ vigilance for invaders. Not only does the immune system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.

“We clearly see shifts in the [microbial] community that precede development of tumors,” says microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote cell proliferation, others appear to protect us against cancerous growth. And in some cases, the conditions that spur one cancer may have the opposite effect in another. “It’s become pretty obvious that the commensal microbiota affect inflammation and, through that or through other mechanisms, affect carcinogenesis,” says Trinchieri. “What we really need is to have a much better understanding of which species, which type of bug, is doing what and try to change the balance.”

Gut feeling

In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic gastritis, an inflammation of the stomach lining that often precedes the development of stomach cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the bacterium, now called Helicobacter pylori, was somehow causing the gastritis.³ So committed was Marshall to demonstrating the microbe’s causal relationship to the inflammatory condition that he had his own stomach biopsied to show that it contained no H. pylori, then infected himself with the bacterium and documented his subsequent experience of gastritis.⁴ Scientists now accept that H. pylori, a common gut microbe that is present in about 50 percent of the world’s population, is responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for stomach cancer.⁵ Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their work.

H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development, but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal evidence that shifts in the microbiome influence the development of diverse tumor types. “You have a mouse model of carcinogenesis. It works beautifully,” says Trinchieri. “You move to another institution. It works completely differently,” likely because the animals’ microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF© AL GRANBERG

Around the turn of the 21st century, cancer researchers began to systematically experiment with the rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouse’s risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice rapidly developed colon tumors, but that germ-free versions of these mice did not.⁶ That same year, an MIT-based group led by the late David Schauer demonstrated that infecting mice with the bacterium Citrobacter rodentium spurred colon tumor development.⁷ And in 2003, MIT’s Susan Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the murine gut microbiome.⁸

More recent work has documented a similar link between colon cancer and the gut microbiome in humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90 people, some with colon cancer, some with precancerous adenomas, and still others with no disease.⁹ The researchers found that the feces of people with cancer tended to have an altered composition of bacteria, with an excess of the common mouth microbes Fusobacterium or Porphyromonas. A few months later, Peer Bork of the European Molecular Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of cancer using the relative abundance of 22 bacterial species, including Porphyromonas andFusobacterium.¹⁰They could also use the method to predict colorectal cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers while yielding false positives less than 10 percent of the time. When the two tests were combined, they caught more than 70 percent of cancers.

Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a consequence of tumor development remained unclear. “What comes first, the change in the microbiome or tumor development?” asks Schloss. To investigate this question, he and his colleagues treated mice with microbiome-altering antibiotics before administering a carcinogen and an inflammatory agent, then compared the outcomes in those animals and in mice that had received only the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome, suggesting that resident bacteria were in some way promoting cancer development. And when the researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota from mice already bearing malignancies developed the most tumors of all.¹¹

Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor formation. The researchers determined that the number of tumors corresponded to the unique ways that each antibiotic cocktail modulated the microbiome.¹²

“We’ve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer,” says Schloss, who hypothesizes that gut bacteria–driven inflammation is to blame for creating an environment that is hospitable to tumor development and growth. Gain or loss of certain components of the resident bacterial community could lead to the release of reactive oxygen species, damaging cells and their genetic material. Inflammation also involves increased release of growth factors and blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)

Recent research has also yielded evidence that the gut microbiota impact the development of cancer in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.

Systemic effects

In the mid-2000s, MIT’s Erdman began infecting a strain of mice predisposed to intestinal tumors withH. hepaticus and observing the subsequent development of colon cancer in some of the animals. To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to develop mammary tumors. “This told us that something really interesting was going on,” Erdman recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more likely to develop mammary tumors than mice not exposed to the bacterium.¹³The researchers showed that systemic immune activation and inflammation could contribute to mammary tumors in other, less cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer development and progression, thanks to their interactions with the immune system. Some microbes, such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while others, such as some Bacteroides species, help protect against tumors by boosting T-cell infiltration.© EYE OF SCIENCE/SCIENCE SOURCE
http://www.the-scientist.com/images/April2016/immune_2.jpg

At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice coming from different vendors—Taconic Biosciences (formerly Taconic Farms) and the Jackson Laboratory—Gajewski takes advantage of the fact that the animals’ different origins result in different gut microbiomes. “We deliberately stayed away from antibiotics, because we had a desire to model how intersubject heterogeneity [in cancer development] might be impacted by the commensals they happen to be colonized with,” says Gajewski in an email to The Scientist.

Last year, the researchers published the results of a study comparing the progression of melanoma tumors implanted under the mice’s skin, finding that tumors in the Taconic mice grew more aggressively than those in the Jackson mice. When the researchers housed the different types of mice together before their tumors were implanted, however, these differences disappeared. And transferring fecal material from the Jackson mice into the Taconic mice altered the latter’s tumor progression.¹⁴

Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mice’s tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria improved their response to the implanted melanoma cells.

“One hypothesis going into the experiments was that we might identify immune-suppressive bacteria, or commensals that shift the immune response towards a character that was unfavorable for tumor control,” says Gajewski. “But in fact, we found that even a single type of bacteria could boost the antitumor immune response.”

http://www.the-scientist.com/images/April2016/immune4.jpg

Drug interactions

Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But cancer cells display “self” molecules that can inhibit immune attack. A new type of immunotherapy, dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking either the tumor cells’ surface molecules or the receptors on T?cells that bind to them.

CANCER THERAPY AND THE MICROBIOME

In addition to influencing the development and progression of cancer by regulating inflammation and other immune pathways, resident gut bacteria appear to influence the effectiveness of many cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide immunotherapy, work by boosting inflammation. If the microbiome is altered in such a way that inflammation is reduced, these therapeutic agents are less effective.
Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer cells are influenced by members of the microbiota that mediate these same cell interactions.
Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1 receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells. While treating Taconic mice with PD-L1–targeting antibodies did improve their tumor responses, they did even better when that treatment was combined with fecal transfers from Jackson mice, indicating that the microbiome and the immunotherapy can work together to take down cancer. And when the researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mice’s tumors virtually disappeared.¹⁴

Gajewski’s group is now surveying the gut microbiota in humans undergoing therapy with checkpoint inhibitors to better understand which bacterial species are linked to positive outcomes. The researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.

Meanwhile, Zitvogel’s group at INSERM is investigating interactions between the microbiome and another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and germ-free mice had poorer responses to a CTLA-4–targeting antibody compared with mice harboring unaltered microbiomes.¹⁵ Particular Bacteroides species were associated with T-cell infiltration of tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals’ responses to the immunotherapy. As an added bonus, treatment with these “immunogenic” Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice transplanted with feces from patients with normal levels of B. fragilis.

“There are bugs that allow the therapy to work, and at the same time, they protect against colitis,” says Trinchieri. “That is very exciting, because not only [can] we do something to improve the therapy, but we can also, at the same time, try to reduce the side effect.”

And these checkpoint inhibitors aren’t the only cancer therapies whose effects are modulated by the microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered microbiomes.¹He and his NCI colleague Goldszmid further found that the platinum chemotherapy oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogel’s group has shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its proper function.

Although the mechanisms by which the microbiome influences the effectiveness of such therapies remains incompletely understood, researchers once again speculate that the immune system is the key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?helper 1 cells and a subtype of T?helper 17 cells referred to as “pathogenic,” both of which destroy tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?helper 17 cells, which translocate to the tumor. When the researchers transferred the “pathogenic” T?helper 17 cells into antibiotic-treated mice, the mice’s response to chemotherapy was partly restored.

Microbiome modification

As the link between the microbiome and cancer becomes clearer, researchers are thinking about how they can manipulate a patient’s resident microbial communities to improve their prognosis and treatment outcomes. “Once you figure out exactly what is happening at the molecular level, if there is something promising there, I would be shocked if people don’t then go in and try to modulate the microbiome, either by using pharmaceuticals or using probiotics,” says Michael Burns, a postdoc in the lab of University of Minnesota genomicist Ran Blekhman.

Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however, molding the microbiome is not simple. “It’s a messy, complicated system that we don’t understand,” says Schloss.

So far, studies of the gut microbiome and colon cancer have turned up few consistent differences between cancer patients and healthy controls. And the few bacterial groups that have repeatedly shown up are not present in every cancer patient. “We should move away from saying, ‘This is a causal species of bacteria,’” says Blekhman. “It’s more the function of a community instead of just a single bacterium.”

But the study of the microbiome in cancer is young. If simply adding one type of microbe into a person’s gut is not enough, researchers may learn how to dose people with patient-specific combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by promoting an anti-inflammatory environment in the gut.¹⁶

“If it is true that, in humans, we can alter the course of the disease by modulating the composition of the microbiota,” says José Conejo-Garcia of the Wistar Institute in Philadelphia, “that’s going to be very impactful.”

Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she became an associate editor at Cancer Today.

GENETIC CONNECTION

The microbiome doesn’t act in isolation; a patient’s genetic background can also greatly influence response to therapy. Last year, for example, the Wistar Institute’s José Garcia-Conejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5 (TLR5) influences clinical outcomes in cancer patients by changing how the patients’ immune cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).

More than 7 percent of people carry a specific mutation in TLR5 that prevents them from mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their colleagues found that estrogen receptor–positive breast cancer patients who carry the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without the mutation. Among patients with ovarian cancer, on the other hand, those with the TLR5 mutation were more likely to live at least six years after diagnosis than patients who don’t carry the mutation.

Investigating the mutation’s contradictory effects, the researchers found that mice with normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17). But when the researchers knocked out the animals’ microbiomes, these differences in cytokine production disappeared, as did the differences in cancer progression between mutant and wild-type animals.

“The effectiveness of depleting specific populations or modulating the composition of the microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,” says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer prognosis may act via microbiome–immune system interactions. “I think that our paper is just the tip of the iceberg.”

References

N. Iida et al., “Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment,” Science, 342:967-70, 2013.
S. Viaud et al., “The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide,” Science, 342:971-76, 2013.
J.R. Warren, B. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,”Lancet, 321:1273-75, 1983.
B.J. Marshall et al., “Attempt to fulfil Koch’s postulates for pyloric Campylobacter,” Med J Aust, 142:436-39, 1985.
J. Parsonnet et al., “Helicobacter pylori infection and the risk of gastric carcinoma,” N Engl J Med, 325:1127-31, 1991.
S. Kado et al., “Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor β chain and p53 double-knockout mice,” Cancer Res, 61:2395-98, 2001.
J.V. Newman et al., “Bacterial infection promotes colon tumorigenesis in ApcMin/+ mice,” J Infect Dis, 184:227-30, 2001.
S.E. Erdman et al., “CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice,” Am J Pathol, 162:691-702, 2003.
J.P. Zackular et al., “The human gut microbiome as a screening tool for colorectal cancer,” Cancer Prev Res, 7:1112-21, 2014.
G. Zeller et al., “Potential of fecal microbiota for early-stage detection of colorectal cancer,” Mol Syst Biol, 10:766, 2014.
J.P. Zackular et al., “The gut microbiome modulates colon tumorigenesis,” mBio, 4:e00692-13, 2013.
J.P. Zackular et al., “Manipulation of the gut microbiota reveals role in colon tumorigenesis,”mSphere, doi:10.1128/mSphere.00001-15, 2015.
V.P. Rao et al., “Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice,” Cancer Res, 66:7395, 2006.
A. Sivan et al., “Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy,” Science, 350:1084-89, 2015.
M. Vétizou et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,”Science, 350:1079-84, 2015.

……..

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells

Koji Atarashi · Takeshi Tanoue · Minoru Ando ·Nobuhiko Kamada ·….. · Yoshinori Umesaki · Kenya Honda ·

ABSTRACT: Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics….. · Sep 2015 · Cell 163(2) https://www.researchgate.net/profile/Shoichi_Kado http://dx.doi.org:/10.1016/j.cell.2015.08.058

MIT Faculty Newsletter Vol. XXII No. 2 Nov / Dec 2009 Memorial Resolution for David B. Schauer

We regretfully acknowledge the sudden death of our friend and colleague, Dr. David Schauer, on June 7, 2009 – one day after his 48th birthday. David Schauer was known not only for his keen scientific mind, but also as a friend whose empathy and compassion touched countless individuals.

David’s research was supported on a continuous basis by the National Institutes of Health throughout his career at MIT. His studies on microbial pathogenesis of gastrointestinal pathogenic bacteria, particularly Citrobacter rodentium, a murine model of enteropathogenic Escherichia coli, and enterohepatic helicobacter are widely known and respected.

David’s research provided important insights into the molecular mechanisms evoked by enteric pathogens and how the infections caused by these bacteria perturb the gastrointestinal barrier, elicit inflammation, and produce clinically relevant disease.

Susan Erdman is a Principal Research Scientist and Assistant Director in the Division of Comparative Medicine at MIT.

Invited Speaker. National Cancer Institute (NCI) Mucosal, Immunosurveillance, Inflammation and Cancer Workshop. April, 2005.
Invited reviewer. National Cancer Institute (NCI) RFA-CA-06-014 Tumor Microenvironment Network. August, 2006.
Invited speaker. Keystone Symposium on Mechanisms Linking Inflammation and Cancer, February, 2007
Alumni Fellow, College of Veterinary Medicine, Mississippi State University, 2007
Invited Speaker. National Cancer Institute (NCI) Changing Concepts in Cancer Etiology: The Role of the Human Microbiome. November, 2009.
Invited speaker. Keystone Symposium on Role of Inflammation in Oncogenesis, February, 2010
Invited reviewer. National Cancer Institute (NCI) Tumor Microenvironment Network. June, 2011
Invited Speaker, National Institute of Health (NIH) Human Microbiome Science Conference: Vision for the Future. Wound healing to longevity: Microbe-induced immune proficiency for human health. July, 2013.
Invited speaker, Wellcome Trust, Human Host-Microbiome Interactions Conference, April 2014.
Invited speaker, Van Andel Institute, Origins of Cancer Conference, July 2014

Peer Bork

Current position: senior group leader (bioinformatics) at the EMBL (Heidelberg);
joint coordinator of the EMBL Structural and Computational Biology unit;
visiting group leader at the MDC (Berlin-Buch)
Advisor: Current and former member of various SABs and evaluation panels (academia + industry)
Cofounder of LION bioscience AG (now SYGNIS Pharma AG), Cellzome AG, Anadys Pharmaceuticals Inc. and biobyte solutions GmbH
Scientific interests: prediction of protein function at different spatial scales,
protein and genome evolution (iTOL), protein domain analysis,
comparative genome and proteome analysis, bridging genotype and phenotype
Some Projects: genome annotation,
comparative analysis of protein modules (SMART),
biochemical pathways and networks (STRING),
networks of chemicals and proteins (STITCH)
bioinformatics applications in molecular medicine
comparative metagenomics
List of group publications

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, …., Julia Tchou, Gabriel A. Rabinovich, Jose R. Conejo-Garcia
Cancer cell 12 Jan 2015; Volume 27, Issue 1, p27–40 http://dx.doi.org/10.1016/j.ccell.2014.11.009

http://www.cell.com/cms/attachment/2023542033/2043769432/fx1.jpg

•TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by γδ T cells
•IL-17 drives malignant progression in IL-6-unresponsive tumors
•TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion
•A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5^R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

HOW THE MICROBIOME PROMOTES CANCER

Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that spur tumor growth and blood vessel formation.

http://www.the-scientist.com/images/April2016/ImmuneInfluence1_640px.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence2_310px1.jpg

Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead to cancerous growth.

HOW THE MICROBIOME STEMS CANCER

Gut bacteria can also produce factors that lower inflammation and slow tumor growth.

Some gut bacteria (e.g., Bifidobacterium)
appear to activate dendritic cells,
which present cancer-cell antigens to T cells that in turn kill the cancer cells.

http://www.the-scientist.com/images/April2016/ImmuneInfluence3_310px1.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence4_310px1.jpg

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Unlocking the Microbiome

Posted in Artificial Intelligence - Breakthroughs in Theories and Technologies, Big Data, Biological Networks, Gene Regulation and Evolution, Gene Regulation, Metabolism, Microbiologial genetics, Microbiology, tagged antimicrobial resistance, Artificial intelligence, feature extraction, Microbiology on February 7, 2016| Leave a Comment »

Unlocking the Microbiome

Larry H. Bernstein, MD, FCAP, Curator

LPBI

3.3.11

3.3.11 Unlocking the Microbiome, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Machine-learning technique uncovers unknown features of multi-drug-resistant pathogen

Relatively simple “unsupervised” learning system reveals important new information to microbiologists

January 29, 201 http://www.kurzweilai.net/machine-learning-technique-uncovers-unknown-features-of-pathogen

http://www.kurzweilai.net/images/Pseudomonas-aeruginosa.jpg

According to the CDC, Pseudomonas aeruginosa is a common cause of healthcare-associated infections, including pneumonia, bloodstream infections, urinary tract infections, and surgical site infections. Some strains of P. aeruginosa have been found to be resistant to nearly all or all antibiotics. (illustration credit: CDC)

A new machine-learning technique can uncover previously unknown features of organisms and their genes in large datasets, according to researchers from the Perelman School of Medicine at the University of Pennsylvania and the Geisel School of Medicine at Dartmouth University.

For example, the technique learned to identify the characteristic gene-expression patterns that appear when a bacterium is exposed in different conditions, such as low oxygen and the presence of antibiotics.

The technique, called “ADAGE” (Analysis using Denoising Autoencoders of Gene Expression), uses a “denoising autoencoder” algorithm, which learns to identify recurring features or patterns in large datasets — without being told what specific features to look for (that is, “unsupervised.”)*

Last year, Casey Greene, PhD, an assistant professor of Systems Pharmacology and Translational Therapeutics at Penn, and his team published, in an open-access paper in the American Society for Microbiology’s mSystems, the first demonstration of ADAGE in a biological context: an analysis of two gene-expression datasets of breast cancers.

Tracking down gene patterns of a multi-drug-resistant bacterium

The new study, published Jan. 19 in an open-access paper in mSystems, was more ambitious. It applied ADAGE to a dataset of 950 gene-expression arrays publicly available at the time for the multi-drug-resistant bacteriumPseudomonas aeruginosa. This bacterium is a notorious pathogen in the hospital and in individuals with cystic fibrosis and other chronic lung conditions; it’s often difficult to treat due to its high resistance to standard antibiotic therapies.

The data included only the identities of the roughly 5,000 P. aeruginosa genes and their measured expression levels in each published experiment. The goal was to see if this “unsupervised” learning system could uncover important patterns in P. aeruginosa gene expression and clarify how those patterns change when the bacterium’s environment changes — for example, when in the presence of an antibiotic.

Even though the model built with ADAGE was relatively simple — roughly equivalent to a brain with only a few dozen neurons — it had no trouble learning which sets of P. aeruginosa genes tend to work together or in opposition. To the researchers’ surprise, the ADAGE system also detected differences between the main laboratory strain of P. aeruginosa and strains isolated from infected patients. “That turned out to be one of the strongest features of the data,” Greene said.

“We expect that this approach will be particularly useful to microbiologists researching bacterial species that lack a decades-long history of study in the lab,” said Greene. “Microbiologists can use these models to identify where the data agree with their own knowledge and where the data seem to be pointing in a different direction … and to find completely new things in biology that we didn’t even know to look for.”

Support for the research came from the Gordon and Betty Moore Foundation, the William H. Neukom Institute for Computational Science, the National Institutes of Health, and the Cystic Fibrosis Foundation.

* In 2012, Google-sponsored researchers applied a similar method to randomly selected YouTube images; their system learned to recognize major recurring features of those images — including cats of course.

Abstract of ADAGE-Based Integration of Publicly Available Pseudomonas aeruginosa Gene Expression Data with Denoising Autoencoders Illuminates Microbe-Host Interactions

The increasing number of genome-wide assays of gene expression available from public databases presents opportunities for computational methods that facilitate hypothesis generation and biological interpretation of these data. We present an unsupervised machine learning approach, ADAGE (analysis using denoising autoencoders of gene expression), and apply it to the publicly available gene expression data compendium for Pseudomonas aeruginosa. In this approach, the machine-learned ADAGE model contained 50 nodes which we predicted would correspond to gene expression patterns across the gene expression compendium. While no biological knowledge was used during model construction, cooperonic genes had similar weights across nodes, and genes with similar weights across nodes were significantly more likely to share KEGG pathways. By analyzing newly generated and previously published microarray and transcriptome sequencing data, the ADAGE model identified differences between strains, modeled the cellular response to low oxygen, and predicted the involvement of biological processes based on low-level gene expression differences. ADAGE compared favorably with traditional principal component analysis and independent component analysis approaches in its ability to extract validated patterns, and based on our analyses, we propose that these approaches differ in the types of patterns they preferentially identify. We provide the ADAGE model with analysis of all publicly available P. aeruginosa GeneChip experiments and open source code for use with other species and settings. Extraction of consistent patterns across large-scale collections of genomic data using methods like ADAGE provides the opportunity to identify general principles and biologically important patterns in microbial biology. This approach will be particularly useful in less-well-studied microbial species.

Abstract of Unsupervised feature construction and knowledge extraction from genome-wide assays of breast cancer with denoising autoencoders

Big data bring new opportunities for methods that efficiently summarize and automatically extract knowledge from such compendia. While both supervised learning algorithms and unsupervised clustering algorithms have been successfully applied to biological data, they are either dependent on known biology or limited to discerning the most significant signals in the data. Here we present denoising autoencoders (DAs), which employ a data-defined learning objective independent of known biology, as a method to identify and extract complex patterns from genomic data. We evaluate the performance of DAs by applying them to a large collection of breast cancer gene expression data. Results show that DAs successfully construct features that contain both clinical and molecular information. There are features that represent tumor or normal samples, estrogen receptor (ER) status, and molecular subtypes. Features constructed by the autoencoder generalize to an independent dataset collected using a distinct experimental platform. By integrating data from ENCODE for feature interpretation, we discover a feature representing ER status through association with key transcription factors in breast cancer. We also identify a feature highly predictive of patient survival and it is enriched by FOXM1 signaling pathway. The features constructed by DAs are often bimodally distributed with one peak near zero and another near one, which facilitates discretization. In summary, we demonstrate that DAs effectively extract key biological principles from gene expression data and summarize them into constructed features with convenient properties.

references:

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In the name of Translation from a food born pathogen to a friendly vaccine: Listeria monocytogenes

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, tagged Demet Sag, food pathogen, L monocytes, Microbiology, vaccine on October 15, 2015| Leave a Comment »

In the name of Translation from a food born pathogen to a friendly vaccine: Listeria monocytogenes

Curator: Demet Sag, PhD, CRA, GCP

Is it a far fetch? Friend or Foe?

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen bacterium. It is used as a prototypes for an experimental model to understand the fundamental processes of adaptive immunity and virulence. 10 species of L. monocytogenes is identified in both humans and animals, L. ivanovii mainly infects ungulates (eg. sheep and cattle), while other species (L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, L. rocourtiae, L. fleischmannii and L. weihenstephanensis) are essentially saprophytes. Within the species of L. monocytogenes, several serovars (e.g., 4b, 1/2a, 1/2b and 1/2c) are highly pathogenic and account for a majority of clinical isolations.

Gram-negative bacteria has inner and outer membranes and they are most studied; yet mechanics of protein secretion across the single cell membrane of Gram-positive is not. The protein secretion in gram positive bacteria is complex not only it requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface but also folding specifically. As a result, protein folding mechanism and stability investigated for the role of PrsA2 and PrsA-like so that optimizing the virulence and protein secretion become possible.

Pathogen: Listeriosis

Listeria monocytogenes is a food-borne pathogen determined in 1980s causing an opportunistic disease called listeriosis which is widespread in nature being part of the faecal flora of many mammals. In addition to contaminated food resources (1-10%), may occur sporadically or in outbreaks. It can be difficult to control and may cause severe clinical outcomes, especially in pregnant women, children and the elderly. The mechanism of pathogenity based on simply altering the actin cytoskeleton structure. Infection causes a spectrum of illness, ranging from febrile gastroenteritis to invasive disease, including bacteraemia, sepsis, and meningoencephalitis.

This organisms copes well with bile acids and acidic environment such as glutamate decarboxylase and arginine deiminase systems to survive in competitive microbiome of GI.

This information may benefit developing effective vaccines, designing pharmabiotics; even including probiotics, prebiotics, or phages.

Nutrition:

Altering dietary habit assumed to control a disease. The effects of various fatty acids on bacterial clearance and disease outcome through suppression or activation of immune responses can’t be simplified down to one or two kinds of fatty acids in foodborne pathogens. Commonly they have a specialized carbohydrate metabolism so they can utilize fatty acids of host and the host may use the end products for an energy resource. The compared food-borne pathogens include Salmonella sp., Campylobacter sp.,Shiga toxin-producing Escherichia coli, Shigella sp., Listeria monocytogenes, and Staphylococcus aureus.

Genetics:

This bacterium has a complex transcriptional machinery to adept, invade several types of cells, and survive. It happens through RNA-based regulation in bacteria in cell biology at the chromatin level during bacterial infection. This includes clathrin, atypical mitochondrial fragmentation, and several hundred non-coding RNAs (ncRNAs) in the Listeria genome. Patho-epigenetics becoming an attractive field. Improved bioinformatics may help to classify these changes under specific regulatory mechanisms and networks to determine their function and use.

The Toxin, Vaccine and Immunotheraphy

The virulence of Listeria monocytogenes mainly depends on a listeriolysin O (LLO) which is a thiol-activated, cholesterol-dependent, pore-forming toxin, and highly immunogenic. In addition, biochemically, LLO, a toxin that belongs to the family of cholesterol-dependent cytolysins (CDCs), exhibits potent cell type-non-specific toxicity and is a source of dominant CD4(+) and CD8(+) T cell epitopes. Hence, it is the major target for innate and adaptive immune responses in different animal models and humans.

As a result, during infection bacteria escape from phagocytosis, allow bacteria to infest the cells and multiply. Thus, due to it’s naturally immunomodulation role this mechanisms is under investigation so that it can be used for cancer immunotherapies for developing immune tolerance. Since it has effective cytotoxicity. Thus, co-administration of this toxin or using as an adjuvant with vaccine vectors are also under research. LLO has diverse biological activities such as cytotoxicity, apoptosis induction, endoplasmic reticulum stress response, modulation of gene expression,

Since FDA approved Sipuleucel-T (Provenge, Dendreon, Seattle, WA), which consists of antigen-loaded dendritic cells (DCs), there is a boom in immunotherapy applications. Yet, there is a shortcoming of this application because of its limited scope in immune response. However, Listeria monocytogenes (Lm) naturally targets DCs in vivo and stimulates both innate and adaptive cellular immunity. Lm-based vaccines engineered to express cancer antigens have demonstrated striking efficacy applications.

Meningitis

On the other hand, there is a caution to be taken in clinics since L. monocytogenes most often presents as acute bacterial meningitis, particularly in weaken immune system of patients such as elderly, already sick patients as secondary infection/opportunistic, and those with already immune fragile state. L. monocytogenes CNS the infections may present as acute bacterial meningitis, meningoencephalitis, or acute encephalitis.

References and Further readings:

PMCID: PMC3574585 PMID: 22595054

Le DT(1), Dubenksy TW Jr, Brockstedt DG. “Clinical development of Listeria monocytogenes-based immunotherapies”. 20. Semin Oncol. 2012 Jun;39(3):311-22. doi: 10.1053/j.seminoncol.2012.02.008.

PMCID: PMC3987759 PMID: 24826075

Liu D(1).“Molecular approaches to the identification of pathogenic and nonpathogenic Listeriae”. 16. Microbiol Insights. 2013 Jul 22;6:59-69. doi: 10.4137/MBI.S10880. eCollection 2013.

PMCID: PMC4385656 PMID: 25874208

Hernández-Flores KG(1), Vivanco-Cid H(2). Biological effects of listeriolysin O: implications for vaccination. Biomed Res Int. 2015;2015:360741. doi: 10.1155/2015/360741. Epub 2015 Mar 22.

PMCID: PMC4369580 PMID: 25241232

Maertens de Noordhout C(1), Devleesschauwer B(2), Angulo FJ(3), Verbeke G(4), Haagsma J(5), Kirk M(6), Havelaar A(7), Speybroeck N(8). “The global burden of listeriosis: a systematic review and meta-analysis”. 2. Lancet Infect Dis. 2014 Nov;14(11):1073-82. doi: 10.1016/S1473-3099(14)70870-9. Epub 2014 Sep 15.

PMID: 24911203

Cossart P(1), Lebreton A(2). “A trip in the “New Microbiology” with the bacterial pathogen Listeria Monocytogenes”. 3. FEBS Lett. 2014 Aug 1;588(15):2437-45. doi: 10.1016/j.febslet.2014.05.051. Epub 2014 Jun 6.

PMCID: PMC4005144 PMID: 24822197

Hernandez-Milian A(1), Payeras-Cifre A(1). “What is new in listeriosis?”. Biomed Res Int. 2014;2014:358051. doi: 10.1155/2014/358051. Epub 2014 Apr 14.

PMCID: PMC4179725 PMID: 25325017

Schultze T(1), Izar B(2), Qing X(1), Mannala GK(1), Hain T(1). “Current status of antisense RNA-mediated gene regulation in Listeria monocytogenes”. 5. Front Cell Infect Microbiol. 2014 Sep 30;4:135. doi: 10.3389/fcimb.2014.00135.

eCollection 2014.

PMCID: PMC3924034 PMID: 24592357

Guariglia-Oropeza V(1), Orsi RH(1), Yu H(2), Boor KJ(1), Wiedmann M(1), Guldimann C(1). “Regulatory network features in Listeria monocytogenes-changing the way we talk”. 6. Front Cell Infect Microbiol. 2014 Feb 14;4:14. doi: 10.3389/fcimb.2014.00014.

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A NEW ERA OF GENETIC MANIPULATION

Posted in Academic Publishing, Bacterial Resistance, Diagnostics and Lab Tests, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gene Regulation, Genetics & Pharmaceutical, Genome Biology, tagged biomarkers, Cas9, CRISPR, CRISPR/Cas9, Demet Sag, Dr. Sag, functional genomics, Functional TransGenomics, gene editing, genome structure and function, germline, Immunology, immunomodulation, innate immune defense, manipulation of gene, Microbiology, scientific, stem cels, translational genomics on April 14, 2015| Leave a Comment »

Demet Sag, PhD, CRA, GCP

Gene engineering and editing specifically are becoming more attractive. There are many applications derived from microbial origins to correct genomes in many organisms including human to find solutions in health.

There are four customizable DNA specific binding protein applications to edit the gene expression in translational genomics. The targeted DNA double-strand breaks (DSBs) could greatly stimulate genome editing through HR-mediated recombination events. We can mainly name these site-specific DNA DSBs:

meganucleases derived from microbial mobile genetic elements (Smith et al., 2006),
zinc finger (ZF) nucleases based on eukaryotic transcription factors (Urnov et al., 2005;Miller et al., 2007),
transcription activator-like effectors (TALEs) from Xanthomonasbacteria (Christian et al., 2010; Miller et al., 2011; Boch et al., 2009; Moscou and Bogdanove, 2009), and
most recently the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (Cong et al., 2013;Mali et al., 2013a).

There is a new ground breaking study published in Science by Valentino Gantz and Ethan Bier of the University of California, San Diego, described an approach called mutagenic chain reaction (MCR).

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

Structural and Metagenomic Diversity of Cas9 Orthologs

(A) Crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA.

(B) Canonical CRISPR locus organization from type II CRISPR systems, which can be classified into IIA-IIC based on their cas gene clusters. Whereas type IIC CRISPR loci contain the minimal set of cas9, cas1, andcas2, IIA and IIB retain their signature csn2 and cas4 genes, respectively.

(C) Histogram displaying length distribution of known Cas9 orthologs as described in UniProt, HAMAP protein family profile MF_01480.

(D) Phylogenetic tree displaying the microbial origin of Cas9 nucleases from the type II CRISPR immune system. Taxonomic information was derived from greengenes 16S rRNA gene sequence alignment, and the tree was visualized using the Interactive Tree of Life tool (iTol).

(E) Four Cas9 orthologs from families IIA, IIB, and IIC were aligned by ClustalW (BLOSUM). Domain alignment is based on the Streptococcus pyogenes Cas9, whereas residues highlighted in red indicate highly conserved catalytic residues within the RuvC I and HNH nuclease domains.

(Cell. Author manuscript; available in PMC 2015 Feb 27.Published in final edited form as:

Cell. 2014 Jun 5; 157(6): 1262–1278.doi: 10.1016/j.cell.2014.05.010)

The uniqueness of this study comes from:

There is a big difference between the new type of mutation and traditional mutation is expressivity of the character since previously mutations were passive and non-transferable at 100% rate. However, in classical Mendelian Genetics, only one fourth f the recessive traits can be presented in new generation. Yet, in this case this can be achieve about 97% plus transferred to new generation.

MCR alterations is active that is they convert matching sequences at the same target site so mutated sites took over the wild type character without degenerating by wild type alleles segregating independently during the breeding process

Therefore, the altered sequences routinely replace the wild type (original) sequences at that site. The data demonstrated that among 92 flies, only one female became wild type but remaining 41 females had yellow eyes yet all 50 males showed wild type eye coloring at the second generation.

The genetic engineering of the genome occurred in a single generation with high efficiency.

Their technique developed by Gantz and Bier had three basic parts:

Both somatic and germline cells expressed a Cas9 gene,

A guide RNA (gRNA) targeted to a genomic sequence of interest,

The Cas9/gRNA cassettes have the flanking homolog arms that matches the two genomic sequences immediately adjacent to either side of the target cut site

There are many applications in translational genomics that requires multiple steps to make it perfect for complicated organisms, such as plants, mosquitoes and human diseases.

Short Walk from Past to the Future of CRISPR/Cas9

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner.

The latest ground-breaking technology for genome editing is based on RNA-guided engineered nucleases, which already hold great promise due to their:

simplicity,
efficiency and
versality

Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987),

their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005).

Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007).

The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

The most widely used system is the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated) system from Streptococcus pyogenes (Jinek et al., 2012).

Then, five independent groups demonstrated that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013, Cong et al., 2013, Hwang et al., 2013, Jinek et al., 2013 and Mali et al., 2013).

Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified colonial cell lines can be derived within 2-3 weeks

Genome editing with site-specific nucleases.

Double-strand breaks induced by a nuclease at a specific site can be repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR). In most cases, NHEJ causes random insertions or deletions (indels), which can result in frameshift mutations if they occur in the coding region of a gene, effectively creating a gene knockout.

Alternatively, when the DSB generates overhangs, NHEJ can mediate the targeted introduction of a double-stranded DNA template with compatible overhangs

Even though the generation of breaks in both DNA strands induces recombination at specific genomic loci, NHEJ is by far the most common DSB repair mechanism in most organisms, including higher plants, and the frequency of targeted integration by HR remains much lower than random integration.

Unlike its predecessors, the CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple gRNAs for each target gene

Unlike ZFNs and TALENs, the CRISPR/Cas9 system can cleave methylated DNA in human cells (Hsu et al., 2013), allowing genomic modifications that are beyond the reach of the other nucleases (Ding et al., 2013).

The main practical advantage of CRISPR/Cas9 compared to ZFNs and TALENs is the ease of multiplexing. The simultaneous introduction of DSBs at multiple sites can be used to edit several genes at the same time (Li et al., 2013; Mao et al., 2013) and can be particularly useful to knock out redundant genes or parallel pathways.

Finally, the open access policy of the CRISPR research community has promoted the widespread uptake and use of this technology in contrast, for example, to the proprietary nature of the ZFN platform.

The community provides access to plasmids (e.g., via the non-profit repository Addgene), web tools for selecting gRNA sequences and predicting specificity:

http://cbi.hzau.edu.cn/ cgi-bin/CRISPR; http://www.genome.arizona.edu/crispr/; and http:// http://www.rgenome.net/cas-offinder,
http://www.e-crisp.org/E-CRISP/index. html
hosts active discussion groups (e.g.: https://groups.google. com/forum/#!forum/crispr).

Downside:

One area that will likely need to be addressed when moving to more complex genomes, for instance, is off-target CRISPR/Cas9 activity since fruit fly has only four chromosomes and less likely to have off-target effects. However, this study provided proof of principle.

Yet, this critics is not new since one of the few criticisms of the CRISPR/Cas9 technology is the relatively high frequency of off-target mutations reported in some of the earlier studies (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jiang et al., 2013a; Mali et al., 2013; Pattanayak et al., 2013).

Several strategies have been developed to reduce off-target genome editing, the most important of which is the considered design of the gRNA.

fusions of catalytically inactive Cas9 and FokI nuclease have been generated, and these show comparable efficiency to the nickases but substantially higher (N140-fold) specificity than the wild-type enzyme (Guilinger et al., 2014; Tsai et al., 2014)

Altering the length of the gRNA can also minimize non-target modifications. Guide RNAs with two additional guanidine residues at the 5′ end were able to avoid off-target sites more efficiently than normal gRNAs but were also slightly less active at on-target sites (Cho et al., 2014)

What more:

The CRISPR/Cas9 system can be used for several purposes in addition to genome editing:

The ectopic regulation of gene expression, which can provide useful information about gene functions and can also be used to engineer novel genetic regulatory circuits for synthetic biology applications.

The external control of gene expression typically relies on the use of inducible or repressible promoters, requiring the introduction of a new promoter and a particular treatment (physical or chemical) for promoter activation or repression.

Disabled nucleases can be used to regulate gene expression because they can still bind to their target DNA sequence. This is the case with the catalytically inactive version of Cas9 which is known as dead Cas9 (dCas9).

Preparing the host for an immunotherapy is possible if it is combined with TLR mechanism:

On the other hand, the host mechanism needs to be review carefully for the design of an effective outcome.

The mechanism of microbial response and infectious tolerance are complex.

During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells.

Uniqueness of TLR comes from four major characteristics of each individual TLR :

ligand specificity,
signal transduction pathways,
expression profiles and
cellular localization.

Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels Specific TLR stimulat ion links innate and acquired responses through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.

Some examples of ligand – TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2, double stranded (ds) RNAs through TLR3, lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5, single stranded RNAs through TLR7/8, synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9.

The specificity is established by correct pairing of a TLR with its proinflammatory cytokine(s), so that these permutations influence creation and maintenance of cell differentiat ion.

Immunotherapy: The immune cells can be used as a sensor to scavenger the circulating malformed cells in vivo diagnostics or attack and remember them, for instance, relapse of cancer, re-infection with a same or similar agent (bacteria or virus) etc.

Not only using unique microbial and other model organism properties but also using the human host defense mechanism during innate immune responses may bring a new combat to create a new method of precision medicine. This can be a new type of immunotherapy, immune cell mediated gene therapy or vaccine even a step for an in vivo diagnostics.

Molecular Genetics took a long road from discovery of restriction enzymes, developing PCR assays, cloning were the beginning. Now, having technology to sequence and compare the sequences between organisms also help to design more sophisticated methods.

Generating mutant lines in Drosophila with the classical genetics methods relies on P elements, a type of transposon and balancers after crossing selected flies with specific markers. This fly pushing is a very tedious work but powerful to identify primary pathways, mechanisms and gene interactions in system and translational genomics.

Thus, Microbial Immunomodulation is an important factor not only using the microorganisms or their mechanisms but also modulating the immune cells based on the host interaction may generate new types of diagnostics and targeted therapy tools.

Microbial immunomodulation. Microbes from the environment, and from the various microbiota, modulate the immune system. Some of this is due to direct effects of defined microbial products on elements of the immune system. But modulation of the immune system also secondarily alters the host–microbiota relationship and leads to changes in the composition of the microbiota, and so to further changes in immunoregulation (shown as indirect pathways). At the end of the day balance is the key for survival.

Graham A. W. Rook,^*,1 Christopher A. Lowry,² and Charles L. Raison³Microbial ‘Old Friends’, immunoregulation and stress resilience Evol Med Public Health. 2013; 2013(1): 46–64. Published online 2013 Apr 9. doi: 10.1093/emph/eot004 PMCID: PMC3868387

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881665/bin/nihms199923f2.jpg

CRISPR-Cas9 mediated NHEJ in transient transfection experiments.

Table 1.

Species	Transformation method	Cas9 codon optimization	Promoters (Cas9, gRNA)	Target	Mutation frequency	Detection method	Off-target (no. of sites analyzed)	Detection method	Multiplex (deletion)	Reference
Arabidopsis thaliana	PEG-protoplast transfection	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3<comma> FLS2	1.1–5.6%	PCR + sequencing				Li et al. (2013)
A. thaliana	Leaf agroinfiltration	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3	2.70%	PCR + sequencing			Yes (48 bp)	Li et al. (2013)
A. thaliana	PEG-protoplast transfection	Arabidopsis (with intron)	CaMV35SPDK, AtU6	RACK1b<comma> RACK1c	2.5–2.7%	PCR + sequencing	No (1 site)	PCR + sequencing		Li et al. (2013)
A. thaliana	Leaf agroinfiltration	C. reinhardtii	CaMV35S, AtU6	Co-transfected GFP	n.a.	Pre-digested PCR + RE				Jiang et al. 2013a and Jiang et al. 2013b
Nicotiana benthamiana	PEG-protoplast transfection	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3	37.7–38.5%	PCR + sequencing				Li et al. (2013)
N. benthamiana	Leaf agroinfiltration	Arabidopsis (with intron)	CaMV35SPDK, AtU6	PDS3	4.80%	PCR + sequencing				Li et al. (2013)
N. benthamiana	Leaf agroinfiltration	Human	CaMV35S, AtU6	PDS	1.8–2.4%	PCR + RE	No (18 sites)	PCR + RE		Nekrasov et al. (2013)
N. benthamiana	Leaf agroinfiltration	C. reinhardtii	CaMV35S, AtU6	Co-transfected GFP	n.a.	pre-digested PCR + RE				Jiang et al. 2013a and Jiang et al. 2013b
N. benthamiana	Leaf agroinfiltration	Human	CaMV35S, CaMV35S	PDS	12.7–13.8%					Upadhyay et al. (2013)
Nicotiana tabacum	PEG-protoplast transfection	Tobacco	2xCaMV35S, AtU6	PDS<comma> PDR6	16.27–20.3%	PCR + RE			Yes (1.8 kb)	Gao et al. (2014)
Oryza sativa	PEG-protoplast transfection	Rice	2xCaMV35S, OsU3	PDS<comma> BADH2<comma> MPK2<comma> Os02g23823	14.5–38.0%	PCR + RE	Noa (3 sites)	PCR + RE		Shan et al. (2013)
O. sativa	PEG-protoplast transfection	Human	CaMV35S, OsU3 or OsU6	MPK5	3–8%	RE + qPCR and T7E1 assay	No (2 sites) Yes (1 site with a mismatch at position 12)	RE + PCR		Xie and Yang (2013)
O. sativa	PEG-protoplast transfection	Rice	CaMV35S, OsU6	SWEET14	n.a.	pre-digested PCR + RE				Jiang et al. 2013a and Jiang et al. 2013b
O. sativa	PEG-protoplast transfection	Rice	ZmUbi, OsU6	KO1 KOL5; CPS4 CYP99A2; CYP76M5 CYP76M6	n.a.	PCR + sequencing			Yes (115<comma> 170<comma> 245 kb)	Zhou et al. (2014)
Triticum aestivum	PEG-protoplast transfection	Rice	2xCaMV35S, TaU6	MLO	28.50%	PCR + RE				Shan et al. (2013)
T. aestivum	PEG-protoplast transfection	Plant	ZmUbi, TaU6	MLO-A1	36%	T7E1				Wang et al. 2014a and Wang et al. 2014b
T. aestivum	Agrotransfection of cells from immature embryos	Human	CaMV35S, CaMV35S	PDS<comma> INOX	18–22%	PCR + sequencing				Upadhyay et al. (2013)
T. aestivum	Agrotransfection of cells from immature embryos	Human	CaMV35S, CaMV35S	INOX		PCR + sequencing	No*	PCR + RE	Yes (53 bp)	Upadhyay et al. (2013)
Zea mays	PEG-protoplast transfection	Rice	2xCaMV35S, ZmU3	IPK	16.4–19.1%	PCR + RE				Liang et al. (2014)
Citrus sinensis	Leaf agroinfiltration	Human	CaMv35S, CaMV35S	PDS	3.2–3.9%	PCR + RE	No (8 sites)	PCR + RE		Jia et al. (2014)

References:

A brief overview of CRISPR-mediated immunity and explain how the emerging new properties of this defense system are being repurposed for genome engineering in bacteria, yeast, human cells, insects, fish, worms, plants, frogs, pigs, and rodents.

Also look at F1000Prime Rep. 2014; 6: 3. For the list of microorganisms use in CRISPR applications.

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Previously Published at Leaders in Pharmaceutical Intelligence:

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

larryhbern

2015/03/27
Published

CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality

TLR ligand	TLR	Reference
Lipopolysaccharide, LPS	TLR4	(96). (112).
Lipopeptides, Pam3Cys	TLR2	(92; 104; 105)
Double stranded (ds) RNAs	TLR3	(106; 107)
Bacterial flagellin	TLR5	(108; 109)
Single stranded RNAs	TLR7/8	(97; 98)
Unmethylated CpG DNA motifs	TLR9	(Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod	TLR7 and TLR8	(Lee J, 2003)

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