The Cross-Border Biotech Blog

An Evolving Concept of Oncolytic Viruses

University of Ottawa ♦ Ottawa Hospital Research Institute ♦ Ontario Cancer Institute ♦ University of Toronto ♦ University of Otago

Published in Molecular Therapy (npg), January 24, 2012

Oncolytic viruses were originally engineered to impose direct damage to tumour cells through infection, replication, and subsequent destruction of cancer cells via cell rupture. Many of today’s oncolytic viruses aim to do this, however it has become apparent that ‘next generation’ oncolytic viruses, those with the greatest efficacy, will be the viruses that not only destroy cancer cells physically but stimulate a strong innate immune response against cancer cells to continue the onslaught following initial infection.

This concept of inducing immune response is not novel. By their nature, oncolytic viruses offer the opportunity to deliver therapeutic genes at the time of infection. Indeed, previous studies have shown that viruses can be engineered to deliver cytokine genes into tumour cells. These cancerous cells then act as factories creating a protein product that leads to their own immune destruction. Suicide.

But researchers have now discovered that it may not be necessary to engineer viruses to do this. Certain viruses are capable of generating such an immune response simply through the presence of the viral particle itself. One example of such a virus is Parapoxvirus ovis, or ORFV. Researchers at the University of Ottawa found that injecting the virus into mice led to a significant upregulation of T cell response, including both CD4+ and CD8+, and an accumulation of B cells, natural killer cells, and various cytokines with anti-tumoural activity.

But there’s more to the story. ORFV infection does not lead to disease in animals, making it an excellent candidate for an oncolytic therapeutic. The real beauty though — the differentiative aspect of this virus — is that ORFV, even in the presence of antibody against the virus, continues to reinfect cancer cells. Animals seem to have a very short-lived immunity against the virus, allowing for an attenuated therapeutic activity unlike any found so far in this field.

Genetic Basis of Jr(a)- Phenotype Discovered

University of Manitoba ♦ Published in Nature Genetics, January 15, 2012

The medical community has been aware of the Jr(a) antigen on red blood cells for quite some time. Roughly 40 years ago it was shown that a small group of individuals created antibodies against this protein motif. In the presence of normal red blood cells the antibodies produced by Jr(a)- individuals react quite vigorously. During transfusion it is important that donor blood does not contain Jr(a) antibodies because they can lead to negative transfusion reactions that are harmful to the recipient. In this recent study, investigators isolated the genetic component responsible for the Jr(a)- phenotype. Carrying out single nucleotide polymorphism analysis, researchers discovered a nearly 400 thousand base pair null region in Jr(a)- individuals that contained the gene ABCG2.

Cutting to the Core of Systemic Immune Response

Ontario Cancer Institute ♦ Published in Science, January 13, 2012

The human body has become adept at regulating pathogens. However, this evolutionary trait also has its down sides. Extreme innate immune response can cause systemic inflammatory reactions that can prove fatal. This recent study out of Tak Mak’s lab identifies a mechanism by which animals induce innate immune response following exposure to foreign pathogens. Tumour necrosis factor alpha (TNF-α) mediates septic shock through its release from cell membranes. This release process is regulated by an enzyme known as TNF-α convertase. Researchers discovered that this enzyme’s maturation and trafficking is controlled by iRhom2. Sure enough, mice deficient in iRhom2 displayed an ability to circumvent lethal doses of bacterial lipopolysaccharide.

Conditions characterized by systemic immune response are some of the hardest to go after in the clinic. As an indication, sepsis has been a graveyard, with only a few of some 40 clinical studies showing any efficacy in the last two decades. Identifying general systemic pathways, like that involving iRhom2, will be critical in creating therapeutics for these complex conditions in the future.

Disruption of Gatekeeper Genes Causes Two-fold Mutation

University of Toronto ♦ Published in EMBO, January 10, 2012

Certain genes have a critical role in maintaining the stability of the genome by exerting a certain control over DNA metabolism. Researchers at the Donnelly Centre in Toronto have discovered that disruption of these so called ‘gatekeeper’ genes has consequences beyond disregulating the direct influence they have on genomic stability. As expected, perturbing gatekeeper genes led to spontaneous DNA damage, however, this damage became amplified downstream. Measurements of ribonucleotide reductase (RNR) in budding yeast showed that expression of this enzyme increased as DNA damage occurred, and that RNR activity was associated with an increase in dNTP pools. A second wave of mutation occurred as yeast cells were then capable of synthesizing DNA in the presence of hydroxyurea in the next S phase.

Movement and Breathing, Where’s the Link?

University of Montreal ♦ Published in PNAS, January 10, 2012

As we exercise the rate at which we respire increases, and can do so dramatically. However, there is much to be elucidated in terms of the neural connections that elicit the body’s breathing response to movement. To interrogate this matter, researchers used the lamprey as a model. They first identified the region of the brain that was associated with an increase in respiration. In lampreys, stimulation of the mesencephalic locomotor region (LMR) increased the rate of respiration. Researchers then used a technique called ‘patch-clamp’, that is capable of taking electrophysiological recordings from single neurons, and used it to show that neurons in the dorsal region of the MLR are directly connected to a respiratory generator. To cross-check their findings, an inhibitor of the innervated region of the respiratory generator was used to show that inhibition at this site did indeed reduce respiration.

MSC Anti-Immune Power

McGill University ♦ Published in Molecular Therapy (npg), January 2012

Mesenchymal stem cells have great potential as a source of therapeutic cell types for transplantation because they are capable of differentiating into bone, fat, cartilage, and muscle. Some reports show they even have the ability to differentiate into neural cells. The research community is beginning to learn, however, that perhaps the most interesting application of MSCs will be as a suppressant for the immune system. MSCs exhibit the unique ability to moderate T cell response. As a result, they are being investigated in clinical studies for treatment of disorders characterized by inflammation and autoimmunity. Rheumatoid arthritis is a model indication for MSC therapy down the line.

Results from clinical trials thus far have been varied. In order to gain a mechanistic understanding of the variability clinicians are observing, researchers at McGill University cross-examined MSCs from normal adult volunteers. Using an in vitro model, 7 different MSC lines were tested for their ability to suppress T cell proliferation. Results of this study indicate that MSCs possessing the most potent anti-immune power upregulate expression of an enzyme known as indoleamine 2,3-dioxygenase (IDO) in response to interferon-α. IDO is the first enzyme in the kynurenine pathway that catalyzes the degradation of trytophan. Several of the metabolites of this pathway are known to activate the stress response kinase CGN2, which causes apoptosis of T cells. Researchers also found that IDO activity is implicated in the differentiation of monocytes into immunosuppressive macrophages that regulate T cell proliferation in an IL-10 dependent fashion.

Elucidating the molecular mechanisms that contribute to the ability of MSCs to temper the immune system will allow us to generate MSC lines that have the most potent anti-immune power for the treatment of inflammatory and autoimmune disease.

Hedgehog Signaling Upholds Integrity of BBB

University of Montreal ♦ Published in Science, December 23, 2011

The blood-brain barrier (BBB) is a crucial boundary within the body that restricts the migration of blood-borne molecules and immune cells from circulation into the brain. Specialized endothelial cells tightly bound together with junctional proteins ensure that only certain small molecules are able to access the brain. Given that Hedgehog (Hh) signaling has been implicated in multiple sclerosis, a disease characterized by autoimmune destruction of myelin cells, researchers at the University of Montreal hypothesized that Hh signaling may have an influence on the formation of the BBB. They found that astrocytes in close proximity to endothelial cells in the BBB release Hh and that endothelial cells express Hh receptors, in keeping with the theory that Hh signaling is important for the integrity of the BBB. In order to demonstrate that Hh signaling also keeps leukocytes out of contact with the brain, researchers injected mice with cyclopamine, a small molecule inhibitor of the Hh pathway. Indeed, pharmacological inhibition of the Hh pathway led to acute disruption of the BBB and passage of leukocytes into the brain. Together these findings implicate the Hh pathway as being critical in the development and structural integrity of the BBB.

Super-Selective Oncolytic Virus

Ottawa Hospital Research Institute ♦ Published in Molecular Therapy (npg), December 20, 2011

More on oncolytic viruses this week but not with VSV this time, but rather the poxvirus JX-594. This particular virus, while having an excellent therapeutic index against multiple solid tumour types, is not that well understood. Researchers seeking to understand the mechanisms underlying its exquisite cancer cell selectivity identified multiple interactions. Three model systems were investigated, including primary normal and cancer cells, surgical explants, and mouse tumour models. It was found that selectivity of JX-594 was driven by multiple factors; the first time selectivity has been attributed to more than one specific mechanism. Among these factors were virus replication activation by the EGFR/Ras signaling pathway, cellular thymidine kinase levels (the virus is engineered to be responsive to TK so this was expected), and similar to VSV, hyporesponsiveness to type-I interferon. These finding will allow for the generation of more selective and potent oncolytic viruses.

Immunomodulators Enhance Polyfunctionality of T Cells Following Vaccination

McMaster Immunology Research Centre ♦ Published in Molecular Therapy (npg), December 20, 2011

Recombinant human adenovirus vaccines are capable of producing memory CD8+ T cell populations, however these populations lack polyfunctionality; in response to antigen stimulation they are not able to produce multiple different cytokines and chemokines. Researchers at McMaster have discovered that inhibiting mammalian target of rapamycin (mTOR) while stimulating OX40 alters not only the magnitude of T cell response, but also its phenotype and functionality. mTOR inhibition modulates differentiation of T cell pools while stimulation of OX40 enhances the process of costimulation. The addition of immunomodulators elicited greater immunity against multiple virus challenges in a mouse model, but was contingent upon sufficiently long transgene expression from the adenovirus under study.

Homeostasis in the Gut, Plasma Cells on Patrol

University of Toronto ♦  McGill University ♦ University of Bern ♦ Memorial University of Newfoundland

Published in Nature, December 11, 2011

The intestine is the largest mucosal surface in the body and is exposed to a diversity of microbes. This microbial life is healthy, although the immune system must still keep bacterial population sizes in check. One mechanism by which this is achieved is through the differentiation of B cells to plasma cells, which in turn secrete the immunoglobulin IgA. New findings indicate that plasma cells have additional roles that keep microbiota at bay. Researchers found that plasma cells secrete tumor-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), both of which exert antimicrobial activity. Microbial co-stimulation is pre-requisite for plasma cell multi-functionality. In order to investigate the effects that TNF-α and iNOS have on regulating flora in the gut, researchers used a double knockout mouse model. Removal of the two mediators led to a change in the composition of intestinal flora and a reduction in secretion of IgA from plasma cells.

VSV 2.0: IL-15 Engineered

McMaster University ♦ Published in Cancer Gene Therapy (npg), December 9, 2011

A popular approach to the development of oncolytic viruses for cancer therapy has been the use of the single-stranded RNA virus known vesicular stomatitis virus (VSV). This particular virus has a hypersensitivity to the protein type I interferon (IFN). Upon exposure to a virus the body releases IFN in attempt to prevent infection. The beauty of VSV is that while normal healthy cells are highly sensitive to IFN, tumour cells remain hyporesponsive. Systemic administration of VSV has little to no effect on normal cells as the virus cannot replicate within them. However, because they fail to respond to IFN, cancer cells remain susceptible to VSV infection, VSV replication, and eventually cell lysis. While VSV alone has been shown to be effective in preclinical models, long-term survival is not greatly increased.

Researchers at McMaster’s Centre for Gene Therapeutics have upped the anti with the development of a genetically engineered VSV that expresses an interleukin-15 (IL-15) transgene. IL-15 is a cytokine that has a critical role in adaptive immune responses to pathogenic entities. The group showed that injections of the IL-15 VSV in a mouse model of colon cancer led to strong localized expression of the cytokine in vivo. The new IL-15-rich microenvironment surrounding tumours increased the adaptive immune response targeted at tumour cells by recruiting and activating natural killer cells and T-cells. This novel virus has the two-fold effect of not only lysing cells, but enhancing anti-tumoral T-cell response. Mice receiving injections exhibited better survival overall.

hiPSC-derived HSCs Engraft Mouse Model

McMaster Stem Cell and Cancer Research Institute ♦ Published in Stem Cells, November 30, 2011

There are several sources currently being used to isolate hematopoietic stem cells (HSCs) for transplantation in humans. Included in these are the bone marrow, peripheral blood, and umbilical cord blood. However, HSCs derived from human embryonic stem cells (hESCs) have, as of yet, failed to be a viable source for transplantation. Although hESCs are capable of hematopoietic differentiation in the petri dish, their HSC progeny are unable to engraft human-mouse xenograft models. This investigation shows that human induced pluripotent stem cells (hiPSCs), which are also capable of hematopoietic differentiation in vitro, produce HSCs that can successfully engraft following transplantation into the mouse model; similar to HSCs from adult sources. This is an advance in terms of studying hematopoietic transplantation in humans from pluripotent sources. Despite the ability of hiPSC-derived HSCs to engraft they were unable to reconstitute hematopoiesis. However, they could establish colonies after being removed from the bone marrow compartment suggesting that a molecular mechanism blocks their capacity to differentiate in vivo. Investigators believe that misexpression of various microRNAs may explain why hiPSCs are unable to reconstitute hematopoiesis.

Breast Cancer Stem Cells, From Where Do They Originate?

British Columbia Cancer Agency ♦ University of Melbourne ♦ University of British Columbia

Published in Stem Cells, November 30, 2011

There has been a lot of talk recently about aldehyde dehydrogenase (ALDH) because it seems to be expressed across such a diversity of different stem cell types. We know that it is expressed by hematopoietic progenitors, mesenchymal progenitors, neural progenitors, and endothelial progenitors. Researchers investigating the expression of ALDH in a number of functionally defined mammary cell types have come across an interesting finding. They found that ALDH expression is actually lower in mammary stem/progenitor cells, that have bilineage differentiation potential, than it is in cells committed to particular lineages. Progenitor cells of the luminal lineage, for example, express relatively high levels of ALDH. It seems that a molecular switch upregulates ALDH, particularly the ALDH1A3 isoform, during the process of lineage commitment. The finding that ALDH is upregulated during differentiation challenges the notion that breast cancer stem cells arise from mammary stem cells. Recent reports have associated ALDH activity with breast cancer stem cells, but if ALDH expression is turned on during differentiation then breast cancer stem cells may in fact be the less primitive luminal progenitor. Further investigation of the temporal and spatial expression of ALDH in the breast should help explain this quandary.

Bacterial Response to Starvation Breeds Resilience

McGill University ♦ Published in Science, November 18, 2011

Biofilms are one of the primary mechanisms by which bacteria evade the toxic effect of antibiotics. Using a process known as quorum sensing bacteria can communicate amongst one another to accumulate in unison on a surface, living, synthetic, or natural. The benefits provided to the bacteria in film-formation are two-fold: firstly, the density of the bacteria reduces bacterial exposure to antibiotics, and secondly, a protective layer eventually encases the film as it exudes heavy polymeric substances.

However, there is a consequence to biofilm formation — nutrient deprivation and starvation. One would think this to be a disadvantage, but it actually further increases bacterial resistance to antibiotics. One hypothesis to explain this phenomenon is that starvation induces growth arrest and that this reduces the activity of factors that antibiotics require to kill bacteria. New findings from McGill indicate that there is more to the explanation than the passive resistance created through growth arrest. Researchers have discovered an active response to starvation, the starvation-signaling stringent response (SR), which increases tolerance to antibiotics. During times of starvation the SR mechanism is activated leading to a reduction in the burden of oxidants inside bacterial cells. A key finding in this study was that bacteria could be sensitized to antibiotics, by several orders of magnitude, by interfering with the SF mechanism.

Cystic fibrosis (CF) is a good example of a disease indication where biofilms contribute to morbidity. Aggressive Pseudomonas aeruginosa infections occur in the lungs and airways of CF patients. These infections can be treated during the early stages of the disease, however resistant biofilms lead to bacterial adaptation and chronic infection, which ultimately causes fatal complications. Future therapies for biofilms will likely involve a multi-faceted approach that target the many pathways and signaling mechanisms that lead to their formation.

Repression of Mitochondrial Translation: New Therapeutic Approach for AML

Ontario Cancer Institute ♦ Published in Cancer Cell, November 15, 2011

A chemical screen has identified the small molecule tigecycline as a potent suppressor of acute myeloid leukemia (AML). The screen involved 312 drugs that had already been approved by the FDA, both on-patent and off-patent, and focused on those that were well characterized antimicrobials or metabolic regulators. Tigecycline exhibited cytotoxic effects on two human AML cell lines, while having little effect on their normal hematopoietic counterparts. The molecule also had antileukemic activity in a mouse model of human leukemia. A genome-wide screen in yeast was carried out to elucidate tigecycline’s mechanism of action. The small molecule was found to inhibit a transcription factor. The interesting finding was that inhibition was not cytoplasmic, but instead mitochondrial. Tigecyline likely interacts with the EF-Tu transcription factor in the mitochondria, as researchers found they could mimic tigecycline’s effects by downregulating expression of EF-Tu using shRNAs.

NF-κβ1 Restrains Maturation of Dendritic Cells

Ontario Cancer Institute ♦ University of Toronto

Published in Nature Medicine, November 13, 2011

Dendritic cells (DCs) are critical for adaptive immune response, however if their proliferation becomes overzealous or their survival too prolonged they can cause autoimmunity. While most researchers have focused on the factors that stimulate DC maturation and autoimmunity, a group at the Ontario Cancer Institute has taken a different approach, and begged the question as to whether there exist factors that put a damper on DC maturation. Sure enough they recently discovered that NF-κβ1 maintains the resting state of DCs. Unstimulated DCs derived from mice lacking the Nfkb1 gene were able to activate CD8+ T lymphocytes. Injecting Nfkb1-/- DCs into mice could also produce diabetes, an autoimmune disease wherein the host’s immune system slowly eats away at insulin producing cells in the pancreas. NF-κβ1 ensures that DC production of TNF-α is regulated. Without NF-κβ1 the production of TNF-α becomes dysregulated leading to upregulation of granzyme B in T cells, which causes autoimmunity.

Human Spinal Cord-Derived NSPCs Survive Transplantation and Differentiate

Toronto Western Research Institute and Krembil Neuroscience Centre

Published in PLoS ONE, November 2, 2011

Researchers have provided a proof-of-principle that adult neural stem/progenitor cells (NSPCs) from the human spinal column can be successfully transplanted in a spinal cord injury (SCI) setting. The ability of NSPCs to engraft and differentiate in vivo was assessed with a mouse model of SCI. Following transplantation NSPCs differentiated into both neurons and glia. In order to establish NSPC lines, neural tissue samples were taken from the spine of organ transplant donors and grown in an adherent cell culture. NSPCs were selected and expanded in the presence of epidermal growth factor and fibroblast growth factor-2. Cultures can be maintained for at least 9 months and exhibit full multipotentiality in that they can differentiate into all three neural cells types, including neurons, oligodendrocytes, and astrocytes. In 2008, a research group successfully established an NSPC cell line, however was not able to scale the cells up for experimentation. This study is the first that shows NSPCs derived from the human spinal column can be scaled up for therapeutic purposes.

Genetically Modified MSCs for Acute Kidney Injury

McGill University ♦ Published in Molecular Therapy (npg), November issue

A research group has taken mesenchymal stem cells (MSCs) and enhanced them with a gene encoding erythropoietin (Epo) to show that they can be used to treat acute kidney injury (AKI). To generate a mouse model of AKI mice were injected with cisplatin, causing injury to the kidney. Epo-enhanced MSCs were then tested against unmodified MSCs for their capacity to regenerate tissue in the kidney and restore kidney function. Mice that received the Epo-enhanced MSCs exhibited improved survival and significantly better kidney function, as measured by blood levels of urea nitrogen, creatinine, and the enzymes amylase and alanine aminotransferase. Histological sections of recipient kidneys displayed less cell death and higher quantities of proliferating cells. Delivery of the Epo gene to modified MSCs causes the cells to secreate Epo protein, which is a known cytoprotective agent. This factor, in addition to the reparative effects of MSCs, produces a particularly strong regenerative effect upon transplantation.

Link Between p53 and Metabolism

York University ♦ University of Toronto ♦ Princess Margaret Hospital

Published in Molecular Cell, November 4, 2011

The tumour suppressor protein p53 has long been known to be the cell’s guardian. The protein can induce cell cycle arrest to allow for DNA repair, induce cellular senescence, or initiate signaling cascades leading to cell death if stress signals become overbearing. New findings suggest p53 is also involved in regulating metabolism on some level. A recent investigation has identified another link between p53 and metabolism, strengthening this hypothesis. Researchers found that the metabolism-regulating gene Lpin1 is partially under the control of p53. The lipin-1 protein controls the degree of fatty acid oxidation in the cell based on p53-signaling. Under normal glucose conditions, where cells can utilize glucose as a source of energy, the lipin-1 protein suppresses fatty acid oxidation. However, during times of nutritional stress lipin-1 promotes the oxidation of fatty acids to provide energy for cellular processes. In this study mouse myoblasts and human fibroblasts were cultured in conditions that induced nutritional stress. During nutrient deprivation reactive oxygen species (ROS) in the cell amass and a protein known as ATM kinase is activated, which in turn causes the activation of p53 and upregulation of Lpin1.

Native Microbiota Stave Off Infection

University of British Columbia ♦ Published in PLoS ONE, October 28, 2011

Genetics can contribute to host susceptibility to microbial infection but are there other causes? A growing body of evidence points to the composition of microbial flora in the intestine as a key factor in how mammals respond to foreign pathogens. In this study out of the University of British Columbia researchers investigated the effect that infection with a common pathogen had on a number of different strains of mice with widely varying genetic backgrounds. As would be expected the mice responded differently after oral exposure. Some mice were resistant to infection while others became very sick, or even died. However, it wasn’t only genetics at work. Transplantation of microbial samples from the intestines of resistant mice into those of lethally susceptible mice prolonged microbial colonization and death, illustrating the importance that the microbial ecosystem has in regulating mucosal immunity and intestinal health.

Selection of Cardiomyocytes with SIRPA

McEwen Centre for Regenerative Medicine (UHN) ♦ University of Toronto ♦ Monash University

Published in Nature Biotechnology, October 23, 2011

Gordon Keller’s lab has discovered a protein, known as signal-regulatory protein alpha (SIRPA), that can enrich for embryonic stem cell and iPS cell-derived cardiomyocytes at purities as high as 98%. Keller, who was the first researcher to produce functional heart cells from human embryonic stem (hES) cells, has spent years investigating the developmental path that leads to the cardiac identity. In order to find this specialized protein marker researchers carried out a screen of cardiomyocytes against a panel of some 380 CD antibodies. SIRPA was found to be expressed specifically on cardiomyocytes that were the progeny of hES cells or iPS cells; the protein will allow for rapid and efficient separation of cardiomyocytes from both primitive cell types.

Engineering Therapeutic Viruses for Greater Impact

Children’s Hospital of Eastern Ontario Research Institute ♦ University of Ottawa ♦ Ottawa Hospital Research Institute ♦ OICR ♦ McGill

Published in Cancer Cell, October 18, 2011

Viruses are now being leveraged in order to target and destroy cancer cells in the body. Termed “oncolytic” viruses, these treatments not only infect and burst cancer cells but also alert the host’s immune system to the presence of the tumour ensuring a continued onslaught following administration. In hopes of improving upon oncolytic viruses currently under development, researchers have implemented functional genomics to glean information related to virus-tumour interactions. A genome-wide RNAi screen identified the endoplasmic reticulum (ER) stress response as a mechanism by which tumour cells resist the effects imposed by oncolytic viruses. Inhibiting the ER stress response pathway sensitized resistant tumour cells and increased the efficacy of oncolytic viruses by up to 10,000 fold in some cases.

CD34: Beyond A Stem Cell Marker

Biomedical Research Centre, Vancouver ♦ University of British Columbia ♦ Sprott Centre for Stem Cell Research, Ottawa ♦ Others..

Published in Stem Cells, October 13, 2011

Despite the fact that the cell surface marker CD34 is typically used to identify a diversity of adult stem cell types, no regeneration-related function has been attributed to it. In attempts to broaden our understanding of the protein’s contribution to regenerative biological processes, researchers at the University of British Columbia probed the effect that it has on muscle regeneration in mice. Knockout mice lacking the protein still developed properly, however exhibited an inability to regenerate muscle following both acute and chronic skeletal muscle injury. Researchers attribute this defect to a shortcoming in satellite cells; a small and rare progenitor cell type found in muscle that migrates to the site of injury to proliferate and restore muscle mass. In the absence of CD34, it appears that satellite cells are unable to migrate along muscle strands to relocate themselves and stimulate the growth of new tissue.

Black Death Reconstructed

McMaster University ♦ University of Tübingen ♦ Max Planck Institute of Evolutionary Anthropology ♦ University of South Carolina ♦ Others..

Published in Nature, October 12, 2011

Scientists working out of McMaster University’s Ancient DNA Centre have managed to reconstruct the genome of Yersinia pestis. The rod-shaped microbe was responsible for the epidemic that killed an estimated 30-50% of the European population between the years 1347 and 1351; the infection was appropriately given the name the Black Death. The advent of high-powered sequencing technologies has allowed us to quickly and accurately map the genomes of ancestral microbes. Not only do these genomes provide valuable insight into their biology, they allow us to plot the evolution of pathogens and adaptations they have gathered over time. DNA samples were collected from the teeth of skeletons unearthed from old burial grounds in London. Contrary to what was originally believed, the sequenced genome revealed that the ancestral microbe was no more virulent than strains in existence today. Researchers believe the severity of the 14th century pandemic was brought on by the generally malnourished and immuno-compromised state of the population at that time. Poor hygiene and particularly wet conditions are likely to have exacerbated the spread of the microbe as well.

Ubiquitin Ligase Sweeps Aggregates

University of British Columbia ♦ Published in Nature Cell Biology, October 9, 2011

It seems that ubiquitin ligase may play a critical role in preventing diseases characterized by protein aggregation. The enzyme acts by fusing a polyubiquitin chain to misfolded proteins that sit in the cytoplasm. Once tagged, these faulty proteins are degraded by other enzymes in the proteasome. Researchers at the University of British Columbia utilized heat-shock to create short-term stores of misfolded proteins in yeast cells and demonstrated that a specific ubiquitin ligase, Hul5, is responsible for ubiquitylation and removal of low-solubility proteins. Pin2, a protein involved in the development of prion disease, was also observed to be removed by Hul5.

Key Determinant of Osteoporosis Discovered

University of Montreal ♦ McGill University ♦ Laval University ♦ Washington University School of Medicine

Published in Cell Metabolism, October 5, 2011

Researchers have discovered a factor that controls the differentiation of osteoclasts, the cell type responsible for bone resorption. Bone resorption is one of two processes that act in concert to ensure a constant bone mass in the human body. When this balance is broken the result is loss of bone density, otherwise known as osteoporosis. When resorption occurs osteoclasts break down releasing calcium into the bloodstream which in turn regulates bone formation. Inppb4α seems to regulate osteoclastogenesis by repressing the differentiation of osteoclasts. Mice deficient in Inppb4α exhibit accelerated osteoclast differentiation and bone loss. An important finding in this study was that the human gene, INPP4B, was found to be a susceptibility locus for osteoporosis, confirming the functional analysis carried out in mice.

Antisense Oligos for Huntington Disease

University of British Columbia ♦ Isis Pharmaceuticals ♦ University of Copenhagen

Published in Molecular Therapy, October 4, 2011

Huntington disease (HD) is characterized by a CAG nucleotide expansion in the Huntington gene (HTT). Hence, targeting the HTT gene has been a desirable therapeutic approach. However, the wild-type (normal and healthy) HTT gene is crucial for neuronal development and survival so down regulating its expression can have deleterious effects. To circumvent this researchers created gene-silencing antisense oligonucleotides (ASOs) that target small variable regions known as single nucleotide polymorphisms (SNPs) found in the HD population. ASOs were modified with S-constrained ethyl (cET) motifs to ensure selectivity of target sites. Delivery of ASOs to the mouse brain demonstrated allele-specific knockdown of the HTT protein in vivo.

Optimizing Lipid Nanoparticles for Delivery of Therapeutic Payload

University of British Columbia ♦ Published in Molecular Therapy, October 4, 2011

One of the most effective means to deliver therapeutic payloads is to drop them into lipid nanoparticles (LNPs) for in vivo delivery. LNPs are easier to manufacture than viral vectors and have lower immunogenicity, making LNP delivery systems an attractive option. In this study researchers investigated the potency of RNAi on primary bone macrophages and dendritic cells using any one of four cationic lipids to build LNPs. Of the four lipids used, DLinkC2-DMA provided the most potent intracellular delivery in vitro and in vivo. To demonstrate the efficacy of DLinkC2-DMA-containing LNPs researchers loaded them with GAPDH-siRNA and administered them intravenously to mice. Significant inhibition of GAPDH and CD45 was observed in spleen and peritoneal macrophages and dendritic cells in relation to LNPs containing other lipid types.

Long Non-Coding RNAs, Dark Matter Comes into Light

British Columbia Cancer Agency Research Centre ♦ University of British Columbia

Published in PLoS ONE, October 3, 2011

Researchers have had a change of heart with respect to the contribution that long non-coding RNAs (LncRNAs) have to cancer formation. LncRNAs do not code for proteins, instead serving structural and functional roles in the cell. Due to the fact that they are not actually translated to proteins, LncRNAs have been dubbed the ‘dark matter’ of the transcriptome. Conservative estimates put their numbers somewhere in the vicinity of 23,000 transcripts. LncRNAs have been implicated in at least two cancer types, breast cancer and lung cancer, but this paper is the first large scale analysis of their contribution to human cancers. By comparing 26 different normal human tissues and 19 human cancers, researchers show that in many cancers LncRNAs are aberrantly expressed and may not be as innocuous as most think. Today’s current commercial microarrays used for transcriptome profiling focus on coding regions of the transcriptome. We may now have to place more emphasis on non-coding regions in order to capture a more comprehensive view of how the transcriptome can contribute to the development of cancer.

Cell Polarity Dictated by Phosphatidylserine

Hospital for Sick Children ♦ Published in Nature Cell Biology, October 2, 2011

The enzyme Cdc42 is responsible in part for cell polarization during asymmetric cellular events. In the case of the yeast S. cerivisiae, polarization is essential during the process of budding and the formation of projections in response to mating factors. Polarization is also crucial to the operation of mammalian cells. In this Nature paper researchers show that phosphatidylserine, a type of phospholipid, is required for the correct dispersal of Cdc42 during polarization events. In an event that precedes the movement of Cdc42, phosphatidylserine is sent via secretory vesicles to the plasma membrane where it accumulates. Yeast mutants lacking phosphatidylserine synthase exhibited impairment in the polarization of Cdc42 and difficulty in budding and mating, suggesting it is at the core of regulating cellular polarization and some very important cellular processes.

Embryonic Stem Cell-Specific Splicing Event Promotes Pluripotency

University of Toronto ♦ Mount Sinai Hospital ♦ Columbia University Medical Centre ♦ Published in Cell, September 30, 2011

There are a host of genes that are upregulated in embryonic stem cells which promote stem cell-like characteristics, namely pluripotency, or the ability to give rise to all cell types in the human body. These genes include Sox2, Oct4, and Nanog, among others, and are associated with primitive cell types. Much time has been invested in researching the transcriptional networks promoting pluripotency, and how the various stem cell-specific transcription factors cross-talk with one another to ensure stem cells do not differentiate. Although these transcription factors can cross-regulate each other, on a more fundamental level they are controlled by the forkhead box (FOX) transcription factors. In this study, researchers discovered that FOX transcription factors do not always operate in the same manner. In stem cells, the FOXP1 mRNA transcript is alternatively spliced in such a manner that the FOXP1 protein has a greater affinity for the DNA promoter regions that control transcription of stem cell factors like Sox2, Oct4, and Nanog. FOXP1 also represses genes that cause differentiation. Interestingly, the FOXP1 isoform studied here could not be found in more differentiated cells, suggesting that alternative splicing is an important mechanism by which stem cells maintain their primitive status.

The Dynamic Immunopeptidome

University of Montreal ♦ Published in Molecular Systems Biology, September 27, 2011

The human body has developed a complex system in which to identify self and non-self. The large majority of nucleated cells in the body display small peptides on the cell surface known as MHC I-associated peptides (MIPs). Although past theories have been conducive to the view that the creation and expression of these peptides remains the same regardless of cellular conditions, researchers now believe that the pattern of MIPs presented on a cell may in fact be dynamic and change according to metabolic conditions. Taking a high-throughput mass spectrometry approach, researchers investigated changes that occurred in the immunopeptidome of a mouse lymphoma cell line. In order to alter metabolism, cells in the treatment group were exposed to rapamycin, a potent inhibitor of the kinase mTOR which is known to regulate cellular homeostasis. Rapamycin induced wide-sweeping changes in the MIP composition on the cell surface. A major finding in this study is that the immunopeptidome displayed on the cell surface actually provides a representation of the various biological events occurring within the cell.

Calcium Channel Key to T Cell Development

University of British Columbia ♦ University of Calgary ♦ Oxford University ♦ Published in Immunity, September 23, 2011

Calcium channels are critical players in physiology, development, and cell metabolism. Researchers have identified a specific calcium channel, CaV1.4, that is essential for channeling calcium ions into the cytoplasm of naive T cells. Intracellular calcium stores control signaling, differentiation, and activation. Researchers also found that CaV1.4 is in part responsible for modulating T cell receptor induced rises in calcium stores. In order to investigate the effects that CaV1.4 has on cell homeostasis researchers used a method known as patch clamp. This technique can record the conductance of single cells. Measurements of thymocytes and peripheral T cells from mice deficient in the CaV1.4 channel indicate that the channel both increases intracellular calcium stores on its own and affects the ability of T cell receptors to elevate intracellular calcium stores. Findings also indicate that CaV1.4 may to a certain degree control survival of developing T cells.

Also see this review published in Trends in Biotechnology, on recent methods used to understand host-virus interactions and identify novel targets for diagnostic and therapeutic applications.