Browsing by Subject "GENE-TRANSFER"
Now showing items 1-17 of 17
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(2019)Horizontal gene transfer is an essential component of bacterial evolution. Quantitative information on transfer rates is particularly useful to better understand and possibly predict the spread of antimicrobial resistance. A variety of methods has been proposed to estimate the rates of plasmid-mediated gene transfer all of which require substantial labor input or financial resources. A cheap but reliable method with high-throughput capabilities is yet to be developed in order to better capture the variability of plasmid transfer rates, e.g. among strains or in response to environmental cues. We explored a new approach to the culture-based estimation of plasmid transfer rates in liquid media allowing for a large number of parallel experiments. It deviates from established approaches in the fact that it exploits data on the absence/presence of transconjugant cells in the wells of a well plate observed over time. Specifically, the binary observations are compared to the probability of transconjugant detection as predicted by a dynamic model. The bulk transfer rate is found as the best-fit value of a designated model parameter. The feasibility of the approach is demonstrated on mating experiments where the RP4 plasmid is transfered from Serratia marcescens to several Escherichia coil recipients. The methods uncertainty is explored via split sampling and virtual experiments.
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(2021)Oncolytic adenoviruses are promising cancer therapeutic agents. Clinical data have shown adenoviruses' ability to transduce tumors after systemic delivery in human cancer patients, despite antibodies. In the present work, we have focused on the interaction of a chimeric adenovirus Ad5/3 with human lymphocytes and human erythrocytes. Ad5/3 binding with human lymphocytes and erythrocytes was observed to occur in a reversible manner, which allowed viral transduction of tumors, and oncolytic potency of Ad5/3 in vitro and in vivo,with or without neutralizing antibodies. Immunodeficient mice bearing xenograft tumors showed enhanced tumor transduction following systemic administration, when Ad5/3 virus was bound to lymphocytes or erythrocytes (P <0.05). In conclusion, our findings reveal that chimeric Ad5/3 adenovirus reaches non-injected tumors in the presence of neutralizing antibodies: it occurs through reversible binding to lymphocytes and erythrocytes.
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(2019)Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.
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(2022)Midbrain dopamine neurons deteriorate in Parkinson's disease (PD) that is a progressive neurodegenerative movement disorder. No cure is available that would stop the dopaminergic decline or restore function of injured neurons in PD. Neurotrophic factors (NTFs), e.g., glial cell line-derived neurotrophic factor (GDNF) are small, secreted proteins that promote neuron survival during mammalian development and regulate adult neuronal plasticity, and they are studied as potential therapeutic agents for the treatment of neurodegenerative diseases. However, results from clinical trials of GDNF and related NTF neurturin (NRTN) in PD have been modest so far. In this review, we focus on cerebral dopamine neurotrophic factor (CDNF), an unconventional neurotrophic protein. CDNF delivered to the brain parenchyma protects and restores dopamine neurons in animal models of PD. In a recent Phase I-II clinical trial CDNF was found safe and well tolerated. CDNF deletion in mice led to age-dependent functional changes in the brain dopaminergic system and loss of enteric neurons resulting in slower gastrointestinal motility. These defects in Cdnf(-/-) mice intriguingly resemble deficiencies observed in early stage PD. Different from classical NTFs, CDNF can function both as an extracellular trophic factor and as an intracellular, endoplasmic reticulum (ER) luminal protein that protects neurons and other cell types against ER stress. Similarly to the homologous mesencephalic astrocyte-derived neurotrophic factor (MANF), CDNF is able to regulate ER stress-induced unfolded protein response (UPR) signaling and promote protein homeostasis in the ER. Since ER stress is thought to be one of the pathophysiological mechanisms contributing to the dopaminergic degeneration in PD, CDNF, and its small-molecule derivatives that are under development may provide useful tools for experimental medicine and future therapies for the treatment of PD and other neurodegenerative protein-misfolding diseases.
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(2016)In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue. (C) 2016 Elsevier Inc. All rights reserved.
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(2017)Prokaryotic evolution is affected by horizontal transfer of genetic material through recombination. Inference of an evolutionary tree of bacteria thus relies on accurate identification of the population genetic structure and recombination-derived mosaicism. Rapidly growing databases represent a challenge for computational methods to detect recombinations in bacterial genomes. We introduce a novel algorithm called fastGEAR which identifies lineages in diverse microbial alignments, and recombinations between them and from external origins. The algorithm detects both recent recombinations (affecting a few isolates) and ancestral recombinations between detected lineages (affecting entire lineages), thus providing insight into recombinations affecting deep branches of the phylogenetic tree. In simulations, fastGEAR had comparable power to detect recent recombinations and outstanding power to detect the ancestral ones, compared with state-of-the-art methods, often with a fraction of computational cost. We demonstrate the utility of the method by analyzing a collection of 616 whole-genomes of a recombinogenic pathogen Streptococcus pneumoniae, for which the method provided a high-resolution view of recombination across the genome. We examined in detail the penicillin-binding genes across the Streptococcus genus, demonstrating previously undetected genetic exchanges between different species at these three loci. Hence, fastGEAR can be readily applied to investigate mosaicism in bacterial genes across multiple species. Finally, fastGEAR correctly identified many known recombination hotspots and pointed to potential new ones. Matlab code and Linux/Windows executables are available at https://users.ics.aalto.fi/similar to pemartti/fastGEAR/ (last accessed February 6, 2017).
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(2021)Dendritic cell (DC)-based vaccines have shown some degree of success for the treatment of prostate cancer (PC). However, the highly immunosuppressive tumor microenvironment leads to DC dysfunction, which has limited the effectiveness of these vaccines. We hypothesized that use of a fully serotype 3 oncolytic adenovirus (Ad3-hTERT-CMV-hCD40L; TILT-234) could stimulate DCs in the prostate tumor microenvironment by expressing CD40L. Activated DCs would then activate cytotoxic T cells against the tumor, resulting in therapeutic immune responses. Oncolytic cell killing due to cancer cell-specific virus replication adds to antitumor effects but also enhances the immunological effect by releasing tumor epitopes for sampling by DC, in the presence of danger signals. In this study, we evaluated the companion effect of Ad3-hTERT-CMV-hCD40L and DC-therapy in a humanized mouse model and PC histocultures. Treatment with Ad3-hTERT-CMV-hCD40L and DC resulted in enhanced antitumor responses in vivo. Treatment of established histocultures with Ad3-hTERT-CMV-hCD40L induced DC maturation and notable increase in proinflammatory cytokines. In conclusion, Ad3-hTERT-CMV-hCD40L is able to modulate an immunosuppressive prostate tumor microenvironment and improve the effectiveness of DC vaccination in PC models and patient histocultures, setting the stage for clinical translation.
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(2020)In this review, we discuss the use of oncolytic viruses in cancer immunotherapy treatments in general, with a particular focus on adenoviruses. These serve as a model to elucidate how versatile viruses are, and how they can be used to complement other cancer therapies to gain optimal patient benefits. Historical reports from over a hundred years suggest treatment efficacy and safety with adenovirus and other oncolytic viruses. This is confirmed in more contemporary patient series and multiple clinical trials. Yet, while the first viruses have already been granted approval from several regulatory authorities, room for improvement remains. As good safety and tolerability have been seen, the oncolytic virus field has now moved on to increase efficacy in a wide array of approaches. Adding different immunomodulatory transgenes to the viruses is one strategy gaining momentum. Immunostimulatory molecules can thus be produced at the tumor with reduced systemic side effects. On the other hand, preclinical work suggests additive or synergistic effects with conventional treatments such as radiotherapy and chemotherapy. In addition, the newly introduced checkpoint inhibitors and other immunomodulatory drugs could make perfect companions to oncolytic viruses. Especially tumors that seem not to be recognized by the immune system can be made immunogenic by oncolytic viruses. Logically, the combination with checkpoint inhibitors is being evaluated in ongoing trials. Another promising avenue is modulating the tumor microenvironment with oncolytic viruses to allow T cell therapies to work in solid tumors. Oncolytic viruses could be the next remarkable wave in cancer immunotherapy.
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(2018)Glial cell line-derived neurotrophic factor (GDNF) is one of the most studied neurotrophic factors. GDNF has two splice isoforms, full-length pre-alpha-pro-GDNF (u-GDNF) and pre-beta-pro-GDNF (beta-GDNF), which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the u-GDNF isoform, and nothing is known about the in vivo effects of the shorter beta-GDNF variant. Here we compare for the first time the effects of overexpressed cx-GDNF and beta-GDNF in non-lesioned rat striatum and the partial 6-hydroxydopamine lesion model of Parkinson's disease. GDNF isoforms were overexpressed with their native pre-pro-sequences in the striatum using an adeno-associated virus (AAV) vector, and the effects on motor performance and dopaminergic phenotype of the nigrostriatal pathway were assessed. In the non-lesioned striatum, both isoforms increased the density of dopamine transporter-positive fibers at 3 weeks after viral vector delivery. Although both isoforms increased the activity of the animals in cylinder assay, only u-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH)-immunoreactivity was decreased in both u-GDNF and 1-GDNF treated animals. In the neuroprotection assay, both GDNF splice isoforms increased the number of TH-immunoreactive cells in the substantia nigra but did not promote behavioral recovery based on amphetamine-induced rotation or cylinder assays. Thus, the shorter GDNF isoform, beta-GDNF, and the full-length alpha-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.
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(2015)The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allowsa timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.
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Systemic Delivery of Oncolytic Adenovirus to Tumors Using Tumor-Infiltrating Lymphocytes as Carriers (2021)Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.
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(2016)Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes ( core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer ( variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total ( the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides ( EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat ( CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core ( shared) proteins matched with the clade distribution obtained from the presence-absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains' core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification.
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(2019)Many functions of eukaryotic cells are compartmentalized within membrane-bound organelles. One or more cis-encoded signals within a polypeptide sequence typically govern protein targeting to and within destination organelles. Perhaps unexpectedly, organelle targeting does not occur with high specificity, but instead is characterized by considerable degeneracy and inefficiency. Indeed, the same peptide signals can target proteins to more than one location, randomized sequences can easily direct proteins to organelles, and many enzymes appear to traverse different subcellular settings across eukaryotic phylogeny. We discuss the potential benefits provided by flexibility in organelle targeting, with a special emphasis on horizontally transferred and de novo proteins. Moreover, we consider how these new organelle residents can be protected and maintained before they contribute to the needs of the cell and promote fitness.
