Response of a chemo-resistant triple-negative breast cancer patient to a combination of p62-encoding plasmid, Elenagen, and CMF chemotherapy.

Triple-negative breast cancers are often characterized by aggressive behavior and short clinical course once they become chemotherapy-resistant. We describe below a patient who has shown a response to combination of chemotherapy with Elenagen, a plasmid encoding p62. Elenagen was tested in a previous phase I/II study in patients with refractory solid tumors and shown to be safe. Also, plasmid ability to halt tumor progression and restore sensitivity to chemotherapy was found. Preclinical data supports effects on tumor grade and change the tumor's microenvironment in spontaneous canine breast cancers. We describe here a 48-year old female with triple-negative and BRCA1/2-negative breast cancer who had a primary resistance to chemotherapy and negative dynamics despite the use of multiple lines of treatments. Elenagen was applied intramuscularly at a dose of 1 mg weekly in combination with standard chemotherapy scheme CMF (cyclophosphamide, methotrexate, fluorouracil). In this patient we observed partial tumor regression (by 33%) and 19 weeks of progression-free survival. This first observed objective response to a combination of Elenagen with chemotherapy demonstrates that even in heavily pretreated chemo-resistant triple-negative tumor, the addition of Elenagen to a chemotherapy regimen can cause an objective response and increase in progression-free survival. Such a regimen is worthy of further study in a larger number of patients.

p62 /SQSTM1 coding plasmid prevents age related macular degeneration in a rat model.

P62/SQSTM1, a multi-domain protein that regulates inflammation, apoptosis, and autophagy, has been linked to age-related pathologies. For example, previously we demonstrated that administration of p62/SQSTM1-encoding plasmid reduced chronic inflammation and alleviated osteoporosis and metabolic syndrome in animal models. Herein, we built upon these findings to investigate effect of the p62-encoding plasmid on an age-related macular degeneration (AMD), a progressive neurodegenerative ocular disease, using spontaneous retinopathy in senescence-accelerated OXYS rats as a model. Overall, the p62DNA decreased the incidence and severity of retinopathy. In retinal pigment epithelium (RPE), p62DNA administration slowed down development of the destructive alterations of RPE cells, including loss of regular hexagonal shape, hypertrophy, and multinucleation. In neuroretina, p62DNA prevented gliosis, retinal thinning, and significantly inhibited microglia/macrophages migration to the outer retina, prohibiting their subretinal accumulation. Taken together, our results suggest that the p62DNA has a strong retinoprotective effect in AMD.

Safety and efficacy of p62 DNA vaccine ELENAGEN in a first-in-human trial in patients with advanced solid tumors.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy.

P62 plasmid can alleviate diet-induced obesity and metabolic dysfunctions.

A high-calorie diet (HCD) induces two mutually exacerbating effects contributing to diet-induced obesity (DIO): impaired glucose metabolism and increased food consumption. A link between the metabolic and behavioral manifestations is not well understood yet. We hypothesized that chronic inflammation induced by HCD plays a key role in linking together the two components of diet-induced pathology. Based on this hypothesis, we tested if a plasmid (DNA vaccine) encoding p62 (SQSTM1) would alleviate DIO including its metabolic and/or food consumption abnormalities. Previously we reported that injections of the p62 plasmid reduce chronic inflammation during ovariectomy-induced osteoporosis. Here we found that the p62 plasmid reduced levels of pro-inflammatory cytokines IL-1ß, IL-12, and INFγ and increased levels of anti-inflammatory cytokines IL-4, IL-10 and TGFß in HCD-fed animals. Due to this anti-inflammatory response, we further tested whether the plasmid can alleviate HCD-induced obesity and associated metabolic and feeding impairments. Indeed, p62 plasmid significantly reversed effects of HCD on the body mass index (BMI), levels of glucose, insulin and glycosylated hemoglobin (HbA1c). Furthermore, p62 plasmid partially restored levels of the satiety hormone, serotonin, and tryptophan, simultaneously reducing activity of monoamine oxidase (MAO) in the brain affected by the HCD. Finally, the plasmid partially reversed increased food consumption caused by HCD. Therefore, the administering of p62 plasmid alleviates both metabolic and behavioral components of HCD-induced obesity.

Mental inertia in the biological sciences.

Often, new discoveries are not made at the moment when all of the necessary techniques and background knowledge become available. Instead, they are delayed as a result of mental inertia unrecognized by the scientist and/or the scientific community. In this paper, I introduce and classify various types of mental inertia that are common in science, using examples from the field of biology.

Importance of mRNA secondary structural elements for the expression of influenza virus genes.

Development of novel vaccines and therapeutics often requires efficient expression of recombinant viral proteins. Here we show that mutations in essential functional regions of conserved influenza proteins NP and NS1, lead to reduced expression of these genes in vitro. According to in silico analysis, these mRNA regions possess distinct secondary structures sensitive to mutations. We identified a novel structural feature within a region in NS1 mRNA that encodes amino acids essential for NS1 function. Mutations altering this mRNA element lead to significantly reduced protein expression. Conversely, expression was not affected by mutations resulting in amino acid substitutions, when they were designed to preserve this secondary RNA structural element. Furthermore, altering this structure significantly reduced RNA transcription without affecting mRNA stability. Therefore, distinct internal secondary structures of viral mRNA may be important for viral gene expression. If such elements encode amino acids essential for the protein function, then early selection against mutations in this region will be beneficial for the virus. This might point at yet another mechanism of viral evolution, especially for RNA viruses. Finally, introducing mutations into viral genes while preserving their secondary RNA structure, suggests a new method for the generation of efficiently expressed recombinants of viral proteins.

Effective expression of recombinant cytotoxic protein via its attachment to a polyglutamine domain.

Inadvertent cytotoxicity may hinder the expression of many recombinant proteins that are of industrial or medicinal importance. Here, we show that covalent binding of the influenza A cytotoxic protein M2 to a polyglutamine domain (polyQ-M2; QM2) results in significant delay of its cytotoxic effects when compared to wild-type protein (M2wt). We also show that while expression of recombinant M2wt from A/WSN/1933 strain could not be attained in vaccinia virus (VV), polyQ-M2 was successfully expressed in this system. Moreover, we demonstrate that in cell culture, the polyQ domain is cleaved off following 48 h of expression, thus releasing free and active M2. Similarly, we show the spontaneous cleavage and polyQ release from fusion with another distinct polypeptide, green fluorescent protein (GFP). Expression of M2 from QM2 construct was more prolonged than one based on M2wt-expressing construct, markedly exceeding it at the later time points. Therefore, cell death caused by a toxic polypeptide may be suppressed via genetic fusion with polyQ, resulting in its enhanced expression, followed by slow release of the free polypeptide from the fusion. Collectively, covalent fusion with polyQ or other aggregate-forming domains presents a novel approach for industrial production of cytotoxic proteins and also holds promise for gene therapy applications.

Four stages of a scientific discipline; four types of scientist.

In this article I propose the classification of the evolutionary stages that a scientific discipline evolves through and the type of scientists that are the most productive at each stage. I believe that each scientific discipline evolves sequentially through four stages. Scientists at stage one introduce new objects and phenomena as subject matter for a new scientific discipline. To do this they have to introduce a new language adequately describing the subject matter. At stage two, scientists develop a toolbox of methods and techniques for the new discipline. Owing to this advancement in methodology, the spectrum of objects and phenomena that fall into the realm of the new science are further understood at this stage. Most of the specific knowledge is generated at the third stage, at which the highest number of original research publications is generated. The majority of third-stage investigation is based on the initial application of new research methods to objects and/or phenomena. The purpose of the fourth stage is to maintain and pass on scientific knowledge generated during the first three stages. Groundbreaking new discoveries are not made at this stage. However, new ways to present scientific information are generated, and crucial revisions are often made of the role of the discipline within the constantly evolving scientific environment. The very nature of each stage determines the optimal psychological type and modus operandi of the scientist operating within it. Thus, it is not only the talent and devotion of scientists that determines whether they are capable of contributing substantially but, rather, whether they have the 'right type' of talent for the chosen scientific discipline at that time. Understanding the four different evolutionary stages of a scientific discipline might be instrumental for many scientists in optimizing their career path, in addition to being useful in assembling scientific teams, precluding conflicts and maximizing productivity. The proposed model of scientific evolution might also be instrumental for society in organizing and managing the scientific process. No public policy aimed at stimulating the scientific process can be equally beneficial for all four stages. Attempts to apply the same criteria to scientists working on scientific disciplines at different stages of their scientific evolution would be stimulating for one and detrimental for another. In addition, researchers operating at a certain stage of scientific evolution might not possess the mindset adequate to evaluate and stimulate a discipline that is at a different evolutionary stage. This could be the reason for suboptimal implementation of otherwise well-conceived scientific policies.

Development of a vaccine against pandemic influenza viruses: current status and perspectives.

The constant threat of a new influenza pandemic, which may be caused by a highly pathogenic avian influenza virus, necessitates the development of a vaccine capable of providing efficient, long-term, and cost-effective protection. Proven avenues for the development of vaccines against seasonal influenza as well as novel approaches have been explored over the past decade. Whereas significant insights are consistently being made, the generation of a highly efficient and cross-protective vaccine against the future pandemic influenza strain remains as the ultimate goal in the field. In this review, we re-examine these efforts and outline the scientific, political, and economic problems that befall this area of biotechnological research.

The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components.

It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency.

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.

Toxicity of influenza A virus matrix protein 2 for mammalian cells is associated with its intrinsic proton-channeling activity.

Molecules of influenza matrix protein 2 (M2) are organized in tetramers that constitute a well-conserved virion component and also form proton channels in the plasma membrane of infected cells. In this report we demonstrate that influenza M2 protein is cytopathic in vitro for mammalian cells. An M2 point-mutant (M2pm) protein was constructed that contained amino acid changes designed to block the proton channel via introduction of large hydrophobic residues. This mutant was significantly less toxic upon transient transfection in vitro than the wild-type M2 (M2wt). To assess the possible correlation between M2 cytotoxicity and its proton channel activity, we monitored changes in mitochondria membrane potential induced by M2wt and M2pm. M2wt rapidly decreased mitochondria membrane potential reflecting the transmembrane proton gradient, while M2pm was markedly less efficient. Thus, M2 is cytotoxic for mammalian cells, likely via its proton channel activity and may therefore contribute to influenza pathogenesis through this previously unknown mechanism.

Protection against mouse and avian influenza A strains via vaccination with a combination of conserved proteins NP, M1 and NS1.

BACKGROUND: Experimental data accumulated over more than a decade indicate that cross-strain protection against influenza may be achieved by immunization with conserved influenza proteins. At the same time, the efficacy of immunization schemes designed along these lines and involving internal influenza proteins, mostly NP and M1, has not been sufficient. OBJECTIVE: To test the immunogenicity and protective efficacy of DNA vaccination with a combination of NP, M1 and NS1 genes of influenza virus. METHODS: The immunogenicity and protective efficacy of DNA vaccination with NP, M1 and NS1 was tested in mice and chickens. Mice were challenged with mouse-adapted viral strains H3N2 and H5N2 and chicken challenged with avian H5N3 virus. RESULTS: In these settings, wild-type NS1 did not impede the cellular and humoral response to NP/M1 immunization in vivo. Moreover, addition of NS1-encoding plasmid to the NP/M1 immunization protocol resulted in a significantly increased protective efficacy in vivo. CONCLUSIONS: The addition of NS1 to an influenza immunization regimen based on conserved proteins bears promise. It is feasible that upon further genetic modification of these and additional conserved influenza proteins, providing for their higher safety, expression and immunogenicity, a recombinant vaccine based on several structural and non-structural proteins or their epitopes will offer broad anti-influenza protection in a wide range of species.