CARG-2020, an oncolytic artificial virus co-delivering three immunomodulators, to regress and cure established tumors in mice.

e14560 Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in humans. Treatment of late-stage CRC remains ineffective, even with the use of latest immunotherapies. Oncolytic viruses have shown limited use for the treatment of cancers, and further improvement of these agents with immune-modulating activities may prove crucial for patients with CRC and other malignancies. To this end, we developed CARG-2020 as an artificial virus for infectious diseases and immuno-oncology (AVIDIO) that employs virus-like vesicles (VLV). VLVs, which are membrane-encapsulated RNA replicons, are oncolytic and can deliver multiple genes resulting in the modulation of several independent immune pathways. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki Forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Unarmed or empty vector (VLV), VLV armed with IL-12 (VLV-IL-12) and a VLV that simultaneously expresses IL-12, a dominant-negative form of IL-17 receptor A (dn-1L17RA) and shRNAs targeting PD-L1 (CARG-2020), were given intratumorally to test their therapeutic potential against established (500-600mm3) MC38 tumors in mice. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with these agents to monitor their antitumor and immunomodulatory activities, respectively. Results: Both VLV-IL-12 and CARG-2020 regressed the tumors to undetectable levels in most mice harboring syngeneic MC38 tumors when given intratumorally. CARG-2020, carrying IL-12, dn-IL17RA and PD-L1 shRNA, exerts broader spectrum of immuno-responses and higher number of complete response rates compared with VLV-IL-12. Treatment of primary tumors with VLV-IL-12 or CARG-2020 also significantly repressed the growth of secondary tumors on the other flank of mouse’s body, suggesting an effective systemic immunity elicited by these two agents against the same type of tumors. In addition to the marked local and systemic activation of Th1 cells and CD8+ T cells by both vectors, CARG-2020 also downregulated PD-L1 expression in tumors, and suppressed expression of IL-17A-activated chemokines CXCL1 and CXCL2 that are known to promote cancer development and therapy resistance. Conclusions: As an oncolytic RNA replicon, AVIDIO platform-derived CARG-2020 encodes IL-12, dn-IL-17RA and PD-L1 shRNAs which are expressed concurrently within the same vector. CARG-2020 modulates IL-12, IL-17RA and PD-L1 signaling, and exerts broad immune modulation in tumor microenvironment. Treatment with CARG-2020 eliminates the majority of grafted large tumors in mice, and is effective against both primary and distal tumors. Based on this impressive efficacy results, further development of CARG-2020 in colorectal patients is warranted.

CRISPR-guided programmable self-assembly of artificial virus-like nucleocapsids

Designer virus-inspired proteins drive the manufacturing of more effective and safer gene-delivery systems as well as simpler models to study viral assembly. However, the self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate was further increased when dCas12a was dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus non-cognate DNA templates by the synthetic polypeptide. Our CRISPR-guided VLPs could help develop programmable bio-inspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.

Coronavirus-19 in the Democratic Republic of Congo: Public Views, Attitudes, and Beliefs in an Unaffected Area: The Case of the City of Mbujimayi

The coronavirus disease (COVID-19) pandemic is rapidly spreading across the world. In Democratic Republic of Congo (DRC), 11 out of 26 provinces have been affected on 19 June 2020. The purpose of this study was to assess the public views, attitudes and beliefs related to the COVID-19 pandemic among the population of an unaffected city.Methods: This is a descriptive observational study conducted in an unaffected city using a questionnaire. Findings: A total of 769 people participated. The average age was 36 ±14 years. The sex ratio (male: female) was 1.6. The majority of respondents (97%) is aware of the existence of the COVID-19 pandemic, it is well informed about the signs of the disease, its severity and barrier measures, however few of them have adopted the change in habit and behavior in respect of the barrier measures. Lack of water for handwashing is the most common difficulty in complying with barrier measures (71%). The COVID-19 is considered as an artificial virus designed to reduce the world population by 26%, God’s punishment (22%). Only 36% of the participants are in favor of vaccination against COVID-19. Vaccine is, a western conspiracy to spread COVID-19 in Africa (38%), testing it on Africans (35%), sterilize Africans’ people (12%).Conclusions: Cities not yet affected by COVID-19 in DRC are aware of the existence of the disease, but insufficiently prepared to deal with it. It is important to increase awareness on barrier measures, combating false information, and improving regular water supply for regular handwashing.

EVALUATION OF ANTITUMOR ACTIVITY OF TUMOR NECROSIS FACTOR ALPHA WITHIN THE ARTIFICIAL VIRUS-LIKE PARTICLE

Introduction. Tumor necrosis factor α (TNF-α) is a natural cytokine, characterized by pronounced antitumor properties. A wide range of side effects serves as an obstacle for the use of TNF-α in clinical practice. One of the ways to improve its therapeutic properties is to increase the tropism of the cytokine to the tumor tissue by incorporating it into the targeted delivery system.The aim of the study was to evaluate the antitumor activity of the preparation containing TNF-α as part of the artificial “virus-like particle” (VLP-TNF-α), developed in SRC VB “Vector” as a transport system for delivering proteins to target cells.Materials and methods. The antitumor effect of VLP-TNF-α preparation was evaluated in experimental B16F10 melanoma model by the change of dynamics of tumor growth (volume, mass) and its morphological structure (presence of necrotic processes, blood vessel destruction). The number of the effector immune cells (CD3+, CD11b+) in the tumor tissue was determined by immunohistochemical method.Results. It has been shown that VLP-TNF-α administered intravenously at the doses of 5 × 104 and 1 × 105 IU/mouse inhibits the growth of the primary tumor. The most pronounced and stable effect was observed with a five-fold administration at the dose of 1 × 105 IU/mouse every other day: tumor growth inhibition was 40 % on the 1st day, and 47 % on the 7 th day upon the treatment. Injections of the preparation resulted in the increase of necrosis number, destruction level of the tumor tissue, development of damage and destruction of the tumor blood vessels and its infiltration with immunocompetent cells.Conclusion. The obtained data indicates that TNF-α within the delivery system exerts antitumor activity, which suggests the possibility of its further use for the treatment of malignant neoplasms, in particular, melanoma.The study was performed in accordance with ethical principles adopted by the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes.

Transport of artificial virus-like nanocarriers through intestinal monolayers via microfold cells

Compared with subcutaneous or intramuscular routes for vaccination, vaccine delivery via the gastrointestinal mucosa has tremendous potential as it is easy to administer and pain-free.