The synthetic compounds, Tilorone and Cridanimod, have the antiviral activity which initially had been ascribed to the capacity to induce interferon. Both drugs induce interferon in mice but not in humans. This study investigates whether these compounds have the antiviral activity in mice and rats since rats more closely resemble the human response. Viral-infection models were created in CD-1 mice and Wistar rats. Three strains of Venezuelan equine encephalitis virus were tested for the performance in these models. One virus strain is the molecularly cloned attenuated vaccine. The second strain has major virulence determinants converted to the wild-type state which are present in virulent strains. The third virus has wild-type virulence determinants, and in addition, is engineered to express green fluorescent protein. Experimentally infected animals received Tilorone or Cridanimod, and their treatment was equivalent to the pharmacopoeia-recomended human treatment regimen. Tilorone and Cridanimod show the antiviral activity in mice and rats and protect the mice from death. In rats, both drugs diminish the viremia. These drugs do not induce interferon-alpha or interferon-beta in rats. The presented observations allow postulating the existence of an interferon-independent and species-independent mechanism of action.

CAR-T cell therapy represents a breakthrough in cancer treatment, yet its implementation in developing countries remains challenging due to technical and infrastructural barriers. This study aimed to establish clinical-scale CAR-T production in Kazakhstan, a country with no prior experience in advanced cell and gene therapies. We implemented a complete CAR-T manufacturing pipeline, including in-house lentiviral vector (LV) production and automated CAR-T cell processing using the CliniMACS Prodigy system. Two anti-CD19 CAR LVs were used, one modeled after FDA-approved Kymriah (4-1BB costimulation) and another replicating Yescarta (CD28 costimulation). The vector produced locally achieved functional titers of 1.5 × 10¹⁰ TU/mL after concentration. Twelve clinical-scale CAR-T products were manufactured, exhibiting a memory-skewed T-cell phenotype. Functional assessments revealed that CD28-based CAR-T cells produced significantly higher Th1 cytokines (IFN-γ, TNF-α, IL-2; p < 0.05) than 4-1BB-based cells, though both demonstrated comparable cytotoxicity against CD19+ targets. These findings demonstrate the feasibility of establishing CAR-T production in resource-limited settings using a decentralized manufacturing framework. This work provides a scalable model of CAR-T therapy production in developing regions, suitable for clinical implementation using the hospital exemption framework. Critical gaps in access to advanced immunotherapies, including CAR-T, in the Central Eurasia region are addressed.

Transient gene expression (TGE) is commonly employed for protein production, but its reliance on plasmid transfection makes it challenging to scale up. In this paper, an alternative TGE method is presented, utilizing pseudoinfectious alphavirus as an expression vector. Pseudoinfectious viruses (PIV) and a replicable helper construct were derived from the genome of the Venezuelan equine encephalitis virus. The PIV carries a mutant capsid protein that prevents packaging into infectious particles, while the replicable helper encodes a wild-type capsid protein but lacks other viral structural proteins. Although PIV and the helper cannot independently spread infection, their combination results in increased titers in cell cultures, enabling easier scale-up of producing cultures. The PIV-driven production of a model protein outperforms that of alphavirus replicon vectors or simple plasmid vectors. Another described feature of the expression system is the modification to immobilized metal affinity chromatography (IMAC), allowing purification of His-tagged recombinant proteins from a conditioned medium in the presence of substances that can strip metal from the IMAC columns. The PIV-based expression system allows for the production of milligram quantities of recombinant proteins in static cultures, without the need for complex equipment such as bioreactors, and complies with regulatory requirements due to its distinction from common recombinant viruses.

The growing demand for high-quality plasmid DNA (pDNA) in cell and gene therapy, including chimeric antigen receptor T-cell therapy (CAR-T) manufacturing, is constrained by the limited availability of transfection-grade plasmids. This study describes a practical, scalable, and cost-effective acidic phenol extraction method based on a modified alkaline lysis protocol. This approach efficiently removes endotoxins and residual genomic DNA without requiring chromatography or ultracentrifugation. When benchmarked against CsCl density gradient ultracentrifugation and a commercial midiprep kit, the acidic phenol method delivered comparable plasmid purity and functional performance while providing superior scalability, yielding 5–10 mg of pDNA per 500 mL culture. Notably, residual endotoxin levels were negligible for downstream applications such as transfection and lentiviral vector packaging. Lentiviral vectors produced with these plasmids reached titers exceeding 5 × 10⁶ TU/mL, with transduction efficiencies statistically indistinguishable from those obtained with CsCl-purified DNA. The acidic phenol extraction method is a robust, scalable, and cost-effective alternative to industry-standard methods, matching them in yield and purity while being readily scalable, making it a practical tool for both academic research and preparative laboratory production.

Transient gene expression (TGE) is commonly employed for protein production, but its reliance on plasmid transfection makes it challenging to scale up. An alternative TGE method is presented, utilizing pseudoinfectious alphavirus as an expression vector. Pseudoinfectious viruses (PIV) and a replicable helper construct were derived from the genome of the Venezuelan equine encephalitis virus. The PIV carries a mutant capsid protein that prevents packaging into infectious particles, while the replicable helper encodes a wild-type capsid protein but lacks other viral structural proteins. Although PIV and the helper cannot independently spread infection, their combination results in increased titers in cell cultures, enabling easier scale-up of producing cultures. The PIV-driven production of a model protein outperforms that of alphavirus replicon vectors or simple plasmid vector. Another described feature of the expression system is the modification to immobilized metal affinity chromatography (IMAC) allowing purification of His-tagged recombinant proteins from conditioned medium in the presence of substances that can strip metal from the IMAC columns. The PIV-based expression system allows for the production of milligram quantities of recombinant proteins in static cultures, without the need for complex equipment such as bioreactors, and complies with regulatory requirements due to its distinction from common recombinant viruses.