This application provides methods and compositions related to constructing nucleic acid hydrogels (e.g., RNA hydrogels) having repetitive monomer units, each monomer unit includes one or more G-quadruplex sequences. These G-quadruplex sequences cross-link the nucleic acid concatemer such that it self-assembles into a hydrogel under appropriate conditions. In some embodiments, each monomeric unit of the nucleic acid concatemer comprises a coding sequence for polypeptide of interest; and the nucleic acid hydrogel formed by the nucleic acid concatemer can be used for expressing the polypeptide in high quantities. In some embodiments, at least two RNA concatemers comprising G-quadruplex sequences are produced, one further comprising a spacer and the other further comprising a sequence encoding a polypeptide of interest. These two RNA concatemers are combined and self assembled to form a single, wideband RNA hydrogel.

Disclosed are compositions for transfecting a nucleic acid molecule into a cell and their applications. Specifically, this relates to a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, including (i) at least one compound of general formula (II) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, wherein Y.sup.1, Y.sup.2, Y.sup.3, Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6, Z.sup.7, X.sub.1, X.sub.2, R.sub.3, P.sup.+, R, T, U and V are as defined in the description. Also disclosed are uses of the composition and to a method for in vitro or ex vivo transfection of live cells.

Disclosed are compositions for transfecting a nucleic acid molecule into a cell and their applications. Specifically, this relates to a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, including (i) at least one compound of general formula (I), preferably of general formula (III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, wherein Y.sup.1, Y.sup.2, Y.sup.3, Z.sup.1, Z.sup.2, Z.sup.3, X.sub.1, X.sub.2, R.sub.3, P.sup.+, R and V are as defined in the description. Also disclosed are uses of the composition and to a method for in vitro or ex vivo transfection of live cells.

A lentiviral vector production system comprises (a) a lentiviral culture supplement to control cell growth, (b) a transfection reagent comprising DHDMS, DOPE, and cholesterol to increase transfection efficiency, (c) a lentiviral production enhancer comprising sodium propionate, sodium butyrate, and caffeine to boost lentiviral production, wherein the lentiviral vector production system is serum-free. A method of lentiviral vector production comprises using the lentiviral production system. Another method for lentiviral vector production comprises (a) culturing eukaryotic cells in a serum-free medium, (b) providing a lentiviral culture supplement to control cell growth, (c) transfecting the cells with a lentiviral vector using a transfection reagent comprising DHDMS, DOPE, and cholesterol to increase transfection efficiency, and (d) providing a lentiviral production using a lentiviral production enhancer comprising sodium propionate, sodium butyrate capable of boosting lentiviral production.

Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding the expression product or products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.

Methods of producing and manufacturing retroviral particles. Such methods may involve the use of an ion-exchange column with an elution buffer comprising one or more salts, wherein the elution buffer has a low total salt concentration (e.g., 400 mM to 800 mM) relative to conventional practice. In some embodiments, the retroviral particles can be generated by host cells transfected with retroviral vectors using polyethylenimine (PEI).

This disclosure provides compositions comprising a modified oncolytic virus that can contain modifications in the viral genome and exogenous nucleic acids coding for proteins. The viral compositions and methods provided herein can be utilized for the treatment of cancer.

The present disclosure relates to a combination of nucleic acids for the production of viral particles, said combination comprising or consisting of (a) a first nucleic acid encoding or being an inhibitory RNA; (b) at least one second nucleic acid comprising helper nucleic acids necessary for production of said viral particles, and/or encoding helper proteins necessary for said production; and (c) a third nucleic acid comprising a binding site for said inhibitory RNA. Furthermore, provided are methods of transfecting a cell with said combination, a production cell obtained by said transfecting or comprising the components of said combination and methods of producing viral particles.

The present invention describes a unique method of treating cancer with the administration of an improved DAGRS™ construct which functions as a humanized agent specifically targeting cancer cells in vivo. A specific DAGRS™ is described constructed of a humanized drug delivery biologic, carboxyl to an Apoptin fragment consisting of Apoptin's proline-rich SH3-binding fragment, a spacer, and a MAP kinase (MAPK) phosphorylation site, in replacement of the SH3-binding domain at HIV-1 TAT's amino terminus. Apoptin is a viral protein with incumbent immunogenicity and toxicity in humans. Improved DAGRS™ constructs are described that replace the viral VP3 peptide with human AKT peptide or derivative, all equivalently spaced 11 amino acids from the initial proline to the beginning of the MAPK phosphorylation site, through which technology the DAGRS™ is fully humanized. DAGRS™ provide for improved bioavailability, enhanced specific activity, and low toxicity for in vivo treatment of cancer. DAGRS™ are a superior method for targeting any oncogene with an inhibitory peptide. An algorithm for “humanization” is described through which human functional equivalent(s) to viral product(s) are identified by alignment of peptides anchored at each end by matching functional motifs that are spaced equivalently distant in the two aligned peptides. The algorithm totally disregards the primary amino acid composition of the spacer, and as such separates from current computer algorithms that prioritize primary amino acid alignments. Accounting for spacing dictates that functional domains be oriented correctly in three dimensions. The invention taught here can be developed into computer algorithms for rapidly identifying these anchored alignments, and thereafter developing safe humanized drugs from disruptive viral activities. Computers once taught the basic rules for anchoring equivalents, can improve on the basic algorithm through artificial intelligence to expand drug development.

Disclosed herein are methods and systems for manufacturing viral vectors (e.g., lentiviral vectors) using a static light scattering device in-line with a manufacturing process.