In some aspects, the disclosure relates to methods for improving titer and yield of viral vector production. In some embodiments, the methods comprise transient silencing of transgene expression during packaging of a viral vector.

Provided herein are, inter alia, oligonucleotides, kits, and methods useful for increasing lentiviral titers.

Provided herein are, inter alia, oligonucleotides, kits, and methods useful for increasing lentiviral titers.

The disclosure relates generally to nucleic acid vectors and packaging cell lines for in vivo expansion of T-cells. More particularly, the disclosure relates to intravenous or intratumoral injection of a lentiviral particle adapted for transduction and expansion of tumor-infiltrating lymphocytes in vivo.

An enveloped viral particle producer or packaging cell, wherein the cell is genetically engineered to decrease expression of MHC-I on the surface of the cell.

The present invention provides a retroviral or lentiviral vector having a viral envelope which comprises: (i) a mitogenic T-cell activating transmembrane protein which comprises a mitogenic domain and a transmembrane domain; and/or (ii) a cytokine-based T-cell activating transmembrane protein which comprises a cytokine domain and a transmembrane domain, wherein the mitogenic or cytokine-based T-cell activating transmembrane protein is not part of a viral envelope glycoprotein. When cells such as T-cells of Natural Killer cells are transduced by such a viral vector, they are simultaneously activated by the mitogenic T-cell activating transmembrane protein and/or the cytokine-based T-cell activating transmembrane protein.

The invention discloses a method for constructing functional exosomes capable of efficiently loading specific miRNA. In order to enable the exosome to carry miRNA with specific regulation function more efficiently so as to play a role in targeted regulation more accurately and efficiently, MS2 phage capsid protein is utilized to edit and construct a capture element of a specific miRNA molecule, and placenta mesenchymal stem cells are reprogrammed to enable the secreted exosome to efficiently load a target miRNA molecule, so that the target miRNA molecule is delivered to tissue cells to play a role in effective regulation, and therefore a new strategy is provided for realizing specific precise treatment in the future.

The invention relates to retroviral producer cell comprising nucleic acid sequences encoding: gag and pol proteins; envelope protein or a functional substitute thereof; amplifiable selection marker; and the RNA genome of the retroviral vector particle, wherein said nucleic acid sequences are all integrated at a single locus within the retroviral producer cell genome. The invention also relates to nucleic acid vectors comprising a non-mammalian origin of replication and the ability to hold at least 25 kilobases (kb) of DNA, characterized in that said nucleic acid vector comprises retroviral nucleic acid sequences encoding: gag and pol proteins, and an env protein or a functional substitute thereof. The nucleic acid vector additionally comprises nucleic acid sequences encoding an amplifiable selection marker. The invention also relates to uses and methods using said nucleic acid vector in order to produce stable retroviral packaging and producer cell lines.

In accordance with the present invention, a method for increasing the yield of rLV vector particles comprising a trans gene encoding a therapeutic protein or fragment thereof is disclosed. In one approach, cells are transfected with plasmids encoding the necessary components for rLV production using a calcium chloride transfection mix at pH 7.1 wherein the calcium chloride and plasmids form a complex which is added to the cells at a constant speed. The cells are then incubated for a suitable time period wherein virus particle media is collected at least twice during the incubation period and stored in a cold storage unit, thereby reducing virus inactivation.

The present invention provides a retroviral or lentiviral vector having a viral envelope which comprises: (i) a mitogenic T-cell activating transmembrane protein which comprises a mitogenic domain and a transmembrane domain; and/or (ii) a cytokine-based T-cell activating transmembrane protein which comprises a cytokine domain and a transmembrane domain, wherein the mitogenic or cytokine-based T-cell activating transmembrane protein is not part of a viral envelope glycoprotein. When cells such as T-cells of Natural Killer cells are transduced by such a viral vector, they are simultaneously activated by the mitogenic T-cell activating transmembrane protein and/or the cytokine-based T-cell activating transmembrane protein.