Described herein are engineered retroviral delivery vesicle generation compositions, systems, and methods to deliver a cargo to a cell. The engineered compositions, systems, and method include one or more polynucleotides encoding one or more elements for forming a delivery vesicle and optionally one or more cargos.

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.

The present disclosure provides a method of generating a stable producer cell line. The generation of stable producer cell lines, such as those provided in accordance with the present invention, increases the reproducibility and ease of creating high titer lentiviral stocks while easing biosafety concerns and the variation in expressed envelope proteins defines the tropism of the generated virus. The present disclosure also provides for a novel lentiviral transfer vector plasmid.

Embodiments relate to methods comprising expressing or overexpressing P-selectin glycoprotein ligand-1 (PSGL-1) in human immunodeficiency virus (HIV) producing cells; isolating HIV particles from the HIV producing cells; and preparing the isolated HIV particles as a HIV vaccine. Embodiments relate to systems comprising a HIV vaccine comprising live attenuated, inactivated, or non-infectious HIV particles. Embodiments relate to systems capable of performing a method comprising administering a vaccine comprising live attenuated, inactivated, or non-infectious HIV particles to a subject in need of the vaccine; and treating or preventing one or more disease states in the subject resulting from HIV infection. Embodiments relate to methods comprising expressing or overexpressing PSGL-1 in virus producing cells; and inhibiting viral infection; or inhibiting viral spreading; or inactivating viruses and virus producing cells; or producing non-infectious virion particles; or allowing the virus producing cells to produce non-infectious virions, isolating the virions, and preparing non-infectious virions, the virions being HIV particles.

The present disclosure provides a method of generating a stable producer cell line. The generation of stable producer cell lines, such as those provided in accordance with the present invention, increases the reproducibility and ease of creating high titer lentiviral stocks while easing biosafety concerns and the variation in expressed envelope proteins defines the tropism of the generated virus. The present disclosure also provides for a novel lentiviral transfer vector plasmid.

The present invention relates to a retro viral system for the transfer of non-viral RNA into target cells and more particularly a retroviral particle capable of delivering multiple RNAs. More particularly, it relates to retroviral particles comprising a protein derived from the Gag polyprotein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest linked to an encapsidation sequence, each encapsidation sequence being recognised by a binding domain introduced into the protein derived from the Gag polyprotein and/or into the integrase.

The present invention relates to a retroviral system for the transfer of non-viral RNA into target cells and more particularly a retroviral particle capable of delivering multiple RNAs. More particularly, it relates to retroviral particles comprising a protein derived from the Gag polyprotein an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest linked to an encapsidation sequence, each encapsidation sequence being recognised by a binding domain introduced into the protein derived from the Gag polyprotein and/or into the integrase.

The present invention provides the use of a packaging cell line for the production of VSV-G pseudotyped retroviral vector particles or virus like particles thereof, wherein said packaging cell line is negative for Low-Density Lipoprotein Receptor (LDLR), optionally said packaging cell line stably expresses VSV-G. A method for producing said VSV-G pseudotyped retroviral vector particles or virus like particles thereof is disclosed as well as said particles obtained by said method.

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.

Disclosed are compositions and methods that relate generally to viruses, and more particularly to the agents and their identification and use of anti-HIV agents which cause latently infected cells to reactivate.