The present disclosure provides methods and compositions for genetically modifying lymphocytes and related methods that include genetically modifying T cells and/or NK cells. The methods use replication incompetent recombinant retroviral particles that comprise a pseudotyping element on their surface and optionally a membrane-bound T cell activation element, such as an anti-CD3, and encode one or more engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR). The methods can include contacting PBMCs with replication incompetent recombinant retroviral particles for various exemplary time periods, such as less than 24 hours or in some illustrative embodiments less than 15 minutes. In some aspects, the present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in whole blood or a component thereof. In some embodiments a lymphodepletion filter assembly is used before or after forming a reaction mixture where lymphocytes are contacted with recombinant retroviral particles in a closed system, to genetically modify the lymphocytes.

Increased viral particle maturation and production can be achieved in various methods for producing viral particles from viral proteins, in general, by inhibiting or preventing Heme Oxygenase 2 (HO-2) from binding to the group-specific antigen (Gag) of the viral proteins, thus allowing delivery of the viral proteins to plasma membranes where they can replicate and mature without interference from HO-2. The increase in viral particle maturation and production can also be achieved by minimizing or eliminating the presence of HO-2 to thus reduce or prevent binding of HO-2 to the group-specific antigen (Gag) of the viral proteins. The invention is particularly applicable to the production of lentiviruses from viral proteins wherein the Matrix domain (MA) of the Gag is myristoylated.

The present invention relates to the industrialization of the production of recombinant lentiviral vectors in order to manufacture sufficient materials for therapeutic applications such as gene therapy and/or DNA vaccination, for use in clinical trials and/or commercial use.

Increased viral particle maturation and production can be achieved in various methods for producing viral particles from viral proteins, in general, by inhibiting or preventing Heme Oxygenase 2 (HO-2) from binding to the group-specific antigen (Gag) of the viral proteins, thus allowing delivery of the viral proteins to plasma membranes where they can replicate and mature without interference from HO-2. The increase in viral particle maturation and production can also be achieved by minimizing or eliminating the presence of HO-2 to thus reduce or prevent binding of HO-2 to the group-specific antigen (Gag) of the viral proteins. The invention is particularly applicable to the production of lentiviruses from viral proteins wherein the Matrix domain (MA) of the Gag is myristoylated.

The invention discloses a method for purifying a retrovirus (retroviral vector), comprising the following steps: A), microfiltrating a cell culture supernatant containing a retrovirus to remove cell debris from the cell culture supernatant and obtain a micro-filtered virus solution; B), treating the micro-filtered virus solution with a nuclease to degrade host DNA residues into small DNA fragments, and obtain an enzyme-digested virus solution; C), ultrafiltering the enzyme-digested virus solution such that the retrovirus is retained in a retentate, collecting the retentate, and obtaining an ultrafiltrated virus solution; and D), performing low speed centrifugation on the ultrafiltered virus solution to precipitate the retrovirus, collecting the precipitate to obtain unsterilized virus. The retrovirus product obtained by this method ensures the purity of the retrovirus solution, and at the same time, the titer of the concentrated virus solution reaches 10.sup.7 IP/ml, which meets the downstream process requirements.

The present invention relates to a stock solution (RLP Stock Solution) of mammalian cell-endogenous retrovirus like particles (RLP, a method of preparing a RLP stock solution, a kit containing a RLP stock solution, and a method of quantifying the amount of RLP removed from a solution. An RLP stock solution will contain a high concentration of RLP and an extremely low amount of any therapeutic proteins of interest. In some instances, an RLP stock solution will contain a very low amount of natural mammalian host cell protein (HCP) and DNA. The method of preparing a RLP stock solution will consist of the production of RLP during fermentation or cell culturing and the subsequent purification of RLP from fermentation or cell culture solution. The kit will comprise at least two containers. One container comprised of RLP stock solution and one comprised of PCR primers or one or more antibodies. The method of quantifying RLP comprises the steps of adding RLP stock solution to an in-process solution containing a recombinant therapeutic of interest, processing the resulting solution through a bioprocess purification technique, and then quantifying the amount of RLP removed.

Disclosed herein are compositions and methods of making and using bat IPSCs (BipS). Also disclosed herein are methods and compositions of virus nucleic acids residing in bat IPSCs. Also disclosed are nucleotides, cells, and methods associated with the compositions including their use as vaccines.

The disclosure provides methods to chemically and photochemically initiate cell membrane blebbing to induce cell vesicle production, modifications thereof, and uses thereof, including for drug delivery, gene therapy, cell-free cell therapy, and molecular therapy.

The disclosure provides compositions comprising bioorthogonally-conjugated induced cellular vesicles (ICVs) derived from mammalian cells that comprise one or more functional moieties that have been conjugated to the surface of the ICVs by using bioorthogonal chemistry, and applications thereof, including methods of treatment and methods of making the bioorthogonally-conjugated ICVs.

The present disclosure provides methods and compositions for genetically modifying lymphocytes and related methods that include genetically modifying T cells and/or NK cells. The methods use replication incompetent recombinant retroviral particles that comprise a pseudotyping element on their surface and optionally a membrane-bound T cell activation element, such as an anti-CD3, and encode one or more engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR). The methods can include contacting PBMCs with replication incompetent recombinant retroviral particles for various exemplary time periods, such as less than 24 hours or in some illustrative embodiments less than 15 minutes. In some aspects, the present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in whole blood or a component thereof. In some embodiments a lymphodepletion filter assembly is used before or after forming a reaction mixture where lymphocytes are contacted with recombinant retroviral particles in a closed system, to genetically modify the lymphocytes.