This invention relates to compositions of matter, methods, modules and automated, end-to-end closed instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.

The subject invention pertains to materials and methods for detecting, preventing and treating retroviral infections in humans and other animals susceptible to infection by retrovirus. It has been discovered that FIV can be transmitted from cats to humans and that the FIV can infect human cells in vivo and that antibodies generated by the infected person cross-react with HIV antigens. Thus, the methods and compositions of the subject invention can be used to detect, prevent and treat FIV infection in humans and other non-feline animals that are susceptible to FIV infection. The methods and compositions of the invention can also be used to prevent and treat infection by HIV in humans.

Provided herein are methods for inducing an immune response against an antigen in a subject. In some embodiments, the methods comprise administering a therapeutically effective amount of a vaccine and an interleukin 10 (IL-10) inhibitor to the subject. In some embodiments, the vaccine is an IL-10-deficient vaccine.

Provided is a method of inserting a polynucleotide sequence into a genome of a cell. The method comprises: generating a double-strand break at a target site of the genome; and introducing into the cell a virus. The virus comprises a nucleic acid comprising the polynucleotide sequence to be inserted or the complementary sequence thereof. The nucleic acid does not comprise a homologous arm or comprises very short (5˜25 bp) homologous arms corresponding to the target site. Also provided herein is a composition for inserting a polynucleotide sequence into a genome of a cell. The composition comprises a site-specific nuclease capable of generating a DNA double-strand break at a target site of the genome and a virus comprising a nucleic acid comprising the polynucleotide sequence or the complementary sequence thereof.

Nucleic acid-guided nuclease editing in mammalian cells may include passaging mammalian cells, in an automated closed cell editing instrument, into smaller aggregates when the aggregates exceed 50-300 microns in size. A library of viral particles may be delivered to the mammalian cells at a multiplicity of infection such that each mammalian cell receives one or no viral particle. The library may include viral vectors with an editing cassette including a pair of gRNA coding sequence and donor DNA. Conditions may be provided to allow a viral vector of the viral vectors to integrate into the mammalian cells. Enriching for mammalian cells may be done with an integrated viral vector. A nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion may be delivered to the enriched mammalian cells and conditions may be provided to allow editing in the mammalian cells.

Artificial target cells lines with improved distinction between background killing and ADCC and/or CAR-mediated killing are presented. In some embodiments, the artificial cells are recombinant SUP-B15 cells expressing target antigens that are recognized by a CAR, and/or a bispecific engager, or a therapeutic antibody.

The invention relates to nucleic acids, including lentiviral vectors and lentiviral vector particles, encoding at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen. The invention encompasses these lentiviral vectors and lentiviral vector particles, methods of making the vectors, and their use, including medicinal uses. The lentiviral vectors and lentiviral vector particles are for use in administering to humans to induce immune responses against the HBV antigens.

A lentiviral vector system for expressing a lentiviral particle is disclosed. The lentiviral vector system includes a therapeutic vector. The lentiviral vector system produces a lentiviral particle for upregulating PAH expression in the cells of a subject afflicted with phenylketonuria (PKU).

The method of generating multipotent stem cells is a method for producing and/or expanding multipotent stem cells by delivering at least one reprogramming protein into somatic cells. The at least one reprogramming protein includes a Master Regulator (MR) protein, which may be BAZ2B, ZBTB20, ZMAT1, CNOT8, KLF12, DMTF1, HBP1, or FLI1. The bromodomain protein BAZ2B, in particular, was identified by first generating bi-species heterokaryons by fusing Tcf7l1.sup.?/? murine embryonic stem cells (ESCs) with human B-cell lymphocytes. Reprogramming of the B-cell nuclei to a multipotent state was tracked by human mRNA transcript profiling at multiple timepoints. Interrogation of a human B-cell regulatory network with gene expression signatures collected from such reprogramming time series identified eight candidate Master Regulator proteins, which were validated in human cord blood-derived hematopoietic progenitor and lineage-committed cells.

The invention provides improved gene therapy methods and compositions.