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 lentivirus-derived particle comprising one or more Cas9-like proteins and at least one optimized sgRNA, wherein the optimized sgRNA comprises a targeting region and a non-targeting region, wherein said non-targeting region comprises a nucleotide sequence corresponding to SEQ ID NO: 1 or sequences having at least 90% sequence identity to SEQ ID NO: 1, said nucleotide sequence further comprising at least the following modifications: an extended repeat-anti-repeat region comprising a first extension of 2-8 base pairs in the repeat-anti-repeat region corresponding to nucleotides 1-12 and 17-30 of SEQ ID NO: 1; and optionally, an extended stem-loop 2 region comprising a second extension of 2-8 base pairs in the stem-loop 2 corresponding to the nucleotides 48-61 of SEQ ID NO: 1; and/or optionally, an A-U flip of the nucleotides corresponding to nucleotides 5 and 36 of SEQ ID NO: 1.

This in invention relates to methods for the nucleic acid amplification of multiple variants (strains) of any pathogen present in a sample, and preferably in a sample from a pathogen infected individual. In preferred embodiments, the pathogen is a retrovirus, such as HIV. The amplified pathogen nucleic acid can be used to identify the pathogen variants present in a sample, to quantitate the pathogen present in a sample, and as a nucleic acid vaccine, or in the preparation of antigen presenting cell vaccines. Nucleic acids produced by the methods of the invention or the proteins encoded thereby can be used to transfect/load antigen presenting cells. The loaded antigen presenting cells can then be used as a vaccine for the treatment of pathogen infection. In another embodiment, nucleic acids produced by the methods of the invention can be used directly as nucleic acid vaccines without prior loading into antigen presenting cells.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

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 direct intratumoral injection of a lentiviral vector adapted for transduction and drug-mediated 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 invention provides improved gene therapy methods and compositions.

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 direct intratumoral injection of a lentiviral vector adapted for transduction and drug-mediated expansion of tumor-infiltrating lymphocytes in vivo.

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 direct intratumoral injection of a lentiviral vector adapted for transduction and drug-mediated expansion of tumor-infiltrating lymphocytes in vivo.

The invention provides improved gene therapy methods and compositions.