The present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells. In some embodiments, the methods include reaction mixtures, and resulting cell formulations, that are created using whole blood, or a component thereof that is not a PBMC, and additionally comprise T cells and recombinant retroviral particles having polynucleotides that encode a CAR. In some embodiments, modified lymphocytes are reintroduced into a subject subcutaneously. In some embodiments, polynucleotides that provide T cells the ability to regulate cell survival and proliferation in response to binding to a CAR, are provided.

Provided is a nucleic acid construct for expressing a gene of interest, the nucleic acid construct comprising, in order from 5′-terminus, each sequence of: (a) a 5′ long terminal repeat (LTR) sequence derived from a retrovirus; (b) a packaging signal sequence (IV) derived from a retrovirus; (c) a sequence or multiple cloning site of the gene of interest; (d) a post-transcriptional regulatory sequence (PRE); (e) an siRNA generating sequence which forms at least one stem-loop structure and in which RNA, which induces RNA interference in mammalian cells, is transcribed; and (f) a 3′ LTR sequence derived form a retrovirus.

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.

A cell for producing retroviral vectors comprising nucleic acid sequences encoding: i) gag-pol; ii) env; iii) the RNA genome of the retroviral vector; and iv) optionally rev, or a functional substitute thereof, wherein at least two nucleic acid sequences are located at the same genetic locus; and wherein the at least two nucleic acid sequences are in reverse and/or alternating orientations.

The present invention discloses a digital-to-analog conversion apparatus having signal calibration mechanism. A DAC circuit includes conversion circuits to generate an output analog signal and an echo-canceling analog signal. An echo transmission circuit performs signal processing on an echo path to generate an echo signal. An echo calibration circuit includes odd and even calibration circuits to perform mapping according to offset tables and perform processing according to response coefficients on odd and even input parts of an input digital signal to generate odd and even calibration parts of an echo-canceling calibration signal. A calibration parameter calculation circuit generates offsets according to an error signal between the echo signal and the echo-canceling calibration signal and path information related to the echo calibration circuit. The echo calibration circuit makes the coefficients converge according to the error signal and pseudo noise transmission path information, and updates the offset tables according to the offset.

The present invention relates to a producer cell which expresses a tagging protein at the cell surface, such that retroviral vectors produced by the cell are tagged with the tagging protein, wherein the tagging protein comprises: i) a binding domain which binds to a capture moiety ii) a spacer; and iii) a membrane targeting domain such that, when incorporated a retroviral vector, the tagging protein facilitates purification of the retroviral vector from cellular supernatant via binding of the tagging protein to the capture moiety. The present invention also relates to a retroviral vector comprising such a producer cell-derived tagging protein.

The present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in shorter times than previously and/or 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.

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 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 methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in shorter times than previously and/or 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.