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.

The present invention relates to a method for purification of viral vectors, more closely it relates to purification of viral vectors from producer cells by using a single automated process. The method comprises the following steps: a) adding producer cells and cell lysis buffer to a processing container; b) mixing said producer cells and cell lysis buffer in said processing container to obtain a mixture; c) flowing said mixture through a chromatography column for purification of viral vectors, wherein the viral vectors are adsorbed on said chromatography column; and d) eluting viral vectors from the chromatography column into a product container.

Provided herein are non-cell particles, e.g. virus particles or virus-like particles, such as pseudotyped lentiviral-like particles, containing an exogenous CD24 or a biologically active portion of CD24. In some embodiments, the non-cell particles, e.g. virus particles or virus-like particles, such as pseudotyped lentiviral-like particles, can further contain an exogenous CD47 or a biologically active portion of CD47. Also provided herein are compositions containing such non-cell particles and methods of making and using the non-cell particles.

The present invention provides a lentiviral vector genome comprising at least one modified viral cis-acting sequence, wherein at least one internal open reading frame (ORF) in the viral cis-acting sequence is disrupted.

The disclosure provides tandem arrays of CRISPR RNAs and methods of use.

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 use of an inhibitor of EXT1 expression and/or activity for the production of a biological entity in a cell. Also, the use of a cell having at least depleted EXT1 expression and/or activity for the production of a biological entity. Further, evidence is provided about the role of glycosylation in rapid dynamism of ER shaping and function. In particular, depletion of EXT1 results in a recomposed ER shaping, which could benefit production of recombinant proteins.

A lentiviral vector production system comprises (a) a lentiviral culture supplement to control cell growth, (b) a transfection reagent comprising DHDMS, DOPE, and cholesterol to increase transfection efficiency, (c) a lentiviral production enhancer comprising sodium propionate, sodium butyrate, and caffeine to boost lentiviral production, wherein the lentiviral vector production system is serum-free. A method of lentiviral vector production comprises using the lentiviral production system. Another method for lentiviral vector production comprises (a) culturing eukaryotic cells in a serum-free medium, (b) providing a lentiviral culture supplement to control cell growth, (c) transfecting the cells with a lentiviral vector using a transfection reagent comprising DHDMS, DOPE, and cholesterol to increase transfection efficiency, and (d) providing a lentiviral production using a lentiviral production enhancer comprising sodium propionate, sodium butyrate capable of boosting lentiviral production.

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 without prior ex vivo stimulation. The methods typically include engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted CAR. Additional elements of such engineered signaling polypeptides are provided herein, as well as vectors, such as retroviral vectors, packaging cell lines and methods of making the same. Furthermore, recombinant retroviruses and methods of making the same are provided. Numerous controls are provided, including riboswitches that are controlled, for example in vivo, by nucleoside analogues.