An inducible expression cassette for controlled expression of multiple genes includes the DNA sequences of a bidirectional promoter inserted between two DNA sequences. Transfection into a host cell and the two DNA sequences encode genes of interest, provides one DNA sequence expressed constitutively and manipulation of the expression of the other DNA sequence. A regulatory DNA cassette containing another bidirectional promoter and two DNA sequences that encode a marker and a regulatory expression product is also disclosed. Both cassettes can be incorporated in a non-viral vector, like the Sleeping Beauty transposon, or a viral vector to induce controlled expression of multiple genes into host cells. A kit containing a package of each above vector type is also disclosed, as is a method of transforming a host cell.

Provided is a recombinant adeno-associated virus (rAAV) having an AAV capsid and a vector genome which comprises a nucleic acid sequence encoding a functional CDKL5 (hCDKLK5). Also provided are a production system useful for producing the rAAV, a pharmaceutical composition comprising the rAAV, and a method of treating a subject having CDD, or ameliorating symptoms of CDD, or delaying progression of CDD via administrating an effective amount of the rAAV to a subject in need thereof.

Edited cell chimerism is currently one of the greatest bottlenecks to clinical efficacy of gene therapies for the hemoglobinopathies. For example, it is difficult to go from low hematopoietic stem cell (HSC) edited cell chimerism in the bone marrow to high edited red blood cell (RBC) chimerism in the bloodstream. The present disclosure provides methods and compositions for genetically modifying hematopoietic stem and progenitor cells (HSPCs), in particular by creating HSPCs that express truncated forms of the EPO.receptor (tEPOR).

The present disclosure provides gene therapy that targets complement pathways for treating dry age-related macular degeneration.

New promoters are described to drive transcription in meningococcus e.g. for over-expression of protein antigens for retention in membrane vesicles. Modified porA promoters lack the wild-type poly-G sequence which can cause phase variation. Meningococcal rRNA-coding genes (e.g. for 16S rRNA) can be used to drive transcription of a protein-coding gene. These approaches can be used in combination.

In accordance with the invention, isolated nucleic acids, expression methods, host cells, expression vectors, and DNA constructs for producing proteins, and proteins produced using the expression methods are described. More particularly, nucleic acids isolated from Pichia pastoris wherein the nucleic acids have promoter activity are described. The invention also relates to expression methods, host cells, expression vectors, and DNA constructs, for using the Pichia pastoris promoters to produce proteins and polypeptides, and to the proteins and polypeptides produced using the expression methods.

Provided are methods for protein engineering, such as engineering proteases or kinases. The methods may utilize yeast display and/or ER sequestration of proteins or substrates. In some aspects, TEV proteases with altered substrate specificity, potency, and/or efficiency are provided.

Provided herein, are compositions based on retroviruses (e.g., lentiviruses) comprising one or more nucleic acid molecules encoding retroviral Pol polyprotein components and a nucleic acid molecule comprising one or more transgene sequences flanked by long terminal repeat sequences, for delivery of the one or more transgenes to a target cell ex vivo or in vivo. The compositions are useful for delivering to a target cell (e.g., hematopoietic stem cells (HSCs), liver cells, ocular cells, muscle cells, epithelial cells, T cells, etc.) and/or stably expressing any transgene (e.g., beta-globin, Factor VIII, RP GTPase regulator (RPGR), dystrophin, cystic fibrosis transmembrane conductance regulator (CFTR), a chimeric antigen receptor, etc.) with a biological effect to treat and/or ameliorate the symptoms associated with any disorder related to gene expression (e.g., sickle cell disease, beta-thalassemia, haemophilia B, retinitis pigmentosa, Duchenne muscular dystrophy, cystic fibrosis, cancer, etc.).

The present invention provides a genetically-modified T cell comprising in its genome a modified human T cell receptor alpha gene. The modified T cell receptor alpha gene comprises an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene that is positioned 5 upstream of TRAC exon 1. The exogenous sequence of interest can comprise an exogenous splice acceptor site and/or a poly A signal, which disrupts expression of the T cell receptor alpha subunit. The sequence of interest can also include a coding sequence for a polypeptide, such as a chimeric antigen receptor. Additionally, the endogenous splice donor site and the endogenous splice acceptor site flanking the intron are unmodified and/or remain functional. The invention further provides compositions and methods for producing the genetically-modified cell, and populations of the cell, and methods for the treatment of a disease, such as cancer, using such cells.

Methods for modifying a genome are provided, wherein the modifications comprise null alleles, conditional alleles and null alleles comprising conditional by inversion elements. Methods are provided which afford the ability in a single targeting step to introduce an allele that can be used to generate a null allele, a conditional allele, or an allele that is a null allele and that further includes a conditional by inversion element. Introduced alleles comprise pairs of cognate recombinase recognition sites, an actuating sequence and/or a drug selection cassette, and a nucleotide sequence of interest, and a conditional by inversion element, wherein upon action of a recombinase a conditional allele with a conditional by inversion element is formed. In a further embodiment, action of a second recombinase forms an allele that contains only a conditional by inversion element in sense orientation. In a further embodiment, action by a third recombinase forms an allele that contains only the actuating sequence in sense orientation.