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 enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

Methods and constructs for engineering circular RNA are disclosed. In some embodiments, the methods and constructs comprise a vector for making circular RNA, the vector comprising the following elements operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, the vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. Methods for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector are also disclosed.

Disclosed are methods and constructs for engineering circular RNA. Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence:

a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. In another embodiment, the vector can comprise the 5′ spacer sequence, but not the 3′ spacer sequence. In yet another embodiment, the vector can comprise the 3′ spacer sequence, but not the 5′ spacer sequence. Also disclosed is a method for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector.

Methods and constructs for engineering circular RNA are disclosed. In some embodiments, the methods and constructs comprise a vector for making circular RNA, the vector comprising the following elements operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, the vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. Methods for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector are also disclosed.

Adeno-associated virus (AAV) Clade F vectors or AAV vector variants (relative to AAV9) for precise editing of the genome of a cell and methods and kits thereof are provided. Targeted genome editing using the AAV Clade F vectors or AAV vector variants provided herein occurred at frequencies that were shown to be 1,000 to 100,000 fold more efficient than has previously been reported. Also provided are methods of treating a disease or disorder in a subject by editing the genome of a cell of the subject via transducing the cell with an AAV Clade F vector or AAV vector variant as described herein and further transplanting the transduced cell into the subject to treat the disease or disorder of the subject. Also provided herein are methods of treating a disease or disorder in a subject by in vivo genome editing by directly administering the AAV Clade F vector or AAV vector variant as described herein to the subject.

Provided herein are methods and compositions to make guide nucleic acids (gNAs), nucleic acids encoding gNAs, collections of gNAs, and nucleic acids encoding for a collection of gNAs from any source nucleic acid. Also provided herein are methods and compositions to use the resulting gNAs, nucleic acids encoding gNAs, collections of gNAs, and nucleic acids encoding for a collection of gNAs in a variety of applications.

The disclosure provides methods and compositions for the integration (insertion) of a donor DNA molecule into a target DNA molecule. In general, the methods include contacting a target DNA molecule with a linear donor DNA molecule and a Cas 1 protein, where the target DNA molecule includes an AT-rich region (e.g., in some cases positioned 5 and within 50 nucleotides of a region that forms a DNA cruciform structure), where the contacting is not in a bacterial or archaeal cell (e.g., the contacting is in vitro outside of a cell, inside of a eukaryotic cell, etc.), and provides for integration of the donor DNA molecule into the target DNA molecule.

There is disclosed an improved, safer and commercially efficient process for developing genetically engineered cells. More specifically, there is disclosed a process comprises introducing a donor DNA construct, a guide RNA, and an RNA-guided nuclease with the host cells to be transfected; and introducing the three components into the host cell. There is further disclosed a donor DNA construct designed for inserting a CAR (chimeric antigen receptor) into a defined genomic site of a host cell. Further, the present disclosure provides a host cell transfected with a CAR that lacks viral vectors that can present a safety concern. The disclosure provides for more efficient and more cost-effective process for engineering T cells to express CAR constructs.

The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8) : (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.