The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.

Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.

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.

A synthetic nucleic acid expression system for production of a transcript of interest in a predefined cell-state is provided, the system comprising (a) a first nucleic acid sequence comprising a first promoter operably linked to a nucleic acid sequence encoding a first trans-spliceable pre-mRNA sequence comprising at least one exon encoding a 5′ fragment of said transcript of interest and a first RNA sequence required for spliceosome-dependent trans-splicing; and (b) a second nucleic acid sequence comprising a second promoter operably linked to a nucleic acid sequence encoding a second trans-spliceable pre-mRNA sequence comprising at least one exon encoding a 3′ fragment of said transcript of interest and a second RNA sequence required for spliceosome-dependent trans-splicing; wherein said first promoter and said second promoter are different and each one is specifically regulated by said predefined cell-state.

Disclosed are methods for performing transcription-dependent directed evolution (TRADE) and novel AAV capsids selected using such methods.

The present disclosure provides nucleic acid molecules encoding for at least two circular RNA (circRNAs), adeno-associated virus (AAV) particles including nucleic acid molecules encoding for at least two circRNAs, pharmaceutical compositions, and methods for delivering such to a subject.

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.

Disclosed are methods for performing transcription-dependent directed evolution (TRADE) and novel AAV capsids selected using such methods. This disclosure also provides novel AAV capsid mutants. TRADE technology was used to identify novel AAV vectors that mediate neuronal transduction in the brain following intravenous administration. Application of TRADE in vivo resulted in the identification of new AAV capsids that can transduce neurons more efficiently and more specifically than AAV9 in the brain following administration of the new AAV capsids. The disclosed methods may be used to identify AAV capsids that target various cell populations.

A new promoter comprising: (i) an hCMV enhancer sequence; (ii) an hCMV promoter sequence; (iii) a splice donor region; (iv) a cell-derived enhancer sequence; and (v) a splice acceptor region.

A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation.