Provided herein are synthetic RNA molecules for reconstitution of RNA molecules, including compositions and methods of using these molecules. For example, such molecules can be used to deliver a protein coding sequence over two or more viral vectors (such as AAVs), resulting in reconstitution of the full-length protein in a cell. Such methods can be used to deliver a therapeutic protein, for example to treat a genetic disease or cancer.

Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

Disclosed herein arm CRISPR/Cas-based genome editing compositions and methods for treating Duchenne Muscular Dystrophy by restoring dystrophin function. The CRISPR/Cas-based genome editing systems may include a guide RNA (gRNA) targeting a fragment of a mutant dystrophin gene, a Cas protein or a fusion protein comprising the Cas protein, and a donor sequence comprising a fragment of a wild-type dystrophin gene.

The invention provides, inter alia, a nucleic acid (e.g. expression vector) that comprises at least a first coding sequence and a second coding sequence. Each conding sequence is under the control of an inducible promoter of defined strength. Different promoters can have different strengths. Each promoter is responsive to the same inducer. The invention also provides: methods of expressing coding regions, methods of making a product of a multi-enzyme pathway, and methods of optimizing the yield of a product of a multi-enzyme metabolic pathway using the nucleic acids provided by the invention. Also disclosed is a method of non-enzymatic gene cloning useful for practicing the invention.

A dual-vector system for expressing an OTOF protein includes two nucleotide sequences, where a first nucleotide sequence includes two ITR sequences and a gene expression cassette inserted between the ITR sequences; a second nucleotide sequence includes two ITR sequences and a gene expression cassette inserted between the ITR sequences; the gene expression cassette of the first nucleotide sequence includes a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of intein, and a polyA; and the gene expression cassette of the second nucleotide sequence includes a promoter, a C-terminal coding sequence of Intein, a C-terminal coding sequence of OTOF, and a polyA. The present disclosure further provides an adeno-associated virus packaged by the vector. The vector and the virus can recover hearing of bilateral ears by a unilateral ear administration in the field of deafness gene therapy large gene dual-vector delivery.

The present invention relates to an expression system for the heterologous expression of a nucleic acid sequence of interest in a mammalian cell, the system comprising: (i) a first genetic entity, comprising: a nucleic acid sequence encoding a functional Epstein Barr virus nuclear antigen 1 (EBNA-1), the nucleic acid sequence being operably linked to regulatory elements that allow for expression of the nucleic acid sequence encoding a functional EBNA-1; (ii) a second genetic entity, comprising: a nucleic acid sequence encoding a functional nucleoside diphosphate kinase A (NDPK-A), the nucleic acid sequence being operably linked to regulatory elements that allow for expression of the nucleic acid sequence encoding a functional NDPK-A; (iii) a third genetic entity, comprising: the nucleic acid sequence of interest being operably linked to regulatory elements that allow for expression of the nucleic acid sequence of interest; and (iv) a four genetic entity, comprising: the Epstein Barr virus OriP sequence or one or more subsequences thereof, wherein the one or more subsequences comprise at least the ‘Family of Repeats’ DNA-binding site for EBNA-1 and the ‘Dyad Symmetry’ DNA-binding site for EBNA-1. The present invention also relates to corresponding mammalian host cells and methods for expressing a nucleic acid sequence of interest by means of such expression system.

This disclosure provides compositions and methods of using these compositions to mediate a targeted trans-splicing event on a pre-mRNA in a cell.

The invention provides, inter alia, a nucleic acid (e.g. expression vector) that comprises at least a first coding sequence and a second coding sequence. Each conding sequence is under the control of an inducible promoter of defined strength. Different promoters can have different strengths. Each promoter is responsive to the same inducer. The invention also provides: methods of expressing coding regions, methods of making a product of a multi-enzyme pathway, and methods of optimizing the yield of a product of a multi-enzyme metabolic pathway using the nucleic acids provided by the invention. Also disclosed is a method of non-enzymatic gene cloning useful for practicing the invention.

The present disclosure provides vectors, methods and kits for for delivery and stable expression of CRISPR/Cas components capable of inducing genetic modification of cells, followed by recombinase-mediated excision of some or all of these components after the cells have been successfully genetically modified. The disclosed vectors and methods provide for reduced immunogenic effects arising from one or more CRISPR/Cas components. The disclosed vectors comprise coding sequences that encode a Cas protein, detectable markers and a guide RNA. The disclosed vectors provide for the subsequent genomic excision of the CRISPR/Cas components after successful genetic modification, as mediated by recombinase recognition of recombination sites flanking one or more of the disclosed coding sequences. The present disclosure further provides methods of generating a population of genetically modified tumor cells for screening a candidate target gene for cancer immunotherapy.