The present disclosure relates to a recombinant nucleic acid molecule and an application thereof in preparation of a circular RNA, and in particular, to a recombinant nucleic acid molecule for preparing a circular RNA, a recombinant expression vector, a circular RNA, a composition, a method for preparing a circular RNA, a method for expressing a target polypeptide in a cell, a method for screening a target coding region sequence, a system for screening a target coding region sequence, and a method for screening a ribozyme recognition site sequence. The recombinant nucleic acid molecule provided by the present disclosure provides a Clean PIE system with a novel structure for preparing a circRNA in vitro, which can avoid introducing additional exon sequences into the circular RNA, improve the sequence accuracy of circular RNA molecules, reduce changes in a secondary structure of the circular RNA, and further reduce the immunogenicity of the circular RNA, and has a good application prospects in the fields of nucleic acid vaccines, expression of therapeutic proteins, gene therapy, and the like.

Disclosed are systems and methods that include or utilize composable mammalian elements of transcription (COMET) including engineered recombinant proteins that regulate transcription and engineered DNA promoter sequences that are regulated by the engineered recombinant proteins. The elements may be composed to form logic gates, gene expression cascades and programs, and cell-based biosensors.

The invention relates to a minimal U6 pol III promoter. The invention also concerns a nucleic acid construct comprising the minimal U6 pol III promoter, a vector comprising the minimal U6 pol III promoter, methods involving the minimal U6 pol III promoter, and uses for the minimal U6 pol III promoter.

The present invention embraces compositions comprising at least two RNA replicons (self-amplifying RNA vectors (saRNAs or rRNAs)) that can be replicated by a replicase of a self-replicating virus, e.g., a replicase of alphavirus origin. Of the at least two replicons, at least one of which optionally comprises an open reading frame encoding for the RNA-dependent RNA polymerase or replicase that is able to replicate each of the at least two replicons. Further, each replicon comprises an open reading frame encoding for different antigens of interest, e.g., different antigens derived from the same or from different pathogenic organisms, for example the glycoprotein and nucleoprotein of Ebola virus.

The present invention relates to nucleic acid regulatory elements that are able to enhance muscle-specific expression of genes, in particular expression in cardiac muscle and/or skeletal muscle, methods employing these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. The present invention is particularly useful for applications using gene therapy, more particularly muscle-directed gene therapy, and for vaccination purposes.

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.

The present invention relates to DNA constructs comprising a novel TIS sequence. The TIS sequence transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript. The DNA construct may further comprise a nucleic acid sequence encoding signal peptide. Additionally, the DNA constructs may also comprise a nucleic acid sequence encoding a recombinant protein or one or more polypeptide chains thereof. The invention further relates to an expression vector, expression cassette and host cell which comprise said DNA constructs. Furthermore, the present invention relates to a recombinant protein expressed by the host cell as well as a method for expressing the recombinant protein.

The described technology pertains to gene therapy methodologies, specifically techniques and systems for delivering therapeutic genes of substantial size that exceed the packaging capacity of adeno-associated virus (AAV) vectors. The disclosed approach employs up to four AAV vectors and the CRE-lox DNA recombination system, utilizing novel lox site embodiments that allow sequence-specific and near-unidirectional recombination. This method supports efficient reconstitution of therapeutic genes up to 16 kb in a predetermined arrangement. Applications include the delivery of genes such as IFT140, PCDH15, CEP290, and CDH23 for addressing genetic disorders, including retinal degeneration. The described technology demonstrates successful production of full-length proteins in mammalian cells and mouse retinas, with therapeutic efficacy observed in an IFT140-associated retinitis pigmentosa mouse model. The CRE-lox approach offers a flexible platform for addressing AAV's packaging constraints, enabling effective gene therapy for large genes.

The application relates to nucleic acid regulatory elements that are able to enhance expression of genes constitutively in a variety of tissues, or in particular tissues including liver, muscle, and the CNS. The application further relates to methods employing these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. These nucleic acid regulatory elements are particularly useful for applications using gene therapy.

Disclosed herein are nucleic acid compositions and methods of use. The nucleic acid compositions may have a therapeutic nucleic acid sequence operably linked to a nuclear targeting sequence that increases expression of the therapeutic nucleic acid in a cell by at least 1.25 fold; at least 80% sequence identity to SEQ ID NO: 6; or a first regulatory element comprising a promoter sequence operably linked to a hemoglobin subunit gamma intron (hBGi) sequence, and a second regulatory element comprising a woodchuck hepatitis posttranscriptional regulatory element (WPRE) sequence. The nucleic acid compositions with these features may enhance therapeutic nucleic acid transfection and expression. Also disclosed herein are transgenes optimized for gene therapy applications, including novel FVIII transgene sequences, in which the B domain may be non-naturally occurring the A1 and/or A3 domain may include at least one amino acid substitution.