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.

There is disclosed a trans-acting functional nucleic acid molecule comprising a eukaryotic target binding sequence comprising a sequence reverse complementary to a target mRNA sequence for which protein translation is to be enhanced, and a regulatory sequence comprising an internal ribosome entry site (IRES) sequence or an internal ribosome entry site (IRES) derived sequence and enhancing translation of the target mRNA sequence, wherein the regulatory sequence is located 3′ of the target binding sequence.

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

This invention is directed to AAV compositions for circular RNA expression and methods of expressing covalently closed, circular RNA.

A tandem DNA element capable of enhancing protein synthesis efficiency, in particular, the nucleic acid construct is formed by an IRES enhancer (such as ScBOI1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, KlBOI1) derived from eukaryotic cells (such as yeast), a Ω sequence, and a yeast-specific Kozak sequence in tandem. The use of the nucleic acid construct in a yeast-based in vitro biosynthesis system (such as a yeast-based in vitro protein synthesis system) can significantly improve protein synthesis efficiency.

Provided herein are recombinant circular RNA (circRNA) molecules comprising an internal ribosome entry site (IRES) operably linked to a protein-coding nucleic acid sequence. The IRES includes at least one RNA secondary structure element; and a sequence region that is complementary to an 18S ribosomal RNA (rRNA). Methods of producing a protein in a cell using the recombinant circRNA molecules are also provided.

This disclosure provides compositions, pharmaceutical preparations, and uses of polyribonucleotides encoding one or more immunogenic polypeptides. In particular, this disclosure features circular polyribonucleotide encoding one or more immunogenic polypeptides.

The present inventions provide eukaryotic cells, such as mammalian cells, that comprise adeno-associated virus (AAV) polynucleotides, including AAV capsid proteins (Cap), and are capable of expressing the polypeptides encoded by the AAV polynucleotides, and thereby are capable of producing AAV, including recombinant AAV. The eukaryotic cells also may comprise adenovirus (Ad) polynucleotides. The present inventions also provide methods of expressing AAV polynucleotides, as well as Ad polynucleotides, in eukaryotic cells, such as CHO cells, HEK 293 and BHK cells. The present inventions further provides other products and methods described herein.

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.

Hoxb5 identifies long-term hematopoietic stem cells. Expression of Hoxb5 distinguishes between LT-HSCs and non-LT-HSCs, and the marker identifies substantially all LT-HSC in the bone marrow. By utilizing fluorescent proteins under the endogenous expression control of Hoxb5, LT-HSC can be monitored and isolated, including without limitation detection and monitoring of HSC in bone morrow; production of LT-HSC from pluripotent stem cells such as iPS cells; for analysis of early stage LT-HSC; in screening methods for expansion and manipulation of LT-HSC, and the like.