The present invention relates to a method and composition for optimized intracellular delivery of nucleic acids, in particular mRNA. In addition to mRNA, the composition, in particular a nanoparticle, may include a glycolipid antigen. Combinations with checkpoint inhibitors are also provided. The method and composition of the invention targets antigen presenting cells and is especially useful for immunotherapy and vaccination purposes.

Circular RNA and transfer vehicles, along with related compositions and methods are described herein. In some embodiments, the inventive circular RNA comprises group I intron fragments, spacers, an IRES, duplex forming regions, and an expression sequence. In some embodiments, the expression sequence encodes a chimeric antigen receptor (CAR). In some embodiments, circular RNA of the invention has improved expression, functional stability, immunogenicity, ease of manufacturing, and/or half-life when compared to linear RNA. In some embodiments, inventive methods and constructs result in improved circularization efficiency, splicing efficiency, and/or purity when compared to existing RNA circularization approaches.

The present invention pertains to extracellular vesicle (EV) therapeutics, wherein the EVs comprise nucleic acid (NA)-based therapeutics such as mRNAs, circular RNAs, miRNAs, shRNAs, circular RNA and/or DNA molecules. The NA therapeutics are loaded into EVs using inventive engineering protein and NA engineering strategies to enhance loading into EVs and to facilitate release of the NA cargo molecules inside target cells.

Provided herein are methods for producing exosomes that contain RNA transcribed from a specific mutant gene or a transgene. In one embodiment, the method comprises the steps of: generating a cell comprising a mutation of a gene by using a site-specific nuclease; culturing the cell in a medium that allows the cell to secrete to the medium an exosome containing an RNA transcribed from the gene and comprising the mutation; and collecting the medium that contains the exosome. The exosomes generated can be used as reference material or therapeutic delivery device.

Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to a parental AAV capsid protein, which, when present in an AAV virion, confer increased infectivity of one or more types of retinal cells as compared to the infectivity of the retinal cells by an AA V virion comprising the unmodified parental AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising a variant AAV capsid protein as described herein, methods of making these rAAV capsid proteins and virions, and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example in, e.g., the delivery of nucleic acid sequences to one or more cells of the retina for the treatment of retinal disorders and diseases.

Circular RNA and transfer vehicles, along with related compositions and methods are described herein. In some embodiments, the inventive circular RNA comprises group I intron fragments, spacers, an IRES, duplex forming regions, and an expression sequence. In some embodiments, the expression sequence encodes a chimeric antigen receptor (CAR). In some embodiments, circular RNA of the invention has improved expression, functional stability, immunogenicity, ease of manufacturing, and/or half-life when compared to linear RNA. In some embodiments, inventive methods and constructs result in improved circularization efficiency, splicing efficiency, and/or purity when compared to existing RNA circularization approaches.

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.

This invention provides a method for the in vivo delivery of oligonucleotides. The invention utilizes the presence of one or plurality of HES linked to an oligonucleotide to deliver a nucleic acid sequence of interest into the cytoplasm of cells and tissues of live organisms. The delivery vehicle is nontoxic to cells and organisms. Since delivery is sequence-independent and crosses membranes in a receptor-independent manner, the delivered oligonucleotide can target complementary sequences in the cytoplasm as well as in the nucleus of live cells. Sequences of bacterial or viral origin can also be targeted. The method can be used for delivery of genes coding for expression of specific proteins, antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, miRNAs, anti-miRNAs or any nucleic acid sequence in a living organism. The latter include mammals, plants, and microorganisms such as bacteria, protozoa, and viruses.

This disclosure relates to mRNA therapy for the treatment of arginase deficiency (AD). mRNAs for use in the invention, when administered in vivo, encode arginase 1 (ARG1). mRNA therapies of the disclosure increase and/or restore deficient levels of ARG1 expression and/or activity in subjects. mRNA therapies of the disclosure further decrease abnormal accumulation of ammonia associated with deficient ARG1 activity in subjects.

The present disclosure provides donor polynucleotides, genome editing systems, methods, pharmaceutical compositions, and kits which correct or induce a mutation that causes Glycogen Storage Disease 1a in a genomic DNA (gDNA) molecule in a cell. In some embodiments the present disclosure provides donor polynucleotides comprising two strands capable of correcting a mutation that causes Glycogen Storage Disease 1a.