Circular RNA compositions and methods
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.
1. A pharmaceutical composition formulated for intravenous administration, the pharmaceutical composition comprising:
a. a circular RNA polynucleotide comprising a non-human post-splicing 3′ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein, and a non-human post-splicing 5′ group I intron fragment,
wherein the post-splicing 3′ group I intron fragment is not a T4 bacteriophage thymidylate synthase (Td) 3′ group I intron fragment, and wherein the post-splicing 5′ group I intron fragment is not a T4 bacteriophage Td 5′ group I intron fragment, and
b. a transfer vehicle comprising (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a human immune cell present in a human subject, such that the CAR or TCR is translated in the human immune cell and expressed on the surface of the human immune cell.
2. The pharmaceutical composition of claim 1 , wherein the IRES is from Salivirus.
3. The pharmaceutical composition of claim 1 , comprising a first internal spacer between the non-human post-splicing 3′ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the non-human post-splicing 5′ group I intron fragment.
4. The pharmaceutical composition of claim 3 , wherein the first and second internal spacers each have a length of about 10 to about 60 nucleotides.
5. The pharmaceutical composition of claim 1 , wherein the transfer vehicle comprises a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, or a biodegradable lipid nanoparticle.
6. The pharmaceutical composition of claim 1 , further comprising a targeting moiety.
7. The pharmaceutical composition of claim 1 , wherein less than 1%, by weight, of the polynucleotides in the composition are double stranded RNA, DNA splints, or triphosphorylated RNA.
8. The pharmaceutical composition of claim 1 , wherein the circular RNA polynucleotide comprises, in the following order, the non-human post-splicing 3′ group I intron fragment, the IRES, the expression sequence encoding a CAR or TCR complex protein, and the non-human post-splicing 5′ group I intron fragment.