Circular rna compositions and methods of use thereof for targeted delivery

A circular ribonucleic acid (circRNA) comprising in order:

• (a) a 5′ exon fragment and a 5′ untranslated region (UTR),
• (b) an Internal Ribosomal Entry Site (IRES) comprising a sequence at least 85% identical to any one of SEQ ID NO:
• 1-11,
• (c) a coding sequence encoding a chimeric antigen receptor (CAR) comprising an anti-CD19 antibody or antigen-binding fragment thereof,
• (d) a 3′ UTR and a 3′ exon fragment, and
• (e) a single microRNA binding site within the 5′ UTR or the 3′ UTR.

A precursor RNA comprising, from 5′ to 3′ end:

• (i) a 5′ intron fragment and a 5′ exon fragment,
• (ii) a 5′ untranslated region (UTR),
• (iii) an IRES,
• (iv) a coding sequence encoding a chimeric antigen receptor (CAR) comprising an anti-CD19 antibody or antigen-binding fragment thereof,
• (v) a 3′ untranslated region (UTR), and
• (vi) a 3′ exon fragment and 3′ intron fragment,
• wherein the 5′ and the 3′ intron fragments are derived from a Twort-ORF142 Group I intron,
• wherein the 3′ UTR comprises a microRNA binding site flanked by an upstream spacer and a downstream spacer, and
• wherein each of the upstream spacer and the downstream spacer comprise no more than five (5) consecutive adenines (As).

A circular RNA therapeutic for targeted delivery comprising:

• (i) a circular RNA (circRNA) comprising an IRES and a coding sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an anti-CD19 antibody or antigen-binding fragment thereof, wherein the circRNA is encapsulated in a lipid nanoparticle (LNP), and
• (ii) a targeting moiety conjugated to the LNP, wherein the targeting moiety comprises an anti-CD5 VHH comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 sequence comprises SEQ ID NO:
• 64, the CDR2 sequence comprises SEQ ID NO:
• 65, and the CDR3 sequence comprises SEQ ID NO:
• 66.

A circRNA therapeutic comprising

• (i) a circRNA comprising a coding sequence encoding an anti-CD19 CAR, wherein the circRNA is encapsulated in an LNP,
• (ii) the LNP, wherein the LNP comprises Compound 7 at 39 mol %, DSPC at 20 mol %, cholesterol at 39 mol %, DMG-PEG 2000 lipid at 1.9 mol %, and DSPE-PEG 2000-maleimide lipid at 0.1 mol %, and
• (iii) a VHH with a conjugation handle comprising the sequence of SEQ ID NO:
• 122, wherein the VHH with the conjugation handle is attached to the LNP via thiol-maleimide conjugation of the cysteine in the conjugation handle with the DSPE-PEG 2000-maleimide lipid.