Circular RNA compositions and uses thereof for diseases caused by varicella-zoster virus

Abstract

The present disclosure relates to circular RNA sequences encoding an antigen from varicella zoster virus (VZV). The circular RNA sequences described herein are used as vaccines and compositions for preventing or treating diseases resulting from VZV, including shingles. Methods of use, including methods of treating or preventing a disease caused by VZV and methods for formulating and preparing the VZV vaccine compositions, are also described.

Description

ATTORNEY DOCKET NO. ORB-018WO1 CIRCULAR RNA COMPOSITIONS AND USES THEREOF FOR DISEASES CAUSED BY VARICELLA-ZOSTER VIRUS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of U.S. Provisional Application Number 63 / 665,949, filed on June 28, 2024, the contents of which are incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed with an electronically filed Sequence Listing in XML format. The sequence listing file entitled ORB-018WO1_SL.XML was created on April 27, 2025, and is 326,296 bytes in size; the information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety. BACKGROUND

[0003] Shingles, also known as herpes zoster (HZ), zoster, or zona, is a viral disease characterized by painful skin lesions (typically, a blistering rash). Shingles is caused by the varicella-zoster virus (VZV).

[0004] Initial infection with VZV causes chickenpox, which generally occurs in children. Once an episode of chickenpox has resolved, the virus is not eliminated from the body. VZV becomes latent in nerve cell bodies and, less frequently, in non-neuronal satellite cells of the dorsal root, cranial nerve, or autonomic ganglion, without causing any symptoms. Years or decades after a chickenpox infection, the virus may reactivate as shingles. The virus breaks out of nerve cell bodies and travels down nerve axons to cause viral infection of the skin in the region of the nerve (i.e., the dermatome), causing painful skin lesions. The risk of developing shingles, which increases with age and with immunosuppression due to disease and / or therapy, appears to be related to a decline in VZV-specific immunity. Shingles can cause pain, itching, or tingling of the skin followed by a painful rash of blister-like sores, fever, headache, chills, and upset stomach. This reactivation of the VZV causes more than five million cases of shingles annually. According to the Centers for Disease Control and Prevention, an estimated one million people get shingles each year in the United States, and about one out of every three people in the United States will develop shingles in their lifetime. Page 1 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0005] Shingles vaccination is the only way to protect against shingles and postherpetic neuralgia, the most common complication of shingles. It has been shown that humans vaccinated with attenuated strains of VZV have received protective immunity from VZV infections. Shingrix® is an FDA-approved vaccine indicated for prevention of herpes zoster (HZ)(shingles). Shingrix® is not indicated for prevention of primary varicella infection (chickenpox). Shingrix® is an adjuvanted recombinant VZV gE protein that unfortunately causes unwanted side effects, such as pain and swelling at injection sites, muscle pain, headache, fever and shivering. According to the Centers for Disease Control and Prevention, it is estimated that about one out of six people receiving the Shingrix® vaccine had symptoms severe enough to prevent them from performing regular activities.

[0006] There is still a strong need for improved vaccines against herpes zoster and related disorders, such as post herpetic neuralgia (PHN). SUMMARY OF THE INVENTION

[0007] The present invention provides, among other things, circular ribonucleic acid (circRNA) molecules encoding a VZV glycoprotein E (gE) polypeptide for use as a VZV vaccine. VZV vaccine formulations, as described herein, are particularly effective in inducing cellular and humoral immune response while maintaining tolerability. This is based, in part, on the surprising discovery that VZV vaccine formulations of the present invention result in significantly increased IgG titers as compared to a commercial VZV vaccine. VZV vaccine formulations of the present invention can include, for example and without limitation, circRNA molecules encoding VZV glycoprotein E (gE) encapsulated by lipid nanoparticles (LNPs).

[0008] In some aspects, the present invention provides a circular RNA (circRNA molecule) encoding a VZV antigen polypeptide or variant thereof. In some embodiments, the VZV antigen polypeptide or variant thereof, is encoded by a codon-optimized coding sequence. In some embodiments, the codon-optimized coding sequence comprises a sequence at least 90% identical to any one of SEQ ID NOs: 11-34.

[0009] In some embodiments, the VZV antigen is a VZV gE polypeptide or a variant thereof. In some embodiments, the VZV gE polypeptide variant is truncated. In some embodiments, the VZV gE polypeptide variant is mutated. In some embodiments, the VZV gE polypeptide variant is truncated and mutated. In some embodiments, the truncated VZV gE polypeptide variant is lacking the carboxy terminal domain.

[0010] In some embodiments, the VZV gE polypeptide comprises the wild type signal Page 2 of 182ATTORNEY DOCKET NO. ORB-018WO1 peptide. In some embodiments, the VZV gE polypeptide comprises a human IgG kappa (IgGκ) signal peptide).

[0011] In some embodiments, the coding sequence comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 95% identical to the sequence of any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 96% identical to the sequence of any one of SEQ ID NOs: 11- 34. In some embodiments, the coding sequence comprises a sequence at least 97% identical to the sequence of any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 98% identical to the sequence of any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 99% identical to the sequence of any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence identical to any one of SEQ ID NOs: 11-34.

[0012] In some embodiments, the circular RNA described herein further comprises an IRES. In some embodiments, the IRES is a viral IRES. In some embodiments, the viral IRES comprises the sequence of an IRES from Canine Kobuvirus, Mouse Kobuvirus, Feline Kobuvirus, Caprine Kobuvirus, CVB3, Apodemus, Agrarius Picornavirus, Human coxsackievirus A7, or Parabovirus. In some embodiments, the viral IRES comprises the sequence of an IRES from Canine Kobuvirus. In some embodiments, the viral IRES comprises the sequence of an IRES from Mouse Kobuvirus. In some embodiments, the viral IRES comprises the sequence of an IRES from Feline Kobuvirus. In some embodiments, the viral IRES comprises the sequence of an IRES from Caprine Kobuvirus. In some embodiments, the viral IRES comprises the sequence of an IRES from CVB3. In some embodiments, the viral IRES comprises the sequence of an IRES from Apodemus. In some embodiments, the viral IRES comprises the sequence of an IRES from Agrarius Picornavirus. In some embodiments, the viral IRES comprises the sequence of an IRES from Human coxsackievirus A7. In some embodiments, the viral IRES comprises the sequence of an IRES from Parabovirus.

[0013] In some embodiments, the IRES comprises the sequence of any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 35. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 36. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 37. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 38. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 39. In some embodiments, the IRES comprises the Page 3 of 182ATTORNEY DOCKET NO. ORB-018WO1 sequence of SEQ ID NO: 40. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 41. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 42. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 43.

[0014] In some embodiments, the circular RNA described herein comprises a sequence at least at least 95% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circular RNA comprises a sequence at least 96% identical to any one of SEQ ID NOs: 76- 108. In some embodiments, the circular RNA described herein comprises a sequence at least at least 97% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circular RNA described herein comprises a sequence at least at least 98% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circular RNA described herein comprises a sequence at least at least 99% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circular RNA described herein comprises a sequence identical to any one of SEQ ID NOs: 76-108.

[0015] In some aspects, the present invention provides a precursor RNA for making the circular RNA described herein. In some embodiments, the precursor RNA encodes a varicella-zoster virus (VZV) antigen or variant thereof. In some embodiments, the precursor RNA comprises, from 5’ to 3’, i) an upstream intron fragment corresponding to the 3’ portion sequence of a Group I intron, ii) an internal ribosome entry site (IRES) as described herein, iii) a codon-optimized coding sequence, as described herein, encoding the VZV antigen polypeptide, or variant thereof, iv) a downstream intron fragment corresponding to the 5’ portion sequence of the Group I intron. In some embodiments, the codon-optimized coding sequence comprises a sequence at least 90% identical to any one of SEQ ID NOs: 11-34.

[0016] In some embodiments, the intron is a Group I intron. In some embodiments, the Group 1 intron is a T4-Td intron. In some embodiments, the upstream intron fragment comprises the sequence of SEQ ID NO: 44, and the downstream intron fragment comprises the sequence of SEQ ID NO: 45. In some embodiments, the intron is a Twort intron. In some embodiments, the upstream intron fragment comprises the sequence of SEQ ID NO: 46, and the downstream intron fragment comprises the sequence of SEQ ID NO: 47.

[0017] In some embodiments, the precursor RNA for making the circular RNA described herein comprises a sequence at least 95% identical to any one of SEQ ID NOs: 1-10 and 53- 75. In some embodiments, the precursor RNA comprises a sequence at least 96% identical to any one of SEQ ID NOs: 1-10 and 53-75. In some embodiments, the precursor RNA comprises a sequence at least 97% identical to any one of SEQ ID NOs: 1-10 and 53-75. In some embodiments, the precursor RNA comprises a sequence at least 98% identical to any Page 4 of 182ATTORNEY DOCKET NO. ORB-018WO1 one of SEQ ID NOs: 1-10 and 53-75. In some embodiments, the precursor RNA comprises a sequence at least 99% identical to any one of SEQ ID NOs: 1-10 and 53-75. In some embodiments, the precursor RNA comprises a sequence identical to any one of SEQ ID NOs: 1-10 and 53-75.

[0018] In some embodiments, the circular RNA described herein is formulated in a delivery vehicle. In some embodiments, the circular RNA is formulated in lipid nanoparticles (LNPs). In some embodiments, the circular RNA is formulated in virus-like particles (VLPs). In some embodiments, the circular RNA is formulated without a delivery vehicle.

[0019] In some embodiments, the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a PEG-modified lipid.

[0020] In some embodiments, the ionizable lipid comprises a compound selected from Table G1, G2, or G3. In some embodiments, the ionizable lipid comprises 3-((((1- ethylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-(((4-(((Z)-oct-5-en-1-yl)oxy)-4-(((Z)-oct-5-en- 1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Compound 1). In some embodiments, the ionizable lipid comprises ((3-hydroxypropyl)azanediyl)bis(heptane- 7,1-diyl) bis(4,4-bis(((E)-oct-5-en-1-yl)oxy)butanoate) (Compound 37). In some embodiments, the ionizable lipid comprises ((2-hydroxyethyl)azanediyl)bis(hexane-6,1-diyl) bis(6,6-bis(hexyloxy)hexanoate) (Compound 49). In some embodiments, the ionizable lipid comprises ((2-hydroxyethyl)azanediyl)bis(heptane-7,1-diyl) bis(4,4-bis(((Z)-oct-5-en-1- yl)oxy)butanoate) (Compound 36). In some embodiments, the ionizable lipid comprises nonyl 8-((6-((4,4-bis(octyloxy)butanoyl)oxy)hexyl)(2-hydroxyethyl)amino)octanoate (Compound 82). In some embodiments, the ionizable lipid comprises nonyl 8-((2- hydroxyethyl)(6-((4-(((Z)-oct-5-en-1-yl)oxy)-4-(((Z)-oct-5-en-1- yl)oxy)butanoyl)oxy)hexyl)amino)octanoate (Compound 81).

[0021] In some embodiments, the ionizable lipid comprises Compound 1:(Compound 1).

[0022] In some embodiments, the ionizable lipid comprises Compound 37: Page 5 of 182ATTORNEY DOCKET NO. ORB-018WO1(Compound 37).

[0023] In some embodiments, the ionizable lipid comprises Compound 49:(Compound 49).

[0024] In some embodiments, the ionizable lipid comprises Compound 36:(Compound 36).

[0025] In some embodiments, the ionizable lipid comprises Compound 82:(Compound 82).

[0026] In some embodiments, the ionizable lipid comprises Compound 81:(Compound 81). Page 6 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0027] In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41% cholesterol, and 1.5-2.5 % PEG-modified lipid, such that the molar ratios combine to 100%.

[0028] In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40% cholesterol, and 2.5 % PEG-modified lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40.25% cholesterol, and 2.25 % PEG-modified lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40.5% cholesterol, and 2 % PEG-modified lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40.75% cholesterol, and 1.75 % PEG-modified lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 41% cholesterol, and 1.5% PEG-modified lipid, such that the molar ratios combine to 100%.

[0029] In some embodiments, the helper lipid is Distearoylphosphatidylcholine (DSPC), and the PEG-modified lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG2K), also known as DMG-PEG2000. In some embodiments, the helper lipid is 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and the PEG-modified lipid is DMG PEG2K.

[0030] In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 49, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 1, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 49, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K, such that the molar ratios combine to 100%.

[0031] In some embodiments, the present invention provides a pharmaceutical composition comprising the circular RNA described herein and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a vaccine composition comprising the circular RNA described herein. In some embodiments, the vaccine composition further comprises an adjuvant.

[0032] In some aspects, the present invention provides a method for stimulating an Page 7 of 182ATTORNEY DOCKET NO. ORB-018WO1 immune response to varicella-zoster virus (VZV) in a human subject. In some embodiments, the method comprises administering to the human subject an effective amount of the circular RNA described herein. In some embodiments, the circular RNA comprises a codon- optimized coding sequence encoding a VZV gE polypeptide or a variant thereof.

[0033] In some embodiments, the circular RNA is formulated in a delivery vehicle. In some embodiments, the circular RNA is not formulated in a delivery vehicle. In some embodiments, the circular RNA is formulated in a LNP as described herein.

[0034] In some embodiments, the immune response is a binding or neutralizing antibody response to the glycoprotein (gE) polypeptide encoded by the circular RNA and / or a cellular immune response to the glycoprotein (gE) polypeptide encoded by the circular RNA. In some embodiments, the immune response is a binding or neutralizing antibody response to the glycoprotein (gE) polypeptide encoded by the circular RNA and a cellular immune response to the glycoprotein (gE) polypeptide encoded by the circular RNA. In some embodiments, the immune response is a binding or neutralizing antibody response to the glycoprotein (gE) polypeptide encoded by the circular RNA. In some embodiments, the immune response is a cellular immune response to the glycoprotein (gE) polypeptide encoded by the circular RNA.

[0035] In some embodiments, the administration is for prophylactic treatment. In some embodiments, the circular RNA is administered with a single dose. In some embodiments, the circular RNA is administered with two doses. In some embodiments, the circular RNA is administered with more than one dose.

[0036] In some embodiments, the circular RNA is administered via intramuscular administration. In some embodiments, the circular RNA is administered via intradermal administration. In some embodiments, the circular RNA is administered via subcutaneous administration.

[0037] In some aspects, the present invention provides a method for treating or preventing a disease caused by varicella-zoster virus (VZV). In some embodiments, the method comprises administering to a human subject in need thereof the circular RNA described herein. In some embodiments, the method comprises administering to a human subject in need thereof the pharmaceutical compositions described herein. In some embodiments, the method comprises administering to a human subject in need thereof the vaccine compositions described herein.

[0038] In some embodiments, the administration is to prevent primary VZV infection in the human subject. In some embodiments, the administration is to prevent herpes zoster (shingles) in an adult human subject. Page 8 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0039] In some embodiments, the circular RNA is administered via intramuscular administration. In some embodiments, the circular RNA is administered via intradermal administration. In some embodiments, the circular RNA is administered via subcutaneous administration. In some embodiments, the pharmaceutical composition is administered via intramuscular administration. In some embodiments, the pharmaceutical composition is administered via intradermal administration. In some embodiments, the pharmaceutical composition is administered via subcutaneous administration. In some embodiments, the vaccine composition is administered via intramuscular administration. In some embodiments, the vaccine composition is administered via intradermal administration. In some embodiments, the vaccine composition is administered via subcutaneous administration. In some embodiments, the administration is a single dose administration.

[0040] In some embodiments, the administration is more than a single dose. In some embodiments, the administration is a double dose administration. In some embodiments, the administration is a multi-dose administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings are for illustration purposes only, and are not meant to be limiting.

[0042] FIG. 1 is a diagram of the gE antigen polypeptide, and shows the extracellular domain, transmembrane domain (TM), and intracellular domain. Some exemplary circRNA VZV vaccines encode a truncated gE antigen polypeptide. For example, the gE antigen polypeptide is truncated at residue 562, 568, or 574 as marked in FIG. 1. In some embodiments, exemplary gE antigen polypeptides comprise an alanine (A) substituted in place of tyrosine (Y) at residue position 569 (Y569A mutation) to disrupt trans-Golgi network targeting in the intracellular domain.

[0043] FIG. 2A displays an exemplary linear precursor to the circRNA VZV vaccines described herein. As shown, an internal ribosomal entry site (IRES) was added 5’ to the codon-optimized coding sequence (CDS) to allow for translation of the circRNA. To promote circularization, an exemplary self-splicing intron was added with part of the intron added 5’ to the IRES (shown as “5’ intron fragment”) and the other part of the intron added 3’ to the coding sequence (shown as “3’ intron fragment”). FIG. 2B displays an exemplary circRNA molecule from the circularization of the linear precursor RNA. The elements flanked by the intron fragments are retained after circularization.

[0044] FIG. 3A and FIG. 3B demonstrate expression of a VZV gE antigen after Page 9 of 182ATTORNEY DOCKET NO. ORB-018WO1 administration of exemplary circRNA VZV constructs to K562 lymphoblast cells. FIG. 3A shows the percent of cells expressing the exemplary VZV gE antigen. FIG. 3B shows the calculated fold-over-control (FOC) values for the exemplary circRNA VZV constructs.

[0045] FIG. 4A – FIG. 4D show the IgG titers from enzyme-linked immunosorbent assays (ELISA) performed on serum samples collected after administration of exemplary circRNA VZV vaccines in mice. The exemplary circRNA VZV vaccines were administered intramuscularly on Day 0 and Day 21, and serum samples were collected on Day 21 and Day 42. The IgG titers include total IgG (FIG. 4A), IgG1 (FIG. 4B), IgG2b (FIG. 4C), and IgGc (FIG. 4D) specific titers.

[0046] FIG. 5A – FIG. 5B show the ratio of IgG2c titers to IgG1 titers in serum collection after administration of exemplary circRNA VZV vaccines in mice at either day 21 (FIG. 5A) or day 42 (FIG. 5B). Higher IgG2c / IgG1 ratios indicate a T helper cell 1 (Th1) focused response in mice.

[0047] FIG. 6 shows the level of gE-specific neutralizing antibody titers (PRNT50) assessed in serum samples collected after administration of exemplary circRNA VZV vaccines in mice.

[0048] FIG. 7A – FIG. 7E summarize the gE-specific CD4 T cell response following administration of exemplary circRNA VZV vaccines in mice (Day 42). Characterized CD4 T cell responses include IFNγ secreting cells (FIG. 7A), TNF secreting cells (FIG. 7B), IL-2 secreting cells (FIG. 7C), IL-4 secreting cells (FIG. 7D), and IL-5 secreting cells (FIG. 7E).

[0049] FIG. 8A – FIG. 8D summarize the gE-specific CD8 T cell response following administration of exemplary circRNA VZV vaccines in mice (Day 42). Characterized CD8 T cell responses include IFNγ secreting cells (FIG. 8A), TNF secreting cells (FIG. 8B), IL-2 secreting cells (FIG. 8C), and polyfunctional cells (FIG. 8D).

[0050] FIG. 9A – FIG. 9B show the body weight (FIG. 9A) and variation in body weight (FIG. 9B) in mice administered the exemplary circRNA VZV vaccines tracked over 42 days.

[0051] FIG. 10A – FIG. 10B summarize injection site reactions following administration of exemplary circRNA VZV vaccines in mice at day 1 (FIG. 10A) and day 22 (FIG. 10B). Increased scores represent more severe injection site reactions, with a score of 0 indicating no observed reaction.

[0052] FIG. 11A – FIG. 11B show the ratio of IgG2c titers to IgG1 titers in serum Page 10 of 182ATTORNEY DOCKET NO. ORB-018WO1 samples collected from mice administered a low dose of exemplary circRNA VZV vaccines formulated in LNP formulations at day 21 (FIG. 11A) and day 35 (FIG. 11B). Higher IgG2c / IgG1 ratios indicate a T helper cell 1 (Th1) response in mice.

[0053] FIG. 12A – FIG. 12B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a low (1 µg) dose of exemplary circRNA VZV vaccines formulated in LNP formulations at day 21 (FIG. 12A) and day 35 (FIG. 12B).

[0054] FIG. 13A – FIG. 13B show the body weight (FIG. 13A) and variation in body weight (FIG. 13B) in mice administered a low dose of exemplary circRNA VZV vaccines formulated in LNP formulation tracked over 35 days.

[0055] FIG. 14A – FIG. 14B summarize injection site reactions following administration of a low dose of exemplary circRNA VZV vaccines formulated with LNP at day 1 (FIG. 14A) and day 22 (FIG. 14B). Increased scores represent more severe injection site reactions, with a score of 0 indicating no observed reaction.

[0056] FIG. 15A – FIG. 15B show the total IgG titers as measured by ELISA in serum samples collected from mice administered a high (10 µg) dose of exemplary circRNA VZV vaccines formulated in LNP formulations at day 21 (FIG. 15A) and day 35 (FIG. 15B). FIG. 15C shows a comparison between the immune response generated by mice administered an exemplary circRNA VZV vaccine at a high (10 µg) dose (Group 1, Construct 1A) and Shingrix® (Group 12) at day 21, and FIG. 15D shows the immune response at day 35.

[0057] FIG. 16 shows the ratio of IgG2c titers to IgG1 titers in serum collection after administration to mice of a high dose of exemplary circRNA VZV vaccines formulated in LNP formulations. Higher IgG2c / IgG1 ratios indicate a T helper cell 1 (Th1) response in mice.

[0058] FIG. 17A – FIG. 17B represent the body weight (FIG. 17A) and variation in body weight (FIG. 17B) in mice administered a high dose of exemplary circRNA VZV vaccines formulated in LNP formulations tracked over 35 days.

[0059] FIG. 18A – FIG. 18B summarize injection site reactions following administration to mice of a high dose of exemplary circRNA VZV vaccines formulated in LNP formulations at day 1 (FIG 18A) and day 22 (FIG. 18B). Increased scores represent more severe injection site reactions, with a score of 0 indicating no observed reaction.

[0060] FIG. 19A – FIG. 19B show the total IgG titers as measured by ELISA in serum samples collected from mice after administration of a single, high (10 µg) dose of exemplary circRNA VZV vaccines formulated in LNP formulations at day 21 (FIG. 19A) and day 35 (FIG. 19B). Page 11 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0061] FIG. 20 shows the ratio of IgG2c titers to IgG1 titers in serum collection after administration to mice of a single, high (10 µg) dose of exemplary circRNA VZV vaccines formulated in LNP formulations. Higher IgG2c / IgG1 ratios are indicative of a T helper cell 1 (Th1) response in mice.

[0062] FIG. 21A – FIG. 21B show the body weight (FIG. 21A) and variation in body weight (FIG. 21B) in mice administered a single, high (10 µg) dose of exemplary circRNA VZV vaccines formulated in LNP formulations tracked over 35 days.

[0063] FIG. 22 summarizes injection site reactions following administration to mice of a single, high (10 µg) dose of exemplary circRNA VZV vaccines formulated in LNP formulations. Increased scores represent more severe injection site reactions, with a score of 0 indicating no observed reaction.

[0064] FIG. 23 shows the total IgG titers as measured by ELISA in serum samples collected from mice in a chickenpox model after administration of single dose of exemplary circRNA VZV vaccines formulated in LNP formulations over the first 56 days of the study.

[0065] FIG. 24A – FIG. 24C shows the total IgG titers measured by ELISA in serum samples collected from mice in a chickenpox model on Days 28 (FIG. 24A), 42 (FIG. 24B), and 56 (FIG. 24C), after administration of single dose of exemplary circRNA VZV vaccines formulated in LNP formulations.

[0066] FIG. 25 shows the body weight in representative mice in a chickenpox model after administration of a single dose of exemplary circRNA VZV vaccines formulated in LNP formulations tracked over the first 56 days of the study.

[0067] FIG. 26A – FIG. 26B summarizes hunched posture (FIG. 26A) and injection site reactions (FIG. 26B) of mice in a chickenpox model following administration of a single dose of exemplary circRNA VZV vaccines formulated in LNP formulations. Increased scores represent more severe injection site reactions or hunched posture, with a score of 0 indicating no observed reaction. FIG. 26C shows body weights for selected groups throughout part 1 of the study (until day 98). Groups receiving Shingrix® (Groups 3 and 4) exhibited high reactogenicity after two boosts (on Day 28 and Day 56) for multiple days and pronounced decrease in body weight from Day 28 to Day 29 and Day 57 to Day 59. FIG. 26D shows that only the groups receiving Shingrix® (groups 3 and 4) exhibited Grade 1 reaction (swollen hindlimbs) or Grade 2 reaction on Day 57; circRNA groups exhibited only Grade 0 reaction. FIG. 26E shows that only the groups receiving Shingrix® (groups 3 and 4) exhibited Grade 1 reaction (swollen hindlimbs) or Grade 2 reaction on Day 59; circRNA groups exhibited only Grade 0 reaction. Page 12 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0068] FIG. 27A shows antibody (IgG) levels in mice challenged with VZV (Webster strain) at a dose of 105pfu on day 238 (at the end of the extended part of the in vivo study). Certain groups of mice (Group 2 (Vehicle Control), Group 4 (Shingrix®), and Group 8 (10 µg Construct 1 formulated with LNP007) were challenged in this study. FIG. 27B shows qPCR data on lung samples collected on Day 240 to detect VZV viral load following the in vivo viral infection. It shows that a single dose of 10 µg of the circRNA VZV vaccine was effective to eliminate the VZV virus in the lung after the viral infection. DEFINITIONS

[0069] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0070] “Adjuvant”: the term “adjuvant” refers to agents which confer immunity by themselves. An adjuvant assists the immune system un-specifically to enhance the antigen- specific immune response by e.g., promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g., modulate the antigen-specific immune response by e.g., shifting the dominating Th2-based antigen specific response to a more Th1-based antigen specific response or vice versa. Accordingly, an adjuvant may favorably modulate cytokine expression / secretion, antigen presentation, type of immune response etc. An adjuvant or an adjuvant component in the broadest sense is typically a (e.g., pharmacological or immunological) agent or composition that may modify, e.g., enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and / or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question. In the context of the present invention an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used interchangeably.

[0071] “Adaptive immune response”: The adaptive immune response is typically understood to be antigen-specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to Page 13 of 182ATTORNEY DOCKET NO. ORB-018WO1 mount these tailored responses is maintained in the body by “memory cells.” Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it. In this context, the first step of an adaptive immune response is the activation of naïve antigen-specific T cells or different immune cells able to induce an antigen-specific immune response by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naïve T cells are constantly passing. Cell types that can serve as antigen- presenting cells are inter alia dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with e.g., a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express major histocompatibility complex (MHC) molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g., of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.

[0072] “Antigen”: The term “antigen” refers to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g., by formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. In the sense of the present invention an antigen may be the product of translation of a provided nucleic acid molecule, preferably an RNA as defined herein. In this context, also fragments, variants and derivatives of peptides and proteins comprising at least one epitope are understood as antigen.

[0073] “Cellular immunity” or “cellular immune response”: the phrase “cellular immunity,” which is used interchangeably with “cellular immune response,” relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T- lymphocytes, and the release of various cytokines in response to an antigen. In a more Page 14 of 182ATTORNEY DOCKET NO. ORB-018WO1 general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. A cellular immune response is characterized e.g. by activating antigen- specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of an antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

[0074] “Circular RNA”: the term “circular RNA” or “circRNA” refer to an RNA that forms a circular structure through covalent or non-covalent bonds. A circular RNA can be endogenous or synthetic. Synthetically created and exogenously delivered circRNAs can be synthesized in vitro using self-splicing permuted introns from phage from in vitro transcribed constructs. Unlike linear mRNAs, circular RNAs are resistant to exonucleolytic decay. In some embodiments, circular RNAs have significantly improved therapeutic properties compared to linear mRNA therapeutics, including enhanced longevity as protein production vectors.

[0075] “Downstream”: As used herein, the term “downstream” refers to sequence that is 3’ to a particular sequence.

[0076] “Humoral immunity” or “humoral immune response”: “Humoral immunity,” which is used interchangeably with “humoral immune response,” refers typically to an immune response that results in antibody production and the accessory processes that may accompany antibody production. A humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0077] “Immune response”: The term “immune response” includes both a specific reaction of the adaptive immune system to a particular antigen (so called “specific” or “adaptive” immune response), and an unspecific reaction of the innate immune system (so called “unspecific” or “innate” immune response). In some aspects, the invention relates to eliciting specific reactions (adaptive immune responses) of the adaptive immune system. Particularly, it relates to adaptive immune responses to infections by viruses like e.g., VZV viruses. Furthermore, the invention also relates to eliciting an innate immune response, since the Page 15 of 182ATTORNEY DOCKET NO. ORB-018WO1 specific (adaptive) immune response can be supported by an additional unspecific reaction (innate immune response). Therefore, in some aspects, the invention also relates to simultaneous stimulation of the innate and the adaptive immune system to evoke an efficient immune response.

[0078] “Innate immune response”: The term “innate immune response,” also known as non-specific immune response, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. The cells of the innate system recognize and respond to pathogens in a generic way, but, unlike the adaptive immune system, the innate immune response does not confer long-lasting or protective immunity to the host. The innate immune system may be activated, for example, by ligands of pathogen- associated molecular patterns (PAMP) receptors, Toll-like receptors (TLRs), or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL- 22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD- like receptor, a ligand of a RIG-1 like receptor, an immunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), an antibacterial agent, or an anti-viral agent. Typically, a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through, a process known as antigen presentation; and / or acting as a physical and chemical barrier to infectious agents.

[0079] “Intron”: Introns in general are non-coding sequences of an RNA transcript. Following transcription, new, immature strands of messenger RNA, called pre-mRNA, may contain both introns and exons (any sequence of DNA or RNA that encode for proteins). The pre-mRNA molecule goes through a modification process in the nucleus called splicing during which the noncoding introns are cut out, and only the coding exons remain. Splicing produces a mature messenger RNA molecule that is then translated into a protein. In the context of the present invention, in some aspects, the introns are self-splicing introns. As used Page 16 of 182ATTORNEY DOCKET NO. ORB-018WO1 herein, the term “self-splicing intron” refers to a group of introns that can act as ribozymes to autocatalytically splice them out from the parent RNA in the absence of any added protein.

[0080] “Spacer”: As used herein, the term “spacer” refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance and / or flexibility between two adjacent polynucleotide regions. The length of a spacer sequence can be pre-determined based on the adjacent sequences. In some embodiments, the nucleic acid for making a circular RNA (e.g., precursor RNA) of the present invention comprises a 5’ spacer and 3’ spacer which are inserted between the circular RNA sequence and the upstream and downstream self-splicing intron sequences.

[0081] “Subject”: the terms “subject,” “individual,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans.

[0082] “Treat” or “Treatment”: As used herein, the terms “treatment,” “treat,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) increasing survival time: (b) decreasing the risk of death due to the disease; (c) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, i.e., arresting its development (e.g., reducing the rate of disease progression); and (e) relieving the disease, i.e., causing regression of the disease.

[0083] “Upstream”: As used herein, the term “upstream” refers to sequence that is 5’ to a particular sequence.

[0084] “Vaccine”: The term “vaccine” refers to a prophylactic or therapeutic material providing at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body’s adaptive immune system to provide, for example, an adaptive immune response.

[0085] “Vehicle”: The term “vehicle” refers to an agent, e.g., a carrier, which may typically be used within a pharmaceutical composition or vaccine for facilitating administering of the components of the pharmaceutical composition or vaccine to an individual. Page 17 of 182ATTORNEY DOCKET NO. ORB-018WO1 DETAILED DESCRIPTION

[0086] The present invention provides, among other things, circular RNA vaccines for treating or preventing herpes zoster. The present invention relates to circular RNA vaccines comprising at least one circular RNA molecule encoding at least one VZV antigen administered in an LNP formulation for use in the treatment and / or prevention of herpes zoster in patients, including, in elderly patients. In some embodiments, the elderly patient preferably exhibits an age of at least 50 years. Accordingly, vaccination of the patient with circRNA vaccines described herein elicits an immune response from the patient to treat herpes zoster. A. Varicella-Zoster Virus and Shingles

[0087] Varicella-zoster virus (VZV) is the cause of both chickenpox and shingles. Chickenpox is associated with the primary infection by VZV. After chickenpox, VZV can permanently rest in the body’s nerves until reactivation leads to shingles. It is estimated that over 99% of Americans born before 1980 had chickenpox and have the potential to develop shingles (also known as herpes zoster). A weakened immune system is one of the most common “triggers” for shingles and can be caused by, for example and without limitation, increased age, immune suppression, and / or stress. Around one in three people in the United States will likely develop shingles in their lifetime.

[0088] After primary infection, VZV establishes latency in ganglionic neurons. Then, when the immune system weakens, VZV reactivates by traveling anterograde towards nerve endings. There, VZV replicates in keratinocytes and epithelial cells, causing the formation of polykaryocytes. This ultimately leads to a dermatomal rash that is typically associated with shingles.

[0089] Shingles often starts with skin sensitivity, tingling, itching, and / or pain, which then develops into a rash of small, red spots that turn into blisters. Other common symptoms include fatigue, headache, and fever, but shingles can cause more serious complications. Post-herpetic neuralgia (PHN) is one such complication defined by a lasting pain sensation in the skin where the shingles rash presented. PHN occurs in around 10-18% of patients with the risk of developing PHN increasing with age. Acute complications of shingles also include, for example and without limitation, meningoencephalitis, myelitis, cranial nerve palsies, vasculopathy, gastrointestinal ulcers, pancreatitis, and hepatitis. Complications associated with shingles also lead to hospitalization in around 1-4% of patients. Increased age and / or a compromised immune system increase the likelihood of hospitalization. Page 18 of 182ATTORNEY DOCKET NO. ORB-018WO1 1. Shingles Treatment

[0090] Once shingles develops in a patient, treatment focuses on reducing pain, speeding healing, and lowering the risks of complications. Likewise, treatment for PHN is centered on relieving pain symptoms. A preferred approach to treating shingles is to prevent it from developing in the first place.

[0091] Vaccines provide a way to prime the immune system to prevent shingles from occurring. The natural immune response to VZV reactivation primarily occurs in the ganglia and in the skin. In the ganglia, local immune response is characterized by infiltration of CD4 T cells, CD8 T cells, natural killer (NK T cells), macrophages, and B cells. Similarly, immune response in the skin is associated with infiltration by CD4 T cells, CD8 T cells, NK T cells, and macrophages accompanied by increased expression of IFN, TNF, and IL-6. Effective vaccines result in the production of memory B and T cells that will more rapidly proliferate upon VZV reactivation.

[0092] Specifically, a vaccine, when injected, for example, intramuscularly, is taken up by dendritic cells and trafficked to the draining lymph node. There, MHC molecules on the dendritic cells present protein antigens from the vaccine and activate T cells. T cells then drive B cell development in the lymph node, resulting in the maturation of the antibody response. Maturation of the antibody response is associated with increases in antibody affinity and induction of different antibody isotypes.

[0093] Short-lived plasma cells will actively secrete antibodies specific for the vaccine protein, producing a rapid rise in serum antibody levels over about 2 weeks after vaccine administration. Memory B cells and CD8 effector T cells are also produced, which are important for the elimination of infected cells. On a longer time scale, vaccines result in long- lived plasma cells that can produce antibodies for decades alongside CD8 memory T cells that can proliferate rapidly when they encounter VZV.

[0094] VZV vaccines include, for example and without limitation, live, attenuated virus vaccines, adjuvanted recombinant protein vaccines, and RNA vaccines. Live, attenuated virus vaccines carry an inherent risk of the attenuated virus reverting to an infectious state, and use of an existing live, attenuated VZV vaccine was halted in the United States as of November 2020. Adjuvanted recombinant protein and RNA vaccines do not carry any infectious material, and as such, do not carry the same risk as live, attenuated virus vaccines. Both can produce a strong immune response to a specific antigen. However, RNA vaccines present the target antigen more naturally, as the target antigens are produced and presented by the body’s Page 19 of 182ATTORNEY DOCKET NO. ORB-018WO1 own antigen presenting cells. RNA vaccines, with the appropriate formulation, also induces more of a cellular immune response than recombinant protein vaccines. B. circRNA Vaccines

[0095] In accordance with the present invention, a circRNA vaccine or composition comprises at least one circRNA molecule encoding a VZV antigen polypeptide. In some embodiments, the circRNA molecule encodes a VZV glycoprotein E (gE), or a variant thereof. The present invention also provides nucleic acid molecules and methods for synthesizing the circRNA molecules disclosed herein. In some embodiments, the circRNA vaccine or composition is delivered using an LNP formulation. 1. circRNA Molecules

[0096] In some embodiments, the present invention provides a nucleic acid molecule for making a circRNA molecule that encodes a VZV antigen polypeptide. In some embodiments, the nucleic acid molecule is a DNA construct (e.g., a vector) that is transcribed into linear RNA, and the linear RNA circularizes into a circular RNA. In some embodiments, the nucleic acid molecule is a linear precursor RNA polynucleotide that circularizes into a circular RNA. The nucleic acid molecule described herein comprises different elements essential for the circular RNA synthesis and function. In some embodiments, the present invention provides a circular RNA encoding a VZV antigen polypeptide that is made from a nucleic acid molecule or by a method described herein.

[0097] In some embodiments, the nucleic acid molecule for making a circular RNA comprises self-splicing intron sequences and a coding sequence. A rational design of a synthetic circular RNA polynucleotide cassette includes at least two self-splicing intron sequences flanking the sequence to be circularized. The upstream and downstream intron sequences are self-spliced to generate a circular RNA comprising the coding sequence. In some embodiments, a 5’ spacer and / or 3’ spacer (e.g., 3’ untranslated region (UTR)) sequences are included between the intron sequences and the coding sequence (i.e., the sequence to be circularized). In some embodiments, at least one internal ribosome entry site (IRES) is added and operably linked to the coding sequence. i. Coding Sequences

[0098] The present invention provides, among other things, circRNA molecules Page 20 of 182ATTORNEY DOCKET NO. ORB-018WO1 comprising coding sequences for circRNA VZV vaccine constructs. The coding sequence, also known as open reading frame (ORF) sequence, is defined herein as the region that encodes the protein of interest.

[0099] In some embodiments, the coding sequence includes a region to initiate translation. In some embodiments, the region includes any translation initiation sequence or signal including a start codon. As a non-limiting example, the coding sequence includes a start codon. In some embodiments, as related to a DNA sequence, the start codon is “ATG,” “ACG,” “AGG,” “ATA,” “ATT,” “CTG,” “GTG,” or “TTG.” In some embodiments, as related to an RNA sequence, the start codon is “AUG,” “AUA,” “AUU,” “CUG,” “GUG,” or “UUG.”

[0100] In some embodiments, the coding sequence further includes a region to stop translation. This region may include any translation termination sequence or signal including a stop codon. As a non-limiting example, the region includes a stop codon. In some embodiments, as related to a DNA sequence, the stop codon is “TGA,” “TAA,” “TGA,” or “TAG.” In some embodiments, as related to an RNA sequence, the stop codon is “UGA,” “UAA,” “UGA” or “UAG.”

[0101] In some embodiments, the circRNA molecule of the present invention comprises a coding sequence. In some embodiments, the circRNA molecule of the present invention comprises a codon-optimized coding sequence (CDS). In some embodiments, the CDS comprises a nucleic acid sequence selected from Table A. Table A. Exemplary coding sequences for VZV gE polypeptide and variants thereof for circRNA VZV vaccines.Page 21 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 22 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 23 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 24 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 25 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 26 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 27 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 28 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 29 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 30 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 31 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0102] In some embodiments, the circRNA molecule encodes a wild-type VZV gE antigen comprising an amino acid sequence of SEQ ID NO: 53, which includes the underlined signal peptide. MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAE SWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQ DLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLR PIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAE SYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGT TYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAP DLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCE ADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVS VDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFL CTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYI KTR (SEQ ID NO: 53).

[0103] In some embodiments, the circRNA molecule encodes a truncated VZV gE Page 32 of 182ATTORNEY DOCKET NO. ORB-018WO1 antigen. In some embodiments, the circRNA molecule encodes a mutated VZV gE antigen. In some embodiments, the circRNA molecule encodes a mutated and truncated VZV gE antigen.

[0104] In some embodiments, the circRNA molecule encodes a VZV gE polypeptide variant. In some embodiments, the VZV gE polypeptide variant is truncated. In some embodiments, the truncated VZV gE polypeptide variant lacks the carboxy terminal domain.

[0105] In some embodiments, the truncated VZV gE antigen comprises residues 1-561 (Δ562) of the wild-type VZV antigen. In some embodiments, the truncated VZV gE antigen comprises residues 1-567 (Δ568) of the wild-type VZV antigen. In some embodiments, the truncated VZV gE antigen comprises residues 1-573 (Δ574) residues of the wild-type VZV antigen. In some embodiments, the mutated VZV antigen comprises a Y569A mutation relative to the wild-type VZV antigen. In some embodiments, the truncated VZV antigen further comprises a Y569A mutation relative to the wild-type VZV antigen.

[0106] In some embodiments, the circRNA molecule encodes a signal peptide. In some embodiments, the circRNA molecule encodes a signal peptide derived from VZV gE. In some embodiments, the circRNA molecule encodes a signal peptide derived from IgGκ. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 54. MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 54)

[0107] In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 55. METPAQLLFLLLLWLPDTTG (SEQ ID NO: 55)

[0108] In some embodiments, the coding sequence comprises a sequence identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 90% identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 95% identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 96% identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 97% identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 98% identical to any one of the sequences in Table A. In some embodiments, the coding sequence comprises a sequence at least 99% identical to any one of the sequences in Table A.

[0109] In some embodiments, the coding sequence comprises a sequence identical to any Page 33 of 182ATTORNEY DOCKET NO. ORB-018WO1 one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 90% identical to any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 95% identical to any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 96% identical to any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 97% identical to any one of SEQ ID NOs: 11-34. In some embodiments, the coding sequence comprises a sequence at least 98% identical to any one of SEQ ID NOs: 11- 34. In some embodiments, the coding sequence comprises a sequence at least 99% identical to any one of SEQ ID NOs: 11-34.

[0110] In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 22. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23. In some Page 34 of 182ATTORNEY DOCKET NO. ORB-018WO1 embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some embodiments, the coding sequence comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34.

[0111] In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 11. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 12. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 13. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 14. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 15. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 16. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 17. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 19. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 20. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 21. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 22. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: Page 35 of 182ATTORNEY DOCKET NO. ORB-018WO1 23. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 24. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 25. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 26. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 27. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 30. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 31. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 32. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 33. In some embodiments, the coding sequence comprises a sequence identical to SEQ ID NO: 34.

[0112] In some embodiments, the coding sequence comprises a sequence at least 75% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 80% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 85% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 90% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 96% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 97% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 98% identical to SEQ ID NO: 18. In some embodiments, the coding sequence comprises a sequence at least 99% identical to SEQ ID NO: 18.

[0113] In some embodiments, the coding sequence comprises a sequence at least 75% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 80% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 85% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 90% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 96% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 97% identical to SEQ ID NO: 28. In some embodiments, the coding sequence comprises a sequence at least 98% identical to SEQ ID Page 36 of 182ATTORNEY DOCKET NO. ORB-018WO1 NO: 28. In some embodiments, the coding sequence comprises a sequence at least 99% identical to SEQ ID NO: 28.

[0114] In some embodiments, the coding sequence comprises a sequence at least 75% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 80% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 85% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 90% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 96% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 97% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 98% identical to SEQ ID NO: 29. In some embodiments, the coding sequence comprises a sequence at least 99% identical to SEQ ID NO: 29.

[0115] In some embodiments, the coding sequence comprises a sequence at least 75% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 80% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 85% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 90% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 96% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 97% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 98% identical to SEQ ID NO: 34. In some embodiments, the coding sequence comprises a sequence at least 99% identical to SEQ ID NO: 34.

[0116] In addition to the coding sequences, the circRNA molecules of the present disclosure comprise regions that are partially or substantially not translatable, e.g., having a noncoding region. Such noncoding regions are different from the non-coding functional sequences and are located in any region of the circRNA molecule. The non-coding regions include, but are not limited to, the spacer and / or the flanking regions. In some embodiments, the noncoding regions are located in more than one region of the circRNA molecule. ii. Codon Optimization Page 37 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0117] The circRNA molecules of the present invention, including their regions, parts, or subregions, may be codon-optimized. In some embodiments, the nucleic acid coding sequences are codon-optimized. In some embodiments, the coding sequences of the circRNA molecules are codon-optimized. In some embodiments, the ORF sequences of the circRNA molecules are codon-optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include, but are not limited to, match codon frequencies in target and host organisms to ensure proper folding, alter GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and RNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide.

[0118] In some embodiments, a codon-optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, and / or (x) systematic variation of codon sets for each amino acid.

[0119] Codon optimization tools, algorithms and services are known in the art, non limiting examples include, but are not limited to, software from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods.

[0120] In some embodiments, the coding sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table B. Table B. Codon Options. Page 38 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0121] In some embodiments, the coding sequences were codon-optimized to increase expression of VZV gE. In some embodiments, the coding sequences were codon-optimized to increase immune response to VZV gE. In some embodiments, the coding sequences were codon-optimized to increase circRNA stability. In some embodiments, the coding sequences were codon-optimized to increase circRNA half-life.

[0122] In some embodiments, the coding sequences were optimized for human codon usage. In some embodiments, the coding sequences were optimized for GC content. In some embodiment, the coding sequences were optimized for human codon usage and GC content. In some embodiments, the coding sequences were optimized for structure stability. In some embodiments, the coding sequences were optimized for human codon usage and structure stability. In some embodiments, the coding sequences were optimized for GC content and structure stability. In some embodiments, the coding sequences were optimized for human codon usage, GC content, and structure stability. iii. Internal Ribosome Entry Site (IRES) Page 39 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0123] In some embodiments, the circRNA molecule described herein comprises an internal ribosome entry site (IRES) sequence. In some embodiments, the nucleic acid molecule encoding the circRNA molecule comprises an IRES. As used herein, the term “internal ribosome entry site” or “IRES” refers to an RNA sequence or structural element ranging in size from 10 nucleotides to 1,000 nucleotides or more which is capable of initiating translation of a polypeptide in the absence of a normal RNA cap structure. In some embodiments, an IRES element engages a eukaryotic ribosome for translation, i.e., the IRES element initiates cap-independent translation and protein synthesis.

[0124] In some embodiments, the IRES has a sequence of an IRES, or is a functional fragment or variant thereof.

[0125] The IRES sequence may be derived from a viral genome or is a cellular IRES. In one embodiment, the nucleic acid molecule described herein comprises a viral IRES.

[0126] In some embodiments, the IRES may include but is not limited to, the encephalomyocarditis virus (EMCV) IRES, poliovirus IRES, Kaposi sarcoma-associated herpesvirus (KSHV) vFLIP IRES, or hepatitis C virus (HCV) IRES. In some embodiments the IRES has a sequence of an IRES from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Human coxsackievirus A7, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPAI, Human AMLI / RUNXI, Drosophila antennapedia, Human AQP4, Human ATIR, Human BAG-I, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEFI, Mouse HIFI alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae Y API, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus EID, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT00I, HRV14, Page 40 of 182ATTORNEY DOCKET NO. ORB-018WO1 HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG-JI, Human Parechovirus 1, Crohivirus B, Y c-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus El 4, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVAI0, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GTI 10, GBV-C Kl 737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDVl, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Apodemus, Agrarius Picornavirus, Caprine Kobuvirus, Canine Kobuvirus, Mouse Kobuvirus, Feline Kobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZl, Salivirus FHB, CVB3, CVBl, Echovirus 7, CVB5, EVA71, CVA3, CVA12, or EV24.

[0127] In some embodiments, the IRES that can be included in a circular RNA is a Type 1 IRES.

[0128] In some embodiments, the IRES that can be included in a circular RNA is a viral IRES sequence.

[0129] In some embodiments, the IRES is an enterovirus IRES. In some embodiments, the IRES is a human rhinovirus (HRV) IRES.

[0130] In some embodiments, the IRES that can be included in a circular RNA is a non viral IRES sequence, including but not limited to, IRES sequences from yeast, the human angiotensin II type 1 receptor IRES, fibroblast growth factor IRESs (e.g., FGF-1 IRES and FGF-2 IRES) vascular endothelial growth factor IRES, and insulin-like growth factor 2 IRES.

[0131] In some embodiments, the IRES that can be included in a circular RNA is a synthetic IRES sequence. A “synthetic IRES” is an IRES that is modified relative to a wildtype IRES in order to modulate its structure and / or activity. For example, in some embodiments, an IRES that is modified to incorporate an aptamer sequence is a synthetic IRES.

[0132] In some embodiments, the IRES sequence in the circular RNA comprises at least one RNA secondary structure element or feature.

[0133] The IRES may be of any length or size. For example, the IRES may be about 100 nucleotides to about 1,000 nucleotides in length (e.g., about 150, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 750, about 800, about 850, or about 900 nucleotides in length, or Page 41 of 182ATTORNEY DOCKET NO. ORB-018WO1 a range defined by any two of the foregoing values). In some embodiments, the IRES may be about 200 nucleotides to about 800 nucleotides in length (e.g., about 150, about 200, about 210, about 220, about 240, about 260, about 280, about 320, about 340, about 360, about 380, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, about 620, about 640, about 660, about 680, about 700, about 720, about 740, about 760, about 780, or about 800 nucleotides in length, or a range defined by any two of the foregoing values). In some embodiments, the IRES may be about 200 to about 400, about 400 to about 600, about 600 to about 700, or about 600 to about 800 nucleotides in length. In some embodiments, the IRES is about 210 nucleotides in length. In some embodiments, the IRES may be about 100 to about 3000 nucleotides in length.

[0134] In some embodiments, as described herein, the IRES sequence is operably linked to a protein-coding sequence. Different IRES elements (and exonic elements in general) affect the strength of protein expression as well as the cell / tissue specificity. Selection of an IRES element depends on the purpose of protein expression.

[0135] In some embodiments, the IRES is “in-frame” with respect to the protein-coding nucleic acid sequence, that is, the IRES is positioned in the circular RNA molecule in the correct reading frame for the encoded protein. In other embodiments, the IRES may be “out of frame” with respect to the protein-coding nucleic acid sequence, such that the position of the IRES disrupts the open reading frame (ORF) of the protein-coding nucleic acid sequence. In other embodiments, the IRES may overlap with one or more ORFs of the protein-coding nucleic acid sequence.

[0136] In some embodiments, the IRES comprises a sequence identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 90% identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 95% identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 96% identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 97% identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 98% identical to any one of the sequences in Table C. In some embodiments, the IRES comprises a sequence at least 99% identical to any one of the sequences in Table C. Table C. Exemplary IRES sequences. Page 42 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 43 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0137] In some embodiments, the IRES comprises a sequence identical to any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises a sequence at least 90% identical to any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises a sequence at least 95% identical to any one of SEQ ID NOs: 35-43In some embodiments, the IRES comprises a sequence at least 96% identical to any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises a sequence at least 97% identical to any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises a sequence at least 98% identical to any one of SEQ ID NOs: 35-43. In some embodiments, the IRES comprises a sequence at least 99% identical to any one of SEQ ID NOs: 35-43.

[0138] In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36. In some Page 44 of 182ATTORNEY DOCKET NO. ORB-018WO1 embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42. In some embodiments, the IRES comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43.

[0139] In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 35. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 36. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 37. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 38. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 39. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 40. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 41. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 42. In some embodiments, the IRES comprises a sequence identical to SEQ ID NO: 43. iv. Exemplary circRNA VZV Vaccines

[0140] In some embodiments, the circRNA molecule of the present invention comprises a coding sequence described herein. In some embodiments, the circRNA molecule of the present invention further comprises an IRES operably linked to the coding sequence described herein. In some embodiments, the circRNA molecule comprises an additional sequence(s) as a result of, and / or to aid in, production, purification, circularization, and / or protein expression.

[0141] In some embodiments, the circRNA of the present invention comprises a sequence identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 75% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 80% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 85% identical to any one of SEQ Page 45 of 182ATTORNEY DOCKET NO. ORB-018WO1 ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 90% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 95% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 96% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 97% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 98% identical to any one of SEQ ID NOs: 76-108. In some embodiments, the circRNA comprises a sequence at least 99% identical to any one of SEQ ID NOs: 76-108.

[0142] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 76. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 77. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 78. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 79. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 80.

[0143] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 81. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 82. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 83. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 85.

[0144] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 88. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89. In some embodiments, the circRNA Page 46 of 182ATTORNEY DOCKET NO. ORB-018WO1 comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90.

[0145] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 91. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 92. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 93. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 94. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 95.

[0146] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 96. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 97. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 98. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 99. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 100.

[0147] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 102. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 105.

[0148] In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 106. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 107. In some embodiments, the circRNA comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 108. Page 47 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0149] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 76. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 77. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 78. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 79. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 80.

[0150] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 81. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 82. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 83. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 84. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 85.

[0151] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 86. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 87. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 88. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 89. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 90.

[0152] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 91. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 92. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 93. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 94. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 95.

[0153] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 96. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 97. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 98. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 99. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 100.

[0154] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 101. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 102. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 103. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 104. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 105.

[0155] In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 106. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 107. In some embodiments, the circRNA comprises a sequence identical to SEQ ID NO: 108. 2. Precursor Molecules for making circRNA VZV Vaccines Page 48 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0156] In some embodiments, the present invention provides nucleic acid molecules for making the circRNA described herein. In some embodiments, the nucleic acid molecule for making circRNA comprises the coding sequence described herein. In some embodiments, the nucleic acid molecule for making circRNA further comprises the IRES described herein.

[0157] In some embodiments, a nucleic acid molecule for making a circRNA VZV vaccine is a precursor linear RNA. In some embodiments, a nucleic acid molecule for making a circRNA VZV vaccine is a DNA vector that is used as a template to transcribe a precursor linear RNA.

[0158] In some embodiments, the nucleic acid molecule for making a circRNA molecule comprises, from 5’ to 3’ end, an upstream intron sequence, an IRES, a coding sequence, and a downstream intron sequence. In some embodiments, the nucleic acid molecule for making a circRNA molecule comprises, from 5’ to 3’ end, an upstream intron sequence, an IRES, a coding sequence, and a downstream intron sequence. i. Self-Splicing Intron Sequences

[0159] In some embodiments, an VZV circRNA vaccine described herein is produced using a permuted intron-exon (PIE) method. In accordance, the nucleic acid molecule for making a circular RNA described herein comprises permuted self-splicing intron sequences. The permutation generates an upstream (i.e., 5’ end) intron sequence corresponding to a 3’ intron splicing fragment of a self-splicing intron and a downstream (i.e., 3’ end) intron sequence corresponding to a 5’ intron splicing fragment of the self-splicing intron. The intron sequences flank the RNA sequence to be circularized.

[0160] In some embodiments, the intron sequences are derived from a Group I self- splicing intron with autocatalytic activity (acting as ribozymes), i.e., self-splicing. Group I introns can be found naturally within the rRNA, tRNA, and mRNA genes of bacteria and non-metazoan eukaryotes (e.g., T4 bacteriophage). A general discussion of the catalytic activity of group I introns can be found in the review article by Hausner et al (Mobile DNA, 2014, vol 5(8)).

[0161] In some embodiments, the intron sequences are derived from a Group II self- splicing intron. Group II introns are naturally found in the bacteria and organelle genomes of lower eukaryotes. A general discussion of the catalytic activity of group II introns can be found in the review article by Pyle (Annu Rev Biophys. 2016, 5(45): 183-205).

[0162] In some embodiments, the nucleic acid molecule for making a circular RNA Page 49 of 182ATTORNEY DOCKET NO. ORB-018WO1 comprises an upstream intron sequence and a downstream intron sequence generated from the cut site or splitting position (“cut site,” “permutation site,” and “splitting position” are used interchangeably herein), wherein the cut site retains the structural integrity of the intron. As used herein, the term “catalytic core” refers to an intron region (e.g., the internal stem structure) needed for the intron to self-splice, which is well-understood in the art. Typically, the catalytic core of an intron, particularly, a Group I intron, is a highly conserved small region of about 70 nucleotides composed of paired regions (e.g., P1-P6) that form elongated domains (e.g., helical domains). More detailed description can be found in, e.g., Michel and Westhof, J. Mol. Bio., 1990, 216: 585-610; Luptak and Doudna, Nucleic Acids Research, 2004, 32(7): 2272-80, the contents of which are incorporated by reference herein. In some embodiments, the cut site is designed to retain the catalytic core of the intron, which is a region of nucleotides containing sequences and structures needed for the intron to self-splice. In some embodiments, a suitable cut site is chosen outside the catalytic core of the intron such that the catalytic core may retain its structural integrity.

[0163] In some embodiments, the intron sequences are derived from T4 bacteriophage td intron. In some embodiments, the intron sequences are derived from Twort ORF142 intron (GenBank: AF132670.1) (referred to herein as Twort intron). In some embodiments, the “upstream intron fragment sequence” and “downstream intron fragment sequence” derived from, for example, the T4Td or Twort intron, are also referred to as “upstream intron-exon fragment sequence” and “downstream intron-exon fragment sequence” in the circRNA that is generated.

[0164] In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence and a downstream intron sequence derived from a Twort intron generated from a cut site or splitting position, wherein the cut site is between any two residues within a region defined by P1, P2, P3, P4, P5, P5a, P6, P6a, P7, P7.1, P7.2, P8, P9, P9.0, P9.1, P10 or P12 as shown, for example, in FIG. 8 in Landthaler and Shub, PNAS, 1999, 96 (12), 7005-7010), which is incorporated herein by reference.

[0165] In some embodiments of the present disclosure, a V1 cut site is between any two residues in a P7 region. In some embodiments, a V2 cut site is between any two residues in a P6a region. In some embodiments, a V3 cut site is between any two residues in a P7.1 region.

[0166] In some embodiments, the V1, V2 and / or V3 cut sites are between any two residues within a loop. In some embodiments, the V1, V2 and / or V3 cut sites are not within an internal stem structure of the intron. In some embodiments, a V1 site is between adenine and uracil (A / U) in a P7 region. Page 50 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0167] In some embodiments, a V2 site is between any two residues in a loop comprising AGUAAU in a P6a region. In some embodiments, a V2 cute site is between adenine and guanine (A / G) within the loop of the intron. In some embodiments, a V2 site is between guanine and uracil (G / U) within the loop of the intron. In some embodiments, a V2 cut site between uracil and adenine (U / A) within the loop of the intron. In some embodiments, a V2 sit is between two adenines within the loop of the intron. In some embodiments, a V2 site is between adenine at position 5 of the loop and uracil at position 6 of the loop. In some embodiments, a V2 cut site between uracil at position 3 of the loop and adenine at position 4 (U / A) of the loop. In some embodiments, a V2 cut site is at an equivalent position in a loop comprising a complementary loop sequence to AGUAAU. In some embodiments, a V2 site is at an equivalent position in a loop comprising one or more variations in the loop sequence of AGUAAU. In some embodiments, a V2 cut site is at an equivalent position in a loop comprising 1, 2, 3, 4, 5 or 6 variations in the loop sequence of AGUAAU. In some embodiments, a V2 site is at a position between a stem residue and the first or last residue of the loop sequence of AGUAAU.

[0168] In some embodiments, a V3 site is between any two residues in a loop comprising GAUA in a P7.1 region. In some embodiments, a V3 site is between guanine and adenine (G / A) within a loop of the intron. In some embodiments, a V3 cut site is between adenine and uracil (A / U) within a loop of the intron. In some embodiments, a V3 site is between uracil and adenine (U / A) within a loop of the intron. In some embodiments, a V3 site is at an equivalent position in a loop comprising a complementary loop sequence to GAUA. In some embodiments, a V3 cut site is at an equivalent position in a loop comprising one or more variations in the loop sequence of GAUA. In some embodiments, a V3 cut site is at an equivalent position in a loop comprising 1, 2, 3 or 4 variations in the loop sequence of GAUA. In some embodiments, a V3 cut site is at a position between a stem residue and the first or last residue of the loop sequence of GAUA.

[0169] In some embodiments, the precursor RNA disclosed herein include exemplary intron sequences that are selected from Table D.

[0170] Table D. Exemplary intron sequences. The bold, underlined sequences are retained after RNA circularization.Page 51 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0171] In some embodiments, the intron sequences are derived from a T4td intron. In some embodiments, the nucleic acid for making a circular RNA for VZV vaccine comprises an upstream intron fragment presented by SEQ ID NO: 44, and a downstream intron fragment presented by SEQ ID NO: 45.

[0172] In some embodiments, the intron sequences are derived from a Twort intron. In some embodiments, the nucleic acid for making a circular RNA for VZV vaccine comprises an upstream intron fragment presented by SEQ ID NO: 46, and a downstream intron fragment presented by SEQ ID NO: 47. ii. Other Sequence Fragments to Facilitate RNA Circularization

[0173] In some embodiments, the nucleic acid molecule for making a circRNA VZV vaccine comprises an additional sequence(s) as a result of, and / or to aid in, production, purification, and / or circularization.

[0174] In some embodiments, the nucleic acid molecule for making a circular RNA comprises a spacer. In some embodiments, the spacer is a 5’ spacer. In some embodiments, the spacer is a 3’ spacer.

[0175] In some embodiments, the nucleic acid molecule for making a circular RNA may comprise a 5’ spacer between the upstream intron sequence and the IRES. In some embodiments, the nucleic acid molecule for making a circular RNA may comprise a 5’ spacer between the upstream intron sequence and the IRES. In some embodiments, the nucleic acid molecule for making a circular RNA may comprise a 3’ spacer between the stop codon of the coding sequence and the downstream intron sequence.

[0176] In some embodiments, the circRNA molecule may comprise a 5’ spacer between the upstream intron sequence and the IRES and a 3’ spacer between the stop codon of the coding sequence and the downstream intron sequence.

[0177] Spacers described herein comprise random sequences and the length of a spacer Page 52 of 182ATTORNEY DOCKET NO. ORB-018WO1 may vary. In some embodiments, the spacer comprises a sequence identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 90% identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 95% identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 96% identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 97% identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 98% identical to any one of the sequences in Table E. In some embodiments, the spacer comprises a sequence at least 99% identical to any one of the sequences in Table E. Table E. Exemplary spacer sequences.

[0178] In some embodiments, the spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence at least 90% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence at least 95% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence at least 96% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence at least 97% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence at least 98% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence at least 99% identical to any one of SEQ ID NOs: 48-52. In some embodiments, the spacer comprises a sequence identical to any one of SEQ ID NOs: 48-52.

[0179] In some embodiments, the spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 48. In some embodiments, the spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49. In some embodiments, the spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 50. In some embodiments, the spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51. In some embodiments, the Page 53 of 182ATTORNEY DOCKET NO. ORB-018WO1 spacer comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52.

[0180] In some embodiments, the spacer comprises a sequence identical to SEQ ID NO: 48. In some embodiments, the spacer comprises a sequence identical to SEQ ID NO: 49. In some embodiments, the spacer comprises a sequence identical to SEQ ID NO: 50. In some embodiments, the spacer comprises a sequence identical to SEQ ID NO: 51. In some embodiments, the spacer comprises a sequence identical to SEQ ID NO: 52.

[0181] In some embodiments, the nucleic acid described herein comprises a 5’ spacer and a 3’ spacer. In some embodiments, the nucleic acid described herein comprises a 5’ spacer. In some embodiments, the nucleic acid described herein comprises a 3’ spacer. In some embodiments, the nucleic acid described herein does not comprise a spacer.

[0182] In some embodiments, the circRNA molecule described herein comprises a 5’ spacer and a 3’ spacer. In some embodiments, the circRNA molecule described herein comprises a 5’ spacer. In some embodiments, the circRNA molecule described herein comprises a 3’ spacer. In some embodiments, the circRNA molecule described herein does not comprise a spacer. iii. Modified Nucleotides

[0183] In some embodiments, the circRNA molecule comprises one or more modified nucleotides. In some embodiments, the circRNA molecule is unmodified.

[0184] In some embodiments, the circRNA molecules of the present invention may include one, two, three, or more modifications. In some embodiments, the modified nucleotides are located in coding region(s). In some embodiments, the modified nucleotides are in the untranslated region(s).

[0185] In some embodiments, the modifications stabilize the circRNA molecule and enhance resistance to degradation as compared to unmodified nucleotides. In some embodiments, modified nucleotides enhance biological functions of nucleic acid molecules, for example, increase binding to a RNA binding protein or increasing translation.

[0186] In some embodiments, the modified nucleotide is one or more of N1 methylpseudouridine, 5-methoxyuridine, N6-methyladenosine, pseudouridine or 5- methylcytosine.

[0187] In some embodiments, the modified nucleotide is N1-methylpseudouridine. In Page 54 of 182ATTORNEY DOCKET NO. ORB-018WO1 some embodiments, the modified nucleotide is 5-methoxyuridine. In some embodiments, the modified nucleotide is N6-methyladenosine. In some embodiments, the modified nucleotide is pseudouridine. In some embodiments, the modified nucleotide is 5-methylcytosine.

[0188] In some embodiments, the modified nucleotide is 100%. In some embodiments, the modified nucleotide is less than 50%. In some embodiments, the modified nucleotide is less than 20%. In some embodiments, the modified nucleotide is less than 10%.

[0189] The circRNA molecules of the present disclosure may contain from about 0% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, T / U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 85% to 95%, from 85% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[0190] In some embodiments, the circRNA molecules are 100% modified. In some embodiments, the mRNA molecules are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the mRNA molecules are at least 75% modified, e.g., at least 75% of the nucleotides are modified. In some embodiments, the mRNA molecules are at least 20% modified, e.g., at least 20% of the nucleotides are modified. In some embodiments, the mRNA molecules are at least 10% modified, e.g., at least 10% of the nucleotides are modified. It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linkage) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification.

[0191] In some embodiments, the modifications are structural modifications and / or chemical modifications. In some embodiments, the chemical modification is a nucleotide and / or nucleoside modification including a nucleobase modification and / or a sugar modification, and a backbone linkage modification (i.e., the internucleoside linkage, e.g., a linking phosphate, a phosphodiester linkage, and a phosphodiester backbone). In some Page 55 of 182ATTORNEY DOCKET NO. ORB-018WO1 embodiments, the structural modification includes a secondary and / or tertiary structural modification.

[0192] In some embodiments, modifications include modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof.

[0193] In some embodiments, one, two, or more (optionally different) nucleoside or nucleotide modifications may be incorporated to the circular polynucleotides of the present disclosure.

[0194] In some embodiments, the circRNA molecule comprises at least one modification described herein. In other embodiments, the circRNA molecules comprise two, three, four, or more (optionally different) chemical modifications described herein. The modifications may be combinations of nucleobase (purine and / or pyrimidine), sugar and backbone (internucleoside) linkage modifications. The modifications may be located on one or more nucleotides of the circular polynucleotide. In some embodiments, all the nucleotides of the circRNA molecule are chemically modified. In some embodiments, all the nucleotides of the nucleic acid sequence with a biological function are chemically modified.

[0195] In some embodiments, the circRNA molecules are at least 10% modified in only one component of the nucleotide, with such component being the nucleobase, sugar, or linkage between nucleosides. For example, modifications may be made to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleobases, sugars, or linkages of a polynucleotide described herein.

[0196] In some embodiments, the circRNA molecules are designed with a patterned array of sugar, nucleobase or linkage modifications.

[0197] In some embodiments, the circRNA molecules comprise modifications to maximize stability.

[0198] In other embodiments, the circRNA molecules comprise modifications to decrease stability.

[0199] In some embodiments, the modified nucleosides and nucleotides include a modified nucleobase. Examples of nucleobases in RNA include, but are not limited to, adenosine(A), guanosine(G), cytosine(C), and uracil(U). Examples of nucleobases in DNA include, but are not limited to, adenine(A), guanine(G), cytosine(C), and thymine(T).

[0200] In some embodiments, the modified nucleobase is a modified uracil(U). Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- Page 56 of 182ATTORNEY DOCKET NO. ORB-018WO1 thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine (I5U) or 5-bromo-uridine (br5U)), 3- methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1- methylpseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), pseudouracil (ψ), 1-methyl-4- thio-pseudouridine (m1, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza- pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, N1-methyl-pseudouridine (also known as 1-methylpseudouridine (m, 3- (3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O- methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2′-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O- methyl-uridine (cmnm5Um), 3,2′-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)- 2′-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'‐F‐ara‐uridine, 2'‐F‐ uridine, 2'‐OH‐ara‐uridine, 5‐(2‐carbomethoxyvinyl) uridine, and 5‐[3‐(1‐E‐ propenylamino)uridine.

[0201] In some embodiments, the modified nucleobase is a modified cytosine(C). Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5- formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine Page 57 of 182ATTORNEY DOCKET NO. ORB-018WO1 (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl- pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O- dimethyl-cytidine (m5Cm), N4-acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl- cytidine (m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm), N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'‐F‐ara‐cytidine, 2'‐F‐cytidine, and 2'‐OH‐ara‐cytidine.

[0202] In some embodiments, the modified nucleobase is a modified adenosine(A). Exemplary nucleobases and nucleosides having a modified adenosine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza- adenosine, 7-deaza-8-aza-adenosine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2- methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6- threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2- methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl- adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thio-adenosine, 2′-O-methyl- adenosine (Am), N6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl-adenosine (m62Am), 1,2′-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2- amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'‐F‐ara‐adenosine, 2'‐F‐ adenosine, 2'‐OH‐ara‐adenosine, and N6‐(19‐amino‐pentaoxanonadecyl)-adenosine.

[0203] In some embodiments, the modified nucleobase is a modified guanosine(G). Exemplary nucleobases and nucleosides having a modified guanosine include inosine (I), 1- methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), Page 58 of 182ATTORNEY DOCKET NO. ORB-018WO1 epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl- guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2′-O-methyl- guanosine (m22Gm), 1-methyl-2′-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2′-O-methyl- guanosine (m2,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m1Im), and 2′-O- ribosylguanosine (phosphate) (Gr(p)).

[0204] In some embodiments, the nucleobase of the nucleotide is independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase and / or analog is each independently selected from adenosine, cytosine, guanosine, uracil, naturally-occurring and synthetic derivatives of a base, including but not limited to pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenosine, 6-methyl and other alkyl derivatives of adenosine and guanosine, 2-propyl and other alkyl derivatives of adenosine and guanosine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenosines and guanosines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanosine and 7-methyladenosine, 8-azaguanosine and 8-azaadenosine, deazaguanosine, 7-deazaguanosine, 3-deazaguanosine, deazaadenosine, 7-deazaadenosine, 3- deazaadenosine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.

[0205] In some embodiments, the circRNA molecule comprises a nucleoside modification. In some embodiments, one or more atoms of a pyrimidine nucleobase is replaced or substituted, for example, with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or groups. Page 59 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0206] In some embodiments, uracil nucleosides of the circRNA molecule of the present disclosure are all modified. In some embodiments, the guanosine nucleosides of the circRNA molecule of the present disclosure are all modified. In some embodiments, the cytosine nucleosides of the circRNA molecule of the present disclosure are all modified. In some embodiments, the thymine nucleosides of circRNA molecule of the present disclosure are all modified. In some embodiments, the adenosine nucleosides of circRNA molecule of the present disclosure are all modified. In some embodiments, the modifications to each nucleobase are the same. In some embodiments, the modifications to each nucleobase are different.

[0207] In some embodiments, modifications of the modified nucleosides and nucleotides are present in the sugar subunit. In some embodiments, the circRNA molecule described herein comprise at least one sugar modification. Generally, circRNA includes the sugar subunit: ribose, which is a 5-membered ring having an oxygen. In some embodiments, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2′OH-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG)-O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); and “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connected by a C1-6alkylene or C1-6 heteroalkylene bridge to the 4’-carbon of the same ribose sugar, where exemplary bridges include methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0208] Other exemplary sugar modifications include replacement of the oxygen(O) in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such Page 60 of 182ATTORNEY DOCKET NO. ORB-018WO1 as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with α-L-threofuranosyl-(3′→2′)) , and peptide nucleic acid (PNA, where 2-amino-ethyl- glycine linkages replace the ribose and phosphodiester backbone).

[0209] In some embodiments, the sugar subunit contains one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. In some embodiments, circRNA molecules as described herein, include nucleotides containing, e.g., arabinose, as the sugar.

[0210] In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the circRNA molecules is substituted with -O- methoxyethyl, referred to as 2’-OMe. In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -F, referred to as 2’-F. In some embodiments, the sugar modification is one or more locked nucleic acids (LNAs). In some embodiments, the polynucleotides are fully 2’-MOE-sugar modified.

[0211] In some embodiments, one or more modifications are present in the internucleoside linkage (the linking phosphate or the phosphodiester linkage or the phosphodiester backbone). In the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably.

[0212] In some embodiments, backbone phosphate groups are modified by replacing one or more of the oxygen atoms with a different substituent. In some embodiments, modified nucleosides and nucleotides include replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonates phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker is also modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0213] The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides through unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules. Page 61 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0214] In some embodiments, the circRNA molecules of the present disclosure comprise at least one phosphorothioate linkage, methylphosphonate linkage between nucleotides, 5′- (E)-vinylphosphonate (5′-E-VP), a phosphate mimic, as a modification.

[0215] In some embodiments, the internucleoside linkages of the circRNA molecules may be partially or fully modified.

[0216] In some embodiments, modified nucleotides incorporated in the circRNA molecules include, for example, 2’-O-Methyl-modified or 2’-O-Methoxyethyl-modified nucleotides (2’-OMe and 2’-MOE modifications, respectively), an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5′- O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)- pseudouridine.

[0217] Additional modifications to circRNA molecules of the present disclosure include, for example, modification or deletion of nucleotides (or codons) encoding one or more N- linked glycosylation site in a translated polypeptide.

[0218] In some embodiments, different sugar modifications, nucleobase modifications, and / or internucleoside linkages (e.g., backbone structures) are introduced at various positions in a polynucleotide described herein. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.

[0219] In some embodiments, the one or more modified nucleotides is a 2′ O-methyl or a phosphorothioate modified nucleotide. Accordingly, in some embodiments, the one or more modified nucleotides comprises a 2′ O-methyl modification. In some embodiments, the one or more modified nucleotides comprises a phosphorothioate modification.

[0220] In some embodiments, the one or more modified nucleotides is selected from 2′-O- methyl 3′-phosphorothioate, 2′O-methyl, 2′-ribo 3′-phosphorothioate, 2′-fluro, 2’-O- methoxyethyl morpholino (PMO), locked nucleic acid (LNA), deoxy, or 5′ phosphate modified nucleotide. Accordingly, in some embodiments, the one or more modified nucleotides is a 2′-O-methyl 3′-phosphorothioate. In some embodiments, the one or more modified nucleotides is a 2′-O-methyl nucleotide. In some embodiments, the one or more modified nucleotides is a 2′-ribo 3′-phosphorothioate. In some embodiments, the one or more modified nucleotides is a 2′-fluro nucleotide. In some embodiments, the one or more modified nucleotides is a locked nucleic acid (LNA). In some embodiments, the one or more modifications comprises a 2’-O-methoxyethyl morpholino (PMO). In some embodiments, the Page 62 of 182ATTORNEY DOCKET NO. ORB-018WO1 one or more modifications comprises a deoxy modification. In some embodiments, the one or more modifications comprises a 5′ phosphate modification.

[0221] Various modified RNA bases are known in the art and include for example, 2′-O- methoxy-ethyl bases (2′-MOE) such as 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2- MethoxyEthoxy G, 2-MethoxyEthoxy T. Other modified bases include for example, 2′-O- Methyl RNA bases, and fluoro bases. Various fluoro bases are known, and include for example, Fluoro C, Fluoro U, Fluoro A, Fluoro G bases. Various 2′OMethyl modifications can also be used with the methods described herein. For example, the following RNA comprising one or more of the following 2′OMethyl modifications can be used with the methods described: 2′-OMe-5-Methyl-rC, 2′-OMe-rT, 2′-OMe-rI, 2′-OMe-2-Amino-rA, Aminolinker-C6-rC, Aminolinker-C6-rU, 2′-OMe-5-Br-rU, 2′-OMe-5-I-rU, 2-OMe-7-Deaza- rG.

[0222] In some embodiments, the circRNA molecule comprises one or more of the following modifications: phosphorothioates, 2′O-methyls, 2′ fluoro (2′F), DNA. In some embodiments, the circRNA molecule comprises one or more of the following modifications: 2′ -O-2-Methoxyethyl (MOE), locked nucleic acids, bridged nucleic acids, unlocked nucleic acids, peptide nucleic acids, morpholino nucleic acids. In some embodiments, the circRNA molecule comprises one or more of the following base modifications: 2,6-diaminopurine, 2- aminopurine, pseudouracil, N1-methyl-psuedouracil, 5′ methyl cytosine, N6- methyladenosine, 2′pyrimidinone (zebularine), thymine. Other modified bases include for example, 2-Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy-C, deoxyInosine, Hydroxymethyl dC, Inverted dT, Iso-dG, Iso-dC, Inverted Dideoxy-T, 5-Methyl dC, 5-Methyl dC, 5-Nitroindole, Super T®, 2′-F-r(C,U), 2′-NH2- r(C,U), 2,2′-Anhydro-U, 3′-Desoxy-r(A,C,G,U), 3′-O-Methyl-r(A,C,G,U), rT, rI, 5-Methyl- rC, 2-Amino-rA, rSpacer (Abasic), 7-Deaza-rG, 7-Deaza-rA, 8-Oxo-rG, 5-Halogenated-rU, N-Alkylated-rN.

[0223] In some embodiments, other chemically modified RNA is used herein. For example, the circRNA molecule can comprise a modified base such as, for example, 5′, Int, 3′ Azide (NHS Ester); 5′ Hexynyl; 5′, Int, 3′ 5-Octadiynyl dU; 5′, Int Biotin (Azide); 5′, Int 6- FAM (Azide); and 5′, Int 5-TAMRA (Azide). Other examples of RNA nucleotide modifications that can be used with the methods described herein include for example phosphorylation modifications, such as 5′-phosphorylation and 3′-phosphorylation. The circRNA molecule can also have one or more of the following modifications: an amino Page 63 of 182ATTORNEY DOCKET NO. ORB-018WO1 modification, biotinylation, thiol modification, alkyne modifier, adenylation, Azide (NHS Ester), Cholesterol-TEG, and Digoxigenin (NHS Ester). iv. Exemplary Nucleic Acid Sequences for Making circRNAs

[0224] In some embodiments, the nucleic acid molecule for making a circular RNA comprises a coding sequence described herein. In some embodiments, the nucleic acid molecule for making a circular RNA further comprises an IRES operably linked to the coding sequence described herein. In some embodiments, the nucleic acid molecule for making a circular RNA comprises intron sequences derived from a self-splicing intron as described herein. In some embodiments, the nucleic acid for making a circular RNA comprises an additional sequence(s) as a result of, and / or to aid in, production, purification, and / or circularization.

[0225] In some embodiments, the nucleic acid molecule for making a circular RNA described herein comprises a sequence identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 75% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 80% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 90% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 95% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 96% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 97% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 98% identical to any one of the sequences in Table F. In some embodiments, the nucleic acid comprises a sequence at least 99% identical to any one of the sequences in Table F. Table F. Exemplary nucleic acid sequences for making a circRNA VZV vaccine. The underlined sequences are retained after circularization. The double underlined sequences are the exemplary coding sequences for the VZV gE polypeptide or variants thereof.Page 64 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 65 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 66 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 67 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 68 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 69 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 70 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 71 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 72 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 73 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 74 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 75 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 76 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 77 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 78 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 79 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 80 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 81 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 82 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 83 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 84 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 85 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 86 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 87 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 88 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 89 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0226] In some embodiments, the nucleic acid described herein comprises a sequence identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 75% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 80% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 90% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 95% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 96% identical to any one of SEQ ID NOs: 1- 10. In some embodiments, the nucleic acid comprises a sequence at least 97% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at Page 90 of 182ATTORNEY DOCKET NO. ORB-018WO1 least 98% identical to any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid comprises a sequence at least 99% identical to any one of SEQ ID NOs: 1-10.

[0227] In some embodiments, the nucleic acid described herein comprises a sequence identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 75% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 80% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 90% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 95% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 96% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 97% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 98% identical to any one of SEQ ID NOs: 53-75. In some embodiments, the nucleic acid comprises a sequence at least 99% identical to any one of SEQ ID NOs: 53-75.

[0228] In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID Page 91 of 182ATTORNEY DOCKET NO. ORB-018WO1 NO: 9. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

[0229] In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 2. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 5. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 6. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 8. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 9. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 10.

[0230] In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 54. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 55. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 56. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 57. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 58. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 59. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 60. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 62.

[0231] In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 63. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 64. In some embodiments, the nucleic acid Page 92 of 182ATTORNEY DOCKET NO. ORB-018WO1 comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 66. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 67. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 68. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 69. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 71. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 72.

[0232] In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 73. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 74. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 75.

[0233] In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 53. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 54. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 55. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 56. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 57. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 58. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 59. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 60. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 61. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 62.

[0234] In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 63. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 64. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID Page 93 of 182ATTORNEY DOCKET NO. ORB-018WO1 NO: 65. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 66. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 67. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 68. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 69. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 71. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 72.

[0235] In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 73. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 74. In some embodiments, the nucleic acid comprises a sequence identical to SEQ ID NO: 75. 3. circRNA Production

[0236] In some embodiments, the nucleic acid encoding the circRNA molecule is synthetic DNA. In some embodiments, the nucleic acid encoding the circRNA molecule is synthetic RNA. In some embodiments, the nucleic acid is chemically synthesized. In some embodiments, the nucleic acid is synthesized in synthesizer machines using nucleotide triphosphate derivatives known as phosphoramidites, which are building blocks of linear oligonucleotides. Nucleoside phosphoramidites use inert substituents to protect reactive moieties such as hydroxyl and amino groups from undesirable reactions and promote phosphodiester bond formation leading to greater homogenous yields. Once synthesis is complete, these groups are removed to generate DNA or RNA oligonucleotides of high purity.

[0237] In some embodiments, the nucleic acid is in vitro transcribed RNA. An in vitro transcription (IVT) reaction typically comprises a double-stranded DNA (dsDNA) template, ribonucleotide triphosphates, and a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase is derived from bacteriophage. In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase.

[0238] In some embodiments, the nucleic acid encoding the circRNA molecule is a DNA template. The DNA template contains a promoter sequence to which the polymerase binds and catalyzes downstream transcription. In some embodiments, the promoter is about 20 to about 40 nucleotides long. In some embodiments, the DNA template is a double-stranded Page 94 of 182ATTORNEY DOCKET NO. ORB-018WO1 PCR product. In some embodiments, the DNA template is a linearized plasmid containing a promoter upstream of the DNA sequence to be transcribed.

[0239] In some embodiments, the nucleic acid encoding the circRNA molecule is circularized into circular RNA.

[0240] Circular RNA may be produced via any method known in the art. In some embodiments, the circular RNA is created via back-splicing using a self-splicing Group I intron. In some embodiments, the circular RNA is created using a self-splicing Group II intron.

[0241] In some embodiments, viral vectors are used to package the constructs for producing the nucleic acids and / or circRNA molecules described herein. In some embodiments, AAV vectors are used to construct the nucleic acids and / or circRNA molecules described herein. In other embodiments, non-viral vectors such as plasmids, cosmids and artificial chromosomes are used to construct the nucleic acids and / or circRNA molecules described herein. C. Formulations and delivery

[0242] In some embodiments, the circRNA of the present invention is formulated to improve delivery. For example, and without limitation, the circRNA can be formulated in lipid nanoparticles (LNPs), polymer-based nanoparticles, lipid-polymer based nanoparticles virus like particles (VLPs), and engineered exosomes. In some embodiments, the circRNA is delivered without any carrier molecules, or naked. In some embodiments, the circRNA is delivered in complex with cationic peptides or polymers. 1. Lipid Nanoparticles (LNPs)

[0243] In some embodiments, a circRNA molecule of the present invention is formulated in lipid nanoparticles (LNPs). LNP components are selected based on the desired target, cargo (e.g., circRNA molecules), size, and / or other desired feature. LNP components include, for example, ionizable lipids, helper lipids, sterols, and / or PEG-lipids. The relative amounts, or molar ratios, of ionizable lipid, helper lipid, cholesterol, and PEG-lipid are optimized for a given target or administration route. In some embodiments, the LNPs do not contain a targeting ligand. In some embodiments, the LNPs contain a targeting ligand.

[0244] In general, LNPs are small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least Page 95 of 182ATTORNEY DOCKET NO. ORB-018WO1 one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.

[0245] The LNPs of the present invention can be prepared with any method commonly known in the art. In some embodiments, the LNPs are stored in a storage buffer. In some embodiments, the storage buffer comprises saline. In some embodiments, the storage buffer does not comprise saline.

[0246] LNP sizes vary. In some embodiments, the LNPs for formulating the circRNA molecules of the present disclosure have an average hydrodynamic diameter of 10-1000 nm (i.e., 10-100 nm, 10-150 nm, 10-200 nm, 50-100 nm, 50-120 nm, 50-150 nm, 60-90 nm, 60- 120 nm, 80-100 nm, 80-120 nm, 100-200 nm, 100-500 nm, 200-800 nm, 100-1000 nm, or 500-1000 nm). In some embodiments, the average hydrodynamic diameter is at least 10 nm, at least 20 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, or at least 150 nm. In some embodiments, the average hydrodynamic diameter is less than 20 nm, less than 50 nm, less than 60 nm, less than 70 nm, less than 80 nm, less than 90 nm, less than 100 nm, less than 110 nm, less than 120 nm, or less than 150 nm. In some embodiments, the average hydrodynamic diameter is about 10 nm, about 20 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, or about 150 nm. i. Ionizable Lipids

[0247] In some embodiments, the LNP comprises at least one ionizable lipid. Ionizable lipids generally contain an amine-containing group on the head group. In some embodiments, the ionizable lipid comprises an ionizable cationic lipid. In some embodiments, the ionizable lipid comprises an ionizable anionic lipid.

[0248] In some embodiments, the ionizable lipid comprises a compound disclosed in WO 2021 / 141969 A1 (Hamilton et al.), the entirety of which is incorporated by reference herein. In some embodiments, the ionizable lipid comprises a compound of Formula (I) of WO 2021 / 141969 A1 (Hamilton et al.).(Formula (I)) Page 96 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0249] In some embodiments, R1in Formula (I) comprises C9-C20alkyl or C9-C20alkenyl with 1-3 units of unsaturation. For example, in some embodiments R1comprises a C9-C20 alkenyl with 2 units of unsaturation, such as, without limitation, a C17 alkenyl with 2 units of unsaturation.

[0250] In some embodiments, X3, X5, and X6in Formula (I) are independently absent.

[0251] In some embodiments, X1is -O-. In some embodiments, X1is absent. -(CH)a-

[0252] In some embodiments, X2isX7. In some embodiments, X2is -(CH2)a- or - CH(OH)-. In some embodiments, a is an integer between 0 and 6. In some embodiments, a is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, a is 0 and X2is absent. In some embodiments, a is 1.

[0253] In some embodiments, X7is independently hydrogen or hydroxyl. In some embodiments, X7is hydroxyl. In some embodiments, X7is hydrogen.

[0254] In some embodiments, X4is a 6-membered heterocyclyl optionally substituted with 1 or 2 C1-C6 alkyl groups. In some embodiments, the heterocyclyl comprises at least one nitrogen. For example, some embodiments, X4is piperidinyl. In some embodiments, X4is ethylpiperidinyl.

[0255] In some embodiments, A1and A2are independently C5-C12 alkyl or C5-C12 alkenyl with 1-3 units of unsaturation. In some embodiments, A1and A2are independently C5-C12alkenyl with 1 unit of unsaturation. In some embodiments, A1is C8alkenyl with 1 unit of unsaturation. In some embodiments, A2is C8 alkenyl with 1 unit of unsaturation.

[0256] In some embodiments, n1 is an integer between 1 and 6. In some embodiments, n1 is 1, 2, 3, 4, 5, or 6. In some embodiments, n1 is 2.

[0257] In some embodiments, the ionizable lipid is a compound selected from Table G1 below. Table G1. Exemplary ionizable lipids of Formula (I).Page 97 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 98 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 99 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 100 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 101 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 102 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0258] In some embodiments, the ionizable lipid is a compound of Formula (I) comprising 3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-(((4-(((Z)-oct-5-en-1-yl)oxy)-4-(((Z)- oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Compound 1). Page 103 of 182ATTORNEY DOCKET NO. ORB-018WO1(Compound 1)

[0259] In some embodiments, the ionizable lipid comprises a compound disclosed in WO 2022 / 140252 A1 (Patwardhan et al.), the entirety of which is incorporated by reference herein. In some embodiments, the ionizable lipid comprises a compound of Formula (III-a-i) of WO 2022 / 140252 A1 (Patwardhan et al.), or its N-oxide:(Formula (III-a-i))

[0260] In some embodiments, R1is hydrogen.

[0261] In some embodiments, L1is C2-C6heteroalkylenyl comprising at least 1 heteroatom. In some embodiments, the heteroatom is oxygen. For example, in some embodiments, L1is a C4 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2CH2-. In some embodiments, L1is a C3heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-.

[0262] In some embodiments, each R is independently C6-C12 alkyl or C6-C12 alkenyl with 1-3 units of unsaturation.

[0263] In some embodiments, each L is independently C1-C5alkylenyl.

[0264] In some embodiments, each L2is independently C4-C8 alkylenyl.

[0265] In some embodiments, the ionizable lipid is a compound selected from Table G2 below. Table G2. Exemplary ionizable lipids of Formula (III-a-i).Page 104 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 105 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 106 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 107 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 108 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 109 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 110 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 111 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 112 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0266] In some embodiments, the ionizable lipid is a compound of Formula (III-a-i) comprising ((3-hydroxypropyl)azanediyl)bis(heptane-7,1-diyl) bis(4,4-bis(((E)-oct-5-en-1- yl)oxy)butanoate) (Compound 37)(Compound 37).

[0267] In some embodiments, the ionizable lipid is a compound of Formula (III-a-i) comprising ((2-hydroxyethyl)azanediyl)bis(hexane-6,1-diyl) bis(6,6-bis(hexyloxy)hexanoate) ((Compound 49).

[0268] In some embodiments, the ionizable lipid is a compound of Formula (III-a-i) comprising ((2-hydroxyethyl)azanediyl)bis(heptane-7,1-diyl) bis(4,4-bis(((Z)-oct-5-en-1- yl)oxy)butanoate) (Compound 36)Page 113 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0269] In some embodiments, the ionizable lipid comprises a compound of Formula (I’’ a) of WO 2022 / 140252 A1 (Patwardhan et al.).(Formula (I’’-a-iii))

[0270] In some embodiments, R1is hydrogen.

[0271] In some embodiments, L1is C2-C6 heteroalkylenyl comprising at least 1 heteroatom. In some embodiments, the heteroatom is oxygen. In some embodiments, L1is a C3heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-.

[0272] In some embodiments, each R is independently C6-C12alkyl or C6-C12alkenyl with 1-3 units of unsaturation.

[0273] In some embodiments, R’’ is C6-C12 alkyl.

[0274] In some embodiments, each L is independently C1-C5 alkylenyl.

[0275] In some embodiments, each L2is independently C4-C8alkylenyl.

[0276] In some embodiments, the ionizable lipid is a compound selected from Table G3 below. Table G3. Exemplary ionizable lipids of Formula (I’’-a-iii).Page 114 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 115 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 116 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 117 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 118 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 119 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 120 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 121 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 122 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 123 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 124 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 125 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 126 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0277] In some embodiments, the ionizable lipid is a compound of Formula (I’’-a-iii) comprising nonyl 8-((6-((4,4-bis(octyloxy)butanoyl)oxy)hexyl)(2- hydroxyethyl)amino)octanoate (Compound 82)(Compound 82).

[0278] In some embodiments, the ionizable lipid is a compound of Formula (I’’-a-iii) comprising nonyl 8-((2-hydroxyethyl)(6-((4-(((Z)-oct-5-en-1-yl)oxy)-4-(((Z)-oct-5-en-1- yl)oxy)butanoyl)oxy)hexyl)amino)octanoate (Compound 81)(Compound 81).

[0279] The ionizable lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, Dlin-DMA, C12-200 and Dlin-KC2-DMA.

[0280] In some embodiments, the ionizable lipid constitutes at least 40, 45, or 50 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 40 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 45 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 50 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 47 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes 47.5 mole percent of the total moles of components in the LNP. Page 127 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0281] In some embodiments, the ionizable lipid constitutes 40 mol% to 50 mol% of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes 45 mol% to 50 mol% of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes 47 mol% to 48 mol% of the total moles of components in the LNP. ii. Sterols

[0282] Sterols can aid in stability and promote membrane fusion of the LNPs. In some embodiments, the LNP comprises at least one sterol. In some embodiments, the sterol comprises cholesterol. In some embodiments, the sterol is unmodified cholesterol. In some embodiments, the sterol comprises a variant of cholesterol. In some embodiments, the LNP comprises a derivative of cholesterol. Cholesterol variants may be side-chain or ring oxidized from enzymes acting on unmodified cholesterol. In some embodiments, the cholesterol is oxidized on the beta-ring structure or on the hydrocarbon tail structure. Cholesterols include, for example and without limitation, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β- hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof.

[0283] In some embodiments, the sterol constitutes 35 mol% to 45 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40 mol% to 45 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40 mol% to 41 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes at least 40 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 41% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40.25% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40.5% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40.75% of the total moles of components in the LNP. iii. PEG-Lipids

[0284] PEG-lipids can reduce LNP aggregation, shield the LNPs from non-specific endocytosis, and reduce opsonization by serum proteins and reticuloendothelial clearance. In some embodiments, the LNP comprises at least one PEG or PEG-modified lipid. In some embodiments, the LNP comprises at least one PEG-modified lipids. In some embodiments, Page 128 of 182ATTORNEY DOCKET NO. ORB-018WO1 the LNP comprises PEG. Alternatively, the PEG-lipids may be referred to as PEGylated lipids or PEG-lipids. In some embodiments, the PEGylation is reversible with the PEG moiety gradually releasing into blood circulation. PEG-lipids include, for example and without limitation, PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG- modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. Additional examples of PEG-lipids include, without limitation, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, or PEG-DSPE lipids. In some embodiments, the PEGylation comprises PEG1k, PEG2k, PEG5k, or PEG10k. In some embodiments, the LNP comprises PEG-DMG. In some embodiments, the PEGylation comprises PEG2k. In some embodiments, the LNP comprises DMG-PEG2k.

[0285] In some embodiments, the PEG-lipid constitutes 0 mol% to 5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1 mol% to 5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1 mol% to 3 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1 mol% to 2 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2 mol% to 3 mol% of the total moles of components in the LNP.

[0286] In some embodiments, the PEG-lipid constitutes 1 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1.25 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1.5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1.75 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2.25 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2.5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2.75 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 3 mol% of the total moles of components in the LNP.

[0287] In some embodiments, the PEG is replaced by a different polymeric compound such as, but not limited to, polyethenes, poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a Page 129 of 182ATTORNEY DOCKET NO. ORB-018WO1 biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine- containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof. iv. Helper Lipid

[0288] Helper lipids in LNPs may contribute to stability and delivery efficiency and / or mitigate any toxicity from the ionizable lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the phospholipid comprises a phospholipid moiety and at least one fatty acid moiety. In some embodiments, the phospholipid comprises one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers.

[0289] Exemplary phospholipid moieties include, but are not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In some embodiments, the fatty acid moiety includes, for example and without limitation, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linoleic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docasohexaenoic acid. Non-natural species or natural species with modification and substitutions, including branching, oxidation, cyclization, and alkynes, are included. For example, a phospholipid may be modified with an alkyne, which could allow for copper-catalyzed cycloaddition, or click-chemistry, with an azide to functionalize the lipid bilayer. In some embodiments, the lipid bilayer is functionalized. In some embodiments, the lipid bilayer is not functionalized.

[0290] In some embodiments, the helper lipid is a lipid having cone-shape geometry, e.g., dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the helper lipid is a cylindrical-shaped lipid such as phosphatidylcholine. Page 130 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0291] Helper lipids include, for example and without limitation, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-(cis-cis-9,12-octadecadienoyl)-sn-glycero-3- phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2- di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecanoyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocoline, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3- phosphoglycerol (DOPG), 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1- hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (POPE), 1,2- Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-octadecanoyl-2-(9Z- octadecenoyl)-sn-glycero-3-phosphocholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE). In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid is DMPC. In some embodiments, the helper lipid is DOPE.

[0292] In some embodiments, the helper lipid constitutes 5 mol% to 15 mol% of the total moles of components in the LNP. In some embodiments, the helper lipid constitutes 5 mol% to 10 mol% of the total moles of components in the LNP. In some embodiments, the helper lipid constitutes 10 mol% to 15 mol% of the total moles of components in the LNP. In some embodiments, the helper lipid constitutes 10 mol% of the total moles of components in the LNP. v. Exemplary LNP Formulations Page 131 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0293] In some embodiments, the LNP formulation comprises about 40 mol% to about 50 mol% ionizable lipid, about 5 mol% to about 15 mol% helper lipid, about 35 mol% to about 45 mol% sterol, and about 0 mol% to about 5 mol% PEG-lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP formulation comprises about 45 mol% to about 50 mol% ionizable lipid, about 5 mol% to about 15 mol% helper lipid, about 40 mol% to about 45 mol% sterol, and about 1 mol% to about 3 mol% PEG-lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises about 45 mol% to about 50 mol% ionizable lipid, about 10 mol% helper lipid, about 40 mol% to about 45 mol% sterol, and about 1 mol% to about 3 mol% PEG-lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises about 47 mol% to about 48 mol% ionizable lipid, about 10 mol% helper lipid, about 40 mol% to about 41 mol% sterol, and about 1 mol% to about 3 mol% PEG-lipid, such that the molar ratios combine to 100%.

[0294] In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 40 mol% sterol, and about 2.5 mol% PEG-lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 40.75 mol% sterol, and about 1.75 mol% PEG-lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 41 mol% sterol, and about 1.5 mol% PEG-lipid, such that the molar ratios combine to 100%. In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 40.5 mol% sterol, and about 2 mol% PEG-lipid, such that the molar ratios combine to 100%.

[0295] In some embodiments, the LNP comprises Compound 1 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 37 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 49 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 36 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 82 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 81 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. Page 132 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0296] In some embodiments, the LNP comprises Compound 1 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 37 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 49 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 36 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 82 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid. In some embodiments, the LNP comprises Compound 81 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2K as the PEG-lipid.

[0297] In some embodiments, the LNP comprises a composition selected from Table H below. Table H. Exemplary LNP compositions.2. Other Delivery Vehicles

[0298] In some embodiments, other delivery vehicles are used to deliver circular RNAs, nucleic acid molecules and compositions thereof described herein. The vehicles may include Page 133 of 182ATTORNEY DOCKET NO. ORB-018WO1 other lipid-based particles such as lipidoids, liposomes, lipoplexes, micelles, multilamellar vesicle (MLV), unicellular vesicle (SUV), polymer-based nanoparticles and exosomes.

[0299] In some embodiments, circular RNA VZV vaccines described herein may also be constructed or altered such that their properties are suitable for different administration routes, such as parenteral (intravenously, intramuscularly or subcutaneously), oral, rectal, opthalmic and / or topical administration.

[0300] In some embodiments, circular RNA VZV vaccines of the present disclosure can be formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a therapeutic outcome. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. In some embodiments, the compositions may be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. i. Engineered Exosomes

[0301] Exosomes are tiny vesicles smaller than 50 nm secreted by mature reticulocytes, which are associated with transferrin receptors and function in antigen presentation during the regulation of immune cells. In some embodiments, engineered exosomes act as cargo carriers and deliver small hydrophilic or lipophilic molecules, including some therapeutic drugs to cells, participating in the regulation of many major diseases. Exosomes can improve bioavailability of some drugs when taken orally, reducing the total dose required for administration, and minimizing side effects. In some embodiments, the circRNA molecules discussed herein are delivered using engineered exosomes. i. Virus Like Particles (VLPs)

[0302] In some embodiments, the circular RNA VZV vaccines discussed herein are delivered using viral delivery particles. Viral particles include recombinant viruses and virus like particles (VLPs). In some embodiments, the circular RNA VZV vaccines discussed herein are delivered using VLPs.

[0303] As used herein, the term “Virus-like particles” (VLPs) are molecules that closely resemble viruses, but are non-infectious. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses Page 134 of 182ATTORNEY DOCKET NO. ORB-018WO1 can be used to create recombinant VLPs. VLPs can be produced from different viruses, such as adeno-associated viruses, retroviruses, lentiviruses and vesiculoviruses. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. VLPs possess diverse applications in therapeutics, immunization, and diagnostics. VLPs have been synthesized in a wide range of expression systems, including prokaryotic (bacteria) and eukaryotic (insect cells, mammalian cell lines, plant cells, or yeast) expression systems. The functionality of VLPs can be increased through modifying their exterior or interior surface by displaying the heterologous epitopes of interest using different methods like peptide conjugation, genetic fusion, and chemical crosslinking.

[0304] In some embodiments, the VLP is derived from a Vesiculovirus. In some embodiments, the VLP is derived from VSV (Indiana vesiculovirus, formerly Vesicular stomatitis Indiana virus (VSIV or VSV). In some embodiments, the virus like particle comprises a mutated VSV-G protein. VSV-G protein is a single transmembrane glycoprotein (G) which plays a critical role during the initial steps of virus infection. it is responsible for virus attachment to specific receptor, LDL-R. In the cell, G protein triggers the fusion between the viral and endosomal membranes, which releases the viral genome in the cytosol for the subsequent steps of infection. In some embodiments, VSV-G protein is mutated to abolish its binding to LDL-R receptor. For example, a VSV-G envelope protein may be a mutated at one or more of any one of H8, K47, Y209, and / or R354. In some embodiments, a VLP may comprise a mutated VSV-G protein described in the PCT patent application Publication No. WO2019057974; the contents of which are incorporated herein by reference in their entireties. In some aspects, the VLP for delivering circular RNA VZV vaccines is a viral particle disclosed in the PCT Publication Nos. WO2020236263 and WO2023107886; the contents of each of which are incorporated herein by reference in their entireties.

[0305] In some embodiments, the virus like particle is pseudotyped. As a non-limiting example, the virus like particle is VSV-G-pseudotyped lentiviruses (VSV-G-LVs).

[0306] In some embodiments, the virus like particle for delivering circular RNA VZV vaccines is a retrovirus, a recombinant AAV, or an adenovirus. D. Compositions

[0307] In one aspect, the present disclosure provides pharmaceutical compositions comprising one or more circRNA molecules described herein. Pharmaceutical compositions may include circRNA molecules as described herein in combination with one or more pharmaceutically or physiologically acceptable carrier, diluents, or excipients. Such Page 135 of 182ATTORNEY DOCKET NO. ORB-018WO1 compositions may include buffers such as neutral buffered saline or phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions which may be used in the pharmaceutical compositions of the disclosure include, for example, DMSO. Compositions can be formulated for any suitable administration, e.g., for intravenous administration.

[0308] Pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., circRNA molecules) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0309] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0310] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) of the circRNA molecules described herein.

[0311] A pharmaceutical composition of the present disclosure may be prepared, packaged, and / or sold in a formulation suitable for administration by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, RNAs, and / or pharmaceutical Page 136 of 182ATTORNEY DOCKET NO. ORB-018WO1 compositions thereof, are administered by systemic intravenous injection. In specific embodiments, circRNA molecules and / or pharmaceutical compositions thereof may be administered intravenously and / or orally. In specific embodiments, circRNA molecules and / or pharmaceutical compositions thereof may be administered intramuscularly.

[0312] Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances. In some embodiments, pharmaceutical compositions of the present invention may optionally comprise one or more additional prophylactic compounds.

[0313] In some embodiments, the therapeutically active substance is an adjuvant. In some embodiments, the prophylactic compound is an adjuvant. In some embodiments, the pharmaceutical composition does not include an adjuvant.

[0314] The adjuvant may be used to enhance antibody response and can comprise any acceptable immunostimulatory compound. For example, adjuvants include cytokines, toxins, or synthetic compositions. Adjuvants may be formulated as oil-in-water emulsions, water-in- oil emulsions, mineral salts, polynucleotides, or natural substances. Additional examples of adjuvants include, without limitation, Freund's adjuvant, oil such as MONTANIDE® ISA51, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, alpha-interferon, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts (such as aluminum hydroxide or other aluminum compound), saponin adjuvant (e.g., QS21), MDP compounds (such as thur-MDP and nor- MDP), CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), lipopeptides (e.g., Pam3Cys), RIBI (which contains three components extracted from bacteria), MPL, trehalose dimycolate (TDM), MHC antigens, and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion.

[0315] In some embodiments, the adjuvant may be encoded by a second RNA molecule. In some embodiments, the pharmaceutical composition comprises a circRNA molecule of the present invention encoding the VZV gE antigen and a second RNA molecule encoding an adjuvant.

[0316] In some embodiments, a biologic response modifier (BRM) is co-administered to enhance immune responses. BRMs can upregulate T cell immunity and downregulate suppressor activity. Exemplary BRMs include, without limitation, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines (such as γ-interferon, IL-2, or IL-12), or genes encoding proteins involved in immune helper functions (such as B-7).

[0317] In accordance with some embodiments, a method of administering pharmaceutical compositions comprising an alternative nucleic acid encoding a VZV antigen to be delivered Page 137 of 182ATTORNEY DOCKET NO. ORB-018WO1 to a subject in need thereof is provided. In some embodiments, compositions are administered to humans. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

[0318] In some embodiments, the compositions and vaccines of the present invention may be formulated into preparations in solid, semi-solid, liquid, lyophilized, frozen, or gaseous forms. In some embodiments, the compositions and vaccines are formulated as a sterile suspension for intramuscular injection. In some embodiments, the compositions and vaccines are lyophilized and reconstituted prior to administration to a patient using a reconstitution liquid, for example, an adjuvant suspension component (liquid). E. Use and Applications

[0319] The circRNAs encoding one or more VZV antigen polypeptides, and compositions and vaccines comprising circRNA encoding one or more VZV antigen polypeptides, can be used to induce an VZV-specific immune response, including any humoral and / or cellular immune response.

[0320] Suitably, the immune response comprises an antibody response to one or more of the herpesvirus polypeptides. The antibody titer produced by pharmaceutical compositions, also referred to herein as vaccines, of the invention may be a neutralizing antibody titer. Antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA); and / or by microneutralization assay for example as described.

[0321] In some embodiments, the circRNAs encoding one or more VZV antigen polypeptides, compositions and vaccines comprising circRNA encoding one or more VZV antigen polypeptides are for prevention of herpes zoster (HZ) (also known as shingles) in a human patient.

[0322] In some embodiments, the patients are adults aged 50 years or older. For examples, the patients are adults aged 60 years or older, adults aged 70 years or older.

[0323] In other embodiments, the patients are adults aged 18 years and older who are or will be at increased risk of HZ due to immunodeficiency or immunosuppression caused by known disease or therapy. Immunocompromised patients may include patients having but not limited to auHSCT recipients, hematologic malignancies, renal transplant, solid malignant tumors, and HIV. Page 138 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0324] In some embodiments, the circRNAs encoding one or more VZV antigen polypeptides, and compositions and vaccines comprising circRNA encoding one or more VZV antigen polypeptides, are for prevention of chickenpox.

[0325] The vaccine may be administered to the patient by any routes of administration. In some embodiments, the vaccine may be administered to the patient by intramuscular, intradermal, or subcutaneous administration.

[0326] In some embodiments, the vaccine is administered by intradermal administration.

[0327] In some embodiments, the vaccine is administered by intramuscular administration.

[0328] In some embodiments, the vaccine is administered by subcutaneous administration.

[0329] In some embodiments, the vaccine is administered in a single dose. In some embodiments, the vaccine is administered intramuscularly in a single dose. In some embodiments, the vaccine is administered intradermally in a single dose. In some embodiments, the vaccine is administered subcutaneously in a single dose.

[0330] In some embodiments, the vaccine is administered in two separate doses. As non- limiting examples, the vaccine is administered according to the following schedules: a first dose at Month 0 followed by a second dose administered 2 to 6 months later. In some embodiments, for patients who are or will be immunodeficient or immunosuppressed and who would benefit from a shorter vaccination schedule: A first dose at Month 0 followed by a second dose administered 1 to 2 months later.

[0331] Accordingly, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 50% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 60% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 70% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 80% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 90% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 95% in subjects aged 50 years and older.

[0332] In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ in patients aged 18 years and older who are or will be immunodeficient or immunosuppressed. Page 139 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0333] In some embodiments. The vaccine efficacy against HZ maintains the same the first year, the second year, the third year, the fourth year and later after vaccination.

[0334] In another aspect, the circRNAs encoding one or more VZV antigen polypeptides, and compositions and vaccines comprising circRNA encoding one or more VZV antigen polypeptides, may be used for the prevention of Postherpetic Neuralgia (PHN) in a patient which can be attributed to the effect of the vaccine on the prevention of HZ.

[0335] In some embodiments, the compositions and vaccines may be administered concomitantly with another vaccine. EXAMPLES

[0336] Various aspects of the invention are described in further detail in the following Examples. The following Examples describe exemplary modes of making and practicing the present invention. However, these Examples are for illustrative purposes only and are not meant to limit the scope of the invention. Example 1. Design of circRNA Constructs and gE Antigen

[0337] Glycoprotein E (gE) is one of the viral binding proteins for varicella-zoster virus (VZV), making gE the target antigen for generating neutralizing antibodies induced by vaccines. This Example describes the design, optimization, and testing of exemplary circRNA VZV vaccines comprising a VZV gE antigen. Antigen Design

[0338] The gE protein comprises a signal domain (also known as a “signal peptide” or “SP”), an extracellular domain, a transmembrane domain, and an intracellular domain. The intracellular domain is responsible for receptor trafficking, including targeting to the trans- Golgi network, endocytosis, and phosphorylation, while the extracellular domain is the domain responsible for viral attachment. Therefore, when optimizing the design of exemplary antigens, the gE protein was truncated at various points to retain the extracellular domain and eliminate the regulatory motifs from the intracellular domain. When applicable, a mutation (Y569A) was included in the intracellular domain. While not bound by any particular mechanism, in some embodiments, the Y569A mutation in the intracellular domain disrupts trans-Golgi network targeting. The truncation points are visualized in FIG. 1. Construct Design

[0339] Exemplary circular RNA (circRNA) constructs were first designed as linear RNA with specific components that promote circularization of the linear precursor RNA to Page 140 of 182ATTORNEY DOCKET NO. ORB-018WO1 circRNA, and translation of the circRNA. As shown in FIG. 2A, in exemplary circRNA VZV vaccine constructs, an internal ribosomal entry site (IRES) was added 5’ to the codon- optimized coding sequences (CDS) to allow for translation of the circRNA. To promote circularization, an exemplary self-splicing intron was then added to the construct, with part of the intron added 5’ to the IRES (shown as the “5’ intron fragment”) and the other part of the intron added 3’ to the coding sequence (shown as the “3’ intron fragment”). In some embodiments, additional elements such as, for example and without limitation, internal homology elements, outer homology elements, and spacers were optionally added to, for example and without limitation, increase circularization efficiency and / or increase expression. After the linear precursor RNA circularizes, it generates the exemplary circRNA construct as shown in FIG. 2B.

[0340] The components of exemplary circRNA VZV vaccine constructs are listed in Table 1 below. Each exemplary construct circularized successfully and resulted in circRNA, as disclosed herein. Table 1. Exemplary circRNA VZV Vaccine Constructs.Codon Optimization

[0341] The open reading frame (ORF) sequences that encode the gE antigen polypeptides were codon-optimized to increase expression of the VZV gE antigen and therefore increase immune response to VZV gE antigen. The ORF sequences were flanked by an exemplary 5’ leader sequence and an exemplary 3’ sequence. The flanking sequences contributed to the secondary structure analysis during codon optimization but were not optimized. The 5’ leader sequence, which contained an exemplary IRES, was specifically added to ensure the secondary structure of the codon-optimized ORF sequences did not interfere with the Page 141 of 182ATTORNEY DOCKET NO. ORB-018WO1 structure and function of the IRES. Codon optimization of the ORF sequences was then performed using one or more of the following strategies: 1) optimization for human codon usage, 2) optimization to increase GC content in addition to optimal codon usage, and 3) optimization for secondary structure stability in addition to optimal codon usage. Construct Production

[0342] The DNA template that contains a DNA-dependent RNA polymerase promoter and the corresponding sequence for the exemplary circRNA VZV gE construct were amplified and the extracted DNA samples were linearized and used as templates for in vitro transcription (IVT) to produce linear RNA. In vitro transcript mixtures were first treated with DNase to degrade the DNA template. The resulting small DNA fragments can then be easily separated from larger RNA molecules by methods known in the art, such as, for example, tangential flow filtration (TFF) during purification. Following the in vitro transcription step, RNA was purified from the impurities and materials used in the previous steps including endotoxins, immunogenic double stranded RNA (dsRNA), residual DNA template, RNA polymerase and elemental impurities. Linear RNA molecules were circularized using methods known in the art such as, for example, co-transcriptional circularization of the precursor RNA by the activity of a self-splicing intron.

[0343] Overall, in this example, exemplary coding sequences encoding a VZV gE antigen were codon-optimized, and exemplary VZV vaccine constructs were designed, produced, and circularized. Example 2. circRNA Translation and Antigen Protein Expression

[0344] Expression of exemplary circRNA VZV vaccine constructs was assessed in K562 lymphoblast cells electroporated with the respective circRNA constructs encoding VZV gE. Expression was determined by measuring VZV fluorescence intensity by flow cytometry. Fold-over-control (FOC) values were calculated for each construct by dividing the geometric mean of the sample by the geometric mean of the negative controls. As shown in FIG. 3A and FIG. 3B, the exemplary constructs displayed expression of the VZV gE antigen in around 90% or more of the parent cells.

[0345] Overall, exemplary circRNA VZV vaccine constructs of the present invention successfully expressed the VZV gE antigen in vitro. Example 3. Exemplary circRNA VZV Vaccine Constructs Induced gE-Specific Immune Response In Vivo Page 142 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0346] This example tested the immunogenicity and tolerability of exemplary circRNA VZV vaccine constructs in a mouse model.

[0347] The study was carried out over 42 days with vaccine doses administered intramuscularly on Day 0 and Day 21 to C57BL / 6J mice. Blood was collected on Days 1, 21, and 42, and spleens were collected on Day 42. Testing groups and dosing can be found in Table 2. Group 1 contained the vehicle, phosphate-buffered saline (PBS). Groups 2-5 tested different exemplary VZV gE antigens encoded by codon-optimized coding sequences. The dose for Shingrix® (group 6) was one tenth of the human dose. Table 2. Study design for circRNA VZV vaccine constructs.

[0348] Total IgG specific to VZV gE protein in serum samples collected on Day 21 and Day 42 was quantified by ELISA. The total IgG titer is shown in FIG. 4A, as well as titers for IgG subclasses including IgG1 (FIG. 4B), IgG2b (FIG. 4C), and IgG2c (FIG. 4D). The exemplary circRNA VZV vaccine constructs of the present invention had comparable or higher IgG titers to Shingrix®, a commercially-available recombinant protein VZV vaccine. The ratio of IgG2c titer to IgG1 titer for each group was calculated on day 21 and day 42, as shown in FIG. 5A and FIG. 5B, respectively. Higher IgG2c / IgG1 ratios are indicative of Th1 type cellular immune response.

[0349] A plaque reduction neutralization test (PRNT) was also performed to assess neutralizing antibodies in the serum samples from Day 21 and Day 42. Serum samples were diluted and mixed with a viral suspension to allow the antibodies to react with the virus. The samples were then poured over a confluent monolayer of host cells. The surface of the cell layer was further covered in a layer of agar or carboxymethyl cellulose to prevent the virus from spreading indiscriminately. The concentration of plaque forming units (PFUs), or regions of infected cells, was estimated after a few days. The PRNT50titer is the Page 143 of 182ATTORNEY DOCKET NO. ORB-018WO1 concentration of serum required to reduce the number of plaques by 50% compared to the serum free virus and represents the effectiveness of the antibodies in serum. The results of the PRNT50 assay are shown in FIG. 6.

[0350] Spleen samples from Day 42 were assessed for VZV gE-specific T cell responses. CD4 and CD8 T cell responses are summarized in FIG. 7A – FIG. 7E and FIG. 8A – FIG. 8D, respectively. The exemplary circRNA VZV vaccine constructs generated higher responses than Shingrix® in both CD4 and CD8 T cells.

[0351] Finally, the tolerability of exemplary circRNA VZV vaccine constructs was assessed. The body weight and variation in body weight were tracked over the duration of the study as shown in FIG. 9A (body weight) and FIG. 9B (variation in body weight). Injection site reactions were also graded on Day 1 and Day 22, the days after each dose. Grades were assigned as described in Table 3. The number of mice per group assigned each grade is summarized at day 1in FIG. 10A and at day 22 in FIG. 10B. The circRNA VZV vaccine constructs had no injection site reactions after either dose while every mouse in the Shingrix® group displayed at least a Grade 1 reaction after each dose. Table 3. Grading scale for injection site reactions.

[0352] Overall, the exemplary circRNA VZV vaccine constructs resulted in higher Th1 cellular immune responses than the recombinant protein vaccine, Shingrix®. The exemplary circRNA VZV vaccine constructs also showed better tolerability than Shingrix® throughout the duration of the study. Example 4. Exemplary circRNA VZV Vaccines in LNP Formulations Induced gE-Specific Immune Response In Vivo

[0353] This example demonstrates the efficacy of exemplary circRNA VZV vaccine constructs in exemplary LNP formulations. LNP Formulations

[0354] Lipid nanoparticles (LNP) formulations with exemplary circRNA VZV vaccine constructs were prepared by methods known in the art. Exemplary LNP compositions are described in Table 4. Page 144 of 182ATTORNEY DOCKET NO. ORB-018WO1 Table 4. Exemplary LNP Formulations.Study A

[0355] Study A was carried out over 35 days with vaccine doses administered intramuscularly on Day 0 and Day 21 to C57BL / 6J mice. Blood was collected on Days 1, 21, and 35, and spleens were collected on Day 35. Testing groups and dosing for Study A can be found in Table 5. Group 13 contained the vehicle, phosphate buffered saline (DPBS). The dose for exemplary circRNA VZV vaccines in Study A was 1 µg (low dose). The dose for Group 12, Shingrix®, was one tenth of the human dose. Construct 1A was dT purified. Construct 1B was RP-HPLC purified. Table 5. Study Design for Study A (Low Dose)

[0356] Total IgG specific to VZV gE protein in serum samples, collected on Day 21 and Day 35, was quantified by ELISA. The ratio of IgG2c titer to IgG1 titer for each group was calculated as shown at day 21 in FIG. 11A and at day 35 in FIG. 11B. Higher IgG2c / IgG1 Page 145 of 182ATTORNEY DOCKET NO. ORB-018WO1 ratios are indicative of Th1 type cellular immune response. Total IgG titers were calculated at day 21 as shown in FIG. 12A and FIG. 12B.

[0357] The tolerability of exemplary circRNA VZV vaccine constructs was also assessed. The body weight and variation in body weight were tracked over the duration of the study. Exemplary results from Group 1, 12, and 13 are shown in FIG. 13A (body weight) and FIG. 13B (variation in body weight). Injection site reactions were graded on Day 1 and Day 22, the days after each dose was administered. Grades were assigned as described in Table 3. The number of mice per group assigned each grade is summarized at day 1 in FIG. 14A and at day 22 in FIG. 14B. The circRNA VZV vaccine constructs had no injection site reactions after either dose, while every mouse in the Shingrix® group displayed at least a Grade 1 reaction after each dose. Study B

[0358] Study B was carried out with the same methodology and testing groups as described in Study A, except the dose for exemplary circRNA VZV vaccines in Study B was 10 µg (high dose). In Study B, the total IgG titers for all groups at Day 21 are shown in FIG. 15A, and at Day 35 are shown in FIG. 15B (at Day 35). Notably, 10 µg of an exemplary circRNA VZV vaccine (Group 1, Construct 1A) generated a higher immune response than Shingrix® (Group 12), as shown in FIG. 15C (at Day 21) and FIG. 15D (at Day 35). The ratio of IgG2c titer to IgG1 titer for each group was calculated as shown in FIG. 16. The body weight and variation in body weight were tracked over the duration of the study. Exemplary results from Group 1, 12, and 13 are shown in FIG. 17A (body weight) and FIG. 17B (variation in body weight). The injection site reactions for each group at day 1 are summarized in FIG. 18A, and at day 22 in FIG. 18B. Study C

[0359] Study C was carried out over 35 days with vaccine doses administered intramuscularly on Day 0 to C57BL / 6J mice. Blood was collected on Days 1, 21, and 35, and spleens were collected on Day 35. Testing groups and dosing for Study C can be found in Table 6. Group 3 contained the vehicle, phosphate buffered saline (DPBS). The dose for exemplary circRNA VZV vaccines in Study C was 10 µg (high dose). The dose for Group 2, Shingrix®, was one tenth of the human dose. Table 6. Study Design for Study C (Single Injection Comparison)Page 146 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0360] The total IgG titers for Study C are shown at day 21 in FIG. 19A and at day 35 FIG. 19B. The ratio of IgG2c titer to IgG1 titer for each group was calculated as shown in FIG. 20. The body weight and variation in body weight were tracked over the duration of the study as shown in FIG. 21A (body weight) and FIG. 21B (variation in body weight). The injection site reactions for each group are summarized in FIG. 22.

[0361] Overall, this example demonstrated that exemplary circRNA VZV vaccines result in higher IgG titers and Th1 cellular response than Shingrix®, a recombinant protein VZV vaccine. Additionally, exemplary circRNA VZV vaccines had comparable tolerability to the vehicle control, as opposed to Shingrix® which consistently resulted in Grade 1 or 2 injection site reactions. Example 5. In Vivo Comparison of Exemplary circRNA VZV Vaccines with Different IRES Sequences

[0362] In this study, exemplary LNP formulations with circRNA VZV vaccines are administered intramuscularly to C57BI / 6J mice. The study is carried out over 35 days, and vaccine doses are administered on Day 0 and Day 21. Serum samples are taken twice on Day 0 (hour 0 and hour 6) and once on Day 21 and Day 35. Spleen samples are collected on Day 35. Testing groups and dosing are described in Table 7. Table 7. Study Design for IRES Comparison in circRNA Constructs

[0363] Serum samples are analyzed for VZV gE-specific IgG titers by ELISAs. Spleen samples are assessed by T-cell Flow Panels to determine VZV gE-specific T-cell responses. The tolerability, as determined by body weight, body weight variation, and injection site reactions, is also assessed over the course of the study. Example 6. In Vivo Efficacy of Exemplary circRNA VZV Vaccines Page 147 of 182ATTORNEY DOCKET NO. ORB-018WO1

[0364] In this study, the efficacy of exemplary circRNA VZV vaccines was tested after a single dose, including in a chickenpox model. Part 1 of the study was carried out over 98 days in C57BI / 6J mice as described in Table 8. Testing groups in the chickenpox model were primed on Day 0 with a live, attenuated varicella vaccine (675 PFU (plaque-forming unit)) to model a population that already had chickenpox. The first boost (“1° boost”) of either an exemplary circRNA VZV vaccine or recombinant protein VZV vaccine (Shingrix®) was administered intramuscularly on Day 28. The second boost (“2° boost”) for groups 3 and 4 (for Shingrix® only) was administered intramuscularly on Day 56. Serum samples were taken on Day 0, Day 28, Day 42, Day 56, Day 70, and Day 98, and blood was collected every two weeks between Day 28 to Day 98. Table 8. Study Design for Efficacy in Chickenpox Model

[0365] Serum samples were analyzed for VZV gE-specific IgG titers by ELISAs. As shown in FIG. 23, administration of an exemplary circRNA VZV vaccine results in higher IgG titers across all time points. IgG titers at Day 28 (FIG. 24A), Day 42 (FIG. 24B), and Day 56 (FIG. 24C) are shown. Before administration of the first boost, the chickenpox model groups had higher IgG titers than those not primed by the live, attenuated varicella vaccine.

[0366] The body weight and variation in body weight were tracked over the duration of the study. As an example, the body weights for select groups are shown in FIG. 25. Tolerability was assessed by injection site reaction and hunched posture. Reactions for each group are summarized at day 29, with hunched posture shown in FIG. 26A and the injection site reaction of swollen hindlimb shown in FIG. 26B.

[0367] At the end of part 1 of the study (at Day 98), the groups receiving Shingrix® (groups 3 and 4) exhibited high reactogenicity after the two boosts (on Day 28 and Day 56) Page 148 of 182ATTORNEY DOCKET NO. ORB-018WO1 for multiple days, as shown in FIG. 26C. FIG. 26C shows the body weights for selected groups throughout part 1 of the study. Specifically, as shown in FIG. 26C, the groups receiving Shingrix® (groups 3 and 4) exhibited a pronounced decrease in body weight from Day 28 to Day 29 and Day 57 to Day 59. Only the groups receiving Shingrix® (groups 3 and 4) exhibited Grade 1 reaction (swollen hindlimbs) or Grade 2 reaction, as shown in FIG. 26D (on Day 57) and FIG. 26E (on Day 59); all circRNAs groups exhibited only Grade 0 reactions. Overall, the groups administered circRNA VZV vaccines better tolerated the vaccine administration better than the groups administered Shingrix®.

[0368] Towards the end of part 1 of the study (at Day 98), the study was extended to include a viral challenge of certain groups of the mice (Group 2 (Vehicle Control), Group 4 (Shingrix®), and Group 8 (10 µg circRNA Construct 1 formulated with LNP007). On Day 238, the mice in these groups were challenged with a VZV virus (Webster strain). A dose of 105pfu of the VZV virus was injected intraperitoneally to each mice. FIG. 27A shows the antibody (IgG) levels in the mice during the study. qPCR was performed on lung samples collected on Day 240 to detect VZV viral load following the in vivo viral infection. As shown in FIG. 27B, a single dose of 10 µg of the circRNA VZV vaccine was effective to eliminate the VZV virus in the lung after the viral infection.

[0369] Overall, this example demonstrated that a single dose of exemplary circRNA VZV vaccines were effective and safe, including in a model population with prior exposure to chickenpox. Table 9. Exemplary circRNA sequences for circRNA VZV vaccines. The double underlined sequences are the exemplary coding sequences for the VZV gE polypeptide or a variant thereof.Page 149 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 150 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 151 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 152 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 153 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 154 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 155 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 156 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 157 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 158 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 159 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 160 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 161 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 162 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 163 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 164 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 165 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 166 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 167 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 168 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 169 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 170 of 182ATTORNEY DOCKET NO. ORB-018WO1Page 171 of 182ATTORNEY DOCKET NO. ORB-018WO1 EQUIVALENTS AND SCOPE

[0370] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. Page 172 of 182

Claims

ATTORNEY DOCKET NO. ORB-018WO1 CLAIMS What is claimed is:

1. A circular RNA encoding a varicella-zoster virus (VZV) antigen or variant thereof comprising a codon-optimized coding sequence encoding the VZV antigen, or variant thereof, wherein the codon-optimized coding sequence comprises a sequence at least 90% identical to any one of SEQ ID NOs: 11-34.

2. The circular RNA of claim 1, wherein the codon-optimized coding sequence comprises a sequence at least 95% identical to any one of SEQ ID NOs: 11-34.

3. The circular RNA of claim 1 or claim 2, wherein the codon-optimized coding sequence comprises a sequence identical to any one of SEQ ID NOs: 11-34.

4. The circular RNA of any one of the previous claims, wherein the VZV antigen is a VZV gE polypeptide or variant thereof.

5. The circular RNA of claim 4, wherein the VZV gE polypeptide variant is a truncated gE polypeptide lacking the carboxy terminal domain.

6. The circular RNA of claim 4 or claim 5, wherein the VZV gE polypeptide variant comprises a human IgG kappa (IgGκ) signal peptide.

7. The circular RNA of any one of the previous claims, wherein the circular RNA further comprises an internal ribosome entry site (IRES).

8. The circular RNA of claim 7, wherein the IRES is a viral IRES.

9. The circular RNA of claim 7 or claim 8, wherein the IRES comprises the sequence of any one of SEQ ID NOs: 35-43.

10. The circular RNA of any one of the previous claims, wherein the circular RNA comprises a sequence at least 95% identical to any one of SEQ ID NOs: 76-108. Page 173 of 182ATTORNEY DOCKET NO. ORB-018WO1 11. The circular RNA of any one of the previous claims, wherein the circular RNA comprises a sequence identical to any one of SEQ ID NOs: 76-108.

12. A precursor RNA for making circular RNA encoding a varicella-zoster virus (VZV) antigen or variant thereof, comprising, from 5’ to 3’, i) an upstream intron fragment corresponding to the 3’ portion sequence of a Group I intron, ii) an internal ribosome entry site (IRES), iii) a codon-optimized coding sequence encoding the VZV antigen, or variant thereof, and iv) a downstream intron fragment corresponding to the 5’ portion sequence of the Group I intron; wherein the codon-optimized coding sequence comprises a sequence at least 90% identical to any one of SEQ ID NOs: 11-34.

13. The precursor RNA of claim 12, wherein the Group I intron is a T4-Td intron or a Twort intron or a variant thereof.

14. The precursor RNA of claim 12 or claim 13, wherein the upstream intron fragment comprises the sequence of SEQ ID NO: 44, and the downstream intron fragment comprises the sequence of SEQ ID NO:

45.

15. The precursor RNA of claim 12 or claim 13, wherein the upstream intron fragment comprises the sequence of SEQ ID NO: 46, and the downstream intron fragment comprises the sequence of SEQ ID NO:

47.

16. The precursor RNA of any one of claims 12-15, wherein the VZV antigen is a VZV gE polypeptide or variant thereof.

17. The precursor RNA of claim 16, wherein the VZV gE polypeptide variant is a truncated gE polypeptide lacking the carboxy terminal domain.

18. The precursor RNA of claim 16 or claim 17, wherein the VZV gE polypeptide variant comprises a human IgG kappa (IgGκ) signal peptide. Page 174 of 182ATTORNEY DOCKET NO. ORB-018WO1 19. The precursor RNA of any one of claims 12-18, wherein the codon-optimized coding sequence comprises a sequence at least 95% identical to any one of SEQ ID NOs: 11- 34.

20. The precursor RNA of any one of claims 12-19, wherein the codon-optimized coding sequence comprises a sequence identical to any one of SEQ ID NOs: 11-34.

21. The precursor RNA of any one of claims 12-20, wherein the IRES is a viral IRES.

22. The precursor RNA of any one of claims 12-21, wherein the IRES comprises the sequence of any one of SEQ ID NOs: 35-43.

23. The precursor RNA of any one of claims 12-22, wherein the precursor RNA comprises a sequence at least 95% identical to any one of SEQ ID NOs: 1-10 and 53-75.

24. The precursor RNA of any one of claims 12-23, wherein the precursor RNA comprises a sequence identical to any one of SEQ ID NOs: 1-10 and 53-75.

25. A circular RNA resulting from circularization of the precursor RNA of any one of claims 12-24, and wherein the precursor RNA is linear.

26. The circular RNA of any one of claims 1-11 or 25, wherein the circular RNA is formulated in lipid nanoparticles (LNPs).

27. The circular RNA of claim 26, wherein the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a PEG-modified lipid, and wherein the ionizable lipid comprises a compound in Table G1, G2, or G3.

28. The circular RNA of claim 27, wherein the ionizable lipid is: Page 175 of 182ATTORNEY DOCKET NO. ORB-018WO1(Compound 49).

29. The circular RNA of any one of claims 26-28, wherein the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41% cholesterol, and 1.5-2.5 % PEG- modified lipid, such that the molar ratios combine to 100%.

30. The circular RNA of claim 29, wherein the LNP comprises a molar ratio of: a) 47.5 % ionizable lipid, 10 % helper lipid, 40% cholesterol, and 2.5 % PEG- modified lipid, b) 47.5 % ionizable lipid, 10 % helper lipid, 40.25% cholesterol, and 2.25 % PEG- modified lipid, c) 47.5 % ionizable lipid, 10 % helper lipid, 40.5% cholesterol, and 2 % PEG- modified lipid, d) 47.5 % ionizable lipid, 10 % helper lipid, 40.75% cholesterol, and 1.75 % PEG- modified lipid, or e) 47.5 % ionizable lipid, 10 % helper lipid, 41% cholesterol, and 1.5% PEG-modified lipid. Page 176 of 182ATTORNEY DOCKET NO. ORB-018WO1 31. The circular RNA of claim 29 or claim 30, wherein the helper lipid is distearoylphosphatidylcholine (DSPC), and wherein the PEG-modified lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

32. The circular RNA of claim 29 or claim 30, wherein the helper lipid is 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and wherein the PEG-modified lipid is DMG-PEG2K.

33. The circular RNA of any one of claims 29-31, wherein the LNP comprises a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K; and wherein Compound 1 is:.

34. A pharmaceutical composition comprising at least one of the circular RNA of any one of claims 1-11 or 25, and a pharmaceutically acceptable carrier.

35. A vaccine composition comprising the circular RNA of any one of claims 1-11 or 25.

36. The vaccine composition of claim 35, further comprising an adjuvant.

37. A method for stimulating an immune response to varicella-zoster virus (VZV) in a human subject comprising administering to the human subject an effective amount of a circular RNA comprising a codon-optimized coding sequence encoding a varicella-zoster virus (VZV) glycoprotein (gE) polypeptide, or a variant thereof, wherein the codon- optimized coding sequence is at least 90%, or at least 95%, or 100% identical to any one of the SEQ ID NOs: 11-34.

38. The method of claim 37, wherein the administration is for prophylactic treatment. Page 177 of 182ATTORNEY DOCKET NO. ORB-018WO1 39. The method of claim 37, wherein the immune response is a neutralizing antibody response to the glycoprotein E (gE) polypeptide encoded by the circular RNA and / or a cellular immune response to the gE polypeptide encoded by the circular RNA.

40. The method of any one of claims 37-39, wherein the codon-optimized coding sequence comprises a sequence at least 95% identical to any one of SEQ ID NOs: 11-34.

41. The method of any one of claims 37-40, wherein the codon-optimized coding sequence comprises a sequence identical to any one of SEQ ID NOs: 11-34.

42. The method of any one of claims 37-41, wherein the VZV antigen is a VZV gE polypeptide or variant thereof.

43. The method of claim 42, wherein the VZV gE polypeptide variant is a truncated gE polypeptide lacking the carboxy terminal domain.

44. The method of claim 42 or claim 43, wherein the VZV gE polypeptide variant comprises a human IgG kappa (IgGκ) signal peptide.

45. The method of any one of claims 37-44, wherein the circular RNA further comprises an IRES.

46. The method of claim 45, wherein the IRES is a viral IRES.

47. The method of claim 45 or claim 46, wherein the IRES comprises the sequence of any one of SEQ ID NOs: 35-43.

48. The method of any one of claims 37-47, wherein the circular RNA comprises a sequence at least 95% identical to any one of SEQ ID NOs: 76-108.

49. The method of any one of claims 37-48, wherein the circular RNA comprises a sequence identical to any one of SEQ ID NOs: 76-108. Page 178 of 182ATTORNEY DOCKET NO. ORB-018WO1 50. The method of any one of claims 37-49, wherein the circular RNA is formulated in a lipid nanoparticle.

51. The method of claim 50, wherein the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a PEG-modified lipid, and wherein the ionizable lipid comprises a compound in Table G1, G2, or G3.

52. The method of claim 51, wherein the ionizable lipid is:(Compound 49).

53. The method of any one of claims 50-52, wherein the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41% cholesterol, and 1.5-2.5 % PEG- modified lipid, such that the molar ratios combine to 100%.

54. The method of claim 53, wherein the LNP comprises a molar ratio of: a) 47.5 % ionizable lipid, 10 % helper lipid, 40% cholesterol, and 2.5 % PEG- modified lipid, b) 47.5 % ionizable lipid, 10 % helper lipid, 40.25% cholesterol, and 2.25 % PEG- modified lipid, c) 47.5 % ionizable lipid, 10 % helper lipid, 40.5% cholesterol, and 2 % PEG- modified lipid, Page 179 of 182ATTORNEY DOCKET NO. ORB-018WO1 d) 47.5 % ionizable lipid, 10 % helper lipid, 40.75% cholesterol, and 1.75 % PEG- modified lipid, or e) 47.5 % ionizable lipid, 10 % helper lipid, 41% cholesterol, and 1.5% PEG-modified lipid.

55. The method of claim 53 or claim 54, wherein the helper lipid is DSPC, and wherein the PEG-modified lipid is DMG-PEG2K.

56. The method of claim 53 or claim 54, wherein the helper lipid is DOPE, and wherein the PEG-modified lipid is DMG-PEG2K.

57. The method of any one of claims 53-55, wherein the LNP comprises a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K; and wherein Compound 1 is:.

58. The method of any one of claims 37-57, wherein the circular RNA is administered with a single dose.

59. The method of any one of claims 37-58, wherein the circular RNA is administered to the human subject via intramuscular administration, intradermal administration, or subcutaneous administration.

60. A method for treating or preventing a disease caused by varicella-zoster virus (VZV) comprising administering to a human subject in need thereof the circular RNA of any one of claims 1-11 or 25-33, or the pharmaceutical composition of claim 34, or the vaccine composition of claim 35 or 36.

61. The method of claim 60, wherein the administration is to prevent primary VZV infection in the human subject. Page 180 of 182ATTORNEY DOCKET NO. ORB-018WO1 62. The method of claim 60, wherein the administration is to prevent herpes zoster (shingles) in an adult human subject.

63. The method of any one of claims 60-62, wherein the circular RNA, pharmaceutical composition, or vaccine composition is administered to the human subject via intramuscular administration, intradermal administration, or subcutaneous administration.

64. The method of any one of claims 60-63, wherein the administration is a single dose administration.

65. The method of any one of claims 60-63, wherein the administration is more than a single dose. Page 181 of 182

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