Production of RNA polynucleotides encoding picornavirus

Abstract

The present disclosure relates to the use of ribozyme to generate a recombinant RNA molecule encoding an oncolytic viral genome, such as a picornavirus, which recombinant RNA molecule has the native 5 '-terminus of the viral genome The disclosure also relates to the design of a corresponding DNA template and the use of the recombinant RNA molecule and / or a corresponding particle for the treatment and prevention of cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 336,601, filed on April 29, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Incorporation of electronically submitted text files

[0004] The contents of the text files submitted electronically here are incorporated herein by reference in their entirety. A computer-readable format copy of the sequence listing (file name: ONCR_031_01WO_SeqList_ST26.xml, creation date: April 28, 2023, file size: 510,929 bytes). Technical Field

[0005] The present disclosure generally relates to the fields of immunology, inflammation and cancer therapy. More specifically, the present disclosure relates to the production of RNA polynucleotides encoding viral genomes of oncolytic viruses such as picornaviruses (e.g., Coxsackieviruses) and the design of recombinant DNA molecules for such viral genome expression. The present disclosure also relates to the treatment and prevention of proliferative disorders such as cancer. Background Art

[0006] Oncolytic viruses are replication-competent viruses with a lytic life cycle that are able to infect and lyse tumor cells. Direct tumor cell lysis not only results in cell death but also generates innate and adaptive immune responses against tumor antigens taken up and presented by local antigen-presenting cells. Thus, oncolytic viruses combat tumor cell growth both by direct cell lysis and by promoting antigen-specific adaptive responses that are able to sustain antitumor responses after viral clearance.

[0007] However, the clinical use of replication-competent viruses faces several challenges. In general, viral exposure activates innate immune pathways, resulting in a broad, nonspecific inflammatory response. If the patient has not been previously exposed to the virus, this initial innate immune response may lead to the development of an adaptive antiviral response and the production of neutralizing antibodies. If the patient has been previously exposed to the virus, existing neutralizing antiviral antibodies may prevent the desired lytic effect. In both cases, the presence of neutralizing antibodies not only prevents the virus from lysing target cells, but also renders re-administration of the viral therapeutic ineffective. These factors limit the use of viral therapeutics in the treatment of metastatic cancers because the efficacy of repeated systemic administrations required to treat such cancers is hindered by naturally occurring antiviral responses.

[0008] One strategy to overcome such challenges is to use artificial particles (e.g., lipid nanoparticles) rather than natural viral particles to deliver polynucleotides encoding viral genomes to target cells. Generating these artificial particles generally requires in vitro production and packaging of recombinant polynucleotides encoding viral genomes, and such recombinant polynucleotides need to be able to effectively generate viral RNA once introduced into cells. However, many viral genomes (e.g., genomes of various picornaviruses) require the natural 5' and / or 3' ends of the viral genome to replicate effectively. Moreover, there is still a long-term and unmet need in the art for compositions and methods related to recombinant polynucleotides containing natural 5' and / or 3' ends of corresponding viral genomes generated in vitro. The present disclosure provides such compositions and methods, etc. Summary of the invention

[0009] In one aspect, the present disclosure provides a recombinant DNA molecule comprising, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, and a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA viral genome, wherein the 5' junction cleavage sequence comprises or consists of an ENV27 ribozyme encoding sequence.

[0010] In one aspect, the present disclosure provides a recombinant DNA molecule, which comprises, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA viral genome, a poly-A tail, and a 3' junction cleavage sequence, wherein the 5' junction cleavage sequence comprises or consists of an ENV27 ribozyme coding sequence.

[0011] In some embodiments, the ENV27 ribozyme coding sequence comprises a polynucleotide sequence (excluding the P3 stem insertion) having at least 80% identity with SEQ ID NO: 132 (excluding its P3 stem insertion corresponding to nucleotides 49-54 of SEQ ID NO: 132) or consists of it. In some embodiments, the polynucleotide sequence (excluding the P3 stem insertion) is 100% identical to SEQ ID NO: 132 (excluding its P3 stem insertion corresponding to nucleotides 49-54 of SEQ ID NO: 132), or has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10 or at most 11 mutations (insertions, deletions or substitutions). In some embodiments, mutations are substitutions. In some embodiments, the ENV27 ribozyme coding sequence comprises a polynucleotide "TTTATT" or "TTTGTT" at a position corresponding to nucleotides 25-30 of SEQ ID NO: 132. In some embodiments, the ENV27 ribozyme coding sequence comprises a polynucleotide "TTTATT" corresponding to the position of nucleotides 25-30 of SEQ ID NO: 132. In some embodiments, the ENV27 ribozyme coding sequence comprises a P3 stem inset of about 1-30, about 1-20, about 6-20, or about 6-10 polynucleotides in length. In some embodiments, the length of the P3 stem inset is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 polynucleotides. In some embodiments, the length of the P3 stem inset is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 polynucleotides. In some embodiments, the P3 stem insertion comprises or consists of a polynucleotide "AGATCT" at a region corresponding to nucleotides 49-54 of SEQ ID NO: 132. In some embodiments, the P3 stem insertion comprises or consists of a polynucleotide "AGAGAAATCT" (SEQ ID NO: 137) at a region corresponding to nucleotides 49-54 of SEQ ID NO: 132. In some embodiments, the P3 stem insertion comprises or consists of a polynucleotide "AGAACGAGAAATCGTTCT" (SEQ ID NO: 138) at a region corresponding to nucleotides 49-54 of SEQ ID NO: 132.

[0012] In some embodiments, the recombinant DNA molecule comprises, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA viral genome, and a poly-A tail.

[0013] In some embodiments, the recombinant DNA molecule comprises, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA viral genome, and a 3' junction cleavage sequence.

[0014] In some embodiments, the recombinant DNA molecule comprises, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA viral genome, a poly-A tail, and a 3' junction cleavage sequence.

[0015] In some embodiments, the synthetic RNA viral genome encodes a picornavirus. In some embodiments, the picornavirus is a coxsackievirus. In some embodiments, the 5' end of the RNA viral genome begins with "UUAAA". In some embodiments, the coxsackievirus is a CVA21 strain. In some embodiments, the CVA21 strain is selected from Kuykendall strain, EF strain and KY strain. In some embodiments, the 5' end of the RNA viral genome comprises a polynucleotide sequence or consisting of a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of nucleotides 1-260 of SEQ ID NO: 1, 5 or 9.

[0016] In some embodiments, the recombinant DNA molecule does not include additional nucleic acid between the 5' junction cleavage sequence and the polynucleotide sequence encoding the RNA molecule. In some embodiments, post-transcriptional cleavage of the 5' junction cleavage sequence and / or the 3' junction cleavage sequence produces the natural 5' and / or 3' end of the synthetic RNA viral genome.

[0017] In some embodiments, the recombinant DNA molecules of the present disclosure further comprise a leader sequence between the promoter sequence and the 5' junction cleavage sequence. In some embodiments, the length of the leader sequence is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 60 bp, less than 50 bp, or less than 40 bp. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity according to SEQ ID NO: 135 or 136. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity according to SEQ ID NO: 135.

[0018] In some embodiments, the recombinant DNA molecule does not comprise additional nucleic acid between the promoter sequence and the leader sequence. In some embodiments, the recombinant DNA molecule does not comprise additional nucleic acid between the leader sequence and the 5' junction cleavage sequence.

[0019] In some embodiments, the promoter sequence is a T7 promoter sequence.In some embodiments, the T7 promoter sequence comprises SEQ ID NO: 91 or consists of it.

[0020] In some embodiments, the poly-A tail is about 50 bp-90 bp in length or about 65 bp-75 bp in length. In some embodiments, the poly-A tail is about 70 bp in length. In some embodiments, the poly-A tail is about 10 bp-50 bp in length or 25 bp-35 bp in length.

[0021] In some embodiments, the 3' junction cleavage sequence comprises or consists of a ribozyme sequence. In some embodiments, the 3' ribozyme sequence is a hepatitis delta virus ribozyme sequence.

[0022] In some embodiments, the 3' connection cleavage sequence comprises or consists of a restriction enzyme recognition sequence. In some embodiments, the 3' connection cleavage sequence comprises or consists of a type IIS restriction enzyme recognition sequence. In some embodiments, the 3' connection cleavage sequence comprises or consists of a BsmBI recognition sequence. In some embodiments, the 3' connection cleavage sequence comprises or consists of a BsaI recognition sequence.

[0023] In some embodiments, the promoter sequence is a T7 promoter sequence, the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 135, the 5' linker cleavage sequence comprises or consists of an ENV27 ribozyme sequence according to any one of SEQ ID NOs: 132-134, the poly-A tail is about 70 bp in length, and the 3' linker cleavage sequence comprises or consists of a BsmBI recognition sequence.

[0024] In some embodiments, the promoter sequence is a T7 promoter sequence, the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 135, the 5' linker cleavage sequence comprises or consists of an ENV27 ribozyme sequence according to any one of SEQ ID NOs: 132-134, the poly-A tail is about 70 bp in length, and the 3' linker cleavage sequence comprises or consists of a BsaI recognition sequence.

[0025] In some embodiments, the recombinant DNA molecule does not include additional nucleic acid within the region spanning the promoter sequence and the 3' junction cleavage sequence.

[0026] In one aspect, the present disclosure provides a method for producing a recombinant RNA molecule, the method comprising in vitro transcription of a recombinant DNA molecule of the present disclosure and purification of the resulting recombinant RNA molecule. In some embodiments, the recombinant RNA molecule comprises the native 5' and 3' ends of the viral genome encoded by the recombinant RNA molecule.

[0027] In one aspect, the disclosure provides recombinant RNA molecules transcribed by recombinant DNA molecules of the disclosure. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of recombinant RNA molecules include natural 5' and 3' ends of the viral genome encoded by the recombinant RNA molecules. In some embodiments, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.5% or no more than 0.1% of recombinant RNA molecules include RNA sequences encoded by ENV27 ribozyme encoding sequences. In some embodiments, at least one recombinant RNA molecule includes RNA sequences encoded by ENV27 ribozyme encoding sequences. In some embodiments, at least 0.0001%, at least 0.001%, at least 0.01%, at least 0.1%, or at least 1% of the recombinant RNA molecules comprise an RNA sequence encoded by the ENV27 ribozyme encoding sequence.

[0028] In one aspect, the present disclosure provides a composition comprising an effective amount of a recombinant RNA molecule of the present disclosure, and a carrier suitable for administration to a mammalian subject.

[0029] In one aspect, the present disclosure provides particles comprising a recombinant RNA molecule of the present disclosure. In some embodiments, the particle is selected from the group consisting of: a nanoparticle, an exosome, a liposome, and a liposome complex. In some embodiments, the particle is a lipid nanoparticle.

[0030] In one aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of particles of the present disclosure. In some embodiments, delivering the composition to a subject delivers the encapsulated recombinant RNA molecules to target cells, and wherein the encapsulated recombinant RNA molecules produce infectious viruses capable of lysing the target cells.

[0031] In one aspect, the present disclosure provides a method for killing cancer cells, the method comprising exposing the cancer cells to particles of the present disclosure or a composition thereof under conditions sufficient to deliver the particles intracellularly to the cancer cells, wherein the replication-competent virus produced by the encapsulated polynucleotides causes the killing of cancer cells. In one aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering an effective amount of particles of the present disclosure or a composition thereof to a subject suffering from cancer. In some embodiments, the method is performed in vivo, in vitro or ex vivo. In some embodiments, the cancer is lung cancer, breast cancer, colon cancer or pancreatic cancer, and wherein the synthetic RNA viral genome comprises a polynucleotide sequence derived from a KY strain. In some embodiments, the cancer is bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer or liver cancer, and wherein the synthetic RNA viral genome comprises a polynucleotide sequence derived from an EF strain. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, colorectal cancer, colon cancer, pancreatic cancer, liver cancer, renal cell carcinoma, gastric cancer, head and neck cancer, thyroid cancer, malignant glioma, glioblastoma, melanoma, B-cell chronic lymphocytic leukemia, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), Merkel cell carcinoma, diffuse large B-cell lymphoma (DLBCL), sarcoma, neuroblastoma, neuroendocrine cancer, rhabdomyosarcoma, medulloblastoma, bladder cancer, and marginal zone lymphoma (MZL). In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer, and liver cancer. In some embodiments, the cancer is renal cell carcinoma, lung cancer, or liver cancer. In some embodiments, the cancer is small cell lung cancer or non-small cell lung cancer (e.g., squamous cell lung cancer or lung adenocarcinoma). In some embodiments, the cancer is hepatocellular carcinoma (HCC) (e.g., HCC associated with hepatitis B virus). In some embodiments, the cancer is a neuroendocrine prostate cancer caused by treatment. In some embodiments, the cancer is lung cancer, liver cancer, prostate cancer (e.g., CRPC-NE), bladder cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, neuroblastoma, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, medulloblastoma, neuroendocrine cancer, Merkel cell carcinoma, or melanoma. In some embodiments, the cancer is neuroblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shown are tumor volumes of SK-MEL-28 tumor-bearing mice after intratumoral administration of PBS or CVA21-RNA molecules formulated with Lipofectamine, or intravenous administration of LNPs containing CVA21-Kuykendall strain RNA molecules (Formulation ID: 70032-6C).

[0033] Figure 2A An overview of an in vitro transcription method for generating authentic 3' ends for picornaviruses using 3' type IIS restriction enzyme recognition sites is shown. Figure 2B Shown are electrophoresis of DNA digested by BsmBI or BsaI restriction enzymes.

[0034] Figure 3 The RNaseH method for generating authentic 5' ends for picornaviruses using a 5' DNA primer and RNaseH enzyme is shown.

[0035] Figure 4 A ribozyme approach for generating authentic 5' ends for picornaviruses is shown.

[0036] Figure 5A – Figure 5B A hammerhead ribozyme is shown for generating discrete 5' ends. Figure 5A Shown is a structural model of a minimal hammerhead ribozyme (HHR) that anneals and cleaves at the 5' end at the arrow (SEQ ID NO: 75). Figure 5B Shown is a structural model of a ribozyme with Stabilized Stem I (STBL) for cleavage at the 5' end at the arrow (SEQ ID NO: 76).

[0037] Figure 6A-6B The pistol ribozyme used to generate discrete 5' ends is shown. Fig. 6A A schematic diagram of the wild-type pistol ribozyme signature is shown (SEQ ID NO:77). Figure 6B The Pistol ribozyme from P. Polymyxa is shown with the addition of a tetraloop to fuse the P3 strand modeled by mFOLD. The dashed box is the region that was mutagenized to preserve the folding of the ribozyme in the context of viral sequences. The "GUC" sequence shown in the dashed box was mutated to "UCA" to generate Pistol 1, and the "GUC" sequence was mutated to "TTA" to generate Pistol 2. (SEQ ID NO: 78)

[0038] Figure 7 Sequence alignment of multiple Pistol ribozyme variants and the location of the P2 motif are shown.

[0039] Figure 8 The in vitro transcription process of CVA21-RNA and Neg-RNA is shown. 5' and 3' ribozymes (Rib) autocatalytically cleave CVA21-RNA, generating CVA21-RNA with discrete 5' and 3' ends required for replication. In contrast, Neg-RNA constructs lack ribozyme sequences and are unable to replicate and produce virions.

[0040] Fig. 9A An overall schematic diagram of the use of junction cleavage sequences to remove non-viral RNA polynucleotides from genomic transcripts to maintain the native 5' and 3' discrete ends of the virus is shown. Fig. 9B A schematic diagram showing the removal of non-viral RNA polynucleotides from genomic transcripts using junction cleavage sequences to maintain the native 5' and 3' discrete ends of the virus, wherein the 3' junction cleavage sequence contains a restriction enzyme recognition site.

[0041] Fig. 10A Schematic representation of CVA21 expression construct design and a corresponding non-limiting example of an in vitro transcription process to generate a synthetic RNA viral genome with precise ends at 5' and 3'. Fig. 10B Shown is the design of a DNA construct to test the cleavage efficiency of the ribozyme as a 5' junction cleavage sequence. Fig. 10C is a gel electrophoresis image showing the cleavage efficiency of the initial candidate ribozymes for either a shorter, approximately 60 nt viral origin or a longer, approximately 260 nt viral origin.

[0042] Fig.11A DNA sequences encoding various ENV27 ribozymes are shown. Fig. 11B Shown are (left) a diagram of the ribozyme secondary structure and (right) base pairing of the ribozyme P2 motif to the 5' end of the viral genome.

[0043] Fig. 12A and Fig. 12B are images of gel electrophoresis showing the cleavage efficiency of the indicated ribozyme constructs. Fig. 12C is a table summarizing the cleavage efficiency of each of the indicated ribozymes incorporated into viral genome expression constructs.

[0044] Fig.13 is a graph showing the viral titers produced by cells transfected with viral genomic RNA produced from the indicated DNA templates.

[0045] Fig.14A is a graph showing the growth of tumors in animals treated with LNPs containing viral genomic RNA produced from the indicated DNA templates. Fig. 14B is a graph of body weight changes in animals treated with LNPs containing viral genomic RNA produced from the indicated DNA templates. Fig. 14C Figure 2 is a graph of tumor growth in the NCI-H1299 animal tumor model treated with LNPs containing viral genomic RNA produced from the indicated DNA templates.

[0046] Fig.15A Shown are UV A280 absorption spectra of CVA21 viral genomes with different poly-A tail lengths using Oligo-dT chromatography. Fig. 15BShown are UV A280 absorbance spectra of SVV viral genomes with different poly-A tail lengths using Oligo-dT chromatography.

[0047] Fig.16A is a graph plotting the change in tumor size over time in animals treated with the indicated LNPs containing RNA viral genomes obtained by in vitro transcription from DNA templates with different designs (e.g., different poly-A tail lengths and 5' ribozyme sequences) using the NCI-H1299 xenograft model. Fig. 16B Shown are graphs plotting tumor size over time in animals treated with the indicated LNPs. DETAILED DESCRIPTION

[0048] The present disclosure provides the design of recombinant polynucleotides encoding viral genomes and template vectors for in vitro viral expression. In particular, recombinant polynucleotides may be required to expose the natural 5' and 3' ends of the viral genome to allow the virus to replicate effectively in cells. Producing such recombinant polynucleotides requires optimizing the design of the vector template and the manufacturing process (e.g., expression, purification, encapsulation and storage).

[0049] In some embodiments, the viral genome has replication capability. The disclosure also provides a viral genome that can be encapsulated in a non-immunogenic particle (such as a lipid nanoparticle, a polymer nanoparticle or an exosome), which can be repeatedly administered to a subject. In some embodiments, the particle also encapsulates a polynucleotide encoding a payload molecule. Therefore, the disclosure can systematically deliver a safe and effective recombinant polynucleotide vector, and provides a method for treating and preventing a variety of proliferative disorders (e.g., cancer).

[0050] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books and papers, are hereby expressly incorporated by reference in their entirety for any purpose. In the case of a term where one or more incorporated documents or portions of documents define a term that contradicts the definition of the term in this application, the definition appearing in this application shall prevail. However, the mention of any reference, article, publication, patent, patent publication and patent application cited herein does not and should not be considered as an admission or any form of suggestion that they constitute effective prior art or form part of the common knowledge of any country in the world.

[0051] definition

[0052] In this specification, unless otherwise stated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and include fractions thereof (such as one-tenth and one-hundredth of integers) where appropriate. It should be understood that, unless otherwise stated, the terms "a" and "an" as used herein refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination thereof in the alternatives. As used herein, the terms "include" and "comprising" are used synonymously. As used herein, "plurality" may refer to one or more components (e.g., one or more miRNA target sequences). In this application, unless otherwise stated, the use of "or" means "and / or".

[0053] As used in this application, the terms "about" and "approximately" are used as equivalents. Any number used in this application, whether or not with about / approximately, is intended to cover any normal fluctuations understood by a person of ordinary skill in the relevant art. In certain embodiments, the term "about" or "approximately" refers to a range of values ​​that fall within 10% in either direction (greater than or less than) of the reference value stated, unless otherwise stated or otherwise apparent from the context (except where such a number would exceed 100% of the possible value). In some embodiments, the term "about" or "approximately" refers to a range of values ​​that fall within 10% or less in either direction (greater than or less than) of the reference value stated (except where such a number would exceed 100% of the possible value).

[0054] "Reduced" or "reduced" means that a specific value is reduced or decreased by at least 5% compared to a reference value, for example 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. A specific value reduction or reduction can also be expressed as a fold change in the value compared to a reference value, for example, a reduction of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 times or more compared to a reference value.

[0055] By "increase" is meant that a particular value is increased by at least 5%, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, 500% or more, compared to a reference value. An increase in a particular value can also be expressed as a fold change in value compared to a reference value, such as an increase of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more compared to a reference value level.

[0056] The term "sequence identity" refers to the percentage of bases or amino acids that are identical and in the same relative position between two polynucleotide or polypeptide sequences. Thus, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, one sequence usually serves as a reference sequence to which a test sequence is compared. The term "reference sequence" refers to the molecule to which the test sequence is compared. Unless otherwise indicated, the term "sequence identity" in the claims refers to the sequence identity as determined by Clustal Sequence identities were calculated using version 1.2.4 with default parameters.

[0057] The term "derived from" refers to a polypeptide or polynucleotide sequence that comprises all or part of a reference polypeptide or polynucleotide sequence. For example, the RNA polynucleotide encoding the SVV or CVA genome described herein may comprise a polynucleotide sequence derived from all or part of a reference SVV or CVA genome (e.g., a naturally occurring or modified SVV or CVA genome). A polypeptide or polynucleotide sequence "derived from" a reference polypeptide or polynucleotide sequence also includes a polypeptide and / or polynucleotide sequence that comprises one or more amino acid or nucleic acid mutations (e.g., substitutions, deletions, and / or insertions) relative to a reference polypeptide or polynucleotide sequence.

[0058] "Complementarity" refers to the ability to pair by base stacking and specific hydrogen bonding between two sequences containing naturally or non-naturally occurring (e.g., modified as described above) bases (nucleotides) or their analogs. For example, if a base at a position on a nucleic acid can hydrogen bond with a base at a corresponding position on a target, then the bases are considered to be complementary to each other at this position. Nucleic acids may contain universal bases or inert abasic spacers that do not provide positive or negative contributions to hydrogen bonding. Base pairing may include canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with and be considered complementary to any A, C, U, or T. Nichols et al., Nature, 1994; 369: 492-493 and Loakes et al., Nucleic Acids Res., 1994; 22: 4039-4043. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33(19): 6258-6267.

[0059] An "expression cassette" or "expression construct" refers to a polynucleotide sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components are in a relationship that permits them to function in the intended manner. For example, a promoter is operably linked to a polynucleotide sequence if the promoter affects transcription or expression of the polynucleotide sequence.

[0060] The term "subject" includes animals, such as, for example, mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is livestock, such as cattle, sheep, goats, cows, pigs, etc.; or domestic animals, such as dogs and cats. In some embodiments (e.g., particularly in a research setting), the subject is a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig (such as an inbred pig), etc. The terms "subject" and "patient" are used interchangeably herein.

[0061] As used herein, "administering" refers to introducing an agent or composition into a subject, or bringing a composition into contact with cells and / or tissues.

[0062] As used herein, "treatment" refers to delivering an agent or composition to a subject to affect a physiological outcome. In some embodiments, treatment refers to treating a disease in a mammal (e.g., a human), including (a) inhibiting the disease, i.e., stopping the development of the disease or preventing the progression of the disease; (b) alleviating the disease, i.e., causing regression of the disease state; and (c) curing the disease.

[0063] The term "effective amount" refers to the amount of an agent or composition required to cause a specific physiological effect (e.g., the amount required to increase, activate and / or enhance a specific physiological effect). The effective amount of a particular agent can be expressed in a variety of ways based on the nature of the agent, such as mass / volume, number of cells / volume, particles / volume, (mass of agent) / (mass of subject), number of cells / (mass of subject), or particles / (mass of subject). The effective amount of a particular agent can also be expressed as a half maximal effective concentration (EC 50 ), the half-maximal effective concentration refers to the concentration of the agent that causes the amplitude of a specific physiological response to be halfway between the reference level and the maximum response level.

[0064] A "population" of cells refers to any number of cells greater than 1, but preferably at least 1 x 10 3 cells, at least 1x10 4 cells, at least 1x10 5 cells, at least 1x10 6 cells, at least 1x10 7 cells, at least 1x10 8 cells, at least 1x10 9 cells, at least 1x10 10 A cell population can refer to an in vitro population (e.g., a cell population in culture) or an in vivo population (e.g., a cell population residing in a specific tissue).

[0065] "Effector function" refers to immune cell function associated with the generation, maintenance and / or enhancement of an immune response against a target cell or target antigen.

[0066] The terms "microRNA," "miRNA," and "miR" are used interchangeably herein and refer to small noncoding endogenous RNAs of approximately 21-25 nucleotides in length that regulate gene expression by directing their target messenger RNAs (mRNAs) for degradation or translational repression.

[0067] The term "composition" as used herein refers to a preparation of the recombinant RNA molecules or particle-encapsulated recombinant RNA molecules described herein that can be administered or delivered to a subject or a cell.

[0068] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0069] As used herein, "pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant and / or emulsifier approved by the U.S. Food and Drug Administration for use in humans and / or livestock.

[0070] The term "replication-competent viral genome" refers to the viral genome that encodes all viral genes necessary for viral replication and production of infectious viral particles.

[0071] The term "oncolytic virus" refers to a virus that has been modified or naturally preferentially infects cancer cells.

[0072] As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring, encoding or transporting another nucleic acid molecule.

[0073] As used herein, the term "corresponding to" or "corresponding to" in relation to an amino acid or nucleic acid position means that when a first polypeptide / polynucleotide sequence is aligned with a reference polypeptide / polynucleotide sequence, a position in the first polypeptide / polynucleotide sequence is aligned with a given amino acid / nucleic acid in the reference polypeptide / polynucleotide sequence. Alignment is performed by a person skilled in the art using software designed for this purpose, for example, Clustal Omega version 1.2.4 and the default parameters of this version.

[0074] The term "encapsulation efficiency" or "EE%" refers to the percentage of target molecules (e.g., synthetic RNA viral genomes) that are successfully encapsulated into LNPs. Encapsulation efficiency can be calculated using the following formula:

[0075] (EE%) = (Wt / Wi) × 100%

[0076] Where Wt is the total amount of drug in the LNP suspension and Wi is the total amount of drug initially added during preparation. As an illustrative example, if 97 mg of the target molecule is encapsulated in the LNP out of a total of 100 mg of the target molecule initially provided to the composition, the encapsulation efficiency may be 97%.

[0077] The term "lipid-nitrogen to phosphorus ratio" or "(N:P)" refers to the ratio of positively charged lipid amine groups to nucleic acid phosphate groups in a lipid nanoparticle.

[0078] The term "half-life" refers to the pharmacokinetic properties of a molecule (e.g., a molecule encapsulated in a lipid nanoparticle). Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known amount of a molecule in the body from a subject's body (e.g., a human patient or other mammal) or its specific compartment (e.g., as measured in serum, i.e., a circulating half-life) or in other tissues after the molecule is administered, by biological processes (e.g., metabolism, excretion, accelerated blood clearance, etc.). In general, an increase in half-life will result in an increase in the average residence time (MRT) of the administered molecule in the circulation.

[0079] The term "accelerated blood clearance" or "ABC" refers to the phenomenon that certain agents (e.g., PEG-containing LNPs) are rapidly cleared from the blood after second and subsequent administrations. ABC has been observed in many lipid delivery vehicles, including liposomes and LNPs.

[0080] As used herein, unless otherwise expressly stated, the term "ratio" when used to refer to lipid composition (e.g., as a percentage of total lipid content) refers to a molar ratio. A molar ratio as a percentage of total lipid content may also be expressed as "mol %. For example, "49:22:28.5:0.5 mol %" means that the molar ratio is 49:22:28.5:0.5.

[0081] As used herein, the term "aliphatic" or "aliphatic group" means a straight chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or contains one or more unsaturated units, or a monocyclic hydrocarbon or bicyclic hydrocarbon (also referred to herein as "carbocycle", "cycloaliphatic" or "cycloalkyl") that is completely saturated or contains one or more unsaturated units but is not aromatic, which has a single point of connection with the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-4 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or contains one or more unsaturated units but is not aromatic, which has a single point of connection with the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0082] As used herein, term "alkyl" is a branched or unbranched saturated hydrocarbon group with a specific number of carbon atoms. In some embodiments, alkyl refers to a branched or unbranched saturated hydrocarbon group with three carbon atoms (C3). In some embodiments, alkyl refers to a branched or unbranched saturated hydrocarbon group with six carbon atoms (C6). In some embodiments, term "alkyl" includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl and hexyl.

[0083] As used herein, the term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n —, wherein n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 2 to 3. Substituted alkylene chains are polymethylene groups in which one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups.

[0084] The term "aryl", used alone or as part of a larger moiety in "aralkyl", "aralkyloxy", or "aryloxyalkyl", refers to monocyclic and bicyclic ring systems having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system that may bear one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, anthracenyl, and the like. Groups in which an aromatic ring is fused to one or more non-aromatic rings (such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like) are also included within the scope of the term "aryl" as used herein.

[0085] The terms "heteroaryl" and "heteroar-", e.g., "heteroaralkyl" or "heteroaralkoxy", used alone or as part of a larger moiety, refer to groups having 5 to 10 ring atoms, preferably 5, 6 or 9 ring atoms; having 6, 10 or 14 pi electrons shared in the ring arrangement; and having 1 to 5 heteroatoms (other than carbon atoms). The term "heteroatom" refers to nitrogen, oxygen or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl and pteridinyl. As used herein, the terms "heteroaryl" and "heteroaryl-" also include groups in which a heteroaromatic ring is fused to one or more aryl rings, cycloaliphatic rings, or heterocyclyl rings, wherein the connecting group or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, and pyrido [2,3-b] -1, 4-oxazine -3 (4H) -one. The heteroaryl group can be a monocyclic or bicyclic ring. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which include optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions are independently optionally substituted.

[0086] The term "haloaliphatic" refers to an aliphatic group substituted with one or more halogen atoms.

[0087] The term "haloalkyl" refers to a straight or branched chain alkyl group substituted with one or more halogen atoms.

[0088] The term "halogen" means F, Cl, Br or I.

[0089] As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic group" and "heterocyclic ring" are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (preferably one to four) heteroatoms as defined above in addition to carbon atoms. When used to refer to the ring atoms of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or +NR (such as in the pyrrolidinyl substituted by TV-). Heterocycle can be connected with its side group on any heteroatom or carbon atom to produce a stable structure, and any ring atom can be optionally substituted. The example of such saturated or partially unsaturated heterocyclic group includes but is not limited to tetrahydrofuranyl, tetrahydrothienylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolane, diazepine, oxazepine, thiazolinyl, morpholinyl and quinuclidine. The terms "heterocycle", "heterocyclic radical", "heterocyclic ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic group" are used interchangeably herein, and also include groups in which the heterocyclic ring is fused with one or more aryl, heteroaryl or alicyclic rings, such as dihydroindole, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolyl, wherein the connecting group or the point of attachment is on the heterocyclic ring. A heterocyclyl group may be monocyclic or bicyclic.The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0090] Heterocycle can be connected with its side group on any heteroatom or carbon atom, so as to produce a stable structure, and any ring atom can be optionally substituted.Examples of such saturated or partially unsaturated heterocyclic groups include but are not limited to tetrahydrofuranyl, tetrahydrothienylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolane, diazepine, oxazepine, thiazepine, morpholinyl and quinuclidine.Term "heterocycle", "heterocyclic radical", "heterocyclic ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic group" are used interchangeably herein, and also include groups in which the heterocyclic ring is fused with one or more aryl, heteroaryl or alicyclic rings, such as dihydroindole, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolyl, wherein the connecting group or the point of attachment is on the heterocyclic ring.Heterocyclic radical groups can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0091] As described herein, the compounds of the present disclosure may contain "optionally substituted" parts. In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the specified part are replaced by suitable substituents. Unless otherwise indicated, the "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a specific group, the substituent at each position can be the same or different. The substituent combinations contemplated by the present disclosure are preferably those substituent combinations that lead to the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is substantially unchanged when subjected to conditions that allow compound production, detection, and in some embodiments, its recovery, purification, and use for one or more purposes disclosed herein.

[0092] Suitable monovalent substituents on the substitutable carbon atoms of the "optionally substituted" group are independently halogen; -(CH2) 0-4 R°; —(CH2) 0-4 OR°; —O(CH2) 0-4 R°, —O—(CH2) 0-4 C(O)OR°; —(CH2) 0-4 CH(OR°)2; —(CH2) 0-4 SR°; —(CH2) 0-4 Ph, which may be substituted by R°; —(CH2) 0-4 O(CH2) 0-1 Ph, which may be substituted by R°; —CH═CHPh, which may be substituted by R°; —(CH2) 0-4 O(CH2) 0-1 -pyridyl, which may be substituted by R°; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R°)2;—(CH2) 0-4 N(R°)C(O)R°;—N(R°)C(S)R°;—(CH2) 0-4 N(R°)C(O)NR°2; —N(R°)C(S)NR°2; —(CH2) 0-4 N(R°)C(O)OR°;—N(R°)N(R°)C(O)R°;—N(R°)N(R°)C(O)NR°2;—N(R°)N(R°)C(O)OR°;—(CH2) 0-4 C(O)R°;—C(S)R°;—(CH2) 0-4 C(O)OR°; —(CH2) 0-4 C(O)SR°; —(CH2) 0-4 C(O)OSiR°3;—(CH2) 0-4OC(O)R°; —OC(O)(CH2) 0-4 SR°, SC(S)SR°; —(CH2) 0-4 SC(O)R°; —(CH2) 0-4 C(O)NR°2; —C(S)NR°2; —C(S)SR°; —SC(S)SR°, —(CH2) 0-4 OC(O)NR°2; —C(O)N(OR°)R°; —C(O)C(O)R°; —C(O)CH2C(O)R°; —C(NOR°)R°; —(CH2) 0-4 SSR°;—(CH2) 0-4 S(O)2R°; —(CH2) 0-4 S(O)2OR°; —(CH2) 0-4 OS(O)2R°;—S(O)2NR°2;—(CH2) 0-4 S(O)R°; —N(R°)S(O)2NR°2; —N(R°)S(O)2R°; —N(OR°)R°; —C(NH)NR°2; —P(O)2R°; —P(O)R°2; —OP(O)R°2; —OP(O)(OR°)2; SiR°3; —(C 1-4 Straight or branched alkylene)O—N(R°)2 or—(C 1-4 linear or branched alkylene) C(O)O—N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C 1-6 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph, -CH2-(5-6 membered heteroaryl ring) or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independent occurrences of R° together with their intervening atoms form a 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

[0093] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with their intervening atoms) are independently halogen, —(CH2) 0-2 R ● 、-(halogenated R ● )、—(CH2) 0-2 OH, —(CH2) 0-2 OR ● 、—(CH2) 0-2 CH(OR ● )2;—O(halogenated R ● )、—CN、—N3、—(CH2)0-2 C(O)R ● 、—(CH2) 0-2 C(O)OH, —(CH2) 0-2 C(O)OR ● 、—(CH2) 0-2 SR ● 、—(CH2) 0-2 SH, —(CH2) 0-2 NH2, —(CH2) 0-2 NHR ● 、—(CH2) 0-2 NR ● 2. —NO2, —SiR ● 3. OSiR ● 3. —C(O)SR ● ,—(C 1-4 Straight or branched alkylene)C(O)OR ● or—SSR ● , where each R ● is unsubstituted or is substituted with one or more halogens and is independently selected from C 1-4 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents on the saturated carbon atom of R° include ═O and ═S.

[0094] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2)) 2-3 O—or—S(C(R*2)) 2-3 S—, wherein each independent occurrence of R* is selected from hydrogen, C 1-6 aliphatic, or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents attached to adjacent substitutable carbons of an "optionally substituted" group include: -O(CR*2) 2-3 O—, wherein each independent occurrence of R* is selected from hydrogen, C 1-6 aliphatic, or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0095] Suitable substituents on the aliphatic group of R* include halogen, -R● 、-(halogenated R ● ),—OH,—OR ● ,—O(halogenated R ● ), —CN, —C(O)OH, —C(O)OR ● 、—NH2、—NHR ● ,—NR ● 2 or -NO2, where each R ● is unsubstituted or, when preceded by "halo", is substituted only by one or more halogens, and is independently C 1-4 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0096] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include or Each of these is independently hydrogen, C which may be substituted as defined below 1-6 aliphatic, unsubstituted -OPh or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or notwithstanding the above definition, two independent occurrences of Together with its intervening atoms, it forms a 3-12 membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0097] Suitable substituents on the aliphatic group are independently halogen, -R ● 、-(halogenated R ● ),—OH,—OR ● ,—O(halogenated R ● ), —CN, —C(O)OH, —C(O)OR ● 、—NH2、—NHR ● ,—NR ● 2 or -NO2, where each R ● is unsubstituted or, when preceded by "halo", is substituted only by one or more halogens, and is independently C 1-4 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0098] As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0099] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in .Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds disclosed herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed of amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts obtained by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

[0100] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N(C 1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Further pharmaceutically acceptable salts include (when appropriate) non-toxic ammonium salts, quaternary ammonium salts and amine cation salts formed using counterions such as halide ions, hydroxide ions, carboxylates, sulfate ions, phosphate ions, nitrate ions, lower alkyl sulfonate ions and aryl sulfonate ions.

[0101] "Pharmaceutically acceptable derivative" means any non-toxic salt, ester, salt of an ester, or other derivative of a disclosed compound that, upon administration to a recipient, is capable of providing, directly or indirectly, a disclosed compound or an active metabolite or residue thereof.

[0102] The term "tertiary amine" is used to describe an amine (nitrogen atom) attached to three carbon-containing groups, each of which is covalently bonded to the amine group through a carbon atom within the group. Tertiary amines can be protonated or form a complex with a Lewis acid.

[0103] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0104] Unless otherwise stated, the structures depicted herein are also intended to include all enantiomers, diastereomers, and geometric (or conformational) forms of the structures, such as R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the present invention are within the scope of the present disclosure. Unless otherwise stated, all tautomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

[0105] General methods in molecular and cellular biochemistry can be found in standard textbooks such as molecular cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al., eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0106] Synthetic RNA viral genome

[0107] In some embodiments, the present disclosure provides recombinant RNA molecules (e.g., RNA genomes) encoding oncolytic viruses. Such recombinant RNA molecules are referred to herein as "synthetic viral genomes" or "synthetic RNA viral genomes". In such embodiments, the synthetic RNA viral genome can produce infectious, lytic viruses when introduced into cells by non-viral delivery vehicles, and no additional exogenous genes or proteins are required in the cells to replicate and produce infectious viruses. In contrast, the endogenous translation mechanism in the host cell mediates the expression of viral proteins from the synthetic RNA viral genome. The expressed viral proteins then mediate viral replication and assemble into infectious viral particles (which may include capsid proteins, envelope proteins, and / or membrane proteins) comprising RNA viral genomes. Therefore, when the RNA polynucleotides described herein (i.e., synthetic RNA viral genomes) are introduced into host cells, viruses capable of infecting another host cell will be produced. In some embodiments, the oncolytic virus is a picornavirus. In some embodiments, the picornavirus is CVA21. In some embodiments, the picornavirus is SVV.

[0108] In some embodiments, the synthetic viral genome is provided in the form of recombinant ribonucleic acid (RNA) (i.e., synthetic RNA viral genome). In some embodiments, the synthetic RNA viral genome comprises one or more nucleic acid analogs. Examples of nucleic acid analogs include 2'-O-methyl substituted RNA, 2'-O-methoxy-ethyl bases, 2' fluoro bases, locked nucleic acids (LNA), non-locked nucleic acids (UNA), bridged nucleic acids (BNA), morpholine and peptide nucleic acids (PNA). In some embodiments, the synthetic RNA viral genome is a replicon, an RNA viral genome encoding a transgenic, an mRNA molecule or a circular RNA molecule (circular RNA). In some embodiments, the synthetic RNA viral genome comprises a single-stranded RNA (ssRNA) viral genome. In some embodiments, the single-stranded genome can be a positive sense genome or a negative sense genome.

[0109] In some embodiments, the recombinant RNA molecule is a circular RNA molecule (circular RNA). Circular RNA molecules lack the free ends necessary for exonuclease-mediated degradation, thereby extending the half-life of RNA molecules and being able to produce more stable proteins over time (see, for example, Wesselhoeft et al., Engineering circular RNA for potent and stable translation in eukaryotic cells. Nature Communications. (2018) 9: 2629). In order to produce functional RNA viruses from circular RNA molecules, once entering the cell, it is necessary to "break" the circular construct so that a linear RNA genome with appropriate 3' and 5' natural ends can be produced. Therefore, in some embodiments, the recombinant RNA molecule encoding an oncolytic virus is provided in the form of a circular RNA molecule, and further comprises one or more additional RNA sequences that promote linearization of circular RNA molecules in cells. Examples of such additional RNA sequences include siRNA target sites, miRNA target sites, and guide RNA target sites. Corresponding siRNA, miRNA, or gRNA can be formulated with circular RNA molecules. Alternatively, miRNA target sites can be selected based on the expression of cognate miRNAs in target cells, such that cleavage of the circular RNA molecule and initial expression of the encoded oncolytic virus is limited to target cells expressing the specific miRNA.

[0110] The synthetic RNA viral genome described herein encodes an oncolytic virus. Examples of oncolytic viruses are known in the art, including but not limited to: picornaviruses (e.g., coxsackieviruses), polioviruses, measles virus, vesicular stomatitis virus, orthomyxoviruses, and Maraba virus. In some embodiments, the oncolytic virus encoded by the synthetic RNA viral genome is a virus in the Picornaviridae family, such as a coxsackievirus, a poliovirus (including chimeric polioviruses such as PVS-RIPO and other chimeric picornaviruses), or a Seneca valley virus, or any virus from a chimeric source of any of a variety of picornaviruses, a virus in the Arenaviridae family, such as a lassa virus, a virus in the Retroviridae family, such as a murine leukemia virus, a virus in the Orthomyxoviridae family, such as an influenza A virus, a virus in the Paramyxoviridae family, such as a Newcastle disease virus or a measles virus, a virus in the Reoviridae family, such as a mammalian orthoreovirus, a virus in the Togaviridae family, such as a sindbis virus, or a virus in the Rhabdoviridae family, such as a vesicular stomatitis virus (VSV) or a Maraba virus.

[0111] Positive-sense single-stranded RNA virus

[0112] In some embodiments, the synthetic RNA viral genome described herein encodes a single-stranded RNA (ssRNA) viral genome. In some embodiments, the ssRNA virus is a positive sense ssRNA (+ sense ssRNA) virus. Exemplary + sense ssRNA viruses include members of Picornaviridae (e.g., Coxsackievirus, poliovirus, and Seneca Valley virus (SVV), including SVV-A), Coronaviridae (e.g., alpha coronavirus, such as HCoV-229E and HCoV-NL63, beta coronavirus, such as HCoV-HKU1, HCoV-OC3, and MERS-CoV), Retroviridae (e.g., murine leukemia virus), and Togaviridae (e.g., alpha virus, such as Semliki Forest virus, Sindbis virus, Ross River virus, or Chikungunya virus). Other exemplary genera and species of positive sense ssRNA viruses are shown in Table 1 below.

[0113] Table 1: Positive-sense ssRNA viruses

[0114]

[0115]

[0116] In some embodiments, the recombinant RNA molecules described herein encode a picornavirus selected from coxsackievirus, poliovirus, and Seneca Valley virus (SVV). In some embodiments, the recombinant RNA molecules described herein encode coxsackievirus. In some aspects of this embodiment, the recombinant RNA molecules are coxsackieviruses and include SEQ ID NO: 2 5'UTR sequence (see, e.g., Brown et al., Complete Genomic Sequencing Shows thatPolioviruses and Members of Human Enterovirus Species C Are Closely Related in the Noncapsid Coding Region. Journal of Virology, (2003) 77: 16, pp. 8973–8984. GenBank Accession No. AF546702). In such embodiments, the 5'UTR sequence of SEQ ID NO: 2 unexpectedly increases the production of functional coxsackieviruses compared to other previously described 5'UTR sequences (see, e.g., Newcombe et al., Cellular receptor interactions of C-cluster human group Acoxsackieviruses Journal of General Virology (2003), 84, 3041-3050. GenBank Accession No. AF465515). In some aspects of this embodiment, the recombinant RNA molecule encodes a coxsackievirus and comprises the sequence of SEQ ID NO: 1.

[0117] In some embodiments, the synthetic RNA viral genome described herein encodes a coxsackievirus. In some embodiments, the coxsackievirus is selected from CVB3, CVA21 and CVA9. The nucleic acid sequences of exemplary coxsackieviruses are provided: GenBank reference number M33854.1 (CVB3), GenBank reference number KT161266.1 (CVA21) and GenBank reference number D00627.1 (CVA9). In some embodiments, the synthetic RNA viral genome described herein encodes a modified CVA21 virus comprising SEQ ID NO: 1, and the modified CVA21 virus is a Kuykendall (Kuyk) strain. In some embodiments, the viral genome sequence of the Kuykendall strain is according to GenBank accession number AF465515.1 or AF546702.1. In some embodiments, the synthetic RNA viral genome described herein encodes a chimeric coxsackievirus. In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21 strain selected from a CVA21-EF strain and a CVA21-KY strain. In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21-EF strain. The exemplary sequence of the EF strain viral genome is according to GenBank accession number EF015029.1. In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21-KY strain. The exemplary sequence of the KY strain viral genome is according to GenBank accession number KY284011.1. As shown in Figures 11-26, EF strains and KY strains provide therapeutic benefits over Kuykendall laboratory strains and previously described synthetic picornavirus compositions.

[0118] Figure 32 provides the domain organization of three CVA21 strains (EF, KY, and Kuykendall), and Table 2 below provides the sequence identity between the various regions of these three strains.

[0119] Table 2: Sequence identities between corresponding regions of different CVA21 strains

[0120]

[0121] Relative to the Kuykendall laboratory strain, one or more specific regions in the viral genome of the CVA21 EF strain or KY strain may contribute to the beneficial therapeutic effects observed for the EF strain and the KY strain. In some embodiments, the one or more specific regions are selected from the group consisting of: 5'UTR (IRES) region, P1 region, and 3D region. The nucleic acid position of each of these specific regions of the virus strains described herein is as follows:

[0122] (a) The 5'UTR (IRES) region of CVA21-Kuykendall encompasses nucleic acid 1-713 of SEQ ID NO: 1. The 5'UTR (IRES) region of CVA21-KY encompasses nucleic acid 1-713 of SEQ ID NO: 5. The 5'UTR (IRES) region of CVA21-EF encompasses nucleic acid 1-748 of SEQ ID NO:9.

[0123] (b) The P1 region of CVA21-Kuykendall encompasses nucleic acid 714-3350 of SEQ ID NO: 1. The P1 region of CVA21-KY encompasses nucleic acid 714-3350 of SEQ ID NO: 5. The P1 region of CVA21-EF encompasses nucleic acid 749-3385 ​​of SEQ ID NO:9.

[0124] (c) The 3D region of CVA21-Kuykendall encompasses nucleic acid 5952-7340 of SEQ ID NO: 1. The 3D region of CVA21-KY encompasses nucleic acid 5952-7340 of SEQ ID NO: 5. The 3D region of CVA21-EF encompasses nucleic acid 5987-7375 of SEQ ID NO:9.

[0125] In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21-KY strain. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a polynucleotide sequence according to SEQ ID NO: 5. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9% or 100% (including all ranges and subranges therebetween) sequence identity with SEQ ID NO: 5. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a polynucleotide sequence having less than 95%, less than 90%, less than 85% or less than 80% identity (including all ranges and subranges therebetween) with SEQ ID NO: 1.

[0126] In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21-KY strain and comprises a 5'UTR (IRES) sequence according to SEQ ID NO: 6 (corresponding to nucleic acids 1-713 of SEQ ID NO: 5). In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21-KY strain and comprises a 5'UTR (IRES) sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) to SEQ ID NO: 6. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a 5'UTR (IRES) sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:2, including all ranges and subranges therebetween.

[0127] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a P1 sequence according to SEQ ID NO:7 (corresponding to nucleic acid 714-3350 of SEQ ID NO:5). In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a P1 sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:7 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a P1 sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:3 (including all ranges and subranges therebetween).

[0128] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a 3D sequence according to SEQ ID NO: 8 (corresponding to nucleic acid 5952-7340 of SEQ ID NO: 5). In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a 3D sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9% or 100% sequence identity (including all ranges and subranges therebetween) with SEQ ID NO: 8. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a 3D sequence that is less than 95%, less than 90%, less than 85%, or less than 80% (including all ranges and subranges therebetween) identical to SEQ ID NO: 4.

[0129] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain. In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a polynucleotide sequence according to SEQ ID NO: 9. In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9% or 100% (including all ranges and subranges therebetween) sequence identity with SEQ ID NO: 9. In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a polynucleotide sequence that is less than 95%, less than 90%, less than 85% or less than 80% (including all ranges and subranges therebetween) identical to SEQ ID NO: 1.

[0130] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a 5'UTR (IRES) sequence according to SEQ ID NO: 10 (corresponding to nucleic acid 1-748 of SEQ ID NO: 9). In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a 5'UTR (IRES) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 10 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a 5'UTR (IRES) sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:2, including all ranges and subranges therebetween.

[0131] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a P1 sequence according to SEQ ID NO: 11 (corresponding to nucleic acid 749-3385 ​​of SEQ ID NO: 9). In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a P1 sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 11 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a P1 sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO: 3 (including all ranges and subranges therebetween).

[0132] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a 3D sequence according to SEQ ID NO: 12 (corresponding to nucleic acid 5987-7375 of SEQ ID NO: 9). In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a 3D sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) with SEQ ID NO: 12. In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a 3D sequence that is less than 95%, less than 90%, less than 85%, or less than 80% (including all ranges and subranges therebetween) identical to SEQ ID NO: 4.

[0133] In some embodiments, the CVA21 RNA virus genome described herein does not contain the nucleotide sequence CGUCUC (SEQ ID NO: 83) or GAGACG (SEQ ID NO: 84). The corresponding complementary DNA sequences CGTCTC (SEQ ID NO: 85) and GAGACG (SEQ ID NO: 86) are BsmBI restriction enzyme recognition sites.

[0134] In some embodiments, the CVA21 RNA virus genome described herein does not contain the nucleotide sequence GGUCUC (SEQ ID NO: 87) or GAGACC (SEQ ID NO: 88). The corresponding complementary DNA sequences GGTCTC (SEQ ID NO: 89) and GAGACC (SEQ ID NO: 90) are BsaI restriction enzyme recognition sites.

[0135] In some embodiments, the synthetic RNA viral genome described herein encodes Seneca Valley virus (SVV). In some embodiments, SVV is selected from wild-type SVV (such as SVV-001, SEQ ID NO: 25) or mutant SVV or chimeric SVV (such as SVV-001-S177A encoded by SEQ ID NO: 26; or SVV-IRES-2-S177A encoded by SEQ ID NO: 68 or SEQ ID NO: 73).

[0136] In some embodiments, SVV is a SVV-S177 mutant. In some embodiments, SVV is a SVV-S177A mutant. As used herein with respect to the SVV viral genome, the term "S177 mutant" refers to an SVV viral genome encoding a VP2 protein, wherein the VP2 protein comprises a mutation at amino acid S177 of the wild-type protein (according to the amino acid numbering of the VP2 protein encoded by SEQ ID NO:25). Therefore, the term "S177A mutant" refers to an SVV mutant having an S177A amino acid substitution of the VP2 protein. In SEQ ID NO:25, the VP2 S177 residue is encoded by the codon "UCU" at nucleic acid positions 1645-1647. Therefore, the SVV-S177 mutant comprises a nucleic acid mutation in the region corresponding to nucleic acid positions 1645-1647 of SEQ ID NO:25. In some embodiments, the SVV-S177A mutant comprises the codon sequence "GCU", "GCC", "GCA" or "GCG" at the region corresponding to nucleic acid positions 1645-1647 of SEQ ID NO: 25. In some embodiments, the SVV-S177A mutant comprises the codon sequence "GCG" at the region corresponding to nucleic acid positions 1645-1647 of SEQ ID NO: 25.

[0137] In some embodiments, the SVV RNA viral genome described herein does not contain the nucleotide sequence GCUCUUC (SEQ ID NO: 79) or GAGAGGC (SEQ ID NO: 80). The corresponding complementary DNA sequences GCTCTTC (SEQ ID NO: 81) and GAGAGGC (SEQ ID NO: 82) are SapI restriction enzyme recognition sites. In some embodiments, the wild-type SVV RNA viral genome comprises SEQ ID NO: 79 at a position corresponding to nucleic acid 1504-1510 and / or nucleic acid 5293-5299 of SEQ ID NO: 25. In some embodiments, the SVV RNA viral genome of the present disclosure comprises at least one nucleotide substitution in the region corresponding to nucleic acid 1504-1510 and / or nucleic acid 5293-5299 of SEQ ID NO: 25 compared to SEQ ID NO: 79. In some embodiments, at least one nucleotide substitution is a silent mutation that does not change the amino acid encoded by the corresponding region of DNA. In some embodiments, the SVV RNA viral genome of the present disclosure comprises a cytidine ("C") at positions corresponding to nucleic acid 1509 and / or 5298 of SEQ ID NO:25.

[0138] In some embodiments, the SVV RNA viral genome described herein does not contain the nucleotide sequence GGUCUC (SEQ ID NO: 87) or GAGACC (SEQ ID NO: 88). The corresponding complementary DNA sequences GGTCTC (SEQ ID NO: 89) and GAGACC (SEQ ID NO: 90) are BsaI restriction enzyme recognition sites.

[0139] In some embodiments, the synthetic RNA viral genomes described herein encode chimeric picornaviruses (e.g., encoding a virus comprising one portion, such as a capsid protein or IRES derived from a first picornavirus, and another portion, such as a nonstructural gene, such as a protease or polymerase derived from a second picornavirus). In some embodiments, the synthetic RNA viral genomes described herein encode chimeric SVVs.

[0140] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising one or more specific regions derived from an SVV strain selected from the group consisting of SVV-001 (SEQ ID NO: 25 or SEQ ID NO: 72 (Genbank ID No.: DQ641257.1)), SVA / BRA / MG2 / 2015 (SEQ ID NO: 69; GenBank ID No.: KR063108.1), SVA / Canada / MB / NCFAD-104 / 2015 (SEQ ID NO: 70; GenBank ID No.: KY486156.1) and SVV-MN 15-308 (SEQ ID NO: 71; GenBank ID No.: KU359214.1). In some embodiments, the one or more specific regions are selected from the group consisting of: 5'UTR (IRES) region, P1 region, and P3 region. The nucleic acid position of each of these specific regions of the virus strains described herein is as follows:

[0141] (a) The 5'UTR (IRES) region of SVV-001 encompasses nucleic acids 1-668 of SEQ ID NO: 25. The 5'UTR (IRES) region of SVA / BRA / MG2 / 2015 encompasses nucleic acids 1-656 of SEQ ID NO: 69. The 5'UTR (IRES) region of SVA / Canada / MB / NCFAD-104 / 2015 encompasses nucleic acids 1-612 of SEQ ID NO: 70. The 5'UTR (IRES) region of SVV-MN15-308 encompasses nucleic acids 1-610 of SEQ ID NO: 71.

[0142] (b) The P1 region of SVV-001 encompasses nucleic acid 669-3477 of SEQ ID NO: 25. The P1 region of SVA / BRA / MG2 / 2015 encompasses nucleic acid 657-3465 of SEQ ID NO: 69. The P1 region of SVA / Canada / MB / NCFAD-104 / 2015 encompasses nucleic acid 613-3421 of SEQ ID NO: 70. The P1 region of SVV-MN15-308 encompasses nucleic acid 611-3419 of SEQ ID NO: 71.

[0143] (c) The P3 region of SVV-001 encompasses nucleic acid 4855-7212 of SEQ ID NO: 25. The P3 region of SVA / BRA / MG2 / 2015 encompasses nucleic acid 4843-7200 of SEQ ID NO: 69. The P3 region of SVA / Canada / MB / NCFAD-104 / 2015 encompasses nucleic acid 4799-7156 of SEQ ID NO: 70. The P3 region of SVV-MN15-308 encompasses nucleic acid 4797-7154 of SEQ ID NO: 71.

[0144] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising a 5'UTR (IRES) region derived from SVA / BRA / MG2 / 2015 (nucleic acids 1-656 of SEQ ID NO: 69). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 1-656 of SEQ ID NO: 69. In some embodiments, except for one or more regions derived from SVA / BRA / MG2 / 2015, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0145] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising a 5'UTR (IRES) region derived from SVA / Canada / MB / NCFAD-104 / 2015 (nucleic acids 1-612 of SEQ ID NO: 70). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 1-612 of SEQ ID NO: 70. In some embodiments, except for one or more regions derived from SVA / Canada / MB / NCFAD-104 / 2015, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0146] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising a 5'UTR (IRES) region derived from SVV-MN15-308 (nucleic acids 1-610 of SEQ ID NO: 71). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 1-610 of SEQ ID NO: 71. In some embodiments, except for one or more regions derived from SVV-MN15-308, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0147] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising the P1 region derived from SVA / BRA / MG2 / 2015 (nucleic acids 657-3465 of SEQ ID NO: 69). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 657-3465 of SEQ ID NO: 69. In some embodiments, except for one or more regions derived from SVA / BRA / MG2 / 2015, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0148] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising the P1 region derived from SVA / Canada / MB / NCFAD-104 / 2015 (nucleic acids 613-3421 of SEQ ID NO:70). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 613-3421 of SEQ ID NO:70. In some embodiments, except for one or more regions derived from SVA / Canada / MB / NCFAD-104 / 2015, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0149] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising the P1 region derived from SVV-MN15-308 (nucleic acids 611-3419 of SEQ ID NO: 71). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 611-3419 of SEQ ID NO: 71. In some embodiments, except for one or more regions derived from SVV-MN15-308, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0150] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising the P3 region derived from SVA / BRA / MG2 / 2015 (nucleic acids 4843-7200 of SEQ ID NO: 69). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 4843-7200 of SEQ ID NO: 69. In some embodiments, except for one or more regions derived from SVA / BRA / MG2 / 2015, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0151] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising the P3 region derived from SVA / Canada / MB / NCFAD-104 / 2015 (nucleic acids 4799-7156 of SEQ ID NO: 70). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 4799-7156 of SEQ ID NO: 70. In some embodiments, except for one or more regions derived from SVA / Canada / MB / NCFAD-104 / 2015, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0152] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV comprising the P3 region derived from SVV-MN15-308 (nucleic acids 4797-7154 of SEQ ID NO: 71). In some embodiments, the synthetic RNA viral genome encoding SVV comprises a polynucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100%, including all ranges and subranges therebetween, to nucleic acids 4797-7154 of SEQ ID NO: 71. In some embodiments, except for one or more regions derived from SVV-MN15-308, the remainder of the SVV viral genome is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant).

[0153] In some embodiments, the synthetic RNA viral genome described herein encodes a chimeric SVV comprising a 5'UTR (IRES) region derived from SVA / Canada / MB / NCFAD-104 / 2015 (SEQ ID NO: 70) and the remainder of the viral genome derived from SVV-001 (SEQ ID NO: 25). In some embodiments, the SVV is a SVV-S177 mutant (e.g., an S177A mutant). In some embodiments, the synthetic RNA viral genome has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% (including all ranges and subranges therebetween) sequence identity to SEQ ID NO: 68.

[0154] In some embodiments, the synthetic RNA viral genome has been engineered and contains less than 100% sequence identity with a wild-type virus (e.g., wild-type CVA21 or wild-type SVV). In some embodiments, the synthetic RNA viral genome contains less than 99.9%, less than 99.8%, less than 99.7%, less than 99.6%, less than 99.5%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91% or less than 90% sequence identity with the corresponding wild-type virus.

[0155] In some embodiments, the synthetic RNA viral genome comprises a microRNA (miRNA) target sequence (miR-TS) box, wherein the miR-TS box comprises one or more miRNA target sequences, and wherein the expression of one or more corresponding miRNAs in the cell inhibits the replication of the oncolytic virus encoded in the cell. In some embodiments, one or more miRNAs are selected from miR-124, miR-1, miR-143, miR-128, miR-219, miR-219a, miR-122, miR-204, miR-217, miR-137, miR-142 and miR-126. In some embodiments, the miR-TS box comprises one or more copies of the miR-124 target sequence, one or more copies of the miR-1 target sequence, and one or more copies of the miR-143 target sequence. In some embodiments, the miR-TS box comprises one or more copies of the miR-128 target sequence, one or more copies of the miR-219a target sequence, and one or more copies of the miR-122 target sequence. In some embodiments, the miR-TS cassette comprises one or more copies of a miR-128 target sequence, one or more copies of a miR-204 target sequence, and one or more copies of a miR-219 target sequence. In some embodiments, the miR-TS cassette comprises one or more copies of a miR-217 target sequence, one or more copies of a miR-137 target sequence, and one or more copies of a miR-126 target sequence.

[0156] In some embodiments, the synthetic RNA viral genome comprises one or more miR-TS boxes, which are incorporated into the 5' untranslated region (UTR) or 3'UTR of one or more essential viral genes. In some embodiments, the synthetic RNA viral genome comprises one or more miR-TS boxes, which are incorporated into the 5' untranslated region (UTR) or 3'UTR of one or more non-essential genes. In some embodiments, the synthetic RNA viral genome comprises one or more miR-TS boxes, which are incorporated into the 5' or 3' of one or more essential viral genes.

[0157] In some embodiments, the synthetic RNA viral genome comprises a heterologous polynucleotide encoding a payload molecule. In such embodiments, the synthetic RNA viral genome drives the production of infectious oncolytic viruses and the expression of payload molecules. In some embodiments, the expression of payload molecules can increase the therapeutic efficacy of oncolytic viruses. In some embodiments, the payload molecule is selected from: IL-12, GM-CSF, CXCL10, IL-36γ, CCL21, IL-18, IL-2, CCL4, CCL5, anti-CD3-anti-FAP BiTE, antigen binding molecules binding to DLL3 or antigen binding molecules binding to EpCAM. In some embodiments, the payload molecule comprises or consists of MLKL 4HB domains. In some embodiments, the payload molecule comprises or consists of Gasdermin D (Gasdermin D) N-terminal fragments. In some embodiments, the payload molecule comprises or consists of Gasdermin E N-terminal fragments. In some embodiments, the payload molecule comprises or consists of HMGB1Box B domains. In some embodiments, the payload molecule comprises SMAC / Diablo or consists of it. In some embodiments, the payload molecule comprises melittin or consists of it. In some embodiments, the payload molecule comprises L-amino acid oxidase (LAAO) or consists of it. In some embodiments, the payload molecule comprises disintegrin or consists of it. In some embodiments, the payload molecule comprises TRAIL (TNFSF10) or consists of it. In some embodiments, the payload molecule comprises nitroreductase (e.g., Escherichia coli (E.coli) NfsB or NfsA) or consists of it. In some embodiments, the payload molecule comprises reovirus FAST protein (e.g., ARV p14, BRV p15 or p14-p15 hybrid) or consists of it. In some embodiments, the payload molecule comprises leptin / FOSL2 or consists of it. In some embodiments, the payload molecule comprises α-1,3-galactosyltransferase or consists of it. In some embodiments, the payload molecule comprises or consists of adenosine deaminase 2 (ADA2). In some embodiments, the payload molecule comprises or consists of a cytokine selected from the group consisting of IL-IL-36γ, IL-7, IL-12, IL-18, IL-21, IL2, or IFNγ. Further description of the types of payload molecules suitable for these embodiments is provided below.

[0158] Methods for producing recombinant RNA viral genomes

[0159] In some embodiments, the present disclosure provides recombinant DNA molecules encoding synthetic RNA viral genomes as described herein. Such recombinant DNA molecules are referred to herein as "DNA templates" or "recombinant DNA templates". In some embodiments, recombinant DNA molecules are used as templates for in vitro transcription of synthetic RNA viral genomes. In some embodiments, recombinant DNA molecules (e.g., DNA templates) include one or more of the following elements from 5' to 3': (i) promoter; (ii) 5' leader sequence; (iii) 5' connection cleavage sequence; (iv) DNA polynucleotide sequence encoding synthetic RNA genome; (v) polyA tail; and / or (vi) 3' connection cleavage sequence. In some embodiments, the recombinant DNA molecules (e.g., DNA templates) encoding recombinant RNA molecules include each of the following elements: (i) promoter; (ii) 5' leader sequence; (iii) 5' connection cleavage sequence; (iv) DNA polynucleotide sequence encoding synthetic RNA genome; (v) polyA tail; and (vi) 3' connection cleavage sequence. Each of these elements is described in detail below. The description provided for each individual element enables specific embodiments of each element to be combined into a final recombinant DNA molecule (eg, DNA template). For example, disclosure of a specific leader sequence can be combined with disclosure of a specific 5' junction cleavage sequence, etc.

[0160] In some embodiments, the recombinant DNA molecule (e.g., DNA template) does not include additional nucleic acid between two adjacent elements, but may include additional nucleic acid upstream of the promoter sequence or downstream of the 3' connection cleavage sequence. In some embodiments, the promoter sequence is a T7 promoter sequence. In some embodiments, the T7 promoter sequence comprises SEQ ID NO: 91 or consists of it.

[0161] In some embodiments, the promoter is suitable for in vitro transcription. In some embodiments, the promoter is a T7 promoter.

[0162] In some embodiments, one or more recombinant DNA templates comprising polynucleotides encoding synthetic RNA viral genomes are used to produce the synthetic RNA viral genomes described herein in vitro. In other words, the recombinant DNA template is a vector comprising polynucleotides encoding synthetic RNA viral genomes. The term "vector" used herein refers to a nucleic acid molecule capable of transferring, encoding or transporting another nucleic acid molecule. The transferred nucleic acid is generally inserted into a vector nucleic acid molecule. The vector may include a sequence that directs autonomous replication in the cell and / or may include a sequence sufficient to allow integration into the host cell DNA. In some embodiments, the recombinant RNA molecules encoding oncolytic viruses described herein are produced using one or more DNA vectors.

[0163] In some embodiments, the synthetic RNA viral genome described herein is produced by introducing a recombinant DNA molecule (e.g., a DNA template) comprising a polynucleotide encoding a recombinant RNA molecule (e.g., by transfection, transduction, electroporation, etc.) into a suitable host cell in vitro. Suitable host cells include insect and mammalian cell lines. The host cells are cultured for an appropriate amount of time to allow the polynucleotide to be expressed and the synthetic RNA viral genome to be produced. The synthetic RNA viral genome is then isolated from the host cell and formulated for therapeutic use (e.g., encapsulated in a particle). Figure 8 A schematic diagram of the in vitro synthesis of the CVA21 RNA virus genome with 3' and 5' ribozymes is shown. The same schematic diagram is also applicable to the synthesis of RNA virus genomes (e.g., CVA21 or SVV virus genomes) using other ligation and cleavage sequence combinations (see, e.g., Fig. 9A ). When the 3' junction cleavage sequence comprises or consists of a restriction enzyme recognition site, the recombinant DNA molecule (e.g., DNA template) can be digested with the corresponding restriction enzyme prior to the in vitro transcription process, such as Fig. 9B shown.

[0164] T7 promoter

[0165] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises a T7 promoter. In some embodiments, the T7 promoter comprises or consists of a polynucleotide sequence of SEQ ID NO: 91. In some embodiments, the T7 promoter comprises or consists of a polynucleotide sequence of SEQ ID NO: 91 with at most 1, 2, 3, or 4 mutations.

[0166] In some embodiments, the T7 promoter is immediately preceding the leader sequence with no additional nucleotides therebetween. In some embodiments, the T7 promoter is immediately preceding the 5' junction cleavage sequence with no additional nucleotides therebetween. In some embodiments, the viral genome encodes CVA21 or SVV.

[0167] Ligation Cutting Sequence

[0168] In some embodiments, the recombinant RNA molecules comprising the synthetic RNA viral genome described herein require the natural discrete 5' and 3' ends of the virus. The RNA transcripts produced by in vitro T7 RNA polymerase or mammalian RNA Pol II contain mammalian 5' and 3' UTRs, but do not contain the discrete, natural ends required for producing infectious RNA viruses. For example, T7 RNA polymerase requires guanosine residues on the 5' end of the template polynucleotide to initiate transcription. However, the 5' end of SVV starts with a uridine residue. Therefore, the T7 leader sequence required for in vitro transcription of the SVV transcript must be removed to generate the natural 5' SVV end required for producing functional infectious SVV. Therefore, in some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genome described herein require additional non-viral 5' and 3' sequences, which enable the generation of natural discrete 5' and 3' ends of the virus. Such sequences are referred to as junction cleavage sequences (JCS) in this article. In some embodiments, the junction cleavage sequence functions to cleave the RNA transcript encoded by T7 RNA polymerase or Pol II at the junction of the viral RNA and the mammalian mRNA sequence, thereby removing the non-viral RNA polynucleotide from the transcript to maintain the native 5' and 3' discrete ends of the virus (see Fig. 9A In some embodiments, the ligation cleavage sequence serves to generate appropriate ends during linearization of the DNA plasmid encoding the synthetic viral genome (e.g., a 3' restriction enzyme recognition sequence is used to generate appropriate 3' ends after linearization of the plasmid template and prior to in vitro transcription of the synthetic RNA genome).

[0169] In some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genomes described herein include at least one 5' connection cleavage sequence and at least one 3' connection cleavage sequence. In some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genomes described herein include one or more 5' connection cleavage sequences and at least one 3' connection cleavage sequence. In some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genomes described herein include at least one 5' connection cleavage sequence and one or more 3' connection cleavage sequences. In some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genomes described herein include one or more 5' connection cleavage sequences and one or more 3' connection cleavage sequences. In some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genomes described herein include two 5' connection cleavage sequences and at least one 3' connection cleavage sequence. In some embodiments, the recombinant DNA molecules (e.g., DNA templates) suitable for producing the synthetic RNA viral genomes described herein include at least one 5' connection cleavage sequence and two 3' connection cleavage sequences.

[0170] The property of connecting the cleavage sequence and removing non-viral RNA from the viral genome transcript can be achieved by a variety of methods. For example, in some embodiments, the connection cleavage sequence is the target of RNA interference (RNAi) molecules. As used herein, "RNA interference molecules" refer to RNA polynucleotides that mediate the degradation of target mRNA sequences by endogenous gene silencing pathways (e.g., Dicer and RNA-induced silencing complexes (RISC)). Exemplary RNA interfering agents include microRNA (miRNA), artificial miRNA (artificial miRNA) (amiRNA), short hairpin RNA (short hairpin RNA) (shRNA) and small interfering RNA (siRNA). In addition, any system for cutting RNA transcripts at specific sites currently known in the art or to be defined in the future can be used to generate the natural discrete ends of viruses.

[0171] In some embodiments, the RNAi molecule is a miRNA. MiRNA refers to a naturally occurring, small non-coding RNA molecule of about 18-25 nucleotides in length, which is at least partially complementary to a target mRNA sequence. In animals, miRNA genes are transcribed into primary miRNAs (pri-miRNAs), which are double-stranded and form a stem-loop structure. Pri-miRNAs are then cut in the nucleus by a microprocessor complex comprising 2 types of RNase III, Drosha, and the microprocessor subunit DCGR8 to form a precursor miRNA (pre-miRNA) of 70–100 nucleotides. Pre-miRNA forms a hairpin structure and is transported to the cytoplasm, where it is processed into a miRNA duplex of about 18-25 nucleotides by the RNase III enzyme Dicer. Although either strand in the duplex has the potential to act as a functional miRNA, typically one strand of the miRNA is degraded and only one strand is loaded onto the Argonaute (AGO) nuclease to generate an effector RNA-induced silencing complex (RISC), in which the miRNA interacts with its mRNA target (Wahid et al., 1803: 11, 2010, 1231-1243). In some embodiments, the 5' and / or 3' junction cleavage sequence is a miRNA target sequence.

[0172] In some embodiments, the RNAi molecule is an artificial miRNA (amiRNA) derived from a synthetic miRNA embedded in a Pol II transcript. (See, e.g., Liu et al., Nucleic Acids Res (2008) 36:9; 2811-2834; Zeng et al., Molecular Cell (2002), 9; 1327-1333; Fellman et al., Cell Reports (2013) 5; 1704-1713). In some embodiments, the 5' and / or 3' junction cleavage sequence is an amiRNA target sequence.

[0173] In some embodiments, RNAi molecules are siRNA molecules. siRNA refers to double-stranded RNA molecules, usually about 21-23 nucleotides in length. Duplex siRNA molecules are processed in the cytoplasm by associating with a multiprotein complex called RNA-induced silencing complex (RISC), during which the "passenger" sense strand is enzymatically cut from the duplex. The antisense "guide" strand contained in the activated RISC then guides RISC to the corresponding mRNA by sequence complementarity, and the AGO nuclease cuts the target mRNA, resulting in specific gene silencing. In some embodiments, siRNA molecules are derived from shRNA molecules. shRNA is a single-stranded artificial RNA molecule of about 50-70 nucleotides in length, and the shRNA forms a stem-loop structure. The expression of shRNA in cells is achieved by introducing a DNA polynucleotide encoding shRNA by a plasmid or viral vector. Then, shRNA is transcribed into a product simulating the pre-miRNA stem-loop structure, and after nuclear export, the hairpin structure is processed by Dicer to form a duplex siRNA molecule, which is then further processed by RISC to mediate target gene silencing. In some embodiments, the 5' and / or 3' junction cleavage sequence is a siRNA target sequence.

[0174] In some embodiments, the ligation cleavage sequence is a guide RNA (gRNA) target sequence. In such embodiments, gRNA can be designed and introduced together with a Cas endonuclease (e.g., Cas13) with RNase activity to mediate the cutting of viral genome transcripts at precise ligation sites. In some embodiments, the 5' and / or 3' ligation cleavage sequence is a gRNA target sequence.

[0175] In some embodiments, the junction cleavage sequence is a pri-miRNA coding sequence. After transcription of the polynucleotide encoding the viral genome (e.g., a recombinant RNA molecule), these sequences form a pri-miRNA stem-loop structure, which is then cut by Drosha in the nucleus to cut the transcript at the precise junction site. In some embodiments, the 5' and / or 3' junction cleavage sequence is a pri-mRNA target sequence.

[0176] In some embodiments, the connection cleavage sequence is a primer binding sequence that promotes cutting by endoribonuclease RNAseH. In such embodiments, the primer that makes 5' and / or 3' connection cleavage sequence annealing is added to the in vitro reaction together with the RNAseH enzyme. The RNAseH specific hydrolysis and the phosphodiester bond of the RNA of DNA hybridization make it possible to cut the synthetic RNA genome intermediate at the accurate connection cleavage sequence to produce required 5' and 3' natural ends.

[0177] In some embodiments, the ligation cleavage sequence comprises or consists of a restriction enzyme recognition site and results in the generation of discrete ends of viral transcripts during linearization of runoff RNA synthesis of the plasmid template with T7 RNA polymerase. In some embodiments, the ligation cleavage sequence is a Type IIS restriction enzyme recognition site. Type IIS restriction enzymes comprise a group of specific enzymes that recognize asymmetric DNA sequences and cut at a defined distance outside their recognition sequence, typically within 1 to 20 nucleotides. Exemplary IIS type restriction enzymes include AcuI, AlwI, BaeI, BbsI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBi, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BstI, CaspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MbolI, MlyI, MmeI, MnlL, NmeAIII, PleI, SapI and SfaNI. The recognition sequences of these IIS type restriction enzymes are known in the art. See the New England Biolabs website at neb.com / tools-and-resources / selection-charts / type-iis-restriction-enzymes. In some embodiments, the ligation cleavage sequence comprises a SapI restriction enzyme recognition site. In some embodiments, the ligation cleavage sequence comprises a BsmBI restriction enzyme recognition site. In some embodiments, the ligation cleavage sequence comprises a BsaI restriction enzyme recognition site. It will be appreciated by those skilled in the art that since the cleavage site of a type IIS restriction enzyme is typically located outside the enzyme recognition site (e.g., offset 1-5 nucleotides), the corresponding ligation cleavage sequence may also comprise additional nucleotides required for the corresponding restriction enzyme to produce discrete ends of the viral transcript (e.g., a poly-A tail at the 3' end).

[0178] In some embodiments, the ligand cleavage sequence is a sequence encoding a ligand-induced self-cleaving ribozyme, called an "aptazyme". Aptazymes are ribozyme sequences that contain an integral aptamer domain that is specific for a ligand. Binding of the ligand to the aptamer domain triggers activation of the enzymatic activity of the ribozyme, resulting in cleavage of the RNA transcript. Exemplary aptazymes include theophylline-dependent aptazymes (e.g., hammerhead ribozymes linked to a theophylline-dependent aptamer described in Auslander et al., Mol BioSyst. (2010) 6, 807-814), tetracycline-dependent aptazymes (e.g., hammerhead ribozymes linked to a Tet-dependent aptamer described by Zhong et al., eLife 2016;5:e18858 DOI:10.7554 / eLife.18858; Win and Smolke, PNAS (2007) 104;14283-14288; Whittmann and Suess, Mol Biosyt (2011) 7;2419-2427; Xiao et al., Chem & Biol (2008) 15;125-1137; and Beilstein et al., ACS Syn Biol (2015) 4; 526-534), guanine-dependent aptamers (e.g., hammerhead ribozymes linked to guanine-dependent aptamers, described by Nomura et al., Chem Commun., (2012) 48(57); 7215-7217). In some embodiments, the 5' and / or 3' junction cleavage sequence is an aptamer encoding sequence.

[0179] In some embodiments, the connection cleavage sequence is a target sequence of an RNAi molecule (e.g., an siRNA molecule, an shRNA molecule, an miRNA molecule, or an amiRNA molecule), a gRNA molecule, or an RNAseH primer. In such embodiments, the connection cleavage sequence is at least partially complementary to the sequence of an RNAi molecule, a gRNA molecule, or a primer molecule. Sequence alignment methods for comparing and determining percent sequence identity and percent complementarity are well known in the art. Optimal alignment of sequences for comparison can be accomplished, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology), by using algorithms known in the art (including BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0180] In some embodiments, the 5' junction cleavage sequence and the 3' junction cleavage sequence are from the same group (e.g., both are RNAi target sequences, both are ribozyme encoding sequences, etc.). For example, in some embodiments, the junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, shRNA, amiRNA, or miRNA target sequence) and is incorporated into the 5' and 3' ends of a polynucleotide encoding a viral genome (e.g., a recombinant RNA molecule). In such embodiments, the 5' and 3' RNAi target sequences can be the same (i.e., the target of the same siRNA, amiRNA, or miRNA) or different (i.e., the 5' sequence is the target of one siRNA, shmiRNA, or miRNA, and the 3' sequence is the target of another siRNA, amiRNA, or miRNA). In some embodiments, the junction cleavage sequence is a guide RNA target sequence and is incorporated into the 5' and 3' ends of a polynucleotide encoding a viral genome (e.g., a recombinant RNA molecule). In such embodiments, the 5' and 3' gRNA target sequences can be the same (i.e., targets of the same gRNA) or different (i.e., the 5' sequence is the target of one gRNA and the 3' sequence is the target of another gRNA). In some embodiments, the ligation cleavage sequence is a pri-mRNA coding sequence and is incorporated into the 5' and 3' ends of the polynucleotide encoding the viral genome (e.g., a recombinant RNA molecule). In some embodiments, the ligation cleavage sequence is a ribozyme coding sequence and is directly incorporated into the 5' and 3' ends of the polynucleotide encoding the viral genome (e.g., a recombinant RNA molecule).

[0181] In some embodiments, the 5' connection cleavage sequence and the 3' connection cleavage sequence are from the same group but are different variants or types. For example, in some embodiments, the 5' and 3' connection cleavage sequences can be target sequences for RNAi molecules, wherein the 5' connection cleavage sequence is a siRNA target sequence, and the 3' connection cleavage sequence is a miRNA target sequence (or vice versa). In some embodiments, the 5' and 3' connection cleavage sequences can be ribozyme coding sequences, wherein the 5' connection cleavage sequence is a hammerhead ribozyme coding sequence, and the 3' connection cleavage sequence is a hepatitis delta virus ribozyme coding sequence.

[0182] In some embodiments, the 5' connection cleavage sequence and the 3' connection cleavage sequence are different types. For example, in some embodiments, the 5' connection cleavage sequence is an RNAi target sequence (e.g., siRNA, amiRNA or miRNA target sequence), and the 3' connection cleavage sequence is a ribozyme sequence, an aptamer sequence, a pri-miRNA sequence or a gRNA target sequence. In some embodiments, the 5' connection cleavage sequence is a ribozyme sequence, and the 3' connection cleavage sequence is an RNAi target sequence (e.g., siRNA, amiRNA or miRNA target sequence), an aptamer sequence, a pri-miRNA encoding sequence or a gRNA target sequence. In some embodiments, the 5' connection cleavage sequence is an aptamer sequence, and the 3' connection cleavage sequence is an RNAi target sequence (e.g., siRNA, amiRNA or miRNA target sequence), a ribozyme sequence, a pri-miRNA sequence or a gRNA target sequence. In some embodiments, the 5' connection cleavage sequence is a pri-miRNA sequence, and the 3' connection cleavage sequence is an RNAi target sequence (e.g., siRNA, amiRNA or miRNA target sequence), a ribozyme sequence, an aptamer sequence or a gRNA target sequence. In some embodiments, the 5' linked cleavage sequence is a gRNA target sequence and the 3' linked cleavage sequence is an RNAi target sequence (eg, a siRNA, amiRNA, or miRNA target sequence), a ribozyme sequence, a pri-miRNA sequence, or an aptazyme sequence.

[0183] Tables 3 and 4 below show exemplary arrangements of ligation cleavage sequences relative to the polynucleotide encoding the synthetic viral genome.

[0184] Table 3: Symmetrical ligation cleavage sequence (JSC) alignment

[0185] 5’ <![CDATA[ JCS ]]> <![CDATA[ JCS ]]> 3’ siRNA TS Synthetic genome siRNA TS miR TS Synthetic genome miR TS AmiR TS Synthetic genome AmiR TS gRNA TS Synthetic genome gRNA TS pri-miR Synthetic genome pri-miR Ribozyme Synthetic genome Ribozyme Aptazyme Synthetic genome Aptazyme RNAseH Primer TS Synthetic genome RNAseH Primer TS

[0186] Table 4: Asymmetric JCS arrangement

[0187]

[0188]

[0189]

[0190] *“Restr Enz RS” refers to restriction enzyme recognition site

[0191] In some embodiments, the ligated cleavage sequence is a ribozyme encoding sequence and mediates self-cleavage of the synthetic RNA genome intermediate to produce the natural discrete 5' and / or 3' ends required for the final synthetic viral RNA genome and subsequent production of infectious RNA virus. Exemplary ribozymes include hammerhead ribozymes (e.g., Figure 5A), Varkud satellite (VS) ribozymes, hairpin ribozymes, GIR1 branched ribozymes, glmS ribozymes, twister ribozymes, twister sister ribozymes (e.g., twister sister 1 or twister sister 2), pistol ribozymes (e.g., Figure 6A-6B and Figure 7 In some embodiments, the 5' and / or 3' junction cleavage sequence is a ribozyme encoding sequence.

[0192] In some embodiments, the 5' junction cleavage sequence comprises or consists of a ribozyme sequence. In some embodiments, the 5' ribozyme sequence is selected from a hammerhead ribozyme sequence, a pistol ribozyme sequence, or a twisted sister ribozyme sequence.

[0193] In some embodiments, the 5' link cleavage sequence comprises or consists of a 5' pistol ribozyme sequence. In some embodiments, the 5' pistol ribozyme sequence is derived from P. polymyxa. In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-19 and 23-24, including all ranges and subranges therebetween. In some embodiments, the 5' pistol ribozyme sequence comprises Fig. 6Aand the P2 motif shown in Figure 6C, wherein the P2 motif is four nucleotides in length and is located in the region corresponding to nucleic acid positions 27-30 of SEQ ID NOs: 16-19 and 23-24. In some embodiments, the 5' pistol ribozyme sequence derived from Paenibacillus polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17, including all ranges and subranges therebetween, wherein the polynucleotide sequence of the P2 motif of the 5' pistol ribozyme sequence is "TTTA". In some embodiments, the 5' pistol ribozyme sequence derived from Paenibacillus polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, wherein the polynucleotide sequence of the P2 motif of the 5' pistol ribozyme sequence is "TTTT". In some embodiments, the 5' pistol ribozyme sequence derived from Paenibacillus polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, wherein the polynucleotide sequence of the P2 motif of the 5' pistol ribozyme sequence is "TTGT". In some embodiments, a 5' pistol ribozyme sequence is incorporated into a recombinant DNA molecule for in vitro transcription of a Coxsackievirus (eg, CVA21) RNA viral genome.

[0194] In some embodiments, the 5' link cleavage sequence comprises or consists of a 5' pistol ribozyme sequence. In some embodiments, the 5' pistol ribozyme sequence is derived from Paenibacillus polymyxa. In some embodiments, the 5' pistol ribozyme sequence derived from Paenibacillus polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64 or 65, including all ranges and subranges therebetween. In some embodiments, the 5' pistol ribozyme sequence comprises a P2 motif that is 4 nucleotides in length and is located in the region corresponding to nucleic acid positions 27-30 of SEQ ID NO: 64 or 65. In some embodiments, the 5' pistol ribozyme sequence derived from Paenibacillus polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, including all ranges and subranges therebetween, wherein the polynucleotide sequence of the P2 motif of the 5' pistol ribozyme sequence is "TCAA". In some embodiments, the 5' pistol ribozyme sequence derived from Paenibacillus polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, including all ranges and subranges therebetween, wherein the polynucleotide sequence of the P2 motif of the 5' pistol ribozyme sequence is "TTAA". In some embodiments, a 5' pistol ribozyme sequence is incorporated into a recombinant DNA molecule for in vitro transcription of an SVV (eg, SVV-IRES-2) RNA viral genome.

[0195] In some embodiments, the 5' linking cleavage sequence comprises or consists of an ENV27 ribozyme coding sequence. In some embodiments, the ENV27 ribozyme coding sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (including all ranges and subranges therebetween) sequence identity with any one of SEQ ID NOs: 130-134. In some embodiments, the ENV27 ribozyme coding sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (including all ranges and subranges therebetween) sequence identity with SEQ ID NO: 132.

[0196] In some embodiments, the ENV27 ribozyme coding sequence contains modification (e.g., insertion) at the P3 stem insertion region, and the P3 stem insertion region corresponds to nucleotides 49-54 of SEQ ID NO:132. Without wishing to be bound by any particular theory, we assume that extending the P3 stem insertion region may promote the folding and / or cutting efficiency of the ENV27 ribozyme. In some embodiments, the ENV27 ribozyme coding sequence comprises a length of about 1-30, about 1-25, about 1-20, about 1-15, about 1-10, about 5-30, about 5-25, about 5-20, about 5-15, about 5-10, about 6-30, about 6-25, about 6-20, about 6-15 or about 6-10 polynucleotides of the P3 stem inset. In some embodiments, the ENV27 ribozyme coding sequence comprises a P3 stem insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 polynucleotides in length. In some embodiments, the P3 stem insertion comprises or consists of a polynucleotide "AGATCT" corresponding to the region of nucleotides 49-54 of SEQ ID NO: 132. In some embodiments, the P3 stem insertion comprises or consists of a polynucleotide "AGAGAAATCT" (SEQ ID NO: 137) corresponding to the region of nucleotides 49-54 of SEQ ID NO: 132. In some embodiments, the P3 stem insertion comprises or consists of a polynucleotide "AGAACGAGAAATCGTTCT" (SEQ ID NO: 138) corresponding to the region of nucleotides 49-54 of SEQ ID NO: 132.

[0197] In some embodiments, the ENV27 ribozyme coding sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all ranges and subranges therebetween) sequence identity to SEQ ID NO: 132 (excluding its P3 stem insertion corresponding to nucleotides 49-54 of SEQ ID NO: 132) (excluding the P3 stem insertion region). In some embodiments, the ENV27 ribozyme coding sequence comprises or consists of a sequence having 100% identity to SEQ ID NO: 132 (excluding its P3 stem insertion corresponding to nucleotides 49-54 of SEQ ID NO: 132) (excluding the P3 stem insertion region). In some embodiments, the ENV27 ribozyme coding sequence comprises or consists of a sequence (excluding the P3 stem inset region) having at most 1, at most 2, at most 3, at most 5, at most 5, at most 6, at most 7, at most 8, at most 8, at most 10 or at most 11 mutations (insertions, deletions or substitutions) compared to SEQ ID NO: 132 (excluding the P3 stem inset region corresponding to nucleotides 49-54 of SEQ ID NO: 132). In some embodiments, such mutations are substitutions.

[0198] In some embodiments, the ENV27 ribozyme coding sequence comprises the polynucleotide "TTTATT" at positions corresponding to nucleotides 25-30 of SEQ ID NO:132.

[0199] In some embodiments, the ENV27 ribozyme coding sequence comprises the polynucleotide "TTTGTT" at positions corresponding to nucleotides 25-30 of SEQ ID NO:132.

[0200] In some embodiments, the ENV27 sequence is incorporated into a recombinant DNA molecule for in vitro transcription of a Coxsackievirus (eg, CVA21) RNA viral genome.

[0201] In some embodiments, the disclosure provides a plurality of recombinant RNA molecules transcribed by the recombinant DNA molecules of the disclosure. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of the recombinant RNA molecules include the natural 5' and 3' ends of the viral genome encoded by the recombinant RNA molecules. In some embodiments, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.5% or no more than 0.1% of the recombinant RNA molecules include the RNA sequence encoded by the ENV27 ribozyme encoding sequence. In some embodiments, at least one recombinant RNA molecule includes the RNA sequence encoded by the ENV27 ribozyme encoding sequence. In some embodiments, at least 0.0001%, at least 0.001%, at least 0.01%, at least 0.1%, or at least 1% of the recombinant RNA molecules comprise the ENV27 ribozyme (encoded by the ENV27 ribozyme encoding sequence).

[0202] In some embodiments, the 5' linker cleavage sequence comprises or consists of the Env25 pistol ribozyme. In some embodiments, the DNA sequence encoding the Env25 pistol ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 96 (including all ranges and subranges therebetween). In some embodiments, the Env25 pistol ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 100 (including all ranges and subranges therebetween).

[0203] In some embodiments, the 5' link cleavage sequence comprises or consists of a pistol ribozyme of Bacillus rotundus. In some embodiments, the DNA sequence encoding the pistol ribozyme of Bacillus rotundus comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 97 (including all ranges and subranges therebetween). In some embodiments, the pistol ribozyme RNA sequence of Bacillus rotundus comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101 (including all ranges and subranges therebetween).

[0204] In some embodiments, the 5' link cleavage sequence comprises or consists of a torsional sister 1 ribozyme. In some embodiments, the DNA sequence encoding the torsional sister 1 ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 98 (including all ranges and subranges therebetween). In some embodiments, the torsional sister 1 ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 102 (including all ranges and subranges therebetween).

[0205] In some embodiments, the 5' link cleavage sequence comprises or consists of a torsional sister 2 ribozyme. In some embodiments, the DNA sequence encoding the torsional sister 2 ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 99 (including all ranges and subranges therebetween). In some embodiments, the torsional sister 2 ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103 (including all ranges and subranges therebetween).

[0206] Preamble sequence

[0207] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises a leader sequence between the promoter sequence and the 5' junction cleavage sequence. In some embodiments, the presence of the leader sequence promotes or ensures the correct folding of the downstream 5' junction cleavage sequence (e.g., 5' ribozyme sequence).

[0208] In some embodiments, the leader sequence length is about 5bp, about 10bp, about 15bp, about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about 95bp or about 100bp, including all ranges and subranges therebetween. In some embodiments, the leader sequence length is at least 5bp, at least 10bp, at least 15bp, at least 20bp, at least 25bp, at least 30bp, at least 35bp, at least 40bp, at least 45bp, at least 50bp, at least 55bp, at least 60bp, at least 65bp, at least 70bp, at least 75bp, at least 80bp, at least 85bp, at least 90bp, at least 95bp or at least 100bp, including all ranges and subranges therebetween. In some embodiments, the leader sequence length is less than 5bp, less than 10bp, less than 15bp, less than 20bp, less than 25bp, less than 30bp, less than 35bp, less than 40bp, less than 45bp, less than 50bp, less than 55bp, less than 60bp, less than 65bp, less than 70bp, less than 75bp, less than 80bp, less than 85bp, less than 90bp, less than 95bp or less than 100bp, including all ranges and subranges therebetween. In some embodiments, the leader sequence length is about 50bp-70bp, about 40bp-60bp, about 60bp-80bp, about 40bp-80bp, about 30bp-70bp, about 50bp-90bp, about 30bp-90bp, about 20bp-60bp or about 60bp-100bp, including all ranges and subranges therebetween. In some embodiments, the leader sequence length is about 57bp or about 55bp-60bp.

[0209] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all ranges and subranges therebetween) according to any one of SEQ ID NOs: 13-15. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all ranges and subranges therebetween) according to any one of SEQ ID NOs: 13-15. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all ranges and subranges therebetween) according to SEQ ID NO: 15. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 15. In some embodiments, the leader sequence is followed or immediately followed by a 5' pistol ribozyme sequence (e.g., a pistol ribozyme from Paenibacillus polymyxa or a variant thereof). In some embodiments, the leader sequence is incorporated into a recombinant DNA molecule (e.g., a DNA template) for in vitro transcription of the CVA21 RNA viral genome.

[0210] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, including all ranges and subranges therebetween, according to any one of SEQ ID NOs: 135-136. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, including all ranges and subranges therebetween, according to SEQ ID NOs: 135. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 135. In some embodiments, the leader sequence is followed or immediately followed by an ENV27 ribozyme sequence (e.g., any one of SEQ ID NO: 130-134 or a variant thereof). In some embodiments, the leader sequence is incorporated into a recombinant DNA molecule (e.g., a DNA template) for in vitro transcription of the CVA21 RNA viral genome.

[0211] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity according to any one of SEQ ID NOs: 53-63, including all ranges and subranges therebetween. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity according to any one of SEQ ID NOs: 53-60 and 62-63, including all ranges and subranges therebetween. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to any one of SEQ ID NOs: 53-60 and 62-63.

[0212] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all ranges and subranges therebetween) sequence identity to SEQ ID NO: 53. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 53.

[0213] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all ranges and subranges therebetween) sequence identity according to SEQ ID NO: 58. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 58.

[0214] In some embodiments, the leader sequence is followed or immediately followed by a 5' pistol ribozyme sequence (e.g., a pistol ribozyme or variant thereof according to SEQ ID NO: 64 or 65). In some embodiments, the leader sequence is incorporated into a recombinant DNA molecule (e.g., a DNA template) for in vitro transcription of an SVV RNA viral genome.

[0215] Poly-A tail

[0216] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises a sequence encoding a polyA tail. In some embodiments, the poly-A tail is connected to the 3' end of the synthetic RNA viral genome. In some embodiments, the poly-A tail length is 2bp-500bp (i.e., 2-500pA). In some embodiments, the poly-A tail length is 2bp-100bp, 2bp-150bp, 2bp-200bp, 2bp-250bp, 2bp-300bp, 2bp-400bp or 2bp-500bp, including all ranges and subranges therebetween. In some embodiments, the poly-A tail is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 bp in length, including all ranges and subranges therebetween. In some embodiments, the poly-A tail length is about 10bp-30bp, 20bp-40bp, 30bp-50bp, 40bp-60bp, 50bp-70bp, 60bp-80bp, 70bp-90bp, 80bp-100bp, 90bp-110bp, 100bp-120bp, 110bp-130bp, 120bp-140bp, 130bp-150bp, 140bp-160bp, 150bp-170bp, 160bp-180bp, 170bp-190bp, or 180bp-200bp, including all ranges and sub-ranges therebetween. In some embodiments, the poly-A tail length is about 65bp-75bp, 60bp-80bp, 55bp-85bp, 50bp-90bp, 45bp-95bp or 40bp-100bp, including all ranges and subranges therebetween. In some embodiments, the poly-A tail length is about 70bp. In some embodiments, a longer poly-A tail (e.g., a length of about 70bp) improves the loading capacity of the synthetic RNA viral genome on the Oligo-dT chromatogram compared to a corresponding synthetic RNA viral genome with a shorter poly-A tail (e.g., a length of about 30bp). In some embodiments, the loading capacity is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, or at least 10-fold compared to a synthetic RNA viral genome having a poly-A tail of about 30p in length.

[0217] Non-limiting examples of recombinant DNA molecule design

[0218] In some embodiments, the synthetic RNA viral genomes described herein are produced in vitro by in vitro RNA transcription (see, e.g., Figure 8 , Fig. 9A , Fig. 9B and Fig. 10A The synthetic RNA viral genome is then purified and formulated for therapeutic use (e.g., encapsulated into lipid nanoparticles).

[0219] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a ribozyme sequence; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a ribozyme sequence. In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' hammerhead ribozyme sequence (e.g., a wild-type HHR or a modified HHR, such as Figure 5A and Figure 5B (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' hepatitis delta virus ribozyme sequence.

[0220] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a ribozyme sequence; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising a restriction enzyme recognition site. In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' hammerhead ribozyme sequence (e.g., a wild-type HHR or a modified HHR, such as Figure 5A and Figure 5B (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0221] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a ribozyme sequence; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a restriction enzyme recognition site. In some embodiments, the DNA template comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' hammerhead ribozyme sequence (e.g., a wild-type HHR or a modified HHR, such as Figure 5A and Figure 5B ); (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0222] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0223] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of an ENVY27 ribozyme sequence; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0224] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a 5' RNAseH primer binding site; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising a restriction enzyme recognition site. In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises a polynucleotide comprising from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a 5' RNAseH primer binding site; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0225] In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a 5' RNAseH primer binding site; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising a restriction enzyme recognition site. In some embodiments, the recombinant DNA molecule (e.g., DNA template) comprises a polynucleotide comprising from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a 5' junction cleavage sequence comprising or consisting of a 5' RNAseH primer binding site; (iii) a polynucleotide encoding a synthetic RNA viral genome; and (iv) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0226] In some embodiments, the synthetic RNA virus genome is a Coxsackievirus (CVA) genome. In some embodiments, the Coxsackievirus is a CVA21 strain. In some embodiments, the CVA21 strain is an EF strain. In some embodiments, the CVA21 strain is a KY strain.

[0227] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) an optional leader sequence; (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a synthetic RNA viral genome; (v) a poly-A tail (e.g., about 20 bp-80 bp in length or about 30 bp-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a restriction enzyme recognition site (e.g., BsmBI or BsaI restriction enzymes).

[0228] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a leader sequence (e.g., SEQ ID NO: 135 or 136); (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail (e.g., about 20 bp-80 bp in length or about 30 bp-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsmBI restriction enzyme recognition site.

[0229] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a leader sequence (e.g., SEQ ID NO: 135 or 136); (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail (e.g., about 20 bp-80 bp in length or about 30 bp-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0230] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a leader sequence according to SEQ ID NO: 135; (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail, and (vi) a 3' junction cleavage sequence comprising or consisting of a BsmBI restriction enzyme recognition site, wherein the combination of the 5' ENV27 ribozyme sequence and the poly-A tail is selected from one of the embodiments E1-E68 provided in Table 5 below.

[0231] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a leader sequence according to SEQ ID NO: 135; (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail, and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site, wherein the combination of the 5' ENV27 ribozyme sequence and the poly-A tail is selected from one of the embodiments E1-E68 provided in Table 5 below.

[0232] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a leader sequence according to SEQ ID NO: 136; (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail, and (vi) a 3' junction cleavage sequence comprising or consisting of a BsmBI restriction enzyme recognition site, wherein the combination of the 5' ENV27 ribozyme sequence and the poly-A tail is selected from one of the embodiments E1-E68 provided in Table 5 below.

[0233] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a promoter sequence (e.g., a T7 polymerase promoter); (ii) a leader sequence according to SEQ ID NO: 136; (iii) a 5' junction cleavage sequence comprising or consisting of an ENV27 ribozyme sequence; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail, and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site, wherein the combination of the 5' ENV27 ribozyme sequence and the poly-A tail is selected from one of the embodiments E1-E68 provided in Table 5 below.

[0234] Table 5: Non-limiting embodiments of leader sequence, 5' ribozyme sequence, and Poly-A tail in DNA template for expressing CVA21 viral genome

[0235]

[0236]

[0237] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a T7 polymerase promoter sequence; (ii) a leader sequence according to SEQ ID NO: 135; (iii) a 5' junction cleavage sequence comprising or consisting of a 5' ENV27 ribozyme sequence according to SEQ ID NO: 132; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail (e.g., a poly-A tail length of about 70 bp, about 60 bp-80 bp, or about 50 bp-90 bp), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsmBI restriction enzyme recognition site.

[0238] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises from 5' to 3': (i) a T7 polymerase promoter sequence; (ii) a leader sequence according to SEQ ID NO: 135; (iii) a 5' junction cleavage sequence comprising or consisting of a 5' ENV27 ribozyme sequence according to SEQ ID NO: 132; (iv) a polynucleotide encoding a CVA21 synthetic RNA virus genome; (v) a poly-A tail (e.g., a poly-A tail length of about 70 bp, about 60 bp-80 bp, or about 50 bp-90 bp), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0239] Table 18 below provides exemplary embodiments of DNA templates encoding the CVA viral genome.

[0240] Table 18: Exemplary DNA template structures

[0241]

[0242] Particles containing synthetic RNA genomes

[0243] In some embodiments, the synthetic RNA genome described herein is encapsulated in a "particle". As used herein, a particle refers to a composition of matter of non-tissue origin, such as a liposome, a liposome complex, a nanoparticle, a nanocapsule, a microparticle, a microsphere (microspheres), a lipid particle, an exosome, a vesicle, etc. In certain embodiments, the particle is non-protein and non-immunogenic. In such embodiments, the encapsulation of the synthetic RNA genome described herein allows the delivery of the viral genome without inducing a systemic antiviral immune response, and mitigates the effects of neutralizing antiviral antibodies. In addition, the encapsulation of the synthetic RNA genome described herein can prevent genome degradation and facilitate its introduction into a target host cell. In some embodiments, the present disclosure provides nanoparticles comprising the synthetic RNA genome described herein. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle further comprises a second RNA molecule encoding a payload molecule.

[0244] In some embodiments, the particle is biodegradable in the subject. In such embodiments, multiple doses of particles can be applied to the subject without accumulating particles in the subject. The example of suitable particles includes: polystyrene particles, polylactic acid-co-glycolic acid PLGA particles, cationic polymer particles based on polypeptides, cyclodextrin particles, chitosan, N, N, N-trimethyl chitosan particles, particles based on lipids, poly (β-amino ester) particles, low molecular weight polyethylene imine particles, polyphosphate particles, disulfide cross-linked polymer particles, polyamidoamine particles, polyethylene imine (PEI) particles and polypropylene sulfide particles stabilized by PLURIONICS.

[0245] In some embodiments, the polynucleotides described herein are encapsulated in inorganic particles. In some embodiments, the inorganic particles are gold nanoparticles (GNPs), gold nanorods (GNRs), magnetic nanoparticles (MNPs), magnetic nanotubes (MNTs), carbon nanohorns (CNHs), carbon fullerenes, carbon nanotubes (CNTs), calcium phosphate nanoparticles (CPNPs), mesoporous silica nanoparticles (MSNs), silica nanotubes (SNTs), or star-shaped hollow silica nanoparticles (SHNPs).

[0246] Preferably, the size of the particles described herein is nanoscopic to improve lysis, avoid phagocytic clearance caused by in vivo aggregation and possible complications and promote pinocytosis. In some embodiments, the average diameter of the particles is about less than about 1000nm. In some embodiments, the average diameter of the particles is less than about 500nm. In some embodiments, the average diameter of the particles is between about 30nm and about 100nm, between about 50nm and about 100nm, or between about 75nm and about 100nm. In some embodiments, the average diameter of the particles is between about 30nm and about 75nm or between about 30nm and about 50nm. In some embodiments, the average diameter of the particles is between about 100nm and about 500nm. In some embodiments, the average diameter of the particles is between about 200nm and about 400nm. In some embodiments, the average size of the particles is about 350nm.

[0247] exosomes

[0248] In some embodiments, the synthetic RNA genome described herein is encapsulated in exosomes. Exosomes are small membrane vesicles of endocytic origin, which are released into the extracellular environment after the plasma membrane fusion of multivesicular bodies and parental cells (e.g., cells releasing exosomes, also referred to herein as donor cells). The surface of the exosomes comprises a lipid bilayer of a cell membrane derived from a parental cell, and may further comprise membrane proteins expressed on the surface of the parental cell. In some embodiments, the exosomes may also contain cytosol from parental cells. Exosomes are produced by many different types of cells, including epithelial cells, B lymphocytes and T lymphocytes, mast cells (MCs) and dendritic cells (DCs), and have been identified in plasma, urine, bronchoalveolar lavage fluid, intestinal epithelial cells and tumor tissues. Since the composition of exosomes depends on the parental cell type from which they are derived, there is no "exosome-specific" protein. However, many exosomes contain proteins associated with the intracellular vesicles from which the exosomes originate in the parent cell (e.g., proteins associated with and / or expressed by endosomes and lysosomes). For example, exosomes can be enriched for antigen presenting molecules such as major histocompatibility complexes I and II (MHC-I and MHC-II), tetraspanins (e.g., CD63), several heat shock proteins, cytoskeletal components (such as actin and tubulin), proteins involved in intracellular membrane fusion, cell-cell interactions (e.g., CD54), signal transduction proteins, and cytoplasmic enzymes.

[0249] Exosomes can mediate the transfer of cellular proteins from one cell (e.g., a parent cell) to a target cell or a recipient cell by fusion of the exosome membrane with the plasma membrane of the target cell. Therefore, the material encapsulated by the modified exosome provides a mechanism by which an exogenous agent (such as a polynucleotide described herein) can be introduced into a target cell. Exosomes that have been modified to contain one or more exogenous agents (e.g., polynucleotides described herein) are referred to herein as "modified exosomes". In some embodiments, modified exosomes are produced by introducing an exogenous agent (e.g., a polynucleotide described herein) into a parent cell. In such embodiments, an exogenous nucleic acid is introduced into a parent, a cell producing an exosome, so that the exogenous nucleic acid itself or a transcript of the exogenous nucleic acid is incorporated into the modified exosome produced by the parent cell. Exogenous nucleic acids can be introduced into parent cells by means known in the art, such as transduction, transfection, transformation, electroporation and / or microinjection of exogenous nucleic acids.

[0250] In some embodiments, modified exosomes are produced by directly introducing the synthetic RNA genome described herein into exosomes. In some embodiments, the synthetic RNA genome described herein is introduced into complete exosomes. "Complete exosomes" refer to exosomes containing proteins and / or genetic material derived from parental cells, and the proteins and / or genetic material are produced from parental cells. Methods for obtaining complete exosomes are known in the art (see, for example, Alvarez-Erviti L. et al., Nat Biotechnol. April 2011; 29(4): 34-5; Ohno S, et al., Mol Ther January 2013; 21(l): 185-91; and European Patent Publication No. 2010663).

[0251] In a specific embodiment, the synthetic RNA genome is introduced into an empty exosome. "Empty exosome" refers to an exosome lacking proteins and / or genetic material (e.g., DNA or RNA) derived from a parental cell. Methods for producing empty exosomes (e.g., lacking genetic material from a parental cell source) are known in the art, including UV exposure, mutations / deletions of endogenous proteins that mediate nucleic acid loading into exosomes, and electroporation and chemical treatment to open holes in the exosome membrane so that endogenous genetic material comes out of the exosome through the opened holes. In some embodiments, the exosome is opened by treating with an aqueous solution having a pH of about 9 to about 14 to obtain an exosome membrane, removing components in the vesicle (e.g., proteins and / or nucleic acids in the vesicle), and reassembling the exosome membrane to form an empty exosome, thereby producing an empty exosome. In some embodiments, components in the vesicle (e.g., proteins and / or nucleic acids in the vesicle) are removed by ultracentrifugation or density gradient ultracentrifugation. In some embodiments, the membrane is reassembled by sonication, mechanical vibration, squeezing through a porous membrane, electric current, or a combination of one or more of these techniques. In specific embodiments, the membrane is reassembled by sonication.

[0252] In some embodiments, the synthetic RNA genomes described herein can be loaded into intact or empty exosomes using conventional molecular biology techniques (such as in vitro transformation, transfection, and / or microinjection) to produce modified exosomes. In some embodiments, the exogenous agent (e.g., a polynucleotide described herein) is directly introduced into intact or empty exosomes by electroporation. In some embodiments, the exogenous agent (e.g., a polynucleotide described herein) is directly introduced into intact or empty exosomes by lipofection (e.g., transfection). Lipofection kits suitable for producing exosomes according to the present disclosure are known in the art and are commercially available (e.g., from Roche). HD transfection reagent and LIPOFECTAMINE from Invitrogen TM2000). In some embodiments, an exogenous agent (e.g., a polynucleotide described herein) is directly introduced into intact or empty exosomes by using heat shock transformation. In such embodiments, exosomes isolated from parent cells are exposed to divalent cations such as Ca 2+ (in CaCl2) is cooled to make the exosome membrane permeable. The exosomes can then be incubated with exogenous nucleic acids and subjected to a brief heat shock (e.g., incubated at 42°C for 30-120 seconds). In a particular embodiment, the exogenous agent (e.g., a polynucleotide described herein) can be loaded into empty exosomes by mixing or co-incubating the agent with the exosome membrane after removing the intravesicular components. Thus, the modified exosomes reassembled by the exosome membrane incorporate the exogenous agent into the intravesicular space. Other methods for producing exosome-encapsulated nucleic acids are known in the art (see, e.g., U.S. Patent Nos. 9,889,210; 9,629,929, and 9,085,778; International PCT Publication Nos. WO 2017 / 161010 and WO 2018 / 039119).

[0253] Exosomes can be obtained from many different parent cells, including cell lines, bone marrow-derived cells, and cells derived from primary patient samples. Exosomes released from parent cells can be separated from the supernatant of parent cell cultures by methods known in the art. For example, the physical properties of exosomes can be used to separate them from culture media or other source materials, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieves, etc.), density (e.g., conventional or gradient centrifugation) and Svedberg constant (e.g., with or without external force, etc.). Alternatively or in addition, separation can be based on one or more biological properties, and includes methods that can use surface markers (e.g., for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non-specific ligand binding, etc.). Analysis of exosome surface proteins can be determined by flow cytometry using fluorescently labeled antibodies to exosome-related proteins (such as CD63). Other markers for characterizing exosomes are described in International PCT Publication No. WO 2017 / 161010. In further contemplated methods, the exosomes may also be fused using chemical and / or physical methods, including PEG-induced fusion and / or ultrasound fusion.

[0254] In some embodiments, size exclusion chromatography can be used to separate exosomes. In some embodiments, exosomes can be further separated after chromatographic separation by centrifugation techniques (one or more chromatographic fractions), as is well known in the art. In some embodiments, the separation of exosomes can involve a combination of multiple methods, including but not limited to differential centrifugation described previously (see Raposo, G. et al., J. Exp. Med. 183, 1161-1172 (1996)), ultracentrifugation, size-based membrane filtration, concentration and / or rate zonal centrifugation.

[0255] In some embodiments, the exosome membrane comprises one or more of phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. In addition, the membrane may comprise one or more polypeptides and one or more polysaccharides, such as glycans. Exemplary exosome membrane compositions and methods for modifying the relative amounts of one or more membrane components are described in International PCT Publication No. WO 2018 / 039119.

[0256] In some embodiments, the particle is an exosome and has a diameter between about 30 nm and about 100 nm, between about 30 nm and about 200 nm, or between about 30 nm and about 500 nm. In some embodiments, the particle is an exosome and has a diameter between about 10 nm and about 100 nm, between about 20 nm and about 100 nm, between about 30 nm and about 100 nm, between about 40 nm and about 100 nm, between about 50 nm and about 100 nm, between about 60 nm and about 100 nm, between about 70 nm and about 100 nm, between about 80 nm and about 100 nm, between about 90 nm and about 100 nm, between about 100 nm and about 200 nm, between about 100 nm and about 150 nm, between about 150 nm and about 200 nm, between about 100 nm and about 250 nm, between about 250 nm and about 500 nm, or between about 10 nm and about 1000 nm. In some embodiments, the particle is an exosome and has a diameter between about 20 nm and 300 nm, between about 40 nm and 200 nm, between about 20 nm and 250 nm, between about 30 nm and 150 nm, or between about 30 nm and 100 nm.

[0257] Compound

[0258] Compounds of formula (I)

[0259] In various embodiments, provided herein are compounds of Formula (I):

[0260]

[0261] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0262] A is –N(CH2R N1 )(CH2R N2 ) or a 4-7 membered heterocyclyl ring containing at least one N, wherein the 4-7 membered heterocyclyl ring is optionally substituted by 0-6 R 3 replace;

[0263] Each X is independently –O–, –N(R 1 )–or–N(R 2 )–;

[0264] R 1 Selected from optionally substituted C1-C 31 a group consisting of aliphatic and steroidal groups;

[0265] R 2 Selected from optionally substituted C1-C 31 a group consisting of aliphatic and steroidal groups;

[0266] R 3 is an optionally substituted C1-C6 aliphatic;

[0267] R N1 and R N2 Each is independently hydrogen, hydroxy-C1-C6 alkyl, C2-C6 alkenyl or C3-C7 cycloalkyl;

[0268] L 1 Selected from optionally substituted C1-C 20 An alkylene chain and a divalent optionally substituted C2-C 20 A group consisting of an alkenylene chain;

[0269] L 2 Selected from optionally substituted C1-C 20 An alkylene chain and a divalent optionally substituted C2-C 20 A group consisting of an alkenylene chain;

[0270] L 3 is a bond, an optionally substituted C1-C6 alkylene chain, or a divalent optionally substituted C3-C7 cycloalkylene group.

[0271] In some embodiments, when A is -N(CH3)(CH3) and X is O, L 3 Not a C1-C6 alkylene chain.

[0272] In some embodiments, the present disclosure includes compounds of Formula (Ia):

[0273]

[0274] or a pharmaceutically acceptable salt or solvate thereof, wherein m is 0, 1, 2, 3, 4, 5 or 6.

[0275] In some embodiments, the present disclosure includes compounds of Formula (Ib):

[0276]

[0277] or a pharmaceutically acceptable salt or solvate thereof, wherein n is 0, 1, 2 or 3; and m is 0, 1, 2, 3, 4, 5 or 6.

[0278] In some embodiments, the present disclosure includes compounds of formula (I-bi):

[0279]

[0280] or a pharmaceutically acceptable salt or solvate thereof.

[0281] In some embodiments, the present disclosure includes compounds of formula (I-bii):

[0282]

[0283] or a pharmaceutically acceptable salt or solvate thereof, wherein m is 0, 1, 2 or 3; and p and q are each 0, 1, 2 or 3, and wherein q+p is less than or equal to 3.

[0284] In some embodiments, the present disclosure includes compounds of formula (I-biii):

[0285]

[0286] or a pharmaceutically acceptable salt or solvate thereof.

[0287] In some embodiments, the present disclosure includes compounds of Formula (Ic):

[0288]

[0289] or a pharmaceutically acceptable salt or solvate thereof.

[0290] In some embodiments, A is -N(CH2R N1 )(CH2R N2 ) or an optionally substituted 4-7 membered heterocyclyl ring containing at least one N.

[0291] In some embodiments, A is -N(CH2R N1 )(CH2R N2 In some embodiments, R N1 and R N2each independently selected from hydrogen, hydroxy-C1-C3 alkylene, C2-C4 alkenyl or C3-C4 cycloalkyl).

[0292] In some embodiments, R N1 and R N2 Each is independently selected from hydrogen, -CH2CH=CH2, -CH2CH2OH, In some embodiments, R N1 and R N2 In some embodiments, R N1 and R N2 In some embodiments, R N1 and R N2 Each is a C2-C4 alkenyl group, for example, -CH2CH=CH2. In some embodiments, R N1 and R N2 Each is hydroxy-C1-C3 alkylene, for example, -CH2CH2OH. In some embodiments, R N1 and R N2 In some embodiments, R N1 and R N2 One of is hydrogen, and the other is C3-C4 cycloalkyl. In some embodiments, R N1 and R N2 One of them is hydrogen and the other is

[0293] In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N. In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing exactly one N. In some embodiments, A is an unsubstituted 4-7 membered heterocyclyl ring containing at least one N. In some embodiments, A is an unsubstituted 4-7 membered heterocyclyl ring containing exactly one N. In some embodiments, A is an optionally substituted 5-6 membered heterocyclyl ring containing at least one N. In some embodiments, A is an unsubstituted 5-6 membered heterocyclyl ring containing at least one N.

[0294] In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N, and the N atom of A is a tertiary amine.

[0295] In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N, further containing one or more S. In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N, further containing exactly one S.

[0296] In some embodiments, A is selected from the group consisting of azetidine, pyrrolidine, piperidine, azepane, and thiomorpholine. In some embodiments, A is selected from the group consisting of pyrrolidine and piperidine.

[0297] In some embodiments, L 1 Selected from optionally substituted C1-C 20 An alkylene chain and a divalent optionally substituted C1-C 20 In some embodiments, L 2 Selected from optionally substituted C1-C 20 The alkylene chain and the divalent optionally substituted C1-C 20 In some embodiments, L 1 is an optionally substituted C1-C 20 In some embodiments, L 2 is an optionally substituted C1-C 20 Alkylene chain.

[0298] In some embodiments, L 1 and L 2 In some embodiments, L 1 and L 2 different.

[0299] In some embodiments, L 1 is an optionally substituted C1-C 10 In some embodiments, L 2 is an optionally substituted C1-C 10 In some embodiments, L 1 is an optionally substituted C1-C5 alkylene chain. 2 is an optionally substituted C1-C5 alkylene chain.

[0300] In some embodiments, L 1 and L 2 Each is -CH2CH2CH2CH2-. In some embodiments, L 1 and L 2 Each is -CH2CH2CH2-. In some embodiments, L 1 and L 2 Each is -CH2CH2-.

[0301] In some embodiments, L 3 is a bond, an optionally substituted C1-C6 alkylene chain, or a divalent optionally substituted C3-C6 cycloalkylene. 3 is a bond. In some embodiments, L 3is an optionally substituted C1-C6 alkylene chain. 3 is an optionally substituted C1-C3 alkylene chain. 3 is an unsubstituted C1-C3 alkylene chain. 3 is -CH2-. In some embodiments, L 3 is -CH2CH2-. In some embodiments, L 3 is -CH2CH2CH2-. In some embodiments, L 3 is a divalent C3-C6 cycloalkylene group. 3 yes

[0302] In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 2-10. In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 2-8. In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 2-5. In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 2-4. In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 2. In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 3. In some embodiments, the number of carbon atoms between S and N in A in the thiolate of formula (I) is 4.

[0303] In some embodiments, R 1 Selected from optionally substituted C1-C 31 In some embodiments, R 2 Selected from optionally substituted C1-C 31 In some embodiments, R 1 is an optionally substituted C1-C 31 In some embodiments, R 2 is an optionally substituted C1-C 31 In some embodiments, R 1 is an optionally substituted C5-C 25 In some embodiments, R 2 is an optionally substituted C5-C 25 In some embodiments, R 1 is an optionally substituted C 10 -C 20 In some embodiments, R 2 is an optionally substituted C10 -C 20 In some embodiments, R 1 is an optionally substituted C 10 -C 20 In some embodiments, R 2 is an optionally substituted C 10 -C 20 In some embodiments, R 1 is unsubstituted C 10 -C 20 In some embodiments, R 2 is unsubstituted C 10 -C 20 alkyl.

[0304] In some embodiments, R 1 is an optionally substituted C 14 -C 16 In some embodiments, R 2 is an optionally substituted C 14 -C 16 In some embodiments, R 1 is unsubstituted C 14 -C 16 In some embodiments, R 2 is unsubstituted C 14 -C 16 alkyl.

[0305] In some embodiments, R 1 is an optionally substituted branched C3-C 31 In some embodiments, R 2 is an optionally substituted branched C3-C 31 In some embodiments, R 1 is an optionally substituted branched C 10 -C 20 In some embodiments, R 2 is an optionally substituted branched C 10 -C 20 In some embodiments, R 1 is an optionally substituted branched C 14 -C 16 In some embodiments, R 2 is an optionally substituted branched C 14 -C 16 In some embodiments, R 1 It is a substituted branched C3-C 31 In some embodiments, R 2 It is a substituted branched C3-C31 In some embodiments, R 1 is a substituted branched C 10 -C 20 In some embodiments, R 2 is a substituted branched C 10 -C 20 In some embodiments, R 1 is a substituted branched C 14 -C 16 In some embodiments, R 2 is a substituted branched C 14 -C 16 alkyl.

[0306] In some embodiments, R 1 and R 2 same.

[0307] In some embodiments, R 1 and R 2 In some embodiments, R 1 is an optionally substituted C6-C 20 Alkenyl and R 2 is an optionally substituted C 10 -C 20 In some embodiments, R 1 It is C6-C 20 Alkenyl and R 2 It is a branched chain C 10 -C 20 alkyl.

[0308] In some embodiments, A is a 4-7 membered heterocyclyl ring containing at least one N and optionally substituted by 0-6 R 3 In some embodiments, R 3 is an optionally substituted C1-C6 aliphatic. 3 is an optionally substituted C1-C3 aliphatic. 3 is an optionally substituted C1-C6 alkyl. 3 is an optionally substituted C1-C3 alkyl. 3 is an unsubstituted C1-C6 alkyl. 3 is an unsubstituted C1-C3 alkyl. 3 is an optionally substituted C1-C6 alkenyl. 3 is an optionally substituted C1-C3 alkenyl. 3is an unsubstituted C1-C6 alkenyl. 3 It is an unsubstituted C1-C3 alkenyl group.

[0309] In some embodiments, R 3 is substituted with 1-3 C3-C6 cycloalkyl groups. 3 is substituted with 1 C3-C6 cycloalkyl. 3 In some embodiments, R 3 is substituted with 1-3 -OH. 3 Replaced by 1 –OH.

[0310] In some embodiments, m is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0311] In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 1 or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0312] In some embodiments, the compound of Formula (I) is a compound selected from Table 21, or a pharmaceutically acceptable salt or solvate thereof.

[0313] Table 21

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320] Compound of formula (A)

[0321] In various embodiments, provided herein are compounds of formula (A):

[0322]

[0323] or a pharmaceutically acceptable salt thereof, wherein:

[0324] n is an integer between 10 and 200, inclusive;

[0325] L P1 Yes – [(CH2) 0-3 –C(O)O] 1-3 –, –(CH2) 0-3 –C(O)O-(CH2) 1-3 –OC(O)– or –C(O)N(H)–;

[0326] R P1 It is C5-C 25 Alkyl or C5-C 25 alkenyl; and

[0327] R P2 It is hydrogen or –CH3.

[0328] In some embodiments, Formula (A) is not HO-(CH2CH2O) n -C(O)N(H)-(CH2) 17 CH3.

[0329] In some embodiments, L P1 is –CH2C(O)O–, –CH2CH2C(O)O–, –CH2C(O)OCH2C(O)O–, –CH2C(O)OCH2CH2OC(O)–, or –C(O)N(H)–.

[0330] In some embodiments, the PEG-lipid is a compound of Formula (Aa), Formula (Ab), Formula (Ac), Formula (Ad), or Formula (Ae):

[0331]

[0332] or a pharmaceutically acceptable salt thereof.

[0333] In some embodiments, R P1 It is C6-C 24 , C 10 -C 20 , C 10 -C 18 , C 10 -C 16 , C 10 -C 14 , C 10 -C 12 , C 12 -C 20 , C 12 -C 18 , C12 -C 16 , C 12 -C 14 , C 14 -C 20 , C 14 -C 18 , C 14 -C 16 , C 16 -C 20 , C 16 -C 18 or C 18 -C 20 In some embodiments, R P1 It is C 14 -C 18 In some embodiments, R P1 It is C 14 -C 16 In some embodiments, R P1 It is C 15 -C 17 In some embodiments, R P1 It is C 16 -C 18 In some embodiments, R P1 It is C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 or C 24 In some embodiments, R P1 It is C6-C 24 , C 10 -C 20 , C 10 -C 18 , C 10 -C 16 , C 10 -C 14 , C 10 -C 12 , C 12 -C 20 , C 12 -C 18 , C 12 -C16 , C 12 -C 14 , C 14 -C 20 , C 14 -C 18 , C 14 -C 16 , C 16 -C 20 , C 16 -C 18 or C 18 -C 20 In some embodiments, R P1 It is C 14 -C 18 In some embodiments, R P1 It is C 14-16 In some embodiments, R P1 It is C 15 -C 17 In some embodiments, R P1 It is C 16-18 In some embodiments, R P1 It is C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 or C 24 Alkenyl.

[0334] In some embodiments, R P2 is hydrogen. In some embodiments, R P2 It is –CH3.

[0335] In some embodiments, n is between 10 and 200, 10 and 180, 10 and 160, 10 and 140, 10 and 120, 10 and 100, 10 and 80, 10 and 60, 10 and 40, 10 and 20, 20 and 200, 20 and 180, 20 and 160, 20 and 140, 20 and 120, 20 and 100, 20 and 80, 20 and 60, 20 and 40, 40 and 200, 40 and 180, 40 and 160, 40 and 140, 40 and 120, 40 and 100, 40 and 80, 40 and 60, 60 and 200, 60 and 200. 0 to 180, 60 to 160, 60 to 140, 60 to 120, 60 to 100, 60 to 80, 80 to 200, 80 to 180, 80 to 160, 80 to 140, 80 to 120, 80 to 100, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 200, 140 to 180, 140 to 160, 160 to 200, 160 to 180 or 180 to 200. In some embodiments, n is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some embodiments, n is about 20 on average. In some embodiments, n is about 40 on average. In some embodiments, n is about 45 on average. In some embodiments, n is about 50 on average. In some embodiments, n is about 68 on average. In some embodiments, n is about 75 on average. In some embodiments, n is about 100 on average.

[0336] In some embodiments, the compound of formula (A) is a compound selected from the group consisting of:

[0337] HO-(CH2CH2O) n -CH2C(O)O-(CH2) 17 CH3, n averages about 45;

[0338] H3CO-(CH2CH2O) n -CH2C(O)O-(CH2) 17 CH3, n averages about 45;

[0339] HO-(CH2CH2O) n -CH2C(O)O-(CH2) 15 CH3, n averages about 45;

[0340] HO-(CH2CH2O) n-CH2C(O)O-(CH2) 13 CH3, n averages about 45; and

[0341] HO-(CH2CH2O) n -C(O)N(H)-(CH2) 17 CH3, n averages about 45;

[0342] or a pharmaceutically acceptable salt thereof.

[0343] Alternative Implementation

[0344] In alternative embodiments, the compounds described herein may also contain one or more isotopic substitutions. For example, hydrogen may be 2 H (D or deuterium) or 3 H (T or tritium); carbon can be, for example, 13 C or 14 C; oxygen can be, for example. 18 O; nitrogen can be, for example, 15 N, etc. In other embodiments, specific isotopes (e.g., 3 H. 13 C. 14 C. 18 O or 15 N) can represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the total isotopic abundance of the element occupying a particular site in the compound.

[0345] Lipid Nanoparticles

[0346] In certain embodiments, synthetic RNA virus genome as described herein is encapsulated in lipid nanoparticle (LNP).In certain embodiments, LNP comprises one or more lipids, such as such as triglyceride (for example tristearin), diglyceride (for example glyceryl behenate), monoglyceride (for example glyceryl monostearate), fatty acid (for example stearic acid), steroid (for example cholesterol) and wax (for example hexadecyl palmitate).In some embodiments, LNP comprises one or more cationic lipids, one or more structural lipids and one or more auxiliary lipids.In some embodiments, LNP comprises one or more cationic lipids, cholesterol and one or more neutral lipids.

[0347] In some embodiments, the compounds of the present disclosure are used to form nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs). In some embodiments, LNPs include PEG-lipids, ionizable lipids, helper lipids, and structural lipids. In some embodiments, LNPs described herein are formulated for delivering therapeutic agents to subjects in need. In some embodiments, LNPs described herein are formulated for delivering nucleic acid molecules to subjects in need.

[0348] The formulation of lipids in LNP has a significant impact on the therapeutic use and efficacy of specific LNPs. For example, LNP formulations (such as SS-OC / cholesterol / DSPC / PEG2k-DPG), for example, in mice, non-human primates (NHPs) and / or humans, typically show increased clearance after repeated intravenous (IV) administration, and the in vivo circulation time after the second dose is much shorter than the in vivo circulation time after the first dose. The shortened circulation time may have a negative impact on the delivery efficiency of LNP, which may be because LNP is less exposed to the target. Therefore, although such formulations may be used to deliver agents that do not require multiple administrations, their use for delivering agents that require subsequent administration may be limited due to this shortened circulation time.

[0349] There remains a need for LNP formulations that exhibit regulated circulation and exposure to target cells, for example, sustained circulation and sustained exposure in vivo after repeated administration. The present disclosure provides such LNP formulations by incorporating the ionizable lipids and / or PEG-lipids of the present disclosure into the lipid formulation of the LNP. Thus, sustained circulation of the LNPs of the present disclosure after repeated administration allows for sustained therapeutic effects of the synthetic RNA viral genomes encapsulated therein.

[0350] In some embodiments, in the absence of the ionizable lipids and / or PEG-lipids of the present disclosure, the rapid clearance of LNPs and their components after repeated administration reduces the delivery efficiency of the encapsulated synthetic RNA viral genome in subsequent doses because the body may clear the LNPs before releasing the synthetic RNA viral genome. In some embodiments, when the ionizable lipids and / or PEG-lipids of the present disclosure are incorporated into LNPs, the clearance of LNPs after repeated administration is delayed, thereby allowing the encapsulated synthetic RNA viral genome to be continuously released and produce a therapeutic effect.

[0351] Polyethylene glycol (PEG)-lipid

[0352] In some embodiments, the PEG-lipids of the present disclosure comprise a hydrophilic head group and a hydrophobic lipid tail. In some embodiments, the hydrophilic head group is a PEG moiety. In some embodiments, the PEG-lipids of the present disclosure comprise a single lipid tail. In some embodiments, the PEG-lipids of the present disclosure comprise a monoalkyl lipid tail, a monoalkenyl lipid tail, a monoalkynyl lipid tail, or a monoacyl lipid tail. In some embodiments, the single lipid tail comprises an ether group, a carbonyl group, or an ester group. In some embodiments, the PEG-lipids of the present disclosure may contain polyoxyethylene alkyl ethers, polyoxyethylene alkenyl ethers, or polyoxyethylene alkynyl ethers (such molecules are also referred to as BRIJ). TM In some embodiments, the PEG-lipids of the present disclosure may contain polyoxyethylene alkyl esters, polyoxyethylene alkenyl esters, or polyoxyethylene alkynyl esters (such molecules are also referred to as MYRJ TM molecular).

[0353] In some embodiments, the PEG-lipid may contain a diacyl lipid tail.

[0354] In some embodiments, the PEG-lipid is a compound of formula (A)

[0355]

[0356] or a pharmaceutically acceptable salt or solvate thereof, wherein the variables are defined herein.

[0357] In some embodiments, the PEG-lipid is a compound of formula (A'):

[0358]

[0359] or a pharmaceutically acceptable salt thereof, wherein:

[0360] n is an integer between 10 and 200, inclusive;

[0361] L P1′ is a bond, –C(O)–, –[(CH2) 0-3 –C(O)O] 1-3 –, –(CH2) 0-3 –C(O)O–(CH2) 1-3 –OC(O)– or –C(O)N(H)–;

[0362] R P1′ It is C5-C 25 Alkyl or C5-C 25 alkenyl; and

[0363] R P2′ It is hydrogen or –CH3.

[0364] In some embodiments, L P1′ is a bond, –C(O)–, –CH2C(O)O–, –CH2CH2C(O)O–, –CH2C(O)OCH2C(O)O–, –CH2C(O)OCH2CH2OC(O)–, or –C(O)N(H)–. In some embodiments, R P1′ YesR P1 In some embodiments, R P2′ YesR P2 .

[0365] In some embodiments, the PEG-lipid is a compound of formula (A''):

[0366]

[0367] or a pharmaceutically acceptable salt thereof, wherein:

[0368] n is an integer between 10 and 200, inclusive;

[0369] L P1″ is a bond, –[(CH2) 0-3 –C(O)O] 1-3 –, –(CH2) 0-3 –C(O)O–(CH2) 1-3 –OC(O)– or –C(O)N(H)–;

[0370] R P1″ It is C5-C 25 Alkyl or C5-C 25 alkenyl; and

[0371] R P2″ It is hydrogen or –CH3.

[0372] In some embodiments, L P1″ is the bond, –CH2C(O)O–, –CH2CH2C(O)O–, –CH2C(O)OCH2C(O)O–, –CH2C(O)OCH2CH2OC(O)–, or –C(O)N(H)–.

[0373] In some embodiments, the PEG-lipid is a compound of Formula (A"-a), Formula (A"-b), Formula (A"-c), Formula (A"-cd), Formula (A"-e), or Formula (A"-f):

[0374]

[0375] or a pharmaceutically acceptable salt thereof.

[0376] In some embodiments, R P1″ YesRP1 In some embodiments, R P2″ YesR P2 In some embodiments, the PEG-lipid is a compound of formula (A″-f1):

[0377]

[0378] or a pharmaceutically acceptable salt thereof.

[0379] In some embodiments, the PEG-lipid is a compound of formula (A"-f2):

[0380]

[0381] or a pharmaceutically acceptable salt thereof.

[0382] In some embodiments, the PEG-lipid is a compound of formula (A″-f3):

[0383]

[0384] or a pharmaceutically acceptable salt thereof.

[0385] In some embodiments, the PEG-lipid of the present disclosure is a compound of formula (B):

[0386] or a pharmaceutically acceptable salt thereof, wherein:

[0387] n is an integer between 10 and 200, inclusive; and

[0388] R B1 It is C5-C 25 Alkyl or C5-C 25 Alkenyl.

[0389] In some embodiments, R B1 YesR P1 .

[0390] In some embodiments, the PEG-lipid is a compound of formula (Ba):

[0391]

[0392] or a pharmaceutically acceptable salt thereof.

[0393] In some embodiments, the PEG-lipid is a compound of formula (Bb):

[0394]

[0395] or a pharmaceutically acceptable salt thereof.

[0396] In some embodiments, n is between 10 and 200, 10 and 180, 10 and 160, 10 and 140, 10 and 120, 10 and 100, 10 and 80, 10 and 60, 10 and 40, 10 and 20, 20 and 200, 20 and 180, 20 and 160, 20 and 140, 20 and 120, 20 and 100, 20 and 80, 20 and 60, 20 and 40, 40 and 200, 40 and 180, 40 and 160, 40 and 140, 40 and 120, 40 and 100, 40 and 80, 40 and 60, 60 and 200, 60 and 200. 0 to 180, 60 to 160, 60 to 140, 60 to 120, 60 to 100, 60 to 80, 80 to 200, 80 to 180, 80 to 160, 80 to 140, 80 to 120, 80 to 100, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 200, 140 to 180, 140 to 160, 160 to 200, 160 to 180 or 180 to 200. In some embodiments, n is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some embodiments, n is about 20 on average. In some embodiments, n is about 40 on average. In some embodiments, n is about 45 on average. In some embodiments, n is about 50 on average. In some embodiments, n is about 68 on average. In some embodiments, n is about 75 on average. In some embodiments, n is about 100 on average.

[0397] In some embodiments, the PEG-lipid comprises a PEG moiety having an average molecular weight of about 500 Daltons to about 10,000 Daltons. In some embodiments, the PEG-lipid comprises an average molecular weight of about 500 Daltons to about 5,000 Daltons, about 500 Daltons to about 4,000 Daltons, about 500 Daltons to about 3,000 Daltons, about 500 Daltons to about 2,000 Daltons, about 500 Daltons to about 1,000 Daltons, about 500 Daltons to about 800 Daltons, about 500 Daltons to about 600 Daltons, about 600 Daltons to about 5,000 Daltons, about 600 Daltons to about 4,000 Daltons, about 600 Daltons to about 3,000 Daltons, about 600 Daltons to about 2,000 Daltons, about 600 Daltons to about 1,000 Daltons, about 600 Daltons to about 800 Daltons, about 800 Daltons to about 5,000 Daltons, about 800 Daltons to about 4,000 Daltons, about 800 Daltons to about to about 3,000 Daltons, about 800 Daltons to about 2,000 Daltons, about 800 Daltons to about 1,000 Daltons, about 1,000 Daltons to about 5,000 Daltons, about 1,000 Daltons to about 4,000 Daltons, about 1,000 Daltons to about 3,000 Daltons, about 1,000 Daltons to about 2,000 Daltons, about 2,000 Daltons to about 5,000 Daltons, In some embodiments, the PEG portion of the PEG-lipid has an average molecular weight of about 1,500 Daltons to about 2,500 Daltons. In some embodiments, the average molecular weight of the PEG portion of the PEG-lipid is about 1,000 Daltons to about 5,000 Daltons. In some embodiments, the PEG-lipid comprises a PEG moiety having an average molecular weight of about 500 Daltons, about 600 Daltons, about 800 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2500 Daltons, about 3,000 Daltons, about 3,500 Daltons, about 4,000 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 8,000 Daltons, about 9,000 Daltons, or about 10,000 Daltons.In some embodiments, the PEG-lipid comprises a PEG moiety having an average molecular weight of at least 500 Daltons, at least 1,000 Daltons, at least 1,500 Daltons, at least 2,000 Daltons, at least 2,500 Daltons, at least 3,000 Daltons, at least 3,500 Daltons, at least 4,000 Daltons, at least 4,500 Daltons, at least 5,000 Daltons, at least 6,000 Daltons, at least 7,000 Daltons, at least 8,000 Daltons, at least 9,000 Daltons, or at least 10,000 Daltons. In some embodiments, the PEG-lipid comprises a PEG moiety having an average molecular weight of no more than 500 Daltons, no more than 1,000 Daltons, no more than 1,500 Daltons, no more than 2,000 Daltons, no more than 2,500 Daltons, no more than 3,000 Daltons, no more than 3,500 Daltons, no more than 4,000 Daltons, no more than 4,500 Daltons, no more than 5,000 Daltons, no more than 6,000 Daltons, no more than 7,000 Daltons, no more than 8,000 Daltons, no more than 9,000 Daltons, or no more than 10,000 Daltons. All values ​​are inclusive of all endpoints.

[0398] In some embodiments, the PEG-lipid is polyoxyethylene (100) stearyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (20) oleyl ether, polyoxyethylene (20) stearyl ether, or a mixture thereof. In some embodiments, the PEG-lipid is polyoxyethylene (100) stearate, polyoxyethylene (50) stearate, polyoxyethylene (40) stearate, polyoxyethylene palmitate, or a mixture thereof.

[0399] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9005-00, and the linear molecular formula is C 18 H 37 (OCH2CH2) n OH, where n is 100. BRIJ TM S100 is also commonly referred to as polyoxyethylene (100) stearyl ether. Thus, in some embodiments, the PEG-lipid is HO-PEG100-CH2(CH2) 16 CH3.

[0400] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9004-95-9, and the linear molecular formula is C 16 H 33 (OCH2CH2) n OH, where n is 20. BRIJ TM C20 also known as BRIJ TM58 and is generally referred to as polyethylene glycol hexadecyl ether, polyoxyethylene (20) hexadecyl ether. Thus, in some embodiments, the PEG-lipid is HO-PEG20-CH2(CH2) 14 CH3.

[0401] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9004-98-2, and the linear molecular formula is C 18 H 35 (OCH2CH2) n OH, where n is 20. BRIJ TM O20 is also commonly referred to as polyoxyethylene (20) oleyl ether. Thus, in some embodiments, the PEG-lipid is HO-PEG20-C 18 H 35 .

[0402] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9005-00-9, and the linear molecular formula is C 18 H 37 (OCH2CH2) n OH, where n is 20. BRIJ TM S20 is also commonly referred to as polyethylene glycol stearyl ether or polyoxyethylene (20) stearyl ether. Thus, in some embodiments, the PEG-lipid is HO-PEG20-CH2(CH2) 16 CH3.

[0403] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9004-99-3, and the linear molecular formula is C 17 H 35 C(O)(OCH2CH2) n OH, where n is 100. MYRJ TM S100 is also commonly referred to as polyoxyethylene (100) stearate. Thus, in some embodiments, the PEG-lipid is HO-PEG100-CH2(CH2) 15 CH3.

[0404] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9004-99-3, and the linear molecular formula is C 17 H 35 C(O)(OCH2CH2) n OH, where n is 50. MYRJ TMS50 is also commonly referred to as polyoxyethylene (50) stearate. Thus, in some embodiments, the PEG-lipid is HO-PEG50-CH2(CH2) 15 CH3.

[0405] In some embodiments of the present disclosure, the PEG-lipid is The CAS number is 9004-99-3, and the linear molecular formula is C 17 H 35 C(O)(OCH2CH2) n OH, where n is 40. MYRJ TM S40 is also commonly referred to as polyoxyethylene (40) stearate. Thus, in some embodiments, the PEG-lipid is HO-PEG40-CH2(CH2) 15 CH3.

[0406] In some embodiments of the present disclosure, the PEG-lipid is

[0407] The CAS number is 1607430-62-04, and the linear molecular formula is C122H242O50. PEG2k-DMG is also known as 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

[0408] In some embodiments of the present disclosure, the PEG-lipid is:

[0409] The alkyl composition is R1COO=C16:0, R2COO=C16:0. PEG2k-DPG is also generally referred to as 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene.

[0410] In some embodiments of the present disclosure, the PEG-lipid can be: PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearoylglyceramide, PEG-cholesterol (l-[8'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl] Carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypoly(ethylene glycol) In some embodiments, the PEG-lipid may be PEG2k-DMG. In some embodiments, the PEG-lipid may be PEG2k-DSG. In other embodiments, the PEG-lipid may be PEG2k-DSPE. In some embodiments, the PEG-lipid may be PEG2k-DMA. In other embodiments, the PEG-lipid may be PEG2k-C-DMA. In some embodiments, the PEG-lipid may be PEG2k-DSA. In other embodiments, the PEG-lipid may be PEG2k-C11. In some embodiments, the PEG-lipid may be PEG2k-C14. In some embodiments, the PEG-lipid may be PEG2k-C16. In some embodiments, the PEG-lipid may be PEG2k-C18.

[0411] In some embodiments, the PEG-lipids with a single lipid tail of the present disclosure (e.g., PEG-lipids of Formula (A), Formula (A′), Formula (A″), or Formula (B)) can reduce accelerated blood clearance (ABC) after administration and / or repeated administration of the LNP compositions of the present disclosure. In some embodiments, the PEG-lipids with a single lipid tail of the present disclosure can reduce or deplete PEG-specific antibodies (e.g., anti-PEG IgM) produced by the immune system of a subject after administration and / or repeated administration of the LNP compositions of the present disclosure.

[0412] In some embodiments, the PEG-lipid comprises a poly(ethylene glycol) chain up to 5 kDa in length covalently attached to a lipid comprising one or more C6-C20 alkyl groups. In some embodiments, the PEG-lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-amine). In some embodiments, the PEG-lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-PEG5K); 1,2-dipalmitoyl-rac-glyceromethoxypolyethylene glycol-2000 (DPG-PEG2K); 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-5000 (DSG-PEG5K); 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DSG-PEG2K); 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-5000 (DMG-PEG5K); and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DMG-PEG2K). In some embodiments, the PEG-lipid is DSPE-PEG5K. In some embodiments, the PEG-lipid is DPG-PEG2K. In some embodiments, the PEG-lipid is DSG-PEG2K. In some embodiments, the PEG-lipid is DMG-PEG2K. In some embodiments, the PEG-lipid is DSG-PEG5K. In some embodiments, the PEG-lipid is DMG-PEG5K.

[0413] In some embodiments, the PEG lipid is a cleavable PEG lipid. Examples of PEG derivatives having cleavable bonds include those modified with peptide bonds (Kulkarni et al. (2014). Mmp-9 responsive PEG cleavable nanostructures for efficient delivery of chemotherapeutics to pancreatic cancer. Mol Pharmaceutics 11: 2390–9; Lin et al. (2015). Drug / dye-loaded, multifunctional peg-chitosan-iron oxide nanocomposites for methotraxatesynergistically self-targeted cancer therapy and dual model imaging. ACS Appl Mater Interfaces 7: 11908–20.), those modified with disulfide bonds (Yan et al. (2014). A method to accelerate the gelation of disulfide-crosslinked hydrogels. Chin J Polym Sci 33: 118–27; Wu & Yan (2015). Copper nanopowder catalyzed cross-coupling of diaryl disulfides with aryl iodides in PEG-400. Synlett 26:537–42), those modified with vinyl ether bonds, hydrazone bonds (Kelly et al. (2016). Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells. Org Biomol Chem 14:9278–86.), and those modified with ester bonds (Xu et al. (2008). Esterase-catalyzed dePEGylation of pH-sensitive vesicles modified with cleavable PEG-lipid derivatives. J Control Release 130:238–45).See also, Fang et al. (2017) Cleaveable PEGylation: strategy for overcoming the “PEG dilemma” inefficient drug delivery. Drug Delivery 24:2, 22-32.

[0414] In some embodiments, the PEG lipid is an activated PEG lipid. Exemplary activated PEG lipids include PEG-NH2, PEG-MAL, PEG-NHS and PEG-ALD. Such functionalized PEG lipids can be used to conjugate the targeting moiety to lipid nanoparticles to direct the particles to specific target cells or tissues (e.g., by the connection of antigen binding molecules, peptides, polysaccharides, etc.). In some embodiments, the functionalized moiety (e.g., -NH2, -MAL, -NHS, -ALD) is added to the PEG-lipid (e.g., BRIJ) of the present disclosure. TM or MYRJ TM The free end of the PEG moiety of the family PEG lipids

[0415] Cationic lipids

[0416] In some embodiments, the LNPs provided herein comprise one or more cationic lipids."Cationic lipid" and "ionizable lipid" are used interchangeably herein.

[0417] Cationic lipids refer to any of a variety of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). For example, cationic lipids that are positively charged at below physiological pH include, but are not limited to, DODAP, DODMA, and DMDMA. In some embodiments, the cationic lipid comprises C 18An alkyl chain, an ether linkage between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0418] In some embodiments, the cationic lipid comprises a protonable tertiary amine head group. Such lipids are referred to herein as ionizable lipids. Ionizable lipids refer to lipid species comprising ionizable amine head groups and generally comprising a pKa of less than about 7. Therefore, in an environment with an acidic pH, the ionizable amine head group is protonated so that the ionizable lipid preferentially interacts with negatively charged molecules (e.g., nucleic acids, such as the recombinant polynucleotides described herein), thereby promoting nanoparticle assembly and encapsulation. Therefore, in some embodiments, ionizable lipids can increase the loading of nucleic acids into lipid nanoparticles. In an environment with a pH value greater than about 7 (e.g., physiological pH≈7.4), the ionizable lipid comprises a neutral charge. When particles comprising ionizable lipids are taken up into the low pH environment of the endosome (e.g., pH<7), the ionizable lipid is protonated again and associates with the anionic endosomal membrane, promoting the release of the contents encapsulated by the particles. In some embodiments, LNPs comprise ionizable lipids, for example, 7.SS cleavable and pH-responsive lipid-like materials (such as SS series).

[0419] In some embodiments, the cationic lipid of the LNP is DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA (MC3), SS-LC (formerly known as SS-18 / 4PE-13), SS-EC (formerly known as SS-33 / 4PE-15), SS-OC, SS-OP, di((Z)-non-2-en-1-yl) 9-((4-dimethylamino) butyryl) oxy) heptadecanedioate (L-319), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a mixture thereof.

[0420] In some embodiments, the cationic lipid of the LNP is a compound of formula (I):

[0421]

[0422] or a pharmaceutically acceptable salt or solvate thereof, wherein the variables are defined herein.

[0423] In some embodiments, the cationic lipid of the present disclosure is a compound selected from Table 21 or a pharmaceutically acceptable salt thereof.

[0424] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-1):

[0425]

[0426] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0427] R la and R 1b each is independently C1-C8 aliphatic or -O(C1-C8 aliphatic)-, wherein the O atom (when present) is bonded to the piperidine ring;

[0428] X a and X b are independently –C(O)O–*, –OC(O)–*, –C(O)N(R x 1 )–*,–N(R x 1 )C(O)–*、–O(C=O)N(R x 1 )–*,–N(R x 1 )(C=O)O–* or –O–, where –* represents the 2a or R 2b The connection points, and each occurrence of R x 1 are independently selected from hydrogen and optionally substituted C1-C4 alkyl; and

[0429] R 2a and R 2b Each is independently a sterol residue, a fat-soluble vitamin residue or C 13 -C 23 Aliphatic.

[0430] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-2):

[0431]

[0432] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0433] R 1a ' and R 1b ' are each independently C1-C8 alkylene or -O(C1-C8 alkylene), wherein the O atom (when present) is bonded to the piperidine ring;

[0434] Y a’ and Y b’ are independently –C(O)O–*, –OC(O)–*, –C(O)N(Rx 1 )–*,–N(R x 1 )C(O)–*、–O(C=O)N(R x 1 )–*,–N(R x 1 )(C=O)O–*、–N(R x 1 )C(O)N(R x 1 )– or –O–, where –* indicates the same as R 2a or R 2b The connection points, and each occurrence of R x 1 independently selected from hydrogen and optionally substituted C1-C4 alkyl;

[0435] Z a’ and Z b’ Each is independently an optionally substituted arylene-C0-C8 alkylene or an optionally substituted arylene-C0-C8 heteroalkylene, wherein the alkylene or heteroalkylene is respectively a ' and Y b 'Bonding;

[0436] R 2a’ and R 2b’ Each is independently a sterol residue, a fat-soluble vitamin residue or C 12 -C 22 Aliphatic.

[0437] In some embodiments, the cationic lipid of the LNP is of formula (II-1a) ( SS-OC) or formula (II-2a) ( SS-OP) compounds:

[0438]

[0439] In some embodiments, the cationic lipid of the LNP is of formula (II-1a) ( SS-OC) compounds. SS-OC is also called SS-18 / 4PE-16.

[0440] In some embodiments, the cationic lipid of the LNP is of formula (II-2a) ( SS-OP) compounds.

[0441] In some embodiments, the cationic lipid of the LNP is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0442] Helper lipids

[0443] In some embodiments, LNP as described herein comprises one or more helper lipids.Term " helper lipid " refers to the lipid that can increase LNP to target (for example, cell) delivery.Do not wish to be bound by any particular theory, helper lipid is expected to enhance the stability and / or fusogenicity of lipid nanoparticles.In some embodiments, helper lipid is phospholipid.In some embodiments, helper lipid is phospholipid substitute (substitute) or substitute (replacement).In some embodiments, helper lipid is alkylresorcinol.

[0444] In some embodiments, the helper lipid is phosphatidylcholine (PC). In some embodiments, the helper lipid is not phosphatidylcholine (PC). In some embodiments, the helper lipid is a phospholipid or a phospholipid substitute. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly) unsaturated phospholipids or phospholipid substitutes or a combination thereof. In general, phospholipids include a phosphate head group and one or more fatty acid tails. In some embodiments, phospholipids may include one or more multiple bonds (e.g., double bonds or triple bonds) (i.e., one or more unsaturated). In some embodiments, the helper lipid is non-cationic.

[0445] The phosphate head group may be selected from, for example, the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0446] The fatty acid tail can be selected from, for example, the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0447] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphophosphingolipids such as sphingomyelin.

[0448] In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0449] In some embodiments, the non-cationic helper lipid is a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) substituted.

[0450] In some embodiments, phospholipids can promote fusion with membrane.For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membrane (for example, cell membrane or intracellular membrane).The fusion of phospholipids with membrane can allow one or more elements of lipid-containing compositions to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to cells.

[0451] In some embodiments, the phosphate head group can be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid tail can be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.

[0452] In some embodiments, the phospholipid is a compound according to formula (III):

[0453]

[0454] Where: R p Represent phosphate head group and R1 and R2 represent with or without unsaturated fatty acid tail, both can be identical or different.Phosphate head group can be selected from the non-limiting group of following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin.Fatty acid tail can be selected from the non-limiting group of following composition: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.Also contemplated non-natural species, including natural species with modification and substitution, including branching, oxidation, cyclization and alkynes.For example, phospholipid can be functionalized with one or more alkynes or cross-linked with one or more alkynes (for example, wherein one or more double bonds are replaced by triple bonds alkenyl). Under appropriate reaction conditions, the alkyne group may undergo a copper-catalyzed cycloaddition reaction upon exposure to azide. Such reactions may be useful for functionalizing the lipid bilayer of LNPs to promote membrane permeation or cell recognition, or for conjugating LNPs with useful components such as targeting or imaging moieties (e.g., dyes).

[0455] In some embodiments, the LNP comprises one or more non-cationic helper lipids (e.g., neutral lipids). Exemplary neutral helper lipids include (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) (DLPE), 1,2-diphytoyl-sn-glycero-3-phosphoethanolamine (DiPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPPE), 1,2-dioleo ...choline (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPPE), E), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), (1,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE ), ceramide and sphingomyelin. In some embodiments, one or more auxiliary lipids are selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the auxiliary lipid of the LNP includes, consists essentially of, or consists of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the LNP comprises DSPC. In some embodiments, the LNP comprises DOPC. In some embodiments, the LNP comprises DLPE. In some embodiments, the LNP comprises DOPE.

[0456] In some embodiments, the phospholipid is selected from the non-limiting group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- -sn-glycero-3-phosphocholine (DPPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine (18:3 (cis) PC), 1,2-diamidonoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6 (cis) PC), 1,2-diaphytoyl-sn-glycero-3-phosphoethanolamine (4ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dialinoleoyl-sn-glycero-3-phosphoethanolamine (PE (18:2 / 18:2), 1,2-diamidonoyl-sn-glycero-3-phosphoethanolamine (PE 18:3 (9Z, 12Z, 15Z), 1,2-diamidonoyl-sn-glycero-3-phosphoethanolamine (DAPE 18:3(9Z,12Z,15Z), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6(cis)PE), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0457] In some embodiments, the helper lipid is selected from the group consisting of: distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, Acylethanolamine, 18-1-trans PE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dimethicone Acylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dioleyl-phosphatidylethanolamine (DEPE), phosphatidylcholine, phosphatidylethanolamine, lysolecithin, lysophatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, hexadecyl phosphate, lysophosphatidylcholine and dilinoleoylphosphatidylcholine.

[0458] In some embodiments, the helper lipid of the present disclosure is DSPC.

[0459] In some embodiments, the LNP includes DSPC. In some embodiments, the LNP includes DOPE. In some embodiments, the LNP includes DMPE. In some embodiments, the LNP includes both DSPC and DOPE.

[0460] In some embodiments, the helper lipid is selected from the group consisting of DSPC, DMPE, and DOPC, or a combination thereof.

[0461] In some embodiments of the present disclosure, the helper lipid lipid is

[0462] The CAS number is 816-94-4 and the linear molecular formula is C44H88NO8P. DSPC is also known as 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0463] In some embodiments, the phospholipids of the present disclosure include modified tails. In some embodiments, the phospholipid is DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine) or an analog thereof, with a modified tail. As described herein, a "modified tail" can be a tail with a shorter or longer aliphatic chain, an aliphatic chain with introduced branches, an aliphatic chain with introduced substituents, an aliphatic chain in which one or more methylene groups are replaced by a cyclic or heteroatom group, or any combination thereof.

[0464] In some embodiments, the helper lipids of the present disclosure are alternative lipids other than phospholipids.

[0465] In some embodiments, the phospholipids useful in the present disclosure include modified tails. In some embodiments, the phospholipids useful in the present disclosure are DSPC or analogs thereof with modified tails. As described herein, a "modified tail" can be a tail with a shorter or longer aliphatic chain, an aliphatic chain with introduced branching, an aliphatic chain with introduced substituents, an aliphatic chain in which one or more methylene groups are replaced by a cyclic or heteroatom group, or any combination thereof.

[0466] In some embodiments, phospholipids useful in the present disclosure comprise a modified phosphorylcholine moiety in which the alkyl chain linking the quaternary amine and the phosphoryl group is not ethylene (eg, n is not 2).

[0467] In some embodiments, LNPs of the present disclosure include oleic acid or oleic acid analogs as helper lipids. In some embodiments, oleic acid analogs include modified oleic acid tails, modified carboxylic acid moieties, or both. In some embodiments, oleic acid analogs are compounds in which the carboxylic acid moieties of oleic acid are replaced by different groups.

[0468] In some embodiments, the LNPs of the present disclosure comprise different zwitterionic groups instead of phospholipids as helper lipids.

[0469] In some embodiments, the auxiliary lipid of the present disclosure is a naturally occurring membrane lipid. In some embodiments, the auxiliary lipid of the present disclosure is 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovose diacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylene hexadecanoyl-sn-glycero-3-phosphocholine (Cyclo PC) or a combination thereof. In some embodiments, the LNP of the present disclosure comprises a combination of auxiliary lipids. In some embodiments, the combination of auxiliary lipids does not comprise DSPC. In some embodiments, the combination of auxiliary lipids comprises DSPC. In some embodiments, compared with LNPs comprising DSPC as the only auxiliary lipid, LNPs comprising one or more naturally occurring membrane lipids (e.g., DGTS) have improved liver transfection / delivery of target molecules encapsulated in LNPs.

[0470] In some embodiments, the helper lipid of the present disclosure is 5-heptadecanylresorcinol or a derivative thereof.

[0471] Structural lipids

[0472] In some embodiments, LNPs of the present disclosure include one or more structural lipids. Incorporation of structural lipids into lipid nanoparticles may help alleviate aggregation of other lipids in the particles. The structural lipids may be, but are not limited to, sterols or lipids containing sterol moieties.

[0473] In some embodiments, the structural lipid of LNP is a sterol (e.g., a plant sterol or an animal sterol). In some embodiments, the sterol is cholesterol or an analog or derivative thereof. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is cholesterol, β-sitosterol, naphthalene sterol, ergosterol, sitosterol, campesterol, stigmasterol, rapeseed sterol, ergosterol, tomatidine, tomatine, ursolic acid, α-tocopherol, including analogs, salts or esters thereof, alone or in combination.

[0474] In some embodiments, the structural lipid of the LNP is cholesterol, a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0475] In some embodiments, the structural lipid of LNP is phytosterol. In some embodiments, phytosterol is sitosterol, stigmasterol, campesterol, sitostanol, campesterol, rapeseed sterol, fucoxantrol, β-sitosterol, stigmasterol, β-sitostanol, ergosterol, lupeol, cycloartenol, A5-avenasterol, A7-avenasterol or A7-stigmasterol, including analogs, salts or esters thereof, alone or in combination.

[0476] In some embodiments, LNP comprises one or more plant sterols. In some embodiments, the plant sterol component of LNP is a single plant sterol. In some embodiments, the plant sterol component of LNP of the present disclosure is a mixture of different plant sterols (e.g., 2,3,4,5 or 6 different plant sterols). In some embodiments, the plant sterol component of LNP of the present disclosure is a blend of one or more plant sterols and one or more animal sterols, such as a blend of plant sterols (e.g., beta-sitosterol, such as beta-sitosterol) and cholesterol.

[0477] In some embodiments of the present disclosure, the structural lipid of the LNP is cholesterol:

[0478] Cholesterol, CAS number 57-88-5, linear molecular formula C 27 H 46 O.

[0479] In some embodiments, the LNP comprises a cationic lipid and one or more helper lipids, wherein the cationic lipid is DOTAP. In some embodiments, the LNP comprises a cationic lipid and one or more helper lipids, wherein the cationic lipid is DLin-MC3-DMA (MC3). In some embodiments, the LNP comprises a cationic lipid and one or more helper lipids, wherein the cationic lipid is In some embodiments, the LNP comprises a cationic lipid and one or more auxiliary lipids, wherein the cationic lipid is In some embodiments, the LNP comprises a cationic lipid and one or more auxiliary lipids, wherein the cationic lipid is In some embodiments, the LNP comprises a cationic lipid and one or more auxiliary lipids, wherein the cationic lipid is SS-OP. In some embodiments, the LNP comprises a cationic lipid and one or more auxiliary lipids, wherein the cationic lipid is L-319. In some embodiments, the LNP further comprises a structural lipid. In some embodiments, the structural lipid is cholesterol.

[0480] In some embodiments, LNP comprises cationic lipids and one or more helper lipids. In some embodiments, LNP comprises cationic lipids and one or more helper lipids, wherein one or more helper lipids comprise DLPE. In some embodiments, LNP comprises cationic lipids and one or more helper lipids, wherein one or more helper lipids comprise DSPC. In some embodiments, LNP comprises cationic lipids and one or more helper lipids, wherein one or more helper lipids comprise DOPE. In some embodiments, LNP comprises cationic lipids and one or more helper lipids, wherein one or more helper lipids comprise DOPC. In some embodiments, LNP further comprises structural lipids. In some embodiments, structural lipids are cholesterol.

[0481] In some embodiments, LNP comprises cationic lipid, auxiliary lipid and structural lipid. In some embodiments, structural lipid is cholesterol. In some embodiments, cationic lipid is DOTAP, and auxiliary lipid is DLPE. In some embodiments, cationic lipid is MC3, and auxiliary lipid is DSPC. In some embodiments, auxiliary lipid is DOPE. In some embodiments, auxiliary lipid is DSPC. In some embodiments, LNP comprises cationic lipid, structural lipid and at least two auxiliary lipids, wherein cationic lipid is DOTAP, and at least two auxiliary lipids comprise DLPE and DSPE. In some embodiments, LNP comprises cationic lipid, structural lipid and at least two auxiliary lipids, wherein cationic lipid is MC3, and at least two auxiliary lipids comprise DSPC and DMG. In some embodiments, at least two auxiliary lipids comprise DOPE and DSPE. In some embodiments, at least two auxiliary lipids comprise DSPC and DMG. In some embodiments, structural lipid is cholesterol. In some embodiments, LNP comprises DOTAP, cholesterol and DLPE. In some embodiments, LNP comprises MC3, cholesterol and DSPC. In some embodiments, LNP comprises DOTAP, cholesterol and DOPE. In some embodiments, LNP comprises DOTAP, cholesterol, DLPE and DSPE. In some embodiments, LNP comprises MC3, cholesterol, DSPC and DMG. In some embodiments, LNP comprises DOTAP, cholesterol, DLPE and DSPE-PEG. In some embodiments, LNP comprises MC3, cholesterol, DSPC and DMG-PEG. In some embodiments, LNP comprises DOTAP, cholesterol, DOPE and DSPE. In some embodiments, LNP comprises DOTAP, cholesterol, DOPE and DSPE-PEG. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol and DPG-PEG (e.g., DPG-PEG2K). In some embodiments, LNP comprises SS-OC, DSPC, cholesterol and PEG-lipid of formula (I) (e.g., BRIJ TM S100).

[0482] Lipid molar ratio in LNP composition

[0483] In some embodiments, LNP of the present disclosure is included in 40mol% to 70mol% of cationic lipids, up to 50mol% of auxiliary lipids, structural lipids between 10mol% to 50mol%, and PEG-lipids between 0.001mol% to 5mol%, including all endpoints. In some embodiments, the total mol% of cationic lipids, auxiliary lipids, structural lipids and PEG-lipids is 100%.

[0484] In some embodiments, the mol% of the cationic lipid in the LNP is 40mol%-70mol%, 40mol%-55mol%, 40mol%-50mol%, 40mol%-45mol%, 44mol%-54mol%, 45mol%-60mol%, 45mol%-55mol%, 45mol%-50mol%, 50mol%-60mol%, 49mol%-64mol%, 50mol%-55mol% or 55mol%-60mol%. In some embodiments, the mol% of the cationic lipid in the LNP is 44mol%-54mol%. In some embodiments, the mol% of cationic lipid in LNP is 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol%, 46mol%, 47mol%, 48mol%, 49mol%, 50mol%, 51mol%, 52mol%, 53mol%, 54mol%, 55mol%, 56mol%, 57mol%, 58mol%, 59mol% or 60mol%. In some embodiments, the mol% of the cationic lipid in the LNP is about 40mol%, about 41mol%, about 42mol%, about 43mol%, about 44mol%, about 45mol%, about 46mol%, about 47mol%, about 48mol%, about 49mol%, about 50mol%, about 51mol%, about 52mol%, about 53mol%, about 54mol%, about 55mol%, about 56mol%, about 57mol%, about 58mol%, about 59mol% or about 60mol%. All values ​​include all endpoints.

[0485] In some embodiments, the mol% of structural lipids in LNP is 10mol%-60mol%, 10mol%-30mol%, 15mol%-35mol%, 20mol%-40mol%, 20mol%-45mol%, 25mol%-33mol%, 24mol%-32mol%, 25mol%-45mol%, 30mol%-50mol%, 35mol%-43mol%, 35mol%-55mol% or 40mol%-60mol%. In some embodiments, the mol% of structural lipids in LNP is 20mol%-45mol%. In some embodiments, the mol% of structural lipids in LNP is 24mol%-32mol%. In some embodiments, the mol% of structural lipids in LNP is 25mol%-33mol%. In some embodiments, the mol% of structural lipids in LNP is 22mol%-28mol%. In some embodiments, the mol% of structural lipids in LNP is 35mol%-45mol%. In some embodiments, the mol% of structural lipids in LNP is 35mol%-43mol%. In some embodiments, the mol% of structural lipids in LNP is 10mol%-60mol%. In some embodiments, the mol% of structural lipids in LNP is 10mol%, 11mol%, 12mol%, 13mol%, 14mol%, 15mol%, 16mol%, 17mol%, 18mol%, 19mol%, 20mol%, 21mol%, 22mol%, 23mol%, 24mol%, 25mol%, 26mol%, 27mol%, 28mol%, 29mol%, 30mol%, 31mol%, 32mol%, 33mol%, 34mol%, 35mol%, 36mol%, 37mol%, 38mol%, 39mol%, 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol%, 46mol%, 47mol%, 48mol%, 49mol%, 50mol%, 51mol%, 52mol%, 53mol%, 54mol%, 55mol%, 56mol%, 57mol%, 58mol%, 59mol%, 60mol%, 61mol%, 62mol%, 63mol%, 64mol%, 65mol%, 66mol%, 67mol%, 68mol%, 69mol%, 70mol%, 71mol%, 72mol%, 73mol%, 74mol%, 75mol%, 76mol%, 77mol%, 78mol%, 79mol%, 80mol%, 81mol%, 82mol%, 83mol%, 84mol%, 85mol%, 86mol%, 87mol%, 88mol%, 89mol%, 90mol%, 91mol%, 92mol%, 93mol%, 94mol%, 95mol%, 96mol%, 97mol%, 98 %, 34mol%, 35mol%, 36mol%, 37mol%, 38mol%, 39mol%, 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol%, 46mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol% or 60 mol%.In some embodiments, the mol% of structural lipids in LNP is about 10mol%, about 11mol%, about 12mol%, about 13mol%, about 14mol%, about 15mol%, about 16mol%, about 17mol%, about 18mol%, about 19mol%, about 20mol%, about 21mol%, about 22mol%, about 23mol%, about 24mol%, about 25mol%, about 26mol%, about 27mol%, about 28mol%, about 29mol%, about 30mol%, about 31mol%, about 32mol%, about 33mol%, about 34mol%, about 35mol%, about 36mol%, about 37mol%, about 38mol%, about 39mol%, about 40mol%, about 41mol%, about 42mol%, about 43mol%, about 44mol%, about 45mol%, about 46mol%, about 47mol%, about 48mol%, about 49mol%, about 50mol%, about 51mol%, about 52mol%, about 53mol%, about 54mol%, about 55mol%, about 56mol%, about 57mol%, about 58mol%, about 59mol%, about 60mol%, about 61mol%, about 62mol%, about 64mol%, about 65mol%, about 66mol%, about 67mol%, about 68mol%, about 69mol%, about 70mol%, about 71mol%, about 72mol%, about 73mol%, about 74mol%, about 75mol%, about 76mol%, about 77mol%, about 78mol%, about 79mol%, about 80mol%, about 81mol%, about 82mol%, about 83mol%, about 84mol%, about 85mol%, about 86mol%, about 87mol%, about 88mol%, about 89mol%, about 90mol%, about 91mol%, about 92mol%, about 9 In some embodiments, the structural lipid is cholesterol.All values ​​include all endpoints.

[0486] In some embodiments, the mol% of the helper lipid in LNP is 1mol%-50mol%. In some embodiments, the mol% of the helper lipid in LNP is up to 29mol%. In some embodiments, the mol% of the helper lipid in LNP is 1mol%-10mol%, 5mol%-9mol%, 5mol%-15mol%, 8mol%-14mol%, 18%-22%, 19mol%-25mol%, 10mol%-20mol%, 10mol%-25mol%, 15mol%-25mol%, 20mol%-30mol%, 25mol%-35mol%, 30mol%-40mol% or 35mol%-50mol%. In some embodiments, the mol% of the helper lipid in LNP is 10mol%-25mol%. In some embodiments, the mol% of the helper lipid in LNP is 5mol%-9mol%. In some embodiments, the mol% of the helper lipid in LNP is 8mol%-14mol%. In some embodiments, the mol% of the helper lipid in the LNP is 18 mol%-22 mol%. In some embodiments, the mol% of the helper lipid in the LNP is 19 mol%-25 mol%. In some embodiments, the mol % of the helper lipid in the LNP is 1 mol %, 2 mol %, 3 mol %, 4 mol %, 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol %, 18 mol %, 19 mol %, 20 mol %, 21 mol %, 22 mol %, 23 mol %, 24 mol %, 25 mol %, 26 mol %, 27 mol %, 28 mol %, 29 mol %, 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol %.In some embodiments, the mol % of the helper lipid in the LNP is about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol %, about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 38 mol %, about 39 mol %, or about 40 mol %. In some embodiments, the helper lipid is DSPC.All values ​​are inclusive.

[0487] In some embodiments, the mol% of PEG-lipid in the LNP is greater than 0 mol% and at most 5 mol% of the total lipid present in the LNP. In some embodiments, the mol% of PEG-lipid is 0.1 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%, 3.1 mol%, 3.2 mol%, 3.3 mol%, 3.4 mol%, 3.5 mol%, 3.6 mol%, 3.7 mol%, 3.8 mol%, 3.9 mol%, 3.1 7mol%, 1.8mol%, 1.9mol%, 2.0mol%, 2.1mol%, 2.2mol%, 2.3mol%, 2.4mol%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%, 3.1 mol%, 3.2 mol%, 3.3 mol%, 3.4 mol%, 3.5 mol%, 4.0 mol%, 4.5 mol% or 5 mol%. In some embodiments, the mol % of PEG-lipid is about 0.1 mol %, about 0.2 mol %, about 0.25 mol %, about 0.3 mol %, about 0.4 mol %, about 0.5 mol %, about 0.6 mol %, about 0.7 mol %, about 0.8 mol %, about 0.9 mol %, about 1.0 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, about 2.0 mol %, about 2.1 mol %, about 2.2 mol %, about 2.3 mol %, about 2.4 mol %, about 2.5 mol %, about 2.6 mol %, about 2.7 mol %, about 2.8 mol %, about 2.9 mol %, about 3.0 mol %, about 3.1 mol %, about 3.2 mol %, about 3.3 mol %, about 3.4 mol %, about 3.5 mol %, about 3.6 mol %, about 3.7 mol %, about 3.8 mol %, about 3.9 mol %, about 3. % about 1.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 4.0 mol%, about 4.5 mol% or about 5 mol%.In some embodiments, the mol % of PEG-lipid is at least 0.1 mol %, at least 0.2 mol %, at least 0.25 mol %, at least 0.3 mol %, at least 0.4 mol %, at least 0.5 mol %, at least 0.6 mol %, at least 0.7 mol %, at least 0.8 mol %, at least 0.9 mol %, at least 1.0 mol %, at least 1.1 mol %, at least 1.2 mol %, at least 1.3 mol %, at least 1.4 mol %, at least 1.5 mol %, at least 1.6 mol %, at least 1.7 mol %, at least 1.8 mol %, at least 1.9 mol %, at least 2.0 mol %, at least 2.1 mol %, at least 2.2 mol %, at least 2.3 mol %, at least 2.4 mol %, at least 2.5 mol %, at least 2.6 mol %, at least 2.7 mol %, at least 2.8 mol %, at least 2 ... %, at least 1.8 mol%, at least 1.9 mol%, at least 2.0 mol%, at least 2.1 mol%, at least 2.2 mol%, at least 2.3 mol%, at least 2.4 mol%, at least 2.5 mol%, at least 2.6 mol%, at least 2.7 mol%, at least 2.8 mol%, at least 2.9 mol%, at least 3.0 mol%, at least 3.1 mol%, at least 3.2 mol%, at least 3.3 mol%, at least 3.4 mol%, at least 3.5 mol%, at least 4.0 mol%, at least 4.5 mol% or at least 5 mol%. In some embodiments, the mol % of PEG-lipid is at most 0.1 mol %, at most 0.2 mol %, at most 0.25 mol %, at most 0.3 mol %, at most 0.4 mol %, at most 0.5 mol %, at most 0.6 mol %, at most 0.7 mol %, at most 0.8 mol %, at most 0.9 mol %, at most 1.0 mol %, at most 1.1 mol %, at most 1.2 mol %, at most 1.3 mol %, at most 1.4 mol %, at most 1.5 mol %, at most 1.6 mol %, at most 1.7 mol %, at most 1.8 mol %, at most 1.9 mol %, at most 2.0 mol %, at most 2.1 mol %, at most 2.2 mol %, at most 2.3 mol %, at most 2.4 mol %, at most 2.5 mol %, at most 2.6 mol %, at most 2.7 mol %, at most 2.8 mol %, at most 2.9 mol %, at most 2.0 In some embodiments, the mol% of PEG-lipid is between 0.1mol%-4mol% of the total lipid present in LNP.In some embodiments, the mol% of PEG-lipid is between 0.1mol%-2mol% of the total lipid present in LNP.In some embodiments, the mol% of PEG-lipid is between 0.2mol%-0.8mol%, 0.4mol%-0.6mol%, 0.7mol%-1.3mol%, 1.2mol%-1.8mol%, or 1mol%-3.5mol% of the total lipid present in the LNP. In some embodiments, the mol% of PEG-lipid is between 0.1mol%-0.7mol%, 0.2mol%-0.8mol%, 0.3mol%-0.9mol%, 0.4mol%-0.8mol%, 0.4mol%-0.6mol%, 0.4mol%-1mol%, 0.5mol%-1.1mol%, 0.6mol%-1.2mol%, 0.7mol%-1.3mol%, 0.8mol%-1.4mol%, 0.9mol%-1.5mol%, 1mol%-3.5mol% of the total lipid present in the LNP. %-3.5mol%, 1mol%-1.6mol%, 1.1mol%-1.7mol%, 1.2mol%-1.8mol%, 1.3mol%-1.9mol%, 1.4mol%-2mol%, 1.5mol%-2.1mol%, 1.6mol%-2.2mol%, 1.7mol%-2.3mol%, 1.8mol%-2.4mol%, 1.9mol%-2.5mol%, 2mol%-2.6mol%, 2.4mol%-3.8mol% or 2.6mol%-3.4mol%. All values ​​include all endpoints.

[0488] In some embodiments, LNP of the present disclosure comprises 44mol%-60mol% of cationic lipids, 19mol%-25mol% of auxiliary lipids, 25mol%-33mol% of structural lipids, and 0.2mol%-0.8mol% of PEG-lipids, including endpoints. In some embodiments, LNP of the present disclosure comprises 44mol%-54mol% of cationic lipids, 19mol%-25mol% of auxiliary lipids, 24mol%-32mol% of structural lipids, and 1.2mol%-1.8mol% of PEG-lipids, including endpoints. In some embodiments, LNP of the present disclosure comprises 44mol%-54mol% of cationic lipids, 8mol%-14mol% of auxiliary lipids, 35mol%-43mol% of structural lipids, and 1.2mol%-1.8mol% of PEG-lipids, including endpoints. In some embodiments, the LNPs of the present disclosure comprise 45 mol%-55 mol% cationic lipids, 5 mol%-9 mol% helper lipids, 36 mol%-44 mol% structural lipids, and 2.5 mol%-3.5 mol% PEG-lipids, inclusive.

[0489] In some embodiments, according to Table 6 below, the LNP of the present disclosure comprises one or more cationic lipids of the present disclosure, one or more auxiliary lipids of the present disclosure, one or more structural lipids of the present disclosure, and one or more PEG-lipids of the present disclosure at a certain mol% of the total lipids in the LNP (or a certain mol% range of the total lipids). In some embodiments, the total mol% of these four lipid components is equal to 100%. In some embodiments, the total mol% of these four lipid components is less than 100%. In some embodiments, the cationic lipid is a compound of formula (I) or a compound selected from Table 21. In some embodiments, the structural lipid is cholesterol. In some embodiments, the auxiliary lipid is DSPC. In some embodiments, the PEG-lipid has formula (A), formula (A′) or formula (A″).

[0490] Table 6: Mol% of lipid components in LNP

[0491]

[0492]

[0493] In some embodiments, the LNPs of the present disclosure comprise 44 mol%-54 mol% of cationic lipids, 19 mol%-25 mol% of auxiliary lipids, 25 mol%-33 mol% of structural lipids, and 0.2 mol%-0.8 mol% of PEG-lipids, including endpoints. In some embodiments, the LNPs of the present disclosure comprise 44 mol%-54 mol% of a compound of formula (II-1a), 19 mol%-25 mol% of DSPC, 25 mol%-33 mol% of cholesterol, and 0.2 mol%-0.8 mol% of a PEG-lipid selected from the group consisting of: HO-PEG100-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 14 CH3、HO-PEG20-C 18 H 35 、HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13 CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3、HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG50-C(O)-CH2(CH2) 15CH3 and HO-PEG40-C(O)-CH2(CH2) 15 CH3, including endpoints.

[0494] In some embodiments, the LNPs of the present disclosure comprise 44 mol%-54 mol% of cationic lipids, 19 mol%-25 mol% of auxiliary lipids, 24 mol%-32 mol% of structural lipids, and 1.2 mol%-1.8 mol% of PEG-lipids, including endpoints. In some embodiments, the LNPs of the present disclosure comprise 44 mol%-54 mol% of a compound of formula (II-1a), 19 mol%-25 mol% of DSPC, 24 mol%-32 mol% of cholesterol, and 1.2 mol%-1.8 mol% of a PEG-lipid selected from the group consisting of: HO-PEG100-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 14 CH3、HO-PEG20-C 18 H 35 、HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13 CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3、HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG50-C(O)-CH2(CH2) 15 CH3 and HO-PEG40-C(O)-CH2(CH2) 15 CH3, including endpoints.

[0495] In some embodiments, the LNPs of the present disclosure comprise 44 mol%-54 mol% of cationic lipids, 8 mol%-14 mol% of auxiliary lipids, 35 mol%-43 mol% of structural lipids, and 1.2 mol%-1.8 mol% of PEG-lipids, including endpoints. In some embodiments, the LNPs of the present disclosure comprise 44 mol%-54 mol% of a compound of formula (II-1a), 8 mol%-14 mol% of DSPC, 35 mol%-43 mol% of cholesterol, and 1.2 mol%-1.8 mol% of a PEG-lipid selected from the group consisting of: HO-PEG100-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 16CH3, HO-PEG20-CH2(CH2) 14 CH3、HO-PEG20-C 18 H 35 、HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13 CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3、HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG50-C(O)-CH2(CH2) 15 CH3 and HO-PEG40-C(O)-CH2(CH2) 15 CH3, including endpoints.

[0496] In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and PEG-lipid, wherein the ratio of SS-OC: DSPC: Chol: PEG-lipid (as a percentage of the total lipid content) is about A: B: C: D, wherein A = 40 mol%-60 mol%, B = 10 mol%-25 mol%, C = 20 mol%-30 mol%, and D = 0 mol%-3 mol%, and wherein A + B + C + D = 100 mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and PEG-lipid, wherein the ratio of SS-OC: DSPC: Chol: PEG-lipid (as a percentage of the total lipid content) is about A: B: C: D, wherein A = 45 mol%-50 mol%, B = 20 mol%-25 mol%, C = 25 mol%-30 mol%, and D = 0 mol%-1 mol%, and wherein A + B + C + D = 100 mol%. In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and PEG-lipid, wherein the ratio of SS-OC:DSPC:Chol:PEG-lipid (as a percentage of the total lipid content) is about 49:22:28.5:0.5. In some embodiments, the PEG-lipid is a compound of formula (A), formula (A′) or formula (A″). In some embodiments, the PEG-lipid is selected from the group consisting of: BRIJ TM S100、BRIJ TM S20, BRIJ TM O20 and BRIJ TM C20. In some embodiments, the PEG-lipid is BRIJ TM S100.

[0497] In some embodiments, LNP comprises DOTAP, cholesterol (Chol) and DLPE, wherein the ratio of DOTAP:Chol:DLPE (percentage of total lipid content) is about 50:35:15. In some embodiments, LNP comprises DOTAP, cholesterol (Chol) and DLPE, wherein the ratio of DOTAP:Chol:DOPE (percentage of total lipid content) is about 50:35:15. In some embodiments, LNP comprises DOTAP, cholesterol (Chol), DLPE, DSPE-PEG, wherein the ratio of DOTP:Chol:DLPE (percentage of total lipid content) is about 50:35:15, and wherein the particles comprise about 0.2 mol% DSPE-PEG. In some embodiments, LNP comprises MC3, cholesterol (Chol), DSPC and DMG-PEG, wherein the ratio of MC3:Chol:DSPC:DMG-PEG (percentage of total lipid content) is about 49:38.5:11:1.5.

[0498] In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K), wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=40mol%-60mol%, B=10mol%-25mol%, C=20mol%-30mol%, and D=0mol%-3mol%, and wherein A+B+C+D=100mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC: DSPC: Chol: DPG-PEG2K (percentage of total lipid content) is about A: B: C: D, wherein A = 45 mol%-50 mol%, B = 20 mol%-25 mol%, C = 25 mol%-30 mol%, and D = 0 mol%-1 mol%, and wherein A + B + C + D = 100 mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC: DSPC: Chol: DPG-PEG2K (percentage of total lipid content) is about 49: 22: 28.5: 0.5.

[0499] In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=40mol%-60mol%, B=10mol%-30mol%, C=20mol%-45mol%, and D=0mol%-3mol%, and wherein A+B+C+D=100mol%. In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=40mol%-60mol%, B=10mol%-30mol%, C=25mol%-45mol%, and D=0mol%-3mol%, and wherein A+B+C+D=100mol%. In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=45mol%-55mol%, B=10mol%-20mol%, C=30mol%-40mol%, and D=1mol%-2mol%, and wherein A+B+C+D=100mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC: DSPC: Chol: DPG-PEG2K (percentage of total lipid content) is about A: B: C: D, wherein A = 45 mol%-50 mol%, B = 10 mol%-15 mol%, C = 35 mol%-40 mol%, and D = 1 mol%-2 mol%, and wherein A + B + C + D = 100 mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC: DSPC: Chol: DPG-PEG2K (percentage of total lipid content) is 49: 11: 38.5: 1.5.

[0500] In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=45mol%-65mol%, B=5mol%-20mol%, C=20mol%-45mol%, and D=0mol%-3mol%, and wherein A+B+C+D=100mol%. In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=50mol%-60mol%, B=5mol%-15mol%, C=30mol%-45mol%, and D=0mol%-3mol%, and wherein A+B+C+D=100mol%. In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC:DSPC:Chol:DPG-PEG2K (as a percentage of the total lipid content) is about A:B:C:D, wherein A=55mol%-60mol%, B=5mol%-15mol%, C=30mol%-40mol%, and D=1mol%-2mol%, and wherein A+B+C+D=100mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC: DSPC: Chol: DPG-PEG2K (percentage of total lipid content) is about A: B: C: D, wherein A = 55 mol%-60 mol%, B = 5 mol%-10 mol%, C = 30 mol%-35 mol%, and D = 1 mol%-2 mol%, and wherein A + B + C + D = 100 mol%. In some embodiments, LNP comprises SS-OC, DSPC, cholesterol (Chol) and DPG-PEG2K, wherein the ratio of SS-OC: DSPC: Chol: DPG-PEG2K (percentage of total lipid content) is 58: 7: 33.5: 1.5.

[0501] In some embodiments, nanoparticles are coated with glycosaminoglycans (GAGs) to regulate or promote the uptake of nanoparticles by target cells. GAGs can be heparin / heparin sulfate, chondroitin sulfate / dermatan sulfate, keratin sulfate or hyaluronic acid (HA). In certain embodiments, the surface of the nanoparticles is coated with HA, and the particles taken up by tumor cells are targeted. In some embodiments, lipid nanoparticles are coated with arginine-glycine-aspartic acid tripeptide (RGD peptide) (see Ruoslahti, Advanced Materials, 24, 2012, 3747-3756; and Bellis et al., Biomaterials, 32 (18), 2011, 4205-4210).

[0502] Characteristics of LNP compositions

[0503] The present disclosure provides compositions (eg, pharmaceutical compositions) comprising a plurality of LNPs as described herein. Also provided herein are compositions comprising LNPs as described herein and encapsulated molecules.

[0504] In some embodiments, the LNPs of the present disclosure can reduce the in vivo immune response compared to the control LNP. In some embodiments, the control LNP is a LNP comprising a PEG-lipid that does not have formula (A), formula (A′) or formula (A″). In some embodiments, the PEG-lipid of the control LNP is PEG2k-DPG. In some embodiments, the PEG-lipid of the control LNP is PEG2k-DMG. In some embodiments, the control LNP has the same PEG-lipid molar ratio as the LNP of the present disclosure. In some embodiments, the control LNP is the same as the LNP of the present disclosure, except that the control LNP comprises a PEG-lipid that does not have formula (A), formula (A′) or formula (A″) (for example, the control LNP may comprise PEG2k-DPG or PEG2k-DMG as the PEG-lipid).

[0505] In some embodiments, the control LNP is a LNP comprising a cationic lipid that does not have formula (I). In some embodiments, the cationic lipid of the control LNP is SS-OC. In some embodiments, the control LNP has the same cationic lipid molar ratio as the LNP of the present disclosure. In some embodiments, the control LNP is identical to the LNP of the present disclosure except that the control LNP comprises a cationic lipid that does not have formula (I) (e.g., the control LNP may comprise SS-OC as the cationic lipid).

[0506] In some embodiments, the weakening of immune response may be the reduction of accelerated blood clearance (ABC). In some embodiments, ABC is related to the secretion of natural IgM and / or anti-PEG IgM. As used herein, the term "natural IgM" refers to the circulating IgM in the serum that exists independently of known immune exposure (for example, being exposed to LNP of the present disclosure). The term "ABC reduction" refers to any reduction in ABC compared with control LNP. In some embodiments, the reduction of ABC may be relative to control LNP, and the removal of LNP after the second or subsequent dose is reduced. In some embodiments, the reduction can be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%. In some embodiments, the reduction is about 10% to about 100%, about 10% to about 50%, about 20% to about 100%, about 20% to about 50%, about 30% to about 100%, about 30% to about 50%, about 40% to about 100%, about 40% to about 80%, about 50% to about 90% or about 50% to about 100%. In some embodiments, the reduction of ABC can be measured by the increase of the detectable level of the synthetic RNA viral genome encapsulated after the second or subsequent administration or continue to be measured. In some embodiments, the reduction of ABC may cause the level of the synthetic RNA viral genome encapsulated to increase (for example, increase 2 times, 3 times, 4 times, 5 times or more times) relative to the level of the synthetic RNA viral genome encapsulated after the control LNP is administered. In some embodiments, the reduced ABC is associated with a lower serum level of anti-PEG IgM.

[0507] In some embodiments, the LNPs of the present disclosure can delay the clearance of LNPs and their components after repeated administration compared to control LNPs, while control LNPs can be cleared before the release of the encapsulated molecules. Therefore, the LNPs of the present disclosure can increase the delivery efficiency of the encapsulated molecules (e.g., synthetic RNA viral genomes) in subsequent doses.

[0508] In some embodiments, the average size (i.e., average outer diameter) of the LNPs is from about 50 nm to about 500 nm. In some embodiments, the average size of the LNPs is from about 50 nm to about 200 nm, from about 100 nm to about 200 nm, from about 150 nm to about 200 nm, from about 50 nm to about 100 nm, from about 50 nm to about 150 nm, from about 100 nm to about 150 nm, from about 200 nm to about 250 nm, from about 250 nm to about 300 nm, from about 300 nm to about 400 nm, from about 150 nm to about 500 nm, from about 200 nm to about 500 nm, from about 300 nm to about 500 nm, from about 350 nm to about 500 nm, from about 400 nm to about 500 nm, from about 425 nm to about 500 nm, from about 450 nm to about 500 nm, or from about 475 nm to about 500 nm. In some embodiments, the average size of LNP is about 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, about 120nm or about 125nm. In some embodiments, the average size of LNP is about 100nm. In some embodiments, the average size of LNP is 50nm to 150nm. In some embodiments, the average size (average outer diameter) of LNP is 50nm to 150nm, 50nm to 125nm, 50nm to 100nm, 50nm to 75nm, 75nm to 150nm, 75nm to 125nm, 75nm to 100nm, 100nm to 150nm, 100nm to 125nm or 125nm to 150nm. In some embodiments, the average size of the LNPs is 70nm to 90nm, 80nm to 100nm, 90nm to 110nm, 100nm to 120nm, 110nm to 130nm, 120nm to 140nm, or 130nm to 150nm. In some embodiments, the average size of the LNPs is 90nm to 110nm. All values ​​include endpoints.

[0509] In some embodiments, the average size (i.e., average outer diameter) of LNP is about 50nm to about 150nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 60nm to about 130nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 70nm to about 120nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 70nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 80nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 90nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 100nm. In some embodiments, the present disclosure provides a therapeutic composition comprising multiple lipid nanoparticles, wherein the average size of multiple LNPs is about 110nm. All values ​​include endpoints.

[0510] In some embodiments, the encapsulation efficiency of LNP to synthetic RNA viral genome is about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%. In some embodiments, about 70%, about 75%, about 80%, about 90%, about 95%, about 97%, about 98% or about 99% of a plurality of LNPs comprise encapsulated synthetic RNA viral genomes. In one embodiment, the encapsulation efficiency of LNP to synthetic RNA viral genomes is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. In some embodiments, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% of a plurality of LNPs comprise an encapsulated synthetic RNA viral genome. In some embodiments, about 70% to 100%, about 75% to 100%, 80% to 100%, about 85% to 100%, about 90% to 100%, about 91% to 100%, about 92% to 100%, about 93% to 100%, about 94% to 100%, about 95% to 100%, about 96% to 100%, about 97% to 100%, about 98% to 100%, about 99% to 100% of a plurality of LNPs comprise an encapsulated synthetic RNA viral genome.

[0511] In some embodiments, LNP is neutrally charged (for example, average zeta potential is between about 0mV and 1mV). In some embodiments, the average zeta potential of LNP is between about 40mV and about -40mV. In some embodiments, the average zeta potential of LNP is between about 40mV and about 0mV. In some embodiments, the average zeta potential of LNP is between about 35mV and about 0mV, about 30mV and about 0mV, about 25mV and about 0mV, about 20mV and about 0mV, about 15mV and about 0mV, about 10mV and about 0mV, or about 5mV and about 0mV. In some embodiments, the average zeta potential of LNP is between about 20mV and about -40mV. In some embodiments, the average zeta potential of LNP is between about 20mV and about -20mV. In some embodiments, the average zeta potential of LNP is between about 10mV and about -20mV. In some embodiments, the average zeta potential of LNP is between about 10mV and about -10mV. In some embodiments, the average zeta potential of the LNP is about 10 mV, about 9 mV, about 8 mV, about 7 mV, about 6 mV, about 5 mV, about 4 mV, about 3 mV, about 2 mV, about 1 mV, about 0 mV, about -1 mV, about -2 mV, about -3 mV, about -4 mV, about -5 mV, about -6 mV, about -7 mV, about -8 mV, about -9 mV, about -9 mV, or about -10 mV.

[0512] In some embodiments, the average zeta potential of the LNP is between about 0 mV and -20 mV. In some embodiments, the average zeta potential of the LNP is less than about -20 mV. For example, in some embodiments, the average zeta potential of the LNP is less than about less than about -30 mV, less than about 35 mV, or less than about -40 mV. In some embodiments, the average zeta potential of the LNP is between about -50 mV and about -20 mV, about -40 mV to about -20 mV, or about -30 mV to about -20 mV. In some embodiments, the average zeta potential of the LNPs is about 0 mV, about -1 mV, about -2 mV, about -3 mV, about -4 mV, about -5 mV, about -6 mV, about -7 mV, about -8 mV, about -9 mV, about -10 mV, about -11 mV, about -12 mV, about -13 mV, about -14 mV, about -15 mV, about -16 mV, about -17 mV, about -18 mV, about -19 mV In some embodiments, the average zeta potential of the LNP is less than about -20mV, less than about -30mV, less than about 35mV, or less than about -40mV.

[0513] In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 10: 1 to about 60: 1. In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 20: 1. In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 30: 1. In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 40: 1. In some embodiments, the LNP comprises a recombinant nucleic acid molecule described herein, and the LNP has a L:N mass ratio of about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 237:1, about 28:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, or about 45:1.

[0514] In some embodiments, the lipid (L) of LNP and nucleic acid molecule (N) mass ratio is between 10: 1 and 60: 1, between 20: 1 and 60: 1, between 30: 1 and 60: 1, between 40: 1 and 60: 1, between 50: 1 and 60: 1, between 10: 1 and 50: 1, between 20: 1 and 50: 1, between 30: 1 and 50: 1, between 40: 1 and 50: 1, between 10: 1 and 40: 1, between 20: 1 and 40: 1, between 30: 1 and 40: 1, between 10: 1 and 30: 1, between 20: 1 and 30: 1, or between 10: 1 and 20: 1, including all endpoints. In some embodiments, the lipid of LNP: nucleic acid molecule mass ratio is between 30: 1 and 40: 1. In some embodiments, the lipid:nucleic acid molecular mass ratio of the LNP is between 30:1 and 36:1.

[0515] In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 10: 1 to about 60: 1. In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 20: 1. In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 30: 1 (L: N). In some embodiments, the LNP comprises a recombinant nucleic acid molecule as described herein, and the mass ratio of the lipid (L) to the nucleic acid (N) of the LNP is about 40: 1 (L: N). In some embodiments, the LNP comprises a recombinant nucleic acid molecule described herein, and the LNP has a L:N mass ratio of about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 237:1, about 28:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, or about 45:1.

[0516] In some embodiments, the LNP comprises a nucleic acid molecule and the lipid-nitrogen to phosphorus ratio (N:P) of the LNP is between 1 and 25. In some embodiments, the N:P is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the N:P is between 1 and 25, between 1 and 20, between 1 and 15, between 1 and 10, between 1 and 5, between 5 and 25, between 5 and 20, between 5 and 15, between 5 and 10, between 10 and 25, between 10 and 20, between 10 and 15, between 15 and 25, between 15 and 20, or between 20 and 25. In some embodiments, the LNP comprises a nucleic acid molecule and the lipid-nitrogen to phosphorus ratio (N:P) of the LNP is 14.

[0517] In some embodiments, LNP comprises a synthetic RNA viral genome encoding an oncolytic virus, wherein the encoded oncolytic virus can reduce the size of a tumor away from the LNP application site of the subject. For example, as shown in the embodiments provided herein, intravenous administration of LNPs described herein results in viral replication in tumor tissue and a reduction in tumor size. These data indicate that the LNPs disclosed herein can be localized to tumors or cancerous tissues away from the LNP application site. Such an effect realizes the use of LNP-encapsulated oncolytic viruses described herein in treating tumors that are not easily accessible and are therefore not suitable for intratumoral delivery therapy.

[0518] LNP preparation method

[0519] In some embodiments, the present disclosure provides a method for preparing a composition of lipid nanoparticles (LNP) containing nucleic acid molecules, the method comprising the following steps:

[0520] (a) diluting the nucleic acid molecule in an aqueous solution to a desired concentration;

[0521] (b) using microfluidic flow to mix an organic lipid phase containing all lipid components of LNP with an aqueous phase containing nucleic acid molecules to form LNP;

[0522] (c) dialyzing the LNPs against a buffer solution to remove the organic solvent;

[0523] (d) concentrating the LNPs to a target volume; and

[0524] (e) Optionally, filtering through a sterile filter.

[0525] In some embodiments, the organic lipid phase and the aqueous phase are mixed in a ratio of 1:1 (volume:volume) to 1:10 (volume:volume). In some embodiments, the organic lipid phase and the aqueous phase are mixed in a ratio of 1:1 (volume:volume), 1:2 (volume:volume), 1:3 (volume:volume), 1:4 (volume:volume), 1:5 (volume:volume), 1:6 (volume:volume), 1:7 (volume:volume), 1:8 (volume:volume), 1:9 (volume:volume) or 1:10 (volume:volume). In some embodiments, the organic lipid phase and the aqueous phase are mixed in a ratio of between 1:1 (vol:vol) and 1:3 (vol:vol), between 1:2 (vol:vol) and 1:4 (vol:vol), between 1:3 (vol:vol) and 1:5 (vol:vol), between 1:4 (vol:vol) and 1:6 (vol:vol), between 1:5 (vol:vol) and 1:7 (vol:vol), between 1:6 (vol:vol) and 1:8 (vol:vol), between 1:7 (vol:vol) and 1:9 (vol:vol), or between 1:8 (vol:vol) and 1:10 (vol:vol). In some embodiments, the organic lipid phase and the aqueous phase are mixed in a ratio of between 1:3 (vol:vol) and 1:5 (vol:vol). In some embodiments, the organic lipid phase and the aqueous phase are mixed in a ratio of 1:3 (vol:vol). In some embodiments, the organic lipid phase and the aqueous phase are mixed in a ratio of 1:5 (volume:volume).

[0526] In some embodiments, the total flow rate of the microfluidic flow is 5mL / min-20mL / min. In some embodiments, the total flow rate of the microfluidic flow is 5mL / min, 6mL / min, 7mL / min, 8mL / min, 9mL / min, 10mL / min, 11mL / min, 12mL / min, 13mL / min, 14mL / min, 15mL / min, 16mL / min, 17mL / min, 18mL / min, 19mL / min or 20mL / min. In some embodiments, the total flow rate of the microfluidic flow is 9mL / min-20mL / min. In some embodiments, the total flow rate of the microfluidic flow is 11mL / min-13mL / min.

[0527] In some embodiments, the solvent in the organic lipid phase in step (b) is ethanol. In some embodiments, heat is applied to the organic lipid phase in step (b). In some embodiments, about 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C is applied to the organic lipid phase in step (b). In some embodiments, 60°C of heat is applied to the organic lipid phase in step (b). In some embodiments, no heat is applied to the organic lipid phase in step (b).

[0528] In some embodiments, the pH of the aqueous solution in step (a) is between 1 and 7. In some embodiments, the pH of the aqueous solution in step (a) is between 1 and 3, between 2 and 4, between 3 and 5, between 4 and 6, or between 5 and 7. In some embodiments, the pH of the aqueous solution in step (a) is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7. In some embodiments, the pH of the aqueous solution in step (a) is 3. In some embodiments, the pH of the aqueous solution in step (a) is 5.

[0529] In some embodiments, the total lipid concentration is between 5mM and 80mM. In some embodiments, the total lipid concentration is about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM or 80mM. In some embodiments, the total lipid concentration is about 20mM. In some embodiments, the total lipid concentration is about 40mM.

[0530] In some embodiments, the LNPs produced by the methods have a lipid-nitrogen to phosphorus ratio (N:P) between 1 and 25. In some embodiments, N:P is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, N:P is between 1 and 25, between 1 and 20, between 1 and 15, between 1 and 10, between 1 and 5, between 5 and 25, between 5 and 20, between 5 and 15, between 5 and 10, between 10 and 25, between 10 and 20, between 10 and 15, between 15 and 25, between 15 and 20, or between 20 and 25. In some embodiments, the LNP comprises a nucleic acid molecule and the lipid-nitrogen to phosphorus ratio (N:P) of the LNP is 14.

[0531] In some embodiments, the buffer in step (c) has a neutral pH (eg, 1xPBS, pH 7.2). In some embodiments, step (d) is concentrated using centrifugal filtration.

[0532] In some embodiments, the encapsulation efficiency of the method of the present disclosure is at least 70%, at least 75%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. In some embodiments, the encapsulation efficiency of the method of the present disclosure is at least 90%. In some embodiments, the encapsulation efficiency of the method of the present disclosure is at least 95%. In some embodiments, the encapsulation efficiency is determined by RiboGreen.

[0533] In some embodiments, the average size (i.e., average outer diameter) of the LNPs produced by the methods of the present disclosure is from about 50 nm to about 500 nm. In some embodiments, the average size of the LNPs is from about 50 nm to about 200 nm, from about 100 nm to about 200 nm, from about 150 nm to about 200 nm, from about 50 nm to about 100 nm, from about 50 nm to about 150 nm, from about 100 nm to about 150 nm, from about 200 nm to about 250 nm, from about 250 nm to about 300 nm, from about 300 nm to about 400 nm, from about 150 nm to about 500 nm, from about 200 nm to about 500 nm, from about 300 nm to about 500 nm, from about 350 nm to about 500 nm, from about 400 nm to about 500 nm, from about 425 nm to about 500 nm, from about 450 nm to about 500 nm, or from about 475 nm to about 500 nm. In some embodiments, the average size of multiple LNPs is about 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, about 120nm or about 125nm. In some embodiments, the average size of multiple LNPs is about 100nm. In some embodiments, the average size of multiple LNPs is 50nm to 150nm. In some embodiments, the average size (average outer diameter) of multiple LNPs is 50nm to 150nm, 50nm to 125nm, 50nm to 100nm, 50nm to 75nm, 75nm to 150nm, 75nm to 125nm, 75nm to 100nm, 100nm to 150nm, 100nm to 125nm or 125nm to 150nm. In some embodiments, the average size of the plurality of LNPs is 70nm to 90nm, 80nm to 100nm, 90nm to 110nm, 100nm to 120nm, 110nm to 130nm, 120nm to 140nm, or 130nm to 150nm. In some embodiments, the average size of the plurality of LNPs is 90nm to 110nm.

[0534] In some embodiments, the polydispersity index of multiple LNPs is between 0.01 and 0.3. In some embodiments, the polydispersity index of multiple LNPs is between 0.1 and 0.15. In some embodiments, the polydispersity index of multiple LNPs is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 016, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29 or about 0.30. In some embodiments, the polydispersity index of the plurality of LNPs is about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15. In some embodiments, the average diameter and / or polydispersity is determined via dynamic light scattering.

[0535] Payload molecule

[0536] In some embodiments, the particle comprises a synthetic RNA viral genome and further comprises a recombinant RNA polynucleotide encoding a payload molecule. In some embodiments, the particle is a lipid nanoparticle and comprises a synthetic RNA viral genome and further comprises a recombinant RNA polynucleotide encoding a payload molecule. In some embodiments, the synthetic RNA viral genome in the particle (e.g., LNP) comprises a recombinant RNA polynucleotide encoding a payload molecule. In some embodiments, the particle (e.g., LNP) comprises 1) a synthetic RNA viral genome (which may or may not encode a payload molecule) and 2) a second recombinant RNA polynucleotide encoding a payload molecule. In some embodiments, the synthetic RNA viral genome and the second recombinant RNA polynucleotide encoding a payload molecule are not connected in the particle (e.g., LNP). In some embodiments, the synthetic RNA viral genome and the second recombinant RNA polynucleotide encoding a payload molecule are non-covalently connected. In some embodiments, the synthetic RNA viral genome and the second recombinant RNA polynucleotide encoding a payload molecule are covalently connected via a covalent bond other than conventional 3', 5' phosphodiester linkages. In some embodiments, one or more miRNA target sequences are incorporated into the 3' or 5' UTR of the RNA polynucleotide encoding a payload molecule. In some embodiments, one or more miRNA target sequences are inserted into a polynucleotide encoding a payload molecule. In such embodiments, translation and subsequent expression of the payload does not occur or is substantially reduced in cells expressing the corresponding miRNA. In some embodiments, the recombinant RNA polynucleotide encoding the payload molecule is a replicon.

[0537] In some embodiments, the payload is a cytotoxic peptide. As used herein, "cytotoxic peptide" refers to a protein that can induce cell death when expressed in a host cell and / or can induce cell death of neighboring cells when secreted by a host cell. In some embodiments, the cytotoxic peptide is a caspase, p53, diphtheria toxin (DT), Pseudomonas exotoxin A (PEA), type I ribozyme inactivating proteins (RIPs) (e.g., saporin and gelonin), type II RIP (e.g., ricin), Shiga-like toxin 1 (Slt1), photosensitive reactive oxygen species (e.g., killer-red). In certain embodiments, the cytotoxic peptide is encoded by a suicide gene (such as a caspase gene) that causes cell death by apoptosis.

[0538] In some embodiments, the payload is an immunomodulatory peptide. As used herein, an "immunomodulatory peptide" is a peptide that can regulate (for example, activate or inhibit) a specific immune receptor and / or pathway. In some embodiments, the immunomodulatory peptide can act on any mammalian cell, including immune cells, tissue cells and stromal cells. In a preferred embodiment, the immunomodulatory peptide acts on immune cells, such as T cells, NK cells, NKT T cells, B cells, dendritic cells, macrophages, basophils, mast cells or eosinophils. Exemplary immunomodulatory peptides include antigen binding molecules, such as antibodies or their antigen binding fragments, cytokines, chemokines, soluble receptors, cell surface receptor ligands, dipeptides and enzymes.

[0539] In some embodiments, the payload is a cytokine, such as IL-1, IL-12, IL-15, IL-18, IL-36γ, TNFα, IFNα, IFNβ, IFNγ or TNFSF14. In some embodiments, the payload is a chemokine, such as CXCL10, CXCL9, CCL21, CCL4 or CCL5. In some embodiments, the payload is a ligand of a cell surface receptor, such as NKG2D ligand, neuropilin ligand, Flt3 ligand, CD47 ligand (e.g., SIRP1α). In some embodiments, the payload is a soluble receptor (such as a soluble cytokine receptor (e.g., IL-13R, TGFβR1, TGFβR2, IL-35R, IL-15R, IL-2R, IL-12R and interferon receptor)) or a soluble innate immune receptor (e.g., Toll-like receptor, complement receptor, etc.). In some embodiments, the payload is a dominant agonist mutant of a protein involved in intracellular RNA and / or DNA sensing (eg, a dominant agonist mutant of STING, RIG-1, or MDA-5).

[0540] In some embodiments, the payload is an antigen binding molecule, such as an antibody or an antigen binding fragment thereof (e.g., a single chain variable fragment (scFv), F(ab), etc.). In some embodiments, the antigen binding molecule specifically binds to a cell surface receptor, such as an immune checkpoint receptor (e.g., PD-1, PD-L1, and CTLA4) or an additional cell surface receptor involved in cell growth and activation (e.g., OX40, CD200R, CD47, CSF1R, TREM2, 4-1BB, CD40, and NKG2D).

[0541] In some embodiments, the payload molecule is a scorpion polypeptide, such as chlorotoxin, BmKn-2, neopladine 1, neopladine 2, and mauriporin. In some embodiments, the payload molecule is a snake polypeptide, such as contortrostatin, apoxin-I, bothropstoxin-I, BJcuL, OHAP-1, rhodostomin, drCT-I, CTX-III, B1L, and ACTX-6. In some embodiments, the payload molecule is a spider polypeptide, such as latarcin and hyaluronidase. In some embodiments, the payload molecule is a honey bee polypeptide, such as melittin and apamin. In some embodiments, the payload molecule is a frog polypeptide, such as PsT-1, PdT-1, and PdT-2.

[0542] In some embodiments, the payload molecule is an enzyme. In some embodiments, the enzyme can regulate the tumor microenvironment by changing the extracellular matrix. In such embodiments, the enzyme may include but is not limited to matrix metalloproteinases (e.g., MMP9), collagenase, hyaluronidase, gelatinase or elastase. In some embodiments, the enzyme is a part of a gene-guided enzyme prodrug therapy (GDEPT) system, such as herpes simplex virus thymidine kinase, cytosine deaminase, nitroreductase, carboxypeptidase G2, purine nucleoside phosphorylase or cytochrome P450. In some embodiments, the enzyme can induce or activate the cell death pathway (e.g., caspase) in the target cell. In some embodiments, the enzyme can degrade extracellular metabolites or information (e.g., adenosine deaminase or arginase or 15-hydroxy prostaglandin dehydrogenase).

[0543] In some embodiments, the payload molecule is MLKL. In some embodiments, the MLKL polypeptide comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 104. In some embodiments, the payload molecule comprises or consists of a MLKL 4HB domain. In some embodiments, the MLKL 4HB domain comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 1-120 of SEQ ID NO: 104.

[0544] In some embodiments, the payload molecule is GSDMD. In some embodiments, GSDMD comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 105. In some embodiments, the payload molecule comprises or consists of a GSDMD N-terminal fragment. In some embodiments, the GSDMD N-terminal fragment comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to amino acids 1-233 of SEQ ID NO: 105. In some embodiments, the payload molecule comprises a mutation corresponding to L192A of SEQ ID NO: 105.

[0545] In some embodiments, the payload molecule is gasdermin E (GSDME). In some embodiments, gasdermin E (GSDME) comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 106. In some embodiments, the payload molecule comprises or consists of a gasdermin E N-terminal fragment. In some embodiments, the gasdermin E N-terminal fragment comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 1-237 of SEQ ID NO: 106.

[0546] In some embodiments, the payload molecule is HMGB1. In some embodiments, the HMGB1 polypeptide comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 107. In some embodiments, the payload molecule comprises or consists of an HMGB1 Box B domain. In some embodiments, the HMGB1 Box B domain comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to amino acids 96-162 of SEQ ID NO: 107.

[0547] In some embodiments, the payload molecule is SMAC / Diablo. In some embodiments, SMAC / Diablo comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108. In some embodiments, the payload molecule comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 56-239 of SEQ ID NO: 108.

[0548] In some embodiments, the payload molecule is melittin. In some embodiments, melittin comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 109.

[0549] In some embodiments, the payload molecule is L-amino acid oxidase (LAAO). In some embodiments, L-amino acid oxidase (LAAO) comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 110.

[0550] In some embodiments, the payload molecule is a disintegrin. In some embodiments, the disintegrin comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 111.

[0551] In some embodiments, the payload molecule is TRAIL (TNFSF10). In some embodiments, TRAIL (TNFSF10) comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 112.

[0552] In some embodiments, the payload molecule is a nitroreductase. In some embodiments, the nitroreductase is NfsB (e.g., from E. coli). In some embodiments, NfsB comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 113. In some embodiments, the nitroreductase is NfsA (e.g., from E. coli). In some embodiments, NfsA comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 114.

[0553] In some embodiments, the payload molecule is a reovirus FAST protein. In some embodiments, the reovirus FAST protein is an ARV p14, a BRV p15, or a p14-p15 hybrid. In some embodiments, the payload molecule is an ARV p14. In some embodiments, the ARV p14 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 115 or consists of it. In some embodiments, the payload molecule is BRV p15. In some embodiments, the BRV p15 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 116 or consists of it. In some embodiments, the payload molecule is a p14-p15 hybrid. In some embodiments, the p14-p15 hybrid comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 117.

[0554] In some embodiments, the payload molecule is leptin / FOSL2. In some embodiments, leptin / FOSL2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118.

[0555] In some embodiments, the payload molecule is adenosine deaminase 2 (ADA2). In some embodiments, adenosine deaminase (ADA2) comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 119.

[0556] In some embodiments, the payload molecule is an alpha-1,3-galactosyltransferase. In some embodiments, the alpha-1,3-galactosyltransferase comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 120.

[0557] In some embodiments, the payload molecule is IL-2. In some embodiments, IL-2 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 121.

[0558] In some embodiments, the payload molecule is IL-7. In some embodiments, IL-7 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 122.

[0559] In some embodiments, the payload molecule is IL12. In some embodiments, the payload molecule comprises an IL-12β subunit comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 123. In some embodiments, the payload molecule comprises an IL-12α subunit comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 124.

[0560] In some embodiments, the payload molecule is IL18. In some embodiments, IL18 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 125.

[0561] In some embodiments, the payload molecule is IL-21. In some embodiments, IL-21 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 126.

[0562] In some embodiments, the payload molecule is IL-36γ. In some embodiments, IL-36γ comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 127.

[0563] In some embodiments, the payload molecule is IFNγ. In some embodiments, IFNγ comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 128.

[0564] In some embodiments, the payload molecule is CCL21. In some embodiments, CCL21 comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 129.

[0565] In some embodiments, the payload molecule is encoded by a polynucleotide molecule according to one of the embodiments provided in Table 20 below.

[0566] Table 20: Non-limiting embodiments of payload configurations

[0567]

[0568]

[0569] *Each 'x' indicates an implementation

[0570] In some embodiments, the payload molecule is a bipartite peptide. As used herein, a "bipartite peptide" refers to a multimeric protein consisting of a first domain capable of binding to a cell surface antigen expressed on a non-cancerous effector cell and a second domain capable of binding to a cell surface antigen expressed by a target cell (e.g., different types of cancer cells, tumor cells, or effector cells). In some embodiments, a single polypeptide domain of the bipartite polypeptide may comprise an antibody or a binding fragment thereof (e.g., a single chain variable fragment (scFv) or F(ab)), a nanobody, a diabody, a flexibody, a DOCK-AND-LOCK TM Antibodies or monoclonal anti-idiotypic antibodies (mAb2). In some embodiments, the structure of the bipartite polypeptide can be a dual variable domain antibody (DVD-Ig TM ), Bispecific T cell engagers (BiTEs TM ), Or dual affinity retargeting (DART) polypeptides. In some embodiments, the bipartite polypeptide is a BiTE and comprises a domain that specifically binds to an antigen shown in Table 8 and / or Table 9. Exemplary BiTEs are shown in Table 7 below.

[0571] Table 7: Validated BiTEs used in preclinical and clinical studies

[0572]

[0573] In some embodiments, the cell surface antigens expressed on the effector cells are selected from Table 8 below. In some embodiments, the cell surface antigens expressed on the tumor cells or effector cells are selected from Table 9 below. In some embodiments, the cell surface antigens expressed on the tumor cells are tumor antigens. In some embodiments, the tumor antigens are selected from CD19, EpCAM, CEA, PSMA, CD33, EGFR, Her2, EphA2, MCSP, ADAM17, PSCA, 17-A1, NKGD2 ligands, CSF1R, FAP, GD2, DLL3, TROP2, Nectin 4 or neuropilin. In other embodiments, the antigen is a viral antigen associated with cancer development. In some embodiments, the viral antigen associated with cancer development is HBV-core (hepatitis B core antigen), HBV-pol, HbS-Ag, HPV E6, HPV E7, Merkel cell polyoma large T antigen or Epstein Barr virus antigen EBNA2 or BZLF1. In some embodiments, the tumor antigen is selected from those listed in Table 9.

[0574] Table 8: Exemplary effector cell target antigens

[0575]

[0576] Table 9: Exemplary target cell antigens

[0577]

[0578]

[0579]

[0580] Pharmaceutical compositions and methods of use

[0581] One aspect of the present disclosure relates to a pharmaceutical composition comprising a recombinant RNA molecule as described herein or a particle comprising a recombinant RNA molecule as described herein, and a method for treating cancer. In some embodiments, the present disclosure provides a method for treating a cancer in a subject in need thereof, the method comprising administering an effective amount of CVA21-EF, CVA21-KY or SVV virus or a corresponding RNA viral genome to the subject. Compositions as described herein can be formulated in any manner suitable for the desired delivery route. Typically, preparations include all physiologically acceptable compositions, including derivatives or prodrugs, solvates, stereoisomers, racemates or tautomers thereof and any pharmaceutically acceptable carriers, diluents and / or excipients.

[0582] As used herein, "pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier approved by the U.S. Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; gum tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered solutions; and any other compatible substances employed in pharmaceutical formulations.

[0583] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and acid addition salts are salts formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and salts formed with organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylaminosulfonic acid (cyclamic acid acid), dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0584] The present disclosure provides a method for killing cancer cells or target cells, the method comprising exposing the cells to RNA polynucleotides or particles described herein or their compositions under conditions sufficient to deliver the composition intracellularly to the cancer cells. As used herein, "cancer cells" or "target cells" refer to mammalian cells selected for treatment or administration with polynucleotides or particles described herein or compositions thereof as described herein. As used herein, "killing cancer cells" specifically refers to the death of cancer cells by apoptosis or necrosis. The killing of cancer cells can be determined by methods known in the art, including but not limited to: tumor size measurement, cell counting, and flow cytometry for detecting cell death markers (such as incorporation of annexin V and propidium iodide).

[0585] The disclosure also provides a method for treating or preventing a cancer in a subject in need, wherein an effective amount of a pharmaceutical composition as described herein is administered to the subject. The route of administration will naturally vary with the position and property of the disease being treated, and may include, for example, intradermal, percutaneous, subcutaneous, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, transdermal (percutaneous), intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration. The polynucleotide compositions of the encapsulation described herein are particularly suitable for treating metastatic cancer, wherein systemic administration may be required to deliver the composition to multiple organs and / or multiple cell types. Therefore, in a specific embodiment, the composition as described herein is systemically administered.

[0586] "Effective amount" or "effective dose" are used interchangeably herein and refer to the amount and or dose of the composition described herein that results in an improvement or remedy in the symptoms of a disease or disorder. An improvement is any improvement or remedy in a disease or disorder or a symptom of a disease or disorder. An improvement is an observable or measurable improvement or may be an improvement in the subject's overall sense of well-being. Thus, those skilled in the art recognize that treatment may improve the disease condition but may not be a complete cure for the disease. Improvements in a subject may include, but are not limited to, a reduction in tumor burden, a reduction in tumor cell proliferation, an increase in tumor cell death, activation of immune p...

Claims

1. A recombinant DNA molecule comprising, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, and a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA virus genome, wherein the 5' junction cleavage sequence comprises or consists of an ENV27 ribozyme coding sequence.

2. A recombinant DNA molecule as described in claim 1, wherein the ENV27 ribozyme coding sequence comprises or consists of a polynucleotide sequence (excluding the P3 stem insertion) that is at least 80% identical to SEQ ID NO: 132 (excluding its P3 stem insertion corresponding to nucleotides 49-54 of SEQ ID NO: 132).

3. A recombinant DNA molecule as described in claim 2, wherein the polynucleotide sequence (excluding the P3 stem insertion) is 100% identical to SEQ ID NO: 132 (excluding its P3 stem insertion corresponding to nucleotides 49-54 of SEQ ID NO: 132), or has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10 or at most 11 mutations (insertions, deletions or substitutions).

4. The recombinant DNA molecule of any one of claims 1 to 3, wherein the polynucleotide sequence is 100% identical to any one of SEQ ID NOs: 130-134, or has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or at most 11 mutations (insertions, deletions, or substitutions).

5. The recombinant DNA molecule of claim 3 or 4, wherein the mutation is a substitution.

6. The recombinant DNA molecule of any one of claims 1 to 5, wherein the ENV27 ribozyme coding sequence comprises a polynucleotide "TTTATT" or "TTTGTT" at a position corresponding to nucleotides 25-30 of SEQ ID NO:

132.

7. The recombinant DNA molecule of claim 6, wherein the ENV27 ribozyme coding sequence comprises a polynucleotide "TTTATT" at positions corresponding to nucleotides 25-30 of SEQ ID NO:

132.

8. The recombinant DNA molecule of any one of claims 2 to 7, wherein the ENV27 ribozyme coding sequence comprises the P3 stem inset of about 1-30, about 1-20, about 6-20, or about 6-10 polynucleotides in length.

9. The recombinant DNA molecule of any one of claims 2 to 8, wherein the P3 stem inset is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 polynucleotides in length.

10. The recombinant DNA molecule of any one of claims 2 to 9, wherein the P3 stem inset is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 polynucleotides in length.

11. The recombinant DNA molecule of any one of claims 8 to 10, wherein the P3 stem insertion comprises or consists of the polynucleotide "AGATCT" at the region corresponding to nucleotides 49-54 of SEQ ID NO:

132.

12. The recombinant DNA molecule of any one of claims 8 to 10, wherein the P3 stem insert comprises or consists of the polynucleotide "AGAGAAATCT" (SEQ ID NO: 137) at the region corresponding to nucleotides 49-54 of SEQ ID NO:

132.

13. The recombinant DNA molecule of any one of claims 8 to 10, wherein the P3 stem insert comprises or consists of the polynucleotide "AGAACGAGAAATCGTTCT" (SEQ ID NO: 138) corresponding to the region of nucleotides 49-54 of SEQ ID NO:

132.

14. The recombinant DNA molecule of any one of claims 1 to 13, comprising from 5' to 3' the promoter sequence, the 5' junction cleavage sequence, the polynucleotide sequence encoding the RNA molecule comprising the synthetic RNA viral genome, and a poly-A tail.

15. The recombinant DNA molecule of claim 14, comprising from 5' to 3' the promoter sequence, the 5' junction cleavage sequence, the polynucleotide sequence encoding the RNA molecule comprising the synthetic RNA viral genome, the poly-A tail, and the 3' junction cleavage sequence.

16. The recombinant DNA molecule of any one of claims 1 to 15, wherein the synthetic RNA viral genome encodes a picornavirus.

17. The recombinant DNA molecule of claim 16, wherein the picornavirus is a coxsackievirus.

18. The recombinant DNA molecule of any one of claims 1 to 17, wherein the 5' end of the RNA viral genome begins with "UUAAA".

19. The recombinant DNA molecule of any one of claims 17 to 18, wherein the Coxsackievirus is a CVA21 strain.

20. The recombinant DNA molecule of claim 19, wherein the CVA21 strain is selected from the group consisting of Kuykendall strain, EF strain and KY strain.

21. A recombinant DNA molecule as described in any one of claims 1 to 20, wherein the 5' end of the RNA viral genome comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to nucleotides 1-260 of any one of SEQ ID NO: 1, 5 or 9.

22. The recombinant DNA molecule of any one of claims 1 to 21, wherein the recombinant DNA molecule does not comprise additional nucleic acid between the 5' junction cleavage sequence and the polynucleotide sequence encoding the RNA molecule.

23. The recombinant DNA molecule of any one of claims 1 to 22, wherein post-transcriptional cleavage of the 5' junction cleavage sequence and / or the 3' junction cleavage sequence produces the native 5' and / or 3' end of the synthetic RNA viral genome.

24. The recombinant DNA molecule of any one of claims 1 to 23, further comprising a leader sequence between the promoter sequence and the 5' junction cleavage sequence.

25. The recombinant DNA molecule of claim 24, wherein the leader sequence is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 60 bp, less than 50 bp, or less than 40 bp in length.

26. The recombinant DNA molecule of any one of claims 24 to 25, wherein the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 135 or 136.

27. The recombinant DNA molecule of any one of claims 24 to 25, wherein the leader sequence comprises or consists of a polynucleotide sequence having a sequence identity of at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO:

135.

28. The recombinant DNA molecule of any one of claims 24 to 27, wherein the recombinant DNA molecule does not comprise additional nucleic acid between the promoter sequence and the leader sequence.

29. The recombinant DNA molecule of any one of claims 24 to 28, wherein the recombinant DNA molecule does not comprise additional nucleic acid between the leader sequence and the 5' junction cleavage sequence.

30. The recombinant DNA molecule of any one of claims 1 to 27, wherein the promoter sequence is a T7 promoter sequence.

31. The recombinant DNA molecule of claim 30, wherein the T7 promoter sequence comprises or consists of SEQ ID NO:

91.

32. The recombinant DNA molecule of any one of claims 1 to 31, wherein the poly-A tail is about 50 bp-90 bp in length or about 65 bp-75 bp in length.

33. The recombinant DNA molecule of claim 32, wherein the poly-A tail is about 70 bp in length.

34. The recombinant DNA molecule of any one of claims 1 to 31, wherein the poly-A tail is about 10 bp-50 bp or 25 bp-35 bp in length.

35. The recombinant DNA molecule of any one of claims 1 to 34, wherein the 3' junction cleavage sequence comprises or consists of a ribozyme sequence.

36. The recombinant DNA molecule of claim 35, wherein the 3' ribozyme sequence is a hepatitis delta virus ribozyme sequence.

37. The recombinant DNA molecule of any one of claims 1 to 34, wherein the 3' junction cleavage sequence comprises or consists of a restriction enzyme recognition sequence.

38. The recombinant DNA molecule of any one of claims 1 to 34, wherein the 3' junction cleavage sequence comprises or consists of a Type IIS restriction enzyme recognition sequence.

39. The recombinant DNA molecule of any one of claims 1 to 38, wherein the 3' junction cleavage sequence comprises or consists of a BsmBI recognition sequence.

40. The recombinant DNA molecule of any one of claims 1 to 38, wherein the 3' junction cleavage sequence comprises or consists of a BsaI recognition sequence.

41. A recombinant DNA molecule as described in any one of claims 1 to 40, wherein the promoter sequence is a T7 promoter sequence, wherein the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 135, wherein the 5' connection cleavage sequence comprises or consists of an ENV27 ribozyme sequence according to any one of SEQ ID NOs: 132-134, wherein the poly-A tail is about 70bp in length, and wherein the 3' connection cleavage sequence comprises or consists of a BsmBI recognition sequence.

42. The recombinant DNA molecule of any one of claims 1 to 40, wherein the promoter sequence is a T7 promoter sequence, wherein the leader sequence is composed of a polynucleotide sequence according to SEQ ID NO: 135, wherein the 5' connection cleavage sequence comprises or consists of an ENV27 ribozyme sequence according to any one of SEQ ID NOs: 132-134, wherein the poly-A tail is approximately 70 bp in length, and wherein the 3' connection cleavage sequence comprises or consists of a BsaI recognition sequence.

43. The recombinant DNA molecule of any one of claims 1 to 42, wherein the recombinant DNA molecule does not comprise additional nucleic acid in the region spanning the promoter sequence and the 3' junction cleavage sequence.

44. A method for producing a recombinant RNA molecule, the method comprising in vitro transcription of a recombinant DNA molecule as claimed in any one of claims 1 to 43 and purification of the resulting recombinant RNA molecule.

45. The method of claim 44, wherein the recombinant RNA molecule comprises the native 5' and 3' ends of the viral genome encoded by the recombinant RNA molecule.

46. ​​A recombinant RNA molecule or a plurality of recombinant RNA molecules transcribed from a recombinant DNA molecule as claimed in any one of claims 1 to 43.

47. The recombinant RNA molecule of claim 46, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of the recombinant RNA molecules comprise the native 5' and 3' ends of the viral genome encoded by the recombinant RNA molecules.

48. The recombinant RNA molecule of claim 46 or 47, wherein no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.5% or no more than 0.1% of the recombinant RNA molecules comprise an RNA sequence encoded by the ENV27 ribozyme encoding sequence.

49. A composition comprising an effective amount of the recombinant RNA molecule of any one of claims 46 to 48, and a carrier suitable for administration to a mammalian subject.

50. A particle comprising the recombinant RNA molecule of any one of claims 46 to 48.

51. The particle of claim 50, wherein the particle is selected from the group consisting of a nanoparticle, an exosome, a liposome, and a liposome complex.

52. The particle of claim 51, wherein the particle is a lipid nanoparticle.

53. A pharmaceutical composition comprising a plurality of particles according to any one of claims 50 to 52.

54. The pharmaceutical composition of claim 53, wherein delivery of the composition to a subject delivers the encapsulated recombinant RNA molecules to target cells, and wherein the encapsulated recombinant RNA molecules produce infectious viruses capable of lysing the target cells.

55. A method of killing cancer cells, the method comprising exposing the cancer cells to the particles or a composition thereof under conditions sufficient to deliver the particles of any one of claims 50 to 52 intracellularly to the cancer cells, wherein replication-competent viruses produced by the encapsulated polynucleotides result in killing of the cancer cells.

56. The method of claim 55, wherein the method is performed in vivo, in vitro or ex vivo.

57. A method of treating cancer in a subject, the method comprising administering to a subject suffering from the cancer an effective amount of a particle according to any one of claims 50 to 52 or a composition thereof.

58. The method of claim 57, wherein the cancer is lung cancer, breast cancer, colon cancer, or pancreatic cancer, and wherein the synthetic RNA viral genome comprises a polynucleotide sequence derived from the KY strain.

59. The method of any one of claims 55 to 58, wherein the cancer is bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer, or liver cancer, and wherein the synthetic RNA viral genome comprises a polynucleotide sequence derived from the EF strain.

60. The method of any one of claims 55 to 58, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, colorectal cancer, colon cancer, pancreatic cancer, liver cancer, renal cell carcinoma, gastric cancer, head and neck cancer, thyroid cancer, malignant glioma, glioblastoma, melanoma, B-cell chronic lymphocytic leukemia, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), Merkel cell carcinoma, diffuse large B-cell lymphoma (DLBCL), sarcoma, neuroblastoma, neuroendocrine carcinoma, rhabdomyosarcoma, medulloblastoma, bladder cancer, and marginal zone lymphoma (MZL).

61. The method of any one of claims 55 to 58, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer, and liver cancer.

62. The method of any one of claims 55 to 58, wherein the cancer is renal cell carcinoma, lung cancer, or liver cancer.

63. The method of any one of claims 55 to 58, wherein the cancer is small cell lung cancer or non-small cell lung cancer (eg, squamous cell lung cancer or lung adenocarcinoma).

64. The method of any one of claims 55 to 58, wherein the cancer is hepatocellular carcinoma (HCC) (eg, hepatitis B virus-associated HCC).

65. The method of any one of claims 55 to 58, wherein the cancer is therapy-induced neuroendocrine prostate cancer.

66. The method of any one of claims 55 to 58, wherein the cancer is lung cancer, liver cancer, prostate cancer (e.g., CRPC-NE), bladder cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, neuroblastoma, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, medulloblastoma, neuroendocrine cancer, Merkel cell carcinoma, or melanoma.

67. The method of any one of claims 55 to 58, wherein the cancer is neuroblastoma.

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