Methods and compositions for circular RNA molecules

Abstract

To provide AAV compositions for circular RNA expression, and to provide methods of expressing covalently closed circular RNA.SOLUTION: Provided is an adeno-associated virus (AAV) genome encoding a circular RNA, the AAV genome comprising, from 5' to 3', the following: (a) a first inverted terminal repeat; (b) a first intron element; (c) a nucleotide sequence of interest; (d) a second intron element; and (e) a second inverted terminal repeat, the (c) being directly linked to the (b) and the (d) respectively, and the first intron element and the second intron element forming at least one pair selected from a specific group. Also provided is a method for expressing a covalently closed circular RNA in a mammalian cell, the method comprising a step of contacting the mammalian cell with an AAV capsid or particle containing the AAV genome.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] [Priority statement] This application is hereby incorporated by reference under 35 U.S.C. § 119(e) into the application filed November 7, 2017. The benefit of U.S. Provisional Patent Application No. 62 / 582,796 is claimed, the entire contents of which are incorporated herein by reference. This document shall form part of the Detailed Description.

[0002] [Statement of government support] This invention was made in part by grant numbers HL089221, HL1127 awarded by the National Institutes of Health. This invention was made with government support under NIH 61, NS099371. have the right.

[0003] [Statement regarding electronic filing of sequence listing] The file is titled 5470-829WO_ST25.txt and is 18,110 bytes in size. 37, generated on November 7, 2018, and filed via EFS-Web. Distributions in ASCII text format submitted under CFR § 1.821 A sequence listing will be submitted in lieu of paper copies. This sequence listing will be used for the purpose of disclosing it. and is incorporated herein by reference.

[0004] The present invention relates to compositions for expressing circular RNA. [Background technology]

[0005] Circular RNA (circRNA) is a non-coding RNA that forms a ring by covalent closure. Circular RNAs are a class of circular RNAs found in a variety of organisms, from archaea to humans. In metazoans, circular RNAs are synthesized by direct backsplicing. They are primarily formed through a process known as ligation, in which donor sites , spliced to an upstream acceptor site (opposite to the normal splicing direction) In many cases, circRNAs consist of circularized exons. Therefore, it is understood that circRNA products and linear mRNA products compete with each other. The ratio of circular to linear products varies depending on the gene, but some In the case of genes, circRNAs are the major RNA products. The signal affecting this is likely encoded within the surrounding intron sequences. revealed that the presence of inverted repeats (e.g., Alu elements) correlates with circularization. Furthermore, RNA-binding proteins such as Quaking (QKI) have been shown to bind to circular RNAs. It is known that it is involved in the regulation of metabolism.

[0006] Recent studies have shown that circRNAs are highly expressed in multiple cell and tissue types. However, the functions of most circRNAs remain unknown. A few exceptions, such as miRNA-7 and MBL protein sponges, respectively, Others that have been characterized include ciRS-7 / Cdr1as and circMbl. Some recent studies suggest that endogenous circRNAs may have coding capabilities. However, the translation efficiency was extremely low at best, and other studies have not yet investigated the relationship between circRNA and ribosomes. In any case, circRNAs are , IRES ( It can be engineered to contain an internal ribosome entry site (circR) sequence. An interesting property of NA is that it lacks free termini, making it more flexible than the linear isoform. Some studies have shown that circRNAs are highly stable due to their demonstrated a half-life at least 2.5 times longer than that of its linear counterpart. The present invention provides a method for the stable and continuous expression of proteins from RNA molecules. Due to these characteristics, circRNAs can be used to identify long-range genes. This makes them useful in gene delivery applications where expression is a major concern. Summary of the Invention

[0007] This summary lists some embodiments of the subject matter disclosed herein and also provides many Where appropriate, variations and permutations of these embodiments are listed. In a given embodiment, one or more representative features may be used. Although certain characteristics are described, they are also exemplary. Such embodiments may be modified to suit the described characteristics. (or plural) may be present in general, but equally, such characteristics are the subject matter disclosed herein, whether or not listed in this Summary. To avoid excessive repetition, this summary will be The present disclosure does not list or suggest all possible combinations of characteristics.

[0008] In one aspect, the present invention provides a method for producing covalently closed circular RNA (circRNA). a) a nucleic acid molecule that is transcribed into non-coding RNA or translatable mRNA; b) a gene of interest that can be expressed in a specific sequence; and b) an intron element associated with said gene of interest, Bax is spliced by the cellular splicing machinery to yield a covalently closed circular RNA. and c) a translatable intron element transcribed from the gene of interest. an internal ribosome entry site (IRES) that drives translation of the mRNA; and d) a 5' untranslated region. Located within the UTR and outside the intron element associated with the gene of interest. e) a promoter region within the 3'UTR and an intron associated with the gene of interest; and a translational control region located outside the element.

[0009] The present invention provides a method for expressing a covalently closed circular RNA molecule in a cell. and producing the covalently closed circular RNA molecule of the present invention in the cell under conditions in which the covalently closed circular RNA molecule is transcribed. the nucleic acid molecule of the present invention, the AAV genome of the present invention, the AAV capsid or particle of the present invention, and / or the The present invention further provides a method comprising introducing the composition of the present invention.

[0010] In another embodiment, the present invention provides a method for covalently binding nucleotides in a tissue-specific and / or cell-specific manner. A method for expressing a covalently closed circular RNA molecule in a The tissues and / or cells are treated with the nucleic acid molecules of the present invention, the AA molecules of the present invention under conditions in which the AA molecules are expressed. V genome, AAV capsid or particle of the invention, and / or a composition of the invention. The present invention provides a method comprising: [Brief explanation of the drawings]

[0011] [Figure 1A]Schematic diagram showing splicing patterns and reporter output. RNA from the IRES-GFP construct is expressed from a CMV promoter and is capped and polyadenylated in its linear isoform. The control IRES-GFP transcript contains the EMCV-IRES followed by GFP. [Figure 1B] Schematic diagram showing splicing patterns and reporter output. RNA from the ZKSCAN1 split-GFP or HIPK3 split-GFP constructs is expressed from a CMV promoter and is capped and polyadenylated in its linear isoform. The precursor split-GFP transcript contains a split-GFP cassette flanked by intronic sequences from the human ZKSCAN1 or HIPK3 gene. The donor and acceptor splice sites are represented by gray triangles, and dotted lines indicate the backsplice pattern. The GFP fragments are separated by an EMCV-IRES, allowing full-length GFP to be expressed upon RNA circularization. [Figure 2A-2B] CircRNA expression in a tissue culture model of glioblastoma. (2A) Representative images of GFP fluorescence from U87 cells expressing IRES-GFP (left), ZKSCAN1 split-GFP (center), or HIPK3 split-GFP (right) 4 days after transduction with recombinant AAV2 vectors at 100,000 vector genomes per cell. (2B) Western blot detecting GFP or loading control actin in lysates of U87 cells transduced as above, as quantified in (2C). [Figure 2C] CircRNA expression in a tissue culture model of glioblastoma. (2B) Western blot detecting GFP or loading control actin in lysates of U87 cells transduced as described above, quantified in (2C). [Figure 2D]Figure 2 shows circRNA expression in a tissue culture model of glioblastoma. (2D) Northern blot of total cellular RNA probed for GFP to characterize the various RNA species expressed from IRES-GFP, ZKSCAN1 split-GFP, or HIPK3 split-GFP constructs. The position of 18S rRNA is indicated as a size marker. A schematic diagram of the expected RNA species is displayed. [Figure 2E] Figure 1 shows circRNA expression in a tissue culture model of glioblastoma. CircRNA bands are quantified relative to IRES-GFP RNA levels. [Figure 3A] (3A) CircRNAs are expressed in mouse cardiac tissue. The indicated constructs were packaged into recombinant AAV9 vectors and intravenously injected into C57 / BL6 mice at a dose of 5.5e11 vector genomes per animal. The mice were harvested 4 weeks post-injection. (3A) Cardiac tissue was sectioned and subsequently immunohistochemically stained to visualize GFP expression. [Figure 3B-3C] (3B) CircRNA expression in mouse heart tissue. The indicated constructs were packaged into recombinant AAV9 vectors and intravenously injected into C57 / BL6 mice at a dose of 5.5e11 vector genomes per animal. The mice were harvested 4 weeks post-injection. (3B) GFP expression levels in stained heart sections were quantified by mean pixel intensity. (3C) Quantification of viral genome copy numbers within each cohort by qPCR using primers specific for the CMV promoter. Normalized to the mouse lamin B2 locus. [Figures 3D-3F]Figure 1 shows the expression of circRNAs in mouse cardiac tissue. The indicated constructs were packaged into recombinant AAV9 vectors and injected intravenously into C57 / BL6 mice at a dose of 5.5e11 vector genomes per animal, with harvest occurring 4 weeks after injection. (3D) RNA was extracted from cardiac tissue, and RT-PCR was performed using primers that amplify across the backsplice junction (see schematic). These primers also amplify full-length GFP generated within the IRES-GFP. (3E) Quantitative RT-PCR was performed using the same samples and primers as specified in (3D). (3F) RNA was treated with RNAse R, and then RT-PCR was performed as in (3D). [Figure 4A] Figure 4A shows the expression of circRNAs in central nervous system tissues. (4A) The indicated constructs were packaged into recombinant AAV9 vectors and injected into the left ventricle of C57 / BL6 mice at a dose of 3.989e10 vector genomes per animal. The mice were harvested 6 weeks after injection. GFP expression was visualized by immunohistochemical staining of brain sections. Representative images of the cortex are shown. [Figure 4B-4C] Figure 4 shows the expression of circRNAs in central nervous system tissues. (4B) Quantification of GFP-positive cells in the samples shown in (4A). (4C) The indicated constructs were packaged into recombinant AAV9 vectors and intravitreally injected into C57 / BL6 mice at a dose of 1e10 vector genomes per animal. The mice were harvested 4 weeks after injection. Representative images of sectioned retinas are shown. Green: immunofluorescent staining for GFP. Blue: DAPI staining of cell nuclei. [Figure 4D-4E] Figure 4 shows the expression of circRNAs in central nervous system tissues. (4D) Quantification of GFP expression by corrected mean fluorescence. (4E) RNA was extracted from injected retinas and quantitative RT-PCR was performed using a primer set that amplifies across back-splice junctions. [Figure 5]CircRNA expression in tissue culture models. Representative images of GFP fluorescence from HEK293 (far left), Huh7 (middle left), U87 (middle right), or Neuro2A (far left) cells expressing IRES-GFP (top), ZKSCAN1 split-GFP (middle), or HIPK3 split-GFP (bottom) after transduction with recombinant AAV2 vectors at 100,000 vector genomes per cell. [Figure 6] CircRNA expression in mouse liver. The indicated constructs were packaged into recombinant AAV9 vectors and injected intravenously into C57 / BL6 mice at a dose of 5.5e11 vector genomes per animal, with the mice harvested 4 weeks post-injection. Liver tissue was sectioned and then immunohistochemically stained to visualize GFP expression. [Figure 7A] Comparison of split and non-split circRNA expression vectors. (7A) The precursor split-GFP transcript contains a split-GFP cassette flanked by intronic sequences derived from the human ZKSCAN1 or HIPK3 gene. The donor and acceptor splice sites are represented by gray triangles, and the dotted line indicates the backsplice pattern. [Figure 7B-7C] Comparison of split and non-split circRNA expression vectors. (7B) Representative images of GFP fluorescence from U87 cells expressing ZKSCAN1 GFP (top) or ZKSCAN1 split GFP (bottom) 4 days after transduction with recombinant AAV2 vectors at 100,000 vector genomes per cell. (7C) Western blot detecting GFP or loading control actin in lysates of U87 cells transduced as above, quantified in (7D). [Figure 7D-7E]Comparison of split and non-split circRNA expression vectors. (7C) Western blot detecting GFP or loading control actin in lysates of U87 cells transduced as described above and quantified in (7D). (7E) RNA was extracted and quantitative RT-PCR was performed using primers that amplify across the backsplice junction. [Figure 7F] Comparison of split and non-split circRNA expression vectors. (7F) The indicated constructs were packaged into recombinant AAV9 vectors and intravenously injected into C57 / BL6 mice at a dose of 5.5e11 vector genomes per animal, with the mice harvested 4 weeks post-injection. Heart tissue was sectioned and then immunohistochemically stained to visualize GFP expression. [Figure 8] Figure 1 shows data summarizing the ability of different intron elements to support circular RNA formation and translation. These data enable and support the modular design of circular RNA-coding constructs. Introns from the ZKSCAN1, HIPK3, laccase2, and EPHB4 genes were used to create CircRNA-generating cassettes. These constructs were packaged into rAAV2 and used to transduce U87 cells at an MOI of 100,000 vg / cell. Images were acquired on day 4. [Figure 9A] (9A) Representative fluorescence image. [Figure 9B-9C] This figure shows that the 3' (right) intron sequence tolerates insertions. Inserting sequences into the right intron of the circRNA generation cassette does not reduce the amount of circRNA produced. (9B) Western blot of GFP translated from circular RNA and control actin. Data are normalized and quantified in (9C). [Figure 9D](9D) Northern blot probed for GFP. The circRNA band is shown. [Figure 9E-9F] (9E) Northern / quantification of circular RNA expression. (9F) Schematic of RNA precursors encoded by AAV vector genomes with insertions within the right (3') intron. Insertions of 100-1000 bases in length do not affect circular RNA levels or translation of circular RNA to GFP. Insertions of 1500 bases improve circular RNA translation, but do not improve circular RNA expression by more than two-fold. [Figure 10A] (10A) Representative fluorescence image. The 5' (left) intron sequence does not tolerate insertions. Inserting a sequence into the left intron of the circRNA generation cassette reduces the amount of circRNA produced. [Figures 10B-10C] (10B) Western blots for GFP and actin, as quantified in (10C). [Figure 10D] (10D) Northern blot probed for GFP. The circRNA band is shown. [Figures 10E-10F](10E) Northern blot quantification. (10F) Schematic of RNA precursors encoded by AAV vector genomes with insertions within the left (5') intron. Insertions of 100-1500 bases in length significantly reduce circular RNA levels or translation of circular RNA to GFP. [Figure 11A] (11A) Representative fluorescence images. (11B) Deletion of intronic sequences in the left intron (while preserving the Alu repeat elements and splice acceptor / donor sites) leads to increased circRNA production. Complete deletion of these sequences in the right intron results in a loss of circRNA formation, but smaller deletions are tolerated. [Figures 11B-11C] (11B) Western blots for GFP and actin, quantified in (11C). [Figure 11D] (11D) Northern blot probed for GFP. CircRNA bands are shown. [Figures 11E-11F](11E) Northern blot quantification. (11F) Schematic diagram. Deletion of intronic sequences in the left intron (while preserving the Alu repeat elements and splice acceptor / donor sites) leads to increased circRNA production. Complete deletion of these sequences in the right intron results in abrogation of circRNA formation, but smaller deletions are tolerated. [Figure 12A] (12A) Representative fluorescence images. Combining deletions in the left and right introns results in a >5-fold increase in circRNA production of the construct. [Figures 12B-12C] Combining deletions in the left and right introns results in a >5-fold increase in circRNA production of the construct. (12B) Western blot for GFP and actin, quantified in (12C). [Figure 12D] (12D) Northern blot probed for GFP. The circRNA band is shown. [Figures 12E-12F] (12E) Northern blot quantification. (12F) Schematic diagram. Combining deletions in the left and right introns results in a >5-fold increase in circRNA production of the construct. [Figure 13A] (13A) Representative fluorescence images. The differences between RΔfull and RΔminimal are not sequence-specific. A deletion (RΔ150) with identical full-length but residual different sequence is also tolerated. [Figures 13B-13D] (13B) Western blots for GFP and actin, quantified as in (13C). (13D) Schematic diagram. [Figure 14A](14A) Alignment of left and right intron sequences for HIPK3 (left: SEQ ID NO: 15; right: SEQ ID NO: 16). Bolded regions indicate repeat sequences containing identified complementarity, and underlined regions indicate permissible deletions resulting in synthetic intron elements. HIPK3 (homeodomain-interacting protein kinase 3) is a protein-coding gene. Its associated pathways include, among others, cellular senescence (KEGG). Gene Ontology (GO) annotations associated with this gene include phosphoryl transferase activity and protein tyrosine kinase activity. [Figure 14B] (14B) Alignment of left and right intron sequences for ZKSCAN1 (left: SEQ ID NO: 13; right: SEQ ID NO: 14). Bolded regions indicate repeat sequences containing identified complementarity, and underlined regions indicate permissible deletions resulting in synthetic intron elements. ZKSCAN1: This gene encodes a member of the Krüppel C2H2-type zinc finger family of proteins. The protein it encodes may function as a transcription factor regulating GABA type A receptor expression in the brain. Transcripts from this gene have been shown to form stable and abundant circular RNA. Increased expression of this gene has been observed in gastric cancer, and the encoded protein can stimulate the migration and invasion of human gastric cancer cells. [Figure 14C] (14C) Sequences of left and right intron sequences for EPHB4 (left: SEQ ID NO: 29; right: SEQ ID NO: 30). Bolded regions indicate repeat sequences containing identified complementarity, and underlined regions indicate permissible deletions resulting in synthetic intron elements. EPHB4: (EPH receptor B4) is a protein-coding gene. Diseases associated with EPHB4 include hydrops fetalis, non-immune and / or atrial septal defect, and hydrops fetalis. Its associated pathways include ERK signaling and Akt signaling, among others. Gene Ontology (GO) annotations associated with this gene include phosphoryl transferase activity and protein tyrosine kinase activity. [Figure 14D] (14D) Alignment of left and right intron sequences for laccase 2 (left: SEQ ID NO: 31; right: SEQ ID NO: 32). Bolded regions indicate repeat sequences containing identified complementarities, and underlined regions indicate permissible deletions resulting in synthetic intron elements. Laccase 2: a protein-coding gene. Its functions include copper ion binding, ferroxidase activity, cuticle expression in Drosophila, and ion transport, among others. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will now be described with reference to the accompanying drawings, which show exemplary embodiments of the invention. However, the invention may be embodied in different forms and the embodiments set forth herein may be modified in various ways. Rather, these embodiments are intended to be exhaustive and not to be limiting. This disclosure is provided so that it is comprehensive and complete, and will fully convey the scope of the invention to those skilled in the art.

[0013] Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present invention. The term "a" has the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. The terminology used in the description of the present invention is limited to the purpose of describing particular embodiments and is not intended to be limiting of the present invention. All publications, patents, and other references cited herein are hereby incorporated by reference in their entirety. Applications, patents, and other references are incorporated herein by reference in their entirety. do.

[0014] [Definition] The following terms may be used in the description and appended claims: The singular forms "a," "an," and "the" also refer to the plural forms unless the context clearly indicates otherwise. It may be intended to be included.

[0015] Furthermore, the term "about" as used herein refers to a measurable number, e.g., a polynucleotide. When expressing the amount of a peptide or polypeptide sequence, such as length, dosage, time, temperature, etc., the amount is expressed as ±2 of the specified amount. Including variations of 0%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. This could mean:

[0016] Also as used herein, "and / or" means the combination of the associated listed items. Any and all possible combinations of one or more of, and alternatively (" When interpreted as "or"), it means the absence of a combination and is inclusive.

[0017] Unless the context indicates otherwise, the various features of the invention described herein may be used in any combination. It is specifically intended that the invention be usable with

[0018] Furthermore, the present invention, in some embodiments of the present invention, includes any of the properties or We also consider whether combinations of features can be excluded or omitted.

[0019] To further illustrate, for example, certain amino acids may be selected from A, G, I, L, and / or V. When the specification suggests that an amino acid may be selected, this expression means that the amino acid is one of these amino acids ( Any subset of the above (may be plural), for example, A, G, I, or L; A, G, I, or V ;A or G;L alone, etc., as if each such subcombination were included in the present specification. Furthermore, such expressions are also intended to be illustrative and not restrictive. It also suggests that one or more of the specified amino acids may be negated. In certain embodiments, the amino acid is not A, G, or I; is not A; is not G or V. etc., as if each such possible contrary were expressly set forth herein. It is denied as such.

[0020] As used herein, the terms "reduce" and "reduce" "reduces," "reduction," and similar terms At most about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, It may mean a decrease or even greater.

[0021] As used herein, the terms "enhance" and "enhance" refer to "enhancements," "enhancement," and similar terms At least about 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 2 This may suggest an increase of 00%, 300%, 400%, 500% or more.

[0022] The term "parvovirus" as used herein includes autonomously replicating parvoviruses and desmoviruses. The family Parvoviridae, which includes the Dependoviruses, may be included. The autonomous parvoviruses are in the genus Parvovirus, Erythrovirus, Densovirus, The genus Iteravirus and Contravirus Representative free-standing parvoviruses include, but are not limited to, members of the genus Parvovirus. Not commonly transmitted by minute virus of mice, bovine parvovirus, or canine parvovirus , Chicken parvovirus, Feline panleukopenia virus, Feline parvovirus, Gacho Muscovy parvovirus, HI parvovirus, Muscovy duck parvovirus, B19 virus, and any other free-standing parvovirus now known or later discovered. Other self-sustaining parvoviruses are known to those skilled in the art, for example, BE RNARD N.FIELDS et al,VIROLOGY,volume 2,c hapter69(4th ed., Lippincott-Raven Publis See hers).

[0023] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, Not specified, but AAV1, AAV2, AAV3 (including 3A and 3B), AA V4 type, AAV5 type, AAV6 type, AAV7 type, AAV8 type, AAV9 type, AAV10 type , AAV11 type, AAV12 type, avian AAV, bovine AAV, canine AAV, equine AAV, human AAV, and any other AAV now known or later discovered. For example, Bernard N. Fields et al., Virology, lume 2, chapter 69 (4th ed., Lippincott-Rave See the AAV Publishers website for some relatively new AAV serotypes and clades. have been identified (e.g., Gao et al. (2004) J Virology 78:6381-6388;Moris et al. (2004) Virology 33-:375-383; and see Table 1).

[0024] The genome sequences of various serotypes of AAV and autonomous parvoviruses, as well as the natural terminal repeats The sequences of the repeat repeat (TR), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases, e.g., GenBan. k® database, etc., for example, GenBank accession number NC_ 044927, NC_002077, NC_001401, NC_001729, NC_ 001863, NC_001829, NC_001862, NC_000883, NC_ 001701, NC_001510, NC_006152, NC_006261, AF0 63497, U89790, AF043303, AF028705, AF028704, J02275, JO1901, J02275, X01457, AF288061, AH0 09962, AY028226, AY028223, NC_001358, NC_001 540, AF513851, AF513852, AY530579; the disclosures of which are The nucleic acid and amino acid sequences of parvoviruses and AAV are taught herein. See, for example, Srivistava et al. 983)J Virology 45:555;Chiorini et al.(19 98) J. Virology 71:6823;Chiorini et al(199 9)J Virology 73:1309;Bantel-Schaal et al. (1999) J. Virology 73:939;Xiao et al. (199) 9) J. Virology 73:3994; Muramatsu et al. (19 96) Virology 221:208;Shade et al. (1986)J Virol.58:921;Gao et al.(2002)Proc.Nat.Ac ad.Sci.USA 99:1 1854;Morris et al.(2004)V irology 33:375-383; WO 00 / 28061, WO 99 / 6 1601, 98 / 11244; and U.S. Pat. No. 6,156,303; The disclosure of See also Table 1, which is incorporated herein by reference.

[0025] The capsid structure of autonomous parvoviruses and AAVs is described by Bernard N. Field. DS et al.,VIROLOGY,volume 2,chapters69 & 70 (4th ed., Lippincott-Raven Publishers) See also the description of the crystal structure of AAV2 (Xi e et al.(2002)Proc.Nat.Acad.Sci.99:10405 -10),AAV4(Padron et al.(2005)J Virol.79: 5047-58),AAV5(Walters et al.(2004)J Viro l.78:3361-71), and CPV (Xie et al. (1996) J Mo l.Biol.6:497-520, and Tsao et al. (1991)Scie nce 251:1456-64).

[0026] The term "tropism" as used herein refers to the ability of a virus to infect specific cells or tissues. and optionally, the sequence(s) carried by the viral genome within the cell. expression (e.g., transcription, and optionally translation) of a desired gene, e.g., in the case of a recombinant virus This may mean that the expression of the heterologous nucleic acid(s) is / are followed by the expression of the heterologous nucleic acid(s). Transcription of the heterologous nucleic acid sequence from the genome can be achieved using, for example, an inducible promoter or other promoters. Initiation may not occur in the absence of trans-acting factors for regulatory nucleic acid sequences. In the case of rAAV genomes, gene expression from the viral genome is stable. from locally integrated proviruses, non-integrated episomes, and viruses It may arise from any other form that is available within the cell.

[0027] As used herein, "systemic tropism" and "systemic transduction" (and the like) are used interchangeably. and the like) refers to the viral capsid or viral vector of the present invention being capable of transmitting the virus to a body-wide system. tissues (e.g., brain, eye, lung, skeletal muscle, heart, liver, kidney, and / or pancreas), These may indicate tropism or may be indicative of transduction. In embodiments, systemic transduction of muscle tissue (e.g., skeletal muscle, diaphragm muscle, and cardiac muscle) is performed. In other embodiments, systemic transduction of skeletal muscle tissue is achieved. For example, in certain embodiments, substantially all skeletal muscle is transduced throughout the body. In particular embodiments, transduction efficiency may be achieved in limb muscles, Systemic transduction of the myocardium and diaphragm muscle is achieved. The drug or viral vector is administered systemically (e.g., intravenously, intra-articularly, or intralymphatically). It can be administered by the systemic route.

[0028] Alternatively, in other embodiments, the capsid or viral vector is delivered locally. In a further embodiment, the capsid or In some embodiments, the capsid or viral vector is delivered to the eye. The drug may be delivered via intravitreal, subretinal, subconjunctival, retrobulbar, anterior chamber, and / or suprachoroidal routes. It is administered.

[0029] Unless otherwise specified, "effective transduction" or "effective tropism" or similar terms are used. The term can be determined by reference to a suitable control (e.g., control At least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or In certain embodiments, The virus vector is distributed in skeletal muscle, cardiac muscle, diaphragm muscle, pancreas (including β-islet cells), spleen, gastrointestinal ducts (e.g., epithelium and / or smooth muscle), cells of the central nervous system, lungs, joint cells, kidneys, and / or or a tropism that is effectively transduced into or effective against a cell or cell layer of the eye. Suitable controls depend on a variety of factors, including the desired tropism profile. Depends.

[0030] Similarly, if a virus does not "transduce effectively" or "effectively" target tissue, For "having no significant tropism" or similar terms, please refer to the appropriate control. In certain embodiments, the viral vector is directed against the liver, kidney, or does not effectively transduce the pancreas, gonads, and / or germ cells (i.e., In certain embodiments, the unwanted tissue(s) ) (e.g., liver) can be transduced into the desired target tissue(s) (e.g., skeletal compared to the level of transduction into cells of the spleen, diaphragm, cardiac muscle, and / or central nervous system Less than 20%, less than 10%, less than 5%, less than 1%, and less than 0.1%.

[0031] As used herein, the term "polypeptide" refers to peptides and The term may encompass both proteins.

[0032] A "polynucleotide" can be a sequence of nucleotide bases and can be RNA, DNA, or DNA-RNA hybrid sequences (naturally occurring and non-naturally occurring nucleotides) In typical embodiments, the DNA sequence is a single-stranded or double-stranded DNA sequence, although the DNA sequence may be either single-stranded or double-stranded. .

[0033] As used herein, an "isolated" polynucleotide (e.g., an "isolated D "RNA" or "isolated RNA" refers to at least one naturally occurring organism or virus. Some other components, e.g., structural components of cells or viruses Nucleotides commonly found in association with polypeptides or other polypeptides or polynucleotides It can refer to a polynucleotide that is at least partially separated from an acid. An "isolated" nucleotide is at least about 10-fold, 100-fold, or 100-fold more abundant than the starting material. 1000-fold, 10,000-fold, or more concentrated.

[0034] Similarly, an "isolated" polypeptide is at least partially isolated from a naturally occurring organism or virus. and some other components, e.g., structural components of cells or viruses. components, or other polypeptides, or nucleic acids commonly found in association with polypeptides. It can refer to a polypeptide that is at least partially separated from an acid. An "isolated" polypeptide is one that is at least about 10-fold, 100-fold, or more abundant than the starting material. , 1000-fold, 10,000-fold, or more concentrated.

[0035] As used herein, to "isolate" or "purify" a viral vector (or (grammatically equivalent term) means that the viral vector is a product of at least some of the components in the starting material. This may mean that the device is at least partially isolated from other components of the device. In embodiments, an "isolated" or "purified" viral vector is a vector that is more purified than the starting material. and at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more concentrated It is shrunk.

[0036] A "therapeutic protein" refers to a protein that is absent or inactive in a cell or subject. The present invention can alleviate, reduce, prevent, delay, and / or stabilize symptoms resulting from a deficiency of and / or otherwise confer a benefit to the subject. It's quality.

[0037] "Therapeutic RNA molecule" or "functional RNA molecule" as used herein refers to a therapeutic RNA molecule that is a therapeutically effective therapeutic agent. As known in the art, antisense nucleic acids, ribozymes (see, e.g., U.S. Pat. No. 5,629,299, and U.S. Pat. No. 5,629,299) and other nucleic acids are useful. ,877,022), spliceosome-mediated trans-splicing RNA that enables ligation (Puttaraju et al. (1999) Nature e Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No. 6,083 ,702), siRNA, shRNA, or miR involved in silencing gene expression Interfering RNA (RNAi) containing NA (Sharp et al., (2000) Science nce 287:2431), and any other non-coding RNA, e.g., "guide ”RNA et al. (Gorman et al. (1998) Proc.Nat.Acad.S ci USA 95:4929; Yuan et al., U.S. Patent No. 5,869,24 No. 8) etc.

[0038] The terms "treatment," "treating," or "treatment of" (and their grammatical variations) is when the severity of the subject's condition is reduced, at least partially improved, or stabilized. and / or any relief, alleviation, reduction, or slight decrease in at least one clinical symptom. It means that a reduction or stabilization of the disease or disorder is achieved and / or a delay in progression of the disease or disorder is observed. You can taste it.

[0039] The terms "prevent", "preventing", and "prevention" (and their grammatical variations) The therapeutic agent (or agents) is intended to prevent or treat the onset of a disease, disorder, and / or clinical condition(s) in a subject. and / or delay, and / or compared to that which would occur in the absence of the method of the present invention. It may refer to a reduction in the severity of the disease, disorder, and / or clinical symptom(s). Prevention may be complete, e.g., the disease, disorder, and / or clinical symptom(s) are completely prevented. Prevention includes preventing or reducing the severity of a disease, disorder, and / or clinical condition(s) in a subject. the occurrence and / or severity of the manifestations is less than that which would occur in the absence of the present invention. It may be partial, as in

[0040] A "therapeutically effective" amount, as used herein, is an amount that provides some improvement or benefit to a subject. In other words, a "therapeutically effective" amount can be an amount sufficient to induce at least It can be an amount that provides some relief, alleviation, reduction, or stabilization of a clinical symptom. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, so long as some benefit is provided to the subject. Recognize that it doesn't have to be objective.

[0041] A "prophylactically effective" amount, as used herein, refers to the amount of a compound that is effective to treat a disease, disorder, and / or condition in a subject. prevents and / or delays the onset of clinical symptoms and / or reduces the severity of a disease in a subject, The severity of the disorder and / or clinical manifestations may be compared to those that would occur in the absence of the methods of the present invention. The level of prevention can be an amount sufficient to reduce and / or delay the progression of the disease compared to the normal progression of the disease. We recognize that a rule need not be perfect as long as some benefit is provided to the subject.

[0042] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein. Heterologous nucleic acid may refer to a nucleic acid sequence that is not naturally occurring within a virus. is an open reading frame encoding a protein or a non-translated RNA of interest. (e.g., for delivery to a cell or subject).

[0043] As used herein, the term "viral vector" "Virus vector," "vector," "virus particle" "particles," "recombinant viral vectors" A "gene delivery vector" or "gene delivery vector" is a molecule capable of functioning as a nucleic acid delivery vehicle. , the vector genome packaged within the virion (e.g., viral DNA [vDN A virus (e.g., AAV) particle containing a nucleotide sequence (e.g., a nucleotide sequence [A]).

[0044] Alternatively, in some contexts, the term "vector" may refer to the vector genome / vDNA alone. It can be used to mean.

[0045] As used herein, the term "capsid" or "viral capsid" refers to a capsid It means a capsid structure made of proteins, in which case the capsid contains the viral genome ( For example, it includes nucleic acid molecules that may be genomes, such as the AAV genome within an AAV capsid.

[0046] A "rAAV vector genome" or "rAAV genome" refers to one or more heterologous nucleic acid sequences The rAAV vector may be an AAV genome (i.e., vDNA) comprising the virus Generally, only the terminal repeats (TRs) in cis are required to generate All other viral sequences are not necessary and may be supplied in trans. Obtain (Muzyczka(1992)Curr.Topics Microbiol.I In general, the rAAV vector genome is To maximize the size of the transgene that can be effectively packaged, one or more The structural and non-structural protein coding sequences are contained in the TR sequence. The sequence may be provided in a form suitable for use in the human genome (e.g., from a vector such as a plasmid, or by packaging the sequence). In some embodiments of the invention, the rAAV vector The genome may contain at least one terminal repeat (TR) sequence (e.g., an AAV-TR sequence), optionally Optionally, two TRs (e.g., two AAV-TRs) are included, which are generally included in the vector. - Located at the 5' and 3' ends of the genome and adjacent to, but not contiguous with, the heterologous nucleic acid sequence The TRs may be identical or may differ from each other.

[0047] The term "terminal repeat" or "TR" refers to a terminal repeat that forms a hairpin structure and functions as an inverted terminal repeat. capable of carrying out the desired functions (i.e., replication, viral packaging, integration, and and / or provirus rescue) The TR may comprise any viral long terminal repeat sequence or synthetic sequence. For example, the TR sequences can be from non-AAVs, such as other parvoviruses (e.g., For example, canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus or any other suitable viral sequence (e.g., SV40 The SV40 hairpin, which serves as the origin of replication, can be used as a TR, and the TR is Further modifications can be made by truncation, substitution, deletion, insertion, and / or addition. The TR may be partially or completely synthetic, e.g., U.S. Pat. No. 5,629,299 to Samulski et al. It may be a "double D arrangement" such as that described in US Pat. No. 4,478,745.

[0048] "AAV terminal repeat" or "AAV-TR" refers to, but is not limited to, serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any AAV currently Derived from any other known or later discovered AAV (see, e.g., Table 1) The AAV terminal repeat sequences may be used to enhance the expression of the AAV virus. Naturally occurring proteins are involved in the packaging, integration, and / or proviral rescue of the virus. It is not necessary to have terminal repeat sequences (e.g., the native AAV-TR sequence may contain insertions, deletions, (This may be altered by truncation, and / or missense mutations).

[0049] The viral vectors of the present invention are further described in International Patent Publication No. WO 00 / 28004. and Chao et al. (2000) Molecular Therapy 2:6 As described in 19, "targeted" viral vectors (e.g., targeted transcription factors) can be used. tropism), and / or "hybrid" parvoviruses (i.e., viruses The TR and viral capsid may be from different parvoviruses.

[0050] The viral vector of the present invention is further described in International Patent Publication No. WO 01 / 92551. (the disclosure of which is incorporated herein by reference in its entirety), Thus, in some embodiments, the parvovirus particles may be double-stranded (double stranded). The duplex genome can be packaged in the viral capsid of the present invention. .

[0051] Additionally, viral capsid or genomic elements may contain insertions, deletions, and / or substitutions. It may contain other modifications.

[0052] As used herein, the term "amino acid" refers to any naturally occurring amino acid, Modified forms and synthetic amino acids may be included. Naturally occurring levorotatory (L-) amino acids The acids are shown in Table 2.

[0053] Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are shown in Table 3). , and / or modified by post-translational modifications (e.g., acetylation, amidation, formylation) , hydroxylation, methylation, phosphorylation, or sulfatation) It may be an amino acid.

[0054] Additionally, non-naturally occurring amino acids can be synthesized by the method of Wang et al. (Annu Rev Biophys Biomol Struct. 35:225-49 (2006) These unnatural amino acids can be used to convert a molecule of interest into an A. It can be advantageously used to chemically link to AV capsid proteins.

[0055] [Nucleic acid molecules, and viral capsids and viral vectors containing the same] In one embodiment, the present invention provides covalently closed circular RNAs (circRNAs). and a nucleic acid molecule encoding a non-coding RNA or a translatable RNA. a) a gene of interest that can be transcribed into a suitable mRNA; b) an interleukin (interleukin) flanking said gene of interest; A transtronic element that is transduced into a cell membrane to produce a covalently closed circular RNA. c) an intron element that is backspliced by the splicing mechanism; Internal ribosome entry sites drive translation of translatable mRNA transcribed from target genes. (IRES); d) an IRES within the 5' untranslated region (UTR) and associated with the gene of interest e) the promoter region, located outside the 3'UTR; and f) the promoter region, located outside the 3'UTR. a translational control region located outside the intronic elements associated with the gene of interest; .

[0056] In some embodiments, the intron element of (b) can be any number of overlaps and / or Consists of any known intron element(s) in any combination of ratios Examples of intronic elements include those described by Rybak-Wolf et al. Mol.Cell 58(5):870-885(2015) , and the circBase circular RNA database (Glazar et al. RNA 20:1666-1670 (2014); and www.circbase.org and US Pat. No. 6,499,099, all of which are incorporated herein by reference in their entirety. There are.

[0057] In some embodiments, the intron element of (b) is SEQ ID NO: 13-24 or Any combination of any of 29 to 32 nucleotide sequences with any overlap number and / or ratio. It may comprise, consist essentially of, or consist of.

[0058] In some embodiments, the IRES in (c) can be any one or any combination thereof. , and / or any number and / or ratio of overlapping viral IRESs listed in Table 5, Table The IRES may be a cellular IRES as described in

[0059] In some embodiments, the translational control region of (e) is a transcription factor, as known in the art. , polyadenylation (polyA) sequences and / or structural elements that stabilize circRNAs It may comprise, consist essentially of, and / or consist of.

[0060] In one embodiment, the present invention provides an AAV inverted terminal repeat (ITR) sequence comprising either Adeno-associated virus (AAV) genomes containing nucleic acid molecules of the invention flanked on one or both sides. We provide a program.

[0061] In one embodiment, the present invention provides an AAV comprising an AAV genome and / or nucleic acid molecule of the present invention. The AAV capsid or particle may be an AAV vector. .

[0062] The present invention relates to a method for producing a nucleic acid molecule of the present invention, an AAV genome of the present invention, a vector or a method for producing a vector comprising the ... and / or compositions comprising the AAV capsids, particles or vectors of the invention. As used herein, the term "pharmaceutically acceptable" means a substance that is not toxic or means a carrier material that is not otherwise repellent, i.e. the material does not contain any repellent biological The compounds can be administered to a subject without causing adverse effects.

[0063] The present invention provides a method for expressing a covalently closed circular RNA molecule in a cell. and producing the covalently closed circular RNA molecule of the present invention in the cell under conditions in which the covalently closed circular RNA molecule is transcribed. a nucleic acid molecule of the invention, an AAV genome of the invention, an AAV capsid or particle of the invention, and / or Further provided is a method comprising the step of introducing a composition of the present invention.

[0064] In another embodiment, the present invention provides a method for covalently binding nucleotides in a tissue-specific and / or cell-specific manner. A method for expressing a covalently closed circular RNA molecule in a The tissues and / or cells are treated with the nucleic acid molecules of the present invention, the AA molecules of the present invention under conditions in which the AA molecules are expressed. V genome, AAV capsid or particle of the invention, and / or a composition of the invention. The present invention provides a method comprising the steps of:

[0065] In some embodiments, the RNA molecule is a therapeutic mRNA molecule (e.g., a protein-stimulating targeting genomic elements, therapeutic RNA molecules encoding guide RNA molecules capable of targeting RNA transcripts; , tRNA molecules, long non-coding RNA molecules, antisense RNA molecules, or any It is a combination of these.

[0066] In some embodiments, the cells and / or tissues are derived from a subject of the present invention, the subject being a mammal. The animal may be a mammal, and in some embodiments is a human.

[0067] In some embodiments of the present invention, the nucleic acid molecules of the present invention are linear nucleic acid molecules or circular nucleic acid molecules. Nucleic acid molecules can be RNA or DNA molecules, which can be single-stranded or double-stranded. The nucleic acid molecule may be present in a cell or tissue of a subject and / or in a subject as a naked nucleic acid. The nucleic acid molecule may be introduced as a nucleic acid construct, a plasmid, a vector, or the ... like. It may be part of a viral vector, capsid, or particle.

[0068] In some embodiments, the circRNA molecules of the present invention encode therapeutic RNAs. and the IRES element can be used to drive translation of the nucleic acid sequence into an amino acid sequence. Not needed.

[0069] In some embodiments, the circRNA molecules of the present invention encode proteins. The IRES element is present in the circRNA molecule and can be used to identify amino acids from the nucleic acid sequence. It is possible to promote translation into the nucleic acid sequence.

[0070] One aspect of the present invention is to provide a method for the production of proteins, e.g., for therapeutic or useful biomanufacturing purposes. , AAV vectors encoding the circRNA molecules of the present invention in mammalian cells or animals The method includes administering a desired gene encoding a polypeptide within the eukaryotic cell. circRNA (which is more resistant to ribonucleases and bases than linear RNA) This is advantageous in achieving the production of a compound (having a longer half-life than the compound described above).

[0071] Another aspect of the invention is directed to the use of nucleotides, e.g., in therapeutics or in the production of proteins or non-coding RNA. The method comprises administering the circRNA molecules of the present invention into mammalian cells or animals for the purpose of producing The circular RNA can be transfected directly or in the form of a DNA vector. The cells can be transfected in a state where they can be transcribed as desired. A polymerase or a nucleic acid encoding the same can be used, or preferably an internal The preferred half-life of circular RNA in eukaryotic cells is , at least 20 as measured by hybridization or quantitative RT-PCR experiments In some embodiments, the temperature may be at least 40 hours, 30 hours, or even at least 40 hours. The present invention provides a method for the production of a compound having a half-life of at least 20 hours in a eukaryotic cell, or The present invention provides a circular RNA that has a half-life at least twice that of the linear equivalent mRNA in the Provide.

[0072] In one embodiment, the present invention provides a method for preparing a recombinant vector comprising an IRES, a 5'UTR, a coding sequence of interest, and a nucleotide sequence encoding the vector. , 3'UTR, and polyadenylation sequence in that order. These RNA elements have translation-enhancing properties and synergistic effects. Many different combinations can be created, including but not limited to: However, IRES-ORF-3'UTR-polyA, IRES-ORF-3'UTR, These include IRES-5'UTR-ORF-3'UTR and the like.

[0073] In some embodiments, the circular RNA molecules of the present invention comprise modified RNA nucleotides. Non-limiting examples of modified ribonucleotide bases include 5-methylcytidine. These nucleotides provide additional stability and immunity. Provides resistance to activation.

[0074] Another embodiment of the present invention is to prepare a DNA template encoding a circular RNA molecule of the present invention. This involves in vitro transcription of the RNA molecule by inverted intron self-sequencing. The lysing sequence promotes the formation of circular RNA without the need for any additional enzymes.

[0075]

[0013] Additional embodiments of the invention include the production of circular RNA inside a cell, said circular RNA comprising: in the cytoplasm by bacteriophage RNA polymerase or by host RNA polymerase II can be transcribed in the nucleus independently of a DNA template.

[0076] In one embodiment of the present invention, a polypeptide encoded by a circular RNA molecule is expressed in an organism. and administering the circular RNA of the present invention to an organism such as a human or an animal so that the circular RNA is expressed in the organism. The polypeptide can be intracellular or can be secreted.

[0077] In another embodiment of the present invention, the circular RNA is capable of expressing a desired polypeptide of interest. In particular, circular RNA can be transfected into cells in tissue culture to Intracellular and membrane proteins can be expressed in the cells of interest.

[0078] In some embodiments of the present invention, an RNA polymerase promoter and a terminator - T7 virus, T6 virus, SP6 virus, T3 virus, or T4 virus It can be derived from.

[0079] In some embodiments, the 3'UTR of the present invention is selected from the group consisting of human beta globin, human alpha globin, Xenopus beta globin, Xenopus alpha globin, human Prolactin, human GAP-43, human eEF1a1, human Tau, human TNFα, Hantavirus small molecule mRNA, Bunyavirus small molecule mRNA, Turnip Yellow mosaic virus, Hepatitis C virus, Rubella virus, Tobacco mosaic virus, Human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorosis spot Virus, woodchuck hepatitis virus post-translationally regulated element, Sindbis virus rus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus It may be derived from Rus.

[0080] In some embodiments, the 5'UTRs of the present invention are those derived from human beta globin, Xenopus laevis Xenopus laevis beta globin, human alpha globin, African clawed frog α-globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus , heat shock protein 70kDa protein 1A, tobacco alcohol dehydrogenase Viruses such as tobacco etch virus, turnip crinkle virus, or adenovirus tripartite fragments It may be derived from an adenovirus tripartite leader.

[0081] In some embodiments, the polyA sequence of the present invention is at least 30 nucleotides long. The length is at least 60 nucleotides.

[0082] Non-limiting examples of IRES of the present invention include those listed in Tables 5-6 herein. , and Taura syndrome virus, Lymphocytic encephalomyelitis virus, Theiler's encephalomyelitis virus lomyelitis virus, Simian virus 40, fire ant (Solenop sis invicta pathogenic virus 1, Rhopalosiphum enteric virus (Rho palosiphum padi virus), reticuloendotheliosis virus, human poliovirus Rus1, Plautia stali intestinal virus tine virus), Kashmir bee virus, human rhinovirus 2, Homalogica Homalodisca coagulata virus-1, human immunodeficiency virus All viruses type 1, Homalogisca coagulatavirus-1, Himetobi P virus (Hime tobi P virus), hepatitis C virus, hepatitis A virus, hepatitis GB virus , foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, ectropis obesity Ectropis obliqua picorna-like virus (Ectropis obliqua picorna-like virus) ike virus), encephalomyocarditis virus, Drosophila C virus, human coxsackievirus Virus B3, Crucifer tobamovirus rus), cricket paralysis virus, bovine viral diarrhea virus 1, black queen disease virus Black queen cell virus, aphid fatal paralysis virus Avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic spot virus, classical Swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Drosophila antennapedia, Human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP 1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HI F1α, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / cs is, human p53, human Pim-1, mouse Rbm3, Drosophila reaper (Dro sophila reaper), Dog cricket, Drosophila Ubx, Human UN R, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless (Dr osophila hairless), S. cerevisiae ) TFIID, S. cerevisiae YAP1, WO0155369, tobacco etch virus Aptamers against IRES from turnip crinkle virus or eIF4G have been reported. It can be obtained.

[0083] In some embodiments, the IRES of the invention is a second IRES or a third IRES. These can be combined to introduce additional initiation factors, bind ribosomal subunits, and ribosomal shunting, ribosomal base pairing, or ribosomal translocation Promote

[0084] In some embodiments, the present invention provides a method for making circular RNA, comprising: thiotriphosphate, inorganic pyrophosphatase, RNase inhibitor, and RNA polymerase adding a ferrocene to the vector of the present invention in an appropriate reaction buffer; transcribing RNA from the vector; and annealing the transcribed RNA to produce a circular mRNA. and allowing the isolated RNA to self-circularize.

[0085] In some embodiments, the ribonucleotides of the present invention are modified ribonucleotides. It may include m5C, m5U, m6A, s2U, .PSI., or 2'-O-methyl-U.

[0086] In some embodiments, the present invention provides a circular RNA and / or vector of the present invention, The present invention provides a gene therapy method comprising introducing into a subject in need thereof

[0087] In some embodiments, the present invention provides a method for detecting a gene encoding a nucleotide sequence by an open reading frame (ORF). The circular RNA and / or vector of the present invention can be used to produce the encoded protein. and a method for biomanufacturing a protein comprising introducing the protein into a mammalian cell or mammal. .

[0088] In particular embodiments, the AAV capsid protein is a naturally occurring AAV capsid protein. or have a sequence that is at least about 90%, 95%, or more identical to the native AAV capsid protein sequence. 97%, 98%, or 99% similar or identical amino acid sequence to the do.

[0089] Methods for determining sequence similarity or identity between two or more amino acid sequences include: Sequence similarity or identity includes, but is not limited to, S mith & Waterman,Adv.Appl.Math.2,482(1981 ) local sequence identity algorithm, Needleman & Wunsch J Mo l.Biol.48,443(1970) sequence identity alignment algorithm, Pe arson & Lipman,Proc.Natl.Acad.Sci.USA85, 2444 (1988) homology search methods, computerized implementation of these algorithms Genetics Computer Group, 575 Science Dr. ive, Madison, WI's Wisconsin Genetics Softwa re Package (GAP, BESTFIT, FASTA, and TFASTA), Devereux et al.Nucl.Acid Res.12,387-395( 1984) and other related art programs. This can be determined using known standard techniques or by testing.

[0090] Another suitable algorithm is that of Altschul et al. J. Mol. Biol. 215,403-410,(1990), and Karlin et al.Proc.N Atl. Acad. Sci. USA 90, 5873-5787 (1993) The BLAST algorithm is used in the BLAST program. ul et al.Methods in Enzymology,266,460-4 80(1996);blast.wustl / edu / blast / README.ht The WU-BLAST-2 program was obtained from the It uses some search parameters that are optionally set to default values. The parameter is a dynamic value and is dependent on the specific sequence composition and the specific database composition. Depending on the composition, the desired sequence is determined by the program itself. However, the values can be adjusted to increase sensitivity. As an algorithm, Altschul et al. (1997) Nucleic Ac Gap introduction as reported by Ids Res.25,3389-3402 BLAST is an example.

[0091] The present invention relates to a method for producing an AAV comprising, consisting essentially of, or consisting of an AAV genome of the present invention. Also provided are viral capsids. In certain embodiments, the viral capsid is a parvovirus. capsid, which can be further divided into autonomous parvovirus capsids or dependvirus capsids. Optionally, the viral capsid is an AAV capsid. In embodiments, the AAV capsid is AAV1, AAV2, AAV3a, AAV3b, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, young or any other AAV shown in Table 1, or which contains one or more insertions, substitutions, and / or derived from any of the above capsids by deletion.

[0092] Viral capsids can be prepared, for example, as described in U.S. Pat. No. 5,863,541. The modified viral capsid can be used as a "capsid vehicle" for Molecules that can be ingested and introduced into cells include heterologous DNA, RNA, and polypeptides. , small organic molecules, metals, or combinations thereof.

[0093] A heterologous molecule is a molecule not naturally found in an AAV infection, e.g., a wild-type AAV genome. Furthermore, therapeutically useful molecules can be defined as molecules that are not encoded by the host. It can assemble with the exterior of the chimeric viral capsid for entry into the primary target cell. Suitable associated molecules include DNA, RNA, small organic molecules, metals, carbohydrates, lipids, and / or polypeptides. In one embodiment of the invention, the therapeutically useful molecule may comprise a capsid protein. Covalently attached (i.e., conjugated or chemically coupled) to a protein Methods for covalently linking molecules are known to those skilled in the art.

[0094] The viral capsid of the present invention is also effective in raising antibodies against the novel capsid structure. As a further alternative, the exogenous amino acid sequence may find use in the treatment of cells that are anti- For example, the exogenous amino acid sequence may be administered to a subject to induce an immune response against the exogenous amino acid sequence. To produce it, it can be inserted into the viral capsid.

[0095] In other embodiments, the viral capsid contains a nucleic acid encoding a polypeptide or a target molecule. before and / or simultaneously with the administration of a viral vector that delivers functional RNA administered simultaneously (e.g., within minutes or hours of each other) to block specific cellular sites. For example, the capsids of the present invention can be administered to block cellular receptors on specific cells. The delivery vector can be administered subsequently or simultaneously, in which case the blocker Reduced transduction of the locked cells and reduced transduction of other targets (e.g., CNS progenitor cells and / or can enhance the transduction of neuroblastoma cells.

[0096] According to an exemplary embodiment, the viral capsid is a vector according to the present invention. The present invention further provides a method for administering the virus capsule of the present invention to a subject. and optionally, the compositions further comprise a virus of the invention. This also includes vectors.

[0097] The present invention provides nucleic acid molecules encoding the viral capsids and capsid proteins of the present invention ( Optionally, an isolated nucleic acid molecule is also provided. Vectors comprising the nucleic acid molecule, as well as the nucleic acid molecule of the present invention, Further provided are cells (in vivo or in culture) containing the described nucleic acid molecules and / or vectors. Suitable vectors include, but are not limited to, viral vectors (e.g., adenovirus, AA V, herpesvirus, vaccinia, poxvirus, baculovirus, etc.), plus Such nucleic acid molecules, vectors, and The cells produce viral capsids or viral vectors as described herein. For example, reagents (e.g., helper packaging constructs or packaging constructs) may be used to These cells can be used as human erythrocytes.

[0098] Viral capsids according to the present invention can be prepared by any method known in the art. For example, by expression from baculovirus (Brown et al. (1994) Vi rology 198:477-488) can be manufactured.

[0099] The capsid proteins and capsids of the present invention may be any now known or later identified. This may include intentional modifications.

[0100] For example, the AAV capsid proteins and viral capsids of the present invention may be used in combination with other viruses, Optionally, for example, as described in International Patent Publication No. WO 00 / 28004. containing all or part of a capsid subunit derived from another parvovirus or AAV. It may be chimeric in that it obtains

[0101] The viral capsid interacts with cell surface molecules present on the desired target tissue(s). A target sequence (e.g., a substituted or unsubstituted viral capsid) that induces the viral capsid to interact with the target sequence. The capsid may be a targeted viral capsid (e.g., a viral capsid containing a sequence inserted into the capsid). See, for example, International Patent Publication No. WO 00 / 28004 and Hauck et al. 003)J Virology 77:2768-2774);Shi et al. Human Gene Therapy 17:353-361(2006) [AAV Cap Integrin receptor binding motifs at positions 520 and / or 584 of the SID subunit and U.S. Patent No. 7,314,912 [which describes the insertion of RGD into AAV2 clones]. RGD after amino acids 447, 534, 573, and 587 of the rhodopsin subunit (See [Describes insertion of PI peptides containing the motif].) Other positions within the AV capsid subunit are known in the art (e.g. , Grifman et al.Molecular Therapy 3:964-9 75 (2001) at positions 449 and 588).

[0102] For example, some of the viral capsids of the present invention are capable of expressing themselves in most target tissues of interest (e.g., For example, liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells, and / or The target sequence is responsible for this low transduction rate. Advantageously, the vector can be incorporated to confer the desired tropism to the viral capsid. , and optionally confer selective tropism to a specific tissue(s). AAV capsid proteins, capsids, and vectors containing target sequences are available from, for example, the International Another possibility is that one or multiple non-naturally occurring amino acids are selected from the group consisting of those described by Wang et al. (Annu Rev Biophys s Biomol Struct. 35:225-49 (2006) directly as a means of redirecting the low-transduction vector to the desired target tissue(s). These non-natural amino acids can be incorporated into AAV capsid subunits at the junction site. The acid binds the molecule of interest to the AAV capsid protein (glycan (mannose-dendritic cell cell targeting); RGD, bombesin, or other agonists for targeted delivery to specific cancer cell types is a neuropeptide; targets specific cell surface receptors, e.g., growth factor receptors, integrins, etc. RNA aptamers or peptides selected from phage display that define These can be advantageously used to chemically link amino acids to other amino acids (including Methods for this are known in the art (e.g., Greg T. Hermanson, Bioconjugate Techniques, 1st Edition, Acade (See mic Press, 1996).

[0103] In an exemplary embodiment, the target sequence is a sequence that induces infection in a particular cell type(s). viral capsid sequences that bind to the virus (e.g., the free-standing parvovirus capsid sequence, AA V capsid sequence, or any other viral capsid sequence).

[0104] As another non-limiting example, to confer a heparin-binding site to the resulting mutant, In addition, a heparin-binding domain (e.g., a respiratory syncytial virus heparin-binding domain) is attached to the H Capsid subunits that do not generally bind to S receptors (e.g., AAV4, AAV5) It can be inserted or substituted into

[0105] B19 infects primary erythroid progenitor cells using globoside as its receptor (B Rowen et al. (1993) Science 262:114). Structure of B19 has been determined at 8 Å resolution (Agbandje-McKenna et al. (1994) Virology 203:106). Binds to globoside The B19 capsid region has been mapped between amino acids 399 and 406 (Cha pman et al. (1993) Virology 194:419), β-barrel structure The region looped out between structures E and F (Chipman et al. (1999) 6) Proc. Nat. Acad. Sci. USA 93:7502). Therefore, B19 The globoside receptor binding domain of the capsid is a globoside receptor binding domain of the viral capsid or viral vector containing it. The vector can be substituted into the AAV capsid protein to target red blood cells.

[0106] In an exemplary embodiment, the exogenous target sequence comprises a modified AAV capsid protein. Encodes a peptide that alters the tropism of the viral capsid or viral vector It can be any amino acid sequence. In certain embodiments, the target peptide or protein is They may be naturally occurring or may be wholly or partially synthetic. Targeting sequences include ligands and other peptides that bind to cell surface receptors and glycoproteins. peptides, e.g., RGD peptide sequences, bradykinin, hormones, peptide growth factors (e.g., For example, epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin phospho-like growth factors I and II, cytokines, melanocyte-stimulating hormones (e.g., α , β, or γ), neuropeptides, and endorphins, etc., and their cognate receptors in cells Other exemplary peptides and fragments thereof that retain the ability to target the peptide to the body include: and proteins such as substance P, keratinocyte growth factor, neuropeptide Y, Gastrin-releasing peptide, interleukin 2, hen egg white lysozyme, erythropoietin cin, gonadoliberin, corticostatin, β-endorphin, leucine enkephalin phenytoin, rimorphin, α-neoenkephalin, angiotensin, pneumidine (pne umadin), vasoactive intestinal peptide, neurotensin, motilin, and As a still further alternative, toxins (e.g., tetanus) can be used. A binding domain derived from a toxin or snake venom, such as α-bungarotoxin, binds to the target sequence. In yet a further exemplary embodiment, the AAV capsid protein may be substituted with a nucleotide sequence similar to that of the nucleotide sequence ... The psid protein is described by Cleves (Current Biology 7:R318( "Non-classical" import / export signal peptides described by (e.g., fibroblast growth factor-1 and -2, interleukin-1, HIV-1 Tat into AAV capsid proteins (e.g., herpesvirus VP22 protein) Peptide motifs that direct uptake by specific cells can also be modified. Included are, for example, the FVFLP peptide motif, which elicits uptake by hepatocytes.

[0107] Phage display technology, as well as other techniques known in the art, can be used to generate vectors for any purpose. These peptides can be used to identify peptides that recognize specific cell types.

[0108] The target sequence may be a receptor (e.g., a protein, carbohydrate, glycoprotein, or proteoglycan). The peptide may encode any peptide that targets a cell surface binding site, including a peptide fragment (e.g., a glycan). Examples of surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate, and other glycosaminoglycans, sialic acid moieties, polysialic acid moieties, glycoproteins, and gangliosides, MHCI glycoproteins, carbohydrate components found on membrane glycoproteins Mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucosa Examples include sugars (including sugars and galactose).

[0109] As a still further alternative, the targeting sequence may be chemically linked to another molecule that targets its entry into the cell. can be used to chemically couple (e.g., chemically couple via its R group) The peptide may be a peptide (which may contain arginine and / or lysine residues which may be linked).

[0110] The above embodiments of the invention involve the delivery of heterologous nucleic acid to a cell or subject, as described herein. Thus, in one embodiment, the present invention provides a method for administering nucleic acid molecules to cells. a method for introducing a virus into a cell, the method comprising contacting the cell with a viral vector and / or composition of the invention. The present invention provides a method comprising the step of:

[0111] A method for delivering a nucleic acid molecule to a subject, comprising administering to the subject a viral vector of the invention and / or Further provided herein are methods comprising administering the compositions of the present invention. In some embodiments, the viral vector or composition is administered to the eye of the subject. In embodiments, administration is intravitreal, subretinal, subconjunctival, retrobulbar, intracameral, and / or suprachoroidal. Depends on each route.

[0112] A method for selectively inducing transduction of cells having polysialic acid on their surface. contacting the cell with a viral vector of the invention and / or a composition of the invention. Additionally presented herein are methods including:

[0113] The present invention provides a method for delivering a nucleic acid molecule of interest to an ocular cell, comprising administering to the ocular cell or layer a nucleic acid molecule of interest, comprising administering to the ocular cell or layer a nucleic acid molecule of interest, the nucleic acid molecule of interest being delivered to the ocular cell or layer ... and contacting the viral vector with the target nucleic acid molecule, In some embodiments of the method, the nucleic acid molecule of interest is encodes a therapeutic protein or a therapeutic RNA. The protein may be a monoclonal antibody or a fusion protein.

[0114] In some embodiments of the above methods, the ocular cell or layer may be in a subject, and in some embodiments, In some embodiments, the subject may be a human subject.

[0115] The present invention provides a method of treating an ophthalmic disorder or defect in a subject, comprising administering to the subject an ophthalmic solution of the present invention. administering a viral vector to treat an ophthalmic disorder or defect, and methods involving nucleic acid molecules encoding therapeutic proteins or therapeutic RNAs effective to treat In some embodiments of this method, the viral vector is delivered to the vitreous, the retina, or the retina. It is administered by the subthecal, subconjunctival, retrobulbar, intracameral, and / or suprachoroidal routes. In some embodiments, the ophthalmic disorder or defect is retinitis pigmentosa, macular degeneration, or Leber's congenital glaucoma. Amaurosis, color vision disorder, X-linked retinoschisis, optic neuritis, choroideremia, optic atrophy, retinal Membrane-cone dystrophy, retinopathy, retinoblastoma, glaucoma, Bardet-Biedl syndrome, or is color blind.

[0116] Delivery of nucleic acid molecules of interest (NOIs) to central nervous system progenitor cells and / or neuroblastoma cells The method further comprises infecting progenitor cells and / or neuroblasts with adeno-associated virus (AAV) serotype 4. (AAV4) capsid protein, the psid protein contains mutations at K492, K503, and N585; and Also provided herein are methods in which the viral vector contains a nucleic acid molecule of interest. In some embodiments of the method, the nucleic acid molecule of interest is a therapeutic protein or a therapeutic The RNA can encode the central nervous system progenitor cells and, in some embodiments, / or the neuroblasts may be in the subject.

[0117] In a further embodiment of the present invention, there is provided a method of treating a neurological disorder or defect in a subject, comprising: administering the viral vector of the present invention and / or the composition of the present invention to a subject, The viral vector may deliver a therapeutic protein or therapeutic agent effective in treating a neurological disorder or defect. In some embodiments of the method, a nucleic acid molecule encoding the RNA is provided. The viral vector is administered intracerebroventricularly, intravenously, or intravenously. Administration may be by intracisternal, intraparenchymal, intracranial, and / or intrathecal routes. In some embodiments, the subject is a human subject.

[0118] The present invention also encompasses viral vectors comprising the nucleic acid molecules of the present invention. The viral vector may be a parvovirus vector (e.g., a parvovirus capsid and and / or vector genome), e.g., AAV vectors (e.g., AAV capsids and and / or vector genome).

[0119] For example, in a representative embodiment, a viral vector comprises (a) a viral capsid (e.g., (b) a nucleic acid comprising a terminal repeat sequence (e.g., AAV-TR); wherein the nucleic acid containing the terminal repeat sequence is encapsidated by the modified viral capsid. The nucleic acid contains two terminal repeat sequences (e.g., two AAV-TRs) It can be optionally included.

[0120] In an exemplary embodiment, the viral vector delivers a protein or functional RNA of interest. A recombinant viral vector comprising a heterologous nucleic acid molecule encoding is described in more detail below.

[0121] [Methods for generating viral vectors] The present invention further provides methods for producing the viral vectors of the present invention. In embodiments, the present invention provides a method for producing a viral vector, the method comprising: In contrast, (a) a nucleic acid template containing at least one TR sequence (e.g., an AAV-TR sequence) (b) replication of the nucleic acid template and its encapsulation into AAV capsids (encapsids) AAV sequences (e.g., encoding the AAV capsid of the present invention) sufficient for apsidation Optionally, providing an AAVrep sequence and an AAVcap sequence corresponding to the AAVrep sequence. In certain embodiments, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. The nucleic acid template contains two AAV-ITR sequences, which (if present) are distinct. It is located 5' and 3' to the heterologous nucleic acid sequence, but does not have to be directly adjacent to the heterologous nucleic acid sequence. There's no need.

[0122] The nucleic acid template and the AAV rep and cap sequences are packaged into the AAV capsid. conditions such that viral vectors containing the ligated nucleic acid template are produced in the cells. The method may further include harvesting the viral vector from the cell. The viral vector can be harvested from the culture medium and / or by lysing the cells. .

[0123] The cells can be any cell permissive for AAV viral replication. Any suitable cell may be employed. In certain embodiments, the cell is a mammalian cell. As a complement, cells express transgenes that provide functions removed from the replication-deficient helper virus. Trans-complementing packaging cell lines, e.g., They may be 293 cells or other E1a transcomplementing cells.

[0124] AAV replication and capsid sequences can be provided by any method known in the art. The current protocol expresses the AAV rep / cap genes on a single plasmid. AAV replication and packaging sequences do not necessarily need to be provided together. However, it may be advantageous to do so. AAVrep and / or cap sequences may be It can be provided by a viral or non-viral vector, e.g., rep / ca The p sequence is provided by a hybrid adenovirus or herpesvirus vector. (e.g., inserted into the E1a or E3 region of a defective adenoviral vector) EBV vectors have also been employed to express the AAV cap and rep genes. One advantage of this method is that the EBV vector is episomal but not transmitted to contiguous cells. The goal is to still maintain high copy number throughout division (i.e., "EBV-based" The nuclear episome is called the "EBV-based nuclear episome." Stably integrated into cells as extrachromosomal elements, Margolski (1992 )See Curr.Top.Microbiol.Immun.158:67).

[0125] As a further alternative, the rep / cap sequences may be stably integrated into the cell. The AAVrep / cap sequences contain the sequences required for rescue and / or packaging. To prevent stumbling, do not place TRs adjacent to each other.

[0126] The nucleic acid template can be provided to the cell using any method known in the art. For example, the template may be a non-viral vector (e.g., a plasmid) or a viral vector. In certain embodiments, the nucleic acid template may be provided by a herpes virus. It is delivered by a viral vector or adenovirus vector (e.g., a defective adenovirus vector). As another example, Palombo e t al. (1998) J. Virology 72:5025 by AAV-TR A baculovirus vector carrying a flanked reporter gene is described. The vector also delivers the template, as described above for the rep / cap genes. It can be adopted to

[0127] In another exemplary embodiment, the nucleic acid template is provided by a replicating rAAV virus. In yet other embodiments, the AAV provirus comprising the nucleic acid template is , stably integrated into the cell's chromosomes.

[0128] To enhance viral titers, helper virus functions that promote productive AAV infection ( For example, an adenovirus or herpesvirus can be provided to the cell. The necessary helper virus sequences are known in the art. Generally, these sequences include: provided by a helper adenovirus vector or herpesvirus vector; Alternatively, adenovirus or herpesvirus sequences can be found, for example, in Ferrari et al. al. (1997) Nature Med. 3:1295; and U.S. Pat. No. 6,044,494. As described by US Pat. Nos. 6,093,570 and 6,183, an efficient AAV Non-infectious adenoviral miniplasmids carrying all of the helper genes that facilitate production The vector may be provided by another non-viral vector or a viral vector.

[0129] Additionally, helper virus functions may be embedded in the chromosome or may be expressed as stable extrachromosomal elements. can be provided by packaging cells with helper sequences maintained as vectors. Generally, helper virus sequences cannot be packaged into AAV virions, e.g. For example, it is not sandwiched by TR.

[0130] AAV replication and capsid sequences, as well as the helper virus, are contained on a single helper construct. It may be advantageous to provide viral sequences (e.g., adenoviral sequences) of the vectors. Those skilled in the art will understand that this helper construct is a non-viral construct. It may be a human or viral construct. The looper construct is a hybrid adenovirus containing the AAVrep / cap genes. The virus may be a herpes simplex virus or a hybrid herpes virus.

[0131] In one particular embodiment, the AAVrep / cap sequence and the adenovirus helper sequence The sequence is supplied by a single adenoviral helper vector. The AAV rep / cap sequence and / or rAAV template may further comprise an acid template. The vector is inserted into a defective region of the adenovirus (e.g., the E1a region or the E3 region). It is possible.

[0132] In a further embodiment, the AAVrep / cap sequences and adenovirus helper sequences are According to this embodiment, the r The AAV template may be provided as a plasmid template.

[0133] In another exemplary embodiment, AAVrep / cap sequences and adenovirus helper sequences is provided by a single adenoviral helper vector, and the rAAV template The rAAV template integrates into the cell as a provirus. Alternatively, the rAAV template is extrachromosomal. EBV vectors that are maintained in cells as nuclear elements (e.g., EBV-based nuclear episomes) Provided by the maker.

[0134] In a further exemplary embodiment, the AAVrep / cap sequence and the adenovirus helper The sequences are provided by a single adenovirus helper. The rAAV template is It can be provided as a separate replicating viral vector. For example, the rAAV template can be , rAAV particle or a second recombinant adenovirus particle.

[0135] According to the above method, the hybrid adenoviral vector can replicate and express the adenovirus. and adenoviral 5' and 3' cis sequences sufficient for packaging (i.e., adenoviral AAV rep / cap sequences and the presence of In this case, the rAAV template is embedded in the adenoviral backbone and and 3' cis sequences, so that these sequences are The product may be packaged in a container.

[0136] As mentioned above, the adenovirus helper sequences and AAV rep / cap sequences are The sequence is generally flanked by TRs to prevent it from being packaged into AAV virions. not present.

[0137] Zhang et al.((2001)Gene Ther.18:704-12) Adenovirus and chimeric helper containing both AAVrep and cap genes This article describes the following.

[0138] Herpesviruses can also be used as helper viruses in AAV packaging methods. A hybrid vector encoding the AAV-Rep protein(s) may be used. Pesviruses may advantageously facilitate scalable AAV vector production schemes. Hybrid herpes simplex virus type 1 (HS) expressing V-2 rep and cap genes V-1) vector was prepared as described in Conway et al. (1999) Gene Therapy y 6:986 and WO 00 / 17377.

[0139] As a further alternative, the viral vectors of the present invention may be prepared using methods such as those described, for example, in Urabe et al. (2002) Human Gene Therapy 13:1935-43 baculoviruses for delivery of the rep / cap genes and rAAV template, The vectors can be produced in insect cells using viral vectors.

[0140] AAV vector stocks free of contaminating helper viruses are available as known in the art. For example, the AAV and helper virus may be prepared by any method. AAVs can also be readily identified based on their affinity for heparin substrates. It can also be isolated from luperviruses (Zolotukhin et al. (1999) Gene Therapy 6:973). Any contaminating helper virus is not a replicative component. A deletion-type replication-deficient helper virus can be used so that the virus is not resistant to replication. Only adenovirus early gene expression is required to mediate AV virus packaging. A further alternative is to use an adenoviral helper that lacks late gene expression, as this is required. Adenovirus mutants defective in late gene expression are known in the art. known (e.g., ts100K and ts149 adenovirus mutants).

[0141] [Recombinant viral vector] The viral vectors, capsids, and particles of the present invention can be used in vitro, ex vivo, and in vivo. Viral vectors are particularly useful for delivering nucleic acids to cells in vivo. , can be advantageously employed to deliver or transfer nucleic acids to animals containing cells.

[0142] Any heterologous nucleic acid sequence(s) of interest may be incorporated into the viral vectors, capsules, or other recombinant vectors of the present invention. The nucleic acid of interest may be a therapeutic (e.g., a medical polypeptides containing human or veterinary (human or veterinary) or immunogenic (e.g., vaccine) proteins; These include nucleic acids encoding a gene, and / or a functional or therapeutic RNA molecule.

[0143] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane conductance regulators (TGF-R1), cystic fibrosis transmembrane conductance regulators (CFR), and cystic fibrosis transmembrane conductance regulators (CFR). cystic fibrosis transmembrane regulat or protein) (CFTR), dystrophin (mini-dystrophin and micro-dystrophin) dystrophin, e.g., Vincent et al. (1993) Nature re Genetics 5:130; U.S. Patent Publication No. 2003 / 017131; International Publication No. WO 2008 / 088895, Wang et al. Proc. Na tl.Acad.Sci.USA 97:13714-13719(2000); and G regorevic et al.Mol.Ther.16:657-64(2008) ), myostatin propeptide, follistatin, activin type II soluble receptor , IGF-1, anti-inflammatory polypeptides, such as IκB dominant mutants, sarcospan, uterine Lofin (Tinsley et al. (1996) Nature 384:349) , mini-utrophin, coagulation factors (e.g., factor VIII, factor IX, factor X, etc.) , erythropoietin, angiostatin, endostatin, catalase, tyrosine hydrochloride xylase, superoxide dismutase, leptin, LDL receptor, lipoprotein Lipase, ornithine transcarbamylase, β-globin, α-globin, spectrum Phosphorus, α1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphatase Phosphingomyelinase, β-glucocerebrosidase, sphingomyelinase , lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein cytokines (e.g., α-interferon, β-interferon, interferon) Interleukin-γ, interleukin-2, interleukin-4, granulocyte macrophage colony knee stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g. For example, somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor Epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain Neurotrophic factor-derived, bone morphogenetic protein tein) [including RANKL and VEGF], glial-derived growth factor, transforming growth factor -α and -β, etc.), lysosomal acid α-glucosidase, α-galactosidase A, receptor (e.g., tumor necrosis growth factor α soluble receptor), S100A1, parvalbumin, adenocarcinoma Nitrinyl cyclase type 6, molecules that regulate calcium handling (e.g., SERCA2 A , PP1 inhibitor 1 and fragments thereof [see, for example, WO 2006 / 029319 and 2007 / 100465]), truncated constitutively active bARKct, and other G-proteins. Anti-inflammatory properties of molecules such as IRAP, which enable protein-coupled receptor kinase type 2 knockdown factor, anti-myostatin protein, aspartoacylase, monoclonal antibody (single chain monoclonal antibody) an exemplary Mab is the Herceptin® Mab. neuropeptides and their fragments (e.g., galanin, neuropeptide Y (US See Patent No. 7,071,172), angiogenesis inhibitors such as vasohibin and other VE GF inhibitors (e.g., Vasohibin 2 [see International Publication No. JP2006 / 073052] Other exemplary heterologous nucleic acid sequences include suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), used in cancer therapy Proteins that confer resistance to drugs used, tumor suppressor gene products (e.g., p53 , Rb, Wt-1), TRAIL, FAS-ligand, and in subjects in need thereof AAV vectors may encode any other polypeptide that has a therapeutic effect. Clonal antibodies and antibody fragments, e.g., antibodies or antibody fragments that target myostatin. It can also be used to achieve (See Biotechnology 23:584-590 (2005)). AAV vectors are One or more components of the CRISPR / Cas complex or other genes It can also be used to provide components for use in editing systems. V vectors are used to deliver secreted therapeutic agents, e.g., fusion proteins (etanercept). When used to deliver a secreted therapeutic agent, the secreted therapeutic agent may be Widespread expression of the virus is not required, as long as it can reach the desired target tissue.

[0144] The heterologous nucleic acid sequence encoding a polypeptide may be a reporter polypeptide (e.g., an enzyme Reporter polypeptides are known in the art and include those that encode These include, but are not limited to, green fluorescent protein, β-galactosidase, alkaline phosphatase, acetyltransferase, luciferase, and chloramphenicol acetyltransferase genes Contains children.

[0145] Optionally, the heterologous nucleic acid encodes a secreted polypeptide (e.g., a polypeptide that is secreted in its natural state). A polypeptide that is a secreted polypeptide, or a polypeptide having a secretory signal sequence known in the art, e.g. A polynucleotide engineered to be secreted by operably associating it with peptide).

[0146] Alternatively, in certain embodiments of the present invention, the heterologous nucleic acid may be an antisense nucleic acid, a ribozyme ( For example, as described in U.S. Pat. No. 5,877,022, spliceosome-mediated RNA (Puttaraju et al. (1 999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487 No. 6,083,702), siRNA involved in gene expression suppression, shR Interfering RNA (RNAi), including miRNA (Sharp et al. (20 00) Science 287:2431), and other RNAs such as "guide" RNAs. Non-coding RNA (Gorman et al. (1998) Proc. Nat. Acad. Sci.USA 95:4929; Yuan et al., U.S. Pat. No. 5,869,2 48) and the like. Exemplary non-coding RNAs include those encoding multidrug resistance (MDR) genes. RNAi against substances (e.g., to treat and / or prevent tumors, and / or to induce chemotherapy) for cardiac administration to prevent damage caused by therapy), RNAi against myostatin (e.g. for Duchenne muscular dystrophy), RNAi against VEGF (for example, tumor RNAi against phospholamban (e.g., for treating and / or preventing cardiovascular disease) For treating diseases, see, for example, Andino et al. J. Gene Med. 10: 132-142(2008), and Li et al.Acta Pharmacol Sin. 26:51-55 (2005); phosphoramidite such as phospholamban S16E ambassador molecules or dominant negative molecules (e.g., for treating cardiovascular disease, e.g., For example, Hoshijima et al. Nat. Med. 8:864-871 (200 2), RNAi against adenosine kinase (e.g., for epilepsy), and pathogenicity Organisms and viruses (e.g., hepatitis B and / or C viruses, human immunodeficiency virus, RNAi targeting viruses such as CMV, herpes simplex virus, and human papillomavirus has been reported. It can be obtained.

[0147] Additionally, nucleic acid sequences that direct alternative splicing can be delivered. Antibodies complementary to the 5' and / or 3' splice sites of strophin exon 51 The sense sequence (or other inhibitory sequence) induces skipping of this exon. , U1, or U7 small nuclear (sn)RNA promoters. , U1 or U7 s located 5' to the antisense / inhibitory sequence(s) A DNA sequence containing an mRNA promoter is packaged into the modified capsid of the present invention. It can be administered and delivered.

[0148] Viral vectors are designed to target heterologous genes that share homology with and recombine with loci on the host chromosome. This approach can be used, for example, to correct a genetic defect in a host cell. It can be used for.

[0149] The present invention provides viral vectors that express immunogenic polypeptides for, e.g., vaccination. Nucleic acids include, but are not limited to, human immunodeficiency virus (HIV), Simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag Immunogens include those derived from proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc. It may encode any immunogen of interest known in the art.

[0150] The use of parvoviruses as vaccine vectors is known in the art (e.g., Miyamura et al.(1994)Proc.Nat.Acad.Sci U SA 91:8507; Young et al., U.S. Pat. No. 5,916,563; Mazzara et al., U.S. Pat. No. 5,905,040, U.S. Pat. No. 5,888 See U.S. Patent No. 2,652 to Samulski et al. and U.S. Patent No. 5,863,541 to Samulski et al. The antigen may be present in the parvovirus capsid. Alternatively, the antigen may be present in a recombinant vector. - can be expressed from heterologous nucleic acid introduced into the genome. Any immunogen of interest known in the art may be provided by the viral vectors of the present invention. obtain.

[0151] Immunogenic polypeptides are polypeptides suitable for and / or not limited to eliciting an immune response. Infection and / or transmission of, but not limited to, microorganisms, bacteria, protozoa, parasites, fungi and / or viruses Any polypeptide suitable for protecting a subject from infection and / or disease, including HIV-1, HIV-1, and HIV-2 diseases. For example, the immunogenic polypeptide may be an orthomyxovirus immunogen (e.g., an immunogen Influenza virus immunogens, such as the influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein, or equine influenza virus rus immunogens, etc.), or lentiviral immunogens (e.g., equine infectious anemia virus immunogens, a simian immunodeficiency virus (SIV) immunogen or a human immunodeficiency virus (HIV) immunogen; For example, the HIV or SIV envelope GP160 protein, HIV or SIV V matrix / capsid protein, and HIV or SIV gag, pol The immunogenic polypeptide may be an arenavirus immunogen. immunogens (e.g., Lassa fever virus immunogens, e.g., Lassa fever virus nucleocapsid protein and and Lassa fever envelope glycoproteins, etc.), poxvirus immunogens (e.g., vaccinia a viral immunogen, such as a vaccinia L1 or L8 gene product, a flavivirus immunogen, immunogens (e.g., yellow fever virus immunogens or Japanese encephalitis virus immunogens), filovirus immunogens, Immunogens (e.g., Ebola virus immunogens or Marburg virus immunogens, NP and GP genes) product, etc.), Bunyavirus immunogens (e.g., RVFV, CCHF and / or SFS viruses), virus immunogen), or coronavirus immunogen (e.g., infectious human coronavirus immunogen, For example, human coronavirus envelope glycoproteins, or transmissible porcine gastroenteritis virus Immunogenic polypeptides may also be used as immunogens (e.g., avian infectious bronchitis virus immunogens). The vaccine also includes polio immunogens, herpes immunogens (e.g., CMV, EBV, HSV immunogens), Mumps immunogen, measles immunogen, rubella immunogen, diphtheria toxin or other diphtheria Hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogens, and / or any other antigens now known in the art or later identified as immunogens. Other vaccine immunogens may be used.

[0152] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor antigen or cancer antigen is expressed on the surface of a cancer cell.

[0153] Exemplary cancer and tumor cell antigens include those described by SA Rosenberg (Immunity 10:281 (1991)). Other exemplary cancer antigens and tumor antigens are described in Examples of the source include, but are not limited to, BRCA1 gene products, BRCA2 gene products, g p100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-E SO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205 , HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakam i et al.(1994)Proc.Natl.Acad.Sci.USA 91: 3515;Kawakami et al.(1994)J.Exp.Med.180: 347;Kawakami et al.(1994)Cancer Res.54:3 124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, C EA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al. l.(1993)J.Exp.Med.178:489);HER-2 / neu gene production (U.S. Patent No. 4,968,603), CA125, LK26, FB5 (endsiali N), TAG72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SP an-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 Protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor Repressor protein (Levine, (1993) Ann. Rev. Biochem. 6 2:623); mucin antigen (International Patent Publication No. WO 90 / 05142); telomere enzyme; nuclear matrix protein; prostatic acid phosphatase; papillomavirus anti- and / or currently known antigens, or the following cancers: melanoma, glandular Cancer, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer Liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer antigens later discovered to be associated with bladder, kidney, pancreatic, and brain cancers; Any other cancer or malignant condition now known or later identified (e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91 (See references below).

[0154] As a further alternative, the heterologous nucleic acid may be expressed in cells in vitro, ex vivo or in vivo. The vector may encode any polypeptide that is desired to be produced in a cell. For example, a virus The vector can be introduced into cultured cells and the expressed gene product isolated therefrom.

[0155] The heterologous nucleic acid(s) of interest may be operably associated with appropriate control sequences. For example, the heterologous nucleic acid may contain expression control elements, e.g., transcription / Translational control signals, replication origins, polyadenylation signals, internal ribosome entry sites (I RES), promoters, and / or enhancers, etc.

[0156] Furthermore, regulated expression of the heterologous nucleic acid(s) of interest can be achieved by, for example, oligonucleotide synthesis. The present invention provides a method for selectively blocking splicing activity at specific sites using peptides, small molecules, and / or Other compounds (e.g., those described in WO 2006 / 119137) that By regulating the alternative splicing of different introns depending on their presence or absence This can be achieved at the post-transcriptional level.

[0157] Various promoter / enhancer elements are available to achieve desired levels and tissue-specific expression. Those skilled in the art will understand that promoters / enhancers may be used depending on the The promoter / enhancer may be constitutive or inducible depending on the desired expression pattern. The sensor may be native or exogenous, and may be a natural sequence or a synthetic sequence. It is intended that the transcription initiation region not be found in the wild-type host into which it is introduced. It is being done.

[0158] In certain embodiments, the promoter / enhancer element is a promoter / enhancer element that is specific to the target cell or the treated cell. In an exemplary embodiment, the promoter / enhancer The elements may be native to the heterologous nucleic acid sequence. The element is generally designed to function in the intended target cell(s). Furthermore, in certain embodiments, the promoter / enhancer element is selected from mammalian Mammalian promoter / enhancer element. Elements may be constitutive or inducible.

[0159] Inducible expression control elements provide regulation over the expression of heterologous nucleic acid sequence(s). This is generally advantageous in applications where it is desirable to provide an inducible promoter for gene delivery. The promoter / enhancer element may be a tissue-specific or preferential promoter / enhancer. -elements, which may be muscle-specific or preferential (cardiac, skeletal, and / or smooth muscle-specific) neural tissue-specific or preferential (including brain-specific or preferential) ), eye-specific or preferential (including retina-specific and cornea-specific), liver-specific or Preferential, bone marrow-specific or preferential, pancreas-specific or preferential, spleen-specific or preferential and lung-specific or -preferential promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible elements and Exemplary inducible promoter / enhancer elements include: Examples of elements include, but are not limited to, Tet on / off elements, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, and The metallothionein promoter is an example.

[0160] In embodiments where the heterologous nucleic acid sequence(s) are transcribed and then translated within the target cell, The specific initiation signal is required for efficient translation of the inserted protein coding sequence. Such exogenous translational control sequences generally include the ATG initiation codon and adjacent sequences. The sequences may be of various origins, both natural and synthetic.

[0161] Viral vectors, capsids, and particles according to the present invention can be used to infect cells, including dividing and non-dividing cells. In some embodiments, the present invention provides a means for delivering heterologous nucleic acids into a wide range of cells, including For example, to produce a polypeptide in vitro or for ex vivo gene therapy. For purposes of this, viral vectors are used to deliver nucleic acids of interest to cells in vitro. Viral vectors can be employed to deliver, for example, immunogenic or therapeutic polypeptides or In a method of delivering a nucleic acid to a subject in need thereof to express a functional RNA Thus, the polypeptide or functional RNA can be administered in vivo in a subject. The subject lacks the polypeptide and therefore does not need it. Alternatively, the polypeptide may be administered to a subject via a gene therapy protocol. The production of polypeptides or functional RNA in elephants may have some beneficial effect. , this method can be practiced.

[0162] The viral vectors, capsids, and particles of the present invention can be used to express the desired vectors in cultured cells or in a subject. It can also be used to produce polypeptides or functional RNAs that are In conjunction with the screening method, for example, to produce a polypeptide or to administer a function to a subject. (using the subject as a bioreactor to observe the effects of functional RNA).

[0163] Generally, the viral vectors, capsids, and particles of the invention contain a polypeptide or functional and delivering a heterologous nucleic acid encoding a target RNA to deliver a therapeutic polypeptide or functional RNA. The invention may be employed to treat and / or prevent any disease state in which it is beneficial to do so. Common disease states include, but are not limited to, cystic fibrosis (cystic fibrosis transmembrane regulation protein) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX) (children), thalassemia (beta-globin), anemia (erythropoietin), and other blood disorders , Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon ), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease ( RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors) ), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL cytokines, including FAS-ligand, interferons; VEGF or multidrug resistance genes RNAi, including RNAi against its product, mir-26a [e.g., related to hepatocellular carcinoma] ]), diabetes mellitus (insulin), Duchenne (dystrophin, minidystrophy insulin-like growth factor I, sarcoglycans [e.g., α, β, γ], and myostatin RNAi against myostatin propeptide, follistatin, and soluble activin II Receptors, anti-inflammatory polypeptides, such as IκB dominant mutants, sarcospan, eutrophin Dystrophin gene expression to induce dystrophin, mini-utrophin, and exon skipping Antisense or RNAi directed against splice junctions in the genome [e.g., International Publication No. No. 2003 / 095647], U7 s to induce exon skipping Antisense to mRNA [see, e.g., WO 2006 / 021724] and antibodies or antibody fragments against myostatin or myostatin propeptide) and Muscular dystrophy including Kerr type, Gaucher disease (glucocerebrosidase), Hurler disease ( α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase) , glycogen storage disorders (e.g., Fabry disease [α-galactosidase] and Pompe disease) [lysosomal acid α-glucosidase]), and other metabolic disorders, congenital emphysema (α1- antitrypsin), Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase) transferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease Tays-Sachs disease (lysosomal hexosaminidase A) , maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (and eye and retina) Other diseases of the membrane; for example, PDGF in macular degeneration, and / or for example, type I diabetes or other inhibitors of VEGF for treating / preventing retinal damage in children - Patents.com , or other angiogenesis inhibitors), diseases of solid organs, such as the brain (Parkinson's disease [G DNF], astrocytoma [response to endostatin, angiostatin, and / or VEGF RNAi targeting glioblastoma [RNAi targeting endostatin, angiostatin, and / or VEGF] including RNAi [including RNAi]), liver, kidney, congestive heart failure, or peripheral arterial disease (PAD) Heart (e.g., protein phosphatase inhibitor I (I-1) and fragments thereof (e.g., I 1C), serca2a, a zinc finger protein that regulates the phospholamban gene , Barkct, P2 adrenergic receptor, p2 adrenergic receptor kinase (BARK ), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin G proteins such as adenylyl cyclase type 6 and truncated constitutively active bARKct A molecule that activates receptor kinase type 2 knockdown: calsarcin cin), RNAi against phospholamban; (by delivering insulin-inhibiting molecules or dominant-negative molecules), arthritis (by delivering insulin-inhibiting molecules or dominant-negative molecules), insulin-like growth factors), joint damage (insulin-like growth factors 1 and / or 2), intimal hyperplasia (e.g. for example, by delivering enos and inos), improved survival of cardiac transplants (superoxide oxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I) , kidney deficiency (erythropoietin), anemia (erythropoietin) rithropoietin), arthritis (anti-inflammatory factors such as IRAP and TNFα soluble receptors), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia ( Ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Spinal cerebral ataxias, including Tennessee disease, SCA1, SCA2, and SCA3 cerebral ataxias), phenylketonuria (phenylalanine hydrochloride) The present invention aims to improve the success rate of transplantation by and / or after organ transplantation to reduce harmful side effects of organ transplantation or adjuvant therapy It may also be used (e.g., immunosuppressants or inhibitory nuclei to block cytokine production). Another example is the administration of bone morphogenetic proteins (BNP2, 7, etc.). RANKL and / or VEGF) are disrupted, for example, in cancer patients. ) or after surgical removal, with bone allograft.

[0164] The present invention can also be used to generate induced pluripotent stem cells (iPS). The viral vectors of the invention can be used to infect non-pluripotent cells, such as adult fibroblasts, skin cells, liver cells, Stem cell-related nucleic acids (complex) are expressed in kidney cells, adipocytes, cardiac cells, nerve cells, epithelial cells, endothelial cells, etc. It can be used to deliver a number of

[0165] Nucleic acids encoding stem cell associated factors are known in the art. Stem Cells and Pluripotency Non-limiting examples of such factors associated with SOX1, SOX2, SOX3, and / or SOX15), Klf family (e.g., Klf1, Klf2, Klf4, and / or Klf5), Myc family (e.g., , C-myc, L-myc, and / or N-myc), NANOG, and / or LIN2 8 is an example.

[0166] The present invention relates to the treatment of epilepsy, stroke, traumatic brain injury, cognitive disorders, behavioral disorders, psychiatric disorders, huntington syndrome, and the like. Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and axon / neuron regeneration any other neurodegenerative condition that would benefit from or require regeneration or repair It may also be practiced for treatment and / or prevention.

[0167] In certain embodiments, the present invention provides methods for the production of, for example, stem cell differentiation and reprogramming factors, e.g., For example, FoxJl, Fox2, NeuroD2, NG2, or Olig2, and / or MicroRNAs, such as miR-137, MiR124, etc., as well as any other factors; or by targeted regulation or overexpression of miRNAs involved in neuronal development and differentiation. promotes axonal regeneration and neuronal repair, restores circuits, and / or repairs lost neurons It can be practiced as a corrective therapy to replenish the body's natural nutrients.

[0168] Gene transfer has substantial potential applications for understanding and providing therapies for disease states. There are several genetic disorders in which the defective gene is known and has been cloned. Generally, the disease conditions described above fall into two classes: those that are generally inherited in a recessive manner, Common conditions that usually involve enzyme deficiencies and regulatory or structural proteins In the case of deficiency diseases, replacement therapy is used. as well as antisense mutations to bring normal genes into affected tissues. Gene transfer can be used to create animal models for diseases. For disease states that are not yet fully understood, gene transfer can be used to create the disease state in a model system. However, this model system can then be used in efforts to combat disease states. These viral vectors make it possible to treat and / or prevent genetic diseases.

[0169] The viral vectors, capsids, and particles of the present invention function in vitro or in vivo. These methods can also be used to provide cells with functional RNA. For example, it can reduce the expression of a particular target protein by a cell. , functional RNA reduces the expression of a specific protein in a subject in need thereof Functional RNAs can also be administered to regulate gene expression and / or cell physiology. for example, to optimize cell or tissue culture systems or in screening methods. and can be administered to cells in vitro.

[0170] Furthermore, the viral vectors, capsids, and particles according to the invention can be used in diagnostic and screening methods. The present invention finds use in a nucleic acid synthesis method, whereby a nucleic acid of interest is synthesized in a cell culture system. or expressed transiently or stably in transgenic animal models.

[0171] The viral vectors, capsids, and particles of the present invention, as will be apparent to those skilled in the art, To assess, but not limited to, gene targeting, clearance, transcription, translation, etc. Viral vectors can be used for a variety of non-therapeutic purposes, including use in protocols for The capsids and particles are also used to assess safety (transmission, toxicity, immunogenicity, etc.). Such data may be used, for example, to assess the efficacy of a drug in the regulatory approval process before clinical efficacy assessment is performed. The drug will be reviewed by the U.S. Food and Drug Administration as part of its regulatory review.

[0172] In a further aspect, the viral vectors, capsids, and particles of the invention are In accordance with this embodiment, the immunogenic polypeptide may be used to generate an immune response. Viral vectors, capsids, and particles containing heterologous nucleic acid sequences encoding and an active immune response is mounted by the subject against the immunogenic polypeptide. The proteolytic polypeptide is as described herein above. In some embodiments, A protective immune response is elicited.

[0173] Alternatively, the viral vectors, capsids, and particles can be administered to cells ex vivo, The modified cells are administered to the subject. and the particles are introduced into cells, and the cells are administered to a subject, where the heterologous antigen encoding the immunogen is The nucleic acid is expressed and can elicit an immune response in a subject against the immunogen. In the example, the cell is an antigen-presenting cell (e.g., a dendritic cell).

[0174] "Active immune response" or "active immunity" refers to the "relationship between host tissues and cells after encountering an immunogen." This is characterized by the synthesis of antibodies or the development of cell-mediated reactivity, or It is involved in the differentiation and proliferation of immunocompetent cells in lymphoreticular tissue, causing both Herbert B. Herscowitz, Immunophysiology:Ce ll Function and Cellular Interactions in Antibody Formation, IMMUNOLOGY:BASIC PRO CESSES 117 (Joseph A. Bellanti ed., 1985). exchange In other words, an active immune response is generated by the host after exposure to an immunogen by infection or vaccination. Active immunization can be contrasted with passive immunization, which is the process of "actively immunizing" a person. from an immunized host to a non-immunized host, Transplantation of the immune system is achieved through the transfer of immune cells (interleukin-2) to the graft (ibid.).

[0175] A "protective" immune response or "protective" immunity, as used herein, refers to a response that is effective in preventing disease. The immune response provides some benefit to the subject in terms of preventing or reducing the incidence of Alternatively, a protective immune response or protective immunity can be achieved by treating a disease, particularly a cancer or tumor. These compounds may be useful in the treatment and / or prevention of cancers (e.g., preventing the formation of cancers or tumors). by preventing cancer or tumor regression and / or by preventing metastasis by preventing metastasis and / or by preventing the growth of metastatic nodules). The protective effect may be complete or partial, provided that the benefits of the treatment outweigh any of its disadvantages. It may be partial.

[0176] In certain embodiments, the viral vector, capsid, particle, or cell comprising the heterologous nucleic acid is , can be administered in an immunogenically effective amount as described herein.

[0177] The viral vectors, capsids, and particles of the present invention may be directed to one or more cancer cell antigens. (or immunologically similar molecules), or any other molecule that generates an immune response against cancer cells. It is administered for the purpose of cancer immunotherapy by administering a viral vector expressing the immunogen For example, to treat a patient with cancer and / or To prevent the onset of cancer, viral vectors containing heterologous nucleic acids encoding cancer cell antigens are administered. By administering the target, an immune response can be generated in the subject against cancer cell antigens. Viral vectors can be used in vivo or ex vivo as described herein. The compound may be administered to a subject by using

[0178] Alternatively, the cancer antigen may be expressed as part of the viral capsid or otherwise integrated into the viral The virion may be associated with the virion capsid (e.g., as described above).

[0179] As another alternative, any other therapeutic nucleic acid known in the art (e.g., RNAi ) or polypeptides (e.g., cytokines) for treating and / or preventing cancer. can be administered.

[0180] As used herein, the term "cancer" can encompass tumor-forming cancers. The term "cancerous tissue" includes tumors. "Cancer cell antigens" includes tumor antigens.

[0181] The term "cancer" is used in its art-recognized sense, e.g., a cancer that spreads to distant sites in the body. It means the uncontrolled growth of tissue that has the ability to metastasize (i.e., metastasize). Examples include, but are not limited to, melanoma, adenocarcinoma, thymoma, lymphoma (e.g., Non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia , uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer cancer, brain cancer, and any other cancer or malignant condition now known or later identified In an exemplary embodiment, the present invention provides a method for treating and / or preventing tumor-forming cancer. A method is provided.

[0182] The term "tumor" is also used in the art to refer to, for example, an abnormal mass of undifferentiated cells within a multicellular organism. It is understood that tumors can be malignant or benign. In exemplary embodiments, the tumors described herein The methods disclosed herein are used to prevent and treat malignant tumors.

[0183] The terms "treating cancer," "treatment of cancer," and equivalent terms refer to the severity of cancer. The disease is prevented or at least partially eliminated and / or the progression of the disease is slowed and / or controlled and / or the disease stabilized. In certain embodiments, these terms refer to a method in which the metastasis of a cancer is prevented, inhibited, or reduced. and / or the growth of metastatic nodules is prevented or suppressed. This means that something is eliminated or at least partially eliminated.

[0184] The terms "cancer prevention" or "preventing cancer" and equivalent terms refer to a method for preventing cancer from occurring. At least partially eliminate, reduce and / or delay the incidence and / or severity of the disease In other words, the development of cancer in a subject is intended to be delayed or delayed. It may be reduced in probability and / or delayed.

[0185] In certain embodiments, cells are removed from a subject with cancer and used to treat cancer cells according to the present invention. The modified cells can then be contacted with a viral vector expressing a cellular antigen. The modified cells are then administered to a subject. This method involves administering the cells to the target cells, thereby eliciting an immune response against the cancer cell antigens. immune system that is unable to mount an adequate immune response (i.e., unable to produce stimulatory antibodies in sufficient quantities) It may be advantageously used in immunocompromised subjects.

[0186] The immune response is mediated by the production of immunomodulatory cytokines (e.g., α-interferon, β-interferon, Interferon, gamma interferon, omega interferon, tau interferon, interferon Interleukin-1α, interleukin-1β, interleukin-2, interleukin Interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7 Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-10 , interleukin-11, interleukin-12, interleukin-13, inter leukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor Death factor-α, tumor necrosis factor-β, monocyte chemoattractant protein-1, granulocyte-macrophage It is well known in the art that the expression of IL-1 can be enhanced by various factors, including colony-stimulating factors and lymphotoxins. Therefore, immunomodulatory cytokines (preferably CTL-inducing cytokines) may be administered to a subject in conjunction with a viral vector.

[0187] Cytokines can be administered by any method known in the art. The cytokine may be administered to a subject, or a nucleic acid encoding the cytokine may be administered via a suitable vector. The cytokines can be produced in vivo by delivery to a subject using the method of the present invention.

[0188] [Subjects, Pharmaceutical Formulations, and Modes of Administration] The viral vectors, capsids, and particles according to the invention are useful for veterinary and medical applications. Suitable subjects include both birds and mammals. The term "avian" as used herein includes, but is not limited to, chickens. , ducks, geese, quail, turkeys, pheasants, parrots, parakeets, etc. The term "mammal" as used herein includes, but is not limited to, humans, Examples of animals include primates other than humans, cattle, sheep, goats, horses, cats, dogs, rabbits, etc. Subjects include neonates, infants, juveniles, adults, and geriatric subjects.

[0189] In an exemplary embodiment, a subject is "in need of" the methods of the present invention.

[0190] In a specific embodiment, the present invention provides a viral vector of the present invention in a pharmaceutically acceptable carrier. and / or capsids and / or particles, and optionally other drugs, pharmaceuticals, Pharmaceutical compositions containing stabilizers, buffers, carriers, adjuvants, diluents, etc. are provided. In other modes of administration, the carrier may be either a solid or a liquid. For inhaled administration, the carrier is respirable and, optionally, a solid or liquid. The compound may be in the form of a granule.

[0191] "Pharmaceutically acceptable" means a material that is not toxic or otherwise objectionable. i.e., the material can be administered to a subject without causing any unwanted biological effects. It means to get.

[0192] One aspect of the present invention is a method for transferring nucleic acid into cells in vitro. The target cells, capsids, or particles can be transduced according to standard transduction methods appropriate for the particular target cells. The viral vector can be introduced into cells at a multiplicity of infection to be administered. This may vary depending on the type and number of target cells and the specific viral vector and may require undue experimentation. In exemplary embodiments, at least about 10 infectious units, optionally at least about 10 5 The infectious units are introduced into the cells.

[0193] The cell(s) into which the viral vectors, capsids, and particles are introduced may be any suitable vector. The cells may be of any type, including but not limited to, nerve cells (cells of the peripheral and central nervous systems), cells, especially brain cells such as neurons and oligodendrocytes), lung cells, and eye cells (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., intestinal and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and / or diaphragm muscle cells) cells), dendritic cells, pancreatic cells (including islet cells), hepatocytes, cardiac myocytes, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells Examples of such cells include cells, germ cells, etc. In exemplary embodiments, the cells can be any progenitor cell. As a further possibility, the cells may be stem cells (e.g., neural stem cells, liver stem cells). As a further alternative, the cells may be cancer cells or tumor cells. As mentioned above, they can come from any species.

[0194] The viral vectors, capsids, and particles can be used to administer the modified cells to a subject. The cells may be introduced into cells in vitro. In certain embodiments, the cells may be cells already removed from a subject. The viral vectors, capsids, and particles are introduced into the cells, and then the The cells are then re-administered into the subject. Cells are removed from the subject for ex vivo manipulation. Methods for subsequent reintroduction into the subject are known in the art (see, e.g., U.S. Pat. No. 6,223,999). (See Patent No. 5,399,346.) Alternatively, viral vectors, capsids, and particles may be , in cells derived from a donor subject, in cultured cells, or from any other suitable source. The cells can be introduced into cells in need thereof (i.e., the "recipient"). It is administered to the subject.

[0195] Suitable cells for ex vivo nucleic acid delivery are as described above. The dosage of the cells will depend on the age, condition and species of the subject, the type of cells, the nucleic acid expressed by the cells, and the mode of administration. Generally, at least 100 mg of benzodiazepine per dose is administered in a pharmaceutically acceptable carrier. Both are about 10 2 ~about 10 8 cells, or at least about 10 3 ~about 10 6 cells are administered In certain embodiments, the cells transduced with the viral vector are combined with a pharmaceutical carrier. The combination is administered to a subject in a therapeutically or prophylactically effective amount.

[0196] In some embodiments, the viral vector is introduced into the cell and the delivered polypeptide is The cells can be administered to a subject (e.g., (either as a transgene or expressed in the capsid). Generally, an immunogenically effective amount A quantity of cells expressing the polypeptide is administered in combination with a pharmaceutically acceptable carrier. An "immunogenically effective amount" is an amount that induces an immunogenic response to a polypeptide in a subject to which the pharmaceutical preparation is administered. The amount of expressed polypeptide is sufficient to elicit an active immune response in a mammalian animal. The dosage is sufficient to generate a protective immune response (as defined above). As long as the benefits of administering the polypeptide outweigh any disadvantages, the protection conferred is acceptable. The degree of control does not have to be complete or permanent.

[0197] A further aspect of the present invention is to administer a viral vector and / or viral capsid to a subject. The viral vector and / or capsid according to the present invention is delivered to a recipient in need thereof. Administration to a human subject or animal can be by any means known in the art. Optionally, the viral vector and / or capsid is in a pharmaceutically acceptable carrier. The compound may be delivered in a therapeutically or prophylactically effective dose.

[0198] The viral vectors and / or capsids of the present invention may be used in a variety of therapeutic applications, including immunotherapy, to elicit an immunogenic response (e.g., Generally, the immunogenic compositions of the invention can be administered in a variety of ways, including as a vaccine. an immunogenically effective amount of a viral vector and / or or capsid. Optionally, the dosage is sufficient to generate a protective immune response (as defined above). The benefits of administering an immunogenic polypeptide are sufficient to eliminate any of its disadvantages. The degree of protection conferred need not be complete or permanent, so long as the benefits outweigh the The immunogenicity and the virulence of the antibody are as described above.

[0199] The dosage of viral vector and / or capsid administered to a subject depends on the mode of administration, the treatment and The disease or condition being prevented and / or treated, the condition of the individual subject, the specific viral vector or The therapeutic effect can be achieved in a conventional manner, depending on the peptide, the nucleic acid to be delivered, etc. An exemplary dose for is at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 1 0 , 10 11 , 10 12 , 10 3 , 10 14 , 10 15 Transducing units (i.e., vectors) -genomic copy number), optionally about 10 8 ~10 13 The titer of transducing units.

[0200] In certain embodiments, two or more administrations (e.g., two, three, four, or more administrations) are administered. The frequency of administration may vary, for example, once a day, once a week, once a month, once a year, etc. It can be used to achieve a desired level of gene expression over an interval period.

[0201] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, and inhalation (e.g., aerosol). via the oral cavity (e.g., sublingually), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraembryo), parenteral Oral (e.g., intravenous, subcutaneous, intradermal, intramuscular [for administration into skeletal muscle, diaphragm muscle, and / or cardiac muscle]) intradermal, intrapleural, intracerebral, transventricular, and intra-articular), localized (e.g., skin and respiratory tract surfaces) For both mucosal surfaces, including the face, and for transdermal administration), intralymphatic, etc., as well as direct tissue administration or injection into an organ (e.g., into the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration can also be to the eye (e.g., intravitreal, subretinal, subconjunctival, retrobulbar, administration via the anterior chamber and / or suprachoroidal routes). (e.g., in or near a tumor or lymph node). The appropriate route will depend on the nature and severity of the condition being treated and / or prevented and the specific The nature of the vector is crucial.

[0202] Delivery to the target tissue involves delivering a depot containing the viral vector and / or capsid. In an exemplary embodiment, the viral vector and / or capsid may be The depot containing the ATP is implanted into skeletal muscle, cardiac muscle, and / or diaphragm muscle tissue, or the tissue is implanted into the skeletal muscle, cardiac muscle, and / or diaphragm muscle tissue. contacted with a film or other matrix containing the virus vector and / or capsid. Such implantable matrices or substrates are described in U.S. Pat. No. 7,201,898. It is listed.

[0203] In a specific embodiment, the viral vector and / or viral capsid according to the invention comprises: administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., muscular dystrophy, heart disease [ For example, to treat and / or prevent PAD or congestive heart failure).

[0204] The present invention provides antisense RNA, RNAi, or other functional RNA ( It can also be practiced to generate nucleic acids (e.g., ribozymes).

[0205] Injectables are liquid solutions or suspensions, and are prepared as solutions or suspensions in liquids prior to injection. It may be prepared in a conventional manner, either as a suitable solid form or as an emulsion. The viral vector and / or viral capsid may be administered in a localized rather than systemic manner, e.g. In addition, viral vectors and / or viral capsuls may be administered as depot or sustained release formulations. The peptides can be delivered attached to a surgically implantable matrix (e.g., rice (See US Patent Publication No. 2004-0013645-A1).

[0206] Viral vectors and viral capsids are administered to tissues of the CNS (e.g., brain, eye). and the ability to produce viral vectors or other viral vectors that are more widely distributed than would be observed in the absence of the present invention. can advantageously provide the capsid.

[0207] In certain embodiments, the delivery vectors of the present invention are used to treat genetic disorders, neurodegenerative diseases, psychiatric disorders, and the like. It can be administered to treat diseases of the CNS, including inflammatory bowel disease and tumors.

[0208] Illustrative diseases of the CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis Amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, Pick's disease, muscular dystrophy - Multiple sclerosis, myasthenia gravis, Binswanger's disease, spinal cord and / or head injury trauma (e.g. traumatic brain injury), Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, brain Stroke, cerebral infarction, psychiatric disorders including mood disorders (e.g., depression, bipolar disorder, persistent affective disorder) disorders, secondary mood disorders), schizophrenia, drug dependence (e.g., alcoholism and its other substance addictions), neuroses (e.g., anxiety disorders, obsessive-compulsive disorders, somatoform disorders, dissociative disorders, grief, postpartum depression), psychosis (e.g., hallucinations and delusions), dementia, paranoia, attention deficit disorders, psychosexual disorders, who would benefit from axonal / neuronal regeneration and / or repair, or any neurodegenerative condition, cognitive disorder, behavioral disorder, sleep disorder, pain disorder, or food allergy that may require Eating or weight disorders (e.g., obesity, cachexia, anorexia nervosa, and bulimia), and These include cancers and tumors of the CNS (e.g., pituitary tumors).

[0209] CNS disorders include ophthalmologic disorders involving the retina, posterior tract, and optic nerve (e.g., retinal pigment epithelium). Degeneration, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

[0210] Most, if not all, ophthalmic diseases and disorders have three types of symptoms: (1) angiogenesis, (2) inflammation, and (3) degeneration. The vector contains anti-angiogenic factors; anti-inflammatory factors; slows cell degeneration and promotes cell sparing. factors that promote cell proliferation, or factors that promote sparing, and combinations of the above. It can be employed to achieve

[0211] For example, diabetic retinopathy is characterized by neovascularization. (e.g., in the vitreous) or periorbital (e.g., in the sub-Tenon's area) Treatment can be by delivering angiogenic factors. One or more neurotrophic factors can also be administered intraocularly. (e.g., intravitreally) or periorbitally.

[0212] Uveitis is associated with inflammation. One or more anti-inflammatory factors may be present in the delivery vectors of the present invention. Administration can be by intraocular (eg, intravitreal or anterior chamber) administration.

[0213] In comparison, retinitis pigmentosa is characterized by retinal degeneration. The present invention relates to a method for treating retinitis pigmentosa by intraocular injection of a delivery vector encoding one or more neurotrophic factors. The treatment can be by administration (e.g., intravitreal administration).

[0214] Age-related macular degeneration involves both neovascularization and retinal degeneration. A delivery vector of the present invention encoding one or more neurotrophic factors is intraocularly (e.g., intravitreally) administered. and / or a delivery vector of the invention encoding one or more anti-angiogenic factors. Treatment can be by administration intraocularly or periorbitally (e.g., into the sub-Tenon's area) .

[0215] Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. The treatment may involve the use of a delivery vector of the invention to induce one or more of the following pathways that protect cells from excitotoxic damage: The administration of a number of neuroprotective agents may include intraocular, optionally intravitreal, administration of Delivered N-methyl-D-aspartate (NMDA) antagonists, cytokines , and neurotrophic factors.

[0216] In other embodiments, the present invention provides a method for treating seizures, e.g., reducing the onset, incidence, or severity of seizures. The effectiveness of therapeutic treatment of seizures can be assessed by behavioral measures. (e.g., eye or mouth tremors, ticks) and / or electrical recording means (most Each seizure can be assessed by its own electrographic abnormality. It may also be used to treat epilepsy characterized by multiple seizures over a long period of time.

[0217] In one exemplary embodiment, somatostatin (or an active fragment thereof) is used to treat pituitary tumors. In order to treat the disease, the delivery vector of the present invention is administered to the brain. A delivery vector encoding somatostatin (or an active fragment thereof) can be administered by microinjection. Similarly, such treatments are used to treat acromegaly (abnormal growth of the pituitary gland). The nucleic acid sequence of somatostatin (e.g., Ge) can be used to treat steroid hormone secretion. GenBank accession number J00306) and amino acid sequence (e.g., GenBank accession number P01166; Processed active peptides somatostatin-28 and somatostatin thiazol-14) are known in the art.

[0218] In certain embodiments, the vector is a vector as described in U.S. Pat. No. 7,071,172. It may contain a secretion signal.

[0219] In an exemplary embodiment of the present invention, the viral vector and / or viral capsid is a CN The viral vector and / or capsid is administered to S (e.g., to the brain or eye). , spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland) ), cerebellum, telencephalon (striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe), cortex, basal ganglia, hippocampus, and the cerebrum, including the amygdala, limbic system, neocortex, striatum, The viral vector and / or capsid may be introduced into the retina, cornea, and / or the inferior colliculus. The virus and / or capsid may also be administered to different regions of the eye, such as the optic nerve. It may be administered by intravitreal, subretinal, subconjunctival, retrobulbar, intracameral, or suprachoroidal routes. obtain.

[0220] Viral vectors and / or capsids are used to better distribute and administer the delivery vector. Viral vectors and / or cerebrospinal fluid (e.g., by lumbar puncture) can be used to deliver the therapeutic agent to the patient. Alternatively, the capsid may further be used in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction). It can be administered intravascularly to the CNS.

[0221] The viral vector and / or capsid may be delivered to, but not limited to, the lateral ventricles, the cisterna magna, or the like. , intraparenchymal, intracranial, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., sugar, e.g., manganese) in the presence of nitrite, etc.), intranasal, intraauricular, intraocular (e.g., intravitreal, subretinal, anterior chamber), and Periorbital (e.g., subtenon's area) delivery, as well as muscle delivery with retrograde delivery to motor neurons The desired region(s) of the CNS may be delivered by any route known in the art, including intravenous delivery. It can be administered at any time.

[0222] In certain embodiments, the viral vector and / or capsid is directed to a desired region within the CNS. or administered in a liquid formulation by direct injection into the compartment (e.g., stereotactic injection). In other embodiments, the viral vector and / or capsid contains a desired region. It can be delivered by topical application to the eyes or by intranasal administration of an aerosol formulation. Administration of the viral vector can be by topical application of drops. The tar and / or capsid may be administered as a solid sustained release formulation (see, e.g., U.S. Pat. No. 6,413,629). (See No. 7,201,898).

[0223] In still further embodiments, the viral vector is used to treat diseases and disorders involving motor neurons. Treating and preventing diseases such as amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc. For example, viral vectors can be used for retrograde delivery to muscle tissue to prevent or treat the disease. from where they can travel into neurons.

[0224] Having described the invention, the same will be more particularly described in the following examples, which are provided to illustrate, and illustrate the principles of the invention. The examples are included herein for illustrative purposes only and are not intended to impose limitations on the invention. It is not intended to be so.

[0225] The subject matter of the present invention will now be described more fully hereinafter with reference to the accompanying examples, The examples illustrate representative embodiments of the presently disclosed subject matter. The subject matter disclosed in may be embodied in different forms, and the embodiments described herein may be Rather, these embodiments are intended to be exhaustive and not to be limiting. and complete in nature and will fully convey the scope of the presently disclosed subject matter to those skilled in the art. will be done. [Example]

[0226] The following examples provide illustrative embodiments. Certain aspects of the following examples are not intended to limit the scope of the present invention. Techniques discovered or contemplated by the inventors to function satisfactorily in the practice of In light of this disclosure and the general level of skill of those skilled in the art, It should be understood that the following examples are intended for illustrative purposes only and are not intended to be limiting unless otherwise stated. Numerous changes, modifications, and variations may be adopted without departing from the scope of the subject matter Recognize.

[0227] [Example 1] Plasmids. Plasmids containing portions of the human ZKSCAN1 and HIPK3 genes. was used in the experiments described herein. Cloning into the plasmid backbone separated by a nucleotide site A running vector was constructed containing EMCV-IRES and GFP (linear or split). A cassette containing the nucleotide sequence α was cloned between these intron sequences. The circGFP cassette was derived from the circGFP plasmid. A GFP cassette was also cloned into the plasmid backbone as a control. All plasmids contained a CMV promoter, an SV40 polyadenylation signal, and an AA It was constructed in a backbone containing terminal inverted repeat sequences derived from the V2 genome.

[0228] Generation of recombinant AAV vectors. Current triple-plasmid transfection protocol. The call was used to generate recombinant AAV vectors. The reaction mixture contained: (i) pXR helper plasmid; (ii) adenovirus helper Plasmid pXX6-80; and (iii) terminal inverted repeat sequences derived from the AAV2 genome. The vector contained the indicated transgene driven by the CMV promoter, flanked by Purification of the protein was performed using an iodixanol gradient ultracentrifugation protocol. Zeba Spin Esalting Column (40K MWCO, Thermo Sc The product was desalted using a centrific (Centific). The primers (forward, 5'-AAC ATG CTA CGC AGA GAG GGA GTG G-3', SEQ ID NO: 1; reverse, 5'-CAT GAG ACA AGG AAC CCC TAG TGA TGG AG-3', sequence Quantitative PCR (Lightcycler 480, Roche Applied Pharma GmbH) was performed using the PCR product number 2. Vg titers were obtained by Pharmacia Biosciences, Pleasanton, CA. I got a good deal.

[0229] Cell culture. HEK293 cells were cultured in 10% FBS (Millipore-Sigma). and Dulbecco's modified Eagle's medium (Gi) supplemented with 1% gentamicin / kanamycin. Huh7 and U87 were cultured in a laboratory (BCO / Life Technologies). Cells were cultured in 5% FBS (Millipore-Sigma) and 1% gentamicin / cannabinol. Dulbecco's modified Eagle's medium (Gibco / Lifetech) supplemented with cephalosporin Neuro2A cells were cultured in 10% FBS (Millipore). MEM supplemented with 1% penicillin / streptomycin and 1% HCl (Sigma-Aldrich) All cells were cultured in a Gibco / Life Technologies facility. were maintained at 37°C and 5% CO2.

[0230] Fluorescence microscopy. Cells were seeded at approximately 70% confluency and then transfected with the indicated rAAV vectors. Transduction was performed using a vector. Cells were transduced at 6, 4, 4, or 13 days after transduction. and imaged (HEK293, Huh7, U87, and Neuro2A cells, respectively). EVOS equipped with a GFP light cube (excitation 470 nm, emission 510 nm) FL epifluorescence cell imaging system (AMC / Life Technologies) ) was used to image the cells.

[0231] RNA extraction. RNA was extracted using Trizol reagent according to the manufacturer's protocol. RNA was extracted from frozen tissues or adherent cells stored at a temperature. , tissue in Trizol using Tissue Lyser II (Qiagen). was first homogenized.

[0232] Western blotting: Lysates were diluted in 1x Passive Lysis Buffer fer (Promega, Madison, WI) and stored at -80°C. The samples were heated to 100°C and then separated on a 10% Tris-glycine gel. The resultant was transferred to a cellulose membrane. The membrane was then incubated overnight at 4°C in TBST containing 5% skim milk. GFP (1:1000 Santa Cruz, SC9996) or The primary antibody against actin (1:10000, GeneTex, GT5512) was used. The membrane was blotted with a stabilized peroxidase-conjugated sheep anti-mouse antibody. Used as the secondary antibody (1:3000, GE Healthcare, NA931V) . SuperSignal West Femto Substrate (Thermo Scienti Develop and quantify blots using a ELISA kit (Fic / Life Technologies). To visualize the chromatin, autoradiography or ChemiDoc XRS+ (Bi Visualization was performed using a chemiluminescence enhanced sensitivity protocol on a fluorochrome plated plate (Rad).

[0233] Northern blotting. 10 μg of RNA was denatured in denaturing buffer (67% deionized HCl). Resuspend in 6% ethanol (6.7% formaldehyde, 1x MOPS running buffer) and The sample was incubated at 0°C for 10 minutes and then cooled on ice. The samples were separated on a gel and then transferred to a Hybond N+ membrane (GE Healthcare Radiolabeled probes were incubated with Prime-It II laser according to the manufacturer's instructions. Random primer labeling kit (Agilent Technologies) The GFP was generated using the following amplification primers (5'-GCATGCTCTTCTCA GGAGCGCACCATCTTCTTCAAGGACGACGG-3', SEQ ID NO: 3, 5'-GCATGCTCTTCTTACCTGGACGTAGCCTTCGGGCATG GC-3', SEQ ID NO: 4) was used for PCR to prepare a DNA template for probe labeling. Radiolabeled probes were prepared according to the manufacturer's protocol. Lustra MicroSpin G-50 column (GE Healthcare) The probe was then purified using Rapid-Hyb buffer (GE Heal) The blots were hybridized to membranes in a 500µL ELISA kit (Litcare). Visualize and detect radiolabeled signals by exposure to a PhosphorImager screen. It was then quantified.

[0234] Intravenous administration. Animal experiments reported in this study were performed at the UNC Institutional Animal Care and Use Facility. Bred and maintained under NIH guidelines approved by the Institutional Animal Control Unit (IACUC) committee. C57 / Bl6 mice were used. 5.5 × 10 11 vg / animal Four weeks after injection, animals were given tribromoethanol (Abatin) (1. Mice were overdosed with 25% solution (0.2 ml per 10 g) by intraperitoneal route. This was followed by transcardial perfusion with phosphate-buffered saline. The remaining part was stored in 4% paraformaldehyde solution (Invitrogen). The tissue was then post-fixed in a cool, dry place.

[0235] ICV administration. The animal experiments reported in this study were approved by the UNC Institutional Animal Care and Use Committee. C57 / was bred and maintained under NIH guidelines approved by the Institutional Assessment and Control Unit (IACUC). The experiment was carried out using Bl6 mice. Pups 1-2 days after birth were quickly anesthetized on ice for 2 minutes. This was followed by stereotaxic ICV injection. The target was placed in the left lateral ventricle (total volume <3 μl) using a KOPF-900 small animal stereotaxic instrument (KO 26s gauge needle connected to a PF Instruments, Tujunga, CA Hamilton 700 series syringe (Sigma-Aldrich, S All neonatal injections were performed into the sagittal sinus using a vena cava (St. Louis, MO). The procedure was performed at a depth of 0.5 mm, 2 mm cranial to the transverse sinus, and 1.5 mm. After administration of the steroid, the mice were allowed to recover under a heat lamp, rubbed against the bedding, and then re-entered under their mother. Six weeks after injection, tribromoethanol (Avertin) (1.25% solution 10 Mice were overdosed with 0.2 ml of phosphate buffered saline (PBS) by intraperitoneal route. This was followed by transcardial perfusion of 4% paraformaldehyde in nitrated saline. Post-fixation was carried out in formaldehyde for 24 hours.

[0236] Tissue processing and immunohistochemistry. Fixed tissues were used with a Leica VT 1,200 S vibrating blade microtome (Leica Biosystems, Buffalo Gro 50 μm thick sections were obtained using a microscope (VE, IL). Immunohistochemistry for GFP expression was performed. Analysis was performed using the Vectastain ABC-HRP kit according to the manufacturer's protocol. (Rabbit IgG PK-4001 Kit, Vector Biolabs, Burlingame, CA) The imaging was performed using a Zeiss CLSM 700 confocal laser (Ngame, CA). Scanning microscope was used to image sections of different organs after immunostaining (Micro (Scopy Services Laboratory, UNC). Quantification was performed using the 16-bit image conversion and inversion technique. This was followed by background subtraction using the ball method. Each section, as well as the unstained control, The integrated density and area were also measured for the intercepts. The density was normalized to the area, Background values (from unstained controls) were subtracted.

[0237] Quantification of vector genomes. Genomic DNA was extracted using the QiaAmp DNA FFPE tissue kit. The viral genome was extracted from fixed tissue sections using a ELISA kit (Qiagen). To calculate the number of repeats, a primer specific for the CMV promoter (5'-CA AGTACGCCCCCTATTGAC-3', SEQ ID NO: 5, and 5'-AAGTCCC Quantitative PCR was performed using the following DNA fragment: GTTGATTTTGGTG-3', SEQ ID NO: 6. The vector genome copy number was determined using the primer 5'-GGACCCAA GGACTACCTCAAGGG-3', SEQ ID NO: 7, and 5'-AGGGCACCTC CATCTCGGAAAC-3', SEQ ID NO: 8) to the mouse lamin B2 locus and normalized it.

[0238] Intravitreal administration. The animal experiments reported in this study were approved by the UNC Institutional Animal Care and Use Committee. C57 was bred and maintained under NIH guidelines approved by the Institutional Animal Care Unit (IACUC). Before injection, 1% atropine and 2.5% phenylephrine were administered. HCl (Akorn Inc., Lake Forest, IL) ophthalmic solution. The eyes were dilated. Mice were treated with 200 mg / kg tribromoethanol (Avertin). A small hole was made in the limbus of the eye using a 30G needle, and then 1 μL of virus was injected. The sputum was slowly delivered using a 34G needle on a Hamilton gas-tight syringe. Four weeks after injection, mice were sacrificed by CO2 inhalation. The limbus was marked at the top of each eye. The eyeballs were enucleated and incubated at 4°C overnight. The corneas were then separated from the eyecups. The eyecups were then immersed in 30% sucrose for 3 hours and treated with optimal cutting temperature compound (Optima l Cutting Temperature compound)(Sakura F The plates were protected from freezing and stored at 20°C in a protective container (Inetek, Torrance, CA). If necessary, the eyecups should be filled with RNAlater (Invitrogen) for later RNA extraction. ) and stored.

[0239] Histoimmunofluorescence. 12 μM retinal sections were cryo-etched using a Leica CM3050 cryostat. (Leica Biosystems Inc., Buffalo Grove, IL) The slides were then resuspended in 1x PBS (Gibco, Gaithersburg, Germany). The retinal sections on the slides were rinsed three times with 0.5% Triton X-100 and and 1% BSA (Fisher Scientific, Waltham, MA). Each was covered for 1 hour. Rabbit anti-GFP (Invitrogen, G10362) was added to Dilute 1:750 in 0.3% BSA + 0.3% Triton X-100 and incubate with sections at 4°C. The slides were then rinsed with 1x PBS and incubated overnight. a Fluorescent goat anti-rabbit 488 (1:500, Invitrogen, A-110 After washing with water, the plates were incubated with ProLong G containing mounting medium at room temperature. old DAPI (Life Technologies, Waltham, MA) The fluorescence was measured using the corrected total fluorescence method. d total fluorescence method) in ImageJ and quantified it.

[0240] RT-PCR. 5 μg of RNA was used for PCR using the Turbo DNA-free kit (Ambion). The DNA was treated with DNase using a DNA fragment size of 1000 nm. Equal amounts of DNase-treated RNA were added to High Capacity RNA-to -cDNA kit (Applied Biosystems / Life techno The product of this reverse transcription reaction was converted into cDNA using a GFP ( 5'-ctgcttgtcggccatgatatagacgttgtggc-3', Row number 9, 5'-caagctgaccctgaagttcatctgcaccacc- 3', SEQ ID NO: 10) and glyceraldehyde 3-phosphate dehydrogenase (GA PDH) (5'-CCACTCCTCCACCTTTGAC-3', SEQ ID NO: 11, 5' -ACCCTGTTGCTGTAGCC-3', SEQ ID NO: 12) The DNA was used as a template for PCR (or quantitative PCR) using primers. Quantitative PCR products were visualized on an agarose gel. For RNAse® experiments, 5 μL of 37 grams of RNA was digested with 5 units of RNase R (Epicentre). The enzyme was inactivated at 95°C for 5 minutes. NA was used for RT-PCR.

[0241] Here, adeno-associated viruses (AAVs) are used to deliver transgenes that express circular RNA in vivo. The reporter design demonstrates that it can be used to deliver gene cassettes. Based on intron sequences derived from the KSCAN1 and HIPK3 genes. Exons 2 and 3 of the mRNA of HIPK1 and exon 2 of the mRNA of HIPK3 are naturally Backspliced to form circRNA. Creating reporter constructs. To achieve this, a portion of the intron sequence associated with the endogenous circularized exon is placed in the vector. Between the intron sequences, a split GFP open reading frame was inserted into the circular R The EMCV-IRES was also designed to be reconstituted only within the circular context. This was done with both intron sequences. To create ZKSCAN1 split-GFP and HIPK3 split-GFP constructs, As a control for IRES-dependent translation, EMCV-IRES and GFP were used. The cassette lacks intron sequences to facilitate backsplicing. The vector was cloned (Fig. 1A-1B).

[0242] In vitro characterization of circRNA expression vectors. To validate different cassettes, To achieve this, the construct was packaged into a recombinant AAV2 vector, the serotype of which was transduces most cells in culture. Among these, the U87 human glioblastoma cell line was used as a viral vector (Fig. 5). The one that showed the most robust expression was selected for further analysis. The control showed the lowest expression, while the two circular cassettes showed nearly equal expression ( This was verified by Western blot analysis of GFP expression (Fig. 2B, Fig. 2C To further characterize the constructs, RNA was extracted from the cells. and used to perform Northern blots. The IRES-GFP construct is expressed in the mRNA transcribed from this construct. In contrast, the following two splits showed a single band at the expected size: The GFP vector contained two bands: unspliced pre-mRNA One band at a size corresponding to A and one band corresponding to the spliced circRNA One band at the corresponding size (Figure 2D). Quantification of the blot revealed that ZKSCAN Levels of circRNAs generated from 1-split GFP and HIPK3-split GFP The levels were not found to be significantly different, confirming the protein level results (Figure 2E Interestingly, the levels of circRNAs were significantly different from those of IRES-GFP RNAs. This suggests that linear IRES-containing RNAs likely undergo cap-dependent translation and I RES-mediated expression was translated less efficiently than circular RNA due to interference during translation. These cell culture experiments suggest that our vectors are translatable circular The relevance of RNA being able to be successfully packaged into AAV for delivery and expression The validity was confirmed.

[0243] circRNA expression after intravenous delivery. Effects observed in cell culture correlate with in vivo Therefore, the following is a summary of the present inventors' findings. The construct was tested in mice. The construct was inserted into a recombinant AAV9 vector. packaged, but the serotype is known to be sufficient to cause transduction in vivo. In the first test, mice were intravenously injected with the viral vector and their heart tissue was analyzed. To confirm the relative levels and localization of GFP expression, Immunohistochemical staining was performed with a control linear IRES-GFP construct. found no GFP-positive cells in the cardiac tissue, which was a surprising result. However, the two circRNA vectors showed expression in cardiomyocytes throughout the heart. ZKSCAN1 split GFP showed robust expression, resulting in a large number of GFP-positive muscle cells. HIPK3 split GFP tissue was also found to be resistant to ZKSCAN1 intron. Although the number of GFP-positive cells was smaller than that observed in the control group (Fig. 3A), this was due to the staining. This was confirmed by mean pixel intensity analysis of color sections (Figure 3B). The difference in expression was further confirmed not to be due to dosage, as the mice received the vector genome. (Figure 3C).

[0244] To assess expression at the RNA level, we also analyzed circRNA splice junctions. RT-PCR was performed using primers that amplify the IRES gene. (Note that this analysis also amplifies IRES-GFP RNA.) No bands were detected for RNA, but none of the split GFP constructs The expression of circRNAs was also observed in the same samples (Fig. 3D). Quantitative RT-PCR was performed on the samples. This analysis confirmed the R of IRES-GFP. NA expression was detectable, albeit at a very low level. 1 Split GFP and HIPK3 Split GFP interact with GAPDH in cardiac tissue. The ZKSCAN1-driven construct showed robust expression at levels comparable to or higher than that of the ZKSCAN1-driven construct. They observed 27-fold higher expression than the HIPK3-driven construct (Figure 3E). This is because both vectors should be transcribed and translated with equal efficiency. The 1 intron sequence promotes circularization more efficiently in cardiac tissue than the HIPK3 intron. Finally, we performed an RNAse R assay to detect split GFP. The RT-PCR bands corresponding to both vectors were resistant to digestion and This provided evidence of circularization (Figure 3F).

[0245] Next, we investigated whether these circRNA expression vectors are expressed in other tissue types. We wanted to investigate this. Liver tissue was collected and processed from the same injections as above. Immunohistochemical staining showed that all constructs expressed very low levels in the liver. Among the three, ZKSCAN1 split GFP showed the most It showed the most expression, followed by HIPK3 split GFP and then IRES-GFP. However, since all three were near background, the differences were not significant (Figure 6) AAV9 is a vector known to cause efficient transduction in the liver. Therefore, this effect is not due to vector tropism. This was consistent with the cell culture data of our colleagues (Fig. 5, Huh7 cells). We have shown that the ZKSCAN1-circRNA is expressed in the liver. This is a somewhat surprising result. There are two possible explanations. First, perhaps Due to the small amount of intron available for circularization, the vector Second, previous studies have shown that other IREs may not be able to cyclize sufficiently. Since it has been shown that EMCV-IRES expression in the liver is lower than that in S. This IRES may not drive translation at high levels.

[0246] The split GFP vector is an accurate representation. This expression vector produces circular The unspliced pre-mRNA also produces a protein, at least in cell culture. Furthermore, ring-specific protein expression was still observed at significant levels in the nucleus (Fig. 2C). To demonstrate this, we have previously used split reporters. To extend to other proteins of interest, it is useful to use non-split ORFs. In this case, the protein is also produced from the linear, unspliced RNA. To test this, the GFP open reading frame was We created another version of the ZKSCAN1 reporter in which the reporter was not split. This reporter contains an IRES-GFP cassette flanked by intron sequences. This vector was named "ZKSCAN1 GFP" and was used in primary cell culture. The ZKS gene was packaged into a rAAV2 vector for culture testing in U87 cells. GFP fluorescence derived from CAN1 GFP and ZKSCAN1 split GFP was significantly did not appear to be different (Fig. 7B); this was confirmed by Western blot analysis. (Figure 7C, quantified in Figure 7D). We also confirmed that there was no significant difference (Figure 7E). Since the ratio is equal between the two vectors, this means there is no significant translational contribution from the pre-mRNA. Next, we also created a non-split version of HIPK3 GFP. Both constructs were used to generate recombinant AAV9 vectors and demonstrated their efficacy in cardiac tissue. The expression was confirmed by intravenous injection. The results reproduced those obtained using the linear GFP construct. Both GFP and HIPK3 GFP showed strong expression in the heart, but ZKSC The expression level of AN1 GFP was higher (Fig. 7F). The level and extent of expression was not noticeably different from the split version, and we This suggests that the split GFP reporter vector shows representative expression.

[0247] circRNA expression in the nervous system. Our cell culture experiments and circRNA expression Both published data on circRNA expression suggest that circRNAs are abundantly expressed in nervous system tissues. Therefore, the viral vector was injected into the left ventricle of mouse pups. Controls were harvested and re-immunohistochemically stained to visualize GFP expression. The IRES-GFP cassette in the split chromosome showed no expression in the brain (Fig. 4A). Both of the GFP vectors showed expression in the brain, although it was restricted to the cerebral cortex. Since AV9 vectors are known to be expressed globally in the brain, this region-specific expression may be intrinsic to circRNAs (e.g., circularization or IRES-mediated translation) Furthermore, GFP expression is predominantly in astrocytes, whereas expression in neurons is limited. In contrast to previous experiments, HIPK3 split GFP expression was barely detectable. ZKSCAN1 splits GFP-expressing tissues in the brain, resulting in more GFP-positive cells than GFP-expressing tissues. (Fig. 4B) This suggests that there are tissue-specific differences in the ability of intron sequences to promote circularization. This is evidence that...

[0248] The final tissue tested for expression was the retina of the eye. The mouse eye was injected into the vitreous. The eyecup was removed and the retina was sectioned for subsequent visualization of expression. Immunofluorescence followed to confirm the identity of the sarcoma. Again, consistent with all other mouse tissues tested. In contrast, the IRES-GFP construct did not show detectable GFP expression. However, neither ZKSCAN1 split GFP nor HIPK3 split GFP was detected in the retina. showed widespread GFP signal throughout the entire length of the nucleus (Figure 4C, quantified in Figure 4D). In this case, HIPK3-split GFP expression was more widespread. In this context, GFP expression is primarily expressed in photoreceptor cells and retinoid pigment epithelium ( retinoid pigment (epithelium) and other cells RNA was prepared for quantitative RT-PCR analysis. Extracted from the second injection cohort (Figure 4E). Although higher than background, IRE The signal for S-GFP RNA was extremely low, corroborating the protein level data. ZKSCAN1 split GFP and HIPK3 split GFP are expressed using IRES-G Compared with FP, all showed a tendency to increase expression, but the latent external factors in each state was not significant due to the values

[0249] These studies suggest that circRNAs can mediate transgene expression in various tissue types. It has been revealed that circRNAs are primarily found in nervous system tissues. Although it has been shown that the gene is expressed in cardiac tissue, our cassette However, expression in the liver was barely observed. This is noteworthy, as AAV vectors are known to target the liver. Even within tissues that showed circRNA expression, cell type specificity was observed. For example, in the brain So, our vector is only in astrocytes, not in neurons. Several studies have demonstrated circRNA expression in neurons. Many circRNAs are thought to function in neuronal processes. This was surprising because circRNAs can be expressed in the nucleus. The cell types involved may be more diverse than previously known. Types of receptors, specifically photoreceptors and retinoid pigment epithelium, have also been targeted for expression within the eye. The recombinant AAV vector used in this study was expressed in most tissues of the animal. Although it is a vector of the same serotype, many vectors with different tropisms have been generated. Therefore, these cir in different vectors as a two-factor system for specificity. It will be interesting to test the cRNA expression constructs. The lactosyltransferases showed tissue-specific differences in expression levels when compared with each other. In this study, only two different intron pairs were examined. Given the large number of known circRNAs, their expression levels and tissue specificity can be examined. There are many more intron pairs available, allowing for targeting to desired tissues and cell types. It may be possible to create a toolkit of intron pairs using expression-driven .

[0250] One of the distinctive properties of circRNAs is that, compared to most linear mRNAs, In this study, tissue was collected only at one time point for expression. However, the increased stability of the RNA compared to the linear message Therefore, it is possible to increase the expression level over time. This will be interesting for future studies. Furthermore, improved RNA stability may lead to robust expression. This can reduce the dose required for expression. is required for transgene expression and induces an inflammatory response that leads to ablation of transgene expression This is particularly important because there is a risk of Therefore, dose-comparison studies are needed to characterize the utility of circRNA expression vectors. .

[0251] For this study, GFP expression was used as a proxy for circRNA expression. The viral IRES used may also restrict expression to specific cell types. The extent of rcRNA expression may be greater than that observed here. It would be interesting to further test the effect of different IRES sequences on the expression of the gene. Apart from protein expression, circRNA vectors can be used to express functional RNAs. There is room for the use of lncRNAs. Various known non-coding RNAs Furthermore, circRNAs, including: It has potential as a platform for expressing designer RNA. These may include designer miRNA sponges (especially those where endogenous circRNAs are Furthermore, RNA has been well characterized as an iRNA sponge. These can be engineered to have binding sites for RNA-binding proteins. Taken together, circRNAs are coding RNAs in various tissues and cell types. It represents a novel method for expressing both RNA and non-coding RNA.

[0252] [Example 2] To investigate the intron requirement for backsplicing, both introns ( A series of constellations with different distances between the Alu element and the splice site on the left and right sides A tract was created. First, a sequence was inserted into the left intron; this The GC content of the HIPK3 intron is similar to that of the endogenous HIPK3 intron, and it has little ability to bind to the HIPK3 intron. The sequences consisted of randomized sequences with matching sequences. The sizes ranged from 100 bp to 1.5 kbp. These insertions were made at a point 100 nt away from the splice site. had a significant effect; even an insertion of 100 nucleotides dramatically reduced circle formation and GFP expression The longer lengths completely abolished expression (Fig. 8).

[0253] Next, into the left intron, located 325 nt from the polypyrimidine tract. A second series of insertions was created. These constructs also showed the same effect (Figures 9A-9C). F) Increasing the distance between the Alu element and the splice site in the left intron results in a circular Importantly, the HIPK3 constitutive A randomized sequence was inserted into the right intron of the tract (Fig. 10A-10F). The effect on the left intron was completely different from that on the right intron. All insertions up to 100 kb have an effect on circular RNA levels or GFP expression. Therefore, the desired sequence was expressed in the right direction without affecting circRNA formation. It is important to point out that different elements, e.g. Other non-coding RNAs, such as miRNAs, in the right intron of the circular RNA construct One of the advantages of this method is that it enables multiplexing of RNAs, etc.

[0254] Next, we wanted to investigate shortening the distance between the Alu element and the splice site. Therefore, the left and right introductory sequences lacking all sequences between the Alu and the splice site were In the left intron, all polypyrimidine tracts were removed. In the right intron, the entire region up to the consensus splice donor site was deleted. A minimal deletion was also made, leaving another approximately 100 nt (polypyrimidin in the left intron). (The left intron was well tolerated, leaving approximately the same amount of the intron). Indeed, a 2- to 3-fold increase in circRNA levels and GFP expression was observed. Consistent with this finding, increasing distance was detrimental (Figs. 8, 9A-9F). Deletion of all genes in the right intron results in the inability to form circRNAs. Minimal deletions in the nuclei result in circRNA and GFP levels equivalent to those without deletions Therefore, in the case of the right intron, a certain minimum distance is required (Fig. 11A~11F).

[0255] Whether sequences within the right intron are important; i.e., due to increased distance or included We also tested whether the minimal construct worked because of the actual sequences included. Therefore, the minimum deletion was to maintain the same distance between the Alu and the splice site. but contains a different sequence (from a different part of the intron) in the right intron. This construct behaved similarly to the original minimal right defect. , with GFP and circRNA levels equivalent to the full-length construct (Figure 1 3A-13D). Both left-sided defects and minimal right-sided defects are acceptable, so these two We decided to rationally combine the two deletions to create a double deletion construct. Importantly, this double deletion construct performed better than either of the single deletions. The circRNA and GFP expression levels were approximately 5-6 times higher than those of the original construct. These findings support circRNA formation. This provides a basis for designing synthetic introns that can

[0256] By using a double deletion construct, the HIPK3 intron was A total of 723 nucleotides were split into 226 nt in the right intron and 497 nt in the left intron. Based on these results, we investigated the role of the circRNA in the formation of circRNAs. We hypothesized that similar deletions could be made in other introns that are involved. A synthetic intron containing deletions within the HB4, ZKSCAN1, and laccase2 intron pairs The theoretical design of the introns is shown in Figure 14A-D. These synthetic introns were then inserted into the AAV vector. Note that this provides more space for incorporating other genomic elements within the It is also important to

[0257] In summary, these basic studies demonstrate that small synthetic backsplicing introns We demonstrate that the insertion of circRNAs can be efficiently generated. or for synthetic intron elements having deletions, the different permutations of the AAV vectors are used to achieve multiplexed expression of different genomic elements. It is possible to achieve this.

[0258] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

[0259] <Representative intron elements> Sequence 1: ZKSCAN1 left [ka] (SEQ ID NO: 13)

[0260] Right side [ka] (SEQ ID NO: 14)

[0261] Sequence 2:HIPK3 left [ka] (SEQ ID NO: 15)

[0262] Right side [ka] (SEQ ID NO: 16)

[0263] <Synthetic intron sequence containing deletion> >HIPK3ΔL [ka] (SEQ ID NO: 17)

[0264] >HIPK3ΔR [ka] (SEQ ID NO: 18)

[0265] >ZKSCAN1ΔL [ka] (SEQ ID NO: 19)

[0266] >ZKSCAN1ΔR [ka] (SEQ ID NO: 20)

[0267] >Laccase 2ΔL [ka] (SEQ ID NO: 21)

[0268] >Laccase 2ΔR [ka] (SEQ ID NO: 22)

[0269] >EPHB4ΔL [ka] (SEQ ID NO: 23)

[0270] >EPHB4ΔR [ka] (SEQ ID NO: 24)

[0271] <Representative IRES elements> Sequence 1: Encephalomyocarditis virus IRES [ka] (SEQ ID NO: 25)

[0272] Sequence 2: Poliovirus IRES [ka] (SEQ ID NO: 26)

[0273] <Representative promoter elements> CMV promoter [ka] (SEQ ID NO: 27)

[0274] <Representative poly(A) sequence elements> SV40 poly-A [ka] (SEQ ID NO: 28)

[0275] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

[0276] [Table 1A] [Table 1B]

[0277] [Table 2]

[0278] [Table 3]

[0279] [Table 4]

[0280] [Table 5A] [Table 5B]

[0281] [Table 6A] [Table 6B]

Claims

1. A nucleic acid molecule encoding a covalently closed circular RNA (circRNA), comprising: a) a gene of interest that can be transcribed into non-coding RNA or translatable mRNA; b) an intron element associated with the gene of interest, which is covalently closed; is back-spliced by the cellular splicing machinery to yield a circular RNA Intron elements and c) an intrasequence ribonucleotide that drives the translation of a translatable mRNA transcribed from the gene of interest; an inomation entry site (IRES); d) an intron element within the 5' untranslated region (UTR) and associated with the gene of interest; a promoter region located outside the ment; e) Within the 3'UTR and outside the intron element associated with the gene of interest and the translation control region located A nucleic acid molecule comprising:

2. (b) the intron element comprises any nucleotide sequence of SEQ ID NO: , in any combination and in any multiple and / or ratio thereof, molecule.

3. (c) IRES is a viral IRES listed in Table 5, a viral IRES listed in Table 6 3. The method of claim 1, further comprising administering to said subject a cellular IRES in any combination thereof and in any number and / or ratio of overlapping IRESs.

1. A nucleic acid molecule according to claim 1.

4. (e) The translation control region includes a polyadenylation (polyA) sequence and / or a circR The nucleic acid molecule of claim 1, wherein the structural element stabilizes the nucleic acid.

5. The nucleic acid sequence according to any one of claims 1 to 4, flanked by AAV inverted terminal repeats (ITRs). An adeno-associated virus (AAV) genome comprising a nucleic acid molecule.

6. An AAV capsid or particle comprising the AAV genome of claim 5.

7. An AAV capsid or particle comprising a nucleic acid molecule according to any one of claims 1 to 4.

8. A nucleic acid molecule according to any one of claims 1 to 4, claim 1, in a pharmaceutically acceptable carrier. 5, and / or the AAV genome according to claim 6 or 7, A composition comprising particles.

9. A method for expressing a covalently closed circular RNA molecule in a cell, comprising: A method according to any one of claims 1 to 4 in a cell under conditions in which a covalently closed circular RNA molecule is transcribed. A method comprising introducing a nucleic acid molecule according to any one of claims 1 to 4.

10. A method for expressing a covalently closed circular RNA molecule in a cell, comprising: The method of claim 5 in the cell under conditions in which a covalently closed circular RNA molecule is transcribed. A method comprising introducing an AAV genome.

11. A method for expressing a covalently closed circular RNA molecule in a cell, comprising: in the cell under conditions in which a covalently closed circular RNA molecule is transcribed, as claimed in claim 6 or 7. A method comprising introducing an AAV capsid or particle described in

12. A method for expressing a covalently closed circular RNA molecule in a cell, comprising:

9. The method of claim 8, wherein the method further comprises the step of: introducing a composition of formula (I) into the tissue containing the compound of formula (I).

13. The present invention provides a method for producing covalently closed circular RNA molecules in a tissue-specific and / or cell-specific manner. a method for expressing a covalently closed circular RNA molecule, the method comprising: Contacting tissues and / or cells with a nucleic acid molecule according to any one of claims 1 to 4. A method comprising:

14. The present invention provides a method for producing covalently closed circular RNA molecules in a tissue-specific and / or cell-specific manner. a method for expressing a covalently closed circular RNA molecule, the method comprising: A method comprising contacting tissues and / or cells with the AAV genome of claim 5.

15. The present invention provides a method for producing covalently closed circular RNA molecules in a tissue-specific and / or cell-specific manner. a method for expressing a covalently closed circular RNA molecule, the method comprising: Contacting tissues and / or cells with the AAV capsid or particle of claim 6 or 7. and a method comprising:

16. The present invention provides a method for producing covalently closed circular RNA molecules in a tissue-specific and / or cell-specific manner. a method for expressing a covalently closed circular RNA molecule, the method comprising: A method comprising contacting tissues and / or cells with the composition of claim 8.

17. The covalently closed circular RNA molecule is a therapeutic mRNA encoding a protein. molecules, RNA silence molecules, guide RNA molecules capable of targeting genomic elements a molecule, a guide RNA molecule capable of targeting an RNA transcript, a tRNA molecule, a long non-covalent a loading RNA molecule, an antisense RNA molecule, or any combination thereof. The method according to any one of claims 9 to 16.

18. Any of claims 9 to 17, wherein the cells and / or tissues are derived from a mammal.

2. The method according to claim 1.

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