Rna adeno-associated virus (raav) vectors and uses thereof

Abstract

The application described herein provides recombinant DNA viral particles comprising a protein shell encapsulating an RNA vector genome, as well as related compositions and uses thereof.

Description

[0001] Citation of relevant applications

[0002] This international patent application claims priority to international patent application number PCT / CN2021 / 075874, filed on February 7, 2021, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Adeno-associated virus (AAV) is a small (approximately 20 nm in diameter) replication-defective, non-enveloped virus that infects humans and other primates. It belongs to the genus *Dependoparvovirus* within the family Parvoviridae. Wild-type AAV can infect both dividing and non-dividing cells and can integrate its genome into the host cell's genome. Its life cycle depends on the presence of helper viruses, such as adenoviruses (AdV), which determines its name and classification.

[0004] AAV has been found in multiple vertebrate species, including humans and non-human primates (NHPs). The current consensus is that AAV does not cause disease in any human, but only elicits a very mild immune response. It consists of an icosahedral protein capsid approximately 20-25 nm in diameter and a single-stranded DNA (ssDNA) genome of approximately 4.7 kb (which can be either a positive (sense) strand or a negative (antense) strand).

[0005] The AAV capsid contains three types of subunits: VP1, VP2, and VP3, with a total of 60 copies in a ratio of 1:1:10 (VP1:VP2:VP3). The genome has two inverted T-terminal repeats (ITRs) at both ends, primarily serving as the origin of viral replication and packaging signals. The rep gene encodes four proteins required for viral replication. These Rep proteins are named according to their molecular weights: Rep78, Rep68, Rep52, and Rep40. The cap gene encodes translation from different start codons. Additionally, a third gene encoding the assembly activation protein (AAP) is located in a different reading frame within the cap coding sequence and has been shown to promote viral particle assembly.

[0006] Currently, thirteen AAV serotypes and many variants have been identified, which recognize different cell receptors, thus exhibiting different histiotropic and cellular tropism profiles. The in vivo histiotropic properties of AAV1-13 have also been thoroughly investigated in numerous animal models.

[0007] Each AAV ITR sequence contains 145 nucleotides. Each ITR sequence can form a hairpin for self-initiation, enabling the synthesis of the second DNA strand independent of primases. It has also been shown that ITRs are necessary for the integration of AAV DNA into the host cell genome (human chromosome 19) and for the rescue and efficient capsidation of AAV DNA to generate fully assembled DNase-resistant AAV particles.

[0008] The AAV2 ITR, serving as the origin of replication, consists of two arm palindromic sequences (B-B' and C-C') embedded within a larger stem palindromic sequence (A-A'). The ITR can acquire two conformations (Flip and Flop). See Figure 1. The Flip and Flop conformations have the B-B' and C-C' palindromic sequences closest to the 3' end, respectively. The 20-nucleotide D sequence, or D region, appears only once at each end of the AAV genome, thus remaining single-stranded.

[0009] The ITR also contains a 22-bp sequence—the Rep-binding element (RBE)—that binds to the AAV Rep78 and Rep68 proteins in a specific orientation. If the ITR is in a palindromic (hairpin) conformation, the Rep protein also contacts the 5-base sequence at the end of one of the short palindromic sequences (RBE'), thereby activating Rep DNA helicase and strand-specific endonuclease activities to aid AAV replication and packaging (see Figure 1).

[0010] The RBE contains a tetranucleotide repeat (e.g., four repeats) with a shared sequence 5'-GNGC-3'. The ATP-dependent DNA helicase activity of Rep78 and Rep68 remodels the A-A' region, generating a stem loop that is located as a single strand atop the terminal dissociation site (trs or TRS). In this conformation, the strand-specific and site-specific endonuclease catalytic domains of Rep78 and Rep68 introduce a nick at the trs. The nucleotide at the apex of the T-shaped structure corresponds to an additional RBE (RBE'), which stabilizes the association between the two largest Rep proteins and the ITR.

[0011] During the AAV lifecycle, when AAV DNA is uncoated in the cell nucleus, the ITR of the imported single-stranded genome snaps into a hairpin, providing a natural 3'-OH primer for second-strand synthesis. This produces a double-stranded molecule with covalently closed (hairpin) ends. A large Rep protein then binds RBE and RBE' within the hairpin, and an activated endonuclease cleaves one strand at a specific site within a recognition sequence called the terminal dissociation site (trs). This generates a new 3'-OH primer, which repairs the ITR to form a normal blunt-ended double-stranded molecule. During cleavage, a molecule of Rep78 or Rep68 is covalently attached to the 5'-terminal phosphate via a tyrosine-phosphate linker. The ITR is then reconfigured into a double hairpin to generate 3'-OH primers, which use cellular complexes to guide strand displacement synthesis along the length of the genome. This displaces the packaged single strand and modifies the covalently closed double-stranded molecule at one end, initiating a new cycle of cleavage, repair, and strand displacement synthesis. Each time this cycle is repeated, a new single chain is generated for packaging. Because the two ends are identical, this process is carried out equally from both ends, generating positive and negative chains for packaging.

[0012] Because AAV can transduce multiple species and tissues in vivo with no evidence of toxicity and generates relatively mild innate and adaptive immune responses, it has been widely used in gene therapy.

[0013] AAV vectors consist of the same capsid sequence and structure found in wild-type AAV (wtAAV). However, AAV vectors will lack all AAV protein-coding sequences and have a genome capsid designed with transgene expression cassettes at their locations. The only sequence of the viral origin is the ITR, which is required to guide genome replication and packaging during vector production. Complete removal of the viral coding sequence maximizes the packaging capacity of the AAV vector and contributes to its low immunogenicity and cytotoxicity during in vivo delivery.

[0014] Since AAV vectors are best suited to accommodate genomes smaller than 4.7 kb, the payload must be carefully designed to take into account not only the transgene sequence but also the regulatory elements required for gene expression (e.g., promoters, enhancers, introns, and polyadenylation signals).

[0015] A popular method for producing AAV vectors involves triple transfecting HEK293T cells containing constitutively expressed AdV E1a and E1b genes into a packaging plasmid expressing the rep and cap genes, a transgenic plasmid to be packaged into an AAV capsid, and a helper plasmid containing other helper AdV genes (such as E2A, E4, and VA RNA genes, which are crucial for replication, information RNA (mRNA) processing, and translation, respectively). Fortunately, by simply exchanging the capsid coding regions in the packaging plasmid or the helper virus, transgenic expression cassettes constructed using AAV2ITR can be packaged into capsids of any serotype.

[0016] AAV vectors recognize and bind to different cellular receptors and enter cells via internalization. Within the endosome, the intact AAV vector particle undergoes a series of pH-dependent structural changes essential for transduction and transport via the cytoskeletal network through the cytosol. Following endosome escape, the AAV vector enters the nucleus through the nuclear pore complex, where it undergoes capsid uncoating to release the genome.

[0017] The single-stranded AAV vector genome released into the cell nucleus is not immediately expressed until it is converted to double-stranded form—a requirement for transcription and a rate-limiting step for transduction.

[0018] Second-strand synthesis begins at the self-initiated ITR at the 3′ end of the genome. Furthermore, the double-stranded genome can be annealed via strand annealing, whereby the positive and negative strands of the genome packaged into individual viral particles anneal via Watson-Crick base pairing once in the cell nucleus. The double-stranded genome is then circularized via intramolecular or intermolecular genomic recombination at the ITR. This circularization and cascading process stabilizes the AAV vector genome into free DNA, leading to sustained gene expression in the cell after mitosis. Figure 2 ).

[0019] CRISPR has brought new momentum to gene therapy. A powerful genome editing tool, CRISPR has shown promise in treating genetic, acquired, and infectious diseases. However, the delivery of CRISPR cellular components remains a major obstacle to its clinical translation. To date, the most successful CRISPR in vivo gene editing has used AAV as the delivery vector.

[0020] However, conventional AAV delivery has encountered many practical difficulties, including 1) increased off-target effects due to prolonged Cas9 expression, 2) stimulating Cas9-specific immune responses, and 3) the high frequency of viral integration in CRISPR-induced double-strand breaks. Recent studies have confirmed pre-existing immunity against Cas9 in the population, which may pose additional challenges to edited cells if Cas9 expression remains persistent.

[0021] Therefore, there is a need to improve existing gene editing tools, such as Cas9-mediated gene editing tools. Summary of the Invention

[0022] One aspect of the present invention provides a ribonucleotide (RNA) sequence capable of being packaged into a DNA virus particle, said RNA sequence comprising: (1) an RNA sequence of interest (RSI), for example, an RNA coding sequence encoding a gene of interest (GOI), RNA encoding a protein (e.g., a therapeutic protein, an antigenic protein, or a gene-editing protein such as a CRISPR / Cas effector enzyme (“Cas protein”), a ZFN protein, a TALEN protein), such as mRNA, or non-coding functional RNA (e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense oligonucleotide, microRNA (miRNA), or an RNA component of a CRISPR-Cas (e.g., Cas9, Cas12, Cas13) system, including guide RNA (or gRNA), such as a single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA, and tracrRNA), or its precursors; and (2) RNA packaging signal (RPS), which is capable of interacting directly or indirectly with, for example, binding to, an RPS-interacting molecule that facilitates the packaging of the RNA sequence into the DNA virus particle; optionally, the DNA sequence encoding or corresponding to the RNA sequence, or the reverse complementary sequence of the DNA sequence, has reduced, weakened, or substantially no ability to be packaged into the DNA virus particle (e.g., the DNA sequence or its reverse complementary sequence lacks a DNA packaging signal, such as a functional AAV ITR for AAV packaging).

[0023] In some implementations, the DNA virus particles are AAV virus particles or oncolytic virus particles.

[0024] In some implementations, the RPS is located at or near the 5' end of the RSI, at or near the 3' end of the RSI, or inside the RSI (e.g., within an intron of the mRNA).

[0025] In some implementations, the RNA sequence contains more than one (e.g., 1, 2, 3, or more) identical or different RPSs.

[0026] In some implementations, two or more (e.g., three) of the more than one RPS are adjacent to or connected in series with each other via the same or different connectors.

[0027] In some implementations, the RNA sequence comprises two or more RPSs that are not adjacent to each other (e.g., each located at or near an end of the RNA sequence of interest (RSI)).

[0028] In some embodiments, the RPS comprises a transcribed modified AAV terminal inverted repeat (ITR), wherein the transcribed modified AAV ITR: (a) comprises a transcribed functional Rep binding element (RBE), optionally further comprising a transcribed functional RBE'; and (b) lacks a transcribed terminal dissociation site (TRS) or a transcribed inverse complementary TRS (rcTRS), or both; optionally, the transcribed modified AAV ITR further comprises a transcribed D region sequence (D sequence or D' sequence); and / or optionally, the RPS interacting molecule is Rep78, Rep68, Rep52, and / or Rep40.

[0029] In some embodiments, the transcribed modified AAV ITR is located within 1000, 800, 500, 300, or 200 nucleotides from the 3' end of the RNA; optionally, the transcribed modified AAV ITR is located at the 5' end of a polyA sequence, a polyA signal sequence (e.g., AAUAAA), or an RNA transcription termination sequence (e.g., a histone downstream element).

[0030] In some embodiments, the transcribed modified AAV ITR is based on the wild-type Flip or Flop ITR modified by transcription; optionally, the wild-type Flip or Flop ITR is derived from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 (optionally, the wild-type Flop ITR has the nucleotide sequence of SEQ ID NO:1).

[0031] In some implementations, the transcribed modified AAV ITR lacks both the transcribed TRS and the transcribed rcTRS.

[0032] In some embodiments, the transcribed modified AAV ITR comprises the transcribed D region sequence (optionally, the modified AAV ITR has the nucleotide sequence of SEQ ID NO:3).

[0033] In some embodiments, the modified AAV ITR of the transcription lacks the D region sequence of the transcription (optionally, the modified AAV ITR has the nucleotide sequence of SEQ ID NO:2).

[0034] In some embodiments, the RNA sequence further comprises a modified AAV ITR of the second transcription, the modified AAV ITR of the second transcription having a functional RBE sequence of the second transcription but lacking the TRS or rcTRS of the second transcription or both; optionally, the modified AAV ITR of the second transcription further comprises a D region sequence of the second transcription.

[0035] In some implementations, the modified AAV ITR of the transcription and the modified AAVITR of the second transcription are the same (or different).

[0036] In some embodiments, the modified AAV ITR of the transcription and the modified AAVITR of the second transcription (if present) contain a deletion in the corresponding wild-type AAV ITR D region sequence or the corresponding wild-type TRS / rcTRS of the transcription, a mutation in the corresponding wild-type AAV ITR D region sequence or the corresponding wild-type TRS / rcTRS of the transcription, or an insertion in the corresponding wild-type AAV ITR D region sequence or the corresponding wild-type TRS / rcTRS of the transcription.

[0037] In some embodiments, the modified AAV ITR of the second transcription is located within 1000, 800, 500, 250, or 150 nucleotides at the 5' end of the RNA sequence.

[0038] In some embodiments, the RPS includes an MS2 sequence, a PP7 binding site, or a com binding site, and the RPS interacting molecule includes an RPS interacting protein (RPSIP) that can interact directly or indirectly with the RPS, such as a phage-derived MS2 coat protein (MCP) for the MS2 sequence, a PP7 phage coat protein (PCP) for the PP7 binding site, or a phage COM protein (COM) for the com binding site.

[0039] In some embodiments, the RPSIP is directly or indirectly associated with (e.g., fused to) the protein components of the viral packaging system of the DNA virus viral particles (e.g., Rep78 and / or Rep68 of adeno-associated virus 2 (AAV2), or assembly activation protein (AAP)).

[0040] In some embodiments, the RNA sequence includes or preferably does not include a transcribed DNA packaging signal, such as a transcribed wild-type AAV ITR sequence (e.g., the RNA sequence includes a transcribed modified AAV ITR sequence having nucleotide additions, deletions, and / or substitutions of the corresponding transcribed wild-type AAV ITR sequence to reduce the DNA packaging capacity of the DNA virus particles).

[0041] In some embodiments, the RNA sequence further comprises: (1) a transcriptional enhancer; (2) a transcriptional intron or exon sequence (e.g., a transcriptional intron or exon sequence for enhancing protein expression); (3) a 5' UTR sequence; (4) a 3' UTR sequence; (5) a poly-A sequence, or a polyadenylation (poly-A) signal sequence, and optionally a GU-rich region downstream of the poly-A signal sequence; (6) a post-transcriptional regulatory element or sequence, such as a transcribed marmot hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) sequence; and / or (7) a transcriptional termination sequence (e.g., a histone downstream element), optionally, the RNA sequence comprising RPS located at the 3' of the post-transcriptional regulatory element or sequence and the 5' of the poly-A sequence or poly-A signal sequence.

[0042] In some implementations, the RNA sequence includes an RSI, optionally a transcribed WPRE sequence, an RPS (e.g., a transcribed modified AAV ITR, MS2 sequence, PP7 binding site, or com binding site), and a polyA sequence or polyA signal sequence in the 5' to 3' direction.

[0043] In some embodiments, GOI comprises proteins (e.g., fluorescent proteins, therapeutic proteins, antigenic proteins, or gene-editing proteins, such as Cas proteins, ZFN proteins, TALEN proteins), enzymes (e.g., Cre proteins or CRISPR / Cas effector enzymes, such as Cas9, Cas12, Cas13, or variants thereof), structural proteins, mRNA, non-coding RNA (ncRNA), siRNA, piRNA, short hairpin RNA or shRNA, microRNA (miRNA) or its precursors (including pre-miRNA and pri-miRNA), ribosomal RNA (rRNA), antisense sequences or oligonucleotides (ASO), RNA components of the CRISPR-Cas system, including guide RNA (or gRNA), such as single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA) and tracr RNA, guide RNA or gRNA, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR for CRISPR / Cas effector enzymes.

[0044] In some embodiments, the RNA sequence is a single-stranded RNA, which is a single-stranded RNA of less than about 8,900 nucleotides, less than about 8,000 nucleotides, less than about 7,000 nucleotides, less than about 6,000 nucleotides, less than about 5,200 nucleotides, less than about 4,000 nucleotides, less than about 3,000 nucleotides, less than about 2,000 nucleotides, about 4,700-5,200 nucleotides, about 4,700-5,000 nucleotides, about 4,700-4,800 nucleotides, or about 4,700 nucleotides.

[0045] Another aspect of the invention provides a polynucleotide comprising a cassette encoding the RNA sequence of the invention; optionally, the polynucleotide is a DNA sequence (e.g., a DNA plasmid) that optionally includes a filler sequence in the backbone of the DNA plasmid and / or optionally does not contain a functional DNA packaging signal, such as AAV ITR.

[0046] In some embodiments, the polynucleotide further comprises a promoter operatively linked to and driving the transcription of an RNA sequence encoded by the box.

[0047] In some implementations, the promoter is a pervasive promoter.

[0048] In some implementations, the promoter is a tissue-specific promoter.

[0049] In some implementations, the promoter is a compositional promoter.

[0050] In some implementations, the promoter is an inducible promoter.

[0051] In some implementations, the polynucleotide further includes an enhancer that enhances transcription of the promoter-driven RNA sequence.

[0052] Another aspect of the present invention provides recombinant DNA virus particles containing an RNA genome (e.g., the RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotide of the present invention) packaged in a protein shell (e.g., capsid) of a DNA virus (e.g., AAV virus or oncolytic virus).

[0053] In some embodiments, the DNA virus is AAV, and the recombinant DNA virus particle is a recombinant RNA adeno-associated virus (rRAAV) particle, the recombinant RNA adeno-associated virus particle comprising: (1) an AAV capsid; and (2) an RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotides of the present invention packaged within the AAV capsid.

[0054] In some implementations, the AAV capsid comprises a capsid from serotypes AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, AAVrh74, or 7m8.

[0055] Another aspect of the invention provides a population of recombinant DNA virus particles (e.g., rRAAV particles) comprising a plurality of recombinant DNA virus particles (e.g., rRAAV particles) of the invention, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the recombinant DNA virus particles (e.g., rRAAV particles) within the population have an RNA sequence of the invention packaged therein or an RNA sequence transcribed from a polynucleotide of the invention.

[0056] Another aspect of the invention provides a host cell comprising the RNA sequence of the invention, the polynucleotide of the invention, the RNA sequence transcribed from the polynucleotide of the invention, the recombinant DNA virus particles of the invention (e.g., rRAAV particles), and / or a population of the recombinant DNA virus particles of the invention (e.g., rRAAV particles).

[0057] In some embodiments, the host cell further comprises a viral packaging system that facilitates the packaging of the RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotide of the present invention into DNA viral particles.

[0058] In some embodiments, the viral packaging system comprises: (1) an AAV rep gene (e.g., coding sequences of Rep78, Rep68, Rep52, and / or Rep40) and an AAV cap gene (e.g., coding sequences of VP1, VP2, VP3, AAP, and / or MAAP) or their expression products, wherein the AAV rep and AAV cap genes are under transcriptional control of one or more promoters driving the transcription of the rep and cap genes; (2) one or more coding sequences of one or more proteins required for AAV packaging, such as adenovirus E2A, E4, and VA genes, or the one or more proteins; and (3) an RPS interacting molecule or its coding sequence; optionally, the ability of the viral packaging system to package DNA sequences into the DNA virus viral particles is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, from the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention; (2) modifying, for example, by mutating the AAV rep gene, the AAV cap gene, or the AAV cap gene. The cap gene, and / or the coding sequences of one or more proteins required for AAV packaging, to reduce, weaken, or substantially eliminate the ability of the corresponding translated protein to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) to enlarge the size of the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention.

[0059] In some implementations, the host cell is a mammalian cell (such as HEK293 cell) or an insect cell (such as Sf9 or Sf21 cell).

[0060] Another aspect of the present invention provides a method for generating a population of recombinant DNA virus particles (e.g., rRAAV particles) or recombinant DNA virus particles (e.g., rRAAV particles) of the present invention, the method comprising: a) culturing host cells of the present invention for a sufficient time, and b) harvesting the recombinant DNA virus particles or the population of the recombinant DNA virus particles.

[0061] In some embodiments, the method further includes isolating or purifying recombinant DNA virus particles or populations of recombinant DNA virus particles.

[0062] Another aspect of the invention provides a method for generating recombinant DNA virus particles (e.g., rRAAV particles) or populations of recombinant DNA virus particles, the method comprising: a) contacting a viral packaging system (e.g., an AAV packaging system) with an RNA sequence of the invention or an RNA sequence transcribed from a polynucleotide of the invention for a period of time sufficient to generate the recombinant DNA virus particles or populations of the recombinant DNA virus particles; b) harvesting the recombinant DNA virus particles or populations of the recombinant DNA virus particles; and optionally, c) isolating or purifying the harvested recombinant DNA virus particles or populations of the recombinant DNA virus particles.

[0063] In some embodiments, the viral packaging system (e.g., an AAV packaging system) comprises: (1) one or more proteins (e.g., VP1, VP2, and / or VP3 for assembling an AAV capsid) for packaging the RNA sequence into the protein shell of the DNA virus viral particle, or one or more coding sequences thereof; (2) one or more proteins (e.g., Rep78, Rep68, Rep52, and / or Rep40 for AAV packaging) for facilitating the assembly of the protein shell and / or the packaging of the RNA sequence into the protein shell of the DNA virus viral particle, or one or more coding sequences thereof (e.g., adenovirus E2a, E4, and VA genes); and (3) an RPS interacting molecule or its coding sequence thereof; optionally, the ability of the viral packaging system to package a DNA sequence into the DNA virus viral particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, from the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention; (2) modifying, for example, by mutating the AAV rep gene, the AAV... The cap gene, and / or the coding sequences of one or more proteins required for AAV packaging, to reduce, weaken, or substantially eliminate the ability of the corresponding translated protein to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) to enlarge the size of the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention.

[0064] Another aspect of the invention provides a system for packaging the RNA sequence of the invention or an RNA sequence transcribed from the polynucleotide of the invention into a DNA virus particle, the system comprising: (1) one or more proteins (e.g., VP1, VP2, and / or VP3 for assembling an AAV capsid) for packaging the RNA sequence into the protein shell of the DNA virus particle, or one or more coding sequences thereof; (2) one or more proteins (e.g., Rep78, Rep68, Rep52, and / or Rep40 for AAV packaging) for facilitating the assembly of the protein shell and / or the packaging of the RNA sequence into the protein shell of the DNA virus particle, or one or more coding sequences thereof (e.g., adenovirus E2a, E4, and VA genes); and (3) an RPS interacting molecule or its coding sequence thereof; optionally, the ability of the viral packaging system to package the DNA sequence into the DNA virus particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, from the polynucleotide encoding the RNA sequence of the invention or the polynucleotide of the invention; (2) modifying, for example, by mutating the AAV. The rep gene, the AAV cap gene, and / or the coding sequences of one or more proteins required for AAV packaging, to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) to enlarge the size of the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention.

[0065] Another aspect of the present invention provides a method for delivering a gene of interest (GOI) to a cell, plant, or animal, the method comprising contacting the cell, plant, or animal with recombinant DNA virus particles (e.g., rRAAV particles) of the present invention, a population of recombinant DNA virus particles (e.g., rRAAV particles) of the present invention, or recombinant DNA virus particles (e.g., rRAAV particles) or a population of recombinant DNA virus particles (e.g., rRAAV particles) generated by the method of the present invention, wherein the GOI is encoded by an RNA sequence of the present invention.

[0066] Another aspect of the present invention provides a method for delivering an RNA sequence of interest (RSI) to a cell, plant, or animal, the method comprising contacting the cell, plant, or animal with recombinant DNA virus particles (e.g., rRAAV particles) of the present invention, a population of recombinant DNA virus particles (e.g., rRAAV particles) of the present invention, or recombinant DNA virus particles (e.g., rRAAV particles) or a population of recombinant DNA virus particles (e.g., rRAAV particles) produced by the method of the present invention.

[0067] Another aspect of the invention provides a method for diagnosing, preventing, or treating a disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount or dose of a population of recombinant DNA virus particles (e.g., rRAAV particles) of the invention or generated by the method of the invention.

[0068] Another aspect of the invention provides the use of the recombinant DNA virus particles (e.g., rRAAV particles) of the invention, a group of recombinant DNA virus particles (e.g., rRAAV particles) of the invention, or recombinant DNA virus particles (e.g., rRAAV particles) or a group of recombinant DNA virus particles (e.g., rRAAV particles) produced by the method of the invention in the manufacture of a medicament for the diagnosis, prevention, or treatment of a disease or disorder in a subject in need.

[0069] It should be understood that any embodiment of the invention described herein, including those described only in the embodiments or claims, or only in one aspect / part below, may be combined with any other one or more embodiments of the invention, unless expressly denied or deemed improper. Attached Figure Description

[0070] Figure 1A The structure and sequence of the wild-type ITR of AAV2 are shown, including the A:A' stem region sequence, B:B' and C:C' T region sequences, and the unpaired D region sequence in the Flip and Flop configurations of the 3' ITR. RBE, RBE', and TRS are also shown.

[0071] Figure 1B and 1C The 5' of AAV1-7 is shown. Figure 1B ) and 3'( Figure 1C Multiple sequence alignment of ITR sequences.

[0072] Figure 2 The lifecycles of AAV vector / virus particles and the tested RAAV vector / virus particles are shown.

[0073] Figure 3This is a schematic diagram of the transgenic plasmids of the RAAV-ITR vector and the control vector, showing the relative positions and orientations of the promoter (such as the CAG promoter or "C"), the GOI coding sequence (such as the coding sequence of the reporter gene tdTomato or "T"), the WPRE sequence (or "W"), the SV40 polyA signal sequence (or "S"), and the wild-type ITR, mutant / optimized ITR (dITR or dITR-D).

[0074] Figure 4 This diagram illustrates the generation of AAV and RAAV-ITR vectors using a three-plasmid system. Recombinant AAV or RAAV viral vectors can be generated by co-transfecting three plasmids (e.g., a transgenic plasmid, a packaging plasmid, and a helper plasmid) into suitable packaging cells such as HEK293 cells. Green ITRs represent wild-type ITRs, and yellow ITRs represent optimized ITRs. pCAG-transgenic, pCAG-transgenic-ITR, and pCAG-ITR-transgenic-ITR are transgenic plasmids; pAAV-rep / cap is a packaging plasmid; and pHelper is a helper plasmid.

[0075] Figure 5A and 5B A representative viral vector titration method is shown. Figure 5A This is a flowchart of RAAV titration. Figure 5B The primers and probes for Q-PCR are shown.

[0076] Figures 6A-6C The titration of the RAAV-ITR carrier is shown. Figure 6A The titration of the CITWS group is shown. Figure 6B The titration of the CTWIS group is shown. Figure 6C The titration of the CITWIS group is shown.

[0077] Figure 7A and 7B The titration and infection of the RAAV-dITR-D vector are shown. Figure 7A The titration of the RAAV-dITR-D carrier is shown. Figure 7B In vitro infection with the RAAV-dITR-D vector is shown. The same volume (5 μL) of purified RAAV-dITR-D vector was used for in vitro infection of 2 × 10⁻⁶ cells. 5 HEK293T cells. Fluorescence images were taken 3 and 5 days post-infection.

[0078] Figure 8A This is a schematic diagram (not to scale) showing different plasmid constructs used to demonstrate the efficient packaging of RNA into RAAV particles.

[0079] Figure 8BThe results of specific DNA and RNA packaging of the AAV-tdTomato and RAAV-tdTomato constructs are shown by detecting the WPRE sequence in the packaged DNA or RNA. Effective RNA packaging occurs when both the heterologous RNA packaging signal (RPS) and its homologous RPS-binding protein (RBP, such as the MCP of MS2) are present.

[0080] Figures 9A-9C The reduced DNA packaging is shown when using an enlarged plasmid backbone. Figure 9A It is used to generate Figure 9B and 9C A schematic diagram of the various plasmids resulting from the study (not to scale), including plasmids with longer backbone sequences due to the inserted filling region (L-CTWM3S). Figure 9B The specific DNA packaging of AAV-tdTomato and RAAV-tdTomato is demonstrated by detecting the presence of the CAG promoter sequence using CAG-specific primer pairs. Figure 9C The specific DNA and RNA packaging of AAV-tdTomato and RAAV-tdTomato was demonstrated by detecting the presence of WPRE sequences using WPRE-specific primer pairs. Results showed that using an amplified / extended plasmid backbone sequence with filler sequences unexpectedly reduced unwanted DNA packaging by approximately 2-fold.

[0081] Figure 10A and 10B The use of the MS2 / MCP packaging system demonstrates the efficient packaging of Cre transgenic cells into RAAVs. Figure 10A The specific DNA packaging of AAV-Cre and RAAV-Cre is shown by detecting the presence of the CAG promoter sequence using CAG-specific primer pairs. Note that the CAG sequence is not present in the RAAV RNA sequence, and the detected RNA signal is a background signal. Figure 10B The specific DNA and RNA packaging of AAV-Cre and RAAV-Cre is shown by detecting the presence of WPRE sequences using WPRE-specific primer pairs.

[0082] Figure 11A-11B The results show that RPS / RBP improves RNA packaging compared to conventional AAV. Figure 11A The results of AAV genome packaging are shown when only DNA packaging signals (i.e., ITRs) are present. Figure 11B The packaging of the AAV genome is shown in the presence of both DNA packaging signal (ITR) and RNA packaging signal (MS2X3).

[0083] Figure 12A-12D The results of optimizing the RAAV system and identifying the performance of the optimized RAAV are shown. Figure 12A This indicates the specific genomic packaging of AAV-Cre and RAAV-Cre by detecting WPRE sequences. Figure 12B This indicates the specific genomic packaging of AAV-Cre and RAAV-Cre by detecting Cre sequences. Figure 12C The silver staining analysis of the composition of AAV and RAAV particles is shown. Figure 12D The morphological analysis of AAV and RAAV particles by TEM is shown, with a scale bar of 100 nm.

[0084] Figure 13A and 13B The results show the reduction in DNA packaging of AAV and RAAV. Figure 13A The engineered Rep demonstrates reduced DNA packaging compared to traditional AAVs. Figure 13B The reduction of DNA packaging in RAAVs was demonstrated by using various mutant MCP fusion proteins, including the double mutant MCP fusion protein DJ-MCPX2.

[0085] Figures 14A-14D The sample shows the expression of a functional transgene-encoded protein in RAAV viral particles. Figures 14A-14C Specify in the same way as in [the previous text]. Figure 14A The time course of Cre mRNA levels in infected cells is shown. Figure 14B The fold change in Cre mRNA levels in infected cells was shown 20 hours after infection. Figure 14C The timeline of Cre DNA infection in cells is shown. Figure 14D The percentage of infected cells quantified by flow cytometry 5 days post-infection is shown, n=2 replicates.

[0086] Figures 15A-15D The results of DNA and mRNA analysis of Ai9-MEF cells infected with AAV or RAAV are shown. Figure 15A The Ct value of Cre mRNA is shown. Figure 15B The Ct value of Cre DNA is shown. Figure 15C The Ct value of GAPDH mRNA is shown. Figure 15D The Ct value of 36B4 DNA is shown.

[0087] Figure 16 Genotyping of Ai9-MEF cells is shown.

[0088] Figures 17A-17B The instantaneous transfer of RAAV particles is shown. Figure 17A Western blot analysis of the lifetime of Cre protein in infected cells following conventional AAV delivery is shown. Figure 17BWestern blot analysis of the lifetime of Cre protein in infected cells after RAAV delivery is shown.

[0089] Figure 18 Additional functional RPS / RBP pairs – PP7 / PCP pairs and com / COM pairs – tested in the RAAV system are shown.

[0090] Figure 19 The RAAV system is shown to be applicable to various AAV serotypes, including AAV-DJ, AAV5, AAV8, and AAV9.

[0091] Figure 20A and 20B The additional AAP and MCP fusion proteins showed that increased RAAV production. Figure 20A This indicates the specific genomic packaging of RAAV-Cre by detecting the Cre sequence. Figure 20B A comparison of RNA packaging efficiency between RAAV and AAP N- or C-terminal fusions (AM or MA fusion constructs) is shown.

[0092] Figures 21A-21C The results of transient transfer of RAAV-Cre into the hippocampus of Ai9- mice are shown. Figure 21A The transfer of high-dose AAV-Cre into the hippocampus of Ai9- mice was demonstrated. Figure 21B The transfer of low-dose AAV-Cre to the hippocampus of Ai9- mice was demonstrated. Figure 21C The transfer of high-dose RAAV-Cre into the hippocampus of Ai9- mice was demonstrated. Figure 21D Results are shown in control mice. Red signal: tdTomato; green signal: Cre; blue signal: DAPI (nuclear staining). Detailed Implementation

[0093] 1. Overview

[0094] The invention described herein provides recombinant viral particles comprising a DNA viral protein shell and a “vector genome” comprising RNA, such as single-stranded RNA (instead of DNA). The “vector genome” may not be typical viral RNA because it may have very few (if any) viral-origin sequences other than the RNA packaging signal (RPS) described below. That is, DNA viruses typically or naturally coat their DNA viral vector genome within a protein shell, while recombinant versions of DNA viral particles as described herein replace the lichenified RNA. “RNA” or “ribonucleic acid” means a ribonucleotide consisting of a phosphate group, a ribose sugar, and a base (A (adenine), U (uracil), G (guanine), or C (cytosine)), each of which may be modified (e.g., base modification, glycosyl modification, phosphate modification, e.g., oxygen modification, fluorine modification, sulfur modification, pseudo-modification (e.g., pseudouridine modification), methylation, end-capping (e.g., 5-end-capping)) or unmodified, and optionally directly or indirectly associated with the respective phosphate group, ribose sugar, and base (A (adenine), U (uracil), G (guanine), or C (cytosine)). A deoxyribose and a base (A (adenine), T (thymine), G (guanine), or C (cytosine)) are fused together, each of which may be modified (e.g., base modification, glycosyl modification, phosphate modification, e.g., oxygen modification, fluorine modification, sulfur modification, pseudo-modification, methylation, end-capping (e.g., 5-end-capping)) or unmodified, for example, an RNA-DNA chimera, a DNA-RNA-DNA chimera, or an RNA-DNA-RNA chimera.

[0095] A typical (non-limiting) example of such recombinant DNA viral particles is adeno-associated virus (AAV), which typically / naturally capsidates a single-stranded DNA (ssDNA) vector genome. Another non-limiting example of such DNA viruses is oncolytic DNA viruses, such as oncolytic herpesviruses (e.g., herpes simplex virus or HSV), oncolytic adenoviruses, vaccinia virus (VACV), vesicular stomatitis virus (VSV), etc.

[0096] This invention is partly based on the surprising discovery that transcribed AAV ITRs in RNA form can facilitate the efficient and direct packaging of transcribed RNA containing such transcribed AAV ITRs into conventional AAV virus particles.

[0097] The invention described herein is also based in part on the surprising discovery that, in addition to transcribed AAV ITR (RNA), certain artificial or heterologous RNA sequences and their homologous / corresponding / natural RNA-binding proteins can also be used as pairs of RNA packaging signals (RPS) and RPS interacting proteins (RPSIPs) to replace the functions of wild-type packaging signal sequences and interacting proteins that are useful for DNA viral packaging, thereby packaging RNA into a DNA viral protein shell, which typically / naturally capsidates the DNA vector genome.

[0098] For example, in wild-type AAV, the ITR sequences at the 5' and 3' ends of the DNA vector genome contain sequence elements, such as Rep-binding elements (RBEs) and RBE's, that can interact with Rep proteins (such as Rep68 and Rep78). The Rep proteins bind to the ITRs and facilitate the packaging of AAV ssDNA vector genomes containing such ITR sequence elements into AAV viral particles.

[0099] The inventors have discovered that by providing a transcribed ITR sequence and / or an artificial or heterologous RNA sequence, such as an MS2 sequence, as an RPS to the RNA sequence of interest (RSI), the resulting RNA sequence consisting of RPS and RSI can be efficiently packaged into the AAV viral protein shell in the presence of a naturally binding MS2 MCP—a bacterial phage-derived MS2 capsid protein (MCP). The ability of artificial RPS / RPSIP pairs—e.g., MS2 / MCP—to facilitate RNA packaging into the DNA viral protein shell is independent of the presence of the natural ITR packaging signal for DNA packaging, but can function independently of it. In a sense, the heterologous MS2-MCP pair constitutes an artificial system of RPS and RPSIP pairs that can effectively replace the natural ITR-Rep DNA packaging system, which effectively facilitates RNA packaging. Such RNA-containing DNA viruses, such as AAV, may be referred to herein as R-DNA viral particles (or RAAV in the case of AAV), or recombinant R-DNA viral particles (or rRAAV in the case of AAV).

[0100] The R-DNA viral particles and RAAV viral particles of the present invention can be used to deliver RNA transcripts of any transgenic or gene of interest (GOI) of appropriate length (e.g., within the packaging limits of various DNA viruses or AAVs) or any guide RNA to host cells tropistically compatible with the protein shell of a DNA virus or the capsid of an AAV virus. As used herein, recombinant DNA viral particles such as recombinant AAV vectors, vector genomes, and recombinant AAV viral particles or recombinant AAV particles are referred to herein as rRAAV vectors (recombinant RNA adeno-associated virus vectors), vector genomes, and recombinant RAAV (rRAAV) viral particles or rRAAV particles (“rRAAV vectors” and “rRAAV particles” are used interchangeably herein).

[0101] In particular, on the one hand, like any normal or conventional AAV vector, the tested RAAV vector can also be composed of any of the same capsids found in any wild-type AAV carrying DNA as viral genetic material. Therefore, the tested RAAV vector possesses all the common advantages derived from AAV capsids, such as specificity / broad tropism and low immunogenicity.

[0102] However, on the other hand, the genome of the tested RAAV vector is composed of short-lived RNA (e.g., mRNA), resulting in transient expression of any gene product encoded on such RNA genetic material.

[0103] Such transient expression is required in at least some cases. For example, the RAAV vector of the present invention is advantageous for in vivo DNA gene editing because time-limited exposure to RAAV-encoded DNA gene editors (such as the mRNA coding sequence of the CRISPR / Cas system effector enzyme Cas9 and its variants fused to a base editor) enables efficient gene editing. Compared to the sustained expression of the same DNA gene editor expressed by conventional DNA-based AAV vectors, this transiently expressed DNA editor also improves the safety profile of gene therapy by reducing off-target gene targeting and decreasing immunogenicity.

[0104] Furthermore, compared to traditional DNA-based AAV vectors, the tested RAAV vectors can carry longer transgenes because they at least exclude the promoter (and any non-transcriptional enhancer sequences that might be required) needed to express the GOI encoded by the DNA-based AAV vector.

[0105] Although the tested rRAAV vectors have different sequence elements and organization compared to traditional DNA-based AAV vectors, the rRAAV viral particles exhibit the same entry and intracellular transport processes as traditional DNA-based AAV vectors. However, they have a very different fate after entering the host cell nucleus. Upon entering the nucleus, the mRNA genome of the tested RAAV vector is released and subsequently transported to the cytoplasm, leading to translation. It is well known that mRNA lifespan is typically short, ranging from minutes to days, and ultimately degrades via numerous cellular mechanisms. However, this limited mRNA lifespan still allows the host cell to complete protein synthesis, generally without delay compared to second-strand cDNA synthesis in DNA-based AAV vectors, and allows the encoded proteins to function rapidly.

[0106] Many such RPS / RPSIP pairs can be used to package RNA into DNA viruses. The inventors have demonstrated at least two additional such pairs, including the PP7 sequence and PP7 bacterial phage coat protein (PCP), and the com sequence and phage COM protein (COM), which effectively package RNA containing heterologous RPS (i.e., PP7 and com sequences, respectively). The three RPS / RPSIP pairs shown cover at least two categories. Unlike the natural viral packaging systems MS2 / MCP and PP7 / PCP, com / COM is not a natural viral packaging system but rather a transcriptional regulator known to play a role in the transcriptional initiation of the bacterial phage Mu mom gene. Many transcribed modified AAV ITR sequences can also be used as RPSs in this invention.

[0107] The invention described herein is not limited to specific serotypes of DNA viruses (e.g., specific AAV serotypes). The inventors have demonstrated that, in each case, the use of a compatible RPSIP in combination with the RNA sequence having a suitable RPS can efficiently package RNA sequences into representative AAV viruses (including AAV5, AAV8, AAV9, and AAV-DJ).

[0108] The invention described herein is also based on the discovery that several independent methods can reduce the efficiency of packaging unwanted DNA into natural DNA virus particles.

[0109] In some implementations, undesirable DNA packaging efficiency can be reduced by increasing the overall size of the DNA vector transcribed from the RNA of interest. For example, in a triple transfection method commonly used for AAV production, the gene of interest (GOI) can be carried by a first plasmid, the required Rep and Cap proteins can be encoded by the rep and cap genes on a second plasmid, and other components required for AAV packaging can be provided by a third plasmid. According to this embodiment of the invention, the RNA sequence to be packaged into a DNA virus can be transcribed from the first plasmid, and the overall size of the first plasmid can be artificially increased by including random filler sequences (e.g., introns), such as filler sequences of at least about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or longer, or by increasing the overall size of the first plasmid by 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or longer filler sequences, for example, increasing to about 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, or longer filler sequences.

[0110] In some other embodiments, undesirable DNA packaging efficiency can be reduced by inhibiting the function of classical elements that promote DNA packaging. Such classical elements for DNA packaging may include DNA sequences (such as elements of AAVITR sequences that promote DNA packaging, including trs sequences, RBE or RBE' sequences, or the entire ITR sequence of AAV); and / or protein elements involved in DNA packaging, such as proteins that interact with DNA sequences (such as mutant Rep68 or Rep78 proteins lacking or having weakened trs-endonuclease activity).

[0111] Therefore, one aspect of the present invention provides a ribonucleotide (RNA) sequence capable of being packaged into a DNA virus particle (such as a DNA virus that naturally packages DNA), wherein the RNA sequence comprises: (1) an RNA sequence of interest (RSI); and (2) an RNA packaging signal (RPS) capable of interacting, for example binding, directly or indirectly with an RPS-interacting molecule (e.g., an RPS-interacting protein or RPSIP) that facilitates the packaging of the RNA sequence into the DNA virus particle.

[0112] Such RNA sequences can contain any RSI (RNA) that can be encoded by a "gene of interest" or "GOI" (DNA).

[0113] As used herein, “gene of interest” or “GOI” includes any coding sequence of a protein or polypeptide, including intron and exon sequences, and / or coding sequences of any non-translated RNA or non-coding RNA (ncRNA, such as siRNA, piRNA, short hairpin RNA or shRNA, microRNA or miRNA or their precursors, including precursor miRNA and primary miRNA, antisense sequences or oligonucleotides (ASO), CRISPR / Cas guide RNA or gRNA, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR, etc.).

[0114] Similarly, representative (non-restrictive) RSIs include, for example, RNA encoding proteins (e.g., therapeutic proteins, antigenic proteins, or gene-editing proteins such as CRISPR / Cas effector enzymes (“Cas proteins”), ZFN proteins, TALEN proteins), such as mRNA, or non-coding functional RNAs (e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), transfer-messenger RNA (tmRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA or oligonucleotides (ASO), microRNA (miRNA), RNA aptamers, or RNA components of CRISPR-Cas (e.g., Cas9, Cas12, Cas13) systems, such as single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA), and tracrRNA. RNA, or its precursors, or RNA components of the RISC complex or RNAi pathway (such as shRNA, miRNA, or siRNA), regulatory RNA, Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (lncRNA) (including intergenic lincRNA, intronic ncRNA, and sense / antisense lncRNA), long intergenic / intergenic non-coding RNA (lincRNA), enhancer RNA, bacterial small RNA (sRNA), snRNA, exRNA, scaRNA, Xist, and HOTAIR and its precursors.

[0115] The RNA sequence or GOI of the present invention may contain one coding sequence or more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) coding sequences. The length of the coding sequence, or the combined length of all coding sequences, may not exceed the maximum length of RNA that can be packaged into a particular or selected DNA virus particle (e.g., AAV virus particle), which may differ between one particular DNA virus (e.g., AAV) virus particle and another.

[0116] In some embodiments, the DNA sequence encoding or corresponding to the RNA sequence of the present invention, or the inverse complementary sequence of said DNA sequence, has reduced, weakened, or substantially no ability to be packaged into DNA virus particles. For example, the DNA sequence may encode the RNA sequence of the present invention (e.g., said DNA sequence has the inverse complementary sequence of said RNA sequence). The DNA sequence may also correspond to the RNA sequence of the present invention because said DNA sequence has the same nucleotide sequence as the RNA sequence of the present invention in other respects, except that said DNA sequence has T instead of U in the RNA sequence of the present invention. In any case, the DNA sequence or its inverse complementary sequence may lack a functional DNA packaging signal for packaging into DNA virus particles, such as the AAV ITR for AAV packaging, such that said DNA sequence or its inverse complementary sequence (DNA) has reduced, weakened, or substantially no ability to be packaged into DNA virus particles.

[0117] In some embodiments, the RNA sequence of the present invention is transcribed from a DNA construct, such as from a DNA plasmid encoding the RNA sequence, wherein the DNA construct / plasmid includes filler sequences (e.g., intron sequences) in its backbone sequence to enhance the packaging of the RNA sequence of the present invention and / or reduce unwanted DNA packaging into DNA viral particles. For example, the RNA sequence of the present invention can be transcribed from a DNA construct / plasmid, and the total size of the DNA construct / plasmid can be artificially increased by including random DNA filler sequences, such as filler sequences at least about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or longer, or by increasing the total size of the DNA construct / plasmid by 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or longer, for example, increasing to about 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, or longer. The filler sequences can be located upstream (e.g., immediately upstream) of the transcription unit containing the coding sequence of the RNA sequence of the present invention (see [link to documentation]). Figure 9A In some embodiments, the long padding sequence >3kb is inserted immediately upstream of the CAG promoter that drives the transcription of the exemplary RNA sequence of the present invention. In some embodiments, the padding sequence is inserted directly upstream of the promoter, which is operatively linked to the codon sequence of the RNA sequence of the present invention. Optionally, in some embodiments, the coding sequence of the RNA sequence of the present invention lacks a functional natural DNA packaging signal for DNA viral particles, such as a functional ITR sequence packaged into AAV viral particles.

[0118] In some embodiments, the RNA sequence of the present invention can be packaged into DNA viral particles, said DNA viral particles being AAV viral particles. Any AAV virus can be used to package the RNA sequence of the present invention, including but not limited to AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, AAVhu32, AAVhu37, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, AAVrh74, or 7m8.

[0119] In some embodiments, the RNA sequence of the present invention can be packaged into DNA viral particles, said DNA viral particles being oncolytic virus particles. Exemplary (non-limiting) oncolytic virus particles include: oncolytic herpesviruses (e.g., herpes simplex virus or HSV), oncolytic adenoviruses, vaccinia virus (VACV), vesicular stomatitis virus (VSV), etc.

[0120] The location of the RPS in the RNA sequence of the present invention can be flexible. In some embodiments, the RPS is located at or near the 5' end of the RNA sequence of the present invention, at or near the 3' end of the RNA sequence of the present invention, or inside the RNA sequence of the present invention. In some embodiments, the RPS is located at or near the 5' end of the RNA sequence of interest (RSI), at or near the 3' end of the RNA sequence of interest (RSI), or inside the RNA sequence of interest (within an intron of the mRNA).

[0121] One or more RPSs may be present in the RNA sequence of the present invention. In some embodiments, the RNA sequence of the present invention contains more than one (e.g., 1, 2, 3, or more) identical or substantially identical RPSs. In some embodiments, the RNA sequence of the present invention contains more than one (e.g., 1, 2, 3, or more) RPSs, and at least two of them are different from each other.

[0122] In the case of more than one RPS on the RNA sequence of the present invention, at least two of the more than one RPS are adjacent to each other, such as in tandem, and have an optional adapter sequence between them. The adapter between any two adjacent RPS sequences can be the same or different. The adapter sequence can be a randomized RNA sequence without substantial secondary structure, without known functional sequences or elements, and / or the GC content can be less than 50%. The length of the adapter can be any length between 1-1 kb, 1-500 bases, 1-200 bases, 1 to about 100 bases, 1 to about 60 bases, about 5 to about 55 bases, about 10 to about 30 bases, or about 15-25 bases.

[0123] In some embodiments, the RNA sequence of the present invention comprises three adjacent RPS sequences separated by two adapter sequences, each adapter sequence being independently about 20 or about 50 bases. For example, the first two of the three identical RPS sequences may be separated by a 20-base adapter, and / or the last two of the RPS sequences may be separated by a 51-base adapter.

[0124] In some embodiments, the RNA sequence of the present invention comprises more than one RPS (e.g., 1, 2, 3, 4, or 5 RPSs), wherein at least two of the more than one RPS are not adjacent to each other. For example, one RPS may be located at the 5' end of the RNA sequence of the present invention, while another RPS may be located at the 3' end of the RNA sequence of the present invention, and optionally a third RPS may be located within an intron of the mRNA as an RSI within the RNA sequence of the present invention. The fourth and / or fifth RPS may be close to or adjacent to any one of the first, second, or third RPSs.

[0125] In some embodiments, the RNA sequence of the present invention comprises at least two (e.g., two or more) RPS sequences that are not adjacent to each other (e.g., each located at or near an end of the RNA sequence of interest (RSI)).

[0126] In some embodiments, the RPS comprises a transcribed modified AAV terminal inverted repeat (ITR), wherein the transcribed modified AAV ITR (a) comprises a transcribed functional Rep binding element (RBE), optionally further comprising a transcribed functional RBE'; and (b) lacks a transcribed terminal dissociation site (TRS) or a transcribed inverse complementary TRS (rcTRS), or both. In some embodiments, the transcribed modified AAV ITR further comprises a transcribed D region sequence (D sequence or D' sequence). In some embodiments, the RPS interacting molecules are Rep78, Rep68, Rep52, and / or Rep40.

[0127] As used herein, “AAV viral particle” includes any viral particle containing a wild-type capsid of adeno-associated virus (AAV) (belonging to the genus Parvovirus, which in turn belongs to the family Parvoviridae), as well as engineered or variants of it having modified sequences and / or tissue or host specificity.

[0128] As used herein, an "intron" refers to a non-coding segment of DNA or RNA, which is typically removed from transcribed RNA by splicing. However, the RNA sequences of the present invention may contain intron sequences, such as intron sequences from a heterologous gene ("heterologous" in relation to the gene of interest or GOI, which is expressed as a transgenic expression delivered to host cells via the rRAAV viral particles of the present invention) to enhance GOI expression. Such intron sequences in the RNA sequences of the present invention may or may not be removed by splicing. Furthermore, such intron sequences may further contain transcribed enhancers or portions thereof, as some enhancers may be located within introns encoding DNA.

[0129] As used herein, "exon" refers to a coding segment of DNA or RNA that is translated into a protein sequence. However, in some embodiments, the exon sequence within the RNA sequence of the present invention may encode part or all of a GOI, which is expressed as a transgenic expression delivered to a host cell via the rRAAV viral particles of the present invention. In other embodiments, the exon sequence within the RNA sequence of the present invention may belong to a heterologous gene (with respect to GOI), and the presence of such exons may enhance GOI expression.

[0130] As used herein, “coding sequence” includes a polynucleotide sequence of DNA or RNA that encodes a product, which may be (a) a protein or polypeptide, or (2) a product other than a protein or polypeptide (e.g., ncRNA, such as siRNA, piRNA, short hairpin RNA or shRNA, microRNA or miRNA or its precursors, including precursor miRNA and primary miRNA, antisense sequences or oligonucleotides (ASO), CRISPR / Cas guide RNA or gRNA, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR, etc.).

[0131] Once the RNA of the RAAV viral particle is separated from the AAV capsid and released into the cell, the ribonucleotide coding sequence of the gene of interest can be further processed within the cell. Processing the coding sequence can produce one or more RNA products, such as siRNA, miRNA, and / or mRNA, which can be further translated into one or more protein products or incorporated into other cellular machinery, such as RISC complexes or CRISPR / Cas effector enzymes (e.g., type II, type V, or type VI effector enzymes).

[0132] As used herein, the term "transcribed" and its grammatical variations refer to a nucleotide sequence containing ribonucleic acid (RNA) nucleotides transcribed from a DNA template (e.g., double-stranded DNA and / or single-stranded DNA). A transcribed RNA molecule can correspond to the positive or negative strand of an AAV ssDNA, wherein the transcribed positive-strand RNA is transcribed from the negative strand of the DNA template, and the transcribed negative-strand RNA is transcribed from the positive strand of the DNA template. In some embodiments, the transcribed RNA molecule can be transcribed from the sense or antisense strand of a double-stranded DNA template. For example, depending on the case, when the dsDNA sequence is represented by the sequence of only one strand (as in SEQ ID NO:1), the transcribed RNA using dsDNA as a template can have the same sequence as the sense or antisense strand. That is, the RNA transcribed from double-stranded DNA as shown in SEQ ID NO:1 may have the same sequence as SEQ ID NO:1 or its reverse complementary sequence, except that U in the RNA replaces T in the DNA.

[0133] The transcribed modified AAV terminal inverted repeat (ITR) sequence of the present invention is an RNA sequence (as opposed to the single-stranded DNA sequence in the conventional AAV viral genome encapsulated within AAV viral particles). As a wild-type AAV ITR DNA sequence, the transcribed modified AAV ITR sequence (RNA) also supports the binding of the RNA sequence of the present invention to the AAV Rep protein, and thus enables the direct packaging of the RNA sequence of the present invention into AAV viral particles. In some embodiments, the transcribed modified ITR sequence comprises a transcribed Rep-binding element (RBE) (e.g., a transcribed functional RBE) and optionally a transcribed RBE' (e.g., a transcribed functional RBE') for Rep binding. In some embodiments, the transcribed modified ITR sequence supports or facilitates the packaging or encapsulation of the RNA sequence into AAV viral particles.

[0134] In some implementations, the modified ITR contains wild-type RBE.

[0135] In some embodiments, the modified ITR comprises a functional RBE that retains at least about 60%, 70%, 80%, 90%, 95%, 100%, or more of the wild-type RBE's ability to support AAV packaging (such as Rep binding). In some embodiments, the functional RBE contains up to about 30%, 25%, 20%, 15%, 10%, or 5% sequence variation compared to the wild-type RBE due to, for example, insertions, deletions, substitutions, and / or other mutations of one or more nucleotides of the RBE.

[0136] In some embodiments, the modified AAV ITR DNA template transcribed from it as an ITR is defective because it lacks one or more functions of the corresponding wild-type AAV ITR, such as the ability to cleave at the TRS (transcriptional terminus, see below). This may be due to, for example, the lack of a functional TRS. In one embodiment, the wild-type TRS is completely absent, so that the modified ITR has no TRS. In one embodiment, the wild-type TRS is mutated by deletion, insertion, substitution, and / or mutation of one or more nucleotides, so that it is no longer recognized and cleaved by Rep during AAV replication.

[0137] In some embodiments, the modified AAV ITR DNA template retains the RBE or a functional variant thereof as described herein, and optionally RBE' or a functional variant thereof. In some embodiments, the RBE and / or RBE' is functional in binding AAV Rep78 / 68.

[0138] The modified AAV terminal inverted repeat (ITR) of the present invention further lacks a transcriptional terminal dissociation site (TRS) or a transcriptional inverse complementary TRS (rcTRS), or both. In some embodiments, the TRS is located at the 5' end of the modified AAVTR. In some embodiments, the TRS is located between the D region sequence and the RBE.

[0139] In some implementations, the transcribed modified AAV ITR lacks both the transcribed TRS and the transcribed rcTRS.

[0140] As used herein, “terminal dissociation site” or “TRS” refers to a single-stranded DNA sequence in the single-stranded AAV vector genome (positive or negative strand) that is recognized by the AAV Rep protein and produces a nick during AAV replication. As used herein, “reverse complementary TRS (rcTRS)” refers to a single-stranded DNA sequence in the single-stranded AAV vector genome (positive or negative strand) that is the reverse complementary sequence of the TRS. The rcTRS pairs with the TRS to form a double-stranded DNA region at one end of the A region stem. See also Figure 1A-1C .

[0141] In AAV2 ITRs, the TRS contains the TTGGC sequence, and the Rep cleavage site is located between the two T's; while the rcTRS contains the GCCAA sequence. A TRS is located at the boundary between the D and A regions, and at the 5' end of the A region sequence (e.g., between the D region sequence and the RBE). For TRS and rcTRS in representative AAV 5' and 3' ITR multiple sequence alignments, please refer to [link to documentation]. Figure 1B and 1C .

[0142] As used in this article, “transcribed TRS” is a single-stranded RNA sequence transcribed from a TRS DNA template. For AAV2 TRS containing TTGGC, transcribed TRS contains GCCAA.

[0143] As used in this article, “transcribed rcTRS” refers to a single-stranded RNA sequence transcribed from an rcTRS DNA template. For AAV2 rcTRS containing GCCAA, transcribed rcTRS contains UUGGC.

[0144] Therefore, if the transcribed modified AAV ITR lacks the GCCAA sequence at the position where the GCCAA sequence normally appears in the corresponding transcribed wild-type AAV2 ITR, for example, due to a complete deletion of the GCCAA sequence, or due to the insertion, deletion, substitution, and / or other mutation of one or more nucleotides in the GCCAA sequence, then the transcribed modified AAV ITR is “lacking transcribed AAV2 TRS”. This could be due to transcription of a modified AAV ITR with a complete deletion of the TRS (TTGGC), or due to the insertion, deletion, substitution, and / or other mutation of one or more nucleotides in the wild-type TRS.

[0145] Therefore, in some embodiments, the RNA sequence or the modified AAV ITR transcribed according to the present invention lacks the functional TRS of transcription.

[0146] Similarly, if the transcribed modified AAV ITR lacks the UUGGC sequence at the position where the UUGGC sequence would normally appear in the corresponding transcribed wild-type AAV2 ITR, for example, due to a complete deletion of the GCCAA sequence, or due to the insertion, deletion, substitution, and / or other mutation of one or more nucleotides in the GCCAA sequence, then that transcribed modified AAV ITR "lacks the transcribed AAV2 rcTRS". This could be due to transcription of a modified AAVTR with a complete deletion of the rcTRS, or due to the insertion, deletion, substitution, and / or other mutation of one or more nucleotides in the wild-type rcTRS.

[0147] In some embodiments, the transcribed modified AAV ITR further comprises a transcribed D region sequence (the D or D' sequence in the wild-type AAV ITR) or a mutant D region sequence (e.g., a sequence having one or more nucleotide insertions, deletions, substitutions, and / or other mutations), the mutant D region sequence substantially preserving the function of the wild-type D region sequence. In other embodiments, the transcribed modified AAV ITR does not contain a transcribed D region sequence, or does not contain a mutant D region sequence (e.g., a sequence having one or more nucleotide insertions, deletions, substitutions, and / or other mutations), the mutant D region sequence substantially preserving the function of the wild-type D region sequence.

[0148] In some embodiments, the transcribed modified AAV ITR comprises a transcribed (functional) D-region sequence. Optionally, the modified AAV ITR DNA template has the nucleotide sequence of SEQ ID NO:3. Optionally, the transcribed modified AAV ITR comprises an RNA equivalent of SEQ ID NO:3 (i.e., the RNA equivalent has the same base sequence as the DNA sequence of SEQ ID NO:3). Optionally, the transcribed modified AAV ITR comprises an RNA equivalent of the inverse complementary sequence of SEQ ID NO:3 (i.e., the RNA equivalent has the same base sequence as the DNA sequence of the inverse complementary sequence of SEQ ID NO:3).

[0149] In some embodiments, the transcribed modified AAV ITR lacks the transcribed (functional) D region sequence. Optionally, the modified AAV ITR DNA template has the nucleotide sequence of SEQ ID NO:2. Optionally, the transcribed modified AAV ITR comprises the RNA equivalent of SEQ ID NO:2 (i.e., the RNA equivalent has the same base sequence as the DNA sequence of SEQ ID NO:2). Optionally, the transcribed modified AAV ITR comprises the RNA equivalent of the inverse complementary sequence of SEQ ID NO:2 (i.e., the RNA equivalent has the same base sequence as the DNA sequence of the inverse complementary sequence of SEQ ID NO:2).

[0150] As used herein, "D-region sequence" refers to the D-sequence or its inverse complementary sequence, the D'-sequence. The location of the D-region sequence depends on whether the ITR adopts a "flip" or "flop" configuration. See also Figure 1A-1C For example, in wild-type AAV2ITR (see Srivastava et al., J. Viol. [Journal of Virology] 45(2):555-564, 1983) Figure 2In the positive-strand ssDNA sequence (incorporated hereby cited), from 5' to 3', there are palindromic sequence fragments named A, B, B', C, C', A', D, ..., D', A, C, C, B, B', and A', where A:A', B:B', C:C', and D:D' are reverse complementary sequences to each other and can form base-pairing stem sequences (although D and D' sequences may not actually pair with each other in the ssDNA AAV vector genome). The B:B' stem of the positive-strand 5' ITR is closer to one end of the sequence (5' end) than the C:C' stem, and is called the Flip ITR. The C:C' stem of the positive-strand 3' ITR is closer to one end of the sequence (3' end) than the B:B' stem, and is called the Flop ITR.

[0151] By, for example, deleting, inserting, substituting, and / or otherwise mutating one or more nucleotides of the transcribed wild-type D region sequence, the modified AAV ITR sequence of the present invention may lack the functional transcribed D region sequence (D or D' sequence).

[0152] In some embodiments, the modified AAV ITR sequence of the RNA or transcription of the present invention comprises a mutated D region sequence of the transcription and / or a mutated TRS sequence of the transcription. In some embodiments, the modified AAV ITR sequence of the RNA or transcription of the present invention does not comprise a mutated D region sequence and / or does not comprise a mutated TRS / rcTRS sequence.

[0153] In some implementations, a modified AAV ITR is transcribed based on a wild-type Flip ITR or a wild-type Flop ITR.

[0154] In some embodiments, the wild-type Flip ITR or wild-type Flop ITR is derived from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, AAVhu32, AAVhu37, AAVPHP.eB, Anc80L65, Anc80L65AAP, AAVrh74, or 7m8. Optionally, the wild-type Flop ITR has the nucleotide sequence of SEQ ID NO:1.

[0155] In some implementations, a transcribed D region sequence is present and is not within the 3' end 50 nucleotides (e.g., 40 nt, 30 nt, 25 nt, or 20 nt) of the RNA.

[0156] In some implementations, a transcribed D region sequence is present and is located within 50 nucleotides (e.g., 40 nt, 30 nt, 25 nt, or 20 nt) of the 3' end of the RNA.

[0157] In some embodiments, the transcribed modified AAV ITR is located within 1000 nucleotides of the 3' end of the RNA. In some embodiments, the transcribed modified AAV ITR is located within 800 nucleotides of the 3' end of the RNA. In some embodiments, the transcribed modified AAV ITR is located within 500 nucleotides of the 3' end of the RNA. In some embodiments, the transcribed modified AAV ITR is located within 300 nucleotides of the 3' end of the RNA. In some embodiments, the transcribed modified AAV ITR is located within 200 nucleotides of the 3' end of the RNA.

[0158] In some implementations, the transcribed modified AAV ITR is located at the 5' of a polyA sequence, a polyA signal sequence (e.g., AAUAAA), or an RNA transcription termination sequence (e.g., a histone downstream element).

[0159] As used herein, a “poly-A sequence” or “poly-A tail” refers to a string of adenine ribonucleotides or adenosine monophosphates (e.g., a string of RNA in which each base is adenine). This poly-A tail is important for nuclear export, translation, and stability of mRNA. The length of the poly-A sequence can vary in different mRNA or RNA sequences of the present invention and can be about 250 nucleotides, about 230 nucleotides, about 200 nucleotides, about 180 nucleotides, about 160 nucleotides, about 140 nucleotides, about 120 nucleotides, about 100 nucleotides, or less.

[0160] As used herein, a “poly-A signal sequence” refers to an RNA sequence (e.g., AAUAAA) located downstream of the last 3' exon and recognized by an RNA cleavage complex, which allows polyadenylation to occur by cleaving the 3' end sequence of newly transcribed RNA with an RNA polymerase (e.g., Pol II). The polyadenylation polymerase then adds and extends the poly(A) tail by adding an adenosine monophosphate unit from ATP to the 3' end of the newly cleaved RNA. Initial RNA cleavage is typically catalyzed by the enzyme CPSF (cleavage / polyadenylation specific factor) and occurs approximately 10–30 nucleotides downstream of its binding site—the poly-A signal sequence (usually AAUAAA on the transcribed RNA). The sequence located / or immediately following the 5' of the RNA cleavage site is often (but not always) CA. The poly-A signal sequence recognized by the RNA cleavage complex varies among different populations of eukaryotic cells, with most polyadenylation sites containing the AAUAAA sequence, although this sequence is less common in plant and fungal mRNA. Additionally, other variants with weaker binding to CPSF exist. All these sequence motifs, recognized by the RNA cleavage complex to enable RNA cleavage and subsequent polyadenylation, fall within the range of the polyA signal sequence.

[0161] As used in this paper, "the GU-rich region of transcription downstream of the polyA site" refers to a sequence (e.g., AAUAAA) that can be used by other proteins (such as cleavage stimulators or CstF) to enhance the binding specificity of CPSF to the polyA signal sequence.

[0162] In some embodiments, the RNA sequence of the present invention further includes a CFI (cleavage factor I) recognition sequence, such as a set of UGAAA sequences in mammals, which can recruit CPSF even if the AAUAAA polyA signal sequence is missing.

[0163] As used herein, “sequences for RNA transcription termination” include RNA sequence motifs present at or near the 3' end of transcribed RNA (e.g., transcribed RNA without a poly(A) tail). Almost all eukaryotic mRNAs, except metazoan replication-dependent histone mRNAs, are polyadenylated, where mRNA processing occurs at sites with a highly conserved stem-loop structure and a downstream purine-rich region of approximately 20 nucleotides. These are a minority (if not the only) of eukaryotic mRNAs that lack a poly(A) tail, end with a stem-loop structure, followed by a purine-rich sequence called a histone downstream element (HDE) or histone 3'UTR stem-loop. The HDE directs the location where the RNA is cleaved during / after transcription, thus forming the 3' end of the histone mRNA. The HDE is involved in nucleocytoplasmic transport of histone mRNAs, as well as the regulation of cytoplasmic stability and translation efficiency.

[0164] In some embodiments, the RNA sequence of the present invention further comprises a modified AAV ITR of the second transcription of the present invention. In some embodiments, the modified AAV ITR of the second transcription has a functional RBE sequence of the transcription, but lacks the TRS or rcTRS of the second transcription or both; optionally, the modified AAV ITR of the second transcription further comprises or lacks the D region sequence of the second transcription. In some embodiments, the modified AAV ITR of the second transcription comprises a mutated D region sequence of the second transcription and / or a mutated TRS sequence of the second transcription.

[0165] In some embodiments, for the RNA sequence of the present invention having two transcribed modified AAV ITRs, the transcribed modified AAV ITR and the second transcribed modified AAV ITR are identical.

[0166] In some embodiments, for the RNA sequence of the present invention having two transcribed modified AAV ITRs, the transcribed modified AAV ITR and the second transcribed modified AAV ITR are different.

[0167] In some implementations, the transcribed modified AAV ITR, the second transcribed modified AAV ITR (if present) contains a deletion in the corresponding wild-type AAV ITR D region sequence or the corresponding wild-type TRS / rcTRS of the transcription, a mutation in the corresponding wild-type AAV ITR D region sequence or the corresponding wild-type TRS / rcTRS of the transcription, or an insertion in the corresponding wild-type AAV ITR D region sequence or the corresponding wild-type TRS / rcTRS of the transcription.

[0168] In some embodiments, for the RNA sequence of the present invention having two transcribed modified AAV ITRs, the second transcribed modified AAV ITR is located within 1000 nucleotides, 800 nucleotides, 500 nucleotides, 250 nucleotides, or 150 nucleotides at the 5' end of the RNA sequence.

[0169] In some embodiments, the RPS includes an MS2 sequence, a PP7 binding site, or a com binding site, and the RPS-interacting molecule includes an RPS-interacting protein (RPSIP) capable of interacting directly or indirectly with the RPS, such as a phage-derived MS2 coat protein (MCP) for the MS2 sequence, a PP7 phage coat protein (PCP) for the PP7 binding site, or a phage COM protein (COM) for the com binding site. The sequences of these RPS / RPSIP pairs are described in the sequence portion of the specification.

[0170] Any one or more RPS sequences described above, including any one of the transcribed modified ITR sequences, and any one of the MS2 sequence, PP7 binding site, and / or com binding site, individually or in combination, can facilitate the packaging of the RNA sequences of the present invention into DNA viral particles in the presence of suitable / compatible homologous RPSIPs.

[0171] In some embodiments, RPSIP is a protein component of the viral packaging system of a DNA virus particle, or is directly or indirectly associated with it. For example, in some embodiments, RPSIP is a protein component of the viral packaging system of a DNA virus, such as Rep78, Rep68, Rep52, and / or Rep40 of AAV. For example, in some embodiments, RPSIP may be directly fused to a protein component of the viral packaging system of a DNA virus. Exemplary protein components of the viral packaging system of AAV include any of Rep proteins (such as Rep78 and / or Rep68 of adeno-associated virus 2 (AAV2)) and / or any of assembly activation proteins (AAP).

[0172] In some implementations, the fusion is an N-terminal fusion, wherein RPSIP (such as MCP, PCP, or COM) is fused to the N-terminus of the Rep68 / 78 protein and / or AAP.

[0173] In some implementations, the fusion is an N-terminal fusion, wherein RPSIP (such as MCP, PCP, or COM) is fused to the C-terminus of the Rep68 / 78 protein and / or AAP.

[0174] In some implementations, the fusion is a direct fusion without a connector sequence.

[0175] In some embodiments, fusion is achieved via one or more adapter sequences, such as flexible peptide adapters that may include Gly and Ser-rich adapters or GS adapters. Representative GS adapters include 1, 2, 3, 4, 5, or more repeats of Gly or Ser, such as GS, GSS, GSSS, GSSSS, and their repeats (e.g., (GS...). p ) n Where p is an integer between 1 and 5, and n is an integer between 1 and 20. A typical GS connector of this type is a GS3 connector or a GS4 connector. In some implementations, p is 3 or 4, and n is 1.

[0176] In some embodiments, the RNA sequence of the present invention may include, but preferably does not include, a transcribed DNA packaging signal, such as a transcribed wild-type AAV ITR sequence. For example, the RNA sequence of the present invention may include a transcribed modified AAV ITR sequence having nucleotide additions, deletions, and / or substitutions of the corresponding transcribed wild-type AAV ITR sequence to reduce the DNA packaging capacity of the viral particles.

[0177] In some embodiments, the RNA sequence of the present invention further comprises one or more of the following: (1) a protein-coding sequence (e.g., mRNA encoding a therapeutic protein or a CRISPR / Cas effector enzyme (including any of the Cas effectors described below, such as Cas9 or a variant thereof, optionally fused with a base editor), non-coding RNA (ncRNA), or functional RNA (e.g., tRNA, ribosomal RNA (rRNA), RNAi reagent or its precursor, siRNA, shRNA, miRNA or its precursor, including precursor miRNA and primary miRNA, antisense RNA (ASO), piRNA, RNA components of the CRISPR-Cas system, such as guide RNA (or gRNA), single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA), or tracr (1) RNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR, etc.; (2) transcription enhancers; (3) intron or exon sequences (e.g., sequences used to enhance protein expression); (4) 5'UTR sequences; (5) 3'UTR sequences; (6) polyA sequences, or (transcribed) polyadenylation (polyA) signal sequences, and optionally, the polyA site of transcription and the downstream GU-rich region transcribed from said polyA site; (7) posttranscriptional regulatory elements or sequences, such as transcribed marmot hepatitis virus (WHP) posttranscriptional regulatory element (WPRE) sequences; and / or (8) transcription termination sequences (e.g., histone downstream elements).

[0178] In some embodiments, the RNA sequence of the present invention comprises RPS located at the 3' of a posttranscriptional regulatory element or sequence and the 5' of a polyA sequence or polyA signal sequence.

[0179] For example, in some embodiments, the RNA sequence of the present invention includes an RSI in the 5' to 3' direction; an optional transcribed WPRE sequence (which may or may not be present); an RPS (e.g., a transcribed modified AAV ITR, MS2 sequence, PP7 binding site, or com binding site); and a polyA sequence or polyA signal sequence.

[0180] In some embodiments, the RNA sequence of the present invention encodes, or GOI comprises, a protein (e.g., a fluorescent protein, a therapeutic protein, an antigenic protein, or a gene-editing protein, such as a Cas protein, a ZFN protein, or a TALEN protein), an enzyme (such as a Cre protein or a CRISPR / Cas effector enzyme, such as Cas9, Cas12, Cas13, or variants thereof), a structural protein, mRNA, non-coding RNA (ncRNA), siRNA, piRNA, short hairpin RNA or shRNA, microRNA (miRNA) or its precursors (including precursor miRNA and primary miRNA), ribosomal RNA (rRNA), antisense sequences or oligonucleotides (ASO), RNA components of the CRISPR-Cas system, including guide RNA (or gRNA), such as single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA), and tracr RNA, guide RNA or gRNA for CRISPR / Cas effector enzymes, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR.

[0181] The total length of the RNA sequence of this invention depends on the packaging capacity of the AAV viral particles. Most AAV viral particles have a packaging capacity of about 4,700-5,200 nucleotides, but some AAV viral particles, such as AAV5 particles, can package up to 8,900 nucleotides.

[0182] Therefore, in some embodiments, the RNA sequence of the present invention to be packaged into AAV virus particles is a single-stranded RNA (ssRNA) with a length of less than about 8,900 nucleotides.

[0183] In some embodiments, the RNA sequence is an ssRNA of less than about 8,000 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of less than about 7,000 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of less than about 6,000 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of less than about 5,200 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of less than about 4,000 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of less than about 3,000 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of less than about 2,000 nucleotides in length.

[0184] In some embodiments, the RNA sequence is an ssRNA of about 4,700-5,200 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of about 4,700-5,000 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of about 4,700-4,800 nucleotides in length. In some embodiments, the RNA sequence is an ssRNA of about 4,700 nucleotides in length.

[0185] Another aspect of the invention provides a polynucleotide comprising a (transcription) cassette encoding an RNA sequence of the invention; optionally, the polynucleotide is a DNA sequence (e.g., a DNA plasmid) which optionally contains filler sequences in the backbone of the DNA plasmid and / or optionally does not contain functional DNA packaging signals, such as AAV ITR.

[0186] In some embodiments, the polynucleotide containing the cassette is a DNA vector encoding the RNA sequence of the present invention. Such a DNA vector and / or its cassette can be used to transcribe and generate the RNA sequence of the present invention for further packaging into, for example, AAV viral particles.

[0187] In some embodiments, the polynucleotide further comprises a promoter operatively linked to and driving the transcription of the cassette-encoded RNA sequence of the present invention to produce the RNA sequence of the present invention.

[0188] In some implementations, the promoter is a pervasive promoter.

[0189] In some implementations, the promoter is a tissue-specific promoter.

[0190] In some implementations, the promoter is a compositional promoter.

[0191] In some implementations, the promoter is an inducible promoter.

[0192] In some implementations, the polynucleotide further includes an enhancer that enhances transcription of the promoter-driven RNA sequence.

[0193] Another aspect of the present invention provides recombinant DNA virus particles containing an RNA genome (e.g., the RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotide of the present invention) packaged in a protein shell (e.g., capsid) of a DNA virus (e.g., AAV virus or oncolytic virus).

[0194] In some embodiments, the DNA virus is AAV, and the recombinant DNA virus particle is a recombinant RNA adeno-associated virus (rRAAV) particle, the recombinant RNA adeno-associated virus particle comprising: (1) an AAV capsid; and (2) an RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotides of the present invention packaged within the AAV capsid.

[0195] In some implementations, the AAV capsid comprises a capsid from serotypes AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, or 7m8.

[0196] A related aspect of the invention provides a population of recombinant DNA virus particles (e.g., rRAAV particles) comprising a plurality of recombinant DNA virus particles (e.g., rRAAV particles) of the invention, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the recombinant DNA virus particles (e.g., rRAAV particles) within the population have a capsidized RNA sequence of the invention or an RNA sequence transcribed from a polynucleotide of the invention packaged therein.

[0197] In some implementations, the population of recombinant viral particles (e.g., rRAAV particles) contains at least 1 × 10⁻⁶ particles. 4 One virus particle, at least 2 × 10 4 One virus particle, at least 5 × 10 4 One virus particle, at least 1×10 5 One virus particle, at least 2 × 10 5 One virus particle, at least 5 × 10 5 One virus particle, at least 1×10 6 One virus particle, at least 2 × 10 6 One virus particle, at least 5 × 10 6 One virus particle, at least 1×10 7 One virus particle, at least 2 × 10 7 One virus particle, at least 5 × 10 7 One virus particle, at least 1×10 8 One virus particle, at least 2 × 10 8 One virus particle, at least 5 × 10 8 One virus particle, at least 1×10 9One virus particle, at least 2 × 10 9 One virus particle, at least 5 × 10 9 One virus particle, at least 1×10 10 One virus particle, at least 2 × 10 10 One virus particle, at least 5 × 10 10 One virus particle, at least 1×10 11 One virus particle, at least 2 × 10 11 One virus particle, at least 5 × 10 11 One virus particle, at least 1×10 12 One virus particle, at least 2 × 10 12 One virus particle, at least 5 × 10 12 One virus particle, at least 1×10 13 One virus particle, at least 2 × 10 13 One virus particle, at least 5 × 10 13 One virus particle, at least 1×10 14 One virus particle, at least 2 × 10 14 One virus particle, at least 5 × 10 14 One virus particle, at least 1×10 15 One virus particle, at least 2 × 10 15 One virus particle, at least 5 × 10 15 One virus particle, at least 1×10 16 One virus particle, at least 2 × 10 16 One virus particle, or at least 5 × 10 16 A virus particle.

[0198] In some implementations, up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.1%, 0.01%, or less of the population of recombinant viral particles are capsidred with non-RNA (e.g., DNA) within the viral particles.

[0199] Another aspect of the invention provides a host cell comprising a population of the RNA sequence of the invention, the polynucleotide of the invention, the RNA sequence transcribed from the polynucleotide of the invention, the recombinant DNA virus particles of the invention (e.g., rRAAV particles), and / or the recombinant DNA virus particles of the invention (e.g., rAAV particles).

[0200] In some embodiments, the host cell further comprises a viral packaging system that facilitates the packaging of the RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotide of the present invention into DNA viral particles.

[0201] In some embodiments, the viral packaging system comprises: (1) an AAV rep gene (e.g., coding sequences of Rep78, Rep68, Rep52, and / or Rep40) and an AAV cap gene (e.g., coding sequences of VP1, VP2, and / or VP3, AAP, and / or MAAP) or their expression products, wherein the AAV rep and AAV cap genes are under transcriptional control of one or more promoters that drive the transcription of the rep and cap genes; (2) one or more coding sequences of one or more proteins required for AAV packaging, such as adenovirus E2A, E4, and VA genes, or the one or more proteins; and (3) an RPS interacting molecule or its coding sequence.

[0202] In some embodiments, the ability of a viral packaging system to package a DNA sequence into a DNA virus particle is reduced, weakened, or substantially eliminated, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, from a polynucleotide encoding the RNA sequence of the present invention or a polynucleotide of the present invention; (2) modifying, for example, by mutating the coding sequences of one or more proteins required for AAV packaging, such as the AAV rep gene, the AAV cap gene, and / or AAV packaging, to reduce, weaken, or substantially eliminate the ability of the corresponding translated protein to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) enlarging the size of the polynucleotide encoding the RNA sequence of the present invention or a polynucleotide of the present invention. In embodiments, the size of the polynucleotide encoding the RNA sequence of the present invention or a polynucleotide of the present invention is enlarged by inserting a filler sequence (e.g., an intron) into a polynucleotide (e.g., a DNA plasmid) (e.g., a backbone).

[0203] In some implementations, the AAV rep gene, AAV cap gene, and / or proteins required for AAV packaging contain mutations that weaken or reduce the ability to promote DNA packaging into DNA viral particles.

[0204] In some embodiments, the Rep68 / Rep78 protein required for DNA packaging contains a mutation that impairs or weakens its trs-endonuclease activity. trs-endonuclease activity is considered necessary for the dissociation of AAV replication (DNA) intermediates at the trs sequence or site, so that individual units of AAV ssDNA can be dissociated before packaging into the AAV capsid.

[0205] In some implementations, the trs-endonuclease mutation includes the Y156F mutation in the common sequence of the Rep78 and Rep68 proteins.

[0206] In some implementations, the Rep78 / Rep68 protein contains a KDE-mu mutation (see the sequence in the sequence section below).

[0207] In some implementations, the Rep78 / Rep68 protein contains the EKE-mu mutation (see the sequence in the sequence section below).

[0208] In some implementations, the Rep78 / Rep68 protein contains two or more mutations selected from the Y156F mutation, the KDE-mu mutation, and the EKE-mu mutation.

[0209] In some embodiments, Rep68 / Rep78 is derived from any of the AAVs having serotypes AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, AAVrh74, or 7m8, and has the corresponding trs-endonuclease mutation with Y156F mutation, KDE-mu mutation, and / or EKE-mu mutation.

[0210] In some embodiments, the host cell further comprises: (1) coding sequences of the AAV rep gene and the AAV cap gene, the AAV rep and AAV cap genes being under transcriptional control of one or more promoters that drive the transcription of the rep and cap genes; and (2) coding sequences of proteins required for AAV packaging, such as adenovirus E2A, E4, and VA genes.

[0211] In some implementations, the host cell is a mammalian cell, such as HEK293 cells or variants thereof (e.g., HEK293T cells), or an insect cell, such as Sf9 or Sf21 cells.

[0212] Another aspect of the present invention provides a method for generating a population of recombinant DNA virus particles (e.g., rRAAV particles) or recombinant DNA virus particles (e.g., rRAAV particles) of the present invention, the method comprising: a) culturing host cells of the present invention for a sufficient time, and b) harvesting the recombinant DNA virus particles or the population of the recombinant DNA virus particles.

[0213] In some embodiments, the method further includes isolating or purifying recombinant DNA virus particles or populations of recombinant DNA virus particles.

[0214] Another aspect of the present invention provides a method for generating recombinant DNA virus particles (e.g., rRAAV particles) or populations of recombinant DNA virus particles, the method comprising: a) contacting a viral packaging system (e.g., an AAV packaging system) with an RNA sequence of the present invention or an RNA sequence transcribed from a polynucleotide of the present invention for a period of time sufficient to generate the recombinant DNA virus particles of the present invention or populations of the recombinant DNA virus particles of the present invention; b) harvesting the recombinant DNA virus particles of the present invention or populations of the recombinant DNA virus particles of the present invention; and optionally, c) isolating or purifying the harvested recombinant DNA virus particles of the present invention or populations of the recombinant DNA virus particles of the present invention.

[0215] In some embodiments, a viral packaging system (e.g., an AAV packaging system) comprises: (1) one or more proteins (e.g., VP1, VP2, and / or VP3 for assembling an AAV capsid) for packaging the RNA sequence into the protein shell of the DNA virus viral particle, or one or more coding sequences thereof; (2) one or more proteins (e.g., Rep78, Rep68, Rep52, and / or Rep40 for AAV packaging) for facilitating the assembly of the protein shell and / or the packaging of the RNA sequence into the protein shell of the DNA virus viral particle, or one or more coding sequences thereof (e.g., adenovirus E2a, E4, and VA genes); and (3) an RPS interacting molecule or its coding sequence thereof. Optionally, the ability of the viral packaging system to package a DNA sequence into the DNA virus particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, from the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention; (2) modifying, for example, by mutating the coding sequences of the AAV rep gene, the AAV cap gene, and / or one or more proteins required for AAV packaging, to reduce, weaken, or substantially eliminate the ability of the corresponding translated protein to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) enlarging the size of the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention.

[0216] Another aspect of the present invention provides a system for packaging the RNA sequence of the present invention or an RNA sequence transcribed from the polynucleotide of the present invention into a DNA virus particle, the system comprising: (1) one or more proteins (e.g., VP1, VP2, and / or VP3 for assembling an AAV capsid) for packaging the RNA sequence into the protein shell of the DNA virus particle, or one or more coding sequences thereof; (2) one or more proteins (e.g., Rep78, Rep68, Rep52, and / or Rep40 for AAV packaging) for facilitating the assembly of the protein shell and / or the packaging of the RNA sequence of the present invention into the protein shell of the DNA virus particle, or one or more coding sequences thereof (e.g., adenovirus E2a, E4, and VA genes); and (3) an RPS interacting molecule or its coding sequence thereof. Optionally, the ability of the viral packaging system to package a DNA sequence into the DNA virus particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, from the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention; (2) modifying, for example, by mutating the coding sequences of the AAV rep gene, the AAV cap gene, and / or one or more proteins required for AAV packaging, to reduce, weaken, or substantially eliminate the ability of the corresponding translated protein to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) enlarging the size of the polynucleotide encoding the RNA sequence of the present invention or the polynucleotide of the present invention.

[0217] Another aspect of the invention provides a method for delivering an RNA sequence of interest (ROI) to a cell, plant, or animal, the method comprising contacting the cell, plant, or animal with recombinant DNA virus particles (e.g., rRAAV particles) of the invention, a population of recombinant DNA virus particles (e.g., rRAAV particles) of the invention, or recombinant DNA virus particles (e.g., rRAAV particles) or a population of recombinant DNA virus particles (e.g., rRAAV particles) generated by the method of the invention, wherein the ROI is optionally encoded by an RNA sequence of the invention.

[0218] Another aspect of the invention provides a method for diagnosing, preventing, or treating a disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount or dose of a population of recombinant DNA virus particles (e.g., rRAAV particles) of the invention or generated by the method of the invention.

[0219] Another aspect of the invention provides the use of the recombinant DNA virus particles (e.g., rRAAV particles) of the invention, a group of recombinant DNA virus particles (e.g., rRAAV particles) of the invention, or recombinant DNA virus particles (e.g., rRAAV particles) or a group of recombinant DNA virus particles (e.g., rRAAV particles) produced by the method of the invention in the manufacture of a medicament for the diagnosis, prevention, or treatment of a disease or disorder in a subject in need.

[0220] Another aspect of the invention provides a fusion protein or conjugate comprising an RPSIP of the invention fused or conjugated to a protein component of a viral packaging system of a DNA virus, wherein the RPSIP interacts / binds to an RPS on an RNA sequence of the invention to facilitate the packaging of the RNA sequence into a DNA virus.

[0221] In some implementations, RPS is MS2 and RPSIP is MCP.

[0222] In some implementations, RPS is the PP7 binding site and RPSIP is PCP.

[0223] In some implementations, RPS is com, and RPSIP is phage COM protein.

[0224] In some embodiments, the fusion or conjugate comprises more than one RPSIP, each of which independently binds to one or more RPSs on the RNA sequence of the present invention. In some embodiments, at least two of the more than one RPSIP are identical. In some embodiments, at least two of the more than one RPSIP are different.

[0225] In some implementations, the fusion or conjugate comprises two MCPs in series.

[0226] In some embodiments, the protein components of the viral packaging system of a DNA virus include the Rep protein of AAV, such as Rep68 or Rep78 of AAV.

[0227] In some embodiments, the Rep protein contains one or more mutations that impair or attenuate trs-endonuclease activity. In some embodiments, the mutations include the Y156F mutation, the KDE-mu mutation, and / or the EKE-mu mutation.

[0228] In some implementations, the protein components of the viral packaging system of a DNA virus include assembly activation protein (AAP).

[0229] In some implementations, RPSIP is directly fused with protein components (e.g., Rep protein or AAP) of the viral packaging system of a DNA virus.

[0230] In some implementations, RPSIP is fused to a protein component (e.g., Rep protein or AAP) of a DNA virus's viral packaging system via a peptide linker.

[0231] In some embodiments, the peptide linker is a flexible linker, such as a linker containing Gly and Ser. In some embodiments, the linker containing Gly and Ser comprises GS. n 1-20 repetitions (e.g., 1-5 or 1-3 repetitions), where n is 1, 2, 3, 4, or 5. In some implementations, GS n The connector is GS2, GS3, or GS4, with 1 to 4 repeats (e.g., 2). In some embodiments, the connector is GSSGSS.

[0232] In some embodiments, the fusion protein comprises an MCP and a Rep, wherein the Rep optionally contains a Y156F mutation, a KDE-mu mutation, and / or an EKE-mu mutation. In some embodiments, the MCP is fused to the N-terminus of the Rep (MCP-Rep). In some embodiments, the Rep fused with the MCP contains a Y156F mutation, a KDE-mu mutation, and / or an EKE-mu mutation. In some embodiments, the MCP-Rep fusion is processed via GS... n Connectors, such as GSSGSS connections. In some implementations, the MCP-Rep comprises two MCPs in series (e.g., no connector between the two MCP sections). In some implementations, the MCP is in another GSSGSS connection. n Connectors, such as the C end of GSSGSS.

[0233] In some embodiments, the fusion protein comprises PCP and Rep, wherein the Rep optionally contains a Y156F mutation, a KDE-mu mutation, and / or an EKE-mu mutation. In some embodiments, PCP is fused to the N-terminus of the Rep (PCP-Rep). In some embodiments, the Rep fused with PCP contains a Y156F mutation, a KDE-mu mutation, and / or an EKE-mu mutation. In some embodiments, the PCP-Rep fusion is processed via GS... n Connectors, such as GSSGSS connections. In some implementations, the PCP-Rep comprises two PCPs in series (e.g., no connector between the two PCP sections). In some implementations, the PCP is connected to another GSSGSS connector. n Connectors, such as the C end of GSSGSS.

[0234] In some embodiments, the fusion protein comprises COM and Rep, wherein the Rep optionally contains a Y156F mutation, a KDE-mu mutation, and / or an EKE-mu mutation. In some embodiments, COM is fused to the N-terminus of Rep (COM-Rep). In some embodiments, the Rep fused with COM contains a Y156F mutation, a KDE-mu mutation, and / or an EKE-mu mutation. In some embodiments, the COM-Rep fusion is processed via GS... n Connectors, such as GSSGSS connections. In some implementations, the COM-Rep contains two COMs in series (e.g., no connector between the two COM sections). In some implementations, the COM is connected to another GSSGSS connector. n Connectors, such as the C end of GSSGSS.

[0235] In some embodiments, the fusion protein comprises MCP and AAP. In some embodiments, MCP is fused to the N-terminus of AAP (MCP-AAP, or MA). In some embodiments, MCP is fused to the C-terminus of AAP (AAP-MCP, or AM). In some embodiments, the MCP-AAP or AAP-MCP fusion is processed via GS. n Connectors, such as GSSGSS connectors. In some implementations, the MCP-AAP fusion compound is connected to another GSSGSS connector. n Connectors, such as the C-terminus of the GSSGSS. In some embodiments, the AAP-MCP fusion is located in another GS. n Connectors, such as the N-terminal of GSSGSS.

[0236] Another aspect of the invention provides a polynucleotide encoding any one of the fusions between the RPSIP of the invention and a protein component (e.g., AAP or Rep protein) of a viral packaging system of a DNA virus.

[0237] With regard to the general aspects of the invention described above, the following sections provide additional details on specific elements of the invention as described herein. Each specific element is contemplated to be able to be combined with any one or more additional elements of the invention, even if not all possible combinations or arrangements of elements are explicitly listed.

[0238] 2. AAV serotype

[0239] The AAV particles packaging the ribopolynucleotides of the present invention may contain or be derived from any natural or recombinant AAV serotype.

[0240] According to this disclosure, AAV particles may utilize or be based on any of the following serotypes and their variants, including but not limited to: AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.40, AAV... 12. AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV13 0.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu.l1 , AAV16.3, AAV16.8 / hu.10, AAV161.10 / hu.60, AAV161.6 / hu.61, AAVl-7 / rh.48, A AVl-8 / rh.49, AAV2, AAV2.5T, AAV2-15 / rh.62, AAV223.1, AAV223.2, AAV223.4, AAV 223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61, AAV24.1, AAV2-4 / rh.50, AAV2-5 / rh .51, AAV27.3, AAV29.3 / bb.l, AAV29.5 / bb.2, AAV2G9, AAV-2-precursor miRNA-101, AAV3 , AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-11 / rh.53, AAV3-3, AAV33.12 / hu.l7, AAV33. 4 / hu.l5, AAV33.8 / hu.l6, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, A AV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-1b, AAV42 -2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV 42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / r 11.64, AAV4-8 / rh.64, AAV4-9 / rh.54, AAV5, AAV52.1 / hu.20, AAV52 / hu.19, AAV5-22 / rh.58, AAV5-3 / rh.57、AAV54.1 / hu.21、AAV54.2 / hu.22、AAV54.4R / hu.27、AAV54.5 / hu.23、AAV54.7 / hu.24、AAV58.2 / hu.25、AAV6、AAV6.1、AAV6.1.2、AAV6.2、AAV7、AAV7.2、AAV7.3 / hu.7、AAV8、AAV-8b、AAV-8h、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAV-b、AAVC1、AAVC2、AAVC5、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5Rl、 AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAV-h、AAVH-1 / hu.l、AAVH2、AAVCy. AVH-5 / hu.3、AAVH6、AAVhE1.1、AAVhER1.14、AAVhErl.16、AAVhErl.18、AAVhER1.23、AAVhErl.35、AAVhErl. 36、AAVhErl.5、AAVhErl.7、AAVhErl.8、AAVhEr2.16、AAVhEr2.29、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AA VhEr2.4、AAVhEr3.1、AAVhu.l、AAVhu.10、AAVhu.ll、AAVhu.l、AAVhu.12、AAVhu.13、AAVhu.14 / 9、AAVhu.15 、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.19、AAVhu.2、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.2 4、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.3、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.3 5、AAVhu.37、AAVhu.39、AAVhu.4、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44Rl、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48Rl、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.5、AAVhu.51、AAVhu.52、AAVhu. u.53、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.6、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.7、AAVhu. u.8、AAVhu.9、AAVhu.t19、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVLG-9 / hu.39、AAV-LK01、AAV-LK02、AAVLK03、AAV-LK04、AAV-LK04 AV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK17、AAV-LK18、AAV-LK19、 AAVN721-8 / rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.l, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, A AVrh.25、AAVrh.2R、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.43、AAVrh.44、AAVrh.45、AAVrh.46、AAVrh.47、AAVrh.48、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.50、AAVrh.51、AAVrh.52. AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl, A AVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, Cow AAV, Goat AAV, Japanese AAV 10, Realistic AAV (ttAAV), UPENN AAV 10, AAV-LK16, AAAV, AAV Remodeling 100-1, AAV Remodeling 100-2, AAV Remodeling 100-3, AAV Remodeling 100-7, AAV Remodeling 10-2, AAV Remodeling 10-6, AAV Remodeling 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and / or AAV SM 10-8.

[0241] In some implementations, the AAV serotype may include mutations in the AAV9 sequence, such as those described by Pulicherla et al. (Molecular Therapy 19(6):1070-1078, 2011), such as AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, and AAV9.84.

[0242] In some embodiments, the AAV serotype may comprise the sequence described in US 6,156,303, such as AAV3B (SEQ ID NO:1 and 10 in US 6,156,303), AAV6 (SEQ ID NO:2, 7 and 11 in US 6,156,303), AAV2 (SEQ ID NO:3 and 8 in US 6,156,303), AAV3A (SEQ ID NO:4 and 9 in US 6,156,303), or derivatives thereof.

[0243] In some embodiments, the serotype may be AAV-DJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8) as described by Grimm et al. (Journal of Virology 82(12):5887-5911, 2008). The amino acid sequence of AAV-DJ8 may contain two or more mutations to remove the heparin-binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO:1 in US7,588,772 may contain two mutations: (1) R587Q (Arg at amino acid 587 is replaced with glutamine Gln), and (2) R590T. As another non-limiting example, the AAV-DJ sequence may contain three mutations: (1) K406R, (2) R587Q, and (3) R590T.

[0244] In some implementations, the AAV serotype may comprise the sequence described in WO 2015 / 121501, such as true AAV (ttAAV) (SEQ ID NO:2 in WO 2015 / 121501), so-called UPenn AAV10 (SEQ ID NO:8 in WO 2015 / 121501), or so-called Japanese AAV10 (SEQ ID NO:9 in WO 2015 / 121501), or variants thereof.

[0245] The selection or use of AAV capsid serotypes may be derived from a variety of species. In some embodiments, the AAV may be avian AAV (aAAV). The aAAV serotype may comprise the sequence described in US 9,238,800, such as aAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 in US 9,238,800) or variants thereof.

[0246] In some embodiments, the AAV may be bovine AAV (bAAV). The bAAV serotype may comprise the sequence described in US 9,193,769, such as bAAV (SEQ ID NO: 1 and 6 in US 9,193,769) or a variant thereof. The bAAV serotype may comprise the sequence described in US 7,427,396, such as bAAV (SEQ ID NO: 5 and 6 in US 7,427,396) or a variant thereof.

[0247] In some implementations, the AAV may be goat AAV. The goat AAV serotype may comprise the sequence described in US 7,427,396, such as goat AAV (SEQ ID NO:3 in US 7,427,396) or a variant thereof.

[0248] In some implementations, AAV can be engineered as a heterozygous AAV derived from two or more parental serotypes.

[0249] In some implementations, AAV may be AAV2G9, which comprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may comprise the sequence described in US 2016-0017005 A1 (incorporated herein by reference).

[0250] In some embodiments, AAV can be a serotype generated from an AAV9 capsid library with mutations in amino acids 390-627 (VP1 number), as described by Pulicherla et al. (Molecular Therapy 19(6):1070-1078, 2011, incorporated herein by reference). The serotype and corresponding nucleotide and amino acid substitutions can be, but are not limited to: AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G120... 3A, G1785T, W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720 T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.4 4(A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W5 09R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50(A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134 Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510 A; P504T), AAV9.80 (G1441A,; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1 464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L 439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

[0251] In some implementations, the AAV may be a CD8 containing at least one AAV shell. + Serotypes of T-cell epitopes. As a non-limiting example, the serotype may be AAV1, AAV2, or AAV8.

[0252] In some implementations, the AAV may be a variant, such as PHP.A or PHP.B as described in Deverman (Nature Biotechnology. 34(2):204-209, 2016, incorporated herein by reference).

[0253] In some embodiments, AAV can be a serotype generated by Cre recombination-based AAV targeted evolution (CREATE) as described by Deverman et al. (Nature Biotechnology 34(2):204-209, 2016, incorporated herein by reference). In some embodiments, AAV serotypes generated in this manner have improved CNS transduction and / or neuronal and astrocyte tropism compared to other AAV serotypes.

[0254] In some embodiments, the AAV serotype can be an AAV9 derivative having a 7-amino acid insertion between amino acids 588 and 589. Non-limiting examples of such 7-amino acid insertions include PHP.A, PHP.B, PHP.B2, PHP.B3, PHP.N, PHP.S, G2A12, G2A15, G2A3, G2B4, and G2B5.

[0255] In some embodiments, AAV may be any serotype selected from SEQ ID NO:4,734-5,302 and Table 2 of WO 2018 / 002719A1 (incorporated herein by reference). In some embodiments, AAV may be encoded by sequences, fragments or variants as described in SEQ ID NO:4,734-5,302 of WO 2018 / 002719A1 (incorporated herein by reference).

[0256] In some implementations, the AAV VP1 capsid sequence is one of the following: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, or 7m8.

[0257] The protein sequences of the representative VP1 capsids mentioned above are provided below.

[0258] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(AAV1;SEQ ID NO:6)

[0259] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(AAV2;SEQ ID NO:7)

[0260] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKGAVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVRGVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTGTVNHQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(AAV3A;SEQ ID NO:8)

[0261] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(AAV3B;SEQ ID NO:9)

[0262] MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQQRLQGDTSFGGNLGRAVFQAKKRVLEPLGLVEQAGETAPGKKRPLIESPQQPDSSTGIGKKGKQPAKKKLVFEDETGAGDGPPEGSTSGAMSDDSEMRAAAGGAAVEGGQGADGVGNASGDWHCDSTWSEGHVTTTSTRTWVLPTYNNHLYKRLGESLQSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGMRPKAMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGNTSQQQTDRNAFYCLEYFPSQMLRTGNNFEITYSFEKVPFHSMYAHSQSLDRLMNPLIDQYLWGLQSTTTGTTLNAGTATTNFTKLRPTNFSNFKKNWLPGPSIKQQGFSKTANQNYKIPATGSDSLIKYETHSTLDGRWSALTPGPPMATAGPADSKFSNSQLIFAGPKQNGNTATVPGTLIFTSEEELAATNATDTDMWGNLPGGDQSNSNLPTVDRLTALGAVPGMVWQNRDIYYQGPIWAKIPHTDGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFSSTPVNSFITQYSTGQVSVQIDWEIQKERSKRWNPEVQFTSNYGQQNSLLWAPDAAGKYTEPRAIGTRYLTHHL(AAV4;SEQ ID NO:10)

[0263] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL(AAV5;SEQ ID NO:11)

[0264] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(AAV6;SEQ ID NO:12)

[0265] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSSVGSGTVAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTIQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQAKNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGKTGATNKTTLENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL(AAV7;SEQ ID NO:13)

[0266] (AAV8;SEQ ID NO:14)

[0267] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(AAV9;SEQ ID NO:15)

[0268] (AAV10;SEQ ID NO:16)

[0269] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPLESPQEPDSSSGIGKKGKQPARKRLNFEEDTGAGDGPPEGSDTSAMSSDIEMRAAPGGNAVDAGQGSDGVGNASGDWHCDSTWSEGKVTTTSTRTWVLPTYNNHLYLRLGTTSSSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGLRPKAMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGIVTGENQNQTDRNAFYCLEYFPSQMLRTGNNFEMAYNFEKVPFHSMYAHSQSLDRLMNPLLDQYLWHLQSTTSGETLNQGNAATTFGKIRSGDFAFYRKNWLPGPCVKQQRFSKTASQNYKIPASGGNALLKYDTHYTLNNRWSNIAPGPPMATAGPSDGDFSNAQLIFPGPSVTGNTTTSANNLLFTSEEEIAATNPRDTDMFGQIADNNQNATTAPITGNVTAMGVLPGMVWQNRDIYYQGPIWAKIPHADGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFTAARVDSFITQYSTGQVAVQIEWEIEKERSKRWNPEVQFTSNYGNQSSMLWAPDTTGKYTEPRVIGSRYLTNHL(AAV11;SEQ ID NO:17)

[0270] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNGRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDKQLEQGDNPYLKYNHADAEFQQRLATDTSFGGNLGRAVFQAKKRILEPLGLVEEGVKTAPGKKRPLEKTPNRPTNPDSGKAPAKKKQKDGEPADSARRTLDFEDSGAGDGPPEGSSSGEMSHDAEMRAAPGGNAVEAGQGADGVGNASGDWHCDSTWSEGRVTTTSTRTWVLPTYNNHLYLRIGTTANSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGLRPKSMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSTYELPYVMDAGQEGSFPPFPNDVFMVPQYGYCGVVTGKNQNQTDRNAFYCLEYFPSQMLRTGNNFEVSYQFEKVPFHSMYAHSQSLDRMMNPLLDQYLWHLQSTTTGNSLNQGTATTTYGKITTGDFAYYRKNWLPGACIKQQKFSKNANQNYKIPASGGDALLKYDTHTTLNGRWSNMAPGPPMATAGAGDSDFSNSQLIFAGPNPSGNTTTSSNNLLFTSEEEIATTNPRDTDMFGQIADNNQNATTAPHIANLDAMGIVPGMVWQNRDIYYQGPIWAKVPHTDGHFHPSPLMGGFGLKHPPPQIFIKNTPVPANPNTTFSAARINSFLTQYSTGQVAVQIDWEIQKEHSKRWNPEVQFTSNYGTQNSMLWAPDNAGNYHELRAIGSRFLTHHL(AAV12;SEQID NO:18)

[0271] MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVEQSPAEPDSSSGIGKSGQQPARKRLNFGQTGDTESVPDPQPLGQPPAAPSGVGSTTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGATNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQTASGTQQSRLLFSQAGPTSMSLQAKNWLPGPCYRQQRLSKQANDNNNSNFPWTGATKYHLNGRDSLVNPGPAMASHKDDKEKFFPMHGTLIFGKEGTNANNADLENVMITDEEEIRTTNPVATEQYGTVSNNLQNSNAGPTTGTVNHQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTNFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(AAV13;SEQ ID NO:19)

[0272] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL(AAV-DJ;SEQ IDNO:20)

[0273] (AAVPHP.eB;SEQ ID NO:21)

[0274] (Anc80L65;SEQ ID NO:22)

[0275] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL(Anc80L65AAP;SEQID NO:23)

[0276] (7m8; SEQ ID NO:24)

[0277] 3. Modified AAV ITR

[0278] As disclosed herein, any transcribed AAV ITR sequence (RNA) can be modified by engineering a coding modified AAV ITR DNA template to, for example, eliminate or inactivate TRS or its equivalent, and / or eliminate its D-region sequence. The transcribed modified AAV ITR obtained by transcribing such a modified AAV ITR DNA template retains the ability to facilitate the packaging of the RNA sequence of the present invention into AAV viral particles.

[0279] During AAV DNA replication, the ITR is cleaved at the terminal dissociation site (TRS) by the virus-encoded Rep protein. This initiation function requires three DNA sequence elements: the Rep-binding element (RBE), a small palindromic sequence containing a single hairpin tip within a terminal repeat (RBE'), and the TRS. During TRS cleavage, Rep is tethered to the RBE (DNA) in a specific orientation. This orientation appears to align Rep to the AAV ITR, allowing specific nucleotides to contact the RBE' and directing cleavage at the TRS. Changes in the polarity or position of the RBE relative to the TRS significantly inhibit Rep cleavage. Substitution within the RBE' also reduces Rep specific activity, but to a lesser extent. During TRS cleavage, the interactions of Rep with the RBE and RBE' are functionally distinct, as the contact of Rep with the RBE is essential for both DNA helicase activity and TRS cleavage. Meanwhile, the interaction of Rep with the RBE' is primarily necessary for ITR unwinding and the formation of the TRS stem-loop structure, rather than for TRS cleavage.

[0280] At least one transcribed modified ITR sequence (RNA) of the present invention is present on the RNA sequence of the present invention. The transcribed modified ITR sequence of the present invention is preferably closer to the 3' end of the RNA sequence of the present invention.

[0281] In some embodiments, the RNA sequence of the present invention comprises two transcribed modified ITR sequences.

[0282] In some implementations, the modified ITR sequences of the two transcriptions may originate from the same AAV serotype.

[0283] In another implementation, the modified ITR sequences of the two transcriptions may originate from two different AAVs of different serotypes.

[0284] In some implementations, one or more transcribed modified ITR sequences include insertions, deletions, and / or mutations.

[0285] In some embodiments, the rRAAV RNA sequence of the present invention comprises a transcribed modified / mutated ITR sequence and a transcribed wild-type ITR sequence.

[0286] In some implementations, one or more transcribed modified ITR sequences are based on wild-type ITRs in the Flip or Flop orientation.

[0287] Based on wild-type ITR sequences known in the art, modified ITR sequences or their encoding DNA sequences of the test transcription can be readily prepared.

[0288] A representative (non-restrictive) wild-type ITR sequence (DNA) includes at least the sequences listed in Table 1. Figure 1B and 1C Multiple sequence alignments of the 5' ITR sequences and the 3' ITR sequences of AAV1-AAV7 are shown, including the common sequence, TRS, RBE and D region sequences.

[0289] Table 1: Representative wild-type AAV terminal inverted repeat (ITR) sequences

[0290]

[0291]

[0292] As used herein, “RBE sequence” or “RBE” refers to the AAV ITR sequence in the A:A' palindromic stem sequence, which forms a stem (typically a double-stranded region of about 21-23 or about 22 bp) when bases pair and facilitates the binding of the ITR to AAV Rep proteins (Rep78 and Rep68). Figure 1A Representative RBE sequences are shown in both the Flip and Flop configurations of wild-type AAV2 ITR.

[0293] Wild-type ITR sequences of many AAV serotypes known in the art are readily available, and each can be compared with other AAV ITRs, such as... Figure 1B and 1C As shown. The alignment results can be used to identify the RBE sequence of any AAV ITR.

[0294] "Transcribed (functional) RBE" refers to RNA transcribed from an RBE DNA template, which is a wild-type RBE or a functional variant thereof having one or more nucleotide insertions, deletions, substitutions, and / or other mutations, provided that the functional variant RBE substantially retains its ability to bind to Rep (e.g., retaining at least about 60%, 70%, 80%, 90%, 95%, or enhanced binding to the same serotype Rep). In some embodiments, the RBE DNA template or transcribed RBE RNA differs from the wild-type sequence by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides. In some embodiments, the functional RBE contains up to about 30%, 25%, 20%, 15%, 10%, or 5% sequence variation compared to the wild-type RBE due to, for example, one or more nucleotide insertions, deletions, substitutions, and / or other mutations in the RBE.

[0295] In some implementations, nucleotide sequence differences do not result in the loss of paired base pairs (e.g., the GC pair in a wild-type RBE can be changed to CG, AT / AU, or TA / UA in a variant RBE without losing the original paired base pairs).

[0296] In some implementations, the transcribed modified ITR sequence (RNA) retains the transcribed Rep-binding element (transcribed RBE) or a functional variant thereof to facilitate Rep-mediated packaging. For example, the RBE DNA sequence of wild-type AAV2 ITR is SEQ ID NO:5.

[0297] In some implementations, the transcribed modified ITR sequence (RNA) further preserves the sequence of the transcribed Rep-binding element (transcribed RBE). For example, in Figure 1A In Flip ITR, the CTTTG DNA sequence that forms a hairpin or loop structure in the B:B' fragment is the RBE' sequence.

[0298] In some implementations, the transcribed modified ITR sequence lacks the transcribed TRS, and / or the transcribed rcTRS, or both.

[0299] In some embodiments, the RNA sequence of the present invention lacks transcribed (functional) TRS sequences because its corresponding DNA sequence lacks certain sequence elements of wild-type TRS, resulting in the loss of wild-type TRS function in the DNA (e.g., the wild-type TRS sequence between the A:A' fragment and the D region sequence, which normally occupies the sequence or inner strand, is usually recognized and cleaved by endonucleases during AAV replication, but is not cleaved if it is present in the ssDNA vector genome of the AAV ITR).

[0300] For example, in some implementations, the inverse complementary sequence of the TRS may be missing or mutated, as in the dITR and dITR-D sequences used in the embodiments.

[0301] Alternatively or additionally, the TRS between the A:A' fragment and the D region sequence may be missing one or more nucleotides, or have one or more nucleotide substitutions or mutations (such as the absence or substitution / mutation of 4 nucleotides in the dITR sequence used in the example).

[0302] In some implementations, all or almost all TRS / rcTRS in the wild-type sequence are missing, resulting in RNA transcripts lacking functional TRS sequences.

[0303] In some embodiments, a portion of the wild-type TRS / rcTRS sequence is altered / mutated, for example, by having insertions, deletions, substitutions, and / or other mutations in the wild-type sequence, such that the mutated TRS / rcTRS produces a corresponding RNA transcript of a functional TRS lacking transcription. For example, in some embodiments, 1, 2, 3, 4, or 5 consecutive or non-consecutive TRS nucleotides and / or rcTRS nucleotides may be deleted or substituted in the mutated sequence.

[0304] In some embodiments, the transcribed modified ITR sequence is transcribed from a modified ITR lacking a D-region sequence or at least lacking a functional D-region sequence (D sequence or D' sequence, depending on the Flip or Flop conformation). For example, in some embodiments, the entire D-region sequence is deleted, resulting in an RNA transcript lacking the functional D-region sequence for transcription. In other embodiments, at least a portion of the D-region sequence is mutated (e.g., with deletion, insertion, substitution, and / or other mutations), resulting in an RNA transcript lacking the functional D-region sequence for transcription. In some embodiments, the mutated D-region sequence has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide of the wild-type sequence.

[0305] In some embodiments, the modified ITR sequence (DNA template) lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the wild-type ITR sequence. For example, both the dITR sequence (SEQ ID NO:2) and the dITR-D sequence (SEQ ID NO:3) lack the 8 nucleotides from the 5' end compared to the wild-type ITR sequence (SEQ ID NO:1).

[0306] The corresponding DNA sequence of any one of the following transcriptions is explicitly considered within the scope of this invention: the RNA coding sequence encoding the above-mentioned transcription (DNA coding sequence of GOI), the modified AAV ITR of transcription (modified AAV ITR), the functional RBE of transcription (functional RBE), the functional D region sequence of transcription (functional D region sequence), and the functional TRS sequence of transcription (functional TRS sequence).

[0307] 4. Introns, exons, UTRs, enhancers, and other elements

[0308] The RNA sequence of the present invention to be capsidated in the rRAAV viral particle of the present invention may further include additional optional sequence elements (such as expression control elements) that can enhance or regulate GOI expression.

[0309] Expression control elements present in the RNA sequences of the present invention promote appropriate heteropolynucleotide (e.g., GOI) transcription and / or translation, including, for example, intron splicing signals, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA and stop codons, etc.

[0310] Typically, expression control elements (some in the RNA sequence of the present invention, while others are present on the DNA encoding the RNA sequence of the present invention) are one or more nucleic acid sequences, such as promoters and enhancers that affect the expression of operatively linked heterologous polynucleotides (e.g., GOIs). These elements typically act in cis, but may also act in trans. Expression control can occur at the levels of transcription, translation, splicing, information stability, etc. Typically, expression control elements regulating transcription are located lateral to the 5' end (i.e., "upstream") of the transcribed polynucleotide. Expression control elements may also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements may be located at a distance from the transcribed gene sequence of interest (e.g., 100 to 500, 500 to 1000, 2000 to 5000 or more nucleotides away from the gene polynucleotide of interest). However, due to the limitation of the multinucleotide length of viral vectors (such as AAV vectors), these expression control elements are typically in the range of 1-1,000, 1-500, 1-250, or 1-100 nucleotides from the gene sequence of interest that is transcribed.

[0311] Some non-restrictive expression control elements that may exist on the RNA sequence of the present invention or on the DNA encoding the RNA sequence of the present invention are described in further detail below.

[0312] Introns

[0313] Introns are known to be post-transcriptional regulatory elements that can effectively induce mRNA to be transported out of the cell nucleus and enhance mRNA stability.

[0314] In some implementations, rRAAV may include one or more introns or fragments thereof. In some implementations, the one or more introns are fragments of the gene of interest. In some implementations, the one or more introns are heterologous to the gene of interest.

[0315] Introns have been reported to influence gene expression levels. This effect is known as intron-mediated enhancement of gene expression (IME) (Lu et al., Mol Genet Genomics 279:563-572, 2008). In some implementations, gene expression levels are increased by approximately 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold compared to gene expression from sequences without one or more introns.

[0316] Non-restrictive introns include the SV40 intron, the β-globulin intron, and the short chimeric intron (CIB). Other introns include the ColE2-RNA-OUT, OIPR, and R6K-RNA-OUT introns described in Lu et al., Hum Gene Ther. 2017; 28(1):125-134 (incorporated by reference); and the human hemoglobin subunit β (HBB2) synthesis intron (Snyder et al., Hum Gene Ther. 8(1997), pp. 1891-1900, incorporated by reference).

[0317] In some embodiments, one or more introns may be less than 25 nucleotides, less than 50 nucleotides, less than 100 nucleotides, less than 150 nucleotides, less than 200 nucleotides, less than 250 nucleotides, less than 300 nucleotides, less than 350 nucleotides, less than 400 nucleotides, less than 450 nucleotides, or less than 500 nucleotides.

[0318] In some embodiments, one or more introns may be greater than 25 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 150 nucleotides, greater than 200 nucleotides, greater than 250 nucleotides, greater than 300 nucleotides, greater than 350 nucleotides, greater than 400 nucleotides, greater than 450 nucleotides, or greater than 500 nucleotides.

[0319] In some embodiments, one or more introns may be about 50 to about 100 nucleotides, about 50 to about 200 nucleotides, about 50 to about 300 nucleotides, about 50 to about 400 nucleotides, about 50 to about 500 nucleotides, about 100 to about 200 nucleotides, about 100 to about 300 nucleotides, about 100 to about 400 nucleotides, about 100 to about 500 nucleotides, about 200 to about 300 nucleotides, about 200 to about 400 nucleotides, about 200 to about 500 nucleotides, about 300 to about 400 nucleotides, about 300 to about 500 nucleotides, or about 400 to about 500 nucleotides.

[0320] enhancer

[0321] As used herein, the term "enhancer" can refer to a sequence located near the gene of interest. Enhancer elements are typically located upstream of promoter elements in the DNA encoding the RNA sequence of the present invention, but can also be located downstream of or within intron sequences (e.g., the gene of interest) and remain functional. Therefore, enhancers or portions thereof can be present in the transcribed RNA sequence of the present invention.

[0322] Non-restricted examples of suitable enhancers include CMV enhancers.

[0323] In some embodiments, enhancer elements may be located 100, 200, or 300 or more base pairs upstream or downstream of the gene of interest (e.g., in the RNA sequence or its DNA coding sequence of the present invention). Enhancer elements typically increase the expression of the gene of interest to a level higher than that provided by promoter elements.

[0324] Untranslated Region (UTR)

[0325] As used herein, a “non-translated region” (“UTR”) refers to RNA that has not been translated after transcription. For example, a 5' UTR is located upstream of the start codon of the gene of interest, and a 3' UTR is located downstream of the stop codon of the gene of interest. In some embodiments, the 5' and / or 3' UTRs may have insertions, deletions, or modifications to enhance the stability of the transcribed gene of interest. For example, a 5' UTR may contain a translation initiation sequence, such as, but not limited to, a kozak sequence and an internal ribosome entry site (IRES). The kozak sequence has a common CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'.

[0326] It is known that a segment of adenosine and uridine is embedded in the 3′ UTR. These AU-rich features are particularly prevalent in high-turnover genes. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this class of AREs include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myopoietin are two well-studied examples of this class. Most proteins that bind to AREs are known to disrupt messenger stability, while members of the ELAV family, especially HuR, have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering HuR-specific binding sites into the 3′ UTR of nucleic acid molecules will result in HuR binding, thereby stabilizing the messenger in vivo. Any of these 5′ and / or 3′ UTR sequences can be present in the RNA sequence of this invention.

[0327] In some embodiments, the 5'UTR and / or 3'UTR may contain sequences heterologous to the gene of interest. In some embodiments, the 5'UTR and / or 3'UTR are natural to the gene of interest.

[0328] In some embodiments, the 5'UTR and / or 3'UTR of mRNA normally expressed in specific tissues or organs such as the lungs, liver, pancreas, endothelial cells, CNS, neurons, astrocytes, skeletal muscle, myocardium, smooth muscle, blood, and hematopoietic cells can be used in the RNA sequence of the present invention, said RNA sequence containing one or more GOIs targeting these tissues.

[0329] Polyadenylation sequence

[0330] In some embodiments, the RNA sequence of the present invention comprises a transcribed modified AAV ITR at the 5' of a polyA sequence, a polyA signal sequence (e.g., AAUAAA), or an RNA transcription termination sequence (e.g., a histone downstream element).

[0331] The above defines "poly-A sequence", "poly-A tail", "poly-A signal sequence" and "sequence for RNA transcription termination".

[0332] In some embodiments, the RNA sequence of the present invention comprises a poly-A tail. This RNA sequence can be packaged into the rRAAV viral particles of the present invention and delivered directly to target cells, and the GOI encoded by the RNA sequence of the present invention can be directly translated.

[0333] In some embodiments, the RNA sequence of the present invention comprises a polyA signal sequence and, optionally, a transcribed GU-rich region downstream of the polyA site. This RNA sequence can be packaged into the rRAAV viral particles of the present invention and delivered directly to target cells. Once inside the target cell, the polyA signal sequence can be recognized by a cytoplasmic polyA-adding enzyme and further processed to generate a polyA tail, followed by translation of the GOI encoded by the RNA sequence of the present invention.

[0334] Representative polyA signal sequences and surrounding sequences include human growth hormone (hGH) polyA sequences (see Liu et al., Gene Ther 20:308-317, 2013, incorporated by reference), bovine growth hormone polyadenylation signal (bGHpA) (Goodwin and Rottman, J Biol Chem. 15 Aug 1992; 267(23):16330-4, incorporated by reference), early or late polyadenylation signals of SV40, and synthetic polyA signals used by Choi et al. (Mol Brain. 2014; 7:17, incorporated by reference).

[0335] Transcription enhancers

[0336] As used herein, a “transcription enhancer” refers to a cis-acting nucleotide sequence that can increase transcription of a gene of interest. In some embodiments, the transcription enhancer may be located in an intron of the RAAV RNA sequence transcribed according to the present invention or partially in an exon region.

[0337] WPRE

[0338] In some embodiments, the RNA sequence of the present invention comprises a transcribed WPRE sequence encoded by a WPRE sequence on encoding DNA.

[0339] The post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHP) is a DNA sequence of about 600 bp that produces a tertiary structure that enhances expression during transcription.

[0340] WPREs are commonly used in molecular biology to increase the expression of genes delivered by viral vectors. They are triadic regulatory elements with γ, α, and β components. The α component is 80 bp long: GCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGT (SEQ ID NO:39). When used alone, the α component exhibits only 9% of the activity of the complete triadic WPRE sequence, and it shares 100% identity with base pairs 1093-1684 of the marmot hepatitis B virus (WHV8) genome.

[0341] In some embodiments, the transcribed WPRE sequence or portions thereof (such as γ, α, and β elements, preferably in a given order) are present in the 3'UTR region of the GOI on the test RNA sequence capsidated in the rRAAV viral particle of the present invention, to significantly improve the stability and protein yield of the RNA sequence of the present invention.

[0342] In some implementations, the WPRE sequence is a short WPRE (WPRE2) containing minimal γ elements and a portion of α-β elements (see Kalev-Zylinska, J Neurosci. 2007, 27:10456-10467, incorporated by reference).

[0343] In some implementations, the WPRE sequence is a short WPRE (WPRE3) containing minimal γ and α elements (see Choi et al., Mol Brain 7, 17 (2014), incorporated by reference).

[0344] In some embodiments, the RNA sequence of the present invention comprises a WPRE sequence and a GOI lacking introns.

[0345] promoter

[0346] As used herein, the term “promoter” is defined as a DNA sequence that is recognized by cellular synthetic machinery or introduced synthetic machinery and is required to initiate specific transcription of a polynucleotide sequence.

[0347] Therefore, the RNA sequence of the present invention does not contain a promoter. On the other hand, the DNA encoding the RNA sequence of the present invention (such as an expression cassette or expression vector encoding the RNA sequence of the present invention) contains a promoter for transcribing the RNA sequence of the present invention.

[0348] As used herein, the term "promoter / regulatory sequence" refers to the nucleic acid sequence required to express a gene product operatively linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence. In other cases, this sequence may also include enhancer sequences and other regulatory elements required to express the gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses a gene product in a tissue or cell type-specific manner (e.g., the RNA sequence of the present invention).

[0349] As used herein, the terms "operably linked" or "operably coupled" refer to the physical or functional juxtaposition of components so as to allow them to function in their intended manner. In instances of expression control elements operably linked to heteropolynucleotides, this relationship enables the control element to regulate the expression of the heteropolynucleotide. More specifically, for example, two operably coupled DNA sequences mean that two DNA sequences are arranged in such a relationship (cis or trans) that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

[0350] In some implementations, the promoter is a constitutive promoter.

[0351] As used in this article, a “constitutive” promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell under most or all physiological conditions of the cell.

[0352] In some embodiments, promoters that can be used to constitutively drive the expression of the RNA sequence encoding the present invention may include: β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) instant-early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof, such as CAG promoter, CB promoter, (human) elongation factor 1α-subunit (EF1α) promoter, and ubiquitin C (UBC) promoter.

[0353] In some implementations, the promoter is an inducible promoter.

[0354] As used in this article, an "inducible" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell essentially only when the inducer corresponding to the promoter is present in the cell.

[0355] In some implementations, the promoter is a tissue-specific promoter, a species-specific promoter, or a cell cycle-specific promoter. See Parr et al., Nat. Med. [Nature Medicine] 3:1145-9, 1997 (the entire contents are incorporated herein by reference).

[0356] As used herein, a “tissue or cell type specific” promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specified by a gene, preferably results in the production of a gene product in a particular cell type or specific cell, for example, because the cell / tissue is the cell or tissue type in which the promoter is typically active.

[0357] Tissue or cell type specific promoters may include neuronal tissue-specific promoters; CNS- or PNS-specific promoters, such as astrocyte, oligodendrocyte, or neuronal promoters; hematopoietic lineage-specific promoters, such as B cell promoters, T cell promoters, NK cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters; endothelial cell promoters; pancreatic promoters; liver / hepatic cell promoters; lung tissue promoters, etc.

[0358] Representative tissue-specific promoters include prion promoters, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptoprotein (Syn), synaptoprotein 1 (Syn1), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabolite glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or neurofilament heavy chain (NFH), β-globulin small gene nβ2, proenkephalinogen (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters.

[0359] Astrocyte-specific promoters include glial fibrillary acidic protein (GFAP) and the EAAT2 promoter.

[0360] Oligodendrocyte-specific promoters include the myelin basic protein (MBP) promoter.

[0361] In some embodiments, the promoter is heterologous to the gene of interest. In some embodiments, the promoter is a natural promoter of the gene of interest. In some embodiments, the heterologous promoter includes insertions, deletions, substitutions, and / or other mutations. In some embodiments, the natural promoter includes insertions, deletions, substitutions, and / or other mutations.

[0362] In some implementations, the promoter is the Pol II promoter. In other implementations, the promoter is the Pol III promoter, such as the U6 promoter.

[0363] 5. Vector (plasmid or rod-like particle)

[0364] As used herein, “carrier” generally refers to a composition containing isolated nucleic acids (DNA or RNA) and which can be used to deliver said isolated nucleic acids into the cell.

[0365] The term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as clomids, plasmids, rod granules (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0366] The rRAAV RNA sequence of the present invention containing GOI is a vector for delivering the GOI to target / host cells via rRAAV viral particles encapsulating a vector.

[0367] In some embodiments, the rRAAV RNA sequence of the present invention is encoded by a DNA expression vector, such as a plasmid or a baculosome (e.g., a vector that can be maintained or replicated like a baculovirus in insect cells). This DNA expression vector can transcribe the RNA sequence of the present invention into suitable host cells, such as mammalian packaging cells (e.g., HEK293T cells) or insect packaging cells (e.g., Sf9 cells), thereby generating the tested rRAAV viral particles in the presence of other elements required for rRAAV packaging (e.g., rep and cap coding sequences).

[0368] Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0369] In some embodiments, the RAAV is transcribed from a plasmid or rod granule. The plasmid or rod granule may include the gene sequence of interest. In some embodiments, a promoter is operatively linked to and located upstream of the gene of interest. In some embodiments, the promoter is not in the transcribed RAAV.

[0370] 6. AAV particles and AAV particle populations

[0371] In some embodiments, the present invention provides isolated rRAAV viral particles, said viral particles comprising any of the RNA sequences of the present invention capsidated within any of the AAV capsids or viral particles described herein.

[0372] In some implementations, the AAV capsid or viral particle is the serotype or a combination of one or more serotypes described herein.

[0373] In the rRAAV vector or RNA sequence of the present invention, the rRAAV genome (RNA) can be a single-stranded (ss) nucleic acid or a double-stranded (ds) or self-complementary (sc) nucleic acid.

[0374] A related aspect of the invention provides a population of recombinant viral particles (e.g., rRAAV particles) comprising a plurality of recombinant viral particles (e.g., rRAAV particles) of the invention, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the recombinant viral particles (e.g., rRAAV particles) within the population have the capsidated RNA sequence of the invention.

[0375] In some embodiments, a population of rRAAV particles contains a plurality of rRAAV viral particles of the present invention, wherein about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the rRAAV particles in the population have the capsidated RNA sequence of the present invention.

[0376] In some implementations, the population of recombinant viral particles (e.g., rRAAV particles) contains at least 1 × 10⁻⁶ particles. 4 One virus particle, at least 2 × 10 4 One virus particle, at least 5 × 10 4 One virus particle, at least 1×10 5 One virus particle, at least 2 × 10 5 One virus particle, at least 5 × 10 5 One virus particle, at least 1×10 6 One virus particle, at least 2 × 10 6 One virus particle, at least 5 × 10 6 One virus particle, at least 1×10 7 One virus particle, at least 2 × 10 7 One virus particle, at least 5 × 10 7 One virus particle, at least 1×10 8 One virus particle, at least 2 × 10 8 One virus particle, at least 5 × 10 8One virus particle, at least 1×10 9 One virus particle, at least 2 × 10 9 One virus particle, at least 5 × 10 9 One virus particle, at least 1×10 10 One virus particle, at least 2 × 10 10 One virus particle, at least 5 × 10 10 One virus particle, at least 1×10 11 One virus particle, at least 2 × 10 11 One virus particle, at least 5 × 10 11 One virus particle, at least 1×10 12 One virus particle, at least 2 × 10 12 One virus particle, at least 5 × 10 12 One virus particle, at least 1×10 13 One virus particle, at least 2 × 10 13 One virus particle, at least 5 × 10 13 One virus particle, at least 1×10 14 One virus particle, at least 2 × 10 14 One virus particle, at least 5 × 10 14 One virus particle, at least 1×10 15 One virus particle, at least 2 × 10 15 One virus particle, at least 5 × 10 15 One virus particle, at least 1×10 16 One virus particle, at least 2 × 10 16 One virus particle, or at least 5 × 10 16 A virus particle.

[0377] In some implementations, up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.1%, 0.01%, or less of the population of recombinant viral particles are capsidred with non-RNA (e.g., DNA) within the viral particles.

[0378] 7. Host cells and AAV production

[0379] The general principles of rAAV production are known in the art. See, for example, the following reviews, Carter (Current Opinions in Biotechnology, 1533-539, 1992); and Muzyczka, Curr. Topics in Microbial and Immunol, 158:97-129, 1992, both of which are incorporated herein by reference). The following references describe various methods: Ratschin et al. (Mol. Cell. Biol. [Journal of Molecular Cell Biology] 4:2072, 1984; Hermonat et al. (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 81:6466, 1984); Tratschin et al. (Mol. Cell. Biol. [Journal of Molecular Cell Biology] 5:3251, 1985); McLaughlin et al. (J. Virol [Journal of Virology] 62:1963, 1988); and Lebkowski et al. (Mol. Cell. Biol. [Journal of Molecular Cell Biology] 7:349, 1988), Samulski et al. (J. Virol [Journal of Virology] 63:3822-3828, 1989); US5,173,414; WO 95 / 13365 and US5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441; WO 97 / 08298; WO 97 / 21825; WO 97 / 06243; WO 99 / 11764; Perrin et al. (Vaccine 13:1244-1250, 1995); Paul et al. (Human Gene Therapy 4:609-615, 1993); Clark et al. (Gene Therapy 3:1124-1132, 1996; US5,786,211; US5,871,982; and US6,258,595).

[0380] AAV vector serotypes can be matched with target cell types. For example, Table 2 of WO 2018002719 A1 lists exemplary cell types that can be transduced by a specified AAV serotype (incorporated herein by reference).

[0381] Packaging cells are used to form viral particles capable of infecting host cells. These cells include HEK293 and Sf9 cells, which can be used to package AAV and adenovirus.

[0382] Viral vectors used in gene therapy are typically generated by producing cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimum viral sequence required for packaging and subsequent integration into host cells (if appropriate), with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions can be provided by the packaging cell lines in trans form, usually as a result of these viral functions / proteins (such as the rep and cap genes of AAV) being expressed either as a transgene integrated into the packaging cells or as a transgene introduced into a second viral vector or expression vector within said packaging cells.

[0383] For example, AAV vectors used in gene therapy typically contain only terminal inverted repeat (ITR) sequences from the AAV genome, which are required for packaging and integration into the host genome. Viral DNA is packaged in cell lines containing helper plasmids encoding other AAV genes, namely rep and cap, but lacking the ITR sequence. These cell lines are also infected with adenovirus as a helper. The helper virus promotes AAV vector replication and AAV gene expression from the helper plasmid. The helper plasmid is not packaged in large quantities due to the lack of the ITR sequence. Adenovirus contamination can be reduced, for example, by performing heat treatments that adenovirus is more sensitive to than AAV.

[0384] In some embodiments, recombinant AAV can be produced using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (containing the gene of interest) to be packaged into AAV particles, an AAV helper vector, and a cofactor vector. The AAV helper vector encodes “AAV helper function” sequences (e.g., rep and cap) that function in trans for productive AAV replication and capsid formation. Preferably, the AAV helper vector supports efficient AAV vector production without generating any detectable wild-type AAV virus particles (e.g., AAV virus particles containing functional rep and cap genes). The cofactor vector encodes nucleotide sequences for non-AAV-derived viral and / or cellular functions (e.g., “cofactor functions”) that AAV depends on for its replication. Cofactor functions include functions required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. The assistive function of a virus can be derived from any of the known assistive viruses, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0385] In some embodiments, the tested rRAAV is generated using a baculovirus expression system packaged in insect cells (such as Sf9 cells). See, for example, WO 2007046703, WO 2007148971, WO 2009014445, WO2009104964, WO 2013036118, WO 2011112089, WO 2016083560, WO 2015137802, and WO2019016349, all of which are incorporated herein by reference.

[0386] Vector titer is typically expressed as viral genome / ml (vg / ml). In some implementations, the viral titer is higher than 1 x 10⁻⁶. 9 Higher than 5x10 10 Higher than 1x10 11 Higher than 5x10 11 Higher than 1x10 12 Higher than 5x10 12 or higher than 1x10 13 vg / ml.

[0387] 8. Gene of Interest (GOI) or RNA Sequence of Interest (RSI)

[0388] The rRAAV viral particles of the present invention can be used to deliver any gene of interest (GOI) or RNA sequence of interest (RSI) to a host cell for any purpose, provided that the GOI is RNA within the packaging limits of the selected AAV viral capsid or AAV viral particle shell, such as about 4,700 nucleotides in total length for most AAV viral particles, and up to about 8,900 nucleotides in total length for some high-capacity AAV viral particles (such as AAV5).

[0389] In some embodiments, representative (non-limiting) RNA sequences of interest (RSIs) include, for example, protein-coding RNA, mRNA, non-coding RNA (ncRNA), tRNA, ribosomal RNA (rRNA), transfer-messenger RNA (tmRNA), antisense oligonucleotides (ASO), RNA aptamers, RNA components of the CRISPR-Cas system such as single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA), tracr RNA, or RNA components of the RISC complex or RNAi pathway (such as shRNA, miRNA, or siRNA), regulatory RNA, Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), long non-coding RNA (lncRNA) (including intergenic lincRNA, intronic ncRNA, and sense / antisense lncRNA), long intergenic / intergenic non-coding RNA (lincRNA), enhancer RNA, bacterial small RNA (sRNA), snRNA, exRNA, scaRNA, Xist, and HOTAIR and its precursors.

[0390] In some embodiments, the RNA sequence of the present invention comprises a protein or polypeptide coding sequence.

[0391] In some embodiments, the protein or polypeptide is a wild-type protein or its functional equivalent or variant (such as an enzyme or structural protein) that can be used to replace a defective protein in a target cell, tissue or organism.

[0392] In some embodiments, the protein or polypeptide is a wild-type protein or its functional equivalent or variant (such as an enzyme or structural protein) that can be used to antagonize compounds (small molecule compounds or macromolecules, such as lipids, fatty acids, proteins, nucleic acids, etc.) in target cells, tissues, or organisms.

[0393] For example, in some embodiments, the RNA sequence of the present invention contains the coding sequence of an effector enzyme of the CRISPR / Cas system.

[0394] In some implementations, the CRISPR-Cas system is a type 1 system, and the effector enzyme is a type I, III, or IV effector enzyme.

[0395] In some implementations, the CRISPR-Cas system is a type 2 system, and the effector enzyme is a type II, V, or VI effector enzyme.

[0396] For example, in some embodiments, the effector enzyme is a type II enzyme, such as Cas9, including Streptococcus pyogenes (SpCas9) or SaCas9 (see WO 2014 / 093622 (PCT / US 2013 / 074667), which is incorporated herein by reference).

[0397] In some implementations, the Cas effector enzymes are two types of V Cas proteins, including Cas12a (formerly known as Cpf1, such as Cas12a of the new culprit Francisella novicida), C2c1, and C2c3.

[0398] In some implementations, the Cas effector enzymes are class 2 type VI Cas proteins, including Cas13a (also known as C2c2), Cas13b, Cas13c, Cas13d, Cas13e, and Cas13f. These Cas proteins use their crRNA to recognize target RNA sequences, rather than target DNA sequences in Cas9 and Cas12a.

[0399] In some embodiments, the Cas effector enzyme is any of the Cas effectors described in WO 2020 / 028555 (the entire contents of which are incorporated herein by reference), including Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, etc.), Cas13 (e.g., Cas13a, Cas13b (e.g., Cas13b-t1, Cas13b-t2, Cas13b-t3), Cas13c, Cas13d, etc.), Cas14, CasX, and CasY.

[0400] In some embodiments, the Cas effector enzyme is fused to a DNA and / or RNA base editor, such as a cytosine or adenine base editor (CBE or ABE). In some embodiments, the base editor preferably edits DNA bases and optionally has reduced or substantially no off-target RNA base editing capability. In some embodiments, the base editor preferably edits RNA bases and optionally has reduced or substantially no off-target DNA base editing capability. In some embodiments, the base editor edits both DNA and RNA bases.

[0401] In some embodiments, the base editor is a first, second (BE2), third (BE3), or fourth-generation (BE4) base editor. In some embodiments, the base editor is a dibase editor.

[0402] In some implementations, the base editor is an RNA adenosine deaminase (ADAR), such as ADAR1, ADAR2, or ADARDD, which includes ADAR2DD (E488Q).

[0403] In any of the above embodiments, the RNA sequence of the present invention may further comprise a guide RNA sequence designed to be loaded into a CRISPR / Cas effector enzyme for binding a target polynucleotide sequence complementary to the guide RNA. After the rRAAV viral particle of the present invention delivers the RNA sequence of the present invention to a target host cell, this gRNA sequence can be treated by a cellular nuclease and released / isolated from the RNA sequence of the present invention. For example, the gRNA may be present in the unpaired 5' or 3' flanking region of a primary miRNA hairpin structure that is part of the RNA sequence of the present invention, and released / isolated from the initial primary miRNA transcript after treatment with a cellular enzyme such as Drosha.

[0404] In some embodiments, the RNA sequence of the present invention comprises the coding sequence of an effector enzyme of a CRISPR / Cas system, and further comprises the coding sequence of a DNA or RNA base editing enzyme or domain, such that the fusion of the Cas effector enzyme and the DNA / RNA base editing enzyme / domain is encoded by the RNA sequence. In some embodiments, the nuclease activity of the Cas effector enzyme is defective, enabling it to bind to the target polynucleotide sequence via its bound guide RNA, but preventing it from cleaving the DNA / RNA target polynucleotide.

[0405] In some embodiments, the RNA sequence of the present invention comprises the coding sequence of a variant or derivative of a CRISPR / Cas system effector enzyme, wherein said variant comprises a deletion (e.g., N- and / or C-terminal deletion, for example, a deletion of no more than 210 N-terminal residues and / or no more than 180 C-terminal residues in Cas13e or Cas13f), insertion, or substitution of the wild-type CRISPR / Cas system effector enzyme, but substantially retains the ability of said wild-type effector enzyme to bind to gRNA and / or cleave target polynucleotides. In some embodiments, said variant lacks the activity of cleaving target polynucleotides.

[0406] In some embodiments, the RNA base editing domain encoded by the RNA sequence of the present invention is an adenosine deaminase, such as a double-stranded RNA-specific adenosine deaminase (e.g., ADAR1 or ADAR2); an apolipoprotein B mRNA editing enzyme; a catalytic polypeptide-like enzyme (APOBEC); or an activation-induced cytidine deaminase (AID).

[0407] In some embodiments, the RNA base editing domain encoded by the RNA sequence of the present invention comprises adenosine deaminase and / or cytidine deaminase, such as RNA-acting cytidine deaminases (CDARs), such as double-stranded RNA-specific adenosine deaminases (ADARs) (e.g., ADAR1 or ADAR2), apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like enzymes (APOBECs, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4), activation-induced cytidine deaminase (AID), cytidine deaminase 1 (CDA1), or mutants thereof.

[0408] In some implementations, ADAR has the E488Q / T375G double mutation or ADAR2DD.

[0409] In some embodiments, the base editing domain is further fused with an RNA-binding domain, such as MS2.

[0410] In some embodiments, the variants or derivatives of the encoded CRISPR / Cas effector enzyme further comprise RNA methyltransferases, RNA demethylases, RNA splicing modifiers, localization factors, or translational modifiers.

[0411] In some embodiments, the Cas effector enzyme, the variant / derivative thereto, or the functional fragment contains a nuclear localization signal (NLS) sequence or a nuclear output signal (NES).

[0412] In some embodiments, the Cas effector enzyme, variant / derivative thereof, or functional fragment thereof is fused with a heterologous functional domain. In some embodiments, the heterologous functional domain comprises: nuclear localization signal (NLS), reporter protein or detection marker (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), localization signal, protein targeting moiety, DNA binding domain (e.g., MBP, Lex A DBD, Gal4 DBD), epitope tag (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc.), transcription activation domain (e.g., VP64 or VPR), transcription repression domain (e.g., KRAB moiety or SID moiety), nuclease (e.g., FokI), deamination domain (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methyltransferase, demethylase, transcription release factor, HDAC, polypeptide with ssRNA cleavage activity, polypeptide with dsRNA cleavage activity, polypeptide with ssDNA cleavage activity, polypeptide with dsDNA cleavage activity, DNA or RNA ligase, or any combination thereof. In some embodiments, the heterologous functional domain is fused to the N-terminus, C-terminus, or interior of the fusion protein.

[0413] In some embodiments, the RNA sequence of the present invention comprises the coding sequence of a CasPR (a CRISPR-associated protein for processing class 1 precursor crRNA) fusion protein, said fusion protein comprising a CasPR (or a homolog, ortholog, paralog, variant, derivative, or functional fragment thereof) fused to a heterologous functional domain; or a functional variant thereof.

[0414] In some implementations, CasPR is Cas5d, Cas6, or Csf5.

[0415] In some implementations, CasPR is MtCas6(IA) (sequence 1), MmCas6(IB) (sequence 2), SpCas5d(I-C1) (sequence 3), BhCas5d(I-C2) (sequence 4), SaCas6(ID) (sequence 5), EcCas6e(IE) (sequence 6), PaCas6f(IF) (sequence 7), MtCas6(III-A) (sequence 8), PfCas6(III-B) (sequence 9), PaCsf5(IV-A1) (sequence 10), or MtCsf5(IV-A2) (sequence 11). All sequences are incorporated herein by reference.

[0416]

[0417]

[0418] MPLIFKIGYNVIPLQDVILPTPSSKVLKYLIQSGKLIPSLKDLITSRDKYKPIFISHLGFNQRRIFQTNGNLKITKGSRLSSIIIAFSTQANVLSEVADEGIFETVYGKFHIMIESIEIVEVEKLKEEVEKHMNDNIRVRFVSPTLLSSKVLLPPSLSERYKKIHAGYSTLPSVGLIVAYAYNVYCNLIGKKEVEVRAFKFGILSNALSRIIGYDLHPVTVAIGEDSKGNLRKARGVMGWIEFDIPDERLKRRALNYLLTSSYLGIGRSRGIGFGEIRLEFRKIEEKEKEG (Sequence 1)

[0419] MDLEYMHISYPNILLNMRDGSKLRGYFAKKYIDEEIVHNHRDNAFVYKYPQIQFKIIDRSPLIIGIGSLGINFLESKRIFFEKELIISNDTNDITEVNVHKDMDHFGTTDKILKYQFKTPWMALNAKNSEIYKNSDEIDREEFLKRVLIGNILSMSKSLGYTIEEKLKVKINLKEVPVKFKNQNMVGFRGEFYINFDIPQYLGIGRNVSRGFGTVVKV (sequence 2)

[0420] MYRSRDFYVRVSGQRALFTNPATKGGSERSSYSVPTRQALNGIVDAIYYKPTFTNIVTEVKVINQIQTELQGVRALLHDYSADLSYVSYLSDVVYLIKFHFVWNEDRKDLNSDRLPAKHEAIMERSIRKGGGRRDVFLGTRECLGLLDDISQEEEYETTVSYYNGVNIDLGIMFHSFAYPKDKKTPLKSYFTKTVMKNGVITFKAQSECDIVNTLSSYAFKAPEEIKSVNDECMEYDAMEKGEN(Sequence 3)

[0421] MRNEVQFELFGDYALFTDPLTKIGGEKLSYSVPTYQALKGIAESIYWKPTIVFVIDELRVMKPIQMESKGVRPIEYGGGNTLAHYTYLKDVHYQVKAHFEFNLHRPDLAFDRNEGKHYSILQRSLKAGGRRDIFLGARECQGYVAPCEFGSGDGFYDGQGKYHLGTMVHGFNYPDETGQHQLDVRLWSAVMENGYIQFPRPEDCPIVRPVKEMEPKIFNPDNVQSAEQLLHDLGGE(sejjn4)

[0422] MPNDPYSLYSIVIELGAAEKGFPTGILGRSLHSQVLQWFKQDNPFLATELHQSQISPFSISPLMGKRHAKLTKAGDRLFFRICLLRGDLLQPLLNGIEQTVNQSVCLDKFRFRLCQTHILPGSHPLAGASHYSLISQTPVSSKITLDFKSTSSFKVDRKIIQVFPLGEHVFNSLLRRWNNFAPEDLHFSQVDWSIPIAAFDVKTIPIHLKKVEIGAQGWVTYIFPNTEQAKIASVLSEFAFFSGVGRKTTMGMQVQVRS (Sequence 5)

[0423] MYLSKVIIARAWSRDLYQLHQGLWHLFPNRPDAARDFLFHVEKRNTPEGCHVLLQSAQMPVSTATVIKTKQVEFQLQVGVPLYFRLRANPIKTILDNQKRLDSKGNIKRCRVPLIKEAEQIAWLQRKLGNAARVEDVHPISERPQYFSGDGKSGKIQTVCFEGVLTINDAPALIDLVQQGIGPAKSMGCGLLSLAPL (Sequence 6)

[0424] MDHYLDIRLPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFPDLDESRSRLGERLRIHASADDLRALLARPLEGLRDHLQFGEPAVVPHPTPYRQVSRVQAKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPLQVTAEEGGFTCYGLSKGGFVPWF (Sequence 7)

[0425] MAARRGGIRRTDLLRRSGQPRGRHRASAAESGLTWISPTLILVGFSHRGDRRMTEHLSRLTLTLEVDAPLERARVATLGPHLHGVLMESIPADYVQTLHTVPVNPYSQYALARSTSLEWKISTLTNEARQQIVGPINDAAFAGFRLRASGIATQVTSR SLEQNPLSQFARIFYARPETRKFRVEFLTPTAFKQSGEYVFWPDPRLVFQSLAQKYGAIVDGEEPDPGLIAEFGQSVRLSAFRWASAPFAVGAARVPGFTGSATFTVRGVDTFASYIAALLWFGEFSGCGIKASMMGAIRVQPLAPREKCVPKP(sejjn8)

[0426] MRFLIRLVPEDKDRAFKVPYNHQYYLQGLIYNAIKSSNPKLATYLHEVKGPKLFTYSLFMAEKREHPKGLPYFLGYKKGFFYFSTCVPEIAEALVNGLLMNPEVRLWDERFYLHEIKVLREPKKFNGSTFVTLSPIAVTVVRKGKSYDVPPMEKEFYSIIKDDLQDKYVMAYGDKPPSEFEMEVLIAKPKRFRIKPGIIYQTAWHLVFRAYGNDDLLKVGYEVGFGEKNSLGFGMVKVEGNKTTKEAEEQEKITFNSREELKTGV (Sequence 9)

[0427] MFVTQVIFNIGERTYPDRARAMVAELMDGVQPGLVATLMNYIPGTSTSRTEFPTVQFGGASDGFCLGFGDGGGAIVRDAVPLIHAALARRMPDRIIQVEHKEHSLSAEARPYVLSYTVPRMVVQKKQRHAERLLHEAEGKAHLEGLFLRSLQRQAAAVGLPLPENLEVEFKGAVGDFAAKHNPNSKVAYRGLRGAVFDVNARLGGIWTAGFMLSKGYGQFNATHQLSGAVNALSE(Sequence 10)

[0428] MHQTLIRINWPKGFKCPPAEFREKLAKSEMFPPEFFHYGTELAVWDKQTAEVEGKIKTVSKEKIIKTFDKPIPLNGRAPVRVIGGQAWAGVIADPEMEGMLIPHLGSILKVASSAAGCAVKIELEQRKFGISYTEYPVKYNLRELVLKRRCEDARSTDIESLIADRIWGGVSGESYYGIDGTCAKFGFEPPSREQLELRIFPMKNIGLHMKSSDGLSKEYMSLIDAEVWMNAKLEGVWQVGNLISRGYGRFIKSIGAQS (Sequence 11)

[0429] Sequence 12: GATAATCTCTTATAGAATTGAAAG

[0430] Sequence 13: CTAAAAGAATAACTTGCAAAATAACAAGCATTGAAAC

[0431] Sequence 14: GTCTCACCCTTCATGGGTGAGTGGATTGAAAT

[0432] Sequence 15: GTCGCACTCTTCATGGGTGCGTGGATTGAAAT

[0433] Sequence 16: GTTTCAGTCCCGTAGTCGGGATTTAGTGGTTGGAAAG

[0434] Sequence 17: GAGTTCCCCGCGCCAGCGGGGATAAACCG

[0435] Sequence 18: GTTCACTGCCGTATAGGCAGCTAAGAAA

[0436] Sequence 19: GTCGTCAGACCCAAAACCCCGAGAGGGGACGGAAAC

[0437] Sequence 20: GTTACAATAAGACTAAAATAGAATTGAAAG

[0438] Sequence 21: GTATTTCCCGCGTGCGCGGGGGTGAGCGG

[0439] Sequence 22: TATTGGATACACCCACTCATTGGTGGGTGGTTAGAAC

[0440] Sequence 23: GAUAAUCUCUUAUAGAAUUGAAAG

[0441] Sequence 24: CUAAAAGAAUAACUUGCAAAAUAACAAGCAUUGAAAC

[0442] Sequence 25: GUCUCACCCUUCAUGGGUGAGUGGAUUGAAAU

[0443] Sequence 26: GUCGCACUCUUCAUGGGUGCGUGGAUUGAAAU

[0444] Sequence 27: GUUUCAGUCCCGUAGUCGGGAUUUAGUGGUUGGAAAG

[0445] Sequence 28: GAGUUCCCCGCGCCAGCGGGGAUAAACCG

[0446] Sequence 29: GUUCACUGCCGUAUAGGCAGCUAAGAAA

[0447] Sequence 30: GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC

[0448] Sequence 31: GUUACAAUAAGACUAAAAUAGAAUUGAAAG

[0449] Sequence 32: GUAUUUCCCGCGUGCGCGGGGGUGAGCGG

[0450] Sequence 33: UAUUGGAUACACCCACUCAUUGGGGGGUGGUUAGAAC

[0451] In some embodiments, the heterologous functional domain fused to CasPR includes: nuclear localization signal (NLS), reporter protein or detection marker (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP), localization signal, protein targeting moiety, DNA binding domain (e.g., MBP, Lex A DBD, Gal4 DBD), epitope tag (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc.), transcription activation domain (e.g., VP64 or VPR), transcription repression domain (e.g., KRAB moiety or SID moiety), nuclease (e.g., FokI), deamination domain (e.g., ADAR1, ADAR2, APOBEC, AID, or TAD), methyltransferase, demethylase, transcription release factor, HDAC, polypeptide with ssRNA cleavage activity, polypeptide with dsRNA cleavage activity, polypeptide with ssDNA cleavage activity, polypeptide with dsDNA cleavage activity, DNA or RNA ligase, or any combination thereof. In some embodiments, the heterologous functional domain comprises an RNA base editor. In some embodiments, the RNA base editor edits an A→G single base change. In some embodiments, the RNA base editor edits a C→U single base change. In some embodiments, the RNA base editor comprises ADAR2DD or a derivative thereof. In some embodiments, the ADAR2DD derivative comprises the E488Q / T375A double mutation.

[0452] In some embodiments, the fusion protein has an amino acid sequence of any one of sequences 45-55.

[0453] MtCas6(IA):

[0454]

[0455] MmCas6(I-B):

[0456]

[0457] SpCas5d(I-C1):

[0458]

[0459] BhCas5d(I-C2):

[0460]

[0461] SaCas6(I-D):

[0462]

[0463] EcCas6e(IE):

[0464]

[0465] PaCas6f(I-F):

[0466]

[0467] MtCas6(III-A):

[0468]

[0469] PfCas6(III-B):

[0470]

[0471] PaCsf5(IV-A1):

[0472]

[0473] MtCsf5(IV-A2):

[0474]

[0475] In some embodiments, the heterologous functional domain is fused to the N-terminus, C-terminus, or interior of the fusion protein.

[0476] In some embodiments, functional variants of the CasPR fusion comprise a protein having the following amino acid sequences: (1) any one of sequences 1-11, except for substitutions, additions, or deletions of amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the protein retains the ability of one of sequences 1-11 to bind to a homologous repeat sequence (e.g., any one of sequences 12-33) of a type I, III, or IV CRISPR system; or (2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of sequence identity with any one of sequences 1-11, wherein the protein retains the ability of one of the CasPR sequences 1-11 to bind to a homologous repeat sequence (e.g., any one of sequences 12-33) of a type I, III, or IV CRISPR system; optionally, provided that the protein is not one of sequences 1-11. All sequences are incorporated herein by reference.

[0477] In some embodiments, the RNA sequence of the present invention further encodes a CasPR guide RNA comprising a guide sequence capable of hybridizing with the target RNA, and a direct repeat (DR) sequence of the guide sequence 3' (or 5'). In some embodiments, the DR sequence has a secondary structure substantially identical to that of any one of sequences 12-33. In some embodiments, the DR sequence is encoded by any one of sequences 12-33 or by the functional portion thereof binding to a homologous wild-type CasPR. In some embodiments, the target RNA is encoded by eukaryotic DNA. In some embodiments, the eukaryotic DNA is non-human mammalian DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, bird DNA, reptile DNA, rodent DNA, fish DNA, worm / nematode DNA, or yeast DNA. In some embodiments, the target RNA is mRNA.

[0478] In some embodiments, the CasPR guide sequence is between 15-120 nucleotides, between 20-100 nucleotides, between 25-80 nucleotides, between 15-55 nucleotides, between 25-35 nucleotides, or about 30 nucleotides.

[0479] In some embodiments, the CasPR guide sequence is 90%-100% complementary to the target RNA. In some embodiments, the CasPR guide RNA is generated by CasPR processing of a precrRNA transcript, and the precrRNA contains two or more guide RNAs, which have different guide sequences for different target RNAs.

[0480] In some embodiments, variants or derivatives of the CasPR fusion compound contain conserved amino acid substitutions of one or more residues of any one of sequences 1-11; optionally, variants or derivatives of the CasPR fusion compound contain only conserved amino acid substitutions. In some embodiments, derivatives of the CasPR fusion compound are capable of binding to the CasPR guide sequence hybridizing to the target RNA, but lack RNase catalytic activity due to a mutation in the RNase catalytic site of the CasPR. In some embodiments, derivatives of the CasPR fusion compound have an N-terminal deletion of no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues, and / or a C-terminal deletion of no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues.

[0481] In some implementations, the CasPR is Cas5d, Cas6, or Csf5, such as MtCas6(IA), MmCas6(IB), SpCas5d(I-C1), BhCas5d(I-C2), SaCas6(ID), EcCas6e(IE), PaCas6f(IF), MtCas6(III-A), PfCas6(III-B), PaCsf5(IV-A1), or MtCsf5(IV-A2).

[0482] In some embodiments, the heterologous functional domain of the CasPR fusion comprises an RNA base editing domain. In some embodiments, the RNA base editing domain comprises adenosine deaminase and / or cytidine deaminase, such as RNA-acting cytidine deaminases (CDARs), such as double-stranded RNA-specific adenosine deaminases (ADARs) (e.g., ADAR1 or ADAR2), apolipoprotein B mRNA editing enzymes, catalytic peptide-like enzymes (APOBECs, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4), activation-induced cytidine deaminase (AID), cytidine deaminase 1 (CDA1), or mutants thereof. In some embodiments, the ADAR comprises the E488Q / T375G double mutation or comprises ADAR2DD. In some embodiments, the base editing domain is further fused with an RNA-binding domain, such as MS2.

[0483] In some embodiments, the derivative of the CasPR fusion further comprises an RNA methyltransferase, an RNA demethylase, an RNA splicing modifier, a localization factor, or a translational modifier.

[0484] In some embodiments, the Cas, homolog, ortholog, paralog, variant, derivative, or functional fragment includes a nuclear localization signal (NLS) sequence or a nuclear output signal (NES).

[0485] In some embodiments, targeting the target RNA results in modification of the target RNA. In some embodiments, the target RNA modification is the cleavage of the target RNA. In some embodiments, the target RNA modification is the deamination of adenosine (A) to inosine (I), and / or the deamination of cytidine (C) to uracil (U).

[0486] In some embodiments, the RNA sequence of the present invention encodes a codon-optimized polynucleotide that encodes a wild-type CasPR (e.g., Cas5d, Cas6, or Csf5), its homologs, its orthologs, its paralogs, its variants or derivatives, or a functional fragment thereof, wherein the polynucleotide is codon-optimized for mammalian (e.g., human) expression, and optionally, the wild-type CasPR has an amino acid sequence of any one of sequences 1-11. In some embodiments, the codon-optimized polynucleotide has an amino acid sequence of any one of sequences 34-44. In some embodiments, the codon-optimized polynucleotide further comprises a sequence encoding a heterologous functional domain. In some embodiments, the heterologous functional domain comprises an RNA base editor.

[0487] MtCas6(IA):

[0488] ATGCCTCTGATCTTCAAGATCGGCTATAACGTGATCCCCCTGCAGGACGTGATCCTGCCCACCCCTTCCAGCAAGGTGCTGAAGTACCTGATCCAGAGCGGCAAGCTGATCCCCAGCCTGAAGGACCTGATCACCAGCCGGGACAAGTACAAGCCAATCTTCATCTCCCACCTGGGCTTCAACCAGCGGAGGATTTTCCAGACCAACGGCAATCTGAAAACCATCACCAAGGGCAGTAGACTGAGCTCCATCATCGCCTTCAGCACCCAGGCCAACGTGCTGTCCGAGGTGGCCGATGAAGGGATCTTCGAAACCGTGTACGGAAAGTTCCACATCATGATCGAAAGCATCGAGATCGTGGAGGTGGAAAAGCTGAAGGAGGAGGTGGAGAAGCACATGAACGACAACATCAGAGTGAGATTCGTGTCTCCCACACTGCTGAGCTCCAAGGTGCTGCTGCCCCCCAGCCTGTCCGAAAGATACAAGAAGATCCACGCCGGGTACAGCACCCTGCCCAGCGTGGGCCTGATCGTGGCCTACGCCTACAACGTGTACTGCAATCTGATCGGCAAGAAGGAAGTGGAAGTGCGGGCCTTCAAGTTTGGAATCCTGAGCAACGCCCTGTCCAGAATCATCGGCTACGACCTGCACCCTGTGACCGTGGCCATCGGCGAGGACAGCAAGGGGAATCTGAGAAAGGCTCGGGGCGTGATGGGCTGGATCGAGTTCGACATCCCCGACGAAAGACTGAAGCGGCGGGCCCTGAACTATCTGCTGACCAGCAGCTACCTGGGCATCGGGAGATCTCGGGGCATCGGCTTCGGCGAGATCCGGCTGGAGTTCCGGAAGATTGAAGAGAAGGAGGGA(Sequence 34)

[0489] MmCas6(I-B):

[0490] ATGGACCTGGAGTACATGCACATCTCCTACCCTAACATCCTGCTGAACATGCGGGACGGCAGCAAGCTGCGGGGCTACTTCGCCAAGAAGTACATCGACGAAGAGATTGTGCACAACCACAGAGACAACGCCTTTGTGTACAAGTACCCCCAGATCCAGTTTAAGATCATCGATAGAAGCCCCCTGATCATCGGCATTGGCTCTCTGGGCATCAATTTCCTGGAGAGCAAGCGGATCTTCTTCGAGAAGGAACTGATTATCAGCAACGACACCAACGACATCACCGAGGTGAACGTGCACAAGGACATGGATCACTTCGGCACGACCGACAAGATCCTGAAGTACCAGTTCAAGACCCCTTGGATGGCACTGAACGCCAAGAATAGCGAGATCTACAAGAACTCTGACGAGATCGACCGGGAGGAGTTCCTGAAGAGAGTGCTGATTGGGAATATCCTGAGCATGTCTAAGAGCCTGGGCTATACCATCGAAGAAAAGCTGAAGGTGAAGATTAACCTGAAGGAAGTGCCCGTGAAGTTCAAGAACCAGAACATGGTGGGCTTTCGGGGCGAGTTCTACATCAACTTCGACATCCCTCAGTATCTGGGCATCGGCCGGAATGTGTCCCGGGGATTCGGCACAGTGGTGAAGGTG(Sequence 35)

[0491] SpCas5d(I-C1):

[0492] ATGAGAAATGAAGTGCAGTTCGAGCTGTTCGGCGACTACGCCCTGTTCACCGACCCCCTGACCAAGATCGGCGGCGAAAAGCTGAGCTACAGCGTGCCTACCTACCAGGCCCTGAAGGGCATCGCCGAGAGCATCTACTGGAAGCCCACCATCGTGTTCGTGATCGACGAACTGCGGGTCATGAAGCCCATTCAGATGGAGTCTAAGGGCGTGAGGCCCATCGAGTACGGCGGCGGCAACACCCTGGCCCACTACACCTACCTGAAGGATGTGCACTACCAGGTGAAGGCCCACTTCGAGTTCAACCTGCACCGGCCCGACCTGGCCTTCGATAGAAACGAGGGCAAGCACTACTCCATCCTGCAGAGAAGCCTGAAGGCCGGCGGCAGAAGAGATATTTTCCTGGGCGCCCGGGAGTGCCAGGGCTACGTGGCCCCCTGCGAGTTCGGCAGCGGCGACGGCTTCTACGACGGCCAGGGCAAGTACCACCTGGGAACCATGGTGCACGGTTTCAACTACCCCGACGAAACCGGACAGCACCAGCTGGATGTGAGACTGTGGTCTGCCGTCATGGAAAACGGCTACATCCAGTTCCCCCGCCCTGAGGACTGCCCCATCGTGCGGCCTGTGAAGGAGATGGAACCCAAGATCTTCAACCCCGACAACGTGCAGTCCGCCGAACAGCTGCTGCACGACCTGGGCGGCGAA (Sequence 36)

[0493] BhCas5d(I-C2):

[0494] ATGTACAGAAGCCGGGACTTCTACGTGAGAGTGTCCGGCCAGCGGGCCCTGTTCACCAACCCCGCCACCAAGGGCGGCTCCGAACGGAGCTCCTACTCCGTGCCTACCCGGCAGGCCCTGAACGGGATTGTGGACGCCATCTACTACAAGCCCACGTTCACCAACATCGTGACCGAGGTGAAGGTGATTAACCAGATCCAGACCGAACTGCAGGGCGTGCGGGCCCTGCTGCATGACTACAGCGCCGACCTGAGCTACGTGTCCTACCTGAGCGACGTGGTGTACCTGATTAAGTTTCATTTCGTGTGGAACGAGGATAGAAAGGACCTGAATAGCGACCGGCTGCCAGCCAAGCATGAGGCCATCATGGAGCGGTCTATCCGGAAGGGCGGCAGACGGGACGTGTTCCTGGGCACCAGAGAATGCCTGGGCCTGCTGGACGACATCAGCCAGGAAGAATACGAAACCACAGTGAGCTATTACAATGGGGTGAACATCGACCTGGGCATCATGTTCCACAGCTTCGCTTACCCCAAGGACAAGAAAACCCCCCTGAAGTCCTACTTCACAAAGACCGTGATGAAGAACGGCGTGATCACCTTCAAGGCCCAGTCCGAATGCGATATTGTGAACACCCTGAGCTCCTACGCCTTCAAGGCCCCCGAGGAGATCAAGAGCGTGAACGACGAGTGCATGGAGTACGACGCCATGGAGAAGGGCGAAAAC (Sequence 37)

[0495] SaCas6(I-D):

[0496] ATGCCCAACGATCCCTACAGCCTGTACTCCATCGTGATCGAACTGGGCGCCGCCGAAAAGGGATTCCCCACAGGCATCCTGGGCAGAAGCCTGCATAGCCAGGTGCTGCAGTGGTTCAAGCAGGATAACCCCTTCCTGGCCACCGAGCTGCACCAGAGCCAGATCTCCCCCTTCTCCATCTCTCCACTGATGGGCAAGCGGCACGCCAAGCTGACCAAGGCCGGCGACCGGCTGTTCTTTCGGATCTGCCTGCTGAGAGGAGATCTGCTGCAGCCCCTGCTGAACGGCATTGAGCAGACCGTGAACCAGAGCGTGTGCCTGGACAAGTTCCGGTTCCGGCTGTGCCAGACCCACATCCTGCCCGGCAGCCACCCTCTGGCTGGCGCCTCCCACTATAGCCTGATCAGCCAGACCCCAGTGAGCTCCAAGATTACCCTGGACTTCAAGAGTTCTACCTCCTTCAAGGTGGACCGGAAGATCATCCAAGTGTTCCCTCTGGGCGAACACGTGTTCAACAGCCTGCTCAGACGCTGGAATAACTTCGCCCCCGAGGACCTGCACTTCTCTCAGGTGGACTGGAGCATCCCCATCGCCGCATTCGACGTGAAAACCATCCCCATCCACCTGAAGAAGGTCGAGATCGGCGCACAGGGCTGGGTGACCTACATCTTCCCCAACACAGAACAGGCCAAGATCGCCTCCGTGCTGAGCGAATTCGCCTTCTTCAGCGGAGTGGGACGGAAAACCACCATGGGCATGGGCCAGGTGCAGGTGCGGTCC (Sequence 38)

[0497] EcCas6e(I-E):

[0498] ATGTACCTGAGCAAGGTGATCATCGCCAGAGCCTGGAGCAGAGACCTGTACCAGCTGCACCAGGGCCTGTGGCACCTGTTCCCCAACCGGCCCGACGCCGCCCGGGATTTCCTGTTCCACGTGGAGAAGAGAAACACCCCGGAAGGCTGCCACGTGCTGCTGCAGAGCGCACAGATGCCTGTGAGCACCGCCGTGGCCACCGTGATCAAGACCAAGCAGGTGGAGTTCCAGCTGCAGGTGGGCGTGCCCCTGTATTTCAGGCTGCGGGCGAATCCCATCAAGACCATCCTGGACAACCAGAAGCGGCTGGACAGCAAGGGCAACATCAAGAGGTGCAGAGTGCCTCTGATCAAGGAGGCCGAACAGATCGCCTGGCTGCAGCGGAAGCTGGGCAATGCCGCCAGAGTGGAGGACGTGCACCCCATCAGCGAGCGGCCCCAGTACTTCTCCGGCGACGGAAAGAGCGGAAAGATCCAGACCGTGTGCTTCGAGGGCGTGCTGACCATCAACGACGCACCCGCCCTGATCGACCTCGTGCAGCAGGGGATCGGCCCTGCCAAGTCCATGGGCTGCGGACTGCTGTCCCTGGCCCCCCTG (Sequence 39)

[0499] PaCas6f(I-F):

[0500] ATGGACCACTACCTGGACATTAGACTGCGCCCTGACCCAGAGTTCCCTCCTGCCCAGCTGATGTCTGTGCTGTTTGGCAAGCTGCACCAGGCCCTGGTGGCCCAGGGCGGTGACAGAATCGGAGTGTCTTTCCCTGATCTGGACGAATCTAGATCTAGACTGGGAGAGAGACTGAGAATCCACGCGTCTGCCGACGACCTGAGAGCTCTGCTGGCCAGACCATGGCTGGAAGGACTGCGCGACCACCTGCAGTTCGGTGAACCTGCCGTGGTGCCTCACCCAACTCCATACAGACAGGTGAGTAGAGTGCAGGCAAAGTCTAATCCAGAGAGACTGAGACGCAGACTGATGAGAAGGCATGACCTGTCCGAAGAAGAAGCCAGAAAGAGAATCCCAGACACAGTGGCCAGAGCCCTGGATCTGCCTTTTGTGACCCTGAGAAGCCAGTCTACCGGCCAGCACTTCAGACTGTTTATTCGCCACGGACCACTGCAGGTGACCGCCGAAGAGGGAGGTTTTACCTGCTACGGACTGAGCAAGGGAGGTTTCGTGCCTTGGTTC (Sequence 40)

[0501] MtCas6(III-A):

[0502] ATGGCCGCCAGAAGAGGCGGAATCCGGAGAACCGACCTGCTGCGGAGGTCTGGCCAGCCTCGGGGCAGACACCGGGCCTCCGCCGCCGAGAGCGGCCTGACATGGATCTCCCCTACCCTGATCCTGGTGGGCTTCAGCCACAGGGGCGATAGGAGAATGACCGAGCACCTGTCCAGACTGACCCTGACCCTGGAAGTGGATGCCCCCCTGGAGAGAGCCCGGGTGGCCACCCTGGGCCCCCACCTGCATGGCGTGCTGATGGAGTCTATCCCCGCCGACTACGTGCAGACACTGCACACAGTGCCGGTGAACCCTTACAGCCAGTACGCTCTGGCCCGGAGCACCACCAGCCTGGAGTGGAAGATCTCCACCCTGACAAATGAGGCCCGGCAGCAGATCGTCGGCCCCATCAACGACGCCGCCTTCGCCGGCTTCCGGCTGCGGGCCAGCGGCATCGCCACCCAGGTGACAAGCAGAAGCCTGGAGCAGAACCCCCTGTCCCAGTTTGCCAGAATCTTCTACGCCAGGCCCGAAACCCGCAAGTTCAGAGTGGAGTTCCTGACCCCCACCGCCTTCAAGCAGAGCGGCGAGTACGTGTTTTGGCCCGATCCCAGACTGGTGTTCCAGTCCCTGGCCCAGAAGTACGGCGCCATCGTGGACGGAGAAGAGCCCGACCCCGGCCTGATCGCCGAGTTTGGCCAGTCCGTGAGACTGAGCGCCTTCAGAGTGGCCAGCGCCCCTTTTGCCGTGGGCGCCGCCAGGGTGCCCGGATTCACCGGCAGCGCCACCTTCACCGTGCGGGGAGTGGACACCTTCGCCAGCTACATCGCCGCTCTGCTGTGGTTCGGCGAGTTCAGCGGATGCGGCATCAAGGCCTCCATGGGAATGGGCGCCATCCGGGTGCAGCCTCTGGCCCCCCGGGAGAAGTGCGTGCCCAAGCCC(Sequence 41)

[0503] PfCas6(III-B):

[0504] ATGAGATTCCTGATCAGACTGGTGCCCGAGGACAAGGACAGAGCCTTCAAGGTGCCTTACAACCACCAGTACTATCTGCAGGGCCTGATCTACAACGCCATCAAGTCCTCCAACCCCAAGCTGGCCACCTACCTGCACGAGGTGAAGGGCCCCAAGCTGTTCACCTACAGCCTGTTCATGGCCGAAAAGCGGGAGCACCCTAAGGGCCTGCCCTACTTTCTGGGCTACAAGAAGGGCTTCTTCTACTTCAGCACCTGCGTGCCCGAGATCGCCGAGGCCCTGGTGAACGGCCTGCTGATGAATCCCGAGGTGCGGCTGTGGGACGAGAGATTCTACCTGCACGAAATCAAGGTCCTGCGGGAGCCCAAGAAGTTCAACGGCAGCACCTTCGTGACCCTGAGCCCCATCGCCGTGACCGTGGTGAGAAAGGGCAAGTCCTACGACGTGCCCCCCATGGAAAAGGAGTTCTACAGCATTATCAAGGATGACCTGCAGGACAAGTACGTGATGGCCTACGGCGACAAGCCCCCCAGTGAGTTCGAGATGGAAGTGCTGATCGCCAAGCCCAAGCGGTTCCGGATCAAGCCCGGCATCTATCAGACCGCCTGGCACCTGGTGTTTCGGGCCTACGGCAATGACGACCTGCTGAAGGTGGGCTACGAAGTGGGATTCGGGGAGAAGAACTCCCTGGGATTCGGAATGGTCAAGGTGGAGGGCAACAAGACCACCAAGGAAGCCGAAGAACAGGAGAAGATCACCTTCAACTCCCGGGAAGAGCTGAAAACAGGCGTG(Sequence 42)

[0505] PaCsf5(IV-A1):

[0506] ATGTTCGTGACCCAGGTGATCTTCAACATCGGCGAACGGACGTACCCCGACAGGGCTCGGGCTATGGTGGCCGAGCTGATGGATGGCGTCCAGCCTGGCCTGGTGGCCACCCTGATGAACTACATCCCCGGCACCAGCACGAGCCGGACAGAGTTCCCCACCGTGCAGTTCGGCGGCGCCAGCGACGGCTTTTGCCTGCTGGGCTTCGGCGACGGCGGCGGCGCCATCGTGAGAGATGCCGTGCCCCTGATCCACGCCGCCCTGGCAAGGCGGATGCCTGATCGGATCATCCAGGTGGAACACAAGGAGCACAGCCTGTCCGCCGAGGCCCGGCCCTACGTGCTGAGCTACACCGTGCCTCGGATGGTGGTGCAGAAGAAGCAGCGGCACGCCGAGAGACTGCTGCACGAAGCCGAGGGAAAGGCTCACCTGGAGGGCCTGTTCCTGCGGAGCCTGCAGAGGCAGGCCGCCGCCGTGGGCCTGCCCCTGCCCGAGAACCTGGAGGTGGAGTTCAAGGGAGCCGTGGGCGACTTCGCCGCAAAGCACAATCCAAATAGCAAGGTGGCCTACCGGGGACTGAGAGGCGCCGTGTTCGATGTGAACGCCAGACTGGGCGGCATCTGGACCGCCGGATTCATGCTGAGCAAGGGCTACGGCCAGTTTAACGCCACCCACCAGCTGAGCGGCGCCGTGAACGCTCTGTCCGAA (Sequence 43)

[0507] MtCsf5(IV-A2):

[0508] ATGCACCAGACCCTGATCCGGATCAACTGGCCCAAGGGATTCAAGTGCCCCCCTGCCGAGTTCCGGGAAAAGCTGGCCAAGAGCGAGATGTTCCCCCCCGAGTTCTTCCACTACGGCACGGAACTGGCCGTGTGGGACAAGCAGACCGCCGAGGTGGAGGGCAAGATCAAGACCGTGTCCAAGGAGAAGATCATCAAGACCTTTGACAAGCCCATCCCCCTGAATGGCCGGGCCCCGGTCAGAGTGATCGGCGGCCAGGCCTGGGCCGGCGTGATCGCCGACCCCGAGATGGAGGGCATGCTGATCCCACACCTGGGGAGCATCCTGAAGGTGGCCAGCAGCGCGGCCGGATGCGCAGTGAAGATCGAACTGGAACAGAGAAAGTTCGGCATCAGCTACACCGAGTACCCCGTGAAGTACAACCTGCGGGAGCTGGTGCTGAAGAGAAGATGCGAGGACGCCCGGTCTACCGATATCGAGAGCCTGATTGCCGATAGAATCTGGGGCGGCGTGTCCGGCGAGAGCTACTATGGCATCGACGGCACATGCGCCAAGTTTGGCTTCGAACCCCCCAGCAGAGAGCAGCTGGAGCTGCGGATCTTCCCCATGAAGAACATCGGACTGCACATGAAGTCCAGCGACGGACTGTCCAAGGAGTACATGAGCCTGATTGACGCCGAGGTGTGGATGAACGCTAAGCTGGAAGGAGTGTGGCAGGTGGGCAACCTGATCAGCAGGGGCTACGGCCGGTTCATCAAGTCTATCGGCGCCCAGTCC (Sequence 44)

[0509] In some embodiments, the RNA sequence of the present invention encodes a non-naturally occurring polynucleotide comprising a derivative of any one of sequences 12-33, wherein the derivative (i) has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotide additions, deletions, substitutions, and / or other mutations compared to any one of sequences 12-33; (ii) has at least 20%, 30%, 40%, 50%, 60%, or 70% of the nucleotides in the sequence compared to any one of sequences 12-33. (iii) Sequence identity of 90%, 80%, 90%, 95%, or 97%; or (iv) hybridization with any one of sequences 12-33 or any one of (i) and (ii) under stringent conditions; or (iv) being a complementary sequence to any one of (i)-(iii), provided that the derivative is not one of sequences 12-33 and that the derivative encodes RNA (or RNA) that retains substantially the same secondary structure (e.g., stem, loop, protrusion, single-stranded region) as any RNA encoded by sequences 12-33. In some embodiments, the derivative is used as a DR sequence for any one of the CasPR of the present invention, its orthologs, its paralogs, its variants, its derivatives, or functional fragments thereof.

[0510] In some embodiments, the RNA sequence of the present invention comprises the coding sequence of an engineered CRISPR-Cas13 effector enzyme, wherein the engineered Cas13: (1) contains a mutation in a region spatially close to the endonuclease catalytic domain of the corresponding wild-type Cas13 effector enzyme; (2) substantially retains the guide sequence-specific endonuclease cleavage activity of wild-type Cas13 for target RNAs complementary to the guide sequence; and (3) substantially lacks the guide sequence-independent appendage endonuclease cleavage activity of wild-type Cas13 for non-target RNAs that do not bind to the guide sequence.

[0511] In some implementations, Cas13 is Cas13a, Cas13b, Cas13c, Cas13d (including CasRx), Cas13e, or Cas13f.

[0512] In some embodiments, the Cas13e has the amino acid sequence of SEQ ID NO:4 in PCT / CN2020 / 119559 (incorporated herein by reference).

[0513] In some embodiments, the region includes any residue within 120, 110, 100, 90, or 80 amino acids of the endonuclease catalytic domain (e.g., the RXXXXH domain) in the primary sequence of Cas13.

[0514] In some embodiments, the region includes any residues more than 100, 110, 120, or 130 residues from any residue in the endonuclease catalytic domain of the primary sequence of Cas13, but spatially located within 1-10 or 5 angstroms of the residues of the endonuclease catalytic domain.

[0515] In some embodiments, the endonuclease catalytic domain is a HEPN domain, optionally comprising an HEPN domain of an RXXXXH motif. In some embodiments, the RXXXXH motif comprises an R{N / H / K}X1X2X3H sequence. In some embodiments, in the R{N / H / K}X1X2X3H sequence, X1 is R, S, D, E, Q, N, G, or Y; X2 is I, S, T, V, or L; and X3 is L, F, N, Y, V, I, S, D, E, or A. In some embodiments, the RXXXXH motif is an N-terminal RXXXXH motif comprising an RNXXXH sequence, such as an RN{Y / F}{F / Y}SH sequence. In some embodiments, the N-terminal RXXXXH motif has an RNYFSH sequence. In some embodiments, the N-terminal RXXXXH motif has an RNFYSH sequence. In some embodiments, the RXXXXH motif is a C-terminal RXXXXH motif containing the sequence R{N / A / R}{A / K / S / F}{A / L / F}{F / H / L}H. In some embodiments, the C-terminal RXXXXH motif has the RN(A / K)ALH sequence. In some embodiments, the C-terminal RXXXXH motif has the RAFFHH or RRAFFH sequence.

[0516] In some embodiments, the region comprises, is substantially composed of, or is composed of residues corresponding to, residues between 2-187, 227-242, or 634-755 of SEQ ID NO:4 of PCT / CN2020 / 119559 (incorporated by reference). In some embodiments, the region comprises, is substantially composed of, or is composed of residues corresponding to, residues between 35-51, 52-67, 156-171, 666-682, or 712-727 of SEQ ID NO:4 of PCT / CN2020 / 119559 (incorporated by reference).

[0517] In some embodiments, the mutation comprises, substantially comprises, or comprises the substitution of one or more charged or polar residues within a 15-20 consecutive amino acid sequence in the region for a charged short-chain aliphatic residue (such as A). In some embodiments, the sequence is about 16 or 17 residues. In some embodiments, substantially all charged and polar residues in the sequence are substituted, except for up to 1, 2, or 3. In some embodiments, a total of about 7, 8, 9, or 10 charged and polar residues in the sequence are substituted. In some embodiments, the N- and C-terminal two residues of the sequence are substituted for amino acids whose coding sequence contains a restriction enzyme recognition sequence. In some embodiments, the two N-terminal residues are VF, the two C-terminal residues are ED, and the restriction enzyme is BpiI. In some embodiments, the one or more charged or polar residues comprise N, Q, R, K, H, D, E, Y, S, and T residues. In some embodiments, the one or more charged or polar residues comprise R, K, H, N, Y, and / or Q residues. In some embodiments, one or more Y residues within the segment are substituted. In some embodiments, the one or more Y residues correspond to Y672, Y676, and / or Y751 of wild-type Cas13e.1 (SEQ ID NO:4 of PCT / CN2020 / 119559 (incorporated by reference)). In some embodiments, the segment is residues 35-51, 52-67, 156-171, 666-682, or 712-727 of SEQ ID NO:4 in PCT / CN2020 / 119559 (incorporated by reference). In some embodiments, the mutation comprises one or more Ala substitutions corresponding to any one or more of SEQ ID NO:37-39, 45, and 48 in PCT / CN2020 / 119559 (incorporated by reference). In some embodiments, the charge-neutral short-chain aliphatic residue is Ala(A). In some embodiments, the mutation comprises, consists essentially of, or consists of substitutions within 2, 3, 4, or 5 segments having 15-20 consecutive amino acids in the region.

[0518] In some implementations, the engineered Cas13 retains at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the guide sequence-specific endonuclease cleavage activity of wild-type Cas13 against the target RNA.

[0519] In some implementations, engineered Cas13 lacks at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the wild-type Cas13’s non-target RNA guide sequence-independent appendage endonuclease cleavage activity.

[0520] In some implementations, engineered Cas13 retains at least about 80%-90% of the guide sequence-specific endonuclease cleavage activity of wild-type Cas13 for target RNA, and lacks at least about 95%-100% of the guide sequence-independent incidental endonuclease cleavage activity of wild-type Cas13 for non-target RNA.

[0521] In some embodiments, the engineered Cas13 of the present invention has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.86% identity with any one of the amino acid sequences in SEQ ID NO: 16, 20, 24, 28, and 32 of PCT / CN2020 / 119559 (incorporated by reference), excluding any one or more regions defined in SEQ ID NO: 16, 20, 24, 28, and 32 of PCT / CN2020 / 119559 (incorporated by reference).

[0522] In some embodiments, the amino acid sequences contain up to 1, 2, 3, 4 or 5 differences in each of one or more regions defined in PCT / CN2020 / 119559 (incorporated by reference) compared to SEQ ID NO:17, 21, 25, 29 and 33.

[0523] In some embodiments, the engineered Cas13 of the present invention has the amino acid sequence of any one of SEQ ID NO: 6-10 in PCT / CN2020 / 119559 (incorporated by reference). In some embodiments, the engineered Cas13 of the present invention has the amino acid sequence of SEQ ID NO: 9 or 10 in PCT / CN2020 / 119559 (incorporated by reference).

[0524] In some embodiments, the engineered Cas13 of the present invention further includes a nuclear localization signal (NLS) sequence or a nuclear output signal (NES). In some embodiments, the engineered Cas13 includes N- and / or C-terminal NLS.

[0525] In some embodiments, the RNA sequence of the present invention encoding the engineered CRISPR / Cas13 effector enzyme of the present invention is codon-optimized for expression in eukaryotes, mammals (such as humans or non-human mammals), plants, insects, birds, reptiles, rodents (such as mice, rats), fish, worms / nematodes, or yeast.

[0526] In some embodiments, the RNA sequence of the present invention comprises the coding sequence of an engineered clustered regularly spaced short palindromic repeat (CRISPR)-Cas13 effector enzyme, said coding sequence (i) having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotide additions, deletions, substitutions, and / or other mutations compared to a wild-type sequence; (ii) having at least 50%, 60%, 70%, 80%, 90%, 95%, or 97% sequence identity with said wild-type sequence; (iii) hybridizing under stringent conditions with said wild-type sequence or any one of (i) and (ii); or (iv) being a complementary sequence to any one of (i)-(iii).

[0527] In some embodiments, the RNA sequence of the present invention comprises the following coding sequences: non-coding RNA (ncRNA), such as siRNA, piRNA, short hairpin RNA or shRNA, microRNA or miRNA or its precursors, including precursor miRNA and primary miRNA, antisense sequences or oligonucleotides (ASO), CRISPR / Cas guide RNA or gRNA, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR, etc.

[0528] 9. How to use

[0529] The rRAAV viral particles and RNA sequences of the present invention can be used to deliver any GOI / RSI to any suitable target cells, tissues or organisms for any gene therapy.

[0530] In some embodiments, the rRAAV viral particles and RNA sequences of the present invention can be used in therapeutic methods, wherein a functional version of a gene can replace a defective or loss-of-function disease gene to restore lost function. For example, in some embodiments, a wild-type coding sequence or a variant coding sequence encoding a wild-type protein and retaining at least one desired function of the wild-type protein can be delivered to target cells / tissues / organs to express the wild-type variant encoded therein to compensate for the lost function of the disease gene.

[0531] In some other embodiments, the rRAAV viral particles and RNA sequences of the present invention can be used in therapeutic methods in which defective or gain-of-function disease genes can be knocked out, knocked down, or otherwise downregulated by gene targeting agents to mitigate the adverse effects of said disease genes. The gene targeting agent may be a CRISPR / Cas effector enzyme (such as the engineered Cas9 or Cas13 effector enzymes described herein), which optionally has guide RNA provided simultaneously (or separately) that co-targets the disease gene. In some embodiments, the gene targeting agent may be a Cas effector enzyme linked to a DNA or RNA base editor for DNA-RNA base editing. In some embodiments, the gene targeting agent is siRNA, shRNA, microRNA, or antisense RNA.

[0532] In some embodiments, the present invention provides a method for modifying target RNA in target cells, the method comprising contacting the target cells with an rRAAV viral particle or RNA sequence of the present invention encoding the CasPR or engineered CRISPR / Cas effector enzyme (or its ortholog, paralog, variant, derivative or functional fragment thereof) described herein, wherein the guide sequence of the CasPR / Cas effector enzyme is complementary to at least 15 nucleotides of the target RNA, and wherein the CasPR / engineered Cas effector enzyme is associated with the guide sequence to form a complex that binds to and modifies the target RNA.

[0533] In some embodiments, the present invention provides a method for treating a condition or disease in a subject in need, the method comprising administering to the subject a composition comprising an rRAAV viral particle or RNA sequence of the present invention encoding the CasPR or engineered CRISPR / Cas effector enzyme (or an ortholog, paralog, variant, derivative, or functional fragment thereof) described herein, wherein a guide sequence of the CasPR / Cas effector enzyme is complementary to at least 15 nucleotides of the target RNA, and wherein the CasPR / engineered Cas effector enzyme is associated with the guide sequence to form a complex that binds to and modifies the target RNA, thereby treating the condition or disease in the subject.

[0534] In some embodiments, the target RNA is modified by cleavage with CasPR or an engineered Cas effector enzyme complex. In some embodiments, the target RNA is modified by deamination with a derivative comprising a double-stranded RNA-specific adenosine and / or cytidine deaminase. In some embodiments, the target RNA is mRNA, tRNA, rRNA, non-coding RNA, lncRNA, or nuclear RNA. In some embodiments, the target RNA is intracellular. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is infected with an infectious agent. In some embodiments, the infectious agent is a virus, prion, protozoan, fungus, or parasite. In some embodiments, the cell is a neuronal cell (e.g., astrocytes, glial cells (e.g., Muller glial cells, oligodendrocytes, ependymal cells, Schwann cells, NG2 cells, or satellite cells)).

[0535] In some embodiments, the condition or disease is cancer or an infectious disease. In some embodiments, the cancer is Wilms' tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or bladder cancer. In some embodiments, the method is an in vitro method, an in vivo method, or an ex vivo method. In some embodiments, after the complex binds to the target RNA, the engineered Cas13 does not exhibit significant (or detectable) associated RNase activity.

[0536] In some embodiments, the condition or disease is a neurological condition such as glaucoma, age-related loss of RGC neurons, optic nerve damage, retinal ischemia, Lieber's hereditary optic neuropathy, neurological conditions associated with RGC neuronal degeneration, neurological conditions associated with functional neuronal degeneration in the striatum of a subject in need, Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, depression, drug addiction, activity disorders such as chorea, choreoathetosis and movement disorders, bipolar disorder, autism spectrum disorder (ASD), or functional impairment.

[0537] In some embodiments, the methods of the present invention result in one or more of the following: (i) in vitro or in vivo induction of cell senescence; (ii) in vitro or in vivo cell cycle arrest; (iii) in vitro or in vivo cell growth inhibition and / or cell growth suppression; (iv) in vitro or in vivo induction of non-responsiveness; (v) in vitro or in vivo induction of apoptosis; and (vi) in vitro or in vivo induction of necrosis.

[0538] Another embodiment of the present invention

[0539] 1. A polynucleotide sequence, including but not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose conformation, α-LNA having an α-L-ribose conformation (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization) or hybrids thereof, capable of being packaged into DNA viral particles, said polynucleotide sequence comprising:

[0540] (1) The polynucleotide sequence of interest (PSI), for example, the RNA coding sequence of the gene of interest (GOI), RNA encoding a protein (e.g., a therapeutic protein, an antigenic protein, or a gene-editing protein such as a CRISPR / Cas effector enzyme (“Cas protein”), a ZFN protein, or a TALEN protein), such as mRNA, or a non-coding functional RNA (e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense oligonucleotide, microRNA (miRNA), or RNA components of the CRISPR-Cas (e.g., Cas9, Cas12, Cas13) system, including guide RNA (or gRNA), such as single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA), and tracrRNA), or a precursor thereof; and,

[0541] (2) A polynucleotide packaging signal (PPS) that can interact directly or indirectly with a PPS interacting molecule, for example, by binding, the PPS interacting molecule facilitating the packaging of the polynucleotide sequence into the DNA virus particle;

[0542] Optionally, the DNA sequence encoding or corresponding to the polynucleotide sequence, or the reverse complementary sequence of the DNA sequence, has reduced, weakened, or substantially no ability to be packaged into the DNA virus particle (e.g., the DNA sequence or its reverse complementary sequence lacks DNA packaging signals, such as a functional AAV ITR for AAV packaging).

[0543] 2. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the DNA virus particle is an AAV virus particle or an oncolytic virus particle.

[0544] 3. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the PPS is located at or near the 5' end of the PSI, at or near the 3' end of the PSI, or inside the PSI (e.g., within an intron of mRNA).

[0545] 4. The polynucleotide sequence as described in any of the foregoing embodiments, which contains more than one (e.g., 1, 2, 3, or more) identical or different PPS.

[0546] 5. A polynucleotide sequence as described in any of the foregoing embodiments, wherein two or more (e.g., three) of the more than one PPS are adjacent to or tandem with each other via the same or different linkers.

[0547] 6. A polynucleotide sequence as described in any of the foregoing embodiments, comprising two or more PPSs that are not adjacent to each other (e.g., each located at or near an end of the polynucleotide sequence of interest (PSI)).

[0548] 7. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the PPS comprises a transcribed modified AAV terminal inverted repeat (ITR), wherein the transcribed modified AAV ITR:

[0549] (a) Contains a functional Rep-binding element (RBE) for transcription, optionally further comprising a functional RBE' for transcription; and,

[0550] (b) Lack of a transcriptional terminal dissociation site (TRS) or a transcriptional reverse complementary TRS (rcTRS), or both;

[0551] Optionally, the transcribed modified AAV ITR further comprises a transcribed D region sequence (D sequence or D' sequence); and / or

[0552] Optionally, the PPS interacting molecules are Rep78, Rep68, Rep52, and / or Rep40.

[0553] 8. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the transcribed modified AAV ITR is located within 1000, 800, 500, 300, or 200 nucleotides from the 3' end of the RNA; optionally, the transcribed modified AAV ITR is located at the 5' end of a polyA sequence, a polyA signal sequence (e.g., AAUAAA), or an RNA transcription termination sequence (e.g., a histone downstream element).

[0554] 9. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the transcribed modified AAV ITR is based on a wild-type Flip or Flop ITR modified by transcription; optionally, the wild-type Flip or Flop ITR is derived from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 (optionally, the wild-type Flop ITR has the nucleotide sequence of SEQ ID NO:1).

[0555] 10. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the transcribed modified AAV ITR lacks both the transcribed TRS and the transcribed rcTRS.

[0556] 11. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the transcribed modified AAV ITR comprises the transcribed D region sequence (optionally, the modified AAV ITR has the nucleotide sequence of SEQ ID NO:3).

[0557] 12. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the transcribed modified AAV ITR lacks the D region sequence of the transcription (optionally, the modified AAV ITR has the nucleotide sequence of SEQ ID NO:2).

[0558] 13. The polynucleotide sequence as described in any of the foregoing embodiments, further comprising a modified AAV ITR of the second transcription having a functional RBE sequence of the second transcription but lacking the TRS or rcTRS of the second transcription or both; optionally, the modified AAV ITR of the second transcription further comprises a D region sequence of the second transcription.

[0559] 14. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the modified AAV ITR of the transcription and the modified AAV ITR of the second transcription are the same (or different).

[0560] 15. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the transcribed modified AAV ITR and the second transcribed modified AAV ITR (if present) comprise a deletion, a mutation, or an insertion in the corresponding transcribed wild-type AAV ITR D region sequence or the corresponding transcribed wild-type TRS / rcTRS.

[0561] 16. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the modified AAVITR of the second transcription is located within 1000, 800, 500, 250, or 150 nucleotides at the 5' end of the polynucleotide sequence.

[0562] 17. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the PPS comprises an MS2 sequence, a PP7 binding site, or a com binding site, and the PPS interacting molecule comprises a PPS interacting protein (PPSIP) capable of interacting directly or indirectly with the PPS, such as a phage-derived MS2 coat protein (MCP) for the MS2 sequence, a PP7 phage coat protein (PCP) for the PP7 binding site, or a phage COM protein (COM) for the com binding site.

[0563] 18. The polynucleotide sequence as described in any of the foregoing embodiments, wherein the PPSIP is directly or indirectly associated with (e.g., fused to) a protein component of the viral packaging system of the DNA virus viral particle (e.g., Rep78 and / or Rep68 of adeno-associated virus 2 (AAV2), or assembly activation protein (AAP)).

[0564] 19. A polynucleotide sequence as described in any of the foregoing embodiments, wherein the polynucleotide sequence includes or preferably does not include a transcribed DNA packaging signal, such as a transcribed wild-type AAV ITR sequence (e.g., the polynucleotide sequence includes a transcribed modified AAV ITR sequence having additions, deletions, and / or substitutions of nucleotides of the corresponding transcribed wild-type AAV ITR sequence to reduce the DNA packaging capacity of the DNA virus particles).

[0565] 20. The polynucleotide sequence as described in any of the foregoing embodiments, further comprising:

[0566] (1) Transcription enhancers;

[0567] (2) Transcriptional intron or exon sequences (e.g., intron or exon sequences transcribed to enhance protein expression);

[0568] (3) 5'UTR sequence;

[0569] (4) 3'UTR sequence;

[0570] (5) A polyA sequence, or a polyadenylated (polyA) signal sequence, and optionally a GU-rich region downstream of the polyA signal sequence;

[0571] (6) Posttranscriptional regulatory elements or sequences, such as sequences of posttranscriptional regulatory elements (WPREs) of marmot hepatitis virus (WHP); and / or,

[0572] (7) Transcription termination sequences (e.g., downstream histone elements),

[0573] Optionally, the polynucleotide sequence includes PPS located at the 3' of the posttranscriptional regulatory element or sequence and the 5' of the polyA sequence or polyA signal sequence.

[0574] 21. The polynucleotide sequence as described in any of the foregoing embodiments, comprising, in the 5' to 3' direction: the PSI, optionally a transcribed WPRE sequence; the PPS (e.g., the transcribed modified AAV ITR, the MS2 sequence, the PP7 binding site, or the com binding site); and the poly-A sequence or the poly-A signal sequence.

[0575] 22. A polynucleotide sequence as described in any of the foregoing embodiments, wherein the GOI comprises a protein (e.g., a fluorescent protein, a therapeutic protein, an antigenic protein, or a gene-editing protein, such as a Cas protein, a ZFN protein, or a TALEN protein), an enzyme (e.g., a Cre protein or a CRISPR / Cas effector enzyme, such as Cas9, Cas12, Cas13, or variants thereof), a structural protein, mRNA, non-coding RNA (ncRNA), siRNA, piRNA, short hairpin RNA or shRNA, microRNA (miRNA) or its precursor (including precursor miRNA and primary miRNA), ribosomal RNA (rRNA), an antisense sequence or oligonucleotide (ASO), an RNA component of the CRISPR-Cas system, including a guide RNA (or gRNA), such as a single guide RNA (or sgRNA, chimeric RNA, RNA chimera), CRISPR RNA (crRNA) and tracr RNA, a guide RNA or gRNA, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, lncRNA, Xist, and HOTAIR for CRISPR / Cas effector enzymes.

[0576] 23. The polynucleotide sequence as described in any of the foregoing embodiments is a single-stranded RNA of length less than about 8,900 nucleotides, less than about 8,000 nucleotides, less than about 7,000 nucleotides, less than about 6,000 nucleotides, less than about 5,200 nucleotides, less than about 4,000 nucleotides, less than about 3,000 nucleotides, less than about 2,000 nucleotides, about 4,700-5,200 nucleotides, about 4,700-5,000 nucleotides, about 4,700-4,800 nucleotides, or about 4,700 nucleotides.

[0577] 24. A polynucleotide comprising a cassette encoding a polynucleotide sequence as described in any of the foregoing embodiments; optionally, the polynucleotide is a DNA sequence (e.g., a DNA plasmid) which optionally includes a filler sequence in the backbone of the DNA plasmid and / or optionally does not contain a functional DNA packaging signal, such as AAV ITR.

[0578] 25. The polynucleotide as described in any of the foregoing embodiments, further comprising a promoter operatively linked to and driving the transcription of the polynucleotide sequence encoded by the cassette.

[0579] 26. The polynucleotide as described in any of the foregoing embodiments, wherein the promoter is a pervasive promoter.

[0580] 27. The polynucleotide as described in any of the foregoing embodiments, wherein the promoter is a tissue-specific promoter.

[0581] 28. The polynucleotide as described in any of the foregoing embodiments, wherein the promoter is a constitutive promoter.

[0582] 29. The polynucleotide as described in any of the foregoing embodiments, wherein the promoter is an inducible promoter.

[0583] 30. The polynucleotide as described in any of the foregoing embodiments, further comprising an enhancer that enhances transcription of the polynucleotide sequence driven by the promoter.

[0584] 31. A recombinant DNA virus particle comprising a polynucleotide genome (e.g., a polynucleotide sequence as described in any of the foregoing embodiments or a polynucleotide sequence transcribed from a polynucleotide as described in any of the foregoing embodiments) packaged in a protein shell (e.g., capsid) of a DNA virus (e.g., AAV virus or oncolytic virus).

[0585] 32. The recombinant DNA virus particle as described in any of the foregoing embodiments, wherein the DNA virus is AAV, and the recombinant DNA virus particle is a recombinant polynucleotide adeno-associated virus (rPAAV) particle, the recombinant polynucleotide adeno-associated virus particle comprising:

[0586] (1) AAV capsid; and,

[0587] (2) A polynucleotide sequence as described in any of the preceding embodiments, or a polynucleotide sequence transcribed from a polynucleotide as described in any of the preceding embodiments, packaged within the AAV capsid.

[0588] 33. The recombinant DNA virus particle as described in any of the foregoing embodiments, wherein the AAV capsid comprises a capsid from serotypes AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, AAVrh74, or 7m8.

[0589] 34. A population of recombinant DNA virus particles (e.g., rPAAV particles), said population comprising a plurality of recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the recombinant DNA virus particles (e.g., rPAAV particles) within said population have a polynucleotide sequence packaged therein as described in any of the foregoing embodiments or a polynucleotide sequence transcribed from a polynucleotide as described in any of the foregoing embodiments.

[0590] 35. A host cell comprising a population of polynucleotide sequences as described in any of the foregoing embodiments, polynucleotides as described in any of the foregoing embodiments, polynucleotide sequences transcribed from polynucleotides as described in any of the foregoing embodiments, recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, and / or recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments.

[0591] 36. The host cell as described in any of the foregoing embodiments, further comprising a viral packaging system that facilitates the packaging of a polynucleotide sequence as described in any of the foregoing embodiments or a polynucleotide sequence transcribed from a polynucleotide as described in any of the foregoing embodiments into the DNA virus particles.

[0592] 37. The host cell as described in any of the foregoing embodiments, wherein the viral packaging system comprises:

[0593] (1) The AAV rep gene (e.g., coding sequences of Rep78, Rep68, Rep52, and / or Rep40) and the AAV cap gene (e.g., coding sequences of VP1, VP2, VP3, AAP, and / or MAAP) or their expression products, wherein the AAV rep and AAV cap genes are under the transcriptional control of one or more promoters that drive the transcription of the rep and cap genes;

[0594] (2) One or more coding sequences of one or more proteins required for AAV packaging, such as the adenovirus E2A, E4, and VA genes, or said one or more proteins; and

[0595] (3) The PPS interacting molecule or its coding sequence;

[0596] Optionally, the ability of the viral packaging system to package a DNA sequence into the DNA virus particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, on a polynucleotide encoding a polynucleotide sequence as described in any of the foregoing embodiments or on a polynucleotide as described in any of the foregoing embodiments; (2) modifying, for example, by mutating the coding sequences of one or more proteins required for AAV packaging, such as the AAV rep gene, the AAV cap gene, and / or AAV packaging proteins, to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) enlarging the size of a polynucleotide encoding a polynucleotide sequence as described in any of the foregoing embodiments or a polynucleotide as described in any of the foregoing embodiments.

[0597] 38. The host cell as described in any of the foregoing embodiments is a mammalian cell (such as HEK293 cell) or an insect cell (such as Sf9 or Sf21 cell).

[0598] 39. A method for generating a population of recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, the method comprising:

[0599] a) Culturing the host cells as described in any of the foregoing embodiments for a sufficient period of time, and

[0600] b) Harvest the recombinant DNA virus particles or a population of the recombinant DNA virus particles.

[0601] 40. The method of any of the foregoing embodiments further includes isolating or purifying the recombinant DNA virus particles or a population of the recombinant DNA virus particles.

[0602] 41. A method for generating recombinant DNA virus particles (e.g., rPAAV particles) or a population of recombinant DNA virus particles, the method comprising:

[0603] a) The time period during which the viral packaging system (e.g., an AAV packaging system) is contacted with a polynucleotide sequence as described in any of the foregoing embodiments, or a polynucleotide sequence transcribed from a polynucleotide as described in any of the foregoing embodiments, sufficient to produce the recombinant DNA virus particles or a population of the recombinant DNA virus particles, and

[0604] b) Harvesting the recombinant DNA virus particles or a population of the recombinant DNA virus particles; and optionally,

[0605] c) Isolate or purify the harvested recombinant DNA virus particles or populations of recombinant DNA virus particles.

[0606] 42. The method of any of the foregoing embodiments, wherein the virus packaging system (e.g., an AAV packaging system) comprises:

[0607] (1) One or more proteins (e.g., VP1, VP2, and / or VP3 for assembling the protein shell of the DNA virus viral particles for packaging the polynucleotide sequence) or one or more coding sequences thereof;

[0608] (2) One or more proteins (e.g., Rep78, Rep68, Rep52, and / or Rep40 for AAV packaging) or one or more coding sequences thereof (e.g., adenovirus E2a, E4, and VA genes) for facilitating the assembly of the protein shell and / or the packaging of the polynucleotide sequence into the protein shell of the DNA virus particle; and

[0609] (3) The PPS interacting molecule or its coding sequence;

[0610] Optionally, the ability of the viral packaging system to package a DNA sequence into the DNA virus particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, on a polynucleotide encoding a polynucleotide sequence as described in any of the foregoing embodiments or on a polynucleotide as described in any of the foregoing embodiments; (2) modifying, for example, by mutating the coding sequences of one or more proteins required for AAV packaging, such as the AAV rep gene, the AAV cap gene, and / or AAV packaging proteins, to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) enlarging the size of a polynucleotide encoding a polynucleotide sequence as described in any of the foregoing embodiments or a polynucleotide as described in any of the foregoing embodiments.

[0611] 43. A system for packaging a polynucleotide sequence as described in any of the foregoing embodiments, or a polynucleotide sequence transcribed from a polynucleotide as described in any of the foregoing embodiments, into a DNA viral particle, the system comprising:

[0612] (1) One or more proteins (e.g., VP1, VP2, and / or VP3 for assembling the protein shell of the DNA virus viral particles for packaging the polynucleotide sequence) or one or more coding sequences thereof;

[0613] (2) One or more proteins (e.g., Rep78, Rep68, Rep52, and / or Rep40 for AAV packaging) or one or more coding sequences thereof (e.g., adenovirus E2a, E4, and VA genes) for facilitating the assembly of the protein shell and / or the packaging of the polynucleotide sequence into the protein shell of the DNA virus particle; and

[0614] (3) The PPS interacting molecule or its coding sequence;

[0615] Optionally, the ability of the viral packaging system to package a DNA sequence into the DNA virus particle is reduced, weakened, or substantially eliminated by, for example, by: (1) removing part or all of the DNA packaging signal, such as AAV ITR, on a polynucleotide encoding a polynucleotide sequence as described in any of the foregoing embodiments or on a polynucleotide as described in any of the foregoing embodiments; (2) modifying, for example, by mutating the coding sequences of one or more proteins required for AAV packaging, such as the AAV rep gene, the AAV cap gene, and / or AAV packaging proteins, to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA sequence into the DNA virus particle (e.g., the Y156F mutation, KDE-mu, or EKE-mu in the common sequence of the Rep78 and Rep68 proteins); and / or (3) enlarging the size of a polynucleotide encoding a polynucleotide sequence as described in any of the foregoing embodiments or a polynucleotide as described in any of the foregoing embodiments.

[0616] 44. A method for delivering a gene of interest (GOI) to a cell, plant, or animal, the method comprising contacting the cell, plant, or animal with recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, a population of recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, or recombinant DNA virus particles (e.g., rPAAV particles) or a population of recombinant DNA virus particles (e.g., rPAAV particles) generated by the method described in any of the foregoing embodiments, wherein the GOI is encoded by the polynucleotide sequence (as described in any of the foregoing embodiments).

[0617] 45. A method for delivering a polynucleotide sequence of interest (PSI) to a cell, plant, or animal, the method comprising contacting the cell, plant, or animal with recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, a population of recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, or recombinant DNA virus particles (e.g., rPAAV particles) or a population of recombinant DNA virus particles (e.g., rPAAV particles) generated by the method described in any of the foregoing embodiments.

[0618] 46. ​​A method for diagnosing, preventing, or treating a disease or disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount or dose of a population of recombinant DNA viral particles (e.g., rPAAV particles) as described in any of the foregoing embodiments or generated by the method described in any of the foregoing embodiments.

[0619] 47. Use of recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, groups of recombinant DNA virus particles (e.g., rPAAV particles) as described in any of the foregoing embodiments, or recombinant DNA virus particles (e.g., rPAAV particles) or groups of recombinant DNA virus particles (e.g., rPAAV particles) produced by the method described in any of the foregoing embodiments in the manufacture of a medicament for the diagnosis, prevention, or treatment of a disease or disorder in a subject in need.

[0620] Example

[0621] The embodiments provided below are used to illustrate several exemplary implementations of the present invention and are not limiting in any way.

[0622] Example 1: Efficient packaging of RNA into RAAV viral particles

[0623] This example demonstrates that RNA vector genomes can be efficiently packaged into AAV viral capsids, especially using modified / recombinant RNAs designed for direct packaging into AAV capsids.

[0624] First, it was unexpectedly shown that, during transcription (as RNA), the AAV packaging signal -ITR (DNA) can facilitate the packaging of the RNA sequence of the present invention (e.g., rRAAV vector genomic RNA) into AAV particles, especially when it is present in certain conformations (e.g., when the transcribed modified AAV ITR sequence is close to the 3' end of the transcribed RNA sequence of the present invention).

[0625] Specifically, wild-type and modified AAV ITR sequences (DNA) from the ends of the AAV vector genome were moved into their respective transgenic expression cassettes to ensure that all transgenic transcripts (RNA) contained candidate packaging signals. To block the production of conventional AAV vectors with ssDNA genomes during RAAV production, optimized ITRs (dITR and dITR-D) were used instead of wild-type ITRs (Table 2).

[0626] Table 2: Nucleic acid sequences of the tested ITRs

[0627]

[0628]

[0629] Specifically, in the wild-type AAV2 ITR (ITR2) sequence in the Flop conformation, the TRS (“TTGGC”) is located at the 5' end of the ITR, and its inverse complementary sequence GCCAA is double-underlined. This sequence can be cloned into a coding plasmid in either orientation (i.e., the sequence shown in SEQ ID NO:1 or its inverse complementary sequence can be used as a template to transcribe the RNA sequence of the present invention). In the experiments described herein, the wild-type AAV2 ITR sequence was cloned in such an orientation that the transcribed RNA had the same sequence as SEQ ID NO:1 (or SEQ ID NO:2 or 3 below), except that the Ts in the transcribed RNA were replaced by Us. In any case, after transcribing this sequence or its inverse transcript, the resulting transcribed RNA of wild-type ITR2 contained a palindromic transcribed RBE (grey shaded). In the experiments described herein, the transcribed RNA contained a transcribed wild-type AAV2 ITR equivalent to SEQ ID NO:1, except that all Ts were replaced by Us. If the reverse complementary sequence of SEQ ID NO:1 is used as a DNA template, the transcribed RNA will contain a transcribed TRS (UUGGC) encoded by GCCAA. The transcribed TRS is located between the transcribed RBE and the transcribed D sequence.

[0630] One of the modified ITR sequences is the “δITR” (or simply “dITR”), which is defective because, except for the first G, the dITR lacks the D region sequence (bold italic), the TRS at the 5' end, and the inverse complementary TRS sequence (“GCCAA”). When this sequence is transcribed, the transcribed RNA of the dITR also contains a palindromic transcribed RBE (grey shading) and a defective transcribed ITR lacking the transcribed TRS (UUGGC) encoded by GCCAA. However, in this experiment, the inverse complementary sequence of SEQ ID NO:2 was used as a DNA template, resulting in transcribed RNA containing a modified AAV2 ITR (transcribed dITR) with the same sequence as SEQ ID NO:2, except that all Ts were replaced by Us.

[0631] Another modified ITR sequence is “dITR-D”, which is also defective because it retains its D sequence (“CTCCATCACTAGGGGTTCCT”, SEQ ID NO:4) but lacks the 5' terminal TRS (TTGGC). Furthermore, only the first G in the inverse complementary TRS (GCCAA) is retained in dITR-D, and the remaining CCAA sequence is replaced by the unrelated ACTAG sequence. In this experiment, the inverse complementary sequence of SEQ ID NO:3 was used as a DNA template, such that the transcribed RNA contained a transcribed modified AAV2 ITR (transcribed dITR) with the same sequence as SEQ ID NO:3, except that all Ts were replaced by Us.

[0632] Note that both the dITR and dITR-D sequences retain the shaded palindromic RBE sequence SEQ ID NO:5 (CTGCGCGCTCGCTCGCTCACTG...CAGTGAGCGAGCGAGCGCGCAG), and their corresponding transcribed modified ITRs also have RBE sequences.

[0633] This optimized ITR coding sequence (DNA) was inserted into two locations in the tdTomato expression cassette: one between the promoter and the tdTomato coding sequence, and the other between the marmot hepatitis virus (WHP) posttranscriptional regulatory element (WPRE) and the SV40 polyA signal.

[0634] Based on the optimized ITR sequence, number, and position used, a series of different ITR-based RAAV vectors were constructed (see [link to documentation]). Figure 3 ).

[0635] A conventional AAV vector with an ssDNA vector genome but lacking ITR sequences at both ends (“CTWS”, representing the sequence elements CAG promoter, tdTomato transgene, WPRE sequence, and SV40 polyA signal sequence) was used as a control. For this experiment, the AAV serotype DJ was chosen because of its excellent transduction efficiency in the cultured cells used. AAV-DJ is a synthetic serotype with a chimeric capsid of AAV-2, 8, and 9. Its capsid contains a heparin-binding domain, which allows for efficient transduction of multiple cell types and evasion of immune neutralization (Grimm et al., J. Virol. [Journal of Virology] 82:5887-5911, 2008).

[0636] All RAAV-ITR viral particles and control viral particles were generated using a three-plasmid transfection system. Figure 4 ).

[0637] Specifically, the RAAV vector was generated by co-transfecting HEK293T cells with a transgenic plasmid, a packaging plasmid, and a helper plasmid (weight ratio 1:1:2). HEK293T cells were cultured in competent DMEM medium and seeded 24 hours prior to transfection. Before transfection, the medium was replaced with fresh DMEM containing 2% FBS. PEI-MAX was used as the transfection reagent. The supernatant was collected on days 2 and 5 post-transfection, and the transfected cells were harvested on day 5. The RAAV vector was purified by density gradient ultracentrifugation using iodixanol.

[0638] Viral titers (DNA titer and RNA titer) were measured using Q-PCR and reverse transcription-PCR (RT-PCT), respectively. Figure 5A The procedure is determined within.

[0639] In summary, the harvested and purified RAAV virus particles were first treated with DNase I and RNase I at 37°C for 2 hours to remove all nucleic acids outside the viral particle protein coat. Next, the nucleases were denatured at 100°C for approximately 30 minutes, and then the RAAV virus particles were denatured and ruptured to release the RAAV nucleic acid contents for further analysis.

[0640] Q-PCR was used to analyze nuclease resistance products to titrate the DNA vector genome capsized within RAAV viral particles. Specifically, one set of Q-PCRs used promoter sequence-specific primer pairs to detect / quantify any functional DNA, and another set of Q-PCRs used WPRE sequence-specific primer pairs to detect / quantify any DNA vector genome capsized within RAAV viral particles. See also Figure 5B .

[0641] Simultaneously, in another sample, any RAAV-capped DNA was first removed by DNase I digestion, then the remaining RNA was reverse transcribed, and the resulting cDNA was used as a Q-PCR template for detecting / quantifying the WPRE sequence transcribed into RNA. To detect / quantify any residual DNA that may remain after incomplete DNA removal, the sample after the DNA removal step was directly amplified using Q-PCR to detect any WPRE (DNA) sequences that may be present in the sample. See also Figure 5A .

[0642] To test the packaging efficiency of the CITWS build (see...) Figure 6A When using a conventional pssDNA construct (with wild-type ITR sequences at both ends) to generate viral particles, the vast majority of viral particles contain a functional DNA vector genome with promoter and WPRE sequences. Very occasionally (by two orders of magnitude, or about 1% of the time), an RNA vector genome is also packaged into the viral particle (see the bar labeled "RNA," which is about two orders of magnitude lower than the bars labeled "DNA" and "functional DNA"). The residual DNA is one order of magnitude lower than the packaged RNA vector genome.

[0643] Meanwhile, removing ITR sequences from both ends of the AAV vector genome essentially eliminates packaging. Figure 6A The CTWS construct (without ITR) produces packaging DNA that is 2-2.5 orders of magnitude lower, or even less RNA.

[0644] Compared to the CTWS control, adding only one optimized ITR sequence (dITR or dITR-D sequence) between the 3' of the promoter and the 5' of the GOI coding sequence did not appear to enhance RNA packaging, although DNA packaging appeared to be slightly improved. See also Figure 6A .

[0645] Interestingly, in Figure 6B Very different results were obtained in the tests of the CTWIS construct. Specifically, regarding the packaging pssDNA construct (compared to...). Figure 6A and Figure 6B The same results were obtained – most packaged viral particles contained DNA (99% or more) and a negligible amount (1% or less) of RNA. However, the addition of an optimized ITR sequence (dITR) between the 3' end of the WPRE sequence and the 5' end of the poly-A sequence significantly reduced or even reversed the difference in packaging efficiency between DNA and RNA. This effect was even more pronounced when using the dITR-D sequence, where the vast majority of packaged nucleic acids were RNA (1-2 orders of magnitude higher than packaged DNA).

[0646] If an additional (i.e., a second) optimized ITR sequence is inserted between the promoter and the GOI encoding sequence in the CITWIS construct, essentially the same result is obtained. See also Figure 6C .

[0647] These results indicate that optimized ITRs (dITR and dITR-D) impair the replication of conventional AAV vectors, resulting in a reduction in the DNA packaged into RAAV viral particles.

[0648] Compared to the control vector CTWS (without ITR) and the RAAV-dITR vector, the RAAV vector with the dITR-D optimized ITR appears to have a better ability to directly capsidate transcribed mRNA into RAAV particles, especially when the dITR-D ITR is located downstream of the mRNA coding sequence and the WPRE sequence (e.g., just at the 5' of the poly-A signal). See also Figures 6A-6C In contrast, dITR-D sequences located upstream of the mRNA coding sequence (e.g., just after the promoter sequence in the expression construct) hardly promote direct packaging of the mRNA into RAAV viral particles. Meanwhile, inserting another dITR-D sequence downstream of the mRNA coding sequence (e.g., just at the 5' of the polyA sequence) similarly significantly increases the packaging of the resulting RAAV mRNA. Figure 6C ).

[0649] In summary, CITWIS-D, carrying dITR-D signals at both ends of its mRNA genome, exhibits the best ability to capsidate-specific mRNAs, although its yield (mRNA-carrying particles) is 20-fold lower than that of conventional AAV vectors with ssDNA vector genomes (pssAAV group). Unlike conventional AAV vectors, the RAAV vector CITWIS-D has compromised DNA packaging, with DNA-carrying particles comprising only about 20% or less of the RAAV vector stock, and the percentage of particles carrying functional DNA is even lower (e.g., less than 10%). Figures 6A-6C ).

[0650] Since AAV packaging capacity is limited (<4700nt), unwanted AAV DNA packaging can be reduced by increasing the size of the transgenic plasmid, and functional DNA packaging can be further reduced by increasing the length of the transgenic cassette, for example by inserting cis-acting elements (such as enhancers, introns, etc.) or non-functional filler sequences into the cassette.

[0651] Example 2: RAAV virus particles have functionality

[0652] This example demonstrates that the tested RAAV-dITR-D vector is infectious and can be used as a gene delivery vector.

[0653] In vitro, 2 × 10⁶ cells were infected with the same volume of purified RAAV-dITR-D (CITWIS-D) vector at the same MOI of approximately 50,000 (the MOI of the CITWIS-D vector is calculated as the sum of the number of DNA particles and the number of mRNA particles). 5 One HEK293T cell.

[0654] Specifically, HEK293T cells were seeded into 24-well plates approximately 24 hours before infection. The RAAV vector was then thoroughly mixed with 1 mL of LDMMEM (containing 2% FBS). The cell culture medium was then removed, and the cells were incubated overnight with the mixed RAAV vector. Fluorescence images were taken 3 and 5 days post-infection.

[0655] The results showed that the CITWIS-D vector expressed tdTomato faster than other vectors, but the expression was also rapidly downregulated (see [link to study]). Figure 7B This rapid expression and degradation phenomenon may be due to the short lifespan of its mRNA genome. Figure 7B ).

[0656] Interestingly, the CTWS construct, lacking any ITR sequence, is clearly packaged to some extent, although the exact mechanism behind this packaging remains unclear. When using the CTWS vector, at least two possibilities could explain the packaging of the mRNA vector genome: overexpressed cellular mRNA may be non-specifically packaged into the RAAV vector, or the CTWS mRNA may possess some RNA structure that interacts with Rep2 or Cap-DJ. Meanwhile, CTWS DNA packaging may be due to the small size of the CTWS plasmid, and increasing the size of the CTWS plasmid might reduce DNA packaging.

[0657] Example 3: Efficient packaging of RNA into RAAV particles

[0658] This example demonstrates that RNA genomes can be efficiently packaged into AAV capsids, particularly using modified / recombinant RNA designed in this paper for direct packaging into AAV capsids to produce RAAV particles.

[0659] 1. Design

[0660] The inventors have devised a strategy that utilizes the strong interaction between the bacterial phage-derived MS2 coat protein (MCP) and its recognition stem-loop MS2 as a novel packaging signal for packaging heterologous RNA into DNA viral particles.

[0661] First, to suppress / reduce the production of conventional AAV particles with packaged ssDNA genomes during RAAV production, the conventional AAV packaging signal—ITR—is removed. Instead, one or three copies of the RNA packaging signal (RPS), namely the MS2 stem-loop (or simply "MS2", whose RNA sequence is listed in the sequence listing below), are inserted into the tdTomato expression cassette, located between the marmot hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) and the SV40 polyA signal, to ensure that all transcribed mRNAs possess the RPS and are thus recognized by the binding protein, namely the bacterial phage-derived MS2 coat protein (or simply "MCP", whose amino acid sequence is shown in the table below) (corresponding to MS2). Figure 8A ).

[0662] Since AAV Rep proteins are non-structural proteins and typically act as a bridge between the ITR of the ssDNA genome and the AAV capsid during AAV packaging, MCP was fused to the N-terminus of both Rep78 and Rep68 proteins from AAV2 (Rep68 is a C-terminal truncated version of Rep78; the amino acid sequences of the two fusions are listed in the sequence listing below). The interaction of these MCP-Rep78 / 68 fusions with the MS2 sequence within the RNA genome and their ability to facilitate the packaging of the RNA genome into the AAV capsid were verified.

[0663] The conventional AAV vector pssAAV-tdTomato (with two wild-type functional ITRs) and CTWS (“CTWS”, representing the sequence elements CAG promoter, tdTomato transgene, WPRE sequence, and SV40 polyA signal sequence) without functional wild-type ITRs were used as controls. Figure 8A In this embodiment, the AAV serotype DJ (“AAV-DJ” or “DJ”) was chosen to be used because it exhibits excellent transduction efficiency in the HEK293T cells used in this embodiment. AAV-DJ is a synthetic serotype with a chimeric capsid of AAV-2, 8, and 9.

[0664] 2. RAAV Packaging and Production

[0665] This study used a traditional three-plasmid transfection system with necessary modifications, co-transfecting the corresponding transgenic plasmid, packaging plasmid, and helper plasmid into HEK293T cells at a mass ratio of 1:1:2, producing RAAV and control AAV particles.

[0666] Specifically, HEK293T cells were cultured in competent DMEM medium and seeded 24 hours prior to transfection. Shortly before transfection, the medium was replaced with fresh DMEM containing 2% FBS. PEI-MAX was used as the transfection reagent. After transfection into infected cells, the transgenic plasmid carrying RPS was transcribed to generate the RNA genome to be packaged. The supernatant was then collected on days 2 and 5 post-transfection, and the transfected cells were harvested on day 5. RAAV and control AAV particles were purified by density gradient ultracentrifugation using iodixanol.

[0667] 3. Detection of the genome in the packaging

[0668] The purified RAAV and control AAV particles were first treated with nucleases (including DNase I and RNase I) at 37°C for 2 hours to remove any nucleic acids that might be present outside the viral particles. Next, the nucleases and RAAV or control AAV particles were digested with proteinase K / SDS and denatured at 65°C for approximately 3 hours to rupture the viral particles and release the genome packaged therein. Then, nuclease-resistant polynucleotides containing the released viral genome were extracted and purified by phenol / chloroform extraction.

[0669] To detect the DNA genomic titer of control AAV and RAAV particles, qPCR was used to directly analyze nuclease-resistant polynucleotides. A pair of WPRE primers specific to the WPRE sequence on the viral genome (as shown in the sequence listing below) were used in qPCR to detect and quantify any DNA genomic material capsized in the control AAV or RAAV particles.

[0670] To detect the RNA genome titer of control AAV and RAAV particles, any control AAV- or RAAV-captured DNA genome was removed by DNase I digestion before reverse transcription of the capsidated RNA genome. The resulting cDNA was used as a Q-PCR template, and the WPRE sequence was detected and quantified using the same WPRE primer pair described above. To detect and quantify any potential residual DNA that may be present due to incomplete DNA removal, the sample was directly amplified (without reverse transcription) using Q-PCR after the DNA removal step, and the WPRE (DNA) sequence was detected using the same WPRE primer pair described above, which was also used for all other WPRE sequence-specific PCR reactions.

[0671] 4. Comparison of Packaging Efficiency

[0672] When control AAV particles were produced using conventional transgenic plasmids containing the pssAAV-tdTomato construct (with wild-type ITRs at both ends) and conventional packaging plasmids for AAV-DJ, the vast majority of particles contained a DNA genome with WPRE sequences. Very occasionally, an RNA genome was also packaged into the particles (see the bar labeled "RNA," which is about 5 orders of magnitude lower than the bar labeled "DNA"). The presence of residual DNA was comparable to the presence of RNA, likely due to the low efficiency of DNase I in digesting the packaged DNA genome prior to reverse transcription.

[0673] Interestingly, when the recombinant packaging plasmid DJ-MCP (an MCP fused to the N-terminus of Rep78 and 68 proteins) was used instead of DJ, the packaging of the DNA genome was slightly reduced (by about 0.5 orders of magnitude), but the pattern of viral genome distribution remained almost identical, with DNA packaging being about 5 orders of magnitude higher than RNA packaging. This result indicates that fusing MCP to the N-terminus of the Rep78 / 68 proteins did not significantly impair their natural function. Figure 8B ).

[0674] Simultaneously, removing ITRs from both ends of the pssAAV-tdTomato construct resulted in the CTWS construct, significantly eliminating DNA packaging. Regardless of the packaging plasmid used (DJ or DJ-MCP), the CTWS construct (ITR-free) produced approximately four orders of magnitude less packaging DNA, and even less packaging RNA.

[0675] Furthermore, by adding one or three copies of RPS(MS2) between the 3' of WPRE and the 5' of the SV40 polyA signal on the ITR-free viral genome, RAAV transgenic plasmids, CTWMS, and CTWM3S were obtained, respectively. Without MCP, the CTWMS and CTWM3S constructs were almost impossible to capsid as DNA or RNA genomes, similar to the CTWS genome distribution pattern. Surprisingly, using DJ-MCP instead of DJ as the packaging plasmid significantly reversed the difference in packaging efficiency between DNA and RNA genomes, and the vast majority of the packaged genome was RNA.

[0676] Compared to CTWS / DJ-MCP, CTWMS / DJ-MCP and CTWM3S / DJ-MCP packaged 100-fold and 400-fold more RNA genomes, respectively, while there was no significant difference in the number of DNA packages among the three. This result indicates that the MCP-Rep78 / 68 fusion can specifically recognize RPS and MS2 embedded in RNA transcripts of CTWMS and CTWM3S plasmids and promote their RNA packaging into RAAV particles. Furthermore, in the CTWM3S construct, three copies of RPS provided better RNA packaging efficiency than one copy. Figure 8B ).

[0677] In summary, introducing the MS2 / MCP pair into a conventional AAV packaging system enables the packaging of the MS2-carrying RNA genome into AAV particles in the presence of the MCP-Rep78 / 68 fusion, thereby generating RAAV particles. Undesirable DNA packaging accounts for only about 10% of the total viral particle population generated using CTWM3S / DJ-MCP.

[0678] In other words, the artificial / heterologous RNA packaging signal (RPS) - MS2 sequence - can be used with its homologous binding protein MCP to replace the natural DNA virus packaging signal pair (i.e., ITR and Rep) to significantly enhance the efficiency of RNA packaging into another DNA virus, while inhibiting its inherent packaging into the same DNA virus.

[0679] Example 4: The expanded plasmid backbone reduced unwanted DNA packaging of RAAV.

[0680] This example demonstrates that increasing the backbone size of the AAV transgenic plasmid by inserting filler sequences into the plasmid backbone can reduce unwanted DNA packaging into RAAV particles.

[0681] Although the CTWMS and CTWM3S constructs used for RAAV particles in Example 3 did not have ITR and there was no reverse packaging in RAAV production, it is speculated that the relatively small size (5-6kb) of the RAAV transgenic plasmid may still lead to unwanted DNA packaging.

[0682] Therefore, a 3266 bp non-coding sequence (filler sequence; see sequence listing below) was inserted upstream of the tdTomato expression cassette of CTWM3S to increase the backbone length of the CTWM3S transgenic plasmid, and the resulting construct was named L-CTWM3S. A schematic diagram of the plasmid is shown below. Figure 9A As shown.

[0683] The conventional AAV genome construct pssAAV-tdTomato and RAAV genome construct CTWM3S used in Example 3 were used as controls in this study. Similar to Example 3, RAAV particles were prepared by co-transfecting CTWM3S or L-CTWM3S transgenic plasmids with packaging plasmid DJ-MCP and helper plasmids into HEK293T cells, and the resulting RAAV particles were purified and the viral genome was quantified. The same pair of WPRE primers was used to detect and quantify any DNA and RNA genomes capsidated in the AAV and RAAV particles. A separate pair of CAG primers specific to the CAG promoter sequence in the viral genome was used in Q-PCR to detect and quantify any functional DNA (i.e., DNA containing the CAG promoter sequence and capable of expressing functional transgenic proteins).

[0684] Note that packaged RNA genomes cannot be detected with CAG primers because they do not contain a CAG promoter, and the RNA bars in the diagram with CAG primers represent background RNA signals (see [link to diagram]). Figure 9B ).

[0685] Surprisingly, regardless of which primer pair was used in Q-PCR, the DNA genomic titer of the L-CTWM3S group was approximately twice that of the CTWM3S group (see [link to Q-PCR]). Figure 9B and 9C The RNA genomic titers of the CTWM3S and L-CTWM3S groups were essentially equivalent (see [link to relevant documentation]). Figure 9C Since the CAG promoter sequence is absent in the RNA transcribed from the CTWM3S and L-CTWM3S transgenic plasmids, the packaged RNA genome can only be detected using a pair of WPRE primers. Figure 9C ).

[0686] In summary, increasing the backbone length of the transgenic plasmid can reduce unwanted DNA packaging in RAAV particles without interfering with their RNA packaging. This suggests that the disassembly of the DNA packaging system and the establishment of the RNA packaging system in AAV particles are two separate processes, and the long filler sequence was used in the RAAV transgenic plasmids in subsequent examples.

[0687] Example 5 uses the RAAV-MS2 / MCP system for additional transgenic applications.

[0688] To verify the general applicability of the RAAV-MS2 / MCP system to additional transgenes and to ensure that the observed RNA packaging is not merely an artifact associated with the reporter gene used, a series of AAV and RAAV transgene plasmids containing Cre recombinase expression cassettes were generated.

[0689] The traditional pssAAV-Cre( Figure 8A(Replace the tdTomate encoding sequence in the pssAAV-tdTomato construct with the Cre encoding sequence) and the corresponding L-CCWS construct ( Figure 8A The tdTomate coding sequence in the CTWS was replaced with the Cre coding sequence, and the filler sequence from Example 4 was inserted as a control, where the second C represents the Cre recombinase transgene. After replacing the tdTomate coding sequence with the Cre coding sequence, the L-CTWM3S construct in Example 4 was also redesigned as the L-CCWM3S construct.

[0690] The Cre transgenic plasmid, along with the packaging plasmid DJ or DJ-MCP and helper plasmids, was co-transfected into HEK293T cells to produce AAV and RAAV particles, respectively. The resulting viral particles were purified, and the viral genome was quantified as described in Example 3.

[0691] For AAV-Cre and RAAV-Cre, the same viral genome distribution results were obtained as for AAV-tdTomato and RAAV-tdTomato. For pssAAV-Cre, most viral particles contained DNA genomes, and the DNA genome titer was approximately 4-5 orders of magnitude higher than the RNA genome titer. For L-CCWS, due to the lack of both DNA and RNA packaging signals, there was almost no capsidation of the DNA and RNA genomes. For L-CCWM3S, RNA packaging was significantly improved by approximately 200-fold in the DJ-MCP case compared to L-CCWS / DJ-MCP, and unwanted DNA-carrying viral particles accounted for only about 1% of the entire viral particle population. Figure 10A and 10B ).

[0692] Since the DJ-MCP fusion not only facilitates RNA packaging but also retains DNA packaging capabilities, its performance was evaluated in a construct containing both a DNA packaging signal (ITR) and an RNA packaging signal (three copies of MS2). This construct was created by inserting three copies of MS2 between the WPRE and SV40 polyA of the pssAAV-Cre construct and named pssAAV-Cre-MS2X3. Results showed that in the absence of MCP, most viral particles contained packaged DNA genome, while only a negligible amount of RNA genome was packaged with or without the RNA packaging signal. When DJ-MCP was used instead of DJ as the packaging plasmid in combination with the RNA binding signal, RNA packaging was significantly improved, and surprisingly, the increased RNA packaging did not significantly interfere with DNA packaging in the pssAAV-Cre-MS2X3 construct. Figure 11BAnother perspective suggests that even without removing the DNA packaging signal—ITR—the introduction of the MS2 / MCP pair can significantly increase RNA packaging. This indicates that the disassembly of the DNA packaging system and the establishment of the RNA packaging system in AAV particles are two separate processes, and that removing the ITR is not a necessary basis for increasing RNA packaging by introducing the RPS / RBP pair.

[0693] In summary, the tested RAAV-MS2 / MCP system can generally be applied to any transgenic Cre recombinase, as shown above. Interestingly, the RAAV-Cre construct produced better yields than the RAAV-tdTomato construct. While not wishing to be bound by any particular theory, this is likely due to the simpler secondary structure of Cre mRNA compared to tdTomato mRNA, based on online RNA secondary structure predictions, such as those found at rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi.

[0694] Example 6: Optimization of the RAAV production system and identification of optimized RAAV particle characteristics

[0695] The endonuclease activity of Rep68 and Rep78 proteins (Rep68 / 78) is crucial for DNA genome replication during the conventional DNA packaging process of AAV particles. Without a functional trs-endonuclease, newly synthesized viral ssDNA cannot be released for packaging. This example investigates whether disrupting trs-endonuclease activity can further reduce unwanted DNA packaging of RAAV particles.

[0696] To investigate this, three trs-endonuclease-negative mutants were constructed: DJ-MCP(Y156F, where the Y156F mutation is located in the common sequence of Rep68 and Rep78 proteins, i.e., Rep68-Y156F and Rep78-Y156F), DJ-MCP(KDE-mu), and DJ-MCP(EKE-mu) (see the sequence listing below).

[0697] First, the DNA and RNA packaging efficiency of DJ-MCP (Y156F) was evaluated using the transgenic plasmid pssAAV-Cre-MS2X3 (as described in Example 5), which contains both DNA and RNA packaging signals. DJ and DJ-MCP were used as packaging plasmid controls. Viral particles were generated, purified, and titrated as described in Example 3.

[0698] The results showed that the Y156F mutation in DJ-MCP significantly reduced ITR-mediated DNA packaging of pssAAV-Cre-MS2X3 without interfering with RNA packaging.

[0699] Therefore, in addition to removing the DNA packaging signal ITR as shown in the previous embodiments, functional proteins involved in the DNA packaging process (such as Rep78 / 68 proteins) can be modified, for example, by mutation, to weaken or eliminate their DNA packaging-related functions. This can serve as another strategy to reduce or inhibit the traditional DNA packaging of AAV particles. Figure 13A ).

[0700] Then, L-CCWM3S from Example 5 was used as the RAAV transgenic plasmid to replace pssAAV-Cre-MS2X3 to provide the viral genome. trs-endonuclease-positive DJ-MCP was used as a control against DJ-MCP(Y156F). Viral particles were generated, purified, and titrated as described in Example 3. Two pairs of primers were used to titrate the viral genome: one pair targeting the WPRE sequence described above, and the other pair (see sequence listing below) targeting the 5' end of the Cre coding sequence.

[0701] The results showed that the Y156F mutation in the Rep78 / 68 protein not only reduced unwanted DNA packaging by approximately 10-fold, but also increased desired RNA packaging by approximately 50%. For the two primer pairs used in Q-PCR (i.e., the WPRE primer pair and the Cre primer pair), the difference in packaging efficiency between packaged DNA and RNA genomes followed a largely similar pattern. Figure 12A-12B ).

[0702] Two other trs-endonuclease mutants, DJ-MCP(KDE-mu) and DJ-MCP(EKE-mu), were also tested and demonstrated to have the same ability as DJ-MCP(Y156F) to reduce unwanted DNA packaging, but only DJ-MCP(Y156F) showed improved RNA packaging. Figure 13B ).

[0703] Further evidence shows that fusing two copies of MCP to the N-terminus of the Rep 78 / 68 protein (MCPx2-Rep78 and MCPx2-Rep68) can also reduce unwanted DNA packaging. Figure 13B ).

[0704] Analysis of the composition of AAV and RAAV particles by silver staining SDS-PAGE revealed that the RAAV capsid is also composed of three VP proteins (VP1, VP2, and VP3), with the VP1 / 2 / 3 ratio similar to that of traditional AAV particles. Figure 12C ).

[0705] To analyze the morphology of RAAV particles, 10 μL of purified AAV and RAAV particles were placed on a copper grid coated with 300 μm carbon-coated polyvinyl acetal (Pioloform) and incubated at room temperature for 2 min. Excess sample was blotted dry with filter paper, immediately replaced with 10 μL of staining agent (3% phosphotungstic acid), allowed to stand for 2 min, and then blotted dry again. The samples were visualized using a Talos L120C transmission electron microscope. The RAAV particles were morphologically similar to conventional AAV vectors, with intact viral particles encapsulating the genome appearing as 25-nm solid spheres, while empty viral particles without a genome encapsulated were 25-nm donut-shaped structures. Figure 12D ).

[0706] In summary, mutations in functional proteins involved in the DNA packaging process during AAV production (including Rep proteins) to weaken or eliminate their DNA packaging-related functions and remove the DNA packaging signaling ITR are the optimal strategy for reducing or inhibiting unwanted DNA packaging in RAAV particles. The resulting RAAV particles have a similar composition and morphology to conventional AAV particles.

[0707] Example 7: RAAV vector for expressing functional proteins

[0708] This example demonstrates that the tested RAAV vector is infectious and can be used as a gene delivery vector.

[0709] The Cre-loxP system is a highly sensitive system used to study the infectivity of the RAAV vector of this invention. Mouse embryonic fibroblasts (MEFs) isolated from homo-Ai9 mice (carrying the loxP-tdTomato-reporter gene system) were incubated overnight with the AAV (pssAAV-Cre / DJ) or RAAV (L-CCWM3S / DJ-MCP(Y156F)) vectors purified in Example 5, and the multiplicity of infection (MOI) (the number of viral particles added per cell during infection) was set, including 7 MOIs for the conventional AAV vector and 3 MOIs for the RAAV vector. The dominant genomic titer, quantified by detecting the Cre coding sequence using the 5' end Cre primers described above, was used as the infection titer. In other words, the DNA genomic titer was used for the conventional AAV vector, while the RNA genomic titer was used for the RAAV vector.

[0710] Specifically, Ai9-MEF cells were injected at a rate of approximately 5 × 10⁻⁶ cells per ... 4Cells / well were seeded into 48-well plates. The AAV or RAAV vector was then thoroughly mixed with 0.5 mL of DMEM containing 2% FBS. The culture medium was removed from the seeded cells, and the cells were incubated overnight at 37°C with the mixed AAV or RAAV vector. Infected cells were collected at different time points for RNA and DNA analysis. As previously described, a pair of primers targeting the 5' end of the Cre coding sequence were used to detect specific Cre-encoded DNA and mRNA derived from the vector. Fluorescence images were taken daily post-infection (pi), and fluorescently positive cells were quantified by flow cytometry 5 days post-infection.

[0711] mRNA analysis showed that specific mRNA was detected in RAAV-infected cells as early as 2 hours post-infection, peaking at 6 hours and then declining. In cells infected with the conventional AAV vector, no significant transcription was detected at 2 hours post-infection, but a rapid increase in transcribed mRNA was observed between 6 and 20 hours post-infection, reaching a plateau at 30 hours. In contrast to the RAAV vector results, mRNA levels in cells infected with the conventional AAV vector did not decrease after reaching the plateau phase. In all samples, Cre mRNA copy number was positively correlated with MOI (Mean Interval of Infection). Figures 14A-14B and Figure 15A Meanwhile, mGAPDH mRNA was used as a reference transcript (housekeeping gene) for testing, and as expected, there was no difference in mGAPDH mRNA levels across all samples. Figure 15C ).

[0712] DNA results differ significantly from mRNA results. Traditional AAV and RAAV vectors exhibit essentially the same DNA copy number pattern, with most of the genomic DNA detected in infected cells as early as 2 hours post-infection, followed by a slight increase from 2 to 20 hours post-infection, very similar to the trend in mRNA levels in RAAV-infected cells. Afterward, DNA levels plateau or decline slowly. Cre DNA copy number is also positively correlated with MOI in all samples, but the amount of Cre DNA detected in RAAV-infected cells is much lower (the DNA copy number in the RAAV-CCWM3S MOI=100 or 300 group is less than that in the AAV-Cre MOI=1 group, and the DNA copy number in the RAAV-CCWM3S MOI=1000 group is less than that in the AAV-Cre MOI=3 group). Figure 14C and Figure 15B Similarly, the DNA level of another housekeeping gene, 36B4, was quantified as a reference gene, and as expected, no significant difference in DNA levels was observed among all samples. Figure 15D ).

[0713] Successful infection with the AAV-Cre or RAAV-CCWM3S vectors resulted in the expression of a functional Cre recombinase and rescued tdTomato expression in Ai9-MEF cells. Therefore, fluorescence images of infected cells were captured and analyzed to assess the infectivity of the viral vectors by counting cells emitting red tdTomato fluorescence. Results showed that the number of fluorescently positive cells generated by RAAV-CCWM3S was comparable to that generated by AAV-Cre, which had a 10-fold lower MOI. Figure 14D The lower fluorescence intensity of tdTomato in RAAV-infected cells may be due to the short lifespan of the Cre mRNA delivered therein, and the inability of a limited amount of Cre recombinase to rescue two copies of the tdTomato expression cassette in homo-Ai9-MEF cells. Figure 16 ).

[0714] Comparison of DNA titer and cell count data showed that most of the tdTomato red fluorescence signal in RAAV-CCWM3S-infected cells was generated by RAAV particles carrying Cre mRNA.

[0715] In summary, the RAAV vector of the present invention can deliver functional Cre mRNA into cells and express functional Cre recombinase.

[0716] Example 8: Transient in vitro transfer of functional genes into cells using RAAV particles.

[0717] To determine the exact lifetime of Cre protein delivered via AAV-Cre or RAAV-CCWM3S as in Example 7, samples were taken at a concentration of 5 × 10⁻⁶ ppm 24 hours prior to infection. 4 Cell confluence at a certain level (cells / well) was seeded with Ai9-MEF cells and then incubated overnight with either AAV-Cre (MOI = 300) or RAAV-CCWM3S (MOI = 10,000) vectors as described above. Cells were collected at several time points after infection, and fluorescence images were taken before cell collection.

[0718] For AAV-Cre, Cre expression increased in the first 4 days but then decreased. In contrast, small amounts of Cre were detected as early as approximately 24 hours after RAAV-CCWM3S transfer and disappeared after day 2. This rapid expression and degradation phenomenon is likely due to the immediate presence and short lifespan of the delivered functional Cre mRNA. Figure 17A and 17B ).

[0719] Example 9: RAAV particles transiently transfer functional genes in vivo into Ai9 mice.

[0720] This example demonstrates that RAAV particles can be used as a tool for in vivo gene delivery and transiently express functional Cre recombinases.

[0721] To investigate the infectivity of RAAV particles in vivo, Ai9 mice (2.5–4 months old) were anesthetized and injected with either 1 μL of AAV-Cre (pssAAV-Cre / DJ) (high dose: 1E9 Vg / mouse; low dose: 3E6 Vg / mouse) or 1 μL of RAAV-Cre (L-CCWM3S / DJ-MCP(Y156F)) (1E9 Vg / mouse) into the right hippocampus (coordinates as follows: anterior / posterior (A / P) = -1.7 mm, mid / lateral (M / L) = -1.0 mm, dorsoventral (D / V) = -2.1 mm). AAV capsid-DJ was used as a control in this assay.

[0722] Six weeks after AAV or RAAV injection, mice were anesthetized and perfused cardiacally with PBS at pH 7.4 at room temperature, followed by perfusion with freshly prepared, ice-cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PB). The brain was post-fixed overnight in 4% PFA. After dehydration, the fixed brain was embedded in OCT for cryosectioning. Brain sections were cut to 20 μm thickness using a cryostat (Leica CM1950), and sections were placed directly onto slides. Slides were baked at 60°C for 1–2 hours, followed by blocking with 5% BSA serum in PBS for 1 hour. Subsequently, slides were incubated overnight at room temperature with anti-Cre primary antibody (10536; Cell Signaling Technology; 1:800 dilution) in PBS (0.1% Triton-X) and 5% BSA. After washing five times with PBS, the slides were incubated in 1% BSA in PBS containing secondary antibodies against the primary antibody and DAPI (D3571, Invitrogen). The secondary antibody used was Alexa Fluor488 donkey anti-rabbit IgG (711-545-152, Jackson ImmunoResearch) (diluted 1:1000). Images were acquired using a Nikon C2si+ confocal microscope.

[0723] Images showing fluorescence from the tdTomato expression system revealed that RAAV-Cre infected cells in the hippocampus of Ai9- mice and rescued tdTomato expression. At the same dose, the number of infected cells in the RAAV-Cre group was less than that in the AAV-Cre group. However, compared to the low-dose AAV-Cre infection group (30-fold lower dose), RAAV-Cre infection generated more tdTomato-positive cells.

[0724] Interestingly, Cre expression was readily detected in both high- and low-dose AAV-Cre infection groups, but no significant Cre expression was detected in RAAV-Cre infected cells, although the detected tdTomato fluorescence indicated that Cre was once present.

[0725] Overall, as shown in the fluorescence images (positive cell counts) of both at the same high dose of 1E9 vg / mouse ( Figure 21A contrast Figure 21C RAAV-Cre exhibits poorer transduction efficiency compared to conventional AAV-Cre because multiple mRNAs for protein translation can be transcribed from a single successfully transduced AAV DNA genome. To further assess Cre expression levels, the transduction efficiency of AAV-Cre was normalized relative to that of RAAV-Cre by reducing a high dose of 1E9 vg / mouse to a low dose of 3E6 vg / mouse. Figure 21B Furthermore, the results showed that although the transduction efficiency of RAAV-Cre was superior to that of the low-dose group of AAV-Cre, as indicated by the fluorescence images (positive cell counts), Cre expression was not detected in RAAV-Cre-infected cells compared to the AAV-Cre group, suggesting that Cre expression in the RAAV-Cre group was transient but functional.

[0726] Example 10 Additional RPS / RBP pairs for RAAV systems

[0727] In addition to the MS2 / MCP pair used in Examples 3-8, two additional pairs of RNA aptamers / aptamer-binding proteins (or RNA packaging signals / RNA binding proteins, referred to herein as "RPS / RBP") were tested for RAAV packaging: (1) PP7 binding site / PP7 bacterial phage coat protein (abbreviated as "PP7 / PCP" or "PCP" or "P", or L-CCW) P (3S "P") and (2) Com binding site / phage COM protein (abbreviated as "com / COM" or "COM", or L-CCW) C(The "C" in 3S). Unlike the natural viral packaging systems MS2 / MCP and PP7 / PCP, com / COM is not a natural viral packaging system, but rather a transcriptional regulator known to play a role in the transcriptional initiation of the bacterial bacteriophage Mu mom gene.

[0728] A transgenic plasmid (L-CCW) carrying three copies of RPS was constructed. P 3S and L-CCW C 3S) and their corresponding packaging plasmids [DJ-PCP(Y156F) having PP7 bacterial phage capsid protein (PCP) fused to the N-terminus of Rep78-Y156F and Rep68-Y156F, and DJ-COM(Y156F) having phage COM protein (COM) fused to the N-terminus of Rep78-Y156F and Rep68-Y156F]. Viral particles were generated, purified, and titrated as described in Example 3.

[0729] The results showed that, similar to the MS2 / MCP pair, which was well demonstrated in all respects in Examples 3-8, the PP7 / PCP and com / COM pairs also resulted in significant RNA packaging of RAAV particles. Figure 18 This expands the range of various RAAV packaging systems.

[0730] Example 11: Application of the RAAV system to various AAV serotypes

[0731] To investigate the application of the RAAV packaging system of the present invention to various AAV serotypes other than AAV-DJ tested in Examples 3-9, two pairs of RPS / RBP (MS2 / MCP and com / COM) and three other different AAV serotypes (AAV5, AAV8, and AAV9) were examined in AAV-DJ. Viral particles were generated, purified, and titrated as described in Example 3.

[0732] The RAAV-MS2 / MCP and RAAV-com / COM systems functioned well in all four serotypes, demonstrating the general applicability of the RAAV packaging system to different AAV serotypes (i.e., not limited to AAV-DJ). In the presence of RBP, Cre RNA genomes containing the corresponding RPS were efficiently capsidated into the corresponding RAAV particles. Although the yield of RNA-packaged RAAV particles varied by serotype, all RAAV5, RAAV8, and RAAV9 particles exhibited higher yields than RAAV-DJ. Figure 19 ).

[0733] Example 12: AAP and MCP fusion protein improves RAAV yield

[0734] Typically, AAV encodes a unique assembly activation protein (AAP) in its native viral genome, which is crucial for capsid assembly. In particular, AAP has been found to be essential for capsid protein stability and the generation of functional AAV particles.

[0735] The AAP-MCP (MCP with the C-terminus fused to AAP) or MCP-AAP (MCP with the N-terminus fused to AAP) fusion protein expression cassettes were inserted backwards into the backbone of the packaging plasmid DJ-MCP(Y156F) used in Examples 6-10, and the resulting constructs were named DJ-MCP(Y156F)-AM and DJ-MCP(Y156F)-MA, respectively. These constructs then expressed both the MCP-Rep78 / 68(Y156F) fusion and the AAP-MCP or MCP-AAP fusion, increasing the amount of RNA-binding protein (RBP) that assists in RNA packaging compared to the MCP-Rep78 / 68 fusion alone. Viral particles were generated, purified, and titrated as described in Example 3.

[0736] The results showed that, compared with the MCP-Rep78 / 68 fusion alone, the yield of RNA-packaged RAAV particles was increased by approximately 65% ​​and approximately 35% in DJ-MCP(Y156F)-MA and DJ-MCP(Y156F)-AM, respectively. Figure 20A and 20B This suggests that RBP can be additionally fused with or associated with any other protein that plays a role in the packaging or assembly of AAV particles to enhance RNA packaging of RAAV particles.

[0737] Using AAP-MCP or MCP-AAP alone, without using MCP-Rep78 / 68, is also within the scope of this invention.

[0738] sequence

[0739] Certain sequences are provided below, including those referenced in the above embodiments.

[0740] Table A: Nucleic acid and amino acid sequences of RBP

[0741]

[0742]

[0743] Table B: Nucleic Acid Sequences of RPS

[0744]

[0745]

[0746] *Sequence elements are matched according to format style (e.g., bold and / or italics, etc.). Table C: Nucleic acid and amino acid sequences of Rep protein.

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760] *Sequence elements are matched according to format style (e.g., double underscores, bold, and / or italics, etc.). Table D: Nucleic acid and amino acid sequences of AAP and MCP fusion proteins.

[0761]

[0762]

[0763]

[0764] * Sequence elements are matched based on formatting styles (e.g., double underscores, bold, and / or italics, etc.).

[0765] Table E: Primer Sequences

[0766] WPRE-F CCCGTATGGCTTTCATTTTCTCC WPRE-R GGCAATGCCCCAACCAGTG Cre-F CCAGTAGATGCCACTAGCGA Cre-R GCCTGGAGATACAGCAGGTA CAG-F CTTCTCCTCCGGGCTGTAAT CAG-R CTTTCACGCAGCCACAGAAA

[0767] *F and R represent the forward and reverse primers, respectively.

[0768] Table F: Filled Sequences

[0769]

[0770]

[0771]

[0772] By invoking and incorporating

[0773] The full disclosure of each patent document, including patent application documents, scientific literature, government reports, websites, and other references cited herein, is incorporated herein by reference in its entirety for all purposes. In the event of a conflict of terminology, this specification shall prevail. All sequence listings or SEQ ID NOs disclosed herein are incorporated herein in their entirety.

[0774] The following references are expressly incorporated herein by reference in such sense as to provide exemplary procedures or other details that supplement the content set forth herein.

[0775] Although illustrative embodiments of the invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes and modifications can be made by those skilled in the art without departing from the scope or spirit of the invention.

Claims

1. A recombinant DNA virus particle, It consists of a protein shell of a DNA virus and an RNA genome packaged within the protein shell of the DNA virus; The RNA genome contains RNA capable of being packaged into DNA viral particles, and the RNA comprises: (1) RNA sequences of interest; and, (2) An RNA packaging signal that can interact directly or indirectly with an RNA packaging signal interacting molecule that promotes the packaging of the RNA into the DNA virus particle; Optionally, the DNA encoding or corresponding to the RNA, or the reverse complementary DNA of the DNA, has a reduced, weakened, or substantially no ability to be packaged into the DNA virus particles.

2. The recombinant DNA virus particle of claim 1, wherein the RNA sequence of interest is selected from the RNA coding sequence of the gene of interest, the RNA coding protein, the non-coding functional RNA, or a precursor thereof.

3. The recombinant DNA virus particle of claim 2, wherein the protein is selected from therapeutic proteins, antigenic proteins, and gene-editing proteins.

4. The recombinant DNA virus particle as described in claim 3, wherein the gene editing protein is selected from CRISPR / Cas effector enzymes, ZFN protein, and TALEN protein.

5. The recombinant DNA virus particle of claim 2, wherein the RNA encoding the protein is mRNA.

6. The recombinant DNA virus particle of claim 2, wherein the non-coding functional RNA is selected from transfer RNA, ribosomal RNA, small interfering RNA, short hairpin RNA, antisense RNA, antisense oligonucleotide, microRNA, and RNA components of the CRISPR-Cas system.

7. The recombinant DNA virus particle of claim 6, wherein the CRISPR-Cas system is selected from CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13 systems.

8. The recombinant DNA virus particle of claim 6, wherein the RNA component of the CRISPR-Cas system is selected from guide RNA, CRISPR RNA, and tracr RNA.

9. The recombinant DNA virus particle of claim 8, wherein the guide RNA is a single guide RNA.

10. The recombinant DNA virus particle of claim 1, wherein the DNA or its reverse complementary DNA lacks a DNA packaging signal.

11. The recombinant DNA virus particle of claim 1, wherein the RNA packaging signal is capable of directly or indirectly binding to RNA packaging signal interacting molecules.

12. The recombinant DNA virus particle as described in claim 1, wherein the DNA virus particle is an AAV virus particle or an oncolytic virus particle.

13. The recombinant DNA virus particle according to any one of claims 1-12, wherein the RNA packaging signal is located at or near the 5' end of the RNA sequence of interest, at or near the 3' end of the RNA sequence of interest, or inside the RNA sequence of interest.

14. The recombinant DNA virus particle according to any one of claims 1-12, wherein the RNA comprises more than one identical or different RNA packaging signal.

15. The recombinant DNA virus particle of claim 14, wherein two or more of the more than one RNA packaging signal are adjacent to or tandem with each other via the same or different adapters.

16. The recombinant DNA virus particle of claim 14, wherein the RNA comprises two or more RNA packaging signals that are not adjacent to each other.

17. The recombinant DNA virus particle of claim 16, wherein the RNA comprises two or more RNA packaging signals that are not adjacent to each other and are each located at or near one end of the RNA sequence of interest.

18. The recombinant DNA virus particle of claim 12, wherein the RNA packaging signal comprises a transcribed modified AAV terminal inverted repeat, wherein the transcribed modified AAV terminal inverted repeat: (a) Contains a functional Rep-binding element for transcription, optionally further comprising a functional Rep-binding element for transcription'; and, (b) Lack of a transcriptional terminal dissociation site or a transcriptional inverse complementary terminal dissociation site, or both; Optionally, the modified AAV-terminal inverted repeat of the transcription further comprises a D-region sequence of the transcription; and / or Optionally, the RNA packaging signaling interaction molecules are Rep78, Rep68, Rep52, and / or Rep40.

19. The recombinant DNA virus particle of claim 18, wherein the transcribed modified AAV terminal inverted repeat is located within 1000 nucleotides of the 3' end of the RNA; optionally, the transcribed modified AAV terminal inverted repeat is located at the 5' end of a polyA sequence, a polyA signal sequence, or an RNA transcription termination sequence.

20. The recombinant DNA virus particle of claim 18 or 19, wherein the transcribed modified AAV terminal inverted repeat is based on a wild-type Flip or Flop ITR; optionally, the wild-type Flip or Flop ITR is derived from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13; optionally, the wild-type Flop ITR has the nucleotide sequence of SEQ ID NO:

1.

21. The recombinant DNA virus particle of claim 18 or 19, wherein the modified AAV terminal inverted repeat of the transcription lacks both the terminal dissociation site of the transcription and the inverted complementary terminal dissociation site of the transcription.

22. The recombinant DNA virus particle of claim 18 or 19, wherein the transcribed modified AAV terminal inverted repeat comprises the D region sequence of the transcription; optionally, the modified AAV terminal inverted repeat has the nucleotide sequence of SEQ ID NO:

3.

23. The recombinant DNA virus particle of claim 18 or 19, wherein the modified AAV terminal inverted repeat of the transcription lacks the D region sequence of the transcription; optionally, the modified AAV terminal inverted repeat has the nucleotide sequence of SEQ ID NO:

2.

24. The recombinant DNA virus particle of claim 18 or 19, wherein the RNA further comprises a modified AAV terminal inverted repeat of the second transcription, the modified AAV terminal inverted repeat of the second transcription having a functional RBE sequence of the second transcription but lacking a terminal dissociation site of the second transcription or a reverse complementary terminal dissociation site of the second transcription or both; optionally, the modified AAV terminal inverted repeat of the second transcription further comprises a D region sequence of the second transcription.

25. The recombinant DNA virus particle of claim 24, wherein the modified AAV terminal inverted repeat of the transcription and the modified AAV terminal inverted repeat of the second transcription are the same or different.

26. The recombinant DNA virus particle of claim 18 or 19, wherein the transcribed modified AAV terminal inverted repeat comprises a deletion in the corresponding wild-type AAV terminal inverted repeat D region sequence or the corresponding wild-type terminal dissociation site / inverted complementary terminal dissociation site, a mutation in the corresponding wild-type AAV terminal inverted repeat D region sequence or the corresponding wild-type terminal dissociation site / inverted complementary terminal dissociation site, or an insertion in the corresponding wild-type AAV terminal inverted repeat D region sequence or the corresponding wild-type terminal dissociation site / inverted complementary terminal dissociation site.

27. The recombinant DNA virus particle of claim 24, wherein the modified AAV terminal inverted repeat of the second transcription comprises a deletion in the corresponding wild-type AAV terminal inverted repeat D region sequence or the corresponding wild-type terminal dissociation site / inverted complementary terminal dissociation site of the transcription, a mutation in the corresponding wild-type AAV terminal inverted repeat D region sequence or the corresponding wild-type terminal dissociation site / inverted complementary terminal dissociation site of the transcription, or an insertion in the corresponding wild-type AAV terminal inverted repeat D region sequence or the corresponding wild-type terminal dissociation site / inverted complementary terminal dissociation site of the transcription.

28. The recombinant DNA virus particle of claim 24, wherein the modified AAV terminal reverse repeat of the second transcription is located within 1000 nucleotides at the 5' end of the RNA.

29. The recombinant DNA virus particle according to any one of claims 1-12, wherein the RNA packaging signal comprises an MS2 sequence, a PP7 binding site, or a com binding site, and the RNA packaging signal interacting molecule comprises an RNA packaging signal interacting protein capable of interacting directly or indirectly with the RNA packaging signal.

30. The recombinant DNA virus particle of claim 29, wherein the RNA packaging signal interacting molecule comprises an RNA packaging signal interacting protein capable of binding directly or indirectly to the RNA packaging signal.

31. The recombinant DNA virus particle of claim 29, wherein the RNA comprises the three said RNA packaging signals.

32. The recombinant DNA virus particle of claim 31, wherein the RNA comprises the same three RNA packaging signals.

33. The recombinant DNA virus particle of claim 29, wherein the RNA packaging signal comprises an MS2 sequence, and the RNA packaging signal interacting molecule comprises a phage-derived MS2 coat protein.

34. The recombinant DNA virus particle of claim 29, wherein the RNA packaging signal comprises a PP7 binding site, and the RNA packaging signal interacting molecule comprises a PP7 phage capsid protein.

35. The recombinant DNA virus particle of claim 29, wherein the RNA packaging signal comprises a COM binding site, and the RNA packaging signal interacting molecule comprises a bacteriophage COM protein.

36. The recombinant DNA virus particle of claim 29, wherein the RNA packaging signal interacting protein is directly or indirectly associated with the protein components of the viral packaging system of the DNA virus particle.

37. The recombinant DNA virus particle of claim 36, wherein the RNA packaging signal interacting protein is fused directly or indirectly to the protein components of the viral packaging system of the DNA virus particle.

38. The recombinant DNA virus particle of claim 36 or 37, wherein the protein components of the viral packaging system of the DNA virus particle are selected from Rep78 and Rep68 of adeno-associated virus 2, and assembly activation protein.

39. The recombinant DNA virus particle according to any one of claims 1-12, wherein the RNA does not contain transcribed DNA packaging signals.

40. The recombinant DNA virus particle of claim 39, wherein the RNA does not contain a transcribed wild-type AAV terminal inverted repeat sequence.

41. The recombinant DNA virus particle of claim 39, wherein the RNA comprises a transcribed modified AAV terminal inverted repeat sequence, the transcribed modified AAV terminal inverted repeat sequence having nucleotide additions, deletions, and / or substitutions of corresponding wild-type AAV terminal inverted repeat sequences to reduce the DNA packaging capacity of the DNA virus particle.

42. The recombinant DNA virus particle according to any one of claims 1-12, wherein the RNA further comprises: (1) Transcription enhancers of transcription; (2) Transcribed intron or exon sequences; (3) 5' UTR sequence; (4) 3' UTR sequence; (5) A polyA sequence, or a polyadenylated signal sequence, and optionally a GU-rich region downstream of the polyA signal sequence; (6) Posttranscriptional regulatory elements or sequences; and / or, (7) Transcription termination sequence, Optionally, the RNA includes the RNA packaging signal located at the 3' of the posttranscriptional regulatory element or sequence and the 5' of the poly-A sequence or polyadenylation signal sequence.

43. The recombinant DNA virus particle of claim 42, wherein the RNA comprises, in the 5' to 3' direction: the RNA sequence of interest, a transcribed marmot hepatitis virus post-transcriptional regulatory element sequence; the RNA packaging signal; and the poly-A sequence or the polyadenylation signal sequence.

44. The recombinant DNA virus particle of claim 2, wherein the gene of interest comprises a gene encoding: a protein, mRNA, non-coding RNA, siRNA, piRNA, short hairpin RNA, microRNA or its precursor, ribosomal RNA, antisense sequence or oligonucleotide, RNA component of the CRISPR-Cas system, rRNA, tRNA, snoRNA, snRNA, exRNA, scaRNA, Xist, or HOTAIR.

45. The recombinant DNA virus particle of claim 44, wherein the protein is selected from fluorescent proteins, therapeutic proteins, antigenic proteins, structural proteins, and gene-editing proteins.

46. ​​The recombinant DNA virus particle of claim 45, wherein the gene editing protein is selected from Cas protein, ZFN protein and TALEN protein.

47. The recombinant DNA virus particle of claim 44, wherein the protein is an enzyme.

48. The recombinant DNA virus particle of claim 47, wherein the enzyme is a Cre protein or a CRISPR / Cas effector enzyme.

49. The recombinant DNA virus particle of claim 48, wherein the CRISPR / Cas effector enzyme is selected from Cas9, Cas12, Cas13, or variants thereof.

50. The recombinant DNA virus particle of claim 44, wherein the RNA component of the CRISPR-Cas system is selected from guide RNA, CRISPR RNA, and tracr RNA.

51. The recombinant DNA virus particle of claim 50, wherein the guide RNA is a single guide RNA.

52. The RNA of any one of claims 1-12, wherein the RNA is a single-stranded RNA of less than 8,900 nucleotides in length.

53. The recombinant DNA virus particle of claim 1, wherein the DNA virus is AAV, and the recombinant DNA virus particle is a recombinant RNA adeno-associated virus particle, the recombinant RNA adeno-associated virus particle comprising: (1) AAV capsid; and, (2) The RNA packaged within the AAV capsid.

54. The recombinant DNA virus particle of claim 53, wherein the AAV capsid comprises a capsid from serotypes AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, Anc80L65, Anc80L65AAP, AAVrh74, or 7m8.

55. A population of recombinant DNA virus particles, the population comprising a plurality of recombinant DNA virus particles as described in any one of claims 1-54, wherein at least 10% of the recombinant DNA virus particles in the population have said RNA packaged therein.

56. A host cell comprising a population of recombinant DNA virus particles as described in any one of claims 1-54 and / or recombinant DNA virus particles as described in claim 55.

57. The host cell of claim 56, further comprising a viral packaging system that facilitates the packaging of RNA as defined in any one of claims 1-54 into the DNA virus particles.

58. The host cell of claim 57, wherein the DNA virus is AAV, and wherein the viral packaging system comprises: (1) The AAV rep gene and the AAV cap gene or their expression products, wherein the AAV rep gene and the AAV cap gene are under the transcriptional control of one or more promoters that drive the transcription of the AAV rep gene and the AAV cap gene; (2) One or more coding sequences of one or more proteins required for AAV packaging; and (3) The RNA packaging signal interaction molecule or its coding sequence; Optionally, the ability of the viral packaging system to package DNA into the DNA virus particles is reduced, weakened, or substantially eliminated by: (1) removing part or all of the DNA packaging signal on the polynucleotide encoding RNA as defined in any one of claims 1-54; (2) modifying the coding sequences of the AAV rep gene, the AAV cap gene, and / or one or more proteins required for AAV packaging to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA into the DNA virus particles; and / or (3) enlarging the size of the polynucleotide encoding RNA as defined in any one of claims 1-54.

59. The host cell of claim 58, wherein modifying the AAV rep gene includes introducing a Y156F mutation, a KDE mutation, or an EKE mutation into a common sequence of the Rep78 and Rep68 proteins.

60. The host cell according to any one of claims 56-59, wherein it is a mammalian cell or an insect cell.

61. The host cell of claim 60, wherein the host cell is a HEK293 cell.

62. The host cell as described in claim 60, wherein it is an Sf9 or Sf21 cell.

63. A method for generating recombinant DNA virus particles, the method comprising: a) Culturing the host cells as described in any one of claims 56-62 for a sufficient time, and b) Harvest the recombinant DNA virus particles or a population of the recombinant DNA virus particles.

64. The method of claim 63, further comprising isolating or purifying the recombinant DNA virus particles or a population of the recombinant DNA virus particles.

65. A method for generating a population of recombinant DNA virus particles according to any one of claims 1-54 or the recombinant DNA virus particles according to claim 55, the method comprising: a) The time period during which the viral packaging system is exposed to the RNA sufficiently to produce the recombinant DNA virus particles or a population of the recombinant DNA virus particles, and b) Harvesting the recombinant DNA virus particles or a population of the recombinant DNA virus particles; and optionally, c) Separate or purify the harvested recombinant DNA virus particles or populations of recombinant DNA virus particles.

66. The method of claim 65, wherein the virus packaging system comprises: (1) One or more proteins, or one or more coding sequences thereof, used to assemble the protein shell of the DNA virus particle for packaging the RNA; (2) One or more proteins, or one or more coding sequences thereof, used to facilitate the assembly of the protein shell and / or the packaging of the RNA into the protein shell of the DNA virus particle; and (3) The RNA packaging signal interaction molecule or its coding sequence; Optionally, the ability of the viral packaging system to package DNA into the DNA virus particles is reduced, weakened, or substantially eliminated by: (1) removing part or all of the DNA packaging signal on the polynucleotide encoding RNA as defined in any one of claims 1-54; (2) modifying one or more coding sequences of the AAV rep gene, the AAV cap gene, and / or one or more proteins required for AAV packaging to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA into the DNA virus particles; and / or (3) enlarging the size of the polynucleotide encoding RNA as defined in any one of claims 1-54.

67. The method of claim 66, wherein modifying the AAV rep gene includes introducing a Y156F mutation, a KDE mutation, or an EKE mutation into a common sequence of the Rep78 and Rep68 proteins.

68. A system for packaging RNA as defined in any one of claims 1-54 into DNA viral particles to produce recombinant DNA viral particles as described in any one of claims 1-54 or a population of recombinant DNA viral particles as described in claim 55, the system comprising: (1) One or more proteins, or one or more coding sequences thereof, used to assemble the protein shell of the DNA virus particle for packaging the RNA; (2) One or more proteins, or one or more coding sequences thereof, used to facilitate the assembly of the protein shell and / or the packaging of the RNA into the protein shell of the DNA virus particle; and (3) The RNA packaging signal interaction molecule or its coding sequence; Optionally, the ability of the viral packaging system to package DNA into the DNA virus particles is reduced, weakened, or substantially eliminated by: (1) removing part or all of the DNA packaging signal on the polynucleotide encoding RNA as defined in any one of claims 1-54; (2) modifying one or more coding sequences of the AAV rep gene, the AAV cap gene, and / or one or more proteins required for AAV packaging to reduce, weaken, or substantially eliminate the ability of the corresponding translated proteins to facilitate the packaging of the DNA into the DNA virus particles; and / or (3) enlarging the size of the polynucleotide encoding RNA as defined in any one of claims 1-54.

69. The system of claim 58, wherein modifying the AAV rep gene includes introducing a Y156F mutation, a KDE mutation, or an EKE mutation into a common sequence of the Rep78 and Rep68 proteins.

70. A non-diagnostic and non-therapeutic method for delivering a gene of interest to a cell, plant, or animal, the method comprising contacting the cell, plant, or animal with a recombinant DNA virus particle as claimed in any one of claims 1-54, a population of recombinant DNA virus particles as claimed in claim 55, or a recombinant DNA virus particle or population of recombinant DNA virus particles produced by the method as claimed in any one of claims 63-67, wherein the gene of interest is encoded by RNA packaged in the recombinant DNA virus particle.

71. The use of any recombinant DNA virus particle as claimed in any one of claims 1-54, a group of recombinant DNA virus particles as claimed in claim 55, or a group of recombinant DNA virus particles or recombinant DNA virus particles produced by the method as claimed in any one of claims 63-67 in the manufacture of a reagent for delivering a gene of interest to a cell, plant, or animal, wherein the gene of interest is encoded by RNA packaged in the recombinant DNA virus particle.

72. A non-diagnostic and non-therapeutic method for delivering RNA of interest to cells, plants, or animals, the method comprising contacting the cells, plants, or animals with recombinant DNA virus particles as claimed in any one of claims 1-54, a population of recombinant DNA virus particles as claimed in claim 55, or recombinant DNA virus particles or a population of recombinant DNA virus particles produced by the method as claimed in any one of claims 63-67, wherein the RNA packaged in the recombinant DNA virus particles contains the RNA of interest.

73. Use of recombinant DNA virus particles as claimed in any one of claims 1-54, a population of recombinant DNA virus particles as claimed in claim 55, or recombinant DNA virus particles or a population of recombinant DNA virus particles produced by the method as claimed in any one of claims 63-67 in the manufacture of a reagent for delivering RNA of interest to cells, plants, or animals, wherein the RNA packaged by the recombinant DNA virus particles contains the RNA of interest.

74. Use of recombinant DNA virus particles as described in any one of claims 1-54, a group of recombinant DNA virus particles as described in claim 55, or recombinant DNA virus particles or a group of recombinant DNA virus particles produced by the method as described in any one of claims 63-67 in the manufacture of a medicament for the diagnosis, prevention, or treatment of a disease or disorder in a subject in need.

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