Recombinant parvovirus vector and its preparation method and use

Abstract

A parvoviral vector carrying a viral genome with covalently blocked ends (ccePV vector), a method for producing the vector and a DNA construct for producing the vector.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 755,144, filed on November 2, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present application relates generally to recombinant viral vectors, and more particularly to parvoviral vectors having a viral genome having a double-stranded region with covalently blocked ends. Summary of the invention

[0004] This application describes a parvoviral vector (ccePV vector) with a viral genome having covalently blocked ends, a method for producing the vector, and a DNA construct for producing the vector. The vector and method are suitable for all gene transfer / therapy applications, such as those requiring delivery of a recombinant gene expression cassette. The ccePV vector is highly flexible, user-friendly, and can be easily modified (by conventional DNA cloning) and used for a variety of applications (by standard PV vector or AAV vector technology). BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Fig. 1, sub-figure (A) shows conventional recombinant AAV (rAAV) vector plasmid, which is used to produce a single-stranded AAV genome of wild-type ITR with an expression cassette flanking a gene of interest (GOI). The single-stranded AAV genome in sub-figure (B) is packaged in AAV particles. Sub-figure (C) shows conventional self-complementary AAV (scAAV) vector plasmid, which is used to produce a double-stranded scAAV genome formed by the single-stranded precursor shown in sub-figure (D) (left side), which is self-annealed or folded back by the self-complementary sequence on either side of mITR. In this case, the single-stranded vector genome includes wild-type ITR (wtITR) at either end. The vector genome packaged in the viral particle is shown on the right side of sub-figure (D), illustrating the annealing of the complementary region to form the double-stranded scAAV genome shown. The two plasmids in sub-figures (A) and (C) can be stably grown in bacterial hosts.

[0006] Figure 2Pathways for transgene expression from different packaged AAV templates are shown. Sub-figure (A) shows the synthesis of the second strand from a single-stranded recombinant AAV (rAAV) DNA to form a double-stranded DNA template for transgene expression; Sub-figure (B) shows a scAAV comprising a duplex DNA with a covalently blocked end (CCE) domain at the 3' end, which can promote transcription of the transgene without the intermediate second strand synthesis step shown in sub-figure (A). Sub-figure (C) shows a cceAAV-ZL with a fully complementary genome, which includes a CCE domain that is not an mTR or shDNA. In sub-figure (C), the CCE domain typically includes a ssDNA region of several nucleotides (2 to 5 nucleotides) to allow the formation of a stable stem structure. Compared with scAAV, the cceAAV-ZL shown in sub-figure (C) does not require mTR or shDNA. This allows for increased packaging capacity in cceAAV-ZL compared to scAAV vectors that are otherwise identical.

[0007] Figure 3 , sub-figure (A) shows an embodiment of a cceAAV comprising a long non-complementary region of single-stranded (ss) DNA. Sub-figure (B) shows another embodiment of a cceAAV comprising a single-stranded loop region (or bulge region) within the complementary double-stranded region. The non-complementary ssDNA can include any DNA sequence, such as a sequence encoding an aptamer / deoxyribozyme, a sequence encoding a polypeptide, a sequence encoding an siRNA, a sequence encoding an miRNA, a sequence encoding an shRNA, a polyadenylation signal, an insulator sequence, etc. The aptamer can be stabilized by its connection / attachment to an adjacent flanking complementary DNA sequence. The ssDNA in cceAAV-LS is an effective template for gene editing or DNA repair applications.

[0008] Figure 4Several parvoviral vectors are depicted, including conventional parvoviral vectors and different cce parvoviral vector embodiments. Sub-figure (A) depicts a traditional parvovirus with a single-stranded (ss) DNA genome. Sub-figure (B) depicts a cce parvovirus with a complementary double-stranded (ds) genome. Sub-figure (C) depicts a cce parvovirus with an ss loop region on one strand of the ds template. Sub-figure (D) depicts a cce parvovirus with an extended ss loop region 3' to a gene of interest (GOI), the loop region defining a CCE domain and including a non-complementary region. Non-complementary ss sequences can include any DNA sequence, such as a sequence encoding an aptamer / deoxyribozyme, a sequence encoding a polypeptide, a sequence encoding a miRNA, a sequence encoding a shRNA, a polyadenylation signal, an insulator sequence, and the like. The aptamer can be stabilized by its connection / attachment to an adjacent flanking complementary DNA sequence. The single-stranded DNA in the ccePV-LS is an effective template for gene editing or DNA repair applications.

[0009] Figure 5 An exemplary ligation strategy for generating a cceAAV-ZL vector is shown. First, a starting plasmid having an AAV ITR and an expression cassette encoding a gene of interest (GOI) is engineered to contain a recognition sequence at the 3' end of the expression cassette to facilitate digestion (or linearization) by an enzyme of interest. The resulting fragment can undergo three different pathways to generate cceAAV-ZL particles containing two ITRs with a 3' CCE domain. The starting plasmid can be: (1) digested in vitro (i.e., linearized), ligated in vitro, and then transfected in an AAV producer cell (e.g., 293 or any other cell with complementary genes provided) transfected with Rep, Cap, and pAd (with additional adenoviral helper genes not provided in the producer cell line); (2) digested in vitro (i.e., linearized), transfected in an AAV producer cell with Rep, Cap, and pAd, and ligated in vivo; or (3) transfected directly in an AAV producer cell with Rep, Cap, and pAd, where the starting plasmid is digested in vivo and ligated in vivo. As a result, cceAAV-ZL genomic DNA is produced (upper right) and packaged into viral particles. The illustration here is for AAV produced in 293 cells using adenovirus assistance. Other auxiliary systems that can be used for the proposed digestion / ligation method include systems based on herpes virus, vaccinia virus and baculovirus. The vector plasmid can also be present in the host cell in a stable free (episomal) form before the digestion / ligation step to form a template molecule corresponding to the ccePV and cceAAV vectors produced.

[0010] Figure 6An exemplary in vivo digestion-ligation strategy for producing a cceAAV-ZL vector is further shown. A starting plasmid including an AAVITR and an expression cassette encoding a gene of interest (GOI) is engineered to contain a recognition sequence at the 3' end of the expression cassette to facilitate in vivo digestion (or linearization) by an enzyme of interest. The plasmid is co-transfected with Rep, Cap, and pAd into an AAV producer cell (e.g., 293) with an enzyme for the linearization reaction. The enzyme can be delivered by a viral vector or stably integrated into the AAV producer cell genome. Figure 6 A production cell with stably integrated Cas9 / gRNA nucleases is depicted. In turn, the plasmid is linearized and then ligated to itself by a host cell ligase to form a template for AAV production, as shown, comprising an expression cassette sequence flanked on both sides by AAV ITRs. Thus, the DNA fragment is modified by in vivo digestion and ligation steps that occur in a host cell environment. In some embodiments, nucleotide insertions or deletions can be engineered into the ligation sites. The closed ends of cceAAV-ZL can have identical sequences or can have minor variations.

[0011] Figure 7An exemplary general digestion-ligation strategy for generating a ccePV-LS vector is shown. Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) containing a parvoviral (PV) left end hairpin (LEH), a PV right end hairpin (REH), and an expression cassette therebetween is cleaved with an enzyme at the 3' end of the expression cassette at position 1, while the second plasmid (i.e., the rightmost plasmid) containing an additional non-complementary region 3' to the expression cassette is similarly cleaved downstream of the non-complementary region at position 2. The second plasmid may be cleaved with the same or a different enzyme. The plasmids may be: (1) digested in vitro, ligated in vitro, and co-transfected with parvoviral helper genes into PV producer cells, such as the 293 cell line (or any other cell line compatible with a particular parvoviral vector); (2) digested in vitro, co-transfected with parvoviral helper genes into producer cells, and ligated in vivo in producer cells; or (3) directly transfected with parvoviral helper genes into PV producer cells, wherein the starting plasmids are digested in vivo and ligated in vivo. As a result of any of these three pathways, the replication template shown is produced, which has LEH and REH sequences flanking the gene of interest (GOI), including a non-complementary region between them, for producing a PV vector in a PV producer cell. Because the ligated fragments include non-complementary regions, the CCE domain will form an extended single-stranded loop or bulge region, as shown in the upper right portion of the figure. Asymmetric digestion can be performed using seamless cloning or other directional cloning techniques to eliminate undesirable self-ligation, which may instead lead to the formation of ccePV-ZL. However, the digestion site in the second plasmid does not have to be at the beginning or end of the non-complementary region; it can be anywhere in the middle of the non-complementary region. In contrast, when the ends of the first plasmid and the second plasmid are identical or nearly identical, the resulting vector ccePV-ZL is produced, which has no obvious loop at the closed end. ccePV can package both polarities of the vector or only one polarity. The illustration here shows only one polarity.

[0012] Figure 8 Shown based on Figure 7An exemplary digestion-ligation strategy for generating a cceAAV-LS vector according to the general method outlined in . Two different starting plasmids are utilized. The first plasmid containing the AAV ITRs and the expression cassette (i.e., the leftmost plasmid shown) is cut with an enzyme at the 3' end of the expression cassette at position 1, while the second plasmid containing an additional non-complementary region 3' to the expression cassette (i.e., the rightmost plasmid) is similarly cut downstream of the non-complementary region at position 2. The second plasmid can be cut with the same or a different enzyme. The plasmid can be: (1) digested in vitro, ligated in vitro, and co-transfected with Rep, Cap, and pAd into an AAV producer cell, such as a 293 cell line or other AAV cell line; (2) digested in vitro, co-transfected with Rep, Cap, and pAd into a producer cell, and ligated in vivo in the producer cell; or (3) directly transfected with Rep, Cap, and pAd in an AAV producer cell, where the starting plasmid is digested in vivo and ligated in vivo. As a result of any of these three approaches, the replication template shown is produced, which has ITRs flanking the gene of interest (GOI) and including a non-complementary region between them for producing an AAV vector in an AAV producer cell. Because the ligated fragments include non-complementary regions, the CCE domain will form an extended single-stranded loop or bulge region, as shown in the upper right portion of the figure. Asymmetric digestion can be performed using seamless cloning or other directional cloning techniques to eliminate undesirable self-ligation, which may instead lead to the formation of cceAAV-ZL. However, the digestion site in the second plasmid does not have to be at the beginning or end of the non-complementary region; it can be anywhere in the middle of the non-complementary region. In contrast, when the ends of the first plasmid and the second plasmid are identical or nearly identical, the resulting vector cceAAV-ZL is produced, which has no obvious loop at the closed end. In addition, a variety of fragment ligation and / or DNA assembly techniques can be used to generate template molecules for producing cceAAV or ccePV.

[0013] Fig. 9A general digestion-ligation strategy for generating a ccePV-BR vector is shown. Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) containing the PV LEH upstream of the expression cassette containing the GOI is cut with an enzyme at the 3' end of the expression cassette at position 1, while the second plasmid (i.e., the rightmost plasmid) containing the REH, the expression cassette, and an additional non-complementary region between the promoter and the GOI is cut downstream of the expression cassette at position 2. The second plasmid can be cut with the same or a different enzyme. The plasmids can be: (1) digested in vitro, ligated in vitro, and co-transfected with parvoviral helper genes into PV producer cells, such as the 293 cell line; (2) digested in vitro, co-transfected with parvoviral helper genes into producer cells, and ligated in vivo in producer cells; or (3) transfected directly into PV producer cells with parvoviral helper genes, where the starting plasmid is digested in vivo and ligated in vivo (not shown). As a result of any of these three approaches, the replication template shown (ccePV-BR, upper right) is generated, which has LEH / REH sequences upstream of the promoter driving the expression of the gene of interest (GOI), and a non-complementary region between them. Because the connected fragments include non-complementary regions, the CCE domain will form an extended single-stranded loop or bulge region, as shown in the upper right portion of the figure, which can accommodate additional sequences, such as aptamer sequences, etc. Seamless cloning or other directional cloning techniques can be used for asymmetric digestion to eliminate undesirable self-ligation, which may instead lead to the formation of symmetrical ligation products from the first plasmid and the second plasmid. By including additional non-complementary sequences, ccePV-BR with multiple ss loop structures can be formed. One or more ss loops in ccePV-BR can contain aptamer coding regions or single-stranded DNA templates for gene editing. The property of parvovirus packaging with limited polarity means that, for example, a construct containing an aptamer sequence will result in a replication template that is not affected by the complementary chain. The aptamer activity can be maintained until the vector genome is degraded / converted by the host cell.

[0014] Fig.10 Shown based on Fig. 9An exemplary digestion-ligation strategy for generating a cceAAV-BR vector according to the general method outlined in . Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) containing the AAV ITRs and the expression cassette is cut with an enzyme at the 3' end of the expression cassette at position 1, while the second plasmid (i.e., the rightmost plasmid) containing an additional non-complementary region between the promoter and the GOI is cut downstream of the otherwise identical expression cassette at position 2. The second plasmid can be cut with the same or different enzyme. The plasmid can be: (1) digested in vitro, ligated in vitro, and co-transfected with Rep, Cap, and pAd into an AAV producer cell, such as a 293 cell line or other compatible cell; (2) digested in vitro, co-transfected with Rep, Cap, and pAd into a producer cell, and ligated in vivo in the producer cell; or (3) directly transfected with Rep, Cap, and pAd into an AAV producer cell, where the starting plasmid is digested in vivo and ligated in vivo (not shown). As a result of any of these three approaches, the replication template shown (cceAAV-BR, upper right) is produced, which has an AAV ITR upstream of the promoter driving the expression of the gene of interest (GOI), and a non-complementary region between them. Because the connected fragments include non-complementary regions, the CCE domain will form an extended single-chain loop or protruding region, as shown in the upper right portion of the figure, which can accommodate additional sequences, such as aptamer sequences, etc. Seamless cloning or other directional cloning techniques can be used for asymmetric digestion to eliminate undesirable self-ligation, which may instead lead to the formation of symmetrical ligation products from the first plasmid and the second plasmid. By including additional non-complementary sequences, cceAAV-BR with multiple ss loop structures can be formed. The AAV vectors herein can be produced in any current AAV production system, including AAV production systems based on herpes virus, vaccinia virus or baculovirus.

[0015] Fig.11A general digestion-ligation strategy for generating ccePV-XP vectors for additional packaging capacity is shown. Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) containing PV LEH upstream of the expression cassette is cut with an enzyme at position 1 at the 3' end of the expression cassette containing the cDNA, while the second plasmid (i.e., the rightmost plasmid) containing PV REH (without cDNA) just upstream of the promoter is cut downstream of the promoter at position 2. The plasmids can be: (1) digested in vitro, ligated in vitro, and then co-transfected with parvoviral helper genes; (2) digested in vitro, co-transfected with parvoviral helper genes into PV producer cells and ligated in vivo; or (3) co-transfected directly into host cells for in vivo digestion and in vivo ligation (not shown). The resulting structure contains PV genomes (ccePV-XP, upper right), each of which contains a non-complementary loop region corresponding to the cDNA that is packaged into PV particles. Thus, the PV genome has a configuration of a double DNA region with promoter activity operably linked to a single-stranded cDNA (loop) region with a coding sequence and a polyadenylation signal. Seamless cloning or other directional cloning techniques can be used to perform asymmetric digestion to eliminate undesired self-ligation, which may instead result in the formation of symmetrical ligation products from the first or second plasmid.

[0016] Fig.12 Shown based on Fig.11Figure 2 is an exemplary digestion-ligation strategy for generating a cceAAV-XP vector for additional packaging capacity based on the general method outlined in Figure 2. Two different starting plasmids are utilized. The first plasmid containing the 5'AAV ITR and the expression cassette (i.e., the leftmost plasmid shown) is cut with an enzyme at the 3' end of the expression cassette containing the cDNA at position 1, while the second plasmid containing only the 5'AAV ITR and the promoter (no cDNA) (i.e., the rightmost plasmid) is cut downstream of the promoter at position 2. The plasmids can be: (1) digested in vitro, ligated in vitro, and then co-transfected with AAV helper genes; (2) digested in vitro and co-transfected into AAV producer cells with parvoviral helper genes; or (3) co-transfected directly into host cells for in vivo digestion and in vivo ligation (not shown). The structure formed thereby comprises an AAV genome (cceAAV-XP, upper right), each genome comprising a non-complementary loop region corresponding to a cDNA, both of which are packaged in an AAV particle, wherein only one of the genomes (the positive strand) is capable of expressing the cDNA. Thus, the AAV positive strand genome has a configuration of a double DNA region with complete promoter activity and a single-stranded cDNA region with a coding sequence and a polyadenylation signal. In contrast, the negative strand is capable of expressing the cDNA after conversion to a double-stranded form. Seamless cloning or other directional cloning techniques can be used for asymmetric digestion to eliminate undesirable self-ligation, which may instead result in the formation of symmetrical ligation products from the first plasmid or the second plasmid.

[0017] Fig.13Another general digestion-ligation strategy for generating ccePV vectors for additional packaging capacity is shown. Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) containing PV LEH and an expression cassette (containing a promoter, cDNA, and a polyadenylation site) is cut with an enzyme at position 1 3' to the polyadenylation site, while the second plasmid (i.e., the rightmost plasmid) containing PVREH and an expression cassette (containing a promoter, cDNA, and a polyadenylation site) is cut with an enzyme at position 2 3' to the poly A site. The plasmids can be: (1) digested in vitro, ligated in vitro, and then co-transfected into PV producer cells with parvoviral helper genes; (2) digested in vitro and transfected into PV producer cells, where the digested plasmids are ligated in vivo; or (3) the plasmids can be directly transfected into PV producer cells with parvoviral helper genes for in vivo digestion and in vivo ligation (not shown). The resulting PV genome packaged into PV particles contains a non-complementary loop region corresponding to the cDNA. Thus, the PV genome has a dual DNA region with a promoter and poly A site upstream of a single long DNA region encoding cDNA. The inclusion of poly A in the second plasmid prevents the production of antisense DNA, which can reduce transgene expression when the PV genome undergoes second-strand DNA synthesis. Alternatively, or in addition, a DNA insulator sequence can be included to further reduce this result and effectively separate the transcription units from each other. This can be important because the self-complementary region includes an enhancer sequence, which can also function in the opposite orientation. Asymmetric digestion can be performed using seamless cloning or other directional cloning techniques to eliminate undesirable self-ligation, which may instead result in the formation of symmetrical ligation products from the first plasmid or the second plasmid.

[0018] Fig.14 Shown based on Fig.13An exemplary digestion-ligation strategy for generating cceAAV vectors for additional packaging capacity according to the general method outlined in . Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) containing the AAV ITRs upstream of the expression cassette (comprising a promoter, cDNA, and a poly A site (if a pol III promoter is used, all poly A sites in this application can be transcription terminators)) is cut with an enzyme at position 1 3' of the poly A site, while the second plasmid (i.e., the rightmost plasmid) containing the AAV ITRs upstream of the expression cassette (comprising a promoter, cDNA, and a poly A site) is cut with an enzyme at position 2 3' of the poly A site. The plasmids can be: (1) digested in vitro, ligated in vitro, and then co-transfected into AAV production cells with Rep, Cap, and pAd; (2) digested in vitro and transfected into AAV production cells, where the digested plasmids are ligated in vivo; or (3) the plasmids can be directly transfected into AAV production cells with Rep, Cap, and pAd for in vivo digestion and in vivo ligation (not shown). The structure formed by it contains two different AAV genomes (upper right), each of which contains a non-complementary loop region corresponding to cDNA, both of which are packaged in AAV particles. However, only one of the genomes (positive chain) can express cDNA immediately after being released from the AAV capsid. The AAV positive genome formed by it has a configuration of a double DNA region, which has a complete promoter activity that drives the expression of the single-stranded cDNA region. In contrast, the negative chain can only express cDNA after the ssDNA genome is converted to a duplex form. Including poly A in the second plasmid prevents the production of antisense DNA, which can reduce transgenic expression when the AAV genome undergoes second-strand DNA synthesis. Alternatively, or in addition, a DNA insulator sequence can be included to further reduce this result and effectively separate the transcription units from each other. This can be very important because the self-complementary region includes an enhancer sequence, which can also work in the opposite orientation. Seamless cloning or other directional cloning techniques can be used for asymmetric digestion to eliminate undesirable self-connections, which may instead lead to the formation of symmetrical connection products from the first plasmid or the second plasmid.

[0019] Fig.15Another general digestion-ligation strategy for producing a ccePV-binary vector for expressing two genes using a single promoter is shown. Two different starting plasmids are used. The first plasmid (i.e., the leftmost plasmid shown) contains a LEH region, which contains a stem-loop structure on the opposite chain, followed by a promoter in the 5' to 3' direction, followed by an expression cassette of the first gene (i.e., luciferase, Luc) in the 3' to 5' orientation. In addition, the second chain covering the Luc region contains a non-complementary region. The first plasmid is cut with the first enzyme at position 1, and the second plasmid containing the GFP expression cassette (i.e., the rightmost plasmid) is cut with the second enzyme at position 2. The plasmid can be: (1) digested in vitro, ligated in vitro, and then co-transfected into PV producer cells with parvoviral helper genes; (2) digested in vitro and transfected into PV producer cells with parvoviral helper genes, where the digested plasmid is ligated in vivo; or (3) the plasmid can be directly transfected into PV producer cells with parvoviral helper genes for in vivo digestion and in vivo ligation (not shown). The resulting PV genome packaged into PV particles contains a non-complementary loop region corresponding to the cDNA (ccePV-diplex, upper right). Therefore, the PV genome has a dual DNA region in which the promoter directs the expression of two genes (Luc and GFP as an example), each with an independent poly A site downstream. The main feature is that the promoter is able to turn on transcription of two genes at the same time. When the PV genome undergoes conversion to a dual form, this can be used to turn off the expression of a second gene, which places the second transgene (such as Luc) downstream of the promoter in the opposite orientation. This is an example of using the PV vector as a gene switch. Asymmetric digestion can be performed using seamless cloning or other directional cloning techniques to eliminate undesired self-ligation, which may otherwise result in the formation of symmetrical ligation products from either the first or second plasmid.

[0020] Fig.16Shown is an exemplary digestion-ligation strategy for producing a cceAAV-duplicate vector for expressing two genes using a single promoter. Two different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) contains AAVI ITR, which includes a stem-loop structure on the relative chain, followed by a promoter in the 5' to 3' direction, followed by an expression cassette of the first gene (i.e., luciferase, Luc) in the 3' to 5' orientation. In addition, the second chain covering the Luc region includes a non-complementary region. The first plasmid is cut with the first enzyme at position 1, and the second plasmid containing the GFP expression cassette (i.e., the rightmost plasmid) is cut with the second enzyme at position 2. The plasmid can be: (1) digested in vitro, ligated in vitro, and then co-transfected with Rep, Cap, and pAd into AAV producer cells; (2) digested in vitro and transfected into AAV producer cells, where the digested plasmid is ligated in vivo; or (3) the plasmid can be directly transfected into AAV producer cells with Rep, Cap, and pAd for in vivo digestion and in vivo ligation (not shown). The structure formed by it contains two different AAV genomes (upper right), each of which contains a promoter that drives the expression of two genes, one of which contains a non-complementary loop region corresponding to one of the genes (Luc), and the other gene (GFP) is encoded in a double-stranded stem upstream of the CCE domain. Both genomes are packaged in AAV particles. The AAV genome formed by it has a configuration of dual DNA regions, which has full promoter activity that drives the expression of two genes, one gene from the single-stranded region and the other gene from the double-stranded region. The main feature is that once the genome is released into the host cell after infection, the promoter is able to turn on the transcription of both genes at the same time. Additionally, when the AAV genome undergoes conversion to a duplex, this can be used to turn off expression of a second gene, which places the second genome (luc) in the opposite orientation downstream of the promoter. This is an example of using AAV as a gene switch. Asymmetric digestion can be performed using seamless cloning or other directional cloning techniques to eliminate undesirable self-ligation, which may instead result in the formation of symmetrical ligation products from the first plasmid or the second plasmid.

[0021] Fig.17A general digestion-ligation strategy for producing another ccePV vector containing a single promoter to express two genes is shown. Three different starting plasmids are used. The first plasmid (i.e., the leftmost plasmid shown) contains the LEH sequence and the expression cassette of the first gene (e.g., Luc) in the 5' to 3' direction, which is cut by the enzyme at position 1. The second plasmid (i.e., the intermediate plasmid) contains a head-to-tail tandem arrangement of poly A sites flanking the GFP-Luc coding region in the 3' to 5' direction, which is cut by the second enzyme at positions 2 and 3. The third plasmid contains the REH sequence upstream of the GFP expression cassette, which is cut by the third enzyme at position 4. In this method, the enzymes can be the same or different. The plasmid can be: (1) digested in vitro, ligated in vitro, and then co-transfected into PV producer cells with parvoviral helper genes; (2) digested in vitro and transfected into PV producer cells with parvoviral helper genes, where the digested plasmid is ligated in vivo; or (3) the plasmid can be directly transfected into PV producer cells with parvoviral helper genes for in vivo digestion and in vivo ligation (not shown). The resulting PV genome packaged in PV particles (upper right) contains a complementary branch region encoding Luc and a dual DNA region encoding GFP, such that a single promoter directs the expression of each gene with independent poly A sites downstream of each gene. Therefore, the main feature of this arrangement is that the promoter is able to drive transcription of two genes simultaneously. In addition, due to this arrangement, both genes will continue to be expressed even after the dual configuration shown by chain foldback.

[0022] Fig.18 Shown based on Fig.17The general method outlined in is an exemplary digestion-ligation strategy for producing a cceAAV vector containing a single promoter to express two genes. Three different starting plasmids are utilized. The first plasmid (i.e., the leftmost plasmid shown) contains an expression cassette for AAV ITR and a first gene (e.g., Luc) in the 5' to 3' direction, which is cut by an enzyme at position 1. The second plasmid (i.e., the intermediate plasmid) contains a poly A site arranged in tandem head to tail with a GFP-Luc coding region flanking the 3' to 5' direction, which is cut by a second enzyme at positions 2 and 3. The third plasmid contains an AAV ITR upstream of the GFP expression cassette, which is cut by a third enzyme at position 4. In this method, the enzymes can be the same or different. The plasmid can be: (1) digested in vitro, ligated in vitro, and then co-transfected into AAV producer cells with parvoviral helper genes; (2) digested in vitro and transfected into AAV producer cells, where the digested plasmid is ligated in vivo; or (3) the plasmid can be directly transfected into AAV producer cells with parvoviral helper genes for in vivo digestion and in vivo ligation (not shown). As a result of any of these three pathways, two different AAV genomes are packaged into AAV particles (upper right). Each genome contains a complementary branch region encoding Luc and a dual DNA region encoding GFP, so that a single promoter directs the expression of each gene, with independent poly A sites downstream of each gene. Therefore, the main feature of this arrangement is that the promoter is able to drive transcription of two genes simultaneously. In addition, due to this arrangement, both genes will continue to be expressed even after the dual structure shown by chain folding back.

[0023] Fig.19 An exemplary method for producing a cceAAV vector using a single plasmid that can operate with the TelN / telRL system is shown. The plasmid contains the AAV ITRs forming a stem-loop structure on both strands, an expression cassette containing the nucleic acid of interest (NAOI or GOI), and a downstream poly A site followed by the telRL site for the TelN enzyme. Cleavage and ligation of the plasmid by the TelN enzyme results in the formation of an intermediate double-stranded closed-end molecule, which is used for packaging when transfected into AAV-producing cells. Fig.19Template molecule for the cceAAV vector shown. Because the AAV packaging signal containing the "D" sequence is configured in the intermediate as 3' to the ITR, rather than 5' to the ITR, only the right side of the intermediate will be compatible with replication and encapsidation, forming the cceAAV vector shown in the upper right of the figure. The TelN enzyme can be delivered using a viral vector (e.g., adenovirus), or expressed from AAV producer cells to avoid the need for in vitro TelN reactions. This design can be further expanded to produce ccePV vectors containing the same ITR at the 5' or 3' end. This approach can also be used to produce scAAV with shDNA when the shDNA sequence matches a telomerase recognition or compatible site.

[0024] Fig. 20 An exemplary method for producing a cceAAV vector using CELiD vector DNA is shown. The disclosed starting CELiD vector DNA can carry a designed TelRL or resT site. After incubation with TelN or resT enzymes, two linear plasmids are formed, which can be used to produce cceAAV when transfected into AAV producer cells. Endless substrates with only one ITR can be used directly as drug products, such as DNA vaccines. When the ITRs are identical, this strategy can also be extended to produce ccePV. Methods for producing linear molecules using pFBGR-bsd or CELiD vectors using baculovirus are described in the literature.

[0025] Fig.21 An exemplary method for producing cceAAV vector template molecules using telomere splitting enzyme (resolvase) ResT is shown. The starting plasmid includes AAV ITR and a double-stranded domain (DS domain), and the double-stranded domain includes an expression cassette containing LL or RR cleavage sites cut by enzyme ResT. This results in the formation of a linear plasmid, which can be used to produce cceAAV when transfected to AAV production cells. ResT can be delivered using a viral vector such as adenovirus, or ResT is expressed from AAV production cells. The left sub-figure shows that the replication of linear molecules leads to the formation of telomeres (L'L, RR') connected or replicated by dimers, which are processed by telomere splitting (resolution) (DNA breakage and aggregation reaction of unique types). Telomere splitting leads to the formation of hairpin telomeres at the ends of linear DNA molecules, and the dimer replication intermediate is separated into the monomer product of DNA replication. Borrelia burgdorferi (B.burgdorferi) enzyme ResT (telomere splitting enzyme) is an example.

[0026] Fig. 22An exemplary method for producing a cceAAV vector template molecule using a linear plasmid comprising a DS domain and AAV ITR cloned in close proximity to the TelR and TelL binding sequences is shown. The linear plasmid can be produced in large quantities in vitro or in bacteria by a loop-mediated isothermal amplification (LAMP) method. The resulting linear plasmid is compatible with producing cceAAV vectors in AAV production host cells.

[0027] Fig.23 An exemplary ligation strategy for producing scAAV with mTR or shDNA closed-end vectors is shown. First, a starting plasmid with AAV ITR and at least half of mTR and shDNA is digested so that the fragment will have half of the mTR or shDNA sequence. Self-ligation will generate a molecule that serves as a scAAV template with mTR or shDNA. The resulting fragments can undergo three different pathways to produce corresponding scAAV particles. The starting plasmid can be: (1) digested in vitro (i.e., linearized), ligated in vitro, and then transfected in AAV production cells (e.g., 293 or any other cell with complementary genes provided) transfected with Rep, Cap, and pAd (with additional adenovirus helper genes not provided in the production cell line); (2) digested in vitro (i.e., linearized), transfected in AAV production cells with Rep, Cap, and pAd, and ligated in vivo; or (3) directly transfected in AAV production cells with Rep, Cap, and pAd, where the starting plasmid is digested in vivo and ligated in vivo. The illustrations presented here are for AAV produced in 293 cells using adenovirus assistance. Other packaging systems include herpes virus, vaccinia virus, or baculovirus-based packaging systems that are compatible with the digestion / ligation method presented. The vector plasmid can also be present in a stable free form in the host cell prior to digestion. This approach represents a more efficient scAAV production system because it employs a wild-type ITR arrangement that is directly compatible with replication and packaging into parvovirus particles. In contrast, conventional methods for producing scAAV rely on indirect schemes to generate template molecules from double-stranded plasmid vectors or shDNA. Indirect conversion via mTR or shDNA is the rate-limiting step in the production of scAAV vectors. According to the methods and compositions described herein, this method can be further extended to other parvoviruses.

[0028] Fig.24An exemplary ligation strategy for producing scAAV in the form of mTR or shDNA closed-end vectors is shown. Two starting plasmids are used, each of which contains AAV ITRs, so that each plasmid is digested and connected to form the structure shown with mTR or shDNA. Here, the fragments are designed to avoid self-ligation, wherein the resulting fragments can undergo three different pathways to produce corresponding scAAV particles. The starting plasmid can be: (1) digested in vitro (i.e., linearized), connected in vitro, and then transfected in AAV production cells (e.g., 293 or any other cell with complementary genes provided) transfected with Rep, Cap and pAd (with other adenovirus auxiliary genes not provided in the production cell line); (2) digested in vitro (i.e., linearized), transfected in AAV production cells with Rep, Cap and pAd, and connected in vivo; or (3) directly transfected in AAV production cells with Rep, Cap and pAd, wherein the starting plasmid is digested in vivo and connected in vivo. The diagram here is an AAV produced in 293 cells using adenovirus assistance. Other packaging systems include herpes virus, vaccinia virus or baculovirus-based packaging systems that are compatible with the proposed digestion / ligation method. Prior to digestion, the vector plasmid can also be present in a stable free form in the host cell. As noted above, this approach represents a more efficient system for scAAV production.

[0029] Fig.25An exemplary ligation strategy for producing scAAV in the form of mTR or shDNA closed-end vectors is shown. The scheme utilizes a single starting plasmid with AAV ITRs, which is digested and connected to a synthetic adapter to form the structure containing mTR or shDNA shown. The fragments are designed to avoid self-ligation. The synthetic adapter can include partial or complete mTR or shDNA. The resulting fragments can undergo three different pathways to produce corresponding scAAV particles. The starting plasmid can be: (1) digested in vitro (i.e., linearized), connected in vitro, and then transfected in AAV production cells (e.g., 293 or any other cell with complementary genes provided) transfected with Rep, Cap, and pAd (with additional adenovirus helper genes not provided in the production cell line); (2) digested in vitro (i.e., linearized), transfected in AAV production cells with Rep, Cap, and pAd, and connected in vivo; or (3) directly transfected in AAV production cells with Rep, Cap, and pAd, where the starting plasmid is digested in vivo and connected in vivo. The illustrations here are for AAV produced in 293 cells using adenovirus helper. Other packaging systems include herpes virus, vaccinia virus, or baculovirus-based packaging systems that are compatible with the proposed digestion / ligation method. Prior to digestion, the vector plasmid can also be present in a stable free form in the host cell. As noted above, this scheme represents a more efficient system for scAAV production.

[0030] Fig.26 An exemplary cceAAV is shown, which expresses acyclic double-stranded AAV RNA (sub-figure A, cceAAV-ZL-dsRNA) or double-stranded AAV RNA with a branch region (sub-figure B, cceAAV-ZL-dsRNA). The main difference is that when the vector is illustrated in a self-complementary configuration, the transcription terminator position is in a reverse orientation. The transcription termination site (here taking poly A as an example) is located before the dsRNA region in the reverse orientation. When the vector is converted to a duplex form, the overall configuration is in the following order: ITR, poly A (reverse), promoter, dsRNA coding region, promoter (reverse), poly A and ITR. In certain specific cases, poly A or transcription termination site can be embedded in the promoter region in a reverse orientation. If introns are used, for example, the transcription termination site can also be embedded in the intron. Poly A or transcription termination site needs to be located at any position after the complementary strand of the dsRNA coding region. Sub-figure B illustrates different configurations of the dsRNA coding region. Similar vectors can be prepared for parvovirus vectors.

[0031] Fig. 27 An exemplary cceAAV expressing double-stranded AAV RNA with long single-stranded regions is shown (panel C).

[0032] Fig.28 An exemplary cceAAV is shown, which expresses an acyclic double-stranded AAV RNA containing only one DNA promoter and only one transcription terminator (panel A, cceAAV-ZL-dsRNA). The capacity of the dsRNA coding sequence is enhanced. When uncoated, the vector does not immediately express the dsRNA. Before expressing the dsRNA, a step such as second-strand DNA synthesis or annealing to the other polarity of the vector DNA is required. DETAILED DESCRIPTION

[0033] definition

[0034] As used herein, the term "parvovirus" is used to refer to any member of the subfamily Parvovirinae, including members of autonomously replicating parvovirus and Dependoparvovirus. Autonomously replicating parvoviruses include members of the genus Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Chapparvovirus, Copiparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice (MVM), bovine parvovirus (BPV), canine parvovirus (CPV), chicken parvovirus, feline panleukopenia virus, feline parvovirus (FPV), goose parvovirus (GPV), porcine parvovirus (PPV), Bocavirus, B19 virus, rat virus (RV), H-1 virus (H-1). Other species of the family Felidae. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., King AMQ, Adams MJ, Carstens EB and Lefkowitz EJ (2012) Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier.

[0035] Dependovirus includes adeno-associated virus (AAV), including but not limited to AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, etc. The parvoviral particles, capsids and genomes of the present invention are preferably derived from AAV.

[0036] The parvovirus used in the present application also includes new variants from genetic engineering, which are further modified in PV capsid genes, nonstructural genes, inverted terminal repeats (ITRs), left-end hairpins (LEH), and right-end hairpins (REH). The parvovirus vector of the present invention can be used to deliver nucleic acids to cells in vitro and in vivo. In particular, the vector of the present invention can be advantageously used to deliver or transfer nucleic acids to animal cells. Nucleic acids of interest (NAOIs) include nucleic acids encoding RNA, peptides and proteins, preferably therapeutic (e.g., for medical or veterinary use) or immunogenic (e.g., for vaccines) peptides or proteins. It can also provide DNA template sequences for gene editing and / or aptamers for targeted delivery.

[0037] As used herein, the term "hybrid parvovirus" refers to a parvoviral genome encapsidated within a different (i.e., another, foreign, exogenous) parvoviral capsid. In other words, the hybrid parvovirus has a parvoviral genome encapsidated within a different parvoviral capsid. As used herein, "different" means that the AAV genome is packaged within another parvoviral capsid, for example, the parvoviral capsid is from another AAV serotype or from an autonomous parvovirus.

[0038] As used herein, the term "parvoviral ITR" refers to an inverted terminal repeat from any parvovirus, whose function is to support parvoviral replication, encapsidation, rescue, integration, etc. When the 5'ITR and 3'ITR of the parvoviral inverted terminal repeat (ITR) are different, the parvoviral inverted terminal repeat is also called the left end hairpin (LEH), the right end hairpin (REH). "AAV ITR" refers to the inverted terminal repeat flanking the AAV genome. Parvoviral ITRs and AAV ITRs may include ITRs from any parvovirus or any parvoviral serotype, and may also include ITRs with mutations that support AAV replication, encapsidation, rescue and / or integration similar to wild-type ITRs.

[0039] As used herein, the term "AAV serotype" refers to a capsid that packages a genome with at least one AAV ITR. It includes any AAV serotype found in nature or any engineered or chemically modified capsid that can package an AAV genome. It includes biologically or chemically modified capsids.

[0040] The terms "short hairpin DNA" and "shDNA" are used interchangeably herein to refer to shDNA as described in US 2018 / 0298380.

[0041] As used herein, the term "scAAV" refers to a single-stranded AAV vector containing a double-stranded region generated by the lack of a terminal split site (TR) in one of the ITRs of AAV, wherein the lack of TR prevents replication from starting at the end of the vector where the TR is not present. The scAAV vector typically comprises a wild-type (wt) AAV TR at each end and a mutant TR (mTR) in the middle, which is connected to the AAV TR via the double-stranded region. The terms "mTR" and "mITR" are used interchangeably herein to represent a mutated inverted terminal repeat as described in US 7,465,583.

[0042] The phrases "covalently blocked end domain", "CCE domain", "single-chain covalently blocked end domain" and "SS-CCE domain" are used interchangeably.

[0043] As used herein, the phrase "covalently blocked end (cce) parvovirus" refers to a linear parvovirus genome packaged into a parvovirus capsid, the parvovirus genome comprising a self-complementary DNA sequence forming a pair of hairpin structures at the 5' and 3' ends, a double-stranded domain (referred to herein as "DS domain") between the 5' and 3' ends, and a SS-CCE end. The DS domain is composed of self-complementary sequences that anneal to each other in the genomic DNA. The SS-CCE end contains a non-complementary sequence comprising a closed single-stranded region connecting the annealed portion in the DS DS domain. The capsid can be from any parvovirus, including any parvovirus serotype. In a preferred embodiment, the cce parvovirus (ccePV) is a cce adeno-associated virus (cceAAV).

[0044] Over the length of the DS domain, the DNA strands in the DS domain may be fully complementary or partially complementary, so that complementary sequences can anneal to each other to form a stable double-stranded region, and a protruding or circular structure may be formed in the non-complementary region. The non-complementary region may include a deletion or insertion in one or both DNA strands, so that a unique one or more single-stranded DNA regions may be formed after the DNA strands anneal to themselves. The resulting one or more stem structures may comprise at least 5% of the length of the DS domain. The difference between cceAAV and scAAV is that cceAAV can be more broadly defined in a manner that does not require a mutant TR (mTR). scAAV represents a species within the larger cceAAV genus described herein, which has a unique cce end in the form of a mutant ITR (mITR) or shDNA sequence. The method for preparing scAAV cannot produce the cceAAV vector defined herein. However, since the new intermediate template molecule employs two fully functional ITRs, which can be replicated more efficiently in production cells as described above, another advantage of the method of the present application is that a more efficient means for producing scAAV is provided.

[0045] In the linear denatured form, cce PV or cce AAV has a 5' parvoviral left terminal hairpin (LEH) or inverted terminal repeat (ITR), a DS domain, a 3' parvoviral right terminal hairpin (REH) or ITR, and a non-complementary region, which explains the formation of the circular structure in the linear non-denatured form. The 5'ITR and 3'ITR can be the same or different. The DS domain can be completely self-complementary or have some non-complementary sequences, as long as they do not prevent the formation of a stable duplex containing the DS domain formed by the self-annealing or folding back of the single strands. When the DNA strands in cccPV anneal to each other, the homology of the DS domain should allow the formation of a stable duplex DNA in at least one region. The DNA strands in the DS domain can share a region with at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% self-complementarity (or reverse complementarity, in this case) on a portion of the DS domain containing the annealed sequence. However, given that DS domains can accommodate insertions or deletions relative to the self-complementary region, including insertions or deletions of thousands of nucleotides in length, the overall level of homology from one end of the DS domain to the other can be as low as 0.1% or as high as 100%.

[0046] The term "aptamer" refers to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers are typically produced by a selection process utilizing a large random sequence library and have a variety of research, industrial and clinical applications. For example, aptamers can be combined with ribozymes to self-cleave in the presence of their target molecules. In addition, natural aptamers are known to be present in riboswitches. In addition to the traditional function of selectively binding to a target ligand, "aptamers" as used herein may more broadly include deoxyribozymes, including DNA enzymes, DNAzymes, and catalytic DNA comprising DNA oligonucleotides capable of performing specific enzymatic reactions.

[0047] The term "guide RNA-based nuclease" refers to a nuclease that works with a guide RNA molecule to perform DNA editing. The terms "guide RNA" or "gRNA" are used interchangeably to refer to an RNA sequence that is used with a guide RNA nuclease, such as Cas9, during the DNA editing process.

[0048] The term "guide DNA-based nuclease" refers to a structure-guided nuclease (SGN) used with a guide DNA molecule for DNA editing. The terms "guide DNA" or "gDNA" are used interchangeably and refer to a DNA sequence used in the DNA editing process with a guide DNA nuclease, (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs)) and a structure-guided nuclease (SGN) (e.g., a fusion protein comprising flap endonuclease 1 (FEN-1) fused to the cleavage domain of a Fok1 endonuclease).

[0049] The terms "transcription activator-like effector nuclease" and "TALEN" are used interchangeably to refer to a fusion protein comprising a TAL effector DNA binding domain fused to a DNA cleavage domain.

[0050] As used herein, the term "meganuclease" refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs) that is not usually present in a given genome. For example, an 18 base pair sequence recognized by the I-SceI meganuclease requires an average of twenty times the size of the human genome to be found once by chance (although a sequence with a single mismatch occurs approximately 3 times in each human-sized genome). Therefore, meganucleases are considered to be the most specific naturally occurring restriction enzymes. Among meganucleases, the LAGLIDADG family of homing endonucleases has become an important tool for studying genomes and genome engineering in the past 15 years. Meganucleases are "molecular DNA scissors" that can be used to replace, eliminate or modify sequences in a highly targeted manner. By modifying their recognition sequences through protein engineering, the target sequence can be changed. Meganucleases are used to modify all genome types, whether bacteria, plants or animals. They open up vast avenues for innovation, particularly in human health, such as using gene therapy to eliminate viral genetic material or “repair” damaged genes.

[0051] The term "hairpin telomere splitting enzyme" and "protelomerase" are used interchangeably and refer to enzymes that promote the formation of covalently sealed hairpin ends on linear DNA molecules in some bacteriophages, bacterial plasmids and bacterial chromosomes. Telomere splitting enzyme is mechanistically related to tyrosine recombinase and IB type topoisomerase, and is also considered to play a role in the genomic plasticity of characterizing Borrelia species. The duplication of DNA molecules causes the telomere (rTel, also referred to as dimer connection) of duplication, which is identified and processed in the DNA breakage and agglomeration reaction promoted by hairpin type telomere splitting enzyme. The reaction product is a covalently sealed hairpin telomere at the two ends of a linear monomeric DNA molecule. Examples of telomere resolvases include, but are not limited to, telomere resolvases from bacteriophages, such as E. coli phage N15, klebsiella oxytoca phage ΦKO2, Yersinia enterocolitica phage PY54, and bacterial species, such as Agrobacterium tumefaciens, Lyme spirochete Borrelia burgdorferi, relapsing fever borreliae B. hermsii, B. parkeri, B. recurrentis, B. turicatae, and avian spirochete B. anserina.

[0052] The present application generally relates to isolated parvoviral (PV) DNA molecules that, when folded back on themselves, have a covalently closed end (cce) configuration that can be packaged into a parvoviral capsid. The present application further relates to methods of producing covalently closed end parvoviral (ccePV) vectors.

[0053] FIG1 , panel (A) shows a conventional double-stranded (ds) parvoviral vector plasmid, specifically, an adeno-associated virus (AAV) vector plasmid, comprising wild-type ITRs flanking an expression cassette comprising a promoter and a 3' poly A signal operably linked to a gene of interest (GOI). Replication of the ds AAV plasmid in panel (A) initially results in the formation of a single-stranded (ss) AAV DNA template shown in panel (B), which is subsequently converted to a double-stranded DNA template for expression in Figure 2 The second strand shown in is synthesized and then transcribed / translated. Sub-figure (C) shows a conventional ds self-complementary AAV (scAAV) vector plasmid, which comprises a wild-type ITR (wtITR) and a mutant ITR (mITR) flanking an expression cassette, wherein the expression cassette comprises a promoter operably connected to a gene of interest (GOI), and the gene of interest is followed by a 3' poly A signal. The replication of the ds scAAV plasmid in sub-figure (C) initially results in the formation of a single-stranded (ss) scAAV DNA template shown in sub-figure (D), which folds back on itself to form a double-stranded DNA template shown on the right side of sub-figure (D), which provides a template for transcription / translation of the transgenic sequence therein. The two plasmids in sub-figures (A) and (C) can be stably grown in a bacterial host.

[0054] Figure 2 The transcription-translation pathways for transgene expression from different AAV particles and their corresponding DNA templates are shown. AAV particles package a single-stranded DNA genome, similar to those depicted in Figure 1, panels (B) and (D). Conventional recombinant AAV genomes are single-stranded DNA templates that undergo a second-strand DNA synthesis step to form a double-stranded DNA template for DNA replication and transgene expression. Figure 2 , sub-figure (A) shows the process of converting ss DNA to ds DNA through the second strand synthesis step. Sub-figure (B) shows a scAAV comprising duplex DNA with a covalently blocked end (CCE) domain at the 3' end, which is able to promote transcription of the transgene without the intermediate second strand synthesis step shown in sub-figure (A). Because the genome in the scAAV particle is completely self-complementary, it folds back on itself to form a mutant ITR (mITR) at the blocked end, as shown in Figure 2As shown in sub-figure (B). Therefore, the CCE domain in sub-figure (B) is in the form of mITR. In some scAAV variants, the closed cece end includes a shDNA sequence (not shown). Sub-figure (C) shows an exemplary cceAAV, cceAAV-zero loop (zero loop; ZL) according to one embodiment of the present application. The specific structure in sub-figure (C) also has a CCE domain and a completely self-complementary genome, which folds back on itself to form a double-stranded AAV, which contains a pair of ITRs at the 5' end, a double-stranded domain (DS domain), and a CCE domain, as shown in FIG. Figure 2 As shown in panel (C). However, in this case, the CCE domain is not mITR or shDNA. In contrast, the CCE domain in cceAAV-ZL is designed without the need for mTR or shDNA, which would reduce the efficiency of AAV (and / or PV) production. The cce end in panel (C) appears to contain a flush end where the DS domain ends. However, due to steric hindrance in forming a duplex, the single-stranded loop includes at least 2 nucleotides.

[0055] Unlike scAAV, the CCE domain in the ccePV or cceAAV vector of the present application does not require, for example, mITR or shDNA. This allows for increased packaging capacity in cceAAV-ZL compared to otherwise identical scAAV vectors, and also provides many advantages over scAAV vectors, as described below. The ccePV or cceAAV genome can be divided into four elements, the 5' end ITR (or LEH / REH), a double-stranded DNA domain ("DS domain"), a 3' ITR (or LEH / REH), and optionally one or more non-complementary regions forming a single-stranded loop region. More particularly, the DS domain and its complementary DNA strand can have any degree of homology, and can include deletions and insertions, thereby allowing the formation of a wide range of ccePV or cceAAV molecules with different structural configurations.

[0056] For example, Figure 3 Sub-figure (A) shows the Figure 2 Panel (C) shows an embodiment of cceAAV with a much longer single-stranded (ss) DNA non-complementary region in the cceAAV-ZL vector. Figure 3Sub-figure (B) shows another exemplary embodiment of cceAAV comprising a single-stranded loop region (or bulge region) within the complementary double-stranded region. Non-complementary ss DNA may include any DNA sequence, such as a sequence encoding an aptamer / DNAzyme, a sequence encoding a polypeptide, a sequence encoding a miRNA, an shRNA encoding sequence, a polyadenylation signal, or an insulator sequence, etc. The aptamer / DNAzyme can be stabilized by its connection / attachment to the adjacent side-joined complementary DNA sequence. Advantageously, the single-stranded DNA in ccePV-LS or cceAAV-LS provides an effective template for gene editing or DNA repair applications.

[0057] In one aspect, a covalently blocked end parvovirus (ccePV) comprises cce PV DNA packaged in a PV capsid. After infection, second strand synthesis is not necessary for transgene transcription. This is even more advantageous for parvovirus vectors that package only one polarity of the vector. The ccePV genome comprises (1) a double-stranded (DS) domain having a first strand having a 3' end and a 5' end, a second strand having a 3' end and a 5' end, (2) a 3' parvoviral ITR linked to the 3' end of the first strand, and (3) a 5' parvoviral ITR linked to the 5' end of the second strand, and (4) a covalently blocked end (cce) domain that covalently links the 5' end of the first strand to the 3' end of the second strand. When the parvovirus is not AAV, the CCE domain can be any sequence.

[0058] The present application includes additional embodiments having a bulge region in the DS domain, or having a long loop segment with mTR or shDNA at the cce end or in the CCE domain. Such vector constructs cannot be produced using existing methods known in the art, and such special molecules / vectors are not possible using existing technology.

[0059] The cce capsid and cce genome of the present application can be derived from the same parvovirus or different parvoviruses. In some embodiments, the cce capsid and cce genome of the present application are derived from the same parvovirus and the same serotype (e.g., capsid based on AAV2 and viral genome based on AAV2). In some embodiments, the cce capsid and cce genome of the present application are derived from the same parvovirus, but have different serotypes (e.g., capsid based on AAV2 and viral genome based on AAV8). In some embodiments, the cce capsid and cce genome of the present application are derived from different parvoviruses (e.g., capsid based on AAV2 and viral genome based on B19). In some embodiments, the cce capsid is an AAV capsid, and the cce genome is a recombinant AAV genome comprising AAV ITR.

[0060] DS domain and CCE domain

[0061] In some embodiments, the first and second strands of the DS domain of the parvovirus of the present application are 100% complementary to each other, and the CCE domain consists of a single-stranded DNA of 0 to 50 nucleotides (cceAAV-ZL). In some other embodiments, the CCE domain consists of 0-5, 0-10, 0-15, 0-20, 0-25, 0-30, 0-35, 0-40, 0-45, 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 10-15, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45, 10-50, 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 20-25, 20-30, 20-35, 20-40, 20-45, 20-50, 25-30, 25-35, 25-40, 25-45, 25-50, 30-35, 30-40, 30-45, 30-50, 35-40, 35-45, 35-50, 40-45, 40-50 or 45-50 nucleotides of single-stranded DNA.

[0062] In some embodiments, the first and second strands of the DS domain of the parvovirus of the present application are 100% complementary to each other, and the CCE domain is dominated by single-stranded DNA (occupying => 50%) (cceAAV-LS). In some embodiments, the CCE domain comprises a DNA aptamer / DNAzyme. In some other embodiments, the CCE domain consists of 51-60, 51-75, 51-100, 51-200, 51-500, 51-700, 51-1000, 51-2000, 51-3000, 51-4000, 60-75, 60-100, 60-200, 60-500, 60-700, 60-1000, 60-2000, 60-3000, 60-4000, 75-100, 75-200, 75-500, 75-700, 75-1000, 75-2000, 75-3000, 75-4000, 100-200, 100-500, 100-700, or 3000-4000 nucleotides.

[0063] In some embodiments, the first and second chains of the DS domain of the parvovirus of the present application are not 100% complementary to each other, but have a certain degree of homology, thereby allowing the formation of a stable duplex in one or more regions when the chains are folded back on each other. In some embodiments, the first and second chains of the DS domain share at least 20%, 30%, 40%, 50%, 60%, 80%, 90% or 95% sequence identity.

[0064] In some embodiments, the first strand in the DS domain of the parvovirus of the present application has one or more regions complementary to the second strand, and one or more regions not complementary to the second strand (cceAAV-BR). In some embodiments, the one or more regions not complementary to the second strand comprise shDNA or a DNA aptamer.

[0065] In some embodiments, the second strand in the DS domain of the parvovirus of the present application has two or more regions complementary to the first strand, and one or more regions not complementary to the first strand (ccePV-BR). In some embodiments, the one or more regions not complementary to the first strand comprise shDNA or a DNA aptamer.

[0066] In some embodiments, one or both of the first and second chains in the DS domain comprise a deletion or insertion relative to the other chain. Such a deletion or insertion allows a raised or circular DNA region to be formed in the DS domain. In some embodiments, the raised DNA region comprises a shDNA sequence or a DNA aptamer. In some embodiments, a plurality of raised or circular DNA regions can be formed. The raised DNA region can be one or more nucleotides, and the length can reach one or more kilobases, provided that it does not unfavorably exceed (with other DNA sequences) the packaging capacity of the ccePV vector. In some embodiments, the raised DNA sequence can be folded into a DNA aptamer. The double-stranded region in the DS domain contributes to stabilizing the DNA aptamer. In some embodiments, the raised DNA comprises regions that are complementary to each other and form double-stranded stems in the raised DNA region. In some embodiments, the single-stranded DNA sequence in the raised DNA region in the DS domain, or the single-stranded DNA sequence in the CCE domain is used as a template for gene editing, DNA repair or DNA recombination. In some embodiments, the length of the raised region is at least 2, 3 or 4 nucleotides.

[0067] In some embodiments, the DS domain encodes a protein. In some embodiments, the DS domain encodes an inhibitory RNA (iRNA) product, such as an shRNA or a microRNA (miRNA). In some embodiments, the DS domain comprises one or more expression cassettes, each independently ranging in size from about 0.2 kb to about 3 kb, with a total of no more than 4 kb.

[0068] ITR

[0069] The 5' ITR and the 3' ITR may be the same or different. In the linear denatured form, the 5' ITR is located at the 5' end of the cce genome and the 3' ITR is located at the 3' end of the cce genome.

[0070] In some embodiments, one of the ITRs may support DNA replication but lose the function of initiating packaging. This would allow one polarity of the vector to be generated, which would be necessary when annealing of both positive and negative polarity of the vector is not desired.

[0071] cceDNA generation

[0072] The carrier DNA of cceDNA is generated by ligation or prototelomerase reaction. Ligation can be performed in vivo or in vitro. There is a difference between the ligation of typical subclones and the ligation here. The ligation here generates template molecules for cce carrier DNA, which are usually unstable in bacteria. Therefore, the in vitro ligation product is directly used for production. Alternatively, PCR or LAMP amplification can be used to further amplify the product of in vitro ligation, which is then used for vector production. In addition, digestion can be performed in vivo (host cells) and ligated using host enzymes, followed by vector production.

[0073] Structural advantages of the ccePV of this application

[0074] As described above, when the DNA chains in the ccePV template are folded / annealed to each other, the CCE parvovirus genome of the present application comprises a double-stranded domain (DS domain) with parvovirus ITR / LEH / REH upstream of the DS domain and a covalently closed end downstream of the DS domain. The genome of the ccePV vector of the present application can have any nucleotides in the CCE domain and can accommodate any length from 0 nucleotides to the allowed length, which is based on the characteristic size / packaging restrictions of a particular parvovirus vector (excluding the length of the ITR and DS domains). In addition, the ccePV of the present application does not require 100% complementarity in the DS domain and allows branched DNA sequences at any point in the DS domain.

[0075] In the case of cceAAV, the genome of the cceAAV vector of the present application is not limited to mTR or shDNA, and can accommodate a wide range of insertions in the CCE domain. The elimination of mTR or shDNA sequences in the CCE domain increases the effective packaging capacity and improves the vector quality by eliminating ineffective conversion of the viral genome caused by mTR or shRNA (which can lead to contamination of plasmid sequences). In addition, the cceAAV of the present application does not require 100% complementarity in the DS domain, and allows branched DNA sequences at any point in the DS domain.

[0076] Functional advantages of ccePV of this application

[0077] Compared to the self-complementary adeno-associated virus (scAAV) containing mTR or shDNA in the CCE domain (which is the only one required for virus production), the CCE domain in the cceAAV of the present application can be fully utilized. The CCE domain can contain any sequence, and can be part of a regulatory element (e.g., intron, promoter), a gene coding region, or an element encoding a functional RNA molecule (e.g., miRNA, antisense RNA, ribozyme, guide DNA). The CCE domain can also encode a functional DNA molecule, such as a single-stranded DNA template for gene editing, a guide DNA for a DNA-guided endonuclease, or a DNA aptamer. In some embodiments, the CCE domain also encodes shDNA, which is separated from the DS domain by a single-stranded DNA region of at least 10 nucleotides at both ends of the shDNA. In some embodiments, the CCE domain contains a promoter that drives the expression of two different expression cassettes. In other embodiments, cceAAV or ccePV can be developed as a gene switch that turns gene expression on and off ( Figure 14-16 ).

[0078] The ccePVs described herein have the advantage of bypassing the requirements for positive and negative strand annealing and second strand DNA synthesis, thereby providing enhanced vector genome stability and improved gene expression.

[0079] Since there is a self-complementary region in the genome of the ccePV of the present application, the ccePV of the present application has enhanced in vivo performance. The genome of the ccePV of the present application is more stable than that of the single-stranded DNA parvoviral vector. In the embodiment where the aptamer / DNAzyme is introduced into the CCE domain of the cce genome, the aptamer is more stable because the aptamer region is connected to the self-complementary DS domain of the cce genome.

[0080] like Figure 4 As shown, ccePV without mismatched nucleotides in the DS region, without protruding loops or stems in the DS region, or without single-stranded loops at the CCE domain has improved gene expression efficiency because the cce genome of the present application bypasses the need for second-strand DNA synthesis. This is particularly advantageous for ccePV carrying DNA aptamers in its genome, because many parvoviruses contain negative-sense DNA and are packaged in the 3' to 5' direction; when there is only one polarity, the DNA aptamer in the ccePV genome does not have a complementary strand, and the action of the aptamer is not adversely affected by the complementary strand.

[0081] The present invention allows the preparation of self-complementary vectors without mutated ITRs. The rescue efficiency of mutated ITRs is low, which also allows packaging of undesired sequences. This new invention not only improves vector yield, but also improves vector quality.

[0082] In this new invention, the production material actually has two ITRs as a normal vector, which is very efficient. In addition, due to the self-complementary nature of the sequence, such DNA cannot be prepared in bacteria because it is unstable in bacteria. In addition, nuclease digestion of cceAAV and ccePV in vivo is very efficient.

[0083] The present invention avoids the use of palindromic sequences or hairpin DNA for rescue. These are similar advantages to mutating ITRs.

[0084] The present invention allows for additional control of gene expression in host cells through changes in the DNA template caused by second strand DNA synthesis or annealing. Second strand DNA synthesis can result in the generation or elimination of a functional template. Many configurations of the AAV genome can only be made by the present invention. Parvoviral vectors can be used to express dsRNA with a single promoter.

[0085] To produce dsRNA in a parvoviral vector, it may be necessary to use two promoters in the vector, one in the negative strand and one in the positive strand. However, this approach reduces vector capacity, and the molecules must be annealed to form the dsRNA. In the present invention, the dsRNA is in a single RNA transcript, and only one promoter is required.

[0086] Method for preparing ccePV

[0087] Another aspect of the present application relates to a method for producing the ccePV of the present application. A major problem with producing ccePV or cceAAV vectors by conventional methods is that, due to long segments of DNA repetition, direct template molecules containing ccePV and cceAAV genomes are unstable and incompatible with reproduction in bacterial host cells. Conventional scAAV vector plasmids avoid this problem by including mTR or shDNA sequences and self-complementary regions during AAV production. However, this method has certain disadvantages. For example, the scAAV vector genome is fully complementary in the stem region (corresponding to the DS domain of the ccePV genome of the present application). There is no possibility of a raised region in the double-stranded stem. In addition, scAAV has mTR or shDNA at the end of the stem region (corresponding to the CCE domain of the ccePV genome of the present application). The presence of mTR or shDNA reduces the capacity of the scAAV vector to accept further insertion of the scAAV vector. The method of the present application eliminates the need for mTR or shDNA in the CCE domain. When used in the conventional sense, mITR or shDNA cannot efficiently convert input DNA (ie, plasmid duplex) into a direct DNA template compatible with replication and encapsidation during scAAV production and often results in contaminants with short vector genomes.

[0088] The one or more starting materials used to prepare ccePV or cceAAV include one or more template vectors in the form of plasmids, yeast shuttle vectors, viruses and / or chemically synthesized templates. In one embodiment, the one or more starting materials include one or more plasmids, at least one of which contains a parvoviral ITR, LEH or REH, followed by a DS domain and one or more endonuclease cleavage sites. Restriction sites can be designed to avoid leaving any unnecessary sequences in the vector. Digestion of the one or more templates generates one or more DNA fragments containing the ITR and DS domains. In some embodiments, the one or more starting materials include one or more plasmids, at least one of which contains a parvoviral ITR, LEH or REH, followed by a DS domain and one or more endonuclease cleavage sites. Fig.17 In the embodiments exemplified in the foregoing, one or more of the plasmids do not comprise the parvoviral ITR, LEH or REH or DS domains described herein.

[0089] To produce ccePV or cceAAV viral particles for nucleic acid transfer applications according to the present application, Figure 5-22 As shown, the ccePV DNA template molecule for replication is first prepared (by recombinant DNA technology), then subjected to extracellular digestion and / or ligation, intracellular digestion and / or ligation, or a combination thereof, and then transfected or stably integrated into suitable PV production cells.

[0090] In a specific embodiment, the method for producing the ccePV or cceAAV particles of the present application (as described herein) comprises the following steps: introducing the PV template molecules into a suitable host cell under conditions that allow parvovirus replication; maintaining the cells under conditions sufficient to produce ccePV vector particles; and harvesting the ccePV from the cultured host cells.

[0091] In order to provide conditions suitable for parvovirus replication, parvovirus helper gene expression plasmids encoding, for example, PV rep and cap genes can be transiently or stably transfected into host cells suitable for PV production, including but not limited to 293 cells. Alternatively, a suitable helper virus can be introduced into the host cell to provide the necessary helper virus functions for PV production.

[0092] In some embodiments, one or more ccePV template molecules can be transiently or stably transfected into host cells suitable for PV production, including but not limited to 293 cells. Alternatively, one or more ccePV template molecules can be introduced into host cells by infection with a suitable vector virus such as vaccinia virus, adenovirus, herpes virus, baculovirus, etc.

[0093] In some embodiments, the in vivo method of gene transfer of ccePV viral particles for expression of one or more proteins and / or delivery of one or more non-coding sequences comprises the step of administering to a host an effective amount of ccePV viral particles, such that the one or more proteins are expressed in the host cells, or the one or more non-coding sequences are delivered to the host cells for their intended purpose, or a combination thereof. In a preferred embodiment, the host is a mammal. In some embodiments, the ccePV viral particles contain a nucleic acid comprising an expression cassette encoding a therapeutic protein or therapeutic RNA in a subject in need thereof.

[0094] 1. In vitro digestion and in vitro ligation of a single DNA template

[0095] In one embodiment, if Figures 5 to 6 As shown, a single ccePV or cceAAV template molecule is digested and ligated extracellularly and then transfected into PV production cells. One or more digested fragments can be self-ligated in vitro using DNA ligase. This can generate a DNA template molecule for the production of cceAV or ccePV with a zero-loop (ZL) sequence (see, e.g. Figures 5 to 6 ).

[0096] The DNA template for PV replication comprises PV or AAV ITR, DS domain, and one or more endonuclease cleavage sites that are cleaved by one or more nucleases. The one or more cleavage sites can be cleaved with restriction enzymes, large-range nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), CRISPR / Cas9 nucleases based on guide RNA, other nucleases, or a combination thereof. The fragments can then be ligated with DNA ligase. The resulting fragments can be used for parvovirus production with or without amplification. The amplification method can be performed in vitro using PCR and rolling circle phi29 type polymerase.

[0097] Transfection of the self-ligated fragments into PV producer cells compatible with PV or AAV vector particle production results in the formation of ccePV or cceAAV according to the inventive subject matter described herein. Other cis or trans functions required for parvovirus / AAV vector production are provided by transfection, viral vector delivery or stable integration of helper genes into the host cells.

[0098] 2. In vitro digestion and in vitro ligation of two or more DNA template molecules

[0099] In some embodiments, the method for producing ccePV or cceAAV comprises using two or more DNA template molecules (including at least one DNA template molecule containing at least one AAV ITR and one DS domain) to cleave with a restriction enzyme, a meganuclease, a transcriptase activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), an RNA-based CRISPR / Cas9 nuclease, other nucleases, or a combination thereof, such as Figures 8 to 18 The use of two or more template molecules allows the production of PV or AAV particles having genomes that contain one or more extended protruding, circular or branched structures in the CCE domain.

[0100] In order to generate ccePV or cceAAV with branches or loops, two or more starting plasmids are usually used. This can be a combination of plasmids, viral vectors or synthetic DNA fragments. The designated sites in the two starting plasmids can be the same or completely different. In order to avoid self-ligation of cce vectors that may cause contamination, asymmetric enzyme digestion or clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 digestion is used. Asymmetric properties and recognition sequence length can effectively prevent undesired self-ligation.

[0101] Since there are two fragments from the plasmid for ligation, common molecular biology techniques must be used to create DNA ends that will not self-ligate (asymmetric digestion using restriction enzymes and CRISPR / Cas9 enzymes will achieve this goal). The fragments can be ligated with DNA ligase. The fragments used for ligation are different. The resulting fragments can be used for parvovirus production with or without amplification. Amplification methods can be performed in vitro using PCR and rolling circle phi29 type polymerase.

[0102] 3. In vitro digestion of a single DNA template without in vitro ligation

[0103] In another embodiment, a single DNA template (e.g., a plasmid) is digested in vitro and the digested fragments are transfected into a host cell suitable for AAV production along with one or more parvoviral helper genes (e.g., Rep, Cap, and pAd (a miniadenovirus that provides additional helper genes). The host cell can be transiently or stably transformed with one or more of these helper genes. The transfected fragments are then ligated intracellularly in vivo using the host's cellular ligases.

[0104] The plasmid carrying at least one AAV ITR and one DS domain is cleaved with restriction enzymes, meganucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), guide RNA-based CRISPR / Cas9 nucleases, other nucleases, or a combination thereof. The resulting fragments will be transfected into host cells for parvovirus production. Host cell ligases will facilitate ligation to generate vector DNA sequences for vector production.

[0105] 4. In vitro digestion of two or more DNA templates without in vitro ligation

[0106] Two plasmids carrying at least one AAV ITR and one DS domain are cleaved with restriction enzymes, or other meganucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or CRISPR / Cas9 nucleases based on guide RNAs. Since there are two fragments from the plasmids for ligation, common molecular biology techniques must be used to create DNA ends that will not self-ligate (asymmetric digestion using restriction enzymes and CRISPR / Cas9 enzymes will achieve this goal). The resulting fragments will be transfected into host cells for parvovirus production. Host cell ligases will facilitate ligation to generate vector DNA sequences for vector production.

[0107] 5. In vivo Digestion and In vivo Ligation of Single DNA Template

[0108] In yet another embodiment, a single DNA template molecule, such as Figures 5 to 6 The template described in is transfected into a host cell, and then the in vivo digestion and in vivo connection of the DNA template therein are performed. The enzyme that cuts the specified site is co-transfected into the host cell. Alternatively, the enzyme can be delivered by a viral vector, such as a vaccinia, herpes virus vector, adenovirus vector or other common viral and non-viral vectors. The enzyme can be delivered to the host cell or integrated into the host cell by physical methods. When the plasmid is connected in vivo, some insertions or deletions are usually observed at the digestion site. Generally, small deletions or insertions will not affect the performance of the vector.

[0109] In a specific embodiment, Fig.19As shown, the method for producing cceAAV or ccePV with a symmetrical genome includes the use of a telomere splitting enzyme (e.g., ResT) delivered to a host cell together with a cce DNA molecule carrying a parvovirus ITR and DS domain. The use of a telomere splitting enzyme can promote the formation of a template molecule for cceAAV or ccePV production, which can be transfected into a parvovirus or AAV production cell line, thereby producing ccePV and cceAAV. Alternatively, a DNA molecule with a parvovirus ITR, DS domain, and a telomere splitting enzyme site can be transfected into a host cell, or delivered to a host cell by a viral vector or stably integrated into a host cell, thereby expressing the telomere splitting enzyme in vivo. Then, the telomere splitting enzyme will act on the starting DNA molecule to generate a molecule for ccePV or cceAAV production.

[0110] Plasmids or viral vectors carrying at least one AAV ITR and one DS domain are transfected or infected into host cells. Restriction enzymes or other large-range nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, or guide RNA-based CRISPR / Cas9 nucleases are co-transfected into host cells, or carried by viral vectors or integrated into host cells and used to digest the plasmid at the desired site. The resulting fragments will be ligated by host cell ligases to generate vector DNA sequences for vector production.

[0111] 6. In vivo ligation of two or more DNA template molecules

[0112] Two plasmids or viral vectors carrying at least one AAV ITR and one DS domain are transfected or infected into a host cell. Restriction enzymes or other large-range nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or CRISPR / Cas9 nucleases based on guide RNAs can be co-transfected into host cells, delivered by viral vectors, or stably integrated into host cells, where these enzymes can digest the plasmid at the desired site. Since there are two fragments from the plasmid for connection, common molecular biology techniques must be used to create DNA ends that will not self-connect (asymmetric digestion using restriction enzymes and CRISPR / Cas9 enzymes will achieve this goal). The resulting fragments will be connected by host cell ligases to generate a vector DNA sequence for vector production.

[0113] 7. In vivo digestion and in vivo ligation of one or more integrated DNA template molecules

[0114] In this method, two DNA fragments are integrated adjacent to each other into the host chromosome. CRISPR / Cas9-mediated digestion will eliminate the space between them and allow the desired vector to be produced.

[0115] In one embodiment, the parvovirus ITR or AAV ITR together with the DS domain and the designated digestion site is delivered via a viral vector (e.g., an adenovirus vector, a herpes virus vector, etc.). The enzyme that digests the template molecule can be transfected into the cell, delivered via a viral vector, or stably integrated into the host cell. After providing the other basic elements of the parvovirus or AAV vector, ccePV or cceAAV can be produced.

[0116] The enzyme used to digest the specified site can be a common restriction nuclease (see: Restriction Enzyme Database (REBASE), zinc finger nuclease, transcription activator-like (TAL) effector nuclease (TALEN), meganuclease and hybrid meganuclease (a large library containing tens of thousands of protein units has been established. These units can be combined to obtain chimeric meganucleases that recognize the target site), clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) system.

[0117] In some embodiments, one or more ccePV template molecules are introduced into a host cell and integrated into a host cell chromosome. In some embodiments, the 5'ITR, the first strand of the DS region, and a specified digestion site are integrated into a site on the host cell chromosome. Downstream, the specified site, the second strand of the DS domain, and the 3'ITR are integrated into the same chromosome. Digestion by a suitable nuclease (e.g., a CRISPR / Cas9 system based on a guide RNA) will remove the spacer sequence between the two regions, which will be compatible with the rescue and production of ccePV.

[0118] Method for preparing scAAV with mTR and shDNA sequences

[0119] The above methods can also be used to generate scAAV with mTR or shDNA. Conventional methods for generating scAAV rely on the inclusion of mTR or shDNA to provide a mechanism that can generate complementary strands during the in vivo rAAV genome rescue step. This conversion efficiency is relatively low, often resulting in the formation of byproducts that reduce vector production and leading to the generation of defective interfering particles or vectors with short genomes.

[0120] The above-described methods for producing cceAAV or ccePV can also be used to produce scAAV vectors using the digestion / ligation strategy outlined herein or using the described telomere splitting enzyme protocol. As described above, since the currently described method already has two fully functional ITRs, the digestion / ligation protocol is superior to conventional scAAV production methods involving mTR or shDNA that utilize template molecules for scAAV production. For example, the present application further provides an improved method for producing scAAV with mTR or shDNA by creating a molecule with the desired closed end for direct transfection. This can be achieved by directly connecting the fragment to the closed end. Alternatively, the closed end can be produced using a telomere splitting enzyme or telomerase. Such molecules provide a more efficient substrate for the production of scAAV vectors with mTR or shDNA (see Figures 23 to 25 and its accompanying drawings).

[0121] Methods of using the ccePV and cceAAV of the present application

[0122] Another aspect of the present application relates to a method for treating a therapeutic condition with the ccePV or cceAAV of the present application, the method comprising the step of administering an effective amount of the ccePV of the present application to a subject in need of such treatment, wherein the ccePV expresses one or more products that have a therapeutic benefit in treating the therapeutic condition.

[0123] Other Implementations

[0124] In some embodiments, the present application relates to a vector having a DNA genome, which DNA genome comprises in a single-stranded form in the 5' to 3' direction: (1) a parvoviral terminal repeat at the 5' end; (2) a first regulatory region comprising (a) a promoter region, or (b) a poly A or poly T sequence, or both (a) and (b), and (3) a first heterologous nucleotide sequence, (4) a covalently blocked end (CCE) domain, (5) a second heterologous nucleotide sequence, (6) a first regulatory region comprising (a) a promoter region, or (b) a poly A or poly T sequence, or both (a) and (b), and (7) a parvoviral terminal repeat at the 3' end; wherein the first regulatory sequence is complementary to the second heterologous sequence and forms a double-stranded region (DS domain) in the vector genome, wherein the CCE domain is (a) a single covalent bond connecting the first heterologous nucleotide sequence and the second nucleotide sequence, or (b) a single-stranded DNA region, and wherein the vector DNA genome is capable of producing a double-stranded (DS) RNA molecule from the DS domain of the vector genome.

[0125] In some embodiments, the DS domain is discontinuous with a self-annealing stem portion comprising more than 20 consecutive nucleotide base pairs from a mutant inverted terminal repeat (mTR) sequence or a short hairpin RNA (shRNA) encoding sequence.

[0126] In some embodiments, the CCE domain does not comprise an mTR sequence or an shRNA encoding sequence, or both.

[0127] The following paragraphs, listed consecutively from 1 to 47, provide various aspects and / or embodiments of the present invention.

[0128] 1. A dual parvoviral particle comprising: a parvoviral capsid; and a vector genome, the vector genome comprising in the 5' to 3' direction: (a) a parvoviral terminal repeat at the 5' end; (b) a parvoviral terminal repeat at the 3' end; (c) a double-stranded (DS) DNA domain between the terminal repeats in (a) and (b), the DS-DNA domain comprising a first heterologous nucleotide sequence that is self-complementary and anneals to a second heterologous nucleotide sequence; and (d) a single-stranded covalently closed end (SS-CCE) domain between the first heterologous nucleotide sequence and the second heterologous nucleotide sequence, the SS-CCE domain comprising a loop structure; wherein the DS domain is discontinuous with a self-annealing stem portion, the self-annealing stem portion comprising more than 20 consecutive nucleotide base pairs from a mutant inverted terminal repeat (mTR) sequence or a short hairpin DNA (shDNA) sequence, and wherein the vector genome is capable of replicating to form the dual parvoviral particle when introduced into a host cell, the host cell expressing a helper function sufficient to produce the dual parvoviral particle.

[0129] 2. The parvoviral particle of paragraph 1, wherein the SS-CCE domain does not contain more than 20 consecutive nucleotides from a mutant inverted terminal repeat (mTR) or short hairpin DNA (shDNA) sequence.

[0130] 3. The parvoviral particle of paragraph 1 or 2, wherein the SS-CCE domain comprises 3 to 4,000 nucleotides, 50 to 4,000 nucleotides, 1,000 to 4,000 nucleotides, 3 to 2,000 nucleotides, 50 to 2,000 nucleotides, 250 to 2,000 nucleotides, or 500 to 2,000 nucleotides.

[0131] 4. The parvoviral particle of any one of paragraphs 1 to 3, wherein the SS-CCE domain comprises a sequence encoding a protein or RNA.

[0132] 5. The parvoviral particle of paragraph 4, wherein the sequence encoding the protein or RNA further comprises a downstream polyadenylation signal operably linked thereto.

[0133] 6. The parvoviral particle of any one of paragraphs 1 to 5, wherein the first heterologous nucleotide sequence has greater than 95%, greater than 99% or 100% reverse complementarity with the second heterologous nucleotide sequence.

[0134] 7. The parvoviral particle of any one of paragraphs 1 to 6, wherein the DS domain comprises a promoter operably linked to a nucleic acid encoding a protein or RNA.

[0135] 8. The parvoviral particle of paragraph 7, wherein the DS domain further comprises a polyadenylation signal operably linked to the nucleic acid encoding the protein or RNA.

[0136] 9. The parvoviral particle of any one of paragraphs 1 to 5, wherein the DS domain is interrupted by circular or branched single-stranded DNA.

[0137] 10. The parvoviral particle of paragraph 9, wherein the circular or branched single-stranded DNA comprises nucleotides encoding a protein or RNA.

[0138] 11. The parvoviral particle of paragraph 10, wherein the sequence encoding the protein or RNA further comprises a downstream polyadenylation signal operably linked thereto.

[0139] 12. The parvoviral particle of any of paragraphs 1 to 11, wherein the parvoviral terminal repeat in (a) comprises a left terminal hairpin (LEH), and the terminal repeat in (b) comprises a right terminal hairpin (REH).

[0140] 13. The parvoviral particle of any one of paragraphs 1 to 11, wherein the parvoviral terminal repeats comprise adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0141] 14. A method for preparing a parvoviral particle as described in paragraph 1, the method comprising: (a) providing a plasmid comprising a fragment, wherein the fragment comprises, in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter operably linked to a nucleic acid encoding a protein or RNA; and (iii) a nuclease cleavage site in the plasmid; (b) providing a host cell comprising one or more auxiliary genes sufficient to replicate the parvoviral terminal repeat comprising the nucleic acid of interest flanking the parvoviral terminal repeat; (c) digesting the plasmid with a nuclease specific for the nuclease cleavage site under conditions sufficient to linearize the plasmid, wherein the plasmid is digested in vitro or in the host cell; (d) ligating the plasmid to itself with a DNA ligase, wherein the plasmid is ligated in vitro or in the host cell; (e) after completing step (d), culturing the host cell containing the plasmid treated according to steps (c) and (d) under conditions suitable for producing parvoviral particles; and (f) recovering the parvoviral particles produced in step (f).

[0142] 15. The method of paragraph 14, wherein the fragment comprises parvovirus LEH.

[0143] 16. The method of paragraph 14, wherein the fragment comprises an AAV ITR.

[0144] 17. The method of any one of paragraphs 14 to 16, wherein the plasmid further comprises a subfragment downstream of the fragment, wherein the subfragment comprises in the 5' to 3' direction: (i) a telR binding site; (b) a TelN nuclease recognition cleavage site; and (iii) a tell binding site, and wherein the plasmid is digested with the TelN enzyme and self-ligated; and wherein the plasmid is digested and self-ligated in vitro, or digested and self-ligated in the host cell.

[0145] 18. A method for preparing a parvoviral particle as described in paragraph 1, the method comprising: (a) providing a first plasmid comprising a first segment, the first segment comprising in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter operably linked to a nucleic acid encoding a protein or RNA; and (iii) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, the second segment comprising in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a right-end hairpin (REH) or an ITR; (ii) a promoter; (iii) a nucleic acid encoding a protein or RNA; and (iv) a second nuclease cleavage site in the second plasmid, wherein the second segment is identical to the first segment except that the second segment further comprises one or more consecutive nucleotide base pairs not present in the first segment; (c) providing a host cell comprising one or more auxiliary genes, the auxiliary genes (d) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(iii) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (e) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(iv) under conditions sufficient to linearize the second plasmid, wherein the second plasmid is digested in vitro or in the host cell; (f) ligating the first plasmid and the second plasmid digested in steps (d) and (e) with a DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell; (g) after completing step (f), culturing the host cell containing the first plasmid and the second plasmid treated according to steps (d) to (f) under conditions suitable for producing parvoviral particles; and (h) recovering the parvoviral particles produced in step (g).

[0146] 19. The method of paragraph 18, wherein the first fragment comprises parvoviral LEH and the second fragment comprises parvoviral REH.

[0147] 20. The method of paragraph 18, wherein the first segment and the second segment each comprise an AAV ITR.

[0148] 21. The method of any of paragraphs 18 to 20, wherein the second fragment comprises a subfragment between (b)(iii) and (b)(iv).

[0149] 22. The method of paragraph 21, wherein the subfragments are 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0150] 23. The method of any of paragraphs 18 to 20, wherein the second fragment comprises a subfragment between (b)(ii) and (b)(iii).

[0151] 24. The method of paragraph 23, wherein the subfragments are 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0152] 25. A method for preparing a parvoviral particle as described in paragraph 1, the method comprising: (a) providing a first plasmid comprising a first segment, the first segment comprising in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter; (iii) a nucleic acid encoding a protein or RNA; (iv) a polyadenylation signal; and (v) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, the second segment comprising in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a right-end hairpin (REH) or an ITR; (ii) a promoter; and (iii) a second nuclease cleavage site in the second plasmid; (c) providing a host cell comprising one or more helper genes sufficient for the replication of a nucleic acid comprising the parvoviral terminal repeat flanking a nucleic acid of interest; (d) Digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(v) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (e) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(iii) under conditions sufficient to linearize the second plasmid, wherein the second plasmid is digested in vitro or in the host cell; (f) ligating the first plasmid and the second plasmid digested in steps (d) and (e) with a DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell; (g) after completing step (f), culturing the host cell containing the first plasmid and the second plasmid treated according to steps (d) to (f) under conditions suitable for producing parvoviral particles; and (h) recovering the parvoviral particles produced in step (g).

[0153] 26. The method of paragraph 25, wherein the first fragment comprises parvoviral LEH and the second fragment comprises parvoviral REH.

[0154] 27. The method of paragraph 25, wherein the first segment and the second segment each comprise an AAV ITR.

[0155] 28. The method of any of paragraphs 25 to 27, wherein the nucleic acid encoding the protein or RNA in (a)(iii) is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0156] 29. A method for preparing a parvoviral particle as described in paragraph 1, the method comprising: (a) providing a first plasmid comprising a first segment, wherein the first segment comprises in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter; (iii) a first polyadenylation signal; (iv) a reverse complementary nucleic acid encoding a first protein or a first RNA, wherein the first protein and the first RNA each have a right-to-left polarity; (v) a nucleic acid encoding a second protein or a second RNA, wherein the first protein and the first RNA each have a right-to-left polarity; The second protein and the second RNA each have a left-to-right polarity; (vi) a second polyadenylation signal; and (vii) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, the second segment comprising in a 5' to 3' direction: (i) a parvoviral terminal repeat comprising a right end hairpin (REH) or an ITR; (ii) the promoter in (a) (ii); (iii) a nucleic acid encoding the second protein or the second RNA, the second protein and the second RNA each have a left-to-right polarity; (iv) a polyadenylation signal; and (viii) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, the second segment comprising in a 5' to 3' direction: (i) a parvoviral terminal repeat comprising a right end hairpin (REH) or an ITR; (ii) the promoter in (a) (ii); (iii) a nucleic acid encoding the second protein or the second RNA, the second protein and the second RNA each have a left-to-right polarity; and (iv) a polyadenylation signal. adenylation signal; and (v) a second nuclease cleavage site in the second plasmid; (c) providing a host cell comprising one or more accessory genes sufficient to replicate a nucleic acid comprising parvoviral terminal repeats flanking a nucleic acid of interest; (d) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(vii) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (e) cleaving the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(v) under conditions sufficient to linearize the second plasmid (f) ligating the first plasmid and the second plasmid digested in steps (d) and (e) with a DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell; (g) after completing step (f), culturing the host cell containing the first plasmid and the second plasmid treated according to steps (d) to (f) under conditions suitable for producing parvoviral particles; and (h) recovering the parvoviral particles produced in step (g).

[0157] 30. The method of paragraph 29, wherein the first fragment comprises parvoviral LEH and the second fragment comprises parvoviral REH.

[0158] 31. The method of paragraph 29, wherein the first segment and the second segment each comprise an AAV ITR.

[0159] 32. The method of any of paragraphs 29 to 31, wherein the nucleic acid encoding the protein or RNA in (a)(iv), (a)(v), or both, is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0160] 33. The method of any of paragraphs 29 to 31, wherein the nucleic acid encoding the protein or RNA in (b)(iii) is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0161] 34. A method for preparing a parvoviral particle as described in paragraph 1, the method comprising: (a) providing a first plasmid comprising a first segment, the first segment comprising in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter having a left-to-right polarity; (iii) a nucleic acid encoding a first protein or a first RNA having a left-to-right polarity; (iv) a polyadenylation signal having a left-to-right polarity, and (v) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, the second segment comprising in the 5' to 3' direction: (i) a first nuclease cleavage site; (ii) a promoter having a right-to-left polarity; (c) providing a third plasmid comprising a third segment, the third segment comprising in a 5' to 3' direction: (i) a parvoviral end fragment comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); terminal repeats; (ii) the promoter with left-to-right polarity in (a) (ii); (iii) the nucleic acid encoding the second protein or the second RNA in (b) (iv), wherein the second protein and the second RNA each have left-to-right polarity; (iv) a polyadenylation signal with left-to-right polarity, and (v) a third nuclease cleavage site unique to the third plasmid, wherein the cleavage site in (b) (iv) is the same as the cleavage site in (c) (v); (d) providing a host cell comprising one or more auxiliary genes, wherein the auxiliary genes are sufficient to replicate the nucleic acid comprising the parvoviral terminal repeats flanking the nucleic acid of interest; (e) under conditions sufficient to linearize the first plasmid, using digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(v), wherein the first plasmid is digested in vitro or in the host cell; (f) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(i) under conditions sufficient to digest the second plasmid and digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(vi), wherein the second plasmid is digested in vitro or in the host cell; (g) digesting the third plasmid with a nuclease specific for the nuclease cleavage site in (c)(v) under conditions sufficient to linearize the third plasmid, wherein the third plasmid is digested in vitro or in the host cell;(h) ligating the linearized first plasmid fragment, the second fragment and the linearized third plasmid fragment together using DNA ligase, wherein the ligation is performed in vitro or in the host cell; (i) after completing step (h), culturing the host cell containing the first plasmid, the second plasmid and the third plasmid treated according to steps (e) to (h) under conditions suitable for producing parvoviral particles; and (j) recovering the parvoviral particles produced in step (i). ;

[0162] 35. The method of paragraph 34, wherein the first segment comprises parvoviral LEH and the third segment comprises parvoviral REH.

[0163] 36. The method of paragraph 34, wherein the first segment and the third segment each comprise an AAV ITR.

[0164] 37. The method of any of paragraphs 34 to 36, wherein the nucleic acid encoding the first protein or first RNA in (a)(iii), (b)(ii), or both, is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0165] 38. The method of any of paragraphs 34 to 36, wherein the nucleic acid encoding the second protein or second RNA in (b)(iv), (c)(iii), or both, is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

[0166] 39. A method for preparing a self-complementary AAV, the method comprising: (a) providing a plasmid comprising a segment, the segment comprising in the 5' to 3' direction: (i) comprising an AAV The invention relates to a method for producing a parvoviral terminal repeat comprising a parvoviral terminal repeat of an ITR; (ii) a promoter operably linked to a nucleic acid encoding a protein or RNA; (iii) at least half of a mutant inverted terminal repeat (mITR) or at least half of a short hairpin DNA (shDNA); and (iv) a nuclease cleavage site; (b) providing a host cell comprising one or more accessory genes sufficient to replicate a nucleic acid comprising parvoviral terminal repeats flanking a nucleic acid of interest; (c) digesting the plasmid with a nuclease specific for the nuclease cleavage site under conditions sufficient to linearize the plasmid, wherein the plasmid is digested in vitro or in the host cell; (d) ligating the plasmid to itself with a DNA ligase, wherein the plasmid is ligated in vitro or in the host cell to form a hairpin comprising an mTR or shDNA sequence; (e) after completing step (d), culturing the host cell comprising the plasmid treated according to steps (c) and (d) under conditions suitable for producing parvoviral particles; and (f) recovering the parvoviral particles produced in step (f).

[0167] 40. A method for preparing a self-complementary AAV, the method comprising: (a) providing a first plasmid comprising a segment, the segment comprising in the 5' to 3' direction: (i) a parvoviral terminal repeat comprising an AAV ITR; (ii) a promoter operably linked to a nucleic acid encoding a protein or RNA; (iii) a portion of an mTR or a portion of an shDNA; and (iv) a nuclease cleavage site; (b) providing a second plasmid comprising a segment, the segment comprising in the 5' to 3' direction: (i) a portion of an AAV (a) (iii) a portion of an mTR or a portion of an shDNA; (b) a nuclease cleavage site; and (c) providing a host cell comprising one or more accessory genes sufficient to replicate a nucleic acid comprising the parvoviral terminal repeats flanking the nucleic acid of interest. (d) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(iii) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell. (e) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b) under conditions sufficient to linearize the second plasmid. (iv) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in step (d) or (iv), wherein the second plasmid is digested in vitro or in the host cell; (f) ligating the first plasmid and the second plasmid digested in steps (d) and (e) with a DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell, thereby forming a hairpin comprising an mTR or shDNA sequence; (g) after completing step (f), culturing the host cell comprising the first plasmid and the second plasmid treated according to steps (d) to (f) under conditions suitable for producing parvoviral particles; and (h) recovering the parvoviral particles produced in step (g).

[0168] 41. A method for preparing a self-complementary AAV, the method comprising: (a) providing a plasmid comprising a segment, the segment comprising in the 5' to 3' direction: (i) comprising an AAV The invention relates to a method for producing a parvoviral terminal repeat comprising at least one half of an mITR or at least one half of an shDNA; (ii) a promoter operably linked to a nucleic acid encoding a protein or RNA; and (iii) a nuclease cleavage site in the plasmid; (b) providing a DNA fragment comprising at least half of an mITR or at least half of an shDNA; (c) providing a host cell comprising one or more accessory genes sufficient to replicate a nucleic acid comprising the parvoviral terminal repeat flanked by a nucleic acid of interest; (d) digesting the plasmid with a nuclease specific for the nuclease cleavage site under conditions sufficient to linearize the plasmid, wherein the plasmid is digested in vitro or in the host cell; (e) ligating the plasmid to the DNA fragment with a DNA ligase, wherein the plasmid is ligated in vitro or in the host cell to form a hairpin comprising an mTR or shDNA sequence; (f) culturing the host cell comprising the plasmid treated according to steps (d) and (t) under conditions suitable for producing parvoviral particles after completing step (e); and (g) recovering the parvoviral particles produced in step (f).

[0169] 42. A vector genome comprising, in the 5' to 3' direction: (1) parvoviral terminal repeats at the 5' end; (2) a first regulatory region comprising (a) a promoter region, or (b) a poly A or poly T sequence, or both (a) and (b), and (3) a first heterologous nucleotide sequence, (4) a covalently blocked end (CCE) domain, (5) a second heterologous nucleotide sequence, (6) a first regulatory region comprising (a) a promoter region, or (b) a poly A or poly T sequence, or both (a) and (b), and (7) parvoviral terminal repeats at the 3' end; wherein the first regulatory sequence is complementary to the second heterologous sequence and forms a double-stranded region (DS domain) in the vector genome, wherein the CCE domain is (a) a single covalent bond linking the first heterologous nucleotide sequence and the second nucleotide sequence, or (b) a single-stranded DNA region, and wherein the vector genome is capable of producing a double-stranded (DS)-RNA molecule from the DS domain of the vector genome.

[0170] 43. The vector genome of paragraph 42, wherein the DS-domain is discontinuous with a self-annealing stem portion comprising more than 20 consecutive nucleotide base pairs from a mutant inverted terminal repeat (mTR) sequence or a short hairpin RNA (shRNA) encoding sequence.

[0171] 44. The vector genome of paragraph 42, wherein the CCE domain does not comprise an mTR sequence, or an shRNA encoding sequence, or both.

[0172] 45. The vector genome of paragraph 42, wherein the CCE domain comprises 0 to 50 nucleotides.

[0173] 46. ​​The vector genome of paragraph 42, wherein the CCE domain comprises 51 to 4000 nucleotides.

[0174] 47. A method for producing a dsRNA molecule, the method comprising: (1) growing a host production cell, (2) introducing the vector genome of paragraph 42 into the host production cell, (3) introducing an auxiliary gene into the host production cell, (4) incubating the host production cell for at least 12 hours, wherein the vector genome expresses the dsRNA molecule.

[0175] Example

[0176] Example 1, Comparison of cceAAV-CB-FIX production methods

[0177] cceAAV-CB-FIX was constructed to express FIX under the control of the CB promoter. It has the configuration of cceAAV-ZL. cceAAV-CB-FIX was constructed using scAAV-CB-FIX as the starting material. First, the mutant ITR was removed from scAAV-CB-FIX and replaced with a fragment containing I-sceI, Not I site and CR1 (cas9 site using guide RNA gCR1) to obtain the plasmid pre-cceAAV-CB-FIX. cceAAV-CB-FIX was produced using the following method. Its yield was then measured and its performance was compared with that of scAAV-CB-FIX.

[0178] Direct ligation, pre-cceAAV-CB-FIX was digested with Not I and EcoRV, and the fragment containing ITR was used for ligation. The resulting ligated fragment was used to co-transfect 293 cells with adenovirus plasmid and pRepCap (AAV helper plasmid). The vector was harvested 96 hours after transfection and purified following standard procedures.

[0179] Using in vivo ligation of I-sceI, the plasmid pre-cceAAV-CB-FIX was used to transfect 293-i-sceI cells together with adenoviral plasmids and pRepCap (AAV helper plasmid). The vector was harvested 96 hours after transfection and purified following standard protocols. 293-i-sceI is a cell line that stably expresses the I-sceI nuclease in the 293 cell line.

[0180] For Cas9-assisted production, pre-cceAAV-CB-FIX was used to transfect 293 cells together with pRepCap (AAV helper plasmid). They were then infected with adenovirus carrying cas9 nuclease and gRNA gCR1. The vector was harvested 48 hours after adenovirus infection and purified following standard procedures.

[0181]

[0182] Example 2, cceAAV for dsRNA expression

[0183] cceAAV-dsRNA expression in Fig.26 As shown in . Obviously, the poly A site is on the antisense strand. Poly A or a poly A functional equivalent element (transcription terminator) can be placed anywhere on the negative strand (for example, before or after the promoter, or in the middle of the antisense strand). When AAV is in a self-complementary configuration, dsRNA is not expressed. The promoter of dsRNA can be any promoter, including class I, class II and class II promoters. After AAV DNA undergoes second-strand DNA synthesis or annealing of the positive and negative strands, dsRNA is expressed. dsRNA can be used to induce cellular responses or regulate target gene expression. In order to prepare cceAAV-GFP-dsRNA, we removed the poly A sequence from pAAV-CB-EGFP and then cloned the poly A sequence in the opposite orientation at the start of the CB promoter to obtain pAAV-antisense-poly A-CB-GFP. To prepare an AAV vector expressing dsRNA of GFP, pAAV-antisense-poly A-CB-GFP was digested with Not I and EcoRV to obtain a fragment with ITR-antisense-poly A-CB-GFP, which was then used to transfect 293 cells together with an AAV helper plasmid and an adenovirus helper plasmid, and the vector cce-AAV-CB-GFP-dsRNA was harvested 96 hours after transfection. The resulting vector was then tested for dsRNA expression. We observed dsRNA expression by qPCR 48 hours after infection. When the dsRNA vector was co-infected with a conventional GFP vector, GFP expression was reduced by 10% to 99% depending on the ratio of the two vectors.

[0184] Example 3. cceAAV for delivering single-stranded DNA template for gene editing

[0185] The basic configuration is Figure 3As shown. To prepare cce-AAV-ssGFP, we prepared two fragments for connection. One fragment contains ITR, 300bp sense strand, and ssDNA box (1300bp) for gene editing. The other fragment contains ITR, 300bp sense strand (no ssDNA box). The two fragments were then connected to prepare the AAV vector before transfection. The cce-AAV-ssGFP was then confirmed by sequencing. Gene editing of cce-AAV-GFP was tested in Cas9-gRNA. Depending on the MOI of cce-AAV-ssGFP, a 5- to 100-fold improvement was observed.

[0186] Example 4. cceAAV for expressing dsRNA and simultaneously delivering ssDNA

[0187] A schematic diagram of a prototype cceAAV for expressing dsRNA and simultaneously delivering ssDNA is shown in Fig. 27 ssDNA is more stable because the flanking regions are duplexes. This approach was achieved by connecting a fragment containing ITR, poly A in antisense orientation, CB promoter, GFP sense strand and 800 bp ssDNA region with another fragment containing ITR, poly A in antisense orientation, CB promoter and GFP sense strand. The vector was prepared by co-transfection with AAV helper plasmid and small adenovirus plasmid. dsRNA expression was confirmed in cells infected with the generated vector.

[0188] Example 5, cceAAV based on TelN

[0189] cceAAV-CB-GFP-miRNA was constructed to express miRNA under the control of the CB promoter. It has the configuration of cceAAV-ZL. scAAV-CB-GFP was used as the starting material to construct cceAAV-CB-GFP-miRNA. First, the mutant ITR was removed from scAAV-CB-GFP and replaced with a miRNA expression fragment containing I-sceI, Not I site, TelRL and CR1 (cas9 site using guide RNA gCR1) to obtain the plasmid pre-cceAAV-CB-GFP-miRNA. When I-sceI, Not I site and CR1 were used, cceAAV-CB-GFP-miRNA was produced using the method described in Example 1. When the TelRL site was used, the precursor plasmid was incubated with TelN (New England Biolabs) for one hour and then transfected. The yield of eceAAV-CB-GFP-miRNA obtained from the new method is 5 to 10 times higher than that of typical scAAV expression miRNA. Contaminant levels are 5 to 10 times lower than typical vector generation methods.

[0190] Example 6, cceAAV carrying DNAzyme

[0191] The construct cceAAV-DNAzyme ( Figure 3 ). The DNAzymes tested included 8-17 and 10-23 DNAzyme (Santoro SW, Joyce GF (April 1997). "A general purpose RNA-cleaving DNA enzyme" Proceedings of the National Academy of Sciences of the United States of America. 94(9):4262-6. doi:10.1073 / pnas.94.9.4262.PMC 20710. PMID 9113977.). 10-23 DNAzyme comprises a 15-nucleotide catalytic core flanked by two substrate recognition domains. The DNAzyme efficiently cleaves complementary RNA between unpaired purines and paired pyrimidines in a sequence-specific manner. 10-23 DNAzyme was cloned into a fragment for ligation. One fragment was configured as 1. ITR, CB-GFP, poly A, -flexible DNA linker (50 bp), 10-23 DNAzyme, and the other fragment was ITR, CB-GFP, poly A, -flexible DNA linker (50 bp). The ligation created a cceAAV genome with 10-23 DNAzyme in the single-stranded DNA region (unpaired). The function of 10-23 DNAzyme was characterized according to the standard procedures published by Joyce's team.

[0192] Example 7, ccePV with two different ITRs (LEH and REH)

[0193] We prepared three versions of the human bocavirus-1 (HBoV1) vector. The first was cceHBoV-LEH2-CB-GFP. It was constructed by self-ligation of the LEH-CB-GFP fragment, which is self-complementary to the two copies of LEH. The second was cceHBoV-REH2-CB-GFP. It was constructed by self-ligation of the REH-CB-GFP fragment, which is self-complementary to the two copies of REH. The third was cceHBoV-REH2-CB-GFP. It was constructed by the REH-CB-GFP fragment and LEH-CB-GFP, which is self-complementary in the transgene cassette but has one REH and one LEH. The resulting plasmids were co-transfected with HBoV1 helper and adenovirus helper, and other reagents promoted replication. The resulting vectors were confirmed by Southern blotting and qPCR and tested for infectivity in 293 cells.

[0194] Example 8, using AAV to express dsRNA after AAV second synthesis or positive and negative polarity annealing.

[0195] The vector is constructed by connecting two fragments, the first fragment comprises AAV-ITR, CB promoter, GFP sequence, and the second fragment comprises complementary GFP sequence, poly A sequence and AAV ITR. The connection of this fragment leads to the molecular configuration of AAV-ITR, CB promoter, GFP sequence, complementary GFP sequence, poly A sequence and AAV ITR (the plasmid cannot be made into a plasmid due to instability in the host cell). After infection, this will generate a GFP sequence as dsRNA. The control of dsRNA of GFP expressed without the need for second DNA synthesis is prepared by connecting the following first fragment comprising AAV-ITR, complementary poly A sequence, CB promoter and GFP sequence (in sequence) and the second fragment comprising complementary GFP sequence, complementary CB promoter sequence, poly A sequence and AAV-ITR (in sequence, it is essentially the first fragment). The final molecular configuration is AAV-ITR, complementary poly A sequence, CB promoter, GFP sequence, complementary GFP sequence, complementary CB promoter sequence, poly A sequence and AAV-ITR. The dsRNA capacity of the control AAV vector is reduced due to the additional complementary sequences of the poly A and promoter. However, it has the advantage of simplified ligation because it uses self-ligation.

[0196] The above description is intended to teach one of ordinary skill in the art how to practice the invention, and is not intended to describe in detail all those obvious modifications and variations that will become apparent to one of ordinary skill in the art after reading this specification. However, it is intended that all such obvious modifications and variations should be included within the scope of the invention as defined by the appended claims. The claims are intended to cover the claimed components and steps in any order that is effective to achieve the intended goal, unless the context specifically indicates otherwise.

Claims

1. A dual adeno-associated virus (AAV) particle comprising: AAV capsid; and A vector genome, the vector genome comprising in the 5' to 3' direction: (a) AAV terminal repeats at the 5' end; (b) AAV terminal repeats at the 3′ end; (c) a double-stranded (DS) DNA domain between the terminal repeats in (a) and (b), the DS-DNA domain comprising a self-complementary first heterologous nucleotide sequence that anneals to a second heterologous nucleotide sequence; and (d) a single-stranded covalently closed end (SS-CCE) domain between the first heterologous nucleotide sequence and the second heterologous nucleotide sequence, wherein the SS-CCE domain comprises a loop structure; wherein the DS domain is discontinuous with the self-annealing stem portion, and wherein the vector genome is capable of replicating to form the dual AAV particles when introduced into a host cell, and the host cell expresses a helper function sufficient to produce the dual AAV particles, wherein the SS-CCE domain comprises a sequence encoding a protein or RNA, The SS-CCE domain does not contain more than 20 consecutive nucleotides from mutant inverted terminal repeat (mTR) and short hairpin DNA (shDNA) sequences.

2. The AAV particle of claim 1, wherein the SS-CCE domain comprises 3 to 4,000 nucleotides, 50 to 4,000 nucleotides, 1,000 to 4,000 nucleotides, 3 to 2,000 nucleotides, 50 to 2,000 nucleotides, 250 to 2,000 nucleotides, or 500 to 2,000 nucleotides.

3. The AAV particle of claim 1, wherein the sequence encoding the protein or RNA further comprises a downstream polyadenylation signal operably linked thereto.

4. The AAV particle of any one of claims 1 to 3, wherein the first heterologous nucleotide sequence has greater than 95%, greater than 99% or 100% reverse complementarity with the second heterologous nucleotide sequence.

5. The AAV particle of any one of claims 1 to 4, wherein the DS domain comprises a promoter operably linked to a nucleic acid encoding a protein or RNA.

6. The AAV particle of claim 5, wherein the DS domain further comprises a polyadenylation signal operably linked to the nucleic acid encoding the protein or RNA.

7. The AAV particle of any one of claims 1 to 6, wherein the AAV terminal repeat in (a) comprises a left-terminal hairpin (LEH), and the terminal repeat in (b) comprises a right-terminal hairpin (REH).

8. The AAV viral particle of any one of claims 1 to 6, wherein the AAV terminal repeats comprise AAV inverted terminal repeats (ITRs).

9. A method for preparing the AAV particle according to claim 1, the method comprising: (a) providing a first plasmid comprising a first segment, wherein the first segment comprises, in the 5' to 3' direction: (i) an AAV terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter operably linked to a nucleic acid encoding a protein or RNA; and (iii) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, the second segment comprising in the 5' to 3' direction: (i) an AAV terminal repeat comprising a right end hairpin (REH) or an ITR; (ii) a promoter; (iii) a nucleic acid encoding a protein or RNA; and (iv) a second nuclease cleavage site in the second plasmid, wherein the second segment is identical to the first segment except that the second segment further comprises one or more consecutive nucleotide base pairs that are not present in the first segment; (c) providing a host cell comprising one or more helper genes sufficient for replication of a nucleic acid comprising the AAV terminal repeats flanking the nucleic acid of interest; (d) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(iii) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (e) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(iv) under conditions sufficient to linearize the second plasmid, wherein the second plasmid is digested in vitro or in the host cell; (f) ligating the first plasmid digested in step (d) and step (e) with the second plasmid using DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell; (g) after completing step (f), culturing the host cell comprising the first plasmid and the second plasmid processed according to steps (d) to (f) under conditions suitable for producing AAV particles; and (h) recovering the AAV particles produced in step (g).

10. The method of claim 9, wherein the first segment comprises AAV LEH and the second segment comprises AAV REH.

11. The method of claim 9, wherein the first segment and the second segment each comprise an AAV ITR.

12. The method of any one of claims 9 to 11, wherein the second fragment comprises a subfragment between (b)(iii) and (b)(iv).

13. The method of claim 12, wherein the subfragments are 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

14. The method of any one of claims 9 to 11, wherein the second fragment comprises a subfragment between (b)(ii) and (b)(iii).

15. The method of claim 14, wherein the subfragments are 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

16. A method for preparing the AAV particle according to claim 1, the method comprising: (a) providing a first plasmid comprising a first segment, wherein the first segment comprises, in the 5' to 3' direction: (i) an AAV terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter; (iii) a nucleic acid encoding a protein or RNA; (iv) a polyadenylation signal; and (v) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, wherein the second segment comprises in the 5' to 3' direction: (i) an AAV terminal repeat comprising a right end hairpin (REH) or an ITR; (ii) a promoter; and (iii) a second nuclease cleavage site in the second plasmid; (c) providing a host cell comprising one or more helper genes sufficient for replication of a nucleic acid comprising the AAV terminal repeats flanking the nucleic acid of interest; (d) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(v) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (e) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(iii) under conditions sufficient to linearize the second plasmid, wherein the second plasmid is digested in vitro or in the host cell; (f) ligating the first plasmid digested in steps (d) and (e) and the second plasmid using DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell; (g) after completing step (f), culturing the host cell comprising the first plasmid and the second plasmid processed according to steps (d) to (f) under conditions suitable for producing AAV particles; and (h) recovering the AAV particles produced in step (g).

17. The method of claim 16, wherein the first segment comprises AAV LEH and the second segment comprises AAV REH.

18. The method of claim 16, wherein the first segment and the second segment each comprise AAVITR.

19. The method of any one of claims 16 to 18, wherein the nucleic acid encoding the protein or RNA in (a)(iii) is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

20. A method for preparing the AAV particle of claim 1, the method comprising: (a) providing a first plasmid comprising a first segment, wherein the first segment comprises in the 5' to 3' direction: (i) an AAV terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter; (iii) a first polyadenylation signal; (iv) a reverse complementary nucleic acid encoding a first protein or a first RNA, wherein the first protein and the first RNA each have a right-to-left polarity; (v) a nucleic acid encoding a second protein or a second RNA, wherein the second protein and the second RNA each have a left-to-right polarity; (vi) a second polyadenylation signal; and (vii) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, wherein the second segment comprises in a 5' to 3' direction: (i) an AAV terminal repeat comprising a right end hairpin (REH) or an ITR; (ii) the promoter in (a)(ii); (iii) a nucleic acid encoding the second protein or the second RNA, wherein the second protein and the second RNA each have a left-to-right polarity; (iv) a polyadenylation signal; and (v) a second nuclease cleavage site in the second plasmid; (c) providing a host cell comprising one or more helper genes sufficient for replication of a nucleic acid comprising the AAV terminal repeats flanking the nucleic acid of interest; (d) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(vii) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (e) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(v) under conditions sufficient to linearize the second plasmid, wherein the second plasmid is digested in vitro or in the host cell; (f) ligating the first plasmid digested in steps (d) and (e) and the second plasmid using DNA ligase, wherein the digested first plasmid and the second plasmid are ligated in vitro or in the host cell; (g) after completing step (f), culturing the host cell comprising the first plasmid and the second plasmid processed according to steps (d) to (f) under conditions suitable for producing AAV particles; and (h) recovering the AAV particles produced in step (g).

21. The method of claim 20, wherein the first segment comprises AAV LEH and the second segment comprises AAV REH.

22. The method of claim 20, wherein the first fragment and the second fragment each comprise AAVITR.

23. The method of any one of claims 20 to 22, wherein the nucleic acid encoding the protein or RNA in (a)(iv), (a)(v), or both, is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

24. The method of any one of claims 20 to 22, wherein the nucleic acid encoding the protein or RNA in (b)(iii) is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

25. A method for preparing the AAV particle of claim 1, the method comprising: (a) providing a first plasmid comprising a first segment, wherein the first segment comprises in the 5' to 3' direction: (i) an AAV terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) a promoter having a left-to-right polarity; (iii) a nucleic acid encoding a first protein or a first RNA having a left-to-right polarity; (iv) a polyadenylation signal having a left-to-right polarity, and (v) a first nuclease cleavage site in the first plasmid; (b) providing a second plasmid comprising a second segment, wherein the second segment comprises in a 5' to 3' direction: (i) a first nuclease cleavage site; (ii) a first polyadenylation signal having a right-to-left polarity; (iii) the nucleic acid encoding the first protein or first RNA in (a) (iii), wherein the first protein or first RNA has a right-to-left polarity; (iv) a nucleic acid encoding a second protein or second RNA, wherein the second protein and the second RNA each have a left-to-right polarity; and (v) a second polyadenylation signal; and (vi) a second nuclease cleavage site, wherein the cleavage site in (b)(i) is the same as the cleavage site in (a)(v); (c) providing a third plasmid comprising a third segment, wherein the third segment comprises in the 5' to 3' direction: (i) an AAV terminal repeat comprising a left-end hairpin (LEH) or an inverted terminal repeat (ITR); (ii) the promoter having a left-to-right polarity in (a)(ii); (iii) the nucleic acid encoding the second protein or the second RNA in (b)(iv), wherein the second protein and the second RNA each have a left-to-right polarity; (iv) a polyadenylation signal having a left-to-right polarity, and (v) a third nuclease cleavage site specific to the third plasmid, wherein the cleavage site in (b)(iv) is the same as the cleavage site in (c)(v); (d) providing a host cell comprising one or more helper genes sufficient for the replication of a nucleic acid comprising the AAV terminal repeats flanking the nucleic acid of interest; (e) digesting the first plasmid with a nuclease specific for the nuclease cleavage site in (a)(v) under conditions sufficient to linearize the first plasmid, wherein the first plasmid is digested in vitro or in the host cell; (f) digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(i) and digesting the second plasmid with a nuclease specific for the nuclease cleavage site in (b)(vi) under conditions sufficient to digest the second plasmid, wherein the second plasmid is digested in vitro or in the host cell; (g) digesting the third plasmid with a nuclease specific for the nuclease cleavage site in (c)(v) under conditions sufficient to linearize the third plasmid, wherein the third plasmid is digested in vitro or in the host cell; (h) ligating the linearized first plasmid fragment, the second fragment and the linearized third plasmid fragment together using DNA ligase, wherein the ligation is performed in vitro or in the host cell; (i) after completing step (h), culturing the host cell comprising the first plasmid, the second plasmid and the third plasmid processed according to steps (e) to (h) under conditions suitable for producing AAV particles; and (j) recovering the AAV particles produced in step (i).

26. The method of claim 25, wherein the first segment comprises AAV LEH and the third segment comprises AAV REH.

27. The method of claim 25, wherein the first segment and the third segment each comprise AAVITR.

28. The method of any one of claims 25 to 27, wherein the nucleic acid encoding the first protein or first RNA in (a)(iii), (b)(ii), or both, is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

29. The method of any one of claims 25 to 27, wherein the nucleic acid encoding the second protein or second RNA in (b)(iv), (c)(iii), or both is 3 to 4,000 base pairs in length, 50 to 4,000 base pairs in length, 1,000 to 4,000 base pairs in length, 3 to 2,000 base pairs in length, 50 to 2,000 base pairs in length, 250 to 2,000 base pairs in length, or 500 to 2,000 base pairs in length.

30. A vector genome, comprising in the 5' to 3' direction: (1) AAV terminal repeats at the 5' end, (2) a first regulatory region comprising (a) a promoter region, or (b) a poly A or poly T sequence, or both (a) and (b), (3) a first heterologous nucleotide sequence, (4) covalently blocked end (CCE) domain, (5) a second heterologous nucleotide sequence, (6) a second regulatory region comprising (a) a promoter region, or (b) a poly A or poly T sequence, or both (a) and (b), and (7) AAV terminal repeats at the 3′ end; wherein the first regulatory region sequence is complementary to the second regulatory region sequence and forms a double-stranded region (DS-domain) in the vector genome, wherein the CCE domain is (a) a single covalent bond connecting the first heterologous nucleotide sequence and the second heterologous nucleotide sequence, or (b) a single-stranded DNA region, and wherein said vector genome is capable of producing a double-stranded (DS)-RNA molecule from said DS-domain of said vector genome, wherein the CCE domain comprises a sequence encoding a protein or RNA, The CCE domain does not contain an mTR sequence and an shRNA coding sequence.

31. The vector genome of claim 30, wherein the CCE domain comprises 5 to 50 nucleotides.

32. The vector genome of claim 30, wherein the CCE domain comprises 51 to 4000 nucleotides.

33. A method for producing a dsRNA molecule, the method comprising: (1) growing host production cells, (2) introducing the vector genome according to claim 30 into the host production cell, (3) introducing auxiliary genes into the host production cell, (4) incubating the host production cell for at least 12 hours, wherein the vector genome expresses the dsRNA molecule.

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