Modified closed ended DNA (CEDNA)

Abstract

To provide modified closed ended DNA.SOLUTION: The ceDNA vectors having a linear and continuous structure can be produced at a high yield and used for effective transfer and expression of a transgene. A ceDNA vector comprises an expression cassette, two different ITR sequences derived from AAV genomes in a specified order. Some ceDNA vectors provided herein further comprise a cis-regulatory element and provide high gene expression efficiencies. Furthermore, a method and a cell line for secure and efficient production of a linear and continuous capsid-free DNA vector are provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Applications Nos. 62 / 556,319, 62 / 556,324, 62 / 556,329, 62 / 556,331, 62 / 556,281, and 62 / 556,335, each filed on September 8, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The said ASCII copy, created on September 7, 2018, is named 080170-090580WOPT_SL.txt and is 205,991 bytes in size.

[0003] The present invention relates to the field of gene therapy, which involves the delivery of exogenous DNA sequences to target cells, tissues, organs or organisms. [Background technology]

[0004] Gene therapy aims to improve clinical outcomes for patients suffering from either genetic mutations or acquired diseases caused by abnormalities in gene expression profiles. Gene therapy includes the treatment or prevention of medical conditions resulting from defective genes or dysregulation or expression, e.g., under- or over-expression, that can result in disorders, diseases, malignancies, etc. For example, diseases or disorders caused by defective genes can be treated, prevented, or ameliorated by delivery of corrective genetic material to the patient, resulting in therapeutic expression of the genetic material in the patient's body. The basis of gene therapy is the provision of an active gene product (sometimes referred to as a transgene) in a transcription cassette, which can result in, for example, a positive gain-of-function effect, a negative loss-of-function effect, or another outcome, e.g., an oncolytic effect. Gene therapy can also be used to treat diseases or malignancies caused by other factors. Human single-gene disorders can be treated by delivery and expression of normal genes to target cells. Delivery and expression of corrective genes in the target cells of the patient can be carried out through a number of methods, including the use of engineered viruses and viral gene delivery vectors. Among the many available virus-derived vectors (e.g., recombinant retroviruses, lentiviruses, adenoviruses, etc.), recombinant adeno-associated viruses (rAAVs) have gained popularity as versatile vectors in gene therapy.

[0005] Adeno-associated viruses (AAV) belong to the Parvoviridae family and more specifically constitute the Dependoparvovirus genus. The AAV genome comprises a linear, single-stranded DNA molecule that contains approximately 4.7 kilobases (kb) and consists of two major open reading frames (ORFs) that encode the nonstructural Rep (replication) and structural Cap (capsid) proteins. A second ORF within the cap gene has been identified that encodes the assembly activating protein (AAP). Flanking the AAV coding region are two cis-acting inverted terminal repeat (ITR) sequences of approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into energetically stable hairpin structures that function as primers for DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmids, and encapsidation of viral nucleic acid in mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0006] AAV-derived vectors (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for delivering genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons; (ii) they lack viral structural genes, thereby reducing host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered non-pathogenic in humans; (iv) in contrast to wild-type AAV, which can integrate into the host cell genome, replication-deficient AAV vectors lack the rep gene and generally persist episomally, thus limiting the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively poor immunogens and therefore do not elicit a significant immune response (see ii), thus resulting in sustained vector DNA and potentially long-term expression of therapeutic transgenes. AAV vectors can also be produced and formulated at high titers and delivered via intra-arterial, intravenous, or intraperitoneal injection, allowing vector distribution and gene transfer to critical muscle regions with a single injection in rodents (Goyenvalle et al., 2004; Fougerousse et al., 2007; Koppanati et al., 2010; Wang et al., 2009) and dogs. In clinical studies to treat spinal muscular atrophy type 1, AAV vectors were delivered systemically with the intention of targeting the brain and resulting in clear clinical improvement.

[0007] However, there are several significant deficiencies in using AAV particles as gene delivery vectors. One significant drawback associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996, Athanasopoulos et al., 2004, Lai et al., 2010). As a result, the use of AAV vectors is limited to protein coding capacity of less than 150,000 Da. A second drawback is that as a result of the prevalence of wild-type AAV infection in the population, rAAV gene therapy candidates need to be screened for the presence of neutralizing antibodies that would eliminate the vector from the patient. A third drawback concerns capsid immunogenicity, which prevents re-administration to patients who were not eliminated from the initial treatment. The immune system in the patient may respond to the vector effectively acting as a "booster" shot, priming the immune system to generate high titer anti-AAV antibodies that would prevent future treatment. Several recent reports have shown a relationship with immunogenicity in high dose situations. Another notable drawback is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA prior to heterologous gene expression. Attempts have been made to circumvent this problem by constructing double-stranded DNA vectors, but this strategy further limits the size of the transgene expression cassette that can be incorporated into the AAV vector (McCarty, 2008; Varenika et al., 2009; Foust et al., 2009).

[0008] Additionally, conventional AAV virions with capsids are produced by introducing one or more plasmids containing the AAV genome, rep gene, and cap gene (Grimm et al., 1998). Upon introduction of these helper plasmids in trans, the AAV genome is "rescued" from the host genome (i.e., amplified after release) and further encapsidated (viral capsid) to produce biologically active AAV vectors. However, such encapsidated AAV viral vectors have been found to transduce certain cell and tissue types inefficiently. The capsid also induces immune responses. Therefore, the use of adeno-associated virus (AAV) vectors for gene therapy is limited due to the single administration to patients (due to patient immune response), the limited range of transgene genetic material suitable for delivery in AAV vectors due to the minimal viral packaging capacity (about 4.5 kb) of the relevant AAV capsid, as well as the slow AAV-mediated gene expression. Application of rAAV for clinical gene therapy is further hindered by patient-to-patient variability that is not predicted by dose-response in syngeneic mouse models or in other model species. Recombinant capsid-free AAV vectors can be obtained as isolated linear nucleic acid molecules that contain an expressible transgene and a promoter region flanked by two wild-type AAV inverted terminal repeats (ITRs) that contain Rep binding sites and terminal resolution sites. These recombinant AAV vectors lack the sequence that codes for AAV capsid proteins and can be single-stranded, double-stranded, or duplexed with one or both ends covalently linked through two wild-type ITR palindromic sequences (e.g., WO2012 / 123430, U.S. Patent No. 9,598,703). They have a much higher transgene capacity, a faster onset of transgene expression, and avoid many of the AAV-mediated gene therapies in that the patient's immune system recognizes the DNA molecule as a virus that should be cleared. However, constant expression of the transgene may not be desirable in all instances, and the AAV canonical wild-type ITRs may not be optimized for ceDNA function. Thus, there remains a significant unmet need for controllable recombinant DNA vectors with improved production and / or expression properties. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2012 / 123430 [Patent Document 2] U.S. Pat. No. 9,598,703 [Non-patent literature]

[0010] [Non-Patent Document 1] Muzyczka,(1992)Curr.Top.Micro.Immunol.158:97-129 Summary of the Invention [Means for solving the problem]

[0011] The invention described herein is a non-viral capsid-free DNA vector with covalently closed ends (referred to herein as a "closed end DNA vector" or "ceDNA vector"). The ceDNA vector described herein is a capsid-free linear double-stranded DNA molecule formed from continuous strands of complementary DNA (linear continuous non-encapsidated structures) with covalently closed ends and containing 5' inverted terminal repeat (ITR) sequences and 3' ITR sequences that are different or asymmetric with respect to each other.

[0012] The technology described herein relates to a ceDNA vector that contains at least one modified AAV inverted terminal repeat (ITR) and an expressible transgene.The ceDNA vector disclosed herein can be produced in eukaryotic cells, and therefore can be devoid of prokaryotic DNA modification and bacterial endotoxin contamination in insect cells.

[0013] In one aspect, a non-viral capsid-free DNA vector with covalently closed ends is obtainable from a vector polynucleotide that is preferably a linear double-stranded molecule and encodes a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), at least one of which comprises a terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as a replicative protein binding site), e.g., a Rep binding site, and one of the ITRs comprises a deletion, insertion, or substitution with respect to the other ITR. That is, one of the ITRs is asymmetric with respect to the other ITR. In one embodiment, at least one of the ITRs is an AAV ITR, e.g., a wild-type AAV ITR or a modified AAV ITR. In one embodiment, at least one of the ITRs is a modified ITR with respect to the other ITR, that is, the ceDNA comprises ITRs that are asymmetric with respect to each other. In one embodiment, at least one of the ITRs is a non-functional ITR.

[0014] In some embodiments, the ceDNA vector comprises (1) an expression cassette comprising a cis-regulatory element, a promoter, and at least one transgene, or (2) a promoter operably linked to at least one transgene, and (3) two self-complementary sequences, e.g., ITRs, flanking the expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, at least one of which comprises an operational Rep-binding element (RBE) (sometimes referred to herein as "RBS") and a terminal resolution site (trs) or a functional variant of the RBE of AAV, and one or more cis-regulatory elements operably linked to a transgene. In some embodiments, the ceDNA vector comprises additional components for regulating expression of the transgene, e.g., a regulatory switch, described herein in the section entitled "Regulatory Switches," for controlling and regulating expression of the transgene, e.g., a regulatory switch that is a kill switch that allows for controlled cell death of cells containing the ceDNA vector.

[0015] In some embodiments, the two self-complementary sequences can be ITR sequences from any known parvovirus, such as a depend virus, such as AAV (e.g., AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, modified AAV2 ITR sequences that retain a Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal resolution site (trs) in addition to a variable palindrome sequence that allows hairpin secondary structure formation. In some embodiments, the ITR is a synthetic ITR sequence that retains a functional Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal resolution site (TRS) in addition to a variable palindrome sequence that allows hairpin secondary structure formation. In some examples, the modified ITR sequence retains the sequence of the RBS, trs, and the structure and position of the Rep binding element that forms the terminal loop portion of one ITR hairpin secondary structure from the corresponding sequence of the wild-type AAV2 ITR.

[0016] Exemplary ITR sequences for use in ceDNA vectors are disclosed in Tables 2-10A and 10B, or in SEQ ID NOs: 2, 52, 101-499, and 545-547, or any one or more of the partial ITR sequences shown in Figures 26A-26B. In some embodiments, the ceDNA vector does not have an ITR comprising any sequence selected from SEQ ID NOs: 500-529.

[0017] In some embodiments, the ceDNA vector may comprise an ITR having an ITR modification corresponding to any of the ITR sequence or ITR subsequence modifications shown in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein.

[0018] As an illustrative example, the disclosure provides a closed-end DNA vector comprising a promoter operably linked to a transgene, the ceDNA being devoid of capsid protein and (a) produced from a ceDNA-plasmid (see, e.g., Examples 1-2 and / or Figures 1A-B) encoding a mutated right AAV2 ITR having the same number of intramolecularly duplexed base pairs as SEQ ID NO:2 or a mutated left AAV2 ITR having the same number of intramolecularly duplexed base pairs as SEQ ID NO:51 in its hairpin secondary configuration (preferably excluding the deletion of any AAA or TTT terminal loops in this configuration compared to these reference sequences), and (b) identified as a ceDNA using the assay for identification of ceDNA by agarose gel electrophoresis under native and denaturing conditions in Example 1. Examples of such modified ITR sequences are provided herein in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B.

[0019] The technology described herein further relates to ceDNA vectors that can deliver and encode one or more transgenes in target cells, for example, the ceDNA vectors contain multicistronic sequences, or the transgene and its native genomic context (e.g., the transgene, introns, and endogenous untranslated regions) are integrated together in the ceDNA vector. The transgene can be a protein-coding transcript, a non-coding transcript, or both. The ceDNA vectors can contain multiple coding sequences, and non-canonical translation initiation sites or two or more promoters for expressing the protein-coding transcript, the non-coding transcript, or both. The transgene can contain sequences that code for two or more proteins, or can be sequences of non-coding transcripts. The expression cassette may, for example, comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and therefore allow delivery of large-sized expression cassettes to result in efficient expression of the transgene. In some embodiments, the ceDNA vector lacks prokaryotic cell-specific methylation.

[0020] The expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments, the ITRs may act as promoters of the transgene. In some embodiments, the ceDNA vector includes additional components for regulating the expression of the transgene. For example, the additional regulatory components may be the regulatory switches disclosed herein, including, but not limited to, kill switches that may kill the ceDNA-infected cells, as well as other inducible and / or repressible elements as necessary.

[0021] The technology described herein further provides a novel method for using ceDNA vector to deliver and efficiently and selectively express one or more transgenes.ceDNA vector has the ability to be incorporated into host cell and transported into the nucleus in the absence of AAV capsid.In addition, the ceDNA vector described herein lacks capsid, thus avoiding the immune reaction that may occur in response to capsid-containing vector.

[0022] Aspects of the present invention relate to the method of producing the ceDNA vector described herein. In other embodiments, the ceDNA vector produced by the method provided herein. In one embodiment, the capsid-free non-viral DNA vector (ceDNA vector) is obtained from a plasmid (referred to as "ceDNA-plasmid" herein) that comprises a polynucleotide expression construct template that comprises a first 5' inverted terminal repeat (e.g., AAV ITR), an expression cassette, and a 3' ITR (e.g., AAV ITR) in that order, and at least one of the 5' and 3' ITR is a modified ITR, or when both the 5' and 3' ITR are modified, they have different modifications from each other and are not the same sequence.

[0023] The ceDNA vectors disclosed herein can be obtained by several means known to the skilled artisan upon reading this disclosure. For example, the polynucleotide expression construct template used to generate the ceDNA vectors of the present invention can be a ceDNA-plasmid (e.g., see Table 12 or FIG. 10B), a ceDNA-bacmid, and / or a ceDNA-baculovirus. In one embodiment, the ceDNA-plasmid contains a restriction cloning site (e.g., SEQ ID NO: 7) operably positioned between the ITRs, e.g., an expression cassette including a promoter operably linked to a transgene, e.g., a reporter gene and / or a therapeutic gene, can be inserted. In some embodiments, the ceDNA vectors are produced from polynucleotide templates (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus) that contain ITRs that are modified compared to the corresponding adjacent AAV3 ITRs or wild-type AAV2 ITR sequences, the modification being any one or more of a deletion, an insertion, and / or a substitution.

[0024] In permissive host cells, for example, in the presence of Rep, a polynucleotide template having at least one modified ITR is replicated to produce a ceDNA vector. ceDNA vector production involves two steps: first, excision ("rescue") of the template from the template backbone (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.), and second, Rep-mediated replication of the excised ceDNA vector. Rep proteins and Rep binding sites of various AAV serotypes are well known to those skilled in the art. Those skilled in the art will understand that Rep proteins are selected from serotypes that bind and replicate nucleic acid sequences based on at least one functional ITR. For example, if a replicable ITR is from AAV serotype 2, the corresponding Rep is from the AAV serotype that cooperates with that serotype, for example, AAV2 ITR cooperates with AAV2 or AAV4 Rep, but not with AAV5 Rep. Upon replication, the covalently closed-end ceDNA vectors continue to accumulate in permissive cells, and the ceDNA vectors preferably accumulate under standard replication conditions and in the presence of Rep proteins sufficiently stably over long periods of time, for example in amounts of at least 1 pg / cell, preferably at least 2 pg / cell, preferably at least 3 pg / cell, more preferably at least 4 pg / cell, and even more preferably at least 5 pg / cell.

[0025] Thus, one aspect of the present invention relates to a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), where the host cells lack viral capsid coding sequences under conditions effective to induce production of ceDNA vectors in the host cells in the presence of Rep proteins, and for a sufficient time, and b) harvesting and isolating the ceDNA vectors from the host cells. The presence of Rep proteins induces replication of vector polynucleotides with modified ITRs to produce ceDNA vectors in the host cells. However, viral particles (e.g., AAV virions) are not expressed. Thus, there is no virion-imposed size limit.

[0026] The presence of a ceDNA vector isolated from a host cell may be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on denaturing and non-denaturing gels to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA.

[0027] In some embodiments, the application may be defined in any of the following paragraphs. 1. A non-viral capsid-free DNA vector (ceDNA vector) having covalently closed ends, said ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site. 2. A ceDNA vector as described in paragraph 1, wherein the ceDNA vector exhibits a characteristic linear, continuous DNA band compared to a linear, discontinuous DNA control when digested with a restriction enzyme having a single recognition site on the ceDNA vector and analyzed by both native and denaturing gel electrophoresis. 3. The ceDNA vector of paragraph 1 or 2, wherein one or more of the asymmetric ITR sequences are derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). 4. The ceDNA vector of paragraph 3, wherein the asymmetric ITRs are derived from different viral serotypes. 5. The ceDNA vector of paragraph 4, wherein one or more asymmetric ITRs are derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 6. A ceDNA vector according to any one of paragraphs 1 to 3, wherein one or more of the asymmetric ITR sequences is synthetic. 7. A ceDNA vector according to any one of paragraphs 1 to 3 and 6, wherein one or more of the ITRs is not a wild-type ITR. 8. The ceDNA vector of any one of paragraphs 1-7, wherein one or more of both of the asymmetric ITRs are modified by a deletion, insertion, and / or substitution in at least one of the ITR regions selected from A, A', B, B', C, C', D, and D'. In some embodiments, one or both of the asymmetric ITRs may have a deletion, insertion, and / or substitution in any combination of the B, B', C, or C' regions listed in Table 1. In some embodiments, one or both of the asymmetric ITRs may have a deletion, insertion, and / or substitution in the B region, and / or the B' region, and / or the C region, and / or the C' region. In some embodiments, one or both of the asymmetric ITRs may have one or more deletions, insertions, and / or substitutions in the A region, and / or the A' region, and / or the B region, and / or the B' region, and / or the C region, and / or the C' region, and / or the D region, and / or the D' region. For example, in some embodiments, a modified ITR may have the removal or deletion of all of a particular arm, e.g., all or part of the A-A' arm, or all or part of the B-B' arm, or all or part of the C-C' arm, or the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form the stem of a loop (see, e.g., ITR-6), so long as there is still a final loop capping the stem (e.g., a single arm). In some embodiments, a modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, a modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm. In some embodiments, the modified ITR may include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. Any combination of base pair removal is envisioned. In some embodiments, the modified ITR lacks the B-B' arm. In some embodiments, the modified ITR lacks the C-C' arm. 9. The ceDNA vector of paragraph 8, wherein the deletion, insertion, and / or substitution results in the deletion of all or part of the stem-loop structure normally formed by the A, A', B, B', C, or C' regions. For example, in some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more complementary base pairs are removed from each of the C and C' portions of the C-C' arm such that the C-C' arm is cleaved, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from each of the B and B' portions of the B-B' arm such that the C-C' arm and / or the B-B' arm are cleaved. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the C portion of the C-C' arm such that only the C' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the C' portion of the C-C' arm such that only the C portion of the arm remains. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the B portion of the B-B' arm such that only the B' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the B' portion of the B-B' arm such that only the B portion of the arm remains. In some embodiments, any deletion in the C, C', B, or B' region still preserves the terminal loop of the stem loop. In some embodiments, the terminal loop is at least three amino acids. In some embodiments, the terminal loop of the C-C' arm and / or the B-B' arm has at least three consecutive TTTs or three consecutive AAAs. 10. A ceDNA vector according to paragraph 8 or paragraph 9, wherein one or both of the asymmetric ITRs are modified by a deletion, insertion and / or substitution resulting in the deletion of all or part of the stem-loop structure normally formed by the B and B' regions. 11. The ceDNA vector according to any one of paragraphs 8 to 10, wherein one or both of the asymmetric ITRs are modified by a deletion, insertion and / or substitution resulting in the deletion of all or part of the stem-loop structure normally formed by the C and C' regions. 12. A ceDNA vector according to paragraph 10 or paragraph 11, wherein one or both of the asymmetric ITRs are modified by a deletion, insertion and / or substitution resulting in the deletion of part of the stem-loop structure normally formed by the B and B' regions and / or part of the stem-loop structure normally formed by the C and C' regions. 13. The ceDNA vector of any one of paragraphs 1 to 12, wherein one or both of the asymmetric ITRs comprise a single stem-loop structure in a region that typically comprises a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions. 14. The ceDNA vector of paragraph 13, wherein one or both of the asymmetric ITRs contain a single stem and two loops in a region that typically includes a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions. 15. The ceDNA vector of paragraph 13 or paragraph 14, wherein one or both of the asymmetric ITRs comprise a single stem and a single loop in a region that typically comprises a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions. 16. The ceDNA vector of any one of paragraphs 1-15, wherein at least one asymmetric ITR is a modified AAV2 ITR comprising a nucleotide sequence selected from an ITR of Figure 26A or 26B, an ITR with at least 95% sequence identity to an ITR of Figure 26A or 26B, and an ITR with at least 95% sequence identity to SEQ ID NOs: 101-499 and 545-547. 17. The ceDNA vector of any one of paragraphs 1-16, wherein at least one asymmetric ITR is a modified AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 2, 52, 63, or 64, or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 52, 63, or 64. 18. The ceDNA vector of any one of paragraphs 1-16, wherein the 5'ITR is a wild-type AAV ITR and the 3'ITR comprises a sequence selected from SEQ ID NOs: 2, 64, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 469-483, and 546, and the ITR sequences shown in Figure 26A, and a sequence having at least 95% sequence identity to any of the sequences. 19. The ceDNA vector of any one of paragraphs 1-16, wherein the 3' ITR is a wild-type AAV ITR and the 5' ITR comprises a sequence selected from SEQ ID NOs: 52, 63, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 484-499, 545, and 547, and the ITR sequences shown in Figure 26B, and a sequence having at least 95% sequence identity to any of the sequences. 20. The 5'ITR comprises a sequence selected from SEQ ID NOs: 52, 63, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 484-499, 545, and 547, and the ITR sequences shown in FIG. 26B, and a sequence having at least 95% sequence identity to any of the sequences; and the 3'ITR comprises a sequence selected from SEQ ID NO: 2, 64, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 469-483, and 546, as well as the ITR sequences shown in Figure 26A, and a sequence having at least 95% sequence identity to any of the sequences. 21. A ceDNA vector described in paragraph 1, comprising at least two asymmetric ITRs selected from (a) SEQ ID NO:1 and SEQ ID NO:52, and (b) SEQ ID NO:2 and SEQ ID NO:51. 22. A ceDNA vector described in paragraph 1, comprising a pair of asymmetric ITRs selected from (a) SEQ ID NO:1 and SEQ ID NO:52, and (b) SEQ ID NO:2 and SEQ ID NO:51. 23. The ceDNA vector of any one of paragraphs 1 to 20, wherein one or both asymmetric ITRs comprise a sequence other than SEQ ID NOs: 2, 52, 63, 64, 113, 114, and 557. 24. A ceDNA vector according to any one of paragraphs 1 to 24, wherein all or part of the heterologous nucleotide sequence is under the control of at least one regulatory switch. 25. The ceDNA vector of paragraph 24, wherein at least one regulatory switch is selected from any or combination of regulatory switches listed in Table 11 or the section entitled "Regulatory Switches" herein. By way of example only, the regulatory switch serves to fine-tune the expression of a heterologous nucleotide sequence, e.g., a transgene, and in some embodiments may serve as a biological containment function of the ceDNA vector. In some embodiments, the regulatory switch is an "on / off" switch. Without being limited by theory, the "on / off" switch is designed to controllably and regulatably start or stop (i.e., shut down) the expression of a heterologous nucleotide sequence or a transgene expressed from the ceDNA vector. In some embodiments, the regulatory switch is a "kill switch" that can instruct a cell containing the ceDNA vector to undergo programmed cell death once the switch is activated. In some embodiments, the regulatory switch is selected from any of the following: a binary switch (e.g., an inducible promoter, cofactor, or exogenous agent that derepresses transcription), a small molecule regulatory switch (e.g., a drug-inducible drug or prodrug activates or turns off transcription), a "passcode" regulatory switch (e.g., a combination of conditions must be present for expression and / or repression of the transgene to occur), a nucleic acid-based regulatory switch (e.g., a nucleic acid-based mechanism that controls expression and / or repression), a post-translational and / or post-transcriptional regulatory switch (e.g., a signal response element (SRE) or destabilization domain (DD) that prevents a functional transgene from being expressed until post-translational modification has occurred), or a kill switch (e.g., a switch that induces programmed cell death as a means of permanently removing an introduced ceDNA vector from a system of interest). 26. A ceDNA vector according to any one of paragraphs 1 to 25, wherein the vector is present in a nanocarrier. 27. The ceDNA vector of paragraph 26, wherein the nanocarrier comprises a lipid nanoparticle (LNP). 28. A non-viral capsid-free DNA vector with covalently closed ends according to any one of paragraphs 1 to 25, wherein the ceDNA vector is obtained from a process comprising the steps of: (a) incubating a population of insect cells harboring a ceDNA expression construct in the presence of at least one Rep protein, the ceDNA expression construct encoding the ceDNA vector, under conditions effective and for a time sufficient to induce production of the ceDNA vector in the insect cells; and (b) isolating the ceDNA vector from the insect cells. 29. A ceDNA vector according to paragraph 28, wherein the ceDNA expression construct is selected from a ceDNA plasmid, a ceDNA bacmid, and a ceDNA baculovirus. 30. A ceDNA vector according to paragraph 28 or paragraph 29, wherein the insect cell expresses at least one Rep protein. 31. The ceDNA vector of paragraph 30, wherein at least one Rep protein is derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). 32. The ceDNA vector of paragraph 31, wherein at least one Rep protein is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 33. A ceDNA expression construct encoding a ceDNA vector according to any one of paragraphs 1 to 25. 34. A ceDNA expression construct according to paragraph 33, which is a ceDNA plasmid, a ceDNA bacmid, or a ceDNA baculovirus. 35. A host cell comprising a ceDNA expression construct according to paragraph 33 or paragraph 34. 36. A host cell according to paragraph 35, which expresses at least one Rep protein. 37. The host cell according to paragraph 36, wherein at least one Rep protein is derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV). 38. The host cell of paragraph 37, wherein at least one Rep protein is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 39. A host cell according to any one of paragraphs 35 to 38, which is an insect cell. 40. The host cell of paragraph 39 which is an Sf9 cell. 41. A method for producing a ceDNA vector, comprising: (a) incubating a host cell described in any one of paragraphs 35 to 40 under conditions effective, and for a time sufficient, to induce production of the ceDNA vector; and (b) isolating the ceDNA from the host cell. 42. A method for treating, preventing, ameliorating, monitoring, or diagnosing a disease or disorder in a subject, the method comprising administering to a subject in need thereof a composition comprising a ceDNA vector described in any one of paragraphs 1-25, wherein at least one heterologous nucleotide sequence is selected to treat, prevent, ameliorate, diagnose, or monitor a disease or disorder. 43. The method of paragraph 42, wherein at least one heterologous nucleotide sequence, when transcribed or translated, corrects an abnormal amount of an endogenous protein in a subject. 44. The method of paragraph 42, wherein at least one heterologous nucleotide sequence, when transcribed or translated, corrects an abnormal function or activity of an endogenous protein or pathway in a subject. 45. The method of any one of paragraphs 42 to 44, wherein at least one heterologous nucleotide sequence encodes or comprises a nucleotide molecule selected from RNAi, siRNA, miRNA, IncRNA, and an antisense oligonucleotide or polynucleotide. 46. ​​The method of any one of paragraphs 42 to 44, wherein at least one heterologous nucleotide sequence encodes a protein. 47. The method of paragraph 42, wherein at least one heterologous nucleotide sequence encodes a marker protein (e.g., a reporter protein). 48. The method of any one of paragraphs 42 to 46, wherein at least one heterologous nucleotide sequence encodes an agonist or antagonist of an endogenous protein or pathway associated with a disease or disorder. 49. The method of any one of paragraphs 42 to 46, wherein at least one heterologous nucleotide sequence encodes an antibody. 50. The method of any one of paragraphs 42 to 49, wherein the disease or disorder is selected from a metabolic disease or disorder, a CNS disease or disorder, an eye disease or disorder, a blood disease or disorder, a liver disease or disorder, an immune disease or disorder, an infectious disease or disorder, a muscle disease or disorder, cancer, and a disease or disorder based on abnormal levels and / or function of a gene product. 51. The method of paragraph 50, wherein the metabolic disease or disorder is selected from diabetes, lysosomal storage disorders, mucopolysaccharidoses, urea cycle diseases or disorders, and glycogen storage diseases or disorders. 52. The method of paragraph 51, wherein the lysosomal storage disorder is selected from Gaucher disease, Pompe disease, metachromatic leukodystrophy (MLD), phenylketonuria (PKU), and Fabry disease. 53. The method of paragraph 51, wherein the urea cycle disease or disorder is ornithine transcarbamylase (OTC) deficiency. 54. The method of paragraph 51, wherein the mucopolysaccharidopathies are selected from Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome, Morquio syndrome, and Maroteaux-Lamy syndrome. 55. The method of paragraph 50, wherein the CNS disease or disorder is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease, Lesch-Nairn disease, epilepsy, cerebral infarction, psychiatric disorders, schizophrenia, drug addiction, neurosis, psychosis, dementia, paranoia, attention deficit disorder, sleep disorders, pain disorders, eating or weight disorders, and cancer and tumors of the CNS. 56. The method of paragraph 50, wherein the ocular disease or disorder is selected from the retina, the posterior thalamic tract, and / or the optic nerve. 57. The method of paragraph 55, wherein the ophthalmologic disorder involving the retina, posterior thalamic tract, and / or optic nerve is selected from diabetic retinopathy, macular degeneration including age-related macular degeneration, geographic atrophy and vascular or "wet" macular degeneration, glaucoma, uveitis, retinitis pigmentosa, Stargardt's disease, Leber's congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), X-linked retinitis pigmentosa (XLRP), X-linked retinoschisis (XLRS), total choroidal atrophy, Leber's hereditary optic neuropathy (LHON), color blindness, cone-rod degeneration, Fuchs' endothelial corneal dystrophy, diabetic macular edema, and eye cancers and tumors. 58. The method of paragraph 50, wherein the blood disease or disorder is selected from hemophilia A, hemophilia B, thalassemia, anemia, and blood cancer. 59. The method of paragraph 50, wherein the liver disease or disorder is selected from progressive familial intrahepatic cholestasis (PFIC), and liver cancer and tumors. 60. The method of paragraph 42, wherein the disease or disorder is cystic fibrosis. 61. The method of paragraphs 42 to 60, wherein the ceDNA vector is administered in combination with a pharma- ceutically acceptable carrier. 62. A method for delivering a therapeutic protein to a subject, the method comprising administering to the subject a composition comprising a ceDNA vector described in any of paragraphs 1-25, wherein at least one heterologous nucleotide sequence encodes a therapeutic protein. 63. The method of paragraph 62, wherein the therapeutic protein is a therapeutic antibody. 64. The method of paragraph 62, wherein the therapeutic protein is selected from an enzyme, erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, cytokine, cystic fibrosis transmembrane conductance regulator (CFTR), peptide growth factor, and hormone. 65. A kit comprising a ceDNA vector and a nanocarrier described in any one of paragraphs 1 to 25, packaged in a container together with an attached instruction sheet. 66. A kit for producing a ceDNA vector, the kit comprising an expression construct comprising at least one restriction site for insertion of at least one heterologous nucleotide sequence, or a regulatory switch, or both, wherein the at least one restriction site is operably positioned between asymmetric inverted repeats (asymmetric ITRs), and at least one of the asymmetric ITRs comprises a functional terminal resolution site and a Rep binding site. 67. A kit according to paragraph 66, suitable for producing a ceDNA vector according to any one of paragraphs 1 to 25. 68. The kit of paragraph 66 or paragraph 67, further comprising a population of insect cells lacking viral capsid coding sequences, which can be induced to produce a ceDNA vector in the presence of Rep proteins. 69. The kit of any one of paragraphs 66 to 68, further comprising a vector comprising a polynucleotide sequence encoding at least one Rep protein, the vector being suitable for expressing said at least one Rep protein in an insect cell.

[0028] In some embodiments, one aspect of the technology described herein relates to a non-viral capsid-free DNA vector (ceDNA vector) having covalently closed ends, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site, optionally wherein the heterologous nucleic acid sequence encodes a transgene, and wherein the vector is not present in a viral capsid.

[0029] These and other aspects of the invention are described in further detail below. [Brief description of the drawings]

[0030] [Figure 1A] The exemplary structure of ceDNA vector is shown.In this embodiment, the exemplary ceDNA vector comprises an expression cassette containing CAG promoter, WPRE and BGHpA.The open reading frame (ORF) encoding luciferase transgene is inserted into the cloning site (R3 / R4) between CAG promoter and WPRE.The expression cassette is flanked by two inverted terminal repeats (ITR)-wild type AAV2 ITR at the upstream (5' end) of the expression cassette and modified ITR at the downstream (3' end), and therefore the two ITRs flanking the expression cassette are asymmetric with respect to each other. [Figure 1B] Figure 1 shows an exemplary structure of a ceDNA vector with an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - a modified ITR upstream (5' end) and a wild-type ITR downstream (3' end) of the expression cassette. [Figure 1C]1 shows an exemplary structure of a ceDNA vector with an expression cassette containing an enhancer / promoter, an open reading frame (ORF) for inserting a transgene, a posttranscriptional element (WPRE), and a polyA signal. The open reading frame (ORF) allows the insertion of a transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) that are asymmetric with respect to each other, a modified ITR at the upstream (5' end) and a modified ITR at the downstream (3' end) of the expression cassette, and both the 5'ITR and the 3'ITR are modified ITRs, but with different modifications (i.e., not the same modifications). [Figure 2A] The T-shaped stem-loop structure of the wild-type left ITR of AAV2 (SEQ ID NO: 538) is provided with the A-A', B-B', C-C' arms, identification of two Rep binding sites (RBE and RBE'), and also shows the terminal resolution site (trs). The RBE contains a series of four double tetramers that are believed to interact with either Rep78 or Rep68. In addition, the RBE' is also believed to interact with the Rep complex assembled on the wild-type or mutated ITR in the construct. The D and D' regions contain transcription factor binding sites and other conserved structures. [Figure 2B] Shown is the proposed Rep catalytic nicking and ligation activity in the wild-type ITR (SEQ ID NO: 539), including the T-shaped stem-loop structure of the wild-type left ITR of AAV2 with identification of the A-A' arm, the B-B' arm, the C-C' arm, two Rep binding sites (RBE and RBE'), and the terminal resolution site (trs), and also showing the D and D' regions which contain several transcription factor binding sites, and other conserved structures. [Figure 3A] 1 provides the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the A-A' arm and the C-C' and B-B' arms of the wild-type left AAV2 ITR (SEQ ID NO:540). [Figure 3B]1 shows an exemplary mutated ITR (also referred to as modified ITR) sequence of the left ITR. The primary structure (left) and predicted secondary structure (right) of the RBE portion of the A-A', C, and B-B' arms of an exemplary mutant left ITR (ITR-1, left) (SEQ ID NO: 113) are shown. [Figure 3C] The primary structure (left) and secondary structure (right) of the A-A' loop and the RBE-containing portions of the B-B' and C-C' arms of the wild-type right AAV2 ITR (sequence number 541) are shown. [Figure 3D] An exemplary right modified ITR is shown. The primary structure (left) and predicted secondary structure (right) of the A-A' arm and the RBE-containing portion of the B-B' and C arms of an exemplary mutant left ITR (ITR-1, right) (SEQ ID NO: 114) are shown. Any combination of left and right ITRs (e.g., AAV2 ITRs or other viral serotypes or synthetic ITRs) can be used, so long as the left ITR is asymmetric or different from the right ITR. Each of the polynucleotide sequences in Figures 3A-3D refers to sequences used in the plasmid or bacmid / baculovirus genome used to produce ceDNA as described herein. The corresponding ceDNA secondary structures deduced from the ceDNA vector configurations and predicted Gibb free energy values ​​in the plasmid or bacmid / baculovirus genome are also included in each of Figures 3A-3D. [Figure 4A] FIG. 4C is a schematic showing an upstream process for generating baculovirus-infected insect cells (BIIC), which are useful for producing ceDNA in the process described in the schematic in FIG. 4B. [Figure 4B] FIG. 1 is a schematic diagram of an exemplary method for ceDNA production. [Figure 4C] 1 shows biochemical methods and processes for confirming ceDNA vector production. [Figure 4D]FIG. 4B is a schematic diagram illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. The ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows the schematic expected bands of an exemplary ceDNA, left uncut or digested with a restriction endonuclease, and then subjected to electrophoresis in either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its double-stranded and uncleaved form, ceDNA exists in at least monomeric and dimeric states and is visible as a faster migrating small monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when ceDNA is cleaved with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original double-stranded DNA is single-stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently linked. Thus, in the second schematic from the right, the digested ceDNA shows a banding distribution similar to that observed on a native gel, but the bands migrate as fragments twice the size of their native gel counterparts. The schematic on the far right shows that uncut ceDNA under denaturing conditions migrates as a single-stranded open circle, and therefore the band observed is twice the size of that observed under native conditions where the circle is not opened.In this figure, "kb" is used to indicate the relative size of nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in a denatured state) or the number of base pairs (e.g., for double-stranded molecules observed in a native state), depending on the context. [Figure 4E]FIG. 4B is a schematic diagram illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process. FIG. 4E shows DNA with a non-contiguous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. FIG. 4E also shows ceDNA with a linear and contiguous structure. The ceDNA vector can be cleaved by a restriction endonuclease, generating two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions but not in denaturing conditions, producing a single strand in which the strands remain connected and migrate as 2 kb and 4 kb. FIG. 4D shows the schematic expected bands of an exemplary ceDNA, left uncut or digested with a restriction endonuclease, and then subjected to electrophoresis in either a native or denaturing gel. The leftmost schematic is a native gel and shows multiple bands suggesting that in its double-stranded and uncleaved form, ceDNA exists in at least monomeric and dimeric states and is visible as a faster migrating small monomer and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when ceDNA is cleaved with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears that corresponds to the expected fragment size remaining after cleavage. Under denaturing conditions, the original double-stranded DNA is single-stranded and migrates as a species twice as large as that observed on a native gel because the complementary strand is covalently linked. Thus, in the second schematic from the right, the digested ceDNA shows a banding distribution similar to that observed on a native gel, but the bands migrate as fragments twice the size of their native gel counterparts. The schematic on the far right shows that uncut ceDNA under denaturing conditions migrates as a single-stranded open circle, and therefore the band observed is twice the size of that observed under native conditions where the circle is not opened.In this figure, "kb" is used to indicate the relative size of nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in a denatured state) or the number of base pairs (e.g., for double-stranded molecules observed in a native state), depending on the context. [Diagram 5] An illustrative diagram of an example denaturing gel run of ceDNA vectors with (+) or without (-) digestion with endonucleases (EcoRI for ceDNA constructs 1 and 2, BamH1 for ceDNA constructs 3 and 4, SpeI for ceDNA constructs 5 and 6, and XhoI for ceDNA vectors 7 and 8). The sizes of the bands highlighted with asterisks were determined and are shown below the diagram. [Figure 6A] Shown are results from an in vitro protein expression assay measuring luciferase activity (y-axis, RQ(Luc)) in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of constructs identified on the x-axis (Construct-1, Construct-3, Construct-5, Construct-7) (Table 12). [Figure 6B] Shown is the luciferase activity (y-axis, RQ(Luc)) measured in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (gray), or 100 ng (white) of the constructs (Construct-2, Construct-4, Construct-6, Construct-8) (Table 12) identified on the x-axis. Also provided is the luciferase activity measured in HEK293 cells treated with Fugene ("Fugene") or untreated ("Untreated") without any plasmid. [Figure 7A] Shown is the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey), or 100 ng (white) of constructs (construct-1, construct-3, construct-5, construct-7) identified on the x-axis. [Figure 7B]Shown is the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey), or 100 ng (white) of constructs (construct-2, construct-4, construct-6, construct-8) identified on the x-axis. [Figure 8A] An exemplary Rep-bacmid in pFBDLSR plasmid containing the nucleic acid sequences of Rep proteins Rep52 and Rep78. This exemplary Rep-bacmid contains the polyhedron promoter sequence of the IE1 promoter fragment (SEQ ID NO:66), the Rep78 nucleotide sequence including the Kozak sequence (SEQ ID NO:67), Rep52 (SEQ ID NO:68) and the Rep58 nucleotide sequence beginning with the Kozak sequence (gccgccacc) (SEQ ID NO:69). [Figure 8B] FIG. 1 is a schematic diagram of an exemplary ceDNA-plasmid-1, carrying a wt-L ITR, a CAG promoter, a luciferase transgene, a WPRE and polyadenylation sequence, and a mod-R ITR. [Figure 9A] 1 shows the predicted lowest energy structure of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified left ITR ("ITR-2 (left)", SEQ ID NO: 101). [Figure 9B] 1 shows the predicted lowest energy structures of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified right ITR ("ITR-2 (right)" SEQ ID NO: 102). They are predicted to form a structure with a single arm (C-C') and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -72.6 kcal / mol. [Figure 10A] 1 shows the predicted lowest energy structure of the RBE-containing portions of the A-A' and B-B' arms of an exemplary modified left ITR ("ITR-3 (left)", SEQ ID NO: 103). [Figure 10B]1 shows the predicted lowest energy structures of the RBE-containing portions of the A-A' and B-B' arms of an exemplary modified right ITR ("ITR-3 (right)" SEQ ID NO: 104). They are predicted to form a structure with a single arm (B-B') and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -74.8 kcal / mol. [Figure 11A] 1 shows the predicted lowest energy structure of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified left ITR ("ITR-4 (left)", SEQ ID NO: 105). [Figure 11B] 1 shows the predicted lowest energy structures of the RBE-containing portions of the A-A' and C-C' arms of an exemplary modified right ITR ("ITR-4 (right)" SEQ ID NO: 106). They are predicted to form a structure with a single arm (C-C') and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -76.9 kcal / mol. [Figure 12A] 1 shows the predicted lowest energy structure of the A-A' arm of an exemplary modified left ITR, as well as the RBE-containing portions of the C-C' and B-B' portions, illustrating complementary base pairing of the C-B' and C'-B portions ("ITR-10 (left)", sequence number 107). [Figure 12B] The predicted lowest energy structures of the RBE-containing portions of the A-A' arm and the B-B' and C-C' portions of an exemplary modified left ITR are shown, showing the complementary base pairing of the B-C' and B'-C portions ("ITR-10 (right)" SEQ ID NO: 108). They are predicted to form a structure with a single arm (C'-B and C-B' portions or B'-C and B-C' portions) and a single unpaired loop. Their Gibbs unfolding free energy is predicted to be -83.7 kcal / mol. [Figure 13A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-17(left)", SEQ ID NO: 109). [Figure 13B]Shown is the predicted lowest energy structure of the RBE-containing portion of the A-A' arm and the C-C' and B-B' portions of an exemplary modified right ITR ("ITR-17(right)" SEQ ID NO: 110). Both ITR-17(left) and ITR-17(right) are predicted to form structures with a single arm (B-B') and a single unpaired loop. Their Gibbs unfolding free energies are predicted to be -73.3 kcal / mol. [Figure 14A] 1 shows the predicted lowest energy structure of the RBE-containing portion of the A-A' arm of an exemplary modified ITR ("ITR-6 (left)", SEQ ID NO: 111). [Figure 14B] 1 shows the predicted lowest energy structure of the RBE-containing portion of the A-A' arm of an exemplary modified ITR ("ITR-6(Right)" SEQ ID NO: 112). Both ITR-6(Left) and ITR-6(Right) are predicted to form structures with a single arm. Their Gibbs unfolding free energies are predicted to be -54.4 kcal / mol. [Figure 15A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C arm and B-B' arm of an exemplary modified left ITR ("ITR-1 (left)", SEQ ID NO: 113). [Figure 15B] Shown are the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C and B-B' arms of an exemplary modified right ITR ("ITR-1 (right)" SEQ ID NO: 114). Both ITR-1 (left) and ITR-1 (right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -74.7 kcal / mol. [Figure 16A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C' and B-B' arms of an exemplary modified left ITR ("ITR-5 (left)" SEQ ID NO: 545). [Figure 16B]Shown are the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C' arms of an exemplary modified right ITR ("ITR-5(right)" SEQ ID NO: 116). Both ITR-5(left) and ITR-5(right) are predicted to form structures with two arms, one of which (e.g., the C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -73.4 kcal / mol. [Figure 17A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-7 (left)", SEQ ID NO: 117). [Figure 17B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-7 (right)" SEQ ID NO: 118). Both ITR-17 (left) and ITR-17 (right) are predicted to form structures with two arms, one of which (e.g., the B-B' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -89.6 kcal / mol. [Figure 18A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-8 (left)", SEQ ID NO: 119). [Figure 18B] Shown are the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-8(Right)" SEQ ID NO: 120). Both ITR-8(Left) and ITR-8(Right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -86.9 kcal / mol. [Figure 19A]1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-9 (left)", SEQ ID NO: 121). [Figure 19B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-9(Right)" SEQ ID NO: 122). Both ITR-9(Left) and ITR-9(Right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -85.0 kcal / mol. [Figure 20A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-11 (left)", SEQ ID NO: 123). [Figure 20B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-11 (right)" SEQ ID NO: 124). Both ITR-11 (left) and ITR-11 (right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -89.5 kcal / mol. [Figure 21A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-12 (left)", SEQ ID NO: 125). [Figure 21B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-12(right)" SEQ ID NO: 126). Both ITR-12(left) and ITR-12(right) are predicted to form structures with two arms, one of which is truncated. Their Gibbs unfolding free energies are predicted to be -86.2 kcal / mol. [Figure 22A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-13 (left)", SEQ ID NO: 127). [Figure 22B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-13 (right)" SEQ ID NO: 128). Both ITR-13 (left) and ITR-13 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -82.9 kcal / mol. [Figure 23A] 1 shows the predicted lowest energy structures of the A-A' arm, and the RBE-containing portions of the C-C' and B-B' arms of an exemplary modified left ITR ("ITR-14 (left)", SEQ ID NO: 129). [Figure 23B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-14 (right)" SEQ ID NO: 130). Both ITR-14 (left) and ITR-14 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -80.5 kcal / mol. [Figure 24A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-C' arms of an exemplary modified left ITR ("ITR-15 (left)" SEQ ID NO: 131). [Figure 24B]Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-15 (right)" SEQ ID NO: 132). Both ITR-15 (left) and ITR-15 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -77.2 kcal / mol. [Figure 25A] 1 shows the predicted lowest energy structures of the A-A' arm and the RBE-containing portions of the C-C' and B-C' arms of an exemplary modified left ITR ("ITR-16 (left)", SEQ ID NO: 133). [Figure 25B] Shown are the predicted lowest energy structures of the RBE-containing portions of the A-A' arm, and the B-B' and C-C' arms of an exemplary modified right ITR ("ITR-16 (right)" SEQ ID NO: 134). Both ITR-16 (left) and ITR-16 (right) are predicted to form structures with two arms, one of which (e.g., the C-C' arm) is truncated. Their Gibbs unfolding free energies are predicted to be -73.9 kcal / mol. [Figure 26A-1] The predicted structures of the RBE-containing portions of the A-A' arms, as well as the modified B-B' arms and / or modified C-C' arms, of exemplary modified right ITRs listed in Table 10A are shown. [Figure 26A-2] Same as above. [Figure 26A-3] Same as above. [Figure 26A-4] Same as above. [Figure 26A-5] Same as above. [Figure 26A-6] Same as above. [Figure 26A-7] Same as above. [Figure 26B-1]Shown are predicted structures of the A-A' arm of an exemplary modified left ITR, and the RBE-containing portion of the modified C-C' arm and / or the modified B-B' arm listed in Table 10B. The structures shown are the predicted lowest free energy structures. Color code: red = >99% probability, orange = 99%-95% probability, beige = 95%-90% probability, dark green 90%-80%, light green = 80%-70%, light blue = 70%-60%, dark blue 60%-50%, and pink = less than 50%. [Figure 26B-2] Same as above. [Figure 26B-3] Same as above. [Figure 26B-4] Same as above. [Figure 26B-5] Same as above. [Figure 26B-6] Same as above. [Figure 26B-7] Same as above. [Figure 26B-8] Same as above. [Figure 27] Figure 1 shows luciferase activity of Sf9 GlycoBac insect cells transfected with asymmetric ITR mutant variants selected from Tables 10A and 10B. The ceDNA vectors had a luciferase gene flanked by wild-type ITRs and modified asymmetric ITRs selected from Tables 10A or 10B. "ITR-50 R no rep" is a known rescuable mutant without coinfection of Rep-containing baculovirus. "Mock" conditions are transfection reagent only with no donor DNA. [Figure 28]A native agarose gel (1% agarose, 1x TAE buffer) of representative crude ceDNA extracts from Sf9 insect cell cultures transfected with ceDNA-plasmids containing the left wt-ITR along with other ITRs selected from the various mutant right ITRs disclosed in Table 10A is shown. 2ug of total extract was loaded per lane. From left to right, lane 1) 1kb+ ladder, lane 2) ITR-18 right, lane 3) ITR-49 right, lane 4) ITR-19 right, lane 5) ITR-20 right, lane 6) ITR-21 right, lane 7) ITR-22 right, lane 8) ITR-23 right, lane 9) ITR-24 right, lane 10) ITR-25 right, lane 11) ITR-26 right, lane 12) ITR-27 right, lane 13) ITR-28 right, lane 14) ITR-50 right, lane 15) 1kb+ ladder. [Figure 29] Denaturing gel (0.8% alkaline agarose) of a representative construct from an ITR mutant library is shown. The ceDNA vector is produced from a plasmid construct containing the left wt-ITR along with another ITR selected from the various mutant right ITRs disclosed in Table 10A. From left to right, lane 1) 1 kb+ DNA ladder, lane 2) ITR-18 right uncut, lane 3) ITR-18 right restriction digest, lane 4) ITR-19 right uncut, lane 5) ITR-19 right restriction digest, lane 6) ITR-21 right uncut, lane 7) ITR-21 right restriction digest, lane 8) ITR-25 right uncut, lane 9) ITR-25 right restriction digest. Extracts were treated with EcoRI restriction endonuclease. Each mutant ceDNA is expected to have a single EcoRI recognition site that produces two characteristic fragments, approximately 2,000 bp and approximately 3,000 bp (reaching approximately 4,000 and 6,000 bp under denaturing conditions, respectively). Untreated ceDNA extracts are expected to be approximately 5,000 bp and migrate at approximately 11,000 bp under denaturing conditions. [Diagram 30]In vitro luciferase activity in HEK293 cells of ITR mutants ITR-18 right, ITR-19 right, ITR-21 right, and ITR-25 right, and ITR-49, where the left ITR in the ceDNA vector is the WT ITR. The "mock" condition is transfection reagent only without donor DNA, and the negative control is untreated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Clinical Chemistry, vol. 10, no. 10, pp. 1111-1115, 2002. and Therapy, 19th Edition, published by Merck Sharp&Dohme Corp., 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, DMand Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference,published by VCH Publishers,Inc.,1995(ISBN 1-56081-569-8)、Immunology by Werner Luttmann,published by Elsevier,2006、Janeway’s Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor&Francis Limited,2014(ISBN 0815345305,9780815345305)、Lewin’s Genes XI,published by Jones&Bartlett Publishers,2014(ISBN-1449659055)、Michael Richard Green and Joseph Sambrook,Molecular Cloning:A Laboratory Manual,4th ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,USA(2012)(ISBN 1936113414)、Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN 044460149X)、Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN 0124199542)、Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,2014(ISBN 047150338X,9780471503385),Current Protocols in Protein Science(CPPS),John E.Coligan(ed.),John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entireties.

[0032] As used herein, "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that can be incorporated into and delivered and expressed by the ceDNA vector disclosed herein. Transgenes of interest include, but are not limited to, nucleic acids encoding a polypeptide, preferably a therapeutic agent (e.g., for medical, diagnostic, or veterinary use), or an immunogenic polypeptide (e.g., for a vaccine). In some embodiments, nucleic acids of interest include nucleic acids that are transcribed into therapeutic RNA. Transgenes included for use in the ceDNA vectors of the present invention include, but are not limited to, those that express or code for one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNAs, RNAi, miRNAs, IncRNAs, antisense oligonucleotides or polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0033] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acid that includes a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not include capsid-encoding sequences, other vector sequences, or inverted terminal repeat regions. An expression cassette may additionally include one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0034] As used herein, the term "terminal repeat" or "TR" includes any viral or synthetic terminal sequence that contains at least one minimally necessary origin of replication and a region that contains a palindromic hairpin structure. The Rep binding sequence ("RBS") (also referred to as an RBE (Rep binding element)) and the terminal resolution site ("TRS") together constitute the "minimally necessary origin of replication," and thus a TR contains at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs," respectively. In the context of a virus, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found in the present invention herein, TRs that are not reverse complements over their entire length can still perform the traditional functions of ITRs, and thus the term ITR is used herein to refer to TRs in a ceDNA genome or ceDNA vector that can mediate replication of the ceDNA vector. It will be understood by those skilled in the art that there may be more than two ITRs or asymmetric ITR pairs in a composite ceDNA vector construct. ITRs may be AAV ITRs or non-AAV ITRs. ITR can be or can be derived from AAV ITR or non-AAV ITR. For example, ITR can be derived from Parvoviridae, which includes parvovirus and dependovirus (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or SV40 hairpin, which serves as origin of SV40 replication, can be used as ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. Parvoviridae viruses are composed of two subfamily, Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvovirus includes the virus family of adeno-associated virus (AAV), which can replicate in vertebrate hosts, including but not limited to human, primate, bovine, canine, equine, and ovine species.

[0035] As used herein, the term "asymmetric ITR" refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are not reverse-complementary throughout their entire length. The sequence difference between the two ITRs can be due to nucleotide addition, deletion, truncation, or point mutation. In one embodiment, one ITR of the pair can be a wild-type AAV sequence, and the other can be a non-wild-type or synthetic sequence. In another embodiment, neither ITR of the pair is a wild-type AAV sequence, and the two ITRs differ in sequence from each other. For convenience, the ITR located 5' (upstream of) the expression cassette in the ceDNA vector is referred to as the "5'ITR" or "left ITR", and the ITR located 3' (downstream of) the expression cassette in the ceDNA vector is referred to as the "3'ITR" or "right ITR".

[0036] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or virus genome as an intermolecular double-stranded polynucleotide of DNA.

[0037] As used herein, the term "ceDNA spacer region" refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. In some embodiments, the ceDNA spacer region holds two functional elements in a desired arrangement for optimal functionality. In some embodiments, the ceDNA spacer region provides or increases the genetic stability of the ceDNA genome, for example, in a plasmid or baculovirus. In some embodiments, the ceDNA spacer region facilitates easy genetic manipulation of the ceDNA genome by providing a convenient location for cloning sites, etc. For example, in certain aspects, an oligonucleotide "polylinker" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to have no known protein (e.g., transcription factor) binding sites, can be positioned in the ceDNA genome to separate cis-acting elements, for example, inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between the polyadenylation signal sequence and the 3' terminal resolution site.

[0038] As used herein, "Rep binding site", "Rep binding element", "RBE", and "RBS" are used interchangeably and refer to a binding site of a Rep protein (e.g., AAV Rep78 or AAV Rep68) that, upon binding by the Rep protein, allows the Rep protein to carry out its site-specific endonuclease activity on a sequence incorporating the RBS. The RBS sequence and its reverse complement together form a single RBS. RBS sequences are known in the art and include, for example, the RBS sequence specified in AAV2, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531). Any known RBS sequence may be used in embodiments of the invention, including other known AAV RBS sequences and other naturally known or synthetic RBS sequences. Without being bound by theory, it is believed that the nuclease domain of the Rep protein binds to the double-stranded nucleotide sequence GCTC, and thus the two known AAV Rep proteins bind directly to the double-stranded oligonucleotide, 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO:531), and stably assemble. In addition, the soluble aggregated conformers (i.e., an indefinite number of interrelated Rep proteins) dissociate and bind to oligonucleotides containing Rep binding sites. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interactions with the nitrogenous bases provide sequence specificity, while the interactions with the phosphodiester backbone are non-sequence specific or low sequence specific, stabilizing the protein-DNA complex.

[0039] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with 5' thymidine that generates a 3'OH that serves as a substrate for DNA elongation via cellular DNA polymerases, such as DNA pol δ or DNA pol ε. Alternatively, the Rep-thymidine complex can participate in a coordinate ligation reaction. In some embodiments, the TRS minimally includes a non-base-paired thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled at least in part by its distance within the same molecule from the RBS. When the acceptor substrate is a complementary ITR, the resulting product is an intermolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' (SEQ ID NO: 45) identified in AAV2. Any known TRS sequence can be used in embodiments of the invention, including other known AAV TRS sequences, other naturally known or synthetic TRS sequences such as AGTT (SEQ ID NO:46), GGTTGG (SEQ ID NO:47), AGTTGG (SEQ ID NO:48), AGTTGA (SEQ ID NO:49), and other motifs such as RRTTRR (SEQ ID NO:50).

[0040] As used herein, the term "ceDNA-plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0041] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby manipulating it as a shuttle vector for baculovirus.

[0042] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0043] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells (including but not limited to insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0044] As used herein, "closed-end DNA vector," "ceDNA vector," and "ceDNA" are used interchangeably and refer to a non-viral capsid-free DNA vector (i.e., an intermolecular duplex) having at least one covalently closed end. In some embodiments, the ceDNA comprises two covalently closed ends.

[0045] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferase causes cells to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0046] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide, or more appropriately, as a polypeptide that kills a cell, such as a toxin, or an agent that makes the cell susceptible to killing with a selected agent or its deletion. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins include, but are not limited to, restriction endonucleases (whether genomic or extrachromosomal) that target host cell DNA sequences, proteases that target polypeptides required for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. In some embodiments, the expression of effector proteins controlled by the synthetic biological circuits described herein can participate as a factor in another synthetic biological circuit, thereby expanding the scope and complexity of the reactivity of the biological circuit system.

[0047] Transcriptional regulators refer to transcriptional activators and repressors that activate or suppress the transcription of a gene of interest. A promoter is a region of nucleic acid that initiates the transcription of a particular gene. Transcriptional activators typically bind near the transcriptional promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to the transcriptional promoter and sterically hinder the initiation of transcription by RNA polymerase. Other transcriptional regulators can serve as either activators or repressors, depending on where they bind and on cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine-zipper proteins.

[0048] As used herein, "repressor protein" or "inducer protein" refers to a protein that binds to a regulatory sequence element and represses or activates the transcription of the sequence operably linked to the regulatory sequence element, respectively.Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input.Preferred proteins described herein are modular in form, for example, comprising separable DNA binding and input agent binding or responsive elements or domains.

[0049] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0050] As used herein, an "input agent responsive domain" is a domain of a transcription factor that responds to a condition or input agent such that it binds to the condition or input agent or otherwise renders the linked DNA-binding fusion domain responsive to the presence of that condition or input. In one embodiment, the presence of the condition or input results in a conformational change in the input agent responsive domain or the protein to which it is fused, which modifies the transcriptional regulatory activity of the transcription factor.

[0051] The term "in vivo" refers to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the embodiments described herein, the method or use may be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses carried out using live cells with intact membranes outside the body of a multicellular animal or plant, such as explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells), among others. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and may refer to introducing a programmable synthetic biological circuit into a non-cellular system, such as a cell or cell system-free medium, such as a cell extract.

[0052] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of the nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled. Promoters can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments of the aspects described herein, a promoter can drive the expression of transcription factors that regulate the expression of itself or of another promoter used in another modular component of the synthetic biological circuit described herein. Within the promoter sequence will be found the transcription initiation site, as well as protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the ceDNA vectors disclosed herein.

[0053] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50-1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream of the gene start site or downstream of the gene start site that they regulate. Enhancers can be located within intronic or exon regions of unrelated genes.

[0054] A promoter can be said to drive expression or drive transcription of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and regulates to control transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in an inverted orientation, whereby what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences are used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0055] A promoter may be one that is naturally associated with a gene or sequence, which may be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon of a given gene or sequence. Such a promoter may be referred to as "endogenous". Similarly, in some embodiments, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.

[0056] In some embodiments, the coding nucleic acid segment is placed under the control of a "recombinant promoter" or a "heterologous promoter", both of which refer to a promoter that is not normally associated with an operably linked encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and synthetic promoters or enhancers that are not "naturally occurring", i.e., may contain different elements of different transcriptional regulatory regions, and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, for the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Additionally, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be used as well.

[0057] As described herein, an "inducible promoter" is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or agent. As defined herein, an "inducer" or "inducing agent" can be endogenous or can be a compound or protein, usually exogenous, that is administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as repressors. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0058] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with the ceDNA vector according to the present invention is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals may be advantageously used as subjects that represent animal models of diseases and disorders. Additionally, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African American, African European, Hispanic, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.

[0059] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the same antigen that the intact antibody binds. In one embodiment, an antibody or antibody fragment comprises an immunoglobulin chain or antibody fragment, and at least one immunoglobulin variable domain sequence. Examples of antibodies and fragments thereof include Fv, scFv, Fab fragments, Fab', F(ab') 2 , Fab'-SH, single domain antibodies (dAbs), heavy chains, light chains, heavy and light chains, complete antibodies (e.g., including each of the Fc, Fab, heavy chain, seq., variable regions, etc.), bispecific antibodies, diabodies, linear antibodies, single chain antibodies, intrabodies, monoclonal antibodies, chimeric antibodies, multispecific antibodies, or multimeric antibodies. The antibody or fragment thereof may be of any class, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, or of any subclass, including, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Additionally, the antibody may be derived from any mammal, such as a primate, human, rat, mouse, horse, goat, etc. In one embodiment, the antibody is human or humanized. In some embodiments, the antibody is a modified antibody. In some embodiments, the components of the antibody may be expressed separately such that the antibody itself assembles following expression of the protein components. In some embodiments, the antibody is "humanized" to reduce immunogenic responses in humans. In some embodiments, the antibody has a desired function, e.g., a desired protein interaction and inhibition, for purposes of treating a disease or disease symptoms. In one embodiment, the antibody or antibody fragment comprises a framework region or F domain. c Includes the area.

[0060] As used herein, the term "antigen-binding domain" of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that is involved in antigen binding. In embodiments, the antigen-binding site is formed by amino acid residues of the variable regions (V) of the heavy (H) and light (L) chains. Three highly branched stretches in the variable regions of the heavy and light chains, called hypervariable regions, are arranged between more conserved adjacent stretches called "framework regions" (FR). FRs are amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface, which is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". Framework regions and CDRs are defined and described, for example, in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically formed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0061] As used herein, the term "full length antibody" refers to an immunoglobulin (Ig) molecule (eg, an IgG antibody) that occurs in nature and is formed by normal immunoglobulin gene fragment recombinatorial processes.

[0062] As used herein, the term "functional antibody fragment" refers to a fragment that binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of a recombinant single-chain polypeptide molecule in which the heavy and light chains, or the light and heavy chain variable regions, are connected by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include portions of an antibody without antigen-binding activity, such as an Fc fragment or a single amino acid residue.

[0063] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence can include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications that correspond to the formation of a protein structure.

[0064] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are essential to the method or composition, but are open to the inclusion of unspecified elements, whether essential or not.

[0065] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0066] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0067] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading this disclosure. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0068] Except in the working examples or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term "about". When used in relation to percentages, the term "about" can mean ±1%. The present invention is further described in detail by the following examples, but the scope of the present invention should not be limited thereto.

[0069] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0070] II. ceDNA Vector Provided herein are novel non-viral capsid-free ceDNA molecules (ceDNA) with covalently closed ends. These non-viral capsid-free ceDNA molecules can be produced in permissive host cells from expression constructs (e.g., ceDNA-plasmids, ceDNA-baculoviruses, or integrative cell lines) that contain a heterologous gene (transgene) located between two different inverted terminal repeat (ITR) sequences, the ITRs being different with respect to each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs contains a functional terminal resolution site (trs) and a Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vectors have covalently closed ends and are therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37°C for, for example, one hour or more.

[0071] The ceDNA vector disclosed herein does not have the packaging constraint imposed by the limited space in the viral capsid.The ceDNA vector represents a versatile eukaryotically produced alternative to the prokaryotically produced plasmid DNA vector, in contrast to the enclosed AAV genome.This allows the insertion of control elements, such as the regulatory switches disclosed herein, large transgenes, multiple transgenes, etc.

[0072] In one aspect, the ceDNA vector comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, where the first ITR and the second ITR are asymmetric with respect to each other, i.e., they are different from each other. In an exemplary embodiment, the first ITR can be a wild-type ITR, and the second ITR can be a mutated or modified ITR. In some embodiments, the first ITR can be a mutated or modified ITR, and the second ITR is a wild-type ITR. In another embodiment, the first ITR and the second ITR are both modified, but are of different sequences, or have different modifications, or are not identical modified ITRs. In other words, the ITRs are asymmetric, where any change in one ITR is not reflected in the other ITR, or the ITRs are different with respect to each other. Exemplary ITRs in ceDNA vectors for use in generating ceDNA-plasmids are described below in the section entitled "ITRs."

[0073] The wild-type or mutant or otherwise modified ITR sequences provided herein represent DNA sequences contained in an expression construct (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus) for the production of a ceDNA vector. Thus, the ITR sequences actually contained in a ceDNA vector produced from a ceDNA-plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0074] In some embodiments, the ceDNA vector described herein comprising an expression cassette with a transgene can be, for example, a regulatory sequence, a sequence encoding a nucleic acid (such as, for example, a miR or antisense sequence), or a sequence encoding a polypeptide (such as, for example, a transgene). In one embodiment, the transgene may be operably linked to one or more regulatory sequence(s) that allow or control expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, and the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric with respect to each other.

[0075] In one embodiment of each of these aspects, the expression cassette is located between two ITRs and includes one or more of a promoter, a post-transcriptional regulatory element, and a polyadenylation and termination signal, in that order, operably linked to the transgene. In one embodiment, the promoter is regulatable-inducible or repressible. The promoter can be any sequence that promotes transcription of the transgene. In one embodiment, the promoter is a CAG promoter (e.g., SEQ ID NO: 03) or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates the expression of the transgene, including, but not limited to, any sequence that creates a tertiary structure that enhances the expression of the transgene.

[0076] In one embodiment, the post-transcriptional regulatory element comprises WPRE (e.g., SEQ ID NO: 08). In one embodiment, the polyadenylation and termination signal comprises BGH polyA (e.g., SEQ ID NO: 09). Any cis-regulatory element known in the art, or combinations thereof, may additionally be used, such as the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements, including but not limited to the thymidine kinase gene of Herpes Simplex Virus or Hepatitis B Virus (HBV). In one embodiment, the expression cassette length in the 5' to 3' orientation exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0077] An expression cassette may comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 500-50,000 nucleotides in length. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 500-75,000 nucleotides in length. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 500-10,000 nucleotides in length. In some embodiments, an expression cassette may comprise a transgene or nucleic acid in the range of 1000-10,000 nucleotides in length. In some embodiments, the expression cassette may contain a transgene or nucleic acid ranging from 500 to 5,000 nucleotides in length. ceDNA vectors do not have the size limitations of encapsidated AAV vectors, thus allowing delivery of large sized expression cassettes resulting in efficient expression of the transgene. In some embodiments, ceDNA vectors lack prokaryotic cell-specific methylation.

[0078] The expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments, the ITRs may act as promoters of the transgene. In some embodiments, the ceDNA vector may include additional components for regulating the expression of the transgene, such as a regulatory switch, described in the section entitled "Regulatory Switches" herein, for controlling and regulating the expression of the transgene, and may include a regulatory switch, which is a kill switch that allows for controlled cell death of cells containing the ceDNA vector, if desired.

[0079] 1A-1C show schematic diagrams of non-limiting exemplary ceDNA vectors, or corresponding sequences of ceDNA plasmids. The ceDNA vector is obtained from a plasmid that is capsid-free and encodes a first ITR, an expressible transgene cassette, and a second ITR in this order, and at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably includes one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA) in this order.

[0080] The expression cassette may include any transgene of interest. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miRs, etc.), preferably therapeutic agents (e.g., for medical, diagnostic, or veterinary applications), or immunogenic polypeptides (e.g., for vaccines). In certain embodiments, the transgene in the expression cassette encodes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof. In some embodiments, the transgene is a therapeutic gene or a marker protein. In some embodiments, the transgene is an agonist or antagonist. In some embodiments, the antagonist is an antibody mimic, or an antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment. In some embodiments, the transgene encodes an antibody, including a full-length antibody or an antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as defined herein.

[0081] In particular, the transgene can encode one or more therapeutic agent(s) including, but not limited to, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen-binding fragments, and variants and / or active fragments thereof, for use in the treatment, prevention, and / or amelioration of one or more symptoms of, for example, a disease, dysfunction, injury, and / or disorder. Exemplary transgenes are described herein in the section entitled "Methods of Treatment."

[0082] There are many structural features of ceDNA vectors that are different from plasmid-based expression vectors. ceDNA vectors may have one or more of the following features: lack of original (i.e., uninserted) bacterial DNA, lack of a prokaryotic replication origin, self-contained (i.e., no sequences other than the two ITRs containing Rep binding and terminal resolution sites (RBS and TRS) or exogenous sequences between the ITRs), presence of hairpin-forming ITR sequences of eukaryotic origin (i.e., they are produced in eukaryotic cells), and absence of bacterial-type DNA methylation, or indeed any other methylation that would be considered abnormal by a mammalian host. In general, it is preferred that the vector does not contain any prokaryotic DNA, although it is contemplated that some prokaryotic DNA may be inserted as exogenous sequences, as a non-limiting example in a promoter or enhancer region. Another important feature that distinguishes ceDNA vectors from plasmid expression vectors is that ceDNA vectors are single-stranded linear DNA with closed ends, whereas plasmids are always double-stranded DNA.

[0083] The ceDNA vectors produced by the methods provided herein preferably have a linear, continuous structure rather than a discontinuous structure, as determined by a restriction enzyme digestion assay (FIG. 4D). A linear, continuous structure is believed to be more stable against attack by cellular endonucleases, while at the same time being less likely to recombine and cause mutagenesis. Thus, a linear, continuous structure is a preferred embodiment. A continuous, linear, single-stranded intramolecular duplex ceDNA vector may be covalently linked at the termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally distinct from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. Complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, whereas, conversely, a ceDNA vector, although having complementary strands, is a single DNA molecule and thus remains a single molecule even when denatured. In some embodiments, the ceDNA vectors described herein, unlike plasmids, can be produced without DNA base methylation of prokaryotic cell types. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (in particular linear vs. circular) and the methods used to produce and purify these different objects (see below), as well as in terms of their DNA methylation, which in the case of ceDNA-plasmids is of prokaryotic cell type and in the case of ceDNA vectors is of eukaryotic cell type.

[0084] Some advantages of the ceDNA vectors described herein over plasmid-based expression vectors include, but are not limited to: 1) Plasmids contain bacterial DNA sequences and are subject to prokaryotic cell-specific methylation, e.g., 6-methyladenosine and 5-methylcytosine methylation, whereas capsid-free AAV vector sequences are of eukaryotic origin and are not subject to prokaryotic cell-specific methylation, and as a result, capsid-free AAV vectors are less likely to induce inflammation and immune responses compared to plasmids. 2) Plasmids require the presence of resistance genes during the production process, whereas ceDNA vectors do not. 3) Circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements, i.e., ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. We hypothesize that the minimal defining elements essential for ITR function are a Rep binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and a terminal resolution site (TRS, 5'-AGTTGG-3' (SEQ ID NO: 48) for AAV2), plus a variable palindromic sequence that allows hairpin formation. 4) ceDNA vectors do not have the over-representation of CpG dinucleotides often found in prokaryotic cell-derived plasmids that reportedly bind members of the Toll-like family of receptors and elicit T cell-mediated immune responses. In contrast, transduction with the capsid-free AAV vectors disclosed herein can use a variety of delivery reagents and efficiently target cells and tissue types that are difficult to transduce with conventional AAV virions.

[0085] III.ITR As disclosed herein, the ceDNA vector contains a heterologous gene located between two inverted terminal repeat (ITR) sequences that are different with respect to each other (i.e., asymmetric ITRs). In some embodiments, at least one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs comprises a functional Rep binding site (RBS, e.g., 5'-GCGCGCTCGCTCGCTC-3', SEQ ID NO:531 for AAV2) and a functional terminal resolution site (TRS, e.g., 5'-AGTT-3', SEQ ID NO:46). In one embodiment, at least one of the ITRs is a non-functional ITR. In one embodiment, the different ITRs are not each wild-type ITR from a different serotype.

[0086] ITRs are illustrated in the specification, and an example herein is the AAV2 ITR, but one of skill in the art will recognize that ITRs from any known parvovirus, e.g., dependovirus, e.g., AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes, see, e.g., NCBI: NC002077; NC001401; NC001729; NC001829; NC006152; NC006260; NC006261), chimeric ITRs, or ITRs from any synthetic AAV, as described above. In some embodiments, AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITRs are derived from B19 parvovirus (GenBank Accession No. NC000883), minute virus of mice (MVM) (GenBank Accession No. NC001510), goose parvovirus (GenBank Accession No. NC001701), snake parvovirus 1 (GenBank Accession No. NC006148).

[0087] In some embodiments, the ITR sequence may be derived from a virus of the Parvoviridae family, which includes two subfamilies, the Parvovirinae subfamily, which infect vertebrates, and the Densovirinae subfamily, which infect insects. The Parvovirinae subfamily (referred to as parvoviruses) includes the Dependovirus family, whose members require co-infection with a helper virus, such as adenovirus or herpesvirus, for productive infection under most conditions. The Dependovirus family includes adeno-associated viruses (AAVs), which normally infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[0088] The skilled artisan will understand that ITR sequences have the general structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure (e.g., Figures 2A and 3A), with each ITR formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), and a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). Based on the exemplary AAV2 ITR sequences provided herein, the skilled artisan can easily determine the corresponding modified ITR sequences from any AAV serotype for use in ceDNA vectors or ceDNA-plasmids. See, for example, structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6). Grimm et al., J. Virology, 2006; 80(1); 426-439, Yan et al., J. Virology, 2005; 364-379, Duan et al., J. Virology, 2005; 364-379, et al., Virology 1999;261;8-14.

[0089] Although certain modifications and mutations in ITRs are described in detail herein, in the context of ITRs, "modified" or "mutated" indicates that nucleotides have been inserted, deleted, and / or substituted relative to the wild-type sequence, reference sequence, or original ITR sequence, and may be modified relative to other adjacent ITRs in a ceDNA vector having two adjacent ITRs. A modified or mutated ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been engineered by the hand of man. For example, a polypeptide is considered to be "engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been engineered by the hand of man and is different from the aspect in which it occurs in nature.

[0090] In some embodiments, the ITR can be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITR does not contain AAV-based sequences. In yet another embodiment, the synthetic ITR preserves the ITR structure described above, but has little or no AAV-origin sequences. In some aspects, the synthetic ITR can preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, is not recognized by wild-type Rep, but only recognized by mutant Rep.

[0091] ITR sequences have the general structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure (e.g., Figures 2A and 3A), with each ITR formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), and a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). Based on the exemplary AAV2 ITR sequences provided herein, one skilled in the art can easily determine ITR sequences or modified ITR sequences from any AAV serotype for use in ceDNA vectors or ceDNA-plasmids. See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1 to AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006; 80(1); 426-439. This shows the percent identity of the left ITR of AAV2 to the left ITRs from other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%).

[0092] Thus, although AAV2 ITRs are used as exemplary ITRs in the ceDNA vectors disclosed herein, the ceDNA vectors disclosed herein can be prepared with or based on ITRs of any known AAV serotype, including, for example, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12). A skilled person can determine the corresponding sequences in other serotypes by known means. For example, determine whether there is a change in the A, A', B, B', C, C', or D region, and determine the corresponding region in another serotype. BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs can be used in default settings to determine corresponding sequences. The present invention further provides a collection and a plurality of ceDNA vectors that include ITRs from a combination of different AAV serotypes. That is, one ITR can be from one AAV serotype and the other ITR can be from a different serotype. Without being bound by theory, in one embodiment, one ITR may be derived from or based on the AAV2 ITR sequence and the other ITR of the ceDNA vector may be derived from or based on any one or more ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[0093] Any parvovirus ITR can be used for modification as an ITR or as a base ITR. Preferably, the parvovirus is a dependovirus. More preferably, it is an AAV. The serotype selected can be based on the tissue tropism of the serotype. AAV2 has a broad tissue tropism, AAV1 preferentially targets neurons and skeletal muscle, and AAV5 targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 preferentially targets skeletal muscle and lung. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue. AAV9 preferentially targets liver, skeletal, and lung tissue. In one embodiment, the modified ITR is based on the AAV2 ITR. For example, it is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:52. In one embodiment of each of these aspects, the vector polynucleotide comprises a pair of ITRs selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:52, and SEQ ID NO:2 and SEQ ID NO:51. In one embodiment of each of these aspects, the vector polynucleotide or non-viral capsid-free DNA vector with covalently closed ends comprises a pair of different ITRs selected from the group consisting of SEQ ID NO: 101 and SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114, and SEQ ID NO: 115 and SEQ ID NO: 116. In some embodiments, the modified ITRs are selected from any of the ITR or partial ITR sequences of SEQ ID NOs: 2, 52, 63, 64, 101-499, or 545-547.

[0094] In some embodiments, the ceDNA vector may comprise an ITR having an ITR sequence or ITR subsequence set forth in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein, or an ITR modification corresponding to any of the modifications of the sequences set forth in Figures 26A or 26B.

[0095] In some embodiments, ceDNA can form an intramolecular double-stranded secondary structure. The secondary structures of the first ITR and the asymmetric second ITR are illustrated in the context of the wild-type ITR (see, e.g., Figures 2A, 3A, and 3C) and modified ITR structures (see, e.g., Figures 2B, and 3B, 3D). The secondary structures are deduced or predicted based on the ITR sequence of the plasmid used to produce the ceDNA vector. Exemplary secondary structures of modified ITRs in which a portion of the stem-loop structure is deleted are shown in Figures 9A-25B and Figures 26A-26B, and also in Tables 10A and 10B. Exemplary secondary structures of modified ITRs that include a single stem and two loops are shown in Figures 9A-13B. Exemplary secondary structures of modified ITRs with a single stem and a single loop are shown in Figure 14. In some embodiments, secondary structure may be predicted using thermodynamic methods based on nearest neighbor rules that predict the stability of a structure as quantified by doubling the free energy change, as presented herein. For example, the structure may be predicted by finding the lowest free energy structure. In some embodiments, Reuter, JS, & Mathews, DH (2010) RNAstructure: software for RNA secondary structure prediction and The algorithm disclosed in the paper "Analysis.BMC Bioinformatics.11,129" and implemented in the RNAstructure software (available at the World Wide Web address: "rna.urmc.rochester.edu / RNAstructureWeb / index.html") can be used to predict ITR structures. This algorithm also includes both free energy change parameters at 37°C, and enthalpy change parameters derived from experimental literature, which may allow prediction of structural stability at any temperature. Using the RNAstructure software, some of the modified ITR structures can be predicted as modified T-shaped stem-loop structures with estimated Gibbs unfolding free energies (ΔG) under physiological conditions shown in Figures 3A-3D. Using the RNAstructure software, three types of modified ITRs are predicted to have higher Gibbs unfolding free energies than the wild-type ITR of AAV2 (-92.9 kcal / mol), as follows: (a) Modified ITRs having a single arm / single unpaired loop structure as provided herein are predicted to have a Gibbs unfolding free energy in the range of -85 to -70 kcal / mol. (b) Modified ITRs having a single hairpin structure as provided herein are predicted to have a Gibbs unfolding free energy in the range of -70 to -40 kcal / mol. (c) Modified ITRs having two arm structures as provided herein are predicted to have a Gibbs unfolding free energy in the range of -90 to -70 kcal / mol. Without wishing to be bound by theory, structures with higher Gibbs free energy are more easily unfolded for replication by Rep 68 or Rep 78 replication proteins. Thus, modified ITRs having higher Gibbs unfolding free energy, e.g., single arm / single unpaired loop structure, single hairpin structure, truncated structure, tend to be replicated more efficiently than wild-type ITRs.

[0096] In one embodiment, the left ITR of the ceDNA vector is modified or mutated with respect to the wild-type (wt) AAV ITR structure, and the right ITR is the wild-type AAV ITR.In one embodiment, the right ITR of the ceDNA vector is modified with respect to the wild-type AAV ITR structure, and the left ITR is the wild-type AAV ITR.In such an embodiment, the modification of the ITR (e.g., the left or right ITR) can be generated by the deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR derived from the AAV genome.

[0097] ITRs as used herein can be degradable and non-degradable and are selected for use in ceDNA vectors, preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, 6, 7, 8, and 9 being preferred. Degradable AAV ITRs do not require wild-type ITR sequences, as long as the terminal repeats mediate the desired function, such as replication, viral packaging, integration, and / or proviral rescue (e.g., endogenous or wild-type AAV ITR sequences can be altered by insertion, deletion, truncation, and / or missense mutation). Typically, but not necessarily, the ITRs are from the same AAV serotype, e.g., both ITR sequences of the ceDNA vector are from AAV2. The ITRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al. Although not necessarily, the ITRs can be from the same parvovirus, e.g., both ITR sequences are from AAV2.

[0098] In one embodiment, the ceDNA may contain an ITR structure that is mutated with respect to one of the wild-type ITRs disclosed herein, but the mutant or modified ITR still retains all operational Rep binding sites (RBE or RBE') and terminal resolution sites (trs). In one embodiment, the mutant ceDNA ITR contains a functional replication protein site (RPS-1), and a replicative protein that binds to the RPS-1 site is used for production.

[0099] In one embodiment, at least one of the ITRs is defective with respect to Rep binding and / or Rep nicking. In one embodiment, the defect is at least 30% relative to the wild-type reduced ITR, and in other embodiments, at least 35%..., 50%..., 65%..., 75%..., 85%..., 90%..., 95%..., 98%..., or completely lacking in function or any point in between. The host cell does not express viral capsid proteins, and the polynucleotide vector template lacks any viral capsid coding sequence. In one embodiment, the polynucleotide vector template and host cell lacking AAV capsid genes, and the resulting proteins also do not encode or express capsid genes of other viruses. Additionally, in certain embodiments, the nucleic acid molecule also lacks AAV Rep protein coding sequence.

[0100] In some embodiments, the structural element of the ITR can be any structural element involved in the functional interaction of the ITR with a large Rep protein (e.g., Rep 78 or Rep 68). In certain embodiments, the structural element provides selectivity for the interaction of the ITR with a large Rep protein, i.e., determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, the structural element physically interacts with the large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleotide sequence of the ITR, the space between two or more elements, or any combination of the above. In one embodiment, the structural element is selected from the group consisting of A and A' arms, B and B' arms, C and C' arms, D arms, Rep binding sites (RBE) and RBE' (i.e., complementary RBE sequences), and terminal resolution sites (trs).

[0101] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleotide sequence of the structural element can be modified compared to the wild-type sequence of the ITR. In one embodiment, the structural elements of the ITR (e.g., A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from a different parvovirus. For example, the replacement structure can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITRs can be AAV2 ITRs and the A or A' arms or RBE can be replaced with structural elements from AAV5. In another embodiment, the ITRs can be AAV5 ITRs and the C or C' arms, RBE, and trs can be replaced with structural elements from AAV2. In another embodiment, the AAV ITRs can be AAV5 ITRs and the B and B' arms are replaced with the AAV2 ITR B and B' arms.

[0102] By way of example only, Table 1 shows exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in a region of a modified ITR, where X indicates a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any of the C and / or C' and / or B and / or B' regions retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. For example, if the modification results in either a single-arm ITR (e.g., a single C-C' arm, or a single B-B' arm), or a modified C-B' arm or C'-B arm, or a two-arm ITR with at least one truncated arm (e.g., a truncated C-C' arm and / or a truncated B-B' arm), at least the single arm, or at least one of the arms of the two-arm ITR (one arm may be truncated), retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In some embodiments, the truncated C-C' arm and / or the truncated B-B' arm have three consecutive T nucleotides (i.e., TTT) in the terminal loop. [Table 1]

[0103] In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1, as well as modifications of at least one nucleotide in any one or more of the regions selected from A' and C, C and C', C' and B, B and B', and B' and A. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the C or C' or B or B' regions still preserves the terminal loop of the stem-loop. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or B and B' retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In alternative embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or B and B' retains three consecutive A nucleotides (i.e., AAA) in at least one terminal loop. In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1 and at least one nucleotide modification (e.g., deletion, insertion, and / or substitution) in one or more of the regions selected from A', A, and / or D. For example, in some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1 and at least one nucleotide modification (e.g., deletion, insertion, and / or substitution) in the A region. In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1 and at least one nucleotide modification (e.g., deletion, insertion, and / or substitution) in the A' region. In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1 and at least one nucleotide modification (e.g., deletion, insertion, and / or substitution) in the A and / or A' regions.In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 1, as well as a modification (e.g., a deletion, insertion, and / or substitution) of at least one nucleotide in the D region.

[0104] In one embodiment, the nucleotide sequence of a structural element can be modified (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein) to produce a modified structural element. In one embodiment, specific modifications to the ITRs are exemplified herein (e.g., SEQ ID NOs: 2, 52, 63, 64, 101-499, or 545-547). In some embodiments, the ITRs can be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein). In other embodiments, the ITR may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to a modified ITR of SEQ ID NO:469-499 or 545-547, or to one of the RBE-containing sections of the A-A' arm and the C-C' and B-B' arms of SEQ ID NO:101-134 or 545-547.

[0105] In some embodiments, the modified ITR may include, for example, the removal or deletion of all of a particular arm, e.g., all or part of the A-A' arm, or all or part of the B-B' arm, or all or part of the C-C' arm, or the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form the stem of a loop (see, e.g., ITR-6), so long as there is still a final loop capping the stem (e.g., a single arm). In some embodiments, the modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, the modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm. In some embodiments, the modified ITR may include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. Any combination of base pair removal is envisioned, for example, six base pairs in the C-C' arm and two base pairs in the B-B' arm may be removed. As an illustrative example, Figures 13A-13B show an exemplary modified ITR with at least seven base pairs deleted from each of the C and C' portions, a substitution of a nucleotide in the loop between the C and C' regions, and a deletion of at least one base pair from each of the B and B' regions, such that the modified ITR includes two arms in which at least one arm (e.g., C-C') is truncated. In this example, the modified ITR contains at least one base pair deletion from each of the B and B' regions, such that arm B-B' is also truncated relative to the WT ITR.

[0106] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more complementary base pairs are removed from each of the C and C' portions of the C-C' arm such that the C-C' arm is cleaved. That is, when a base is removed in the C portion of the C-C' arm, the complementary base pair in the C' portion is removed, thereby cleaving the C-C' arm. In such embodiments, 2, 4, 6, 8, or more base pairs are removed from the C-C' arm such that the C-C' arm is cleaved. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the C portion of the C-C' arm such that only the C' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the C' portion of the C-C' arm such that only the C portion of the arm remains.

[0107] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more complementary base pairs are removed from each of the B and B' portions of the B-B' arm such that the B-B' arm is cleaved. That is, when a base is removed in the B portion of the B-B' arm, the complementary base pair in the B' portion is removed, thereby cleaving the B-B' arm. In such embodiments, 2, 4, 6, 8, or more base pairs are removed from the B-B' arm such that the B-B' arm is cleaved. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the B portion of the B-B' arm such that only the B' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs are removed from the B' portion of the B-B' arm such that only the B portion of the arm remains.

[0108] In some embodiments, the modified ITR may have a 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR may have a 1-30 nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR has a 2-20 nucleotide deletion relative to the full-length wild-type ITR sequence.

[0109] In some embodiments, the modified ITR forms two opposing longitudinally asymmetric stem-loops, e.g., the C-C' loop is of a different length relative to the B-B' loop. In some embodiments, one of the opposing longitudinally asymmetric stem-loops of the modified ITR has a C-C' and / or B-B' stem portion in the range of 8-10 base pairs in length, and a loop portion having 2-5 unpaired deoxyribonucleotides (e.g., between C and C' or between B and B'). In some embodiments, one longitudinally asymmetric stem-loop of the modified ITR has a C-C' and / or B-B' stem portion less than 8 or less than 7, 6, 5, 4, 3, 2, 1 base pairs in length, and a loop portion having 0-5 nucleotides (e.g., between C and C' or between B and B'). In some embodiments, the modified ITR with a longitudinally asymmetric stem-loop has a C-C' and / or B-B' stem portion less than 3 base pairs in length.

[0110] In some embodiments, the modified ITRs do not contain any nucleotide deletions in the RBE-containing portions of the A or A' regions so as not to interfere with DNA replication (e.g., binding to the RBE by Rep proteins or nicking at the terminal resolution site). In some embodiments, modified ITRs encompassed for use herein have one or more deletions in the B, B', C, and / or C regions described herein. Some non-limiting examples of modified ITRs are shown in Figures 9A-26B.

[0111] In some embodiments, the modified ITR may include a deletion of the B-B' arm such that a C-C' arm remains. See, for example, the exemplary ITR-2 (left) and ITR-2 (right), and ITR-4 (left) and ITR-4 (right) shown in Figures 9A-9B (Figures 11A-11B). In some embodiments, the modified ITR may include a deletion of the C-C' arm such that a B-B' arm remains. See, for example, the exemplary ITR-3 (left) and ITR-3 (right), shown in Figures 10A-10B. In some embodiments, the modified ITR may include a deletion of the B-B' and C-C' arms such that a single stem loop remains. See, for example, the exemplary ITR-6 (left) and ITR-6 (right), and ITR-21 and ITR-37 shown in Figures 14A-14B. In some embodiments, the modified ITR may include a deletion of the C' region, such that a truncated C loop and B-B' arm remain. See, for example, exemplary ITR-1 (left) and ITR-1 (right) shown in Figures 15A-15B. Similarly, in some embodiments, the modified ITR may include a deletion of the C region, such that a truncated C loop and B-B' arm remain. See, for example, exemplary ITR-5 (left) and ITR-5 (right) shown in Figures 16A-16B.

[0112] In some embodiments, the modified ITRs can include deletions of base pairs in any one or more of the C, C', B, or B' portions, which allows complementary base pairing between the C-B' and C'-B portions to produce a single arm, see, e.g., ITR-10(right) and ITR-10(left) (Figures 12A-12B).

[0113] In some embodiments, in addition to modifications in one or more nucleotides in the C, C', B, and / or B' regions, modified ITRs for use herein may include modifications (e.g., deletions, substitutions, or additions) of at least 1, 2, 3, 4, 5, 6 nucleotides in any one or more of the regions selected from between A' and C, between C and C', between C' and B, between B and B', between B' and A. For example, the nucleotides between B' and C in the modified right ITR may be substituted from nA to G, C, or A, or deleted, or one or more nucleotides may be added. The nucleotides between C' and B in the modified left ITR may be changed from T to G, C, or A, or deleted, or one or more nucleotides may be added.

[0114] In certain embodiments of the present invention, the ceDNA vector does not have a modified ITR consisting of a nucleotide sequence selected from any of SEQ ID NOs: 550 to 557. In certain embodiments of the present invention, the ceDNA vector does not have a modified ITR comprising a nucleotide sequence selected from any of SEQ ID NOs: 550 to 557.

[0115] In some embodiments, the ceDNA vector comprises a regulatory switch as disclosed herein and a selected modified ITR having a nucleotide sequence selected from the group consisting of any of SEQ ID NOs: 550-557.

[0116] In another embodiment, the structure of the structural element may be modified. For example, the structural element may be modified in the height of the stem and / or the number of nucleotides in the loop. For example, the height of the stem may be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. In one embodiment, the stem height may be about 5 nucleotides to about 9 nucleotides and functionally interacts with Rep. In another embodiment, the stem height may be about 7 nucleotides and functionally interacts with Rep. In another example, the loop may be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0117] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites in an RBE or extended RBE can be increased or decreased. In one example, an RBE or extended RBE can contain 1, 2, 3, 4, 5, 6 or more GAGY binding sites, or any range therein. Each GAGY binding site can independently be the exact GAGY sequence or a sequence similar to GAGY, so long as the sequence is sufficient to bind to the Rep protein.

[0118] In another embodiment, the spacing between two elements (such as, but not limited to, an RBE and a hairpin) can be altered (e.g., increased or decreased) to alter functional interactions with a large Rep protein. For example, the spacing can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any range therein.

[0119] The ceDNA vectors described herein may contain ITR structures that are modified with respect to the wild-type AAV2 ITR structure disclosed herein, but still retain operational RBE, trs, and RBE' portions. Figures 2A and 2B show one possible mechanism for the manipulation of the trs site in the wild-type ITR structure of the ceDNA vector. In some embodiments, the ceDNA vector contains one or more functional ITR polynucleotide sequences that include a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO:531) for AAV2) and a terminal resolution site (TRS, 5'-AGTT (SEQ ID NO:46)). In some embodiments, at least one ITR (wild-type or modified ITR) is functional. In alternative embodiments where the ceDNA vector contains two modified ITRs that are different or asymmetric from each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0120] In some embodiments, the ceDNA vector does not have a modified ITR selected from any sequence provided herein consisting of, or consisting essentially of, SEQ ID NOs: 500-529. In some embodiments, the ceDNA vector does not have an ITR selected from any sequence selected from SEQ ID NOs: 500-529.

[0121] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vectors described herein has a modification in the loop arm, cleavage arm, or spacer. Exemplary sequences of ITRs with modifications in the loop arm, cleavage arm, or spacer are listed in Table 2.

[0122] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vector described herein has modifications in the loop arm and cleavage arm. Exemplary sequences of ITRs with modifications in the loop arm and cleavage arm are listed in Table 3.

[0123] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vectors described herein has modifications in the loop arms and spacer. Exemplary sequences of ITRs with modifications in the loop arms and spacer are listed in Table 4.

[0124] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vector described herein has a modification in the cleavage arm and spacer. Exemplary sequences of ITRs with modifications in the cleavage arm and spacer are listed in Table 5.

[0125] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vectors described herein has modifications in the loop arm, cleavage arm, and spacer. Exemplary sequences of ITRs with modifications in the loop arm, cleavage arm, and spacer are listed in Table 6.

[0126] In some embodiments, an ITR (e.g., a left or right ITR) is modified such that it contains the lowest unfolding energy ("low energy structure"). A low energy will have a reduced Gibbs free energy compared to the wild-type ITR. Exemplary sequences of ITRs modified to lower (i.e., reduced) unfolding energies are presented herein in Tables 7-9.

[0127] In some embodiments, the modified ITRs are selected from any of those shown in Tables 2-9, 10A or 10B, or a combination thereof. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

Table 2-5

Table 3-1

Table 3-2

Table 4-1

Table 4-2

Table 4-3

Table 5-1

Table 5-2

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

[0128] As disclosed herein, modified ITRs can be generated to include one or more nucleotide deletions, insertions, or substitutions from the wild-type ITRs derived from the AAV genome. Modified ITRs can be generated by genetic modification during propagation in a plasmid in Escherichia coli, or as a baculovirus genome in Spodoptera frugiperda cells, or by other biological methods, for example, using the polymerase chain reaction or chemical synthesis in vitro.

[0129] In some embodiments, the modified ITR comprises a deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR of AAV2 (left) (SEQ ID NO: 51) or the wild-type ITR of AAV2 (right) (SEQ ID NO: 1). Specifically, one or more nucleotides are deleted, inserted, or substituted from B-C' or C-C' of the T-shaped stem-loop structure. Additionally, the modified ITR does not contain modifications to the Rep binding element (RBE) and terminal resolution site (trs) of the wild-type ITR of AAV2, although the RBE' (TTT) may or may not be present depending on whether the template has undergone a round of replication, thereby converting the AAA triplet to the complementary RBE'-TTT.

[0130] Three types of modified ITRs are exemplified: (1) modified ITRs with the lowest energy structure containing a single arm and a single unpaired loop ("single arm / single unpaired loop structure"), (2) modified ITRs with the lowest energy structure with a single hairpin ("single hairpin structure"), and (3) modified ITRs with the lowest energy structure with two arms, one of which is truncated ("truncated structure").

[0131] Modified ITR with a single arm / single unpaired loop structure Wild-type ITRs can be modified to form secondary structures that include a single arm and a single unpaired loop (i.e., "single arm / single unpaired loop structures"). The Gibbs unfolding free energies (ΔG) of the structures can range from -85 kcal / mol to -70 kcal / mol. Exemplary structures of modified ITRs are provided.

[0132] Modified ITRs that are predicted to form a single arm and a single unpaired loop structure may contain a deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR in the sequences forming the B and B' arms and / or the C and C' arms. Modified ITRs may be generated by genetic modification or biosynthesis and / or chemical synthesis.

[0133] For example, ITR-2 left and right (SEQ ID NOs: 101 and 102) provided in Figures 9A-9B are generated to have a deletion of two nucleotides from the C-C' arm and a deletion of 16 nucleotides from the B-B' arm in the wild-type ITR of AAV2. The three nucleotides remaining in the B-B' arm of the modified ITR do not make complementary pairing. Thus, ITR-2 left and right have the lowest energy structure with a single C-C' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -72.6 kcal / mol.

[0134] ITR-3 left and right (SEQ ID NO: 103 and 104) provided in Figures 10A and 10B are generated to contain a 19 nucleotide deletion in the C-C' arm from the wild-type ITR of AAV2. The three nucleotides remaining in the B-B' arm of the modified ITR do not make a complementary pairing. Thus, ITR-3 left and right have the lowest energy structure with a single B-B' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be about -74.8 kcal / mol.

[0135] ITR-4 left and right (SEQ ID NO: 105 and 106) provided in Figures 11A and 11B are generated to contain a 19 nucleotide deletion in the B-B' arm from the wild-type ITR of AAV2. The three nucleotides remaining in the B-B' arm of the modified ITR do not make complementary pairing. Thus, ITR-4 left and right have the lowest energy structure with a single C-C' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be about -76.9 kcal / mol.

[0136] ITR-10 left and right (SEQ ID NOs: 107 and 108) provided in Figures 12A and 12B are generated to contain an 8 nucleotide deletion in the B-B' arm from the wild-type ITR of AAV2. The nucleotides remaining in the B-B' and C-C' arms create new complementary bonds between the B and C' motifs (ITR-10 left) or between the C and B' motifs (ITR-10 right). Thus, ITR-10 left and right have the lowest energy structure with a single B-C' or C-B' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -83.7 kcal / mol.

[0137] ITR-17 left and right (SEQ ID NOs: 109 and 110) provided in Figures 13A and 13B are generated to contain a 14 nucleotide deletion in the C-C' arm from the wild type ITR of AAV2. The remaining 8 nucleotides in the C-C' arm do not create a complementary bond. As a result, ITR-17 left and right have the lowest energy structure with a single B-B' arm and a single unpaired loop. The Gibbs free energy of unfolding the structure is predicted to be approximately -73.3 kcal / mol.

[0138] The sequences of the wild-type ITR left or right (top) and various modified ITR left or right (bottom) that are predicted to form a single arm / single unpaired loop structure are aligned and provided in Table 7 below. [Table 7-1] [Table 7-2] [Table 7-3]

[0139] Modified ITR with a single hairpin structure The wild-type ITR can be modified to have a lowest energy structure that includes a single hairpin structure. The Gibbs unfolding free energy (ΔG) of the structure can range from -70 kcal / mol to -40 kcal / mol. Exemplary structures of modified ITRs are provided in Figures 14A and 14B.

[0140] Modified ITRs predicted to form a single hairpin structure may contain deletions, insertions, or substitutions of one or more nucleotides from the wild-type ITR in the sequences forming the B and B' arms and / or the C and C' arms. Modified ITRs may be generated by genetic modification or biosynthesis and / or chemical synthesis.

[0141] For example, ITR-6 left and right (SEQ ID NOs: 111 and 112) provided in Figures 14A and 14B contain 40 nucleotide deletions in the B-B' and C-C' arms from the wild-type ITR of AAV2. The nucleotides remaining in the modified ITRs are predicted to form a single hairpin structure. The Gibbs free energy of unfolding the structure is approximately -54.4 kcal / mol.

[0142] The sequences of the wild-type ITR and ITR-6 (both left and right) were aligned and are provided in Table 8 below. [Table 8]

[0143] Modified ITR with a truncated structure The wild-type ITRs can be modified to have a lowest energy structure containing two arms, one of which is cleaved. Their Gibbs unfolding free energies (ΔG) are in the range of -90 to -70 kcal / mol. Thus, their Gibbs unfolding free energies are lower than those of the wild-type ITR of AAV2.

[0144] The modified ITR may include a deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR in the sequences forming the B and B' arms and / or the C and C' arms. In some embodiments, the modified ITR may include, for example, the removal of all of a particular loop, such as the A-A' loop, the B-B' loop, or the C-C' loop, or alternatively, the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form the stem of the loop, so long as the final loop at the end of the stem is still present. The modified ITR may be generated by genetic modification or biosynthesis and / or chemical synthesis.

[0145] Exemplary structures of modified ITRs having truncated structures are provided in Figures 15A-15B.

[0146] The sequences of various modified ITRs predicted to form cleavage structures are aligned with the sequence of the wild-type ITR and are provided in Table 9 below. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0147] Additional exemplary modified ITRs of each of the above classes for use herein are provided in Tables 10A and 10B. The predicted secondary structures of the right-modified ITRs of Table 10A are shown in Figure 26A, and the predicted secondary structures of the left-modified ITRs of Table 10B are shown in Figure 26B.

[0148] Tables 10A and 10B show exemplary right- and left-modified ITRs. [Table 10A-1] [Table 10A-2] [Table 10B-1] [Table 10B-2]

[0149] In an embodiment of the present invention, the ceDNA vector disclosed herein does not have a modified ITR having a nucleotide sequence selected from any of the group of SEQ ID NOs: 550, 551, 552, 553, 553, 554, 555, 556, 557.

[0150] To the extent that the ceDNA vector has modified ITRs, one of the modifications in the B, B', C, or C' regions set forth in SEQ ID NOs:550-557, as defined in any one or more of the claims of this application, or within any invention defined in any amended claims that may be hereafter filed in this application or in any patent derived therefrom, and to the extent applicable under the law of any relevant country(ies) to that claim(s), we hereby reserve the right to disclaim such disclosure from the claims of this application or any patent derived therefrom to the extent necessary to prevent the invalidation of this application or any patent derived therefrom.

[0151] For example, without limitation, we reserve the right to disclaim any one of the following subject matter from any claim of this application (as currently or hereafter amended) or any patent proceeding therefrom:

[0152] A. A modified ITR selected from any of the group consisting of SEQ ID NOs: 2, 52, 63, 64, 113, 114, 550, 551, 552, 553, 553, 554, 555, 556, 557 for use in a ceDNA vector without a regulatory switch

[0153] B. Modified ITRs as specified in A. above in a ceDNA vector lacking regulatory sequences. The heterologous nucleic acid encodes ABCA4, USA2A var1, VEGFR, CEP290, BDD factor VIII (FVIII), factor VIII, vWF_His, vWF, lecithin cholesterol acetyltransferase, PAH, G6PC, or CFTR.

[0154] Without limitation, the above reservation of disclaimer is expressly stated to apply to at least all paragraphs of claims 1 through 57 of this application, including, but not limited to, paragraphs recited in

[0027] and

[0340] .

[0155] IV. Regulatory Elements ceDNA vectors can be produced from expression constructs that further comprise a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments, ITRs can act as promoters of transgenes. In some embodiments, ceDNA vectors comprise additional components for regulating the expression of transgenes, such as the regulatory switches described herein, and regulate the expression of transgenes, or kill switches that can kill cells that contain ceDNA vectors.

[0156] The ceDNA vectors can be produced from expression constructs that further include a specific combination of cis-regulatory elements, such as the WHP post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) and BGH polyA (SEQ ID NO: 9). Suitable expression cassettes for use in expression constructs are not limited by the packaging constraints imposed by the viral capsid. The expression cassettes of the present invention include promoters that can affect cell specificity as well as overall expression levels. In the case of transgene expression, they can include highly active virus-derived immediate early promoters. The expression cassettes can contain tissue-specific eukaryotic promoters to restrict transgene expression to specific cell types and reduce toxic effects and immune responses resulting from unregulated ectopic expression. In a preferred embodiment, the expression cassettes can contain synthetic regulatory elements, such as the CAG promoter (SEQ ID NO: 3). The CAG promoter includes (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and first intron of the chicken β-actin gene, and (iii) a splice acceptor of the rabbit β-globulin gene. Alternatively, the expression cassette may contain an alpha-1-antitrypsin (AAT) promoter (SEQ ID NO: 4 or SEQ ID NO: 74), a liver-specific (LP1) promoter (SEQ ID NO: 5 or SEQ ID NO: 16), or a human elongation factor-1 alpha (EF1a) promoter (e.g., SEQ ID NO: 6 or SEQ ID NO: 15). In some embodiments, the expression cassette comprises one or more constitutive promoters, such as the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), or the cytomegalovirus (CMV) immediate early promoter (optionally with the CMV enhancer, e.g., SEQ ID NO: 22). Alternatively, an inducible promoter, the native promoter of the transgene, a tissue-specific promoter, or various promoters known in the art can be used.

[0157] Suitable promoters, including those mentioned above, may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to direct the expression of any RNA polymerase (e.g., pol Expression can be driven by SV40 early promoter, mouse mammary tumor virus long terminal sequence (LTR) promoter, adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, e.g., CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 micronucleus promoter (U6, e.g., SEQ ID NO: 18 (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep.1;31(17)), human H1 promoter (H1) (e.g., SEQ ID NO: 19), CAG promoter, human alpha 1-antitypsin (HAAT) promoter (e.g., SEQ ID NO: 21), and the like. In embodiments, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites. In embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.

[0158] A promoter can include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also include distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viral, bacterial, fungal, plant, insect, and animal. A promoter can constitutively or differentially regulate the expression of a gene component with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to an external stimulus, such as a physiological stress, a pathogen, a metal ion, or an inducer. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter, and CMV IE promoter, as well as the promoters listed below. Such promoters and / or enhancers can be used for the expression of any gene of interest (e.g., gene editing molecules, donor sequences, therapeutic proteins, etc.). For example, the vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein. The promoter operably linked to a sequence encoding a therapeutic protein can be a promoter from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.The promoter can also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionine. The promoter can also be a tissue-specific promoter, such as a liver-specific promoter, such as human alpha 1 antitypsin (HAAT) (natural or synthetic). In one embodiment, delivery to the liver can be achieved using endogenous ApoE-specific targeting of the composition comprising the ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0159] In one embodiment, the promoter used is the native promoter of the gene that codes for therapeutic protein.The promoter and other regulatory sequences of each gene that codes for therapeutic protein are known and characterized.The promoter region used may further comprise one or more additional regulatory sequences (e.g., native), such as enhancers (e.g., SEQ ID NO:22 and SEQ ID NO:23).

[0160] Non-limiting examples of suitable promoters for use in accordance with the present invention include, for example, the CAG promoter (SEQ ID NO: 3), the HAAT promoter (SEQ ID NO: 21), the human EF1-α promoter (SEQ ID NO: 6), or a fragment of the EF1a promoter (SEQ ID NO: 15), the IE2 promoter (e.g., SEQ ID NO: 20), and the rat EF1-α promoter (SEQ ID NO: 24).

[0161] Polyadenylation sequence: A sequence encoding a polyadenylation sequence may be included in a ceDNA vector to stabilize the mRNA expressed from the ceDNA vector and to aid in nuclear transport and translation. In one embodiment, the ceDNA vector does not contain a polyadenylation sequence. In other embodiments, the vector contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments, the polyadenylation sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0162] The expression cassette may include a polyadenylation sequence known in the art or a variant thereof, such as a naturally occurring sequence isolated from bovine BGHpA (e.g., SEQ ID NO: 74) or viral SV40pA (e.g., SEQ ID NO: 10), or a synthetic sequence (e.g., SEQ ID NO: 27). Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, USE may be used in combination with SV40pA or a heterologous polyA signal.

[0163] The expression cassette may also include post-transcriptional elements to increase expression of the transgene. In some embodiments, the Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) is used to increase expression of the transgene. Other post-transcriptional processing elements can be used, such as the post-transcriptional elements from the thymidine kinase gene of Herpes Simplex Virus or Hepatitis B Virus (HBV). Secretory sequences can be linked to the transgene, such as the VH-02 and VK-A26 sequences, e.g., SEQ ID NO: 25 and SEQ ID NO: 26.

[0164] V. Adjustment switch A molecular regulatory switch is one that responds to a signal to produce a measurable change in state. Such regulatory switches can be usefully combined with the ceDNA vectors described herein to control the output of the ceDNA vector. In some embodiments, the ceDNA vector includes a regulatory switch that serves to fine-tune the expression of a transgene. For example, it can serve as a biological containment function for the ceDNA vector. In some embodiments, the switch is an "on / off" switch designed to start or stop (i.e., shut down) the controllable and regulatable expression of a gene of interest in the ceDNA vector. In some embodiments, the switch can include a "kill switch" that can instruct a cell containing the ceDNA vector to undergo programmed cell death once the switch is activated.

[0165] A. Binary Regulatory Switch In some embodiments, the ceDNA vector comprises a regulatory switch that can be useful for controllably adjusting the expression of the transgene. In such an embodiment, the expression cassette located between the ITRs of the ceDNA vector may additionally comprise a regulatory region, such as a promoter, cis-element, repressor, enhancer, etc., operably linked to the gene of interest, which is regulated by one or more cofactors or exogenous agents. Thus, in one embodiment, transcription and expression of the gene of interest from the ceDNA vector occurs only when one or more cofactor(s) or exogenous agents are present in the cell. In another embodiment, transcription and expression of the gene of interest can be derepressed using one or more cofactor(s) or exogenous agents.

[0166] Any nucleic acid regulatory region known by those skilled in the art can be used in the ceDNA vector designed to contain a regulatory switch. By way of example only, the regulatory region can be regulated by a small molecule switch or an inducible or repressible promoter. Non-limiting examples of inducible promoters are hormone-inducible or metal-inducible promoters. Other exemplary inducible promoter / enhancer elements include, but are not limited to, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters. (Fussenegger et al., 2003). Classical tetracycline-based or other antibiotic-based switches are encompassed for use, including those disclosed in (E. et al., Nature Biotechnol. 18:1203-1208 (2000)).

[0167] B. Small Molecule Regulatory Switches Various small molecule-based regulatory switches known in the art are known in the art and can be combined with the ceDNA vectors disclosed herein to form a ceDNA vector controlled by a regulatory switch. In some embodiments, the regulatory switch is an orthogonal ligand / nuclear receptor pair, such as retinoid receptor mutant / LG335 and GRQCIMFI (with an artificial promoter controlling the expression of an operably linked transgene, such as those disclosed in Taylor, et al. BMC Biotechnology 10 (2010): 15); an engineered steroid receptor, such as a modified progesterone receptor with a C-terminal truncation that cannot bind progesterone but binds RU486 (mifepristone) (U.S. Patent No. 5,364,791); an ecdysone receptor from Drosophila and their ecdysteroid ligands (Saez, et al., PNAS, 97 (26) (2000), 14512-14517), or a ceDNA vector controlled by a regulatory switch. rd;Nat Methods. 2013, 10(11):1085-8, and any one or combination of switches controlled by the antibiotic trimethoprim (TMP).

[0168] Other small molecule-based regulatory switches known to the skilled artisan are also envisioned for use in controlling transgene expression in ceDNA, such as those disclosed in Buskirk et al., Cell; Chem and Biol., 2005; 12(2); 151-161; Liang, F.-S., et al., (2011) Science Signaling, 4(164), exogenous L-arginine-sensitive on-switches such as those disclosed in Hartenbach,et al. Nucleic Acids Research, 35(20),2007, synthetic bile acid-sensitive on-switches such as those disclosed in Rossger et al., Metab Eng. 2014,21:81-90, biotin-sensitive on-switches such as those disclosed in Weber et al., Metab. Eng. 2009 Mar;11(2):117-124, dual-input food additive benzoate / vanillin-sensitive regulatory switches such as those disclosed in Xie et al., Nucleic Acids Research,2014;42(14);e116, 4-hydroxytamoxifen-sensitive switches such as those disclosed in Giuseppe et al., Molecular Therapy,6(5),653 -663, and Gitzinger et al. al., Proc. Natl. Acad. Sci. USA. 2009 Jun 30;106(26):10638-10643.

[0169] In some embodiments, the regulatory switch for controlling the transgene expressed by the ceDNA vector is a prodrug activation switch, such as those disclosed in U.S. Pat. Nos. 8,771,679 and 6,339,070.

[0170] Exemplary regulatory switches for use in ceDNA vectors include, but are not limited to, those in Table 11.

[0171] C. "Passcode" adjustment switch In some embodiments, the regulatory switch may be a "passcode switch" or "passcode circuit." A passcode switch allows fine tuning of the control of expression of a transgene from a ceDNA vector when certain conditions occur. That is, a combination of conditions must be present for expression and / or repression of the transgene to occur. For example, at least conditions A and B must occur for expression of the transgene to occur. A passcode regulatory switch may be any number of conditions, for example, at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or more conditions are present for expression of the transgene to occur. In some embodiments, at least two conditions (e.g., A, B conditions) must occur, and in some embodiments, at least three conditions must occur (e.g., A, B, and C or A, B, and D). By way of example only, conditions A, B, and C must be present for gene expression to occur from a ceDNA with a passcode "ABC" regulatory switch. Conditions A, B, and C may be as follows: Condition A is the presence of a pathology or disease, condition B is a hormonal response, and condition C is a response to the expression of the transgene. As merely an illustrative example, if the transgene is insulin, condition A occurs when the subject has diabetes, condition B is when blood glucose levels are high, and condition C is when endogenous insulin levels are not expressed in the required amount. Once blood glucose levels drop or a desired insulin level is reached, the transgene (e.g., insulin) is turned off again and turned back on until three conditions occur. In another illustrative example, if the transgene is EPO, condition A is the presence of chronic kidney disease (CKD), condition B is when the subject has hypoxia in the kidney, and condition C is when erythropoietin-producing cell (EPC) mobilization in the kidney is insufficient or alternatively when HIF-2 activation is insufficient. Once oxygen levels increase or a desired EPO level is reached, the transgene (e.g., EPO) is turned off again and turned back on until three conditions occur.

[0172] The passcode regulatory switch is useful for fine-tuning transgene expression from a ceDNA vector. For example, the passcode regulatory switch can be modular in that it contains multiple switches, e.g., a tissue-specific inducible promoter that is turned on only in the presence of a certain level of a metabolite. In such an embodiment, for transgene expression from the ceDNA vector to occur, an inducing agent must be present (condition A) in the desired cell type (condition B), and the metabolite is at, above, or below a certain threshold (condition C). In an alternative embodiment, the passcode regulatory switch can be designed such that transgene expression is on when conditions A and B are present, but off when condition C is present. Such an embodiment is useful when condition C occurs as a direct result of an expressed transgene. That is, condition C serves to turn off transgene expression from the ceDNA vector as a positive feedback to the loop when the transgene has had a sufficient amount of the desired therapeutic effect.

[0173] In some embodiments, the passcode regulatory switch included for use in ceDNA vectors is disclosed in WO2017 / 059245, which describes a synthetic biological circuit that uses hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival, referred to as a "passcode switch" or "passcode circuit" or "passcode kill switch". The passcode regulatory switches described in WO2017 / 059245 are particularly useful for use in ceDNA vectors because they are modular and customizable in both the environmental conditions that control the activation of the circuit and the output module that controls cell fate. Furthermore, passcode circuits have particular utility for use in ceDNA vectors because, without the appropriate "passcode" molecule, they only allow transgene expression in the presence of the required predefined conditions. If something goes wrong with the cell or further transgene expression is not desired for some reason, the associated kill switch (i.e., a deadman switch) can be triggered.

[0174] In some embodiments, the passcode regulatory switches or "passcode circuits" included for use in ceDNA vectors include hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biological containment conditions. In contrast to deadman switches, which trigger cell death in the presence of predefined conditions, the "passcode circuits" allow cell survival or transgene expression in the presence of specific "passcodes" and can be easily reprogrammed to allow transgene expression and / or cell survival only when a given environmental condition or passcode is present.

[0175] In one embodiment, a "passcode" system that restricts cell growth to the presence of a predetermined set of at least two selected drugs comprises one or more nucleic acid constructs encoding expression modules comprising: i) a toxin expression module encoding a toxin that is toxic to host cells, where the toxin-encoding sequence is operably linked to a promoter P1 that is repressed by binding of a first hybrid repressor protein; and ii) a first hybrid repressor protein expression module encoding a first hybrid repressor protein, hRP1, where expression of hRP1 is controlled by an AND gate formed by two hybrid transcription factors hTF1 and hTF2, whose binding or activity is responsive to drugs A1 and A2, respectively, such that both drugs A1 and A2 are required for expression of hRP1, where in the absence of either A1 or A2, hRP1 expression is insufficient to repress the toxin promoter module P1 and toxin production, thereby killing the host cell. In this system, the hybrid factors hTF1, hTF2, and hRP1 each contain an environmental sensing module from one transcription factor and a DNA recognition module from a different transcription factor that sensitizes the binding of the respective pathway regulatory switch to the presence of an environmental agent A1 or A2 that is different to the one that each subunit normally binds in nature.

[0176] Thus, the ceDNA vector may contain a "passcode regulatory circuit" that requires the presence and / or absence of a specific molecule to activate the output module. In some embodiments, where a gene encoding a cytotoxin is placed in the output module, this passcode regulatory circuit may not only be used to regulate transgene expression, but may also be used to create a kill switch mechanism where the circuit kills the cell if it behaves in an undesirable manner (e.g., leaves a specific environment defined by a sensor domain or differentiates into a different cell type). In one non-limiting example, the modularity of the hybrid transcription factor, the circuit architecture, and the output module allow the circuit to be reconfigured to sense and respond to other environmental signals in other ways, and control other functions in the cell in addition to induced cell death, as understood in the art.

[0177] Any and all combinations of the regulatory switches disclosed herein, such as small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulatory radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches known to those skilled in the art disclosed herein, can be used in the passcode regulatory switches disclosed herein. Regulatory switches encompassed for use are also discussed in the review article Kis et al., JR Soc Interface. 12:20141000 (2015) and summarized in Table 1 of Kis. In some embodiments, the regulatory switches for use in the passcode system can be selected from any of the switches in Table 11, or combinations thereof.

[0178] D. Nucleic Acid-Based Regulatory Switches for Controlling Transgene Expression In some embodiments, the regulatory switch for controlling the transgene expressed by ceDNA is based on a nucleic acid-based control mechanism. Exemplary nucleic acid control mechanisms are known in the art and are envisioned for use. For example, such mechanisms include riboswitches, such as those disclosed in US2009 / 0305253, US2008 / 0269258, US2017 / 0204477, WO2018 / 026762A1, U.S. Patent No. 9,222,093, and European Patent Application No. EP288071, and those disclosed in the review by Villa JK et al., Microbiol Spectr.2018 May;6(3). Metabolite-responsive transcriptional biosensors are also included, such as those disclosed in WO2018 / 075486 and WO2017 / 147585. Other mechanisms known in the art that may be used include silencing transgenes by siRNA or RNAi molecules (e.g., miR, shRNA). For example, a ceDNA vector may contain a regulatory switch that encodes an RNAi molecule that is complementary to the transgene expressed by the ceDNA vector. If such an RNAi is expressed even when expressed by the ceDNA vector, the transgene is silenced by the complementary RNAi molecule, and if the RNAi is not expressed when the transgene is expressed by the ceDNA vector, the transgene is not silenced by the RNAi. Such examples of RNAi molecules that control gene expression or as regulatory switches are disclosed in US2017 / 0183664. In some embodiments, the regulatory switch comprises a repressor that blocks the expression of the transgene from the ceDNA vector. In some embodiments, the on / off switch is a small transcription activating RNA (STAR) based switch, such as those disclosed in Chappell J. et al., Nat Chem Biol. 2015 Mar;11(3):214-20, and Chappell et al., Microbiol Spectr. 2018 May;6(3).In some embodiments, the adjustment switch is a toe hold switch, such as those disclosed in US2009 / 0191546, US2016 / 0076083, WO2017 / 087530, US2017 / 0204477, WO2017 / 075486, and Green et al., Cell, 2014;159(4);925-939.

[0179] In some embodiments, the regulatory switch is a tissue-specific self-inactivating regulatory switch, e.g., as disclosed in US2002 / 0022018, whereby the regulatory switch purposefully switches off transgene expression at sites where transgene expression would otherwise be detrimental. In some embodiments, the regulatory switch is a recombinase reversible gene expression system, e.g., as disclosed in US2014 / 0127162 and U.S. Patent No. 8,324,436.

[0180] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by ceDNA vector is a hybrid of nucleic acid-based control mechanism and small molecule regulatory system.Such systems are well known to those skilled in the art and are envisaged for use herein.Examples of such regulatory switches include but are not limited to LTRi system or "Lac-Tet-RNAi" system, as disclosed in, for example, US2010 / 0175141, and Deans T.et al., Cell.,2007,130(2);363-372, WO2008 / 051854 and US Patent No. 9,388,425.

[0181] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the ceDNA vector requires circular permutation as disclosed in US Pat. No. 8,338,138. In such embodiments, the molecular switch is multistable, i.e., can switch at least two states, or bistable, i.e., the state is either "on" or "off", e.g., can emit light or not, can bind or not, can catalyze or not, can transfer electrons or not, etc. In another aspect, the molecular switch uses fusion molecules, so that the switch can switch more than two states. For example, in response to a particular threshold state exhibited by the insert sequence or the acceptor sequence, each other sequence of the fusion can exhibit a range of states (e.g., a range of binding activity, a range of enzyme catalysis, etc.). Thus, rather than switching "on" or "off", the fusion molecule can exhibit a graded response to a stimulus.

[0182] In some embodiments, the nucleic acid based regulatory switch may be selected from any of the switches in Table 11, or a combination thereof.

[0183] E. Post-transcriptional and post-translational regulatory switches In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the ceDNA vector is a post-transcriptional modification system. For example, such regulatory switches are described in US2018 / 0119156, GB2011 / 07768, WO2001 / 064956A3, EP2707487, and Beilstein The riboswitch may be an aptazyme riboswitch sensitive to tetracycline or theophylline, as disclosed in Zhong et al., ACS Synth.Biol., 2015,4(5),pp 526-534; Zhong et al., Elife.2016 Nov 2;5.pii:e18858. In some embodiments, it is envisioned that one skilled in the art can code both the transgene and an inhibitory siRNA that contains a ligand-sensitive (off-switch) aptamer, the end result of which is a ligand-sensitive on-switch.

[0184] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by ceDNA vector is a post-translational modification system.In alternative embodiments, the gene or protein of interest is expressed as a pro- or pre-pro-protein, or has a signal response element (SRE) or destabilization domain (DD) attached to the expressed protein, thereby preventing correct protein folding and / or activity until post-translational modification occurs.In the case of a destabilization domain (DD) or SRE, the destabilization domain is cleaved after translation in the presence of an exogenous drug or small molecule.Those skilled in the art can utilize the control method as disclosed in U.S. Pat. No. 8,173,792 and PCT Application No. WO2017 / 180587. Other post-transcriptional regulatory switches envisioned for use in ceDNA vectors to control functional transgene activity are disclosed in Rakhit et al., Chem Biol. 2014;21(9):1238-52 and Navarro et al., ACS Chem Biol. 2016;19;11(8):2101-2104A.

[0185] In some embodiments, the genetic regulatory switch for controlling the transgene or gene of interest expressed by the ceDNA vector is a post-translational modification system that incorporates a ligand-sensitive intein into the transgene coding sequence, thereby inhibiting the transgene or expressed protein prior to splicing. For example, this has been demonstrated using both 4-hydroxytamoxifen and thyroid hormone (e.g., U.S. Pat. Nos. 7,541,450, 9,200,045, 7,192,739; Buskirk, et al, Proc Natl Acad Sci USA. 2004 Jul 20; 101(29):10505-10510; ACS Synth Biol. 2016 Dec 16;5(12):1475-1484, and 2005 Feb;14(2):523-532). In some embodiments, the post-transcriptional regulatory switch can be selected from any of the switches in Table 11, or a combination thereof.

[0186] F. Other Exemplary Adjustment Switches Any known regulatory switch can be used in the ceDNA vector to control gene expression of the transgene expressed by the ceDNA vector, including those triggered by environmental changes. Additional examples include, but are not limited to, the BOC method of Suzuki et al., Scientific Reports 8;10051 (2018), genetic code expansion and non-physiological amino acids, radiation-regulated or ultrasound-regulated on / off switches (see, e.g., Scott S et al., Gene Ther. 2000 Jul;7(13):1121-5, U.S. Patent Nos. 5,612,318, 5,571,797, 5,770,581, 5,817,636, and WO1999 / 025385A1). In some embodiments, the regulatory switch is controlled by an implantable system, e.g., as disclosed in U.S. Patent No. 7,840,263, US2007 / 0190028A1, and gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activate a promoter operably linked to a transgene in a ceDNA vector.

[0187] In some embodiments, the regulatory switch envisioned for use in ceDNA vector is hypoxia-mediated or stress-activated switch, such as those disclosed in WO1999 / 060142A2, US Patent No. 5,834,306, US Patent No. 6,218,179, US Patent No. 6,709,858, US2015 / 0322410, Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD, and NRSE elements, and conditionally inducible silence elements, including hypoxia response element (HRE), inflammatory response element (IRE), and shear stress activation element (SSAE), as disclosed in US Patent No. 9,394,526. Such an embodiment is useful for turning on the expression of transgenes from ceDNA vector after ischemia or in ischemic tissue and / or tumor.

[0188] In some embodiments, the regulatory switches contemplated for use in the ceDNA vectors are optogenetic (e.g., light-controlled) regulatory switches, such as those described in Polesskaya et al. al., BMC Neurosci. 2018;19(Suppl 1):12, which are also contemplated for use herein. In such embodiments, the ceDNA vector may contain a genetic element that is light sensitive and can regulate a transgene in response to visible wavelengths (e.g., blue, near infrared). ceDNA vectors containing optogenetic regulatory switches are useful when expressing transgenes in locations of the body that can receive such a light source, e.g., skin, eyes, muscles, etc., and can also be used when the ceDNA vector is expressing a transgene in internal organs and tissues, where the light source can be provided by suitable means (e.g., an implantable device as disclosed herein). Such optogenetic regulatory switches include the use of light-responsive elements, or light-inducible transcriptional effectors (LITEs) (disclosed, for example, in 2014 / 0287938), the light-on system (disclosed, for example, in Wang et al., Nat Methods. 2012 Feb 12;9(3):266-9, which is reported to allow in vivo control of insulin transgene expression), the Cry2 / CIB1 system (disclosed, for example, in Kennedy et al., Nature Methods;7,973-975(2010)), and the FKF1 / GIGANTEA system (disclosed, for example, in Yazawa et al., Nat Biotechnol. 2009 Oct;27(10):941-5).

[0189] G. Kill Switch Another embodiment of the present invention relates to a ceDNA vector comprising a kill switch. The kill switch disclosed herein can cause cells comprising the ceDNA vector to be killed or undergo programmed cell death as a means of permanently removing the introduced ceDNA vector from the subject's system. It will be understood by those skilled in the art that the use of a kill switch in the ceDNA vector of the present invention is usually coupled with targeting of the ceDNA vector to a limited number of cells that the subject can tolerately lose, or to a cell type (e.g., cancer cells) where apoptosis is desired. In all aspects, the "kill switch" disclosed herein is designed to provide rapid and robust cell killing of cells comprising the ceDNA vector in the absence of an input survival signal or other specified condition. In other words, the kill switch encoded by the ceDNA vector herein can restrict cell survival of cells comprising the ceDNA vector to an environment defined by a particular input signal. Such a kill switch serves as a biological containment function when it is desirable to remove the ceDNA vector from the subject or ensure that the encoded transgene is not expressed. Therefore, kill switch is a synthetic biological circuit in ceDNA vector that connects environmental signals with the conditional survival of cells that contain ceDNA vector.In some embodiments, different ceDNA vectors can be designed to have different kill switches.This allows control which transgene-expressing cells are killed when a cocktail of ceDNA vectors is used.

[0190] In some embodiments, the ceDNA vector may include a kill switch, which is a modular biological containment circuit. In some embodiments, a kill switch contemplated for use in a ceDNA vector is disclosed in WO2017 / 059245, which describes a switch referred to as a "deadman kill switch" that includes a mutually inhibitory sequence of at least two repressible sequences, whereby an environmental signal represses the activity of a second molecule in the construct (e.g., using a small molecule binding transcription factor, resulting in a "live" state due to the repression of toxin production). In a cell containing a ceDNA vector that includes a deadman kill switch, loss of the environmental signal causes the circuit to permanently switch to a "dead" state, where the toxin is derepressed, resulting in toxin production that kills the cell. In another embodiment, a synthetic biological circuit, referred to as a "passcode circuit" or "passcode kill switch," is provided that uses hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival. The deadman and passcode kill switches described in WO2017 / 059245 are particularly useful for use in ceDNA vectors because they are modular and customizable in both the environmental conditions that control the activation of the circuit and the output module that controls cell fate.By appropriately selecting a toxin, including but not limited to an endonuclease, such as EcoRI, the passcode circuit present in the ceDNA vector can be used to not only kill the host cell containing the ceDNA vector, but also degrade its genome and associated plasmids.

[0191] Other kill switches known to those of skill in the art include those disclosed herein, e.g., in US2010 / 0175141, US2013 / 0009799, US2011 / 0172826, US2013 / 0109568, as well as those disclosed in Jusiak et al, Reviews in Cell Biology and molecular Medicine;2014;1-56, Kobayashi et al., PNAS,2004;101;8419-9, Marchisio et al., Int. Journal of Biochem and Cell Biol.,2011;43;310-319, and Reinshagen et al., Science The kill switches disclosed in Translational Medicine, 2018, 11 are also included for use in the kill switches disclosed in Translational Medicine, 2018, 11.

[0192] Thus, in some embodiments, the ceDNA vector may include a kill switch nucleic acid construct, which includes a nucleic acid encoding an effector toxin or a reporter protein, and the expression of the effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, the predetermined condition may be the presence of an environmental agent, such as an exogenous agent, without which the cells will by default express the effector toxin (e.g., death protein) and be killed. In an alternative embodiment, the predetermined condition is the presence of two or more environmental agents, for example, the cells will only survive when two or more required exogenous agents are provided, and in the absence of any of them, the cells containing the ceDNA vector will be killed.

[0193] In some embodiments, the ceDNA vector is modified to incorporate a kill switch to destroy cells containing the ceDNA vector, effectively terminating the in vivo expression of the transgene expressed by the ceDNA vector (e.g., therapeutic gene, protein, or peptide, etc.). Specifically, the ceDNA vector is further engineered to express a switch protein that is not functional in mammalian cells under normal physiological conditions. Only upon administration of a drug or environmental condition that specifically targets this switch protein, will the cells expressing the switch protein be destroyed, thereby terminating the expression of the therapeutic protein or peptide. For example, it has been reported that cells expressing HSV-thymidine kinase can be killed upon administration of drugs such as ganciclovir and cytosine deaminase. See, e.g., Dey and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in Targets in Gene Therapy, edited by You (2011), and Beltinger et al., Proc. Natl. Acad. Sci. USA 96(15):8699-8704 (1999). In some embodiments, the ceDNA vector can include a siRNA kill switch called DISE (Death Induced by Survival gene Elimination) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[0194] In some aspects, a deadman kill switch is a biological circuit or system that renders a cellular response sensitive to a predetermined condition, such as an agent in the cell growth environment, e.g., the loss of an exogenous agent. Such a circuit or system can include a nucleic acid construct that includes an expression module that forms a deadman regulatory circuit that is sensitive to the predetermined condition, the construct includes an expression module that forms the regulatory circuit, the construct includes i) a first repressor protein expression module, wherein the first repressor protein binds to a first repressor protein nucleic acid binding element and represses transcription from a coding sequence comprising the first repressor protein binding element, and the repression activity of the first repressor protein is sensitive to inhibition by a first exogenous agent, the presence or absence of the first exogenous agent establishing a predetermined condition; and ii) a second repressor protein expression module, wherein the second repressor protein binds to a second repressor protein nucleic acid binding element and represses transcription from a coding sequence that contains the second repressor protein binding element, the second repressor protein being different from the first repressor protein; and iii) an effector expression module comprising a nucleic acid sequence encoding an effector protein operably linked to a genetic element comprising a binding element for a second repressor protein, whereby expression of the second repressor protein causes repression of effector expression from the effector expression module, the second expression module comprising a first repressor protein nucleic acid binding element that permits repression of transcription of the second repressor protein when the element is bound by the first repressor protein, each module being operable to inhibit transcription of the first repressor protein produced from the first repressor protein expression module in the absence of a first exogenous agent; and an effector expression module which is coupled to a first exogenous agent to form a regulatory circuit to repress transcription from a second repressor protein expression module, whereby repression of the effector expression by the second repressor protein is relieved, resulting in expression of the effector protein, but in the presence of a first exogenous agent, activity of the first repressor protein is inhibited, allowing expression of the second repressor protein, which maintains expression of the effector protein expression in an "off" state, whereby the first exogenous agent is required by the circuit to maintain the effector protein expression in an "off" state, and removal or absence of the first exogenous agent defaults to expression of the effector protein.

[0195] In some embodiments, the effector is a toxin or protein that induces a cell death program. Any protein that is toxic to the host cell can be used. In some embodiments, the toxin only kills the cell in which it is expressed. In other embodiments, the toxin kills other cells of the same host organism. Any of a number of products that lead to cell death can be used for the dead man kill switch. Agents that inhibit DNA replication, protein translation, or other processes that degrade, for example, host cell nucleic acids are particularly useful. To identify an efficient mechanism for killing the host cell upon activation of the circuit, several toxin genes that directly damage the DNA or RNA of the host cell were tested. Endonuclease ecoRI21 , DNA gyrase inhibitor ccdB 22 , and the ribonuclease-type toxin mazF 23 were tested because they are well characterized, native to E. coli, and offer a range of killing mechanisms. To increase the robustness of the circuit and provide an independent method of circuit-dependent cell death, the system can be further adapted to express targeted proteases or nucleases that further interfere with, for example, the repressors that maintain the death genes in the "off" state. Upon loss or withdrawal of the survival signal, death gene repression is even more efficiently removed, for example, by active degradation of the repressor protein or its message. As a non-limiting example, mf-Lon protease was used to not only degrade LacI, but also to target proteins essential for degradation. The mf-Lon degradation tag pdt#1 was tagged with the five essential genes (whose protein products are particularly sensitive to mf-Lon degradation) and the five essential genes (whose protein products are particularly sensitive to mf-Lon degradation). 20 ) and cell viability was measured following removal of ATc. Among the essential gene targets tested, the peptidoglycan biosynthesis gene murC resulted in the strongest and fastest cell death phenotype (viability of 1 × 10 within 6 h). -4 less than).

[0196] As used herein, a "predetermined input" refers to an agent or disease state that affects the activity of a transcription factor polypeptide in a known manner. In general, such agents can modify the activity of a transcription factor polypeptide by binding to and / or altering the structure of the transcription factor polypeptide. Examples of predetermined inputs include, but are not limited to, environmental input agents that are not required for the survival of a given host organism (i.e., in the absence of the synthetic biological circuit described herein). For example, conditions that may provide a predetermined input include, for example, temperature (e.g., when the activity of one or more factors is temperature sensitive), the presence or absence of light (including light of a given wavelength spectrum), and concentrations of gases, salts, metals, or minerals. Environmental input agents include, for example, small molecules, biological agents, such as pheromones, hormones, growth factors, metabolites, nutrients, etc., and their analogs; chemical concentrations, environmental by-products, metal ions, and other such molecules or agents; light levels; temperature; mechanical stress or pressure; or electrical signals, such as current and voltage.

[0197] In some embodiments, reporters are used to quantify the strength or activity of signals received by the modules or programmable synthetic biological circuits of the present invention. In some embodiments, reporters can be fused in frame to other protein coding sequences to identify where the proteins are located in the cell or organism. Luciferase can be used as an effector protein in various embodiments described herein, for example, to measure low levels of gene expression, since cells have little or no background luminescence in the absence of luciferase. In other embodiments, enzymes that produce colored substances can be quantified using other instruments that can make absorbance measurements, including spectrophotometers or plate readers. Like luciferase, enzymes such as β-galactosidase tend to amplify low signals, so such enzymes can be used to measure low levels of gene expression. In some embodiments, the effector protein can be an enzyme that can degrade or otherwise destroy a given toxin. In some embodiments, the effector protein can be an odorant enzyme that converts a substance into an odor-producing product. In some embodiments, the effector protein can be an enzyme that phosphorylates or dephosphorylates either small molecules or other proteins, or that methylates or demethylates other proteins or DNA.

[0198] In some embodiments, the effector protein can be a receptor, a ligand, or a soluble protein. Receptors tend to have three domains, an extracellular domain for binding to a ligand such as a protein, peptide, or small molecule, a transmembrane domain, and an intracellular or cytoplasmic domain that may frequently be involved in some type of signaling event, such as phosphorylation. In some embodiments, transporter, channel, or pump gene sequences are used as effector proteins. Non-limiting examples and sequences of effector proteins for use with the kill switches described herein can be found on the World Wide Web at the Registry of Standard Biological Parts at parts.igem.org.

[0199] As used herein, a "modulator protein" is a protein that regulates expression from a target nucleic acid sequence. Modulator proteins include, for example, transcription factors, including transcription activators and repressors, among others, proteins that bind to or modify transcription factors and affect their activity. In some embodiments, modulator proteins include, for example, proteases that degrade protein factors involved in regulating expression from a target nucleic acid sequence. Preferred modulator proteins include, for example, modular proteins in which the DNA-binding and input agent binding or responsive elements or domains are separable and mobile, such that, for example, fusion of the DNA-binding domain of a first modulator protein to the input agent responsive domain of a second modulator protein results in a new protein that binds to the DNA sequence recognized by the first protein, but is usually sensitive to the input agent to which the second protein responds. Thus, as used herein, the term "modulator polypeptide," and more specifically, "repressor polypeptide," includes, in addition to the designated polypeptide, for example, "LacI (repressor) polypeptide," variants of such polypeptides that respond to different or variant input agents, or derivatives. Thus, in the case of LacI polypeptides, LacI mutants or variants are included that bind agents other than lactose or IPTG, and a wide range of such agents are known in the art. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0200] VI. Detailed Methods for the Production of ceDNA Vectors A. General production As described herein, ceDNA vectors can be obtained by a process that includes a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), where the host cells lack viral capsid coding sequences under conditions and for a sufficient time to induce the production of ceDNA vectors in the host cells in the presence of Rep proteins, and b) harvesting and isolating the ceDNA vectors from the host cells. The presence of Rep proteins induces the replication of vector polynucleotides with modified ITRs to produce ceDNA vectors in the host cells. However, viral particles (e.g., AAV virions) are not expressed. Thus, there is no size restriction such as that naturally imposed in AAV or other viral vectors.

[0201] The presence of a ceDNA vector isolated from a host cell may be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA.

[0202] In yet another aspect, the present invention provides the use of host cell lines that stably integrate a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, for example as described in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cell at an MOI of about 3. When the host cell line is a mammalian cell line, for example, HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpes virus, may be used to introduce Rep protein into the cell, allowing excision and amplification of ceDNA in the presence of Rep and a helper virus.

[0203] In one embodiment, the host cells used to make the ceDNA vectors described herein are insect cells, and a baculovirus is used to deliver both the polynucleotides encoding the Rep proteins and the non-viral DNA vector polynucleotide expression construct template for ceDNA, e.g., as described in Figures 4A-4C and Example 1. In some embodiments, the host cells are engineered to express the Rep proteins.

[0204] The ceDNA vector is then harvested and isolated from the host cells. The time for harvesting the ceDNA vector described herein from the cells can be selected and optimized to achieve high yield production of the ceDNA vector. For example, the harvest time can be selected taking into consideration cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after sufficient time has passed since baculovirus infection to produce the ceDNA vector, but before a portion of the cells begin to die due to the toxicity of the baculovirus. The DNA-vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation can also be adapted for DNA-vectors. In general, any nucleic acid purification method can be employed.

[0205] DNA vector can be purified by any means known to those skilled in the art for DNA purification.In one embodiment, ceDNA vector is purified as DNA molecule.In another embodiment, ceDNA vector is purified as exosome or microparticle.

[0206] The presence of ceDNA vectors can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme that has a single recognition site on the DNA vector, and using gel electrophoresis to analyze both digested and undigested DNA material to confirm the presence of characteristic linear and continuous DNA compared to linear and discontinuous DNA. Figures 4C and 4E show one embodiment for identifying the presence of closed-end ceDNA vectors produced by the process herein. For example, Figure 5 is a gel confirming the production of ceDNA from multiple plasmid constructs using one embodiment for producing these vectors described in the Examples.

[0207] B. ceDNA plasmid ceDNA-plasmid is a plasmid used for the later production of ceDNA vector. In some embodiments, ceDNA-plasmid can be constructed using known techniques to provide at least the following as components operably linked in the direction of transcription: (1) 5'ITR sequence, (2) expression cassette containing cis-regulatory elements, such as promoter, inducible promoter, regulatory switch, enhancer, etc., and (3) 3'ITR sequence (3'ITR sequence is asymmetric with respect to 5'ITR sequence). In some embodiments, the expression cassette flanked by ITRs contains cloning sites for introducing exogenous sequences. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0208] In one aspect, the ceDNA vector is derived from a plasmid, referred to herein as a "ceDNA-plasmid," that encodes, in this order, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette comprising a transgene, and a mutated or modified AAV ITR, the ceDNA-plasmid lacking an AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes, in this order, a first (or 5') modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3') wild-type AAV ITR, the ceDNA-plasmid lacking an AAV capsid protein coding sequence, the 5' and 3' ITRs being asymmetric with respect to each other. In an alternative embodiment, the ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutated or modified AAV ITR, wherein the ceDNA-plasmid lacks the AAV capsid protein coding sequence and the 5' and 3' modified ITRs are different and do not have the same modifications.

[0209] In further embodiments, the ceDNA-plasmid system lacks viral capsid protein coding sequences (i.e., lacks not only AAV capsid genes, but also capsid genes of other viruses). Additionally, in certain embodiments, the ceDNA-plasmid also lacks AAV Rep protein coding sequences. Thus, in preferred embodiments, the ceDNA-plasmid lacks functional AAV cap and AAV rep genes (GG-3' for AAV2) as well as variable palindrome sequences that allow hairpin formation.

[0210] The ceDNA-plasmids of the present invention can be generated using the native nucleotide sequence of the genome of any AAV serotype known in the art. In one embodiment, the ceDNA-plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes. For example, see Kotin and Smith, The Springer, available at NCBI: NC002077, NC001401, NC001729, NC001829, NC006152, NC006260, NC006261, URLs maintained by Springer. Index of Viruses (www web address: oesys.springer.de / viruses / database / mkchapter.asp?virID=42.04) (Note - references to a URL or database refer to the contents of the URL or database as of the effective filing date of this application). In a particular embodiment, the ceDNA-plasmid backbone is derived from the AAV2 genome. In another particular embodiment, the ceDNA-plasmid backbone is a synthetic backbone engineered to include 5' and 3' ITRs from one of these AAV genomes.

[0211] The ceDNA-plasmid may optionally contain a selectable or selectable marker for use in establishing a ceDNA vector producing cell line. In one embodiment, the selectable marker may be inserted downstream (i.e., 3') of the 3'ITR sequence. In another embodiment, the selectable marker may be inserted upstream (i.e., 5') of the 5'ITR sequence. Suitable selectable markers include, for example, those that confer drug resistance. The selectable marker may be, for example, the Blasticidin S resistance gene, kanamycin, geneticin, etc. In a preferred embodiment, the drug selectable marker is the Blasticidin S resistance gene.

[0212] Exemplary ceDNA (e.g., rAAV0) is produced from an rAAV plasmid. A method for producing an rAAV vector can include (a) providing a host cell with an rAAV plasmid as described above, where both the host cell and the plasmid depict a capsid protein-encoding gene, (b) culturing the host cell under conditions that allow for the production of a ceDNA genome, and (c) harvesting the cell and isolating the produced AAV genome from the cell.

[0213] C. Exemplary Methods for Producing ceDNA Vectors from ceDNA Plasmids Also provided herein are methods for generating capsid-free ceDNA vectors, particularly those that have yields high enough to provide sufficient vector for in vivo experiments.

[0214] In some embodiments, the method for producing ceDNA vectors includes the steps of (1) introducing a nucleic acid construct containing an expression cassette and two asymmetric ITR sequences into a host cell (e.g., Sf9 cells), (2) optionally establishing a clonal cell line, for example, by using a selection marker present on the plasmid, (3) introducing a Rep-encoding gene into said insect cells (either by transfection or infection with a baculovirus carrying said gene), and (4) harvesting the cells and purifying the ceDNA vector. The nucleic acid construct containing the expression cassette and two ITR sequences described above for the production of capsid-free AAV vectors can be in the form of a cfAAV-plasmid, or a bacmid or baculovirus produced with a cfAAV-plasmid as described below. The nucleic acid construct can be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

[0215] D. Cell lines: The host cell line used in the production of ceDNA vectors can include insect cell lines derived from Spodoptera frugiperda (Sf9, Sf21, etc.) or Trichoplusia ni cells, or other eukaryotic cell lines, including other invertebrate, vertebrate, or mammalian cells. Other cell lines known to the skilled artisan can also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes, and mature and immature dendritic cells. The host cell line can be transfected for stable expression of ceDBA-plasmid for high-yield ceDNA vector production.

[0216] The ceDNA-plasmid can be introduced into Sf9 cells by transient transfection using reagents known in the art (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation). Alternatively, stable Sf9 cell lines that stably integrate the ceDNA-plasmid into their genome can be established. Such stable cell lines can be established by incorporating a selection marker into the above ceDNA-plasmid. If the ceDNA-plasmid used to transfect the cell line contains a selection marker such as an antibiotic, cells that are transfected with the ceDNA-plasmid and have integrated the ceDNA-plasmid DNA into their genome can be selected by adding the antibiotic to the cell growth medium. Resistant clones of cells can then be isolated and propagated by single cell dilution or colony transfer techniques.

[0217] Isolate and purify E. ceDNA vector Examples of processes for obtaining and isolating ceDNA vectors are described in Figures 4A-4E and in the specific examples below. The ceDNA-vectors disclosed herein can be obtained from producer cells expressing AAV Rep protein(s) and further transformed with ceDNA-plasmids, ceDNA-bacmids, or ceDNA-baculoviruses. Plasmids useful for producing ceDNA vectors include those shown in Figure 8A (useful for Rep BIIC production), Figure 8B (plasmids used to obtain ceDNA vectors).

[0218] In one embodiment, the polynucleotide encodes an AAV Rep protein (Rep78 or 68) delivered to a producer cell in a plasmid (Rep-plasmid), a bacmid (Rep-bacmid), or a baculovirus (Rep-baculovirus). The Rep-plasmid, Rep-bacmid, and Rep-baculovirus can be generated by the methods described above.

[0219] Methods for producing ceDNA vectors, which are exemplary ceDNA vectors, are described herein. The expression constructs used to generate the ceDNA vectors of the present invention can be plasmids (e.g., ceDNA-plasmids), bacmids (e.g., ceDNA-bacmids), and / or baculoviruses (e.g., ceDNA-baculoviruses). By way of example only, ceDNA vectors can be generated from cells co-infected with ceDNA-baculoviruses and Rep-baculoviruses. Rep proteins produced from the Rep-baculoviruses replicate the ceDNA-baculoviruses to generate ceDNA vectors. Alternatively, ceDNA vectors can be AAV delivered in Rep-plasmids, Rep-bacmids, or Rep-baculoviruses. It can be generated from cells stably transfected with a construct containing a sequence encoding the Rep protein (Rep78 / 52). The ceDNA-baculovirus can be transiently transfected into cells and replicated by the Rep protein to produce the ceDNA vector.

[0220] The bacmid (e.g., ceDNA-bacmid) can be transfected into permissive insect cells, such as Sf9, Sf21, Tni (Trichoplusia ni) cells, High Five cells, and the like, to generate ceDNA-baculovirus, which is a recombinant baculovirus that contains sequences that include asymmetric ITRs and an expression cassette. The ceDNA-baculovirus can be reinfected into insect cells to obtain the next generation of recombinant baculovirus. Optionally, this step can be repeated once or multiple times to produce larger amounts of recombinant baculovirus.

[0221] The time for harvesting the ceDNA vector described herein from cells can be selected and optimized to achieve high yield production of ceDNA vector. For example, the harvest time can be selected taking into consideration cell viability, cell morphology, cell growth, etc. Usually, cells can be harvested after sufficient time has passed since baculovirus infection to produce ceDNA vector (e.g., ceDNA vector), but before a portion of the cells begin to die due to viral toxicity. ceDNA vectors can be isolated from Sf9 cells using a plasmid purification kit, such as Qiagen ENDO-FREE PLASMID® kit. Other methods developed for plasmid isolation can also be adapted for ceDNA vectors. In general, any nucleic acid purification method known in the art can be employed, as well as commercially available DNA extraction kits.

[0222] Alternatively, purification can be implemented by subjecting the cell pellet to an alkaline lysis process, centrifuging the resulting lysate, and performing chromatographic separation.As one non-limiting example, this process can be performed by loading the supernatant onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acid, then eluting (e.g., with 1.2M NaCl solution), and performing further chromatographic purification on a gel filtration column (e.g., 6 Fast Flow GE).The capsid-free AAV vector is then recovered, for example, by precipitation.

[0223] In some embodiments, ceDNA vectors can also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein / nucleic acid cargoes through shedding of membrane microvesicles (Cocucci et al, 2009, EP10306226.1). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles), both of which contain proteins and RNA as cargo. Microvesicles are generated from direct budding of the plasma membrane, and exosomes are released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane. Thus, microvesicles and / or exosomes containing ceDNA vectors can be isolated from cells transduced with ceDNA plasmids, or bacmids or baculoviruses generated with ceDNA plasmids.

[0224] Microvesicles can be isolated by subjecting the culture medium to filtration or ultracentrifugation at 20,000×g and exosomes at 100,000×g. The optimal duration of ultracentrifugation can be experimentally determined and depends on the particular cell type from which the vesicles are isolated. Preferably, the culture medium is first cleared by low speed centrifugation (e.g., 2000×g for 5-20 minutes) and subjected to spin concentration, for example, using AMICON® spin columns (Millipore, Watford, UK). Microvesicles and exosomes can be further purified via FACS or MACS by using specific antibodies that recognize specific surface antigens present on the microvesicles and exosomes. Other microvesicles and exosome purification methods include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. During purification, the vesicles are washed, for example, with phosphate buffered saline. One advantage of using microvesicles or exosomes to deliver ceDNA-containing vesicles is that these vesicles can be targeted to various cell types by including on them membrane proteins that are recognized by specific receptors on each cell type. (See also EP10306226)

[0225] Another aspect of the invention herein relates to a method for purifying ceDNA vector from host cell lines that stably integrate ceDNA constructs into their own genome.In one embodiment, ceDNA vector is purified as DNA molecule.In another embodiment, ceDNA vector is purified as exosome or microparticle.

[0226] Figure 5 shows a gel confirming the production of ceDNA from multiple ceDNA plasmid constructs using the methods described in the Examples. The ceDNA is confirmed by a characteristic band pattern in the gel, as discussed with respect to Figure 4D in the Examples. Other characteristics of the ceDNA production process and intermediates are summarized in Figures 6A and 6B and Figures 7A and 7B, as described in the Examples.

[0227] VII. Pharmaceutical Compositions In another embodiment, a pharmaceutical composition is provided, comprising a ceDNA vector disclosed herein and a pharma- ceutically acceptable carrier or diluent.

[0228] The DNA disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises the ceDNA vector disclosed herein and a pharma- ceutically acceptable carrier. For example, the ceDNA vector described herein can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high pressure intravenous or intra-arterial injection, as well as intracellular injection, e.g., intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentration. Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compounds in a suitable buffer with one or a combination of the above-listed components, and filter sterilization.

[0229] The ceDNA vector-containing pharma- ceutically active composition can be formulated to deliver the transgene in the nucleic acid to the recipient's cells and cause therapeutic expression of the transgene therein. The composition can also include a pharma- ceutically acceptable carrier.

[0230] The ceDNA vectors disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, bronchial, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, anterior chamber, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intra-arterial injection, as well as intracellular injection, e.g., intranuclear microinjection or intracytoplasmic injection, are also contemplated.

[0231] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under the conditions of manufacture and storage.Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentration.Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compounds in a suitable buffer with one or a combination of the components listed above, and sterilizing by filtration.

[0232] Various techniques and methods for delivering nucleic acid to cells are known in the art.For example, nucleic acid such as ceDNA can be formulated in lipid nanoparticle (LNP), lipidoid, liposome, lipid nanoparticle, lipoplex, or core-shell nanoparticle.Typically, LNP is composed of nucleic acid (e.g., ceDNA) molecule, one or more ionized or cationic lipids (or their salts), one or more non-ionic or neutral lipids (e.g., phospholipids), molecules that prevent aggregation (e.g., PEG or PEG-lipid complex), and optionally sterol (e.g., cholesterol).

[0233] Another method for delivering nucleic acid such as ceDNA to cells is by complexing the nucleic acid with a ligand that is internalized by the cell. For example, the ligand can bind to a receptor on the cell surface and be internalized via endocytosis. The ligand can be covalently bound to the nucleotide in the nucleic acid. Exemplary complexes for delivering nucleic acid into cells are described in, for example, WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515, and WO2017 / 177326.

[0234] Nucleic acids such as ceDNA can also be delivered to cells by transfection. Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS™ P protein transfection reagent (New York, NY). England Biolabs), CHARIOT™ Protein Delivery Reagent (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMD Millipore), 293fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific), LIPOFECTAMINE(TM)(Thermo Fisher Scientific), LIPOFECTIN(TM)(Thermo Fisher Scientific), DMRIE-C, CELLFECTIN(TM)(Thermo Fisher Scientific), OLIGOFECTAMINE(TM)(Thermo Fisher Scientific), LIPOFECTACE(TM), FUGENE(TM)(Roche, Basel, Switzerland), FUGENE(TM) HD(Roche), TRANSFECTAM(TM)(Transfectam, Promega, Madison, Wis.), TFX-10(TM)(Promega), T FX-20(TM)(Promega), TFX-50(TM)(Promega), TRANSFECTIN(TM)(BioRad, Hercules,Calif.), SILENTFECT(TM)(Bio-Rad), Effectene(TM)(Qiagen,Valencia,Calif.), DC-chol(Avanti Polar Lipids), GENEPORTER(TM)(Gene Therapy Systems, San Diego, Calif.), DHARMAFECT 1(TM)(Dharmacon, Lafayette, Colo.), DHARMAFECT Examples of suitable delivery systems include, but are not limited to, DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, Mo.), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids, such as ceDNA, can also be delivered to cells via microfluidics methods known to those of skill in the art.

[0235] Methods for non-viral delivery of nucleic acids in vivo or ex vivo include electroporation, lipofection (see U.S. Pat. Nos. 5,049,386, 4,946,787, and commercially available reagents such as Transfectam™ and Lipofectin™), microinjection, biolistics, virosomes, liposomes (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820(1992), U.S. Patent No. 4,186,183, U.S. Patent No. 4,217,344, U.S. Patent No. 4,235,871, U.S. Patent No. 4,261,975, U.S. Patent No. 4,485,054, U.S. Patent No. 4,501,728, U.S. Patent No. 4,774,085, U.S. Patent No. 4,837,028 and U.S. Patent No. 4,946,787), immunoliposome, polycation or lipid:nucleic acid complex, naked DNA and drug-enhanced uptake of DNA.Sonoporation, for example, using Sonitron 2000 system (Rich-Mar) can also be used for the delivery of nucleic acid.

[0236] The ceDNA vector described herein can also be directly administered to organism for in vivo cell transduction.Administration can be by any of the routes that are usually used to finally bring molecules into contact with blood or tissue cells, including but not limited to infusion, injection, topical application, and electroporation.Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and more than one route can be used to administer a particular composition, although a particular route can often produce a more immediate and effective response than another route.

[0237] Methods for Introduction of Nucleic Acid Vectors The ceDNA vectors disclosed herein can be delivered into hematopoietic stem cells, for example, by the methods described in US Pat. No. 5,928,638.

[0238] The ceDNA vector according to the present invention can be added to liposomes for delivery to cells or target organs in a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of formulation development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery are composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0239] In some embodiments, the present disclosure provides liposomal formulations that include one or more compounds with polyethylene glycol (PEG) functional groups (so-called "PEGylated compounds"), which can reduce immunogenicity / antigenicity, provide hydrophilicity and hydrophobicity to the compound(s), and reduce dosing frequency. Alternatively, the liposomal formulations simply include polyethylene glycol (PEG) polymers as additional components. In such embodiments, the molecular weight of the PEG or PEG functional group can be from 62 Da to about 5,000 Da.

[0240] In some embodiments, the present disclosure provides liposomal formulations that deliver APIs with extended or controlled release profiles over a period of hours to weeks. In some related embodiments, the liposomal formulations can include aqueous chambers bounded by lipid bilayers. In other related embodiments, the liposomal formulations encapsulate APIs with components that undergo physical transitions at elevated temperatures that release the API over a period of hours to weeks.

[0241] In some embodiments, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein. In some embodiments, the liposomal formulation comprises an Optisome.

[0242] In some aspects, the present disclosure provides a method for the preparation of a lipid-rich lipid complex comprising administering to a subject ... The present invention provides a liposome formulation comprising one or more lipids selected from: dioleoylserine; POPC (palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxypolyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dioleoylphosphatidylcholine); DOPE (dioleoyl-sn-glycero-phosphoethanolamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphatidylcholine), or any combination thereof.

[0243] In some embodiments, the present disclosure provides a liposomal formulation comprising a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 56:38:5. In some embodiments, the total lipid content of the liposomal formulation is 2-16 mg / mL. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. In some embodiments, the present disclosure provides a liposomal formulation comprising a phosphatidylcholine functional group, an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol. In some embodiments, the PEGylated lipid is PEG-2000-DSPE. In some embodiments, the present disclosure provides a liposome formulation comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[0244] In some embodiments, the present disclosure provides a liposomal formulation comprising one or more lipids containing phosphatidylcholine functional groups and one or more lipids containing ethanolamine functional groups.In some embodiments, the present disclosure provides a liposomal formulation comprising one or more lipids containing phosphatidylcholine functional groups, lipids containing ethanolamine functional groups, and a sterol, such as cholesterol.In some embodiments, the liposomal formulation comprises DOPC / DEPC and DOPE.

[0245] In some embodiments, the present disclosure provides liposomal formulations further comprising one or more pharmaceutical excipients, such as sucrose and / or glycine.

[0246] In some embodiments, the present disclosure provides liposomal formulations that are either unilamellar or multilamellar structures. In some embodiments, the present disclosure provides liposomal formulations that include multivesicular particles and / or effervescent particles. In some embodiments, the present disclosure provides liposomal formulations that are larger in relative size to typical nanoparticles, about 150-250 nm in size. In some embodiments, the liposomal formulation is a lyophilized powder.

[0247] In some embodiments, the present disclosure provides liposomal formulations made and loaded with ceDNA vectors disclosed or described herein by adding a weak base to a mixture with isolated ceDNA outside the liposome. This addition increases the pH outside the liposome to approximately 7.3, driving the API into the liposome. In some embodiments, the present disclosure provides liposomal formulations having an acidic pH inside the liposome. In such cases, the inside of the liposome can be pH 4-6.9, more preferably pH 6.5. In other embodiments, the present disclosure provides liposomal formulations made by using intraliposomal drug stabilization techniques. In such cases, a polymeric or non-polymeric highly charged anion and an intraliposomal entrapment agent, such as polyphosphate or sucrose octasulfate, is utilized.

[0248] In another aspect, the present disclosure provides a liposome formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0249] For example, delivery agents such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, nucleic acids can be formulated for delivery or encapsulated in lipid particles, liposomes, vesicles, nanoparticles, gold particles, etc. Such formulations may be preferred for the introduction of pharma- ceutically acceptable formulations of the nucleic acids disclosed herein.

[0250] ceDNA vector can be delivered in vitro or in vivo using various delivery methods or their modifications known in the art.For example, in some embodiments, ceDNA vector is delivered by creating temporary penetration in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic or laser-based energy, thereby facilitating DNA entry into targeted cells.For example, ceDNA vector can be delivered by squeezing cells through size-restricted channels or by other means known in the art to temporarily disrupt cell membrane.In some cases, ceDNA vector is directly injected into skin, thyroid, cardiac muscle, skeletal muscle or liver cells as naked DNA alone.

[0251] In some cases, ceDNA vectors are delivered by gene gun: gold or tungsten spherical particles (1-3 μm diameter) coated with capsid-free AAV vectors are accelerated to high velocities by pressurized gas and can penetrate into target tissue cells.

[0252] In some embodiments, electroporation is used to deliver ceDNA vector.Electroporation causes the temporary destabilization of cell membrane target cell tissue by inserting electrode pair into tissue, so that DNA molecules in the medium surrounding the destabilized membrane can penetrate into the cytoplasm and nucleoplasm of cell.Electroporation has been used in vivo for many tissue types, such as skin, lung, and muscle.

[0253] In some cases, ceDNA vectors are delivered by hydrodynamic injection, a simple and highly efficient method for the direct intracellular delivery of any water-soluble compound and particle to skeletal muscles throughout the viscera and limbs.

[0254] In some cases, ceDNA vectors are delivered by ultrasound, which creates nanoscopic holes in the membrane to facilitate intracellular delivery of DNA particles to cells of internal organs or tumors, so the size and concentration of plasmid DNA play a major role in the efficiency of this system. In some cases, ceDNA vectors are delivered by magnetofection, which uses a magnetic field to concentrate particles containing nucleic acids in target cells.

[0255] In some cases, chemical delivery systems can be used, for example, by using nanomer complexes including the compression of negatively charged nucleic acids with cationic liposomes / micelles or polycationic nanomer particles commonly known as cationic polymers.Cationic lipids used in delivery methods include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol-derivative compounds, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.

[0256] A. Exosomes: In some embodiments, the ceDNA vectors disclosed herein are delivered by being packaged in exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell, they contain cytosol from the s cell that produced the exosomes, and exhibit membrane proteins from the parent cell on the surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). In some embodiments, exosomes with diameters of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, 50 nm to 100 nm are envisioned for use. Exosomes can be isolated for delivery to target cells, either by using their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing the capsid-free AAV vectors of the invention.

[0257] B. Microparticles / Nanoparticles: In some embodiments, the ceDNA vector disclosed herein is delivered by lipid nanoparticles. Generally, lipid nanoparticles include ionized amino lipids (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and coating lipids (polyethylene glycol-dimyristol glycerol, PEG-DMG), as disclosed, for example, by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceutical 5(3):498-507.

[0258] In some embodiments, the lipid nanoparticles have an average diameter between about 10 and about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter between about 10 and about 300 nm. In some embodiments, the lipid nanoparticles have a diameter less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter between about 25 and about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution, with an average size (e.g., diameter) of about 70 nm to about 200 nm, and more typically, the average size is about 100 nm or less.

[0259] Various lipid nanoparticles known in the art can be used to deliver the ceDNA vector disclosed herein.For example, various delivery methods using lipid nanoparticles are described in US Patent No. 9,404,127, US Patent No. 9,006,417 and US Patent No. 9,518,272.

[0260] In some embodiments, the ceDNA vector disclosed herein is delivered by gold nanoparticles.Generally, nucleic acid can be covalently bound to gold nanoparticles or non-covalently bound to gold nanoparticles (e.g., bound by charge-charge interaction) as described, for example, by Ding et al.(2014).Gold Nanoparticles for Nucleic Acid Delivery.Mol.Ther.22(6);1075-1083.In some embodiments, gold nanoparticle-nucleic acid complexes are produced using the method described, for example, in U.S. Patent No. 6,812,334.

[0261] C. Liposomes The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulation half-life have been developed (US Pat. No. 5,741,516). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (US Pat. Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868 and 5,795,587).

[0262] Liposomes have been used successfully with several cell types that are normally resistant to transfection by other procedures. In addition, liposomes do not contain the DNA length constraints typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed that examine the efficacy of liposome-mediated drug delivery.

[0263] Liposomes are formed from phospholipids dispersed in an aqueous medium, which simultaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 ANG, which contain an aqueous solution in their core.

[0264] In some embodiments, the liposome comprises cationic lipid. The term "cationic lipid" includes lipids and synthetic lipids that have both polar and non-polar domains, can be positively charged at or near physiological pH, bind polyanions such as nucleic acids, and facilitate the delivery of nucleic acids to cells. In some embodiments, the cationic lipid comprises saturated and unsaturated alkyl, and alicyclic ethers and esters of amines, or their derivatives. In some embodiments, the cationic lipid comprises linear, branched alkyl, alkenyl groups, or any combination thereof. In some embodiments, the cationic lipids contain from 1 to about 25 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms). In some embodiments, the cationic lipids contain more than 25 carbon atoms. In some embodiments, the straight or branched chain alkyl or alkene group has 6 or more carbon atoms. The cationic lipids may also, in some embodiments, contain one or more alicyclic groups. Non-limiting examples of alicyclic groups include cholesterol and other steroid groups. In some embodiments, the cationic lipids are prepared with one or more counterions. Exemplary counterions (anions) include Cl, ... - , Br - , I - , F - , acetate, trifluoroacetate, sulfate, nitrite, and nitrate salts.

[0265] Non-limiting examples of cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) star dendrimers, lipofectin (a combination of DOTMA and DOPE), lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectin (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide (DDAB). The nucleic acid (eg, CELiD) can also be complexed, for example, with poly(L-lysine) or avidin, and a lipid may or may not be included in the mixture, for example, steryl-poly(L-lysine).

[0266] In some embodiments, the ceDNA vectors disclosed herein are delivered using cationic lipids described in U.S. Pat. No. 8,158,601 or lipids described in U.S. Pat. No. 8,034,376.

[0267] D. complex In some embodiments, the ceDNA vectors disclosed herein are conjugated (e.g., covalently linked to an agent that increases cellular uptake). An "agent that increases cellular uptake" is a molecule that facilitates the transport of nucleic acids through lipid membranes. For example, nucleic acids can be conjugated to lipophilic compounds (e.g., cholesterol, tocopherol, etc.), cell penetrating peptides (CPPs) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine). Further examples of agents that increase cellular uptake can be found, for example, in Winkler (2013). Oligonucleotide conjugates for Ther. Deliv. 4(7);791-809.

[0268] In some embodiments, the ceDNA vector disclosed herein is conjugated to a polymer (e.g., a polymer molecule) or a folate molecule (e.g., a folic acid molecule). In general, delivery of nucleic acids conjugated to polymers is known in the art, for example, as described in WO2000 / 34343 and WO2008 / 022309. In some embodiments, the ceDNA vector disclosed herein is conjugated to a poly(amide) polymer, for example, as described in U.S. Pat. No. 8,987,377. In some embodiments, the nucleic acid described by the present disclosure is conjugated to a folic acid molecule, for example, as described in U.S. Pat. No. 8,507,455.

[0269] In some embodiments, the ceDNA vectors disclosed herein are conjugated to carbohydrates, for example, as described in U.S. Pat. No. 8,450,467.

[0270] E. Nanocapsules Alternatively, nanocapsule formulations of ceDNA vectors disclosed herein can be used. Nanocapsules can generally capture substances in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such fine particles (approximately 0.1 μm in size) should be designed using polymers so that they can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0271] VIII. Methods for delivering ceDNA vectors In some embodiments, ceDNA vector can be delivered to target cells in vitro or in vivo by various suitable methods.ceDNA vector can be applied or injected alone.ceDNA vector can be delivered to cells without the aid of transfection reagent or other physical means.Alternatively, ceDNA vector can be delivered using any transfection reagent known in the art that facilitates DNA entry into cells or other physical means known in the art, such as liposome, alcohol, polylysine-rich compound, arginine-rich compound, calcium phosphate, microvesicle, microinjection, electroporation, etc.

[0272] In contrast, transduction with the capsid-free AAV vectors disclosed herein can use a variety of delivery reagents and efficiently target cells and tissue types that are difficult to transduce with conventional AAV virions.

[0273] In another embodiment, the ceDNA vector is administered to the CNS (e.g., brain or eye). The ceDNA vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (cerebrum including striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. The ceDNA vector may also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve. The ceDNA vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture). The ceDNA vector may also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0274] In some embodiments, the ceDNA vector may be administered to the desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's area) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0275] In some embodiments, the ceDNA vector is administered in a liquid formulation by direct injection (e.g., stereotactic injection) into the desired region or compartment in the CNS. In other embodiments, the ceDNA vector can be provided by topical application to the desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be by topical application of liquid drops. As a further alternative, the ceDNA vector can be administered as a solid sustained release formulation (see, e.g., U.S. Pat. No. 7,201,898). In yet further embodiments, the ceDNA vector can be used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, the ceDNA vector can be delivered to muscle tissue and translocated from there into neurons.

[0276] VIII. Additional Uses of ceDNA Vectors The compositions and ceDNA vectors provided herein can be used to deliver transgenes for a variety of purposes. In some embodiments, the transgene encodes a protein or functional RNA intended to be used for research purposes, e.g., to create a somatic transgenic animal model harboring the transgene, e.g., to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA intended to be used to create an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating, preventing, or ameliorating a disease state or disorder in a mammalian subject. The transgene can be introduced (e.g., expressed) into a subject in an amount sufficient to treat a disease associated with reduced expression, lack of expression, or dysfunction of the gene. In some embodiments, the transgene can be introduced (e.g., expressed) into a subject in an amount sufficient to treat a disease associated with increased expression, activity of a gene product, or inappropriate upregulation of a gene (wherein the transgene suppresses or otherwise reduces its expression).

[0277] IX.How to use The ceDNA vector of the present invention can also be used in the method for the delivery of nucleotide sequence of interest to target cell.This method can be a method for delivering therapeutic gene of interest to the cell of a subject who needs it.The present invention allows the in vivo expression of polypeptide, protein or oligonucleotide encoded by therapeutic exogenous DNA sequence in cell, thereby expressing therapeutic level of polypeptide, protein or oligonucleotide.These results are seen in both in vivo and in vitro forms of ceDNA vector delivery.

[0278] The method for delivery of nucleic acid of interest in cells of a subject can comprise administration of the ceDNA vector of the present invention comprising the nucleic acid of interest to the subject.In addition, the present invention provides a method for delivery of nucleic acid of interest in cells of a subject in need thereof, comprising multiple administration of the ceDNA vector of the present invention comprising the nucleic acid of interest.Since the ceDNA vector of the present invention does not induce immune reaction, such multiple administration strategy is not hindered by host immune system reaction against the ceDNA vector of the present invention, in contrast to what is observed with encapsidation vector.

[0279] The ceDNA vector nucleic acid(s) is administered in an amount sufficient to transfect cells of the desired tissue and to provide sufficient levels of gene transfer and expression without excessive adverse effects.Conventional pharmacologic acceptable administration routes include, but are not limited to, intravenous (e.g., liposomal formulations), direct delivery to selected organs (e.g., intraportal delivery to the liver), intramuscular, and other administration routes.If desired, administration routes can be combined.

[0280] CeDNA vector delivery is not limited to one type of ceDNA vector.As such, in another embodiment, multiple ceDNA vectors containing different exogenous DNA sequences can be delivered simultaneously or sequentially to target cells, tissues, organs, or subjects.Thus, this strategy can allow the expression of multiple genes.Delivery can also be carried out for gene therapy in clinical situations at multiple times, and importantly, at increasing or decreasing doses later, assuming the lack of anti-capsid host immune response due to the absence of viral capsid.Since capsid is absent, anti-capsid response is expected not to occur.

[0281] The present invention also provides a method for treating a disease in a subject, comprising introducing a therapeutically effective amount of a ceDNA vector, optionally with a pharma- ceutically acceptable carrier, into a target cell (particularly a muscle cell or tissue) of a subject in need thereof. The ceDNA vector may be introduced in the presence of a carrier, but such a carrier is not required. The ceDNA vector as implemented comprises a nucleotide sequence of interest useful for treating a disease. In particular, the ceDNA vector may comprise a desired exogenous DNA sequence operably linked to a control element capable of directing the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into a subject. The ceDNA vector may be administered via any suitable route as provided above and elsewhere herein.

[0282] X. Treatment Methods The technology described herein also demonstrates methods for making the disclosed ceDNA vectors and how to use them in a variety of methods (e.g., ex situ, in vitro and in vivo applications, methodologies, diagnostic procedures, and / or gene therapy regimens).

[0283] Provided herein is a method of treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of a ceDNA vector, optionally with a pharma- ceutically acceptable carrier, into a target cell (e.g., a muscle cell or tissue, or other diseased cell type) of a subject in need thereof. The ceDNA vector may be introduced in the presence of a carrier, but such a carrier is not required. The ceDNA vector as implemented comprises a nucleotide sequence of interest useful for treating a disease. In particular, the ceDNA vector may comprise a desired exogenous DNA sequence operably linked to a control element that can direct the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into a subject. The ceDNA vector may be administered via any suitable route as provided above and elsewhere herein.

[0284] Any transgene can be delivered by the ceDNA vector disclosed herein.Transgenes of interest include nucleic acids that code for polypeptides or non-coding nucleic acids (e.g., RNAi, miRs, etc.), preferably therapeutic agents (e.g., for medical, diagnostic, or veterinary use), or immunogenic polypeptides (e.g., for vaccines).

[0285] In certain embodiments, the transgenes expressed by the ceDNA vectors described herein express or encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0286] In particular, the transgene can encode one or more therapeutic agent(s) including, but not limited to, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen-binding fragments, and variants and / or active fragments thereof, agonists, antagonists, mimetics, for use in the treatment, prevention, and / or amelioration of one or more symptoms of, for example, a disease, dysfunction, injury, and / or disorder. In one aspect, the disease, dysfunction, injury, injury, and / or disorder is a human disease, dysfunction, injury, injury, and / or disorder.

[0287] As described herein, the transgene can encode a therapeutic protein or peptide, or a therapeutic nucleic acid sequence, or a therapeutic agent, including, but not limited to, one or more agonists, antagonists, anti-apoptotic factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietin agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, enzymes, receptor binding proteins, transport proteins or one or more inhibitors thereof, serotonin receptors or one or more uptake inhibitors thereof, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, cytotoxins, or any combination thereof.

[0288] In some embodiments, the transgene in the expression cassette, expression construct, or ceDNA vector described herein can be codon optimized for the host cell. As used herein, the term "optimized codons" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a substantial number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of a vertebrate of interest, for enhanced expression in the cells of the vertebrate of interest, such as a mouse or human (e.g., humanized). Various species exhibit a particular bias toward certain codons of a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc.) or another publicly available database.

[0289] In some embodiments, the ceDNA vector expresses the transgene in a host cell of interest. In some embodiments, the host cell of interest is a human host cell, such as a blood cell, stem cell, hematopoietic cell, CD34 + The subject host cells may be hepatocytes, cancer cells, vascular cells, muscle cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other cells of mammalian origin, including, without limitation, hepatic (i.e., liver) cells, lung cells, cardiac cells, pancreatic cells, intestinal cells, diaphragm cells, renal (i.e., kidney) cells, neuronal cells, blood cells, bone marrow cells, or any one or more selected tissues of the subject for which gene therapy is contemplated. In one embodiment, the subject host cells are human host cells.

[0290] Disclosed herein is a ceDNA vector composition and formulation, comprising one or more of the ceDNA vectors of the present invention together with one or more pharma- ceutically acceptable buffers, diluents, or excipients.Such compositions can be included in one or more diagnostic or therapeutic kits for diagnosing, preventing, treating, or improving one or more symptoms of disease, injury, disorder, trauma, or dysfunction.In one embodiment, the disease, injury, disorder, trauma, or dysfunction is a human disease, injury, disorder, trauma, or dysfunction.

[0291] Another aspect of the technology described herein provides a method for providing a diagnostically or therapeutically effective amount of a ceDNA vector to a subject in need thereof, comprising providing an amount of a ceDNA vector disclosed herein to a cell, tissue, or organ of a subject in need thereof for a time effective to allow expression of a transgene from the ceDNA vector, thereby providing a diagnostically or therapeutically effective amount of a protein, peptide, nucleic acid expressed by the ceDNA vector to the subject. In a further aspect, the subject is a human.

[0292] Another aspect of the technology described herein provides a method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject.In a general and general sense, the method comprises administering at least one or more of the disclosed ceDNA vectors to a subject in need thereof in an amount and for a time period to diagnose, prevent, treat, or ameliorate one or more symptoms of disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject.In a further aspect, the subject is a human.

[0293] Another aspect is the use of ceDNA vectors as a tool to treat or reduce one or more symptoms of a disease or disease state. There are several genetic diseases with known defective genes, typically divided into two classes: enzyme deficiency states, which are usually inherited recessively, and imbalance states, which may involve regulatory or structural proteins, but are typically not always inherited dominantly. In the case of deficiency disease, ceDNA vectors can be used to deliver transgenes to carry normal genes into affected tissues for replacement therapy, and in some embodiments, antisense mutations can be used to create animal models of the disease. In the case of imbalance disease states, ceDNA vectors can be used to create pathologies in model systems, which can then be used to try to address the pathologies. Thus, the ceDNA vectors and methods disclosed herein allow for the treatment of genetic diseases. As used herein, a pathology is treated by partially or fully repairing the deficiency or imbalance that causes the disease or makes it more severe.

[0294] In general, the ceDNA vectors disclosed herein can be used to deliver any transgene to treat, prevent, or ameliorate symptoms associated with any disorder related to gene expression. Exemplary conditions include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes, muscular dystrophies (e.g., Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh's syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments, the ceDNA vectors disclosed herein may be advantageously used in the treatment of individuals with metabolic disorders (e.g., omegaly transcarbamylase deficiency).

[0295] In some embodiments, the ceDNA vectors described herein can be used to treat, improve, and / or prevent diseases or disorders caused by mutations in genes or gene products. Exemplary diseases or disorders that can be treated with ceDNA vectors include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII, Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC)); blood diseases or disorders (e.g., hemophilia (A and B), thalassemia, and anemia); cancer and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).

[0296] In yet a further embodiment, the ceDNA vectors disclosed herein can be used to deliver heterologous nucleotide sequences in situations where it is desirable to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor, as described herein).

[0297] Thus, in some embodiments, the ceDNA vectors described herein can be used to correct abnormal levels and / or functions of gene products (e.g., absence or deficiency in protein) that result in disease or disorder. The ceDNA vectors can produce functional proteins and / or modify the levels of proteins to alleviate or reduce symptoms or confer benefits resulting from a particular disease or disorder caused by the absence or deficiency in protein. For example, treatment of OTC deficiency can be achieved by producing functional OTC enzyme. Treatment of hemophilia A and B can be achieved by modifying the levels of factor VIII, factor IX, and factor X. Treatment of PKU can be achieved by modifying the levels of phenylalanine hydroxylase enzyme. Treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha galactosidase or beta glucocerebrosidase, respectively. Treatment of MLD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively. Treatment of cystic fibrosis may be achieved by producing a functional cystic fibrosis transmembrane conductance regulator. Treatment of glycogen storage disease may be achieved by restoring functional G6Pase enzyme function. Treatment of PFIC may be achieved by producing a functional ATP8B1, ABCB11, ABCB4, or TJP2 gene.

[0298] In alternative embodiments, antisense nucleic acid can be provided to cells in vitro or in vivo using ceDNA vector disclosed herein.For example, when transgene is RNAi molecule, expression of antisense nucleic acid or RNAi in target cell reduces expression of specific protein by cells.Therefore, transgene that is RNAi molecule or antisense nucleic acid can be administered to reduce expression of specific protein in subject that needs it.Antisense nucleic acid can also be administered to cells in vitro to regulate cell physiology, for example, optimize cell or tissue culture system.

[0299] In some embodiments, exemplary transgenes encoded by the ceDNA vector include transgenes encoding lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease, or genes encoding ... II-related iduronate sulfatase), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, and the like), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial-derived growth factor (GDNF), transforming growth factor-alpha and -beta, and the like), receptors (e.g., tumor necrosis factor receptor). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, two or more transgenes are encoded by a ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments, the transgene encodes an antibody, including a full-length antibody or an antibody fragment, as defined herein. In some embodiments, the antibody is an antigen-binding domain or an immunoglobulin variable domain sequence, as defined herein. Other exemplary transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer treatment, and tumor suppressor gene products.

[0300] In a representative embodiment, the transgene expressed by ceDNA vector can be used for the treatment of muscular dystrophy in a subject who needs to be treated.The method includes administering an effective amount of the ceDNA vector described herein for treatment, improvement, or prevention, and the ceDNA vector includes a heterologous nucleic acid encoding dystrophin, mini-dystrophin, micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptide, such as IκB dominant mutant, sarcospan, utrophin, micro-dystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, antibody or antibody fragment against myostatin or myostatin polypeptide, and / or RNAi against myostatin.In a specific embodiment, the ceDNA vector can be administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle, as described elsewhere herein.

[0301] In some embodiments, the gene is selected for the purpose of producing a polypeptide (e.g., an enzyme) or functional RNA (e.g., RNai, microRNA, antisense RNA) that normally circulates in the blood, or for the purpose of inhibiting a disorder (e.g., metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., VIII), mucopolysaccharidosis (e.g., Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteaux-Lamy syndrome, etc.) or lysosomal dysfunction (e.g., myelopathy, pulmonary edema ... The ceDNA vectors can be used to deliver transgenes to skeletal, cardiac, or diaphragm muscles for systemic delivery to other tissues to treat, ameliorate, and / or prevent glycogen storage diseases (Gaucher disease [glucocerebrosidase], Pompe disease [lysosomal acid alpha-glucosidase], or Fabry disease [alpha-galactosidase A]) or glycogen storage diseases (Pompe disease [lysosomal acid alpha-glucosidase). Other suitable proteins for treating, ameliorating, and / or preventing metabolic disorders are described above.

[0302] In other embodiments, the ceDNA vector disclosed herein can be used to deliver transgenes in a method for treating, improving, and / or preventing metabolic disorders in a subject in need thereof.Exemplary metabolic disorders and transgenes encoding polypeptides are described herein.Optionally, the polypeptide is secreted (e.g., a polypeptide that is a secreted polypeptide in its native state, or a polypeptide that is engineered to be secreted by operable association with a secretion signal sequence, e.g., as known in the art).

[0303] Another aspect of the present invention relates to a method of treating, ameliorating, and / or preventing congenital heart disease or PAD in a subject in need thereof, the method comprising administering to the mammalian subject a ceDNA vector as described herein, the ceDNA vector being capable of, for example, targeting sarcoplasmic endoplasmic reticulum Ca2+ receptors. 2+ RNAi of phospholamban, such as phospholamban inhibitors or dominant negative molecules, such as phospholamban S16E, a zinc finger protein that regulates the phospholamban gene, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcin, β-adrenergic receptor kinase inhibitor (βARKct), inhibitor of protein phosphatase 1, S100A1, parvalbumin, adenylyl cyclase type 6, molecules that affect G-protein-linked receptor kinase type 2 knockdown, such as truncated constitutively active βARKct, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206, and / or mir-208.

[0304] The ceDNA vector disclosed herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles containing the ceDNA vector, which the subject inhales.The respirable particles can be liquid or solid.The aerosol of liquid particles containing the ceDNA vector can be produced by any suitable means, such as by using a pressure-driven aerosol nebulizer or ultrasonic nebulizer, as known to those skilled in the art.See, for example, US Patent No. 4,501,729.The aerosol of solid particles containing the ceDNA vector can be produced similarly by using any solid particle drug aerosol generator, by techniques known in the pharmaceutical field.

[0305] In some embodiments, the ceDNA vectors may be administered to tissues of the CNS (e.g., brain, eye). In certain embodiments, the ceDNA vectors disclosed herein may be administered to treat, ameliorate, or prevent diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Exemplary diseases of the CNS include Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal or head injury, Ty-Sac disease, Leish-Nairn disease, epilepsy, cerebral infarction, mood disorders (e.g., depression, bipolar disorder, persistent affective disorder, secondary mood disorder). These conditions include, but are not limited to, psychiatric disorders, including schizophrenia, drug addiction (e.g., alcoholism and other drug addiction), neuroses (e.g., anxiety disorders, obsessive-compulsive disorder, somatoform disorders, dissociative disorders, grief, postpartum depression), psychoses (e.g., hallucinations and delusions), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleep disorders, pain disorders, eating or weight disorders (e.g., obesity, cachexia, anorexia, and bulimia), and cancers and tumors of the CNS (e.g., pituitary tumors).

[0306] Ocular disorders that may be treated, ameliorated, or prevented with the ceDNA vectors of the present invention include ophthalmic disorders involving the retina, the posterior thalamic tract, and / or the optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy, and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma). Many ocular diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. In some embodiments, the ceDNA vectors disclosed herein can be used to deliver anti-angiogenic factors, anti-inflammatory factors, factors that slow cell degeneration, promote cell sparing, or promote cell proliferation, and combinations thereof. Diabetic retinopathy, for example, is characterized by angiogenesis. Diabetic retinopathy may be treated by delivering one or more anti-angiogenic factors either intraocularly (e.g., intravitreally) or periocularly (e.g., in the subtenon's area). One or more neurotrophic factors may also be co-delivered either intraocularly (e.g., intravitreally) or periocularly. Additional eye diseases that may be treated, ameliorated, or prevented with the ceDNA vectors of the present invention include geographic atrophy, vascular or "wet" macular degeneration, Stargardt's disease, Leber's congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), X-linked retinitis pigmentosa (XLRP), X-linked retinoschisis (XLRS), total choroidal atrophy, Leber's hereditary optic neuropathy (LHON), color blindness, cone-rod degeneration, Fuchs' endothelial corneal dystrophy, diabetic macular edema, and eye cancers and tumors.

[0307] In some embodiments, inflammatory eye diseases or disorders (e.g., uveitis) may be treated, ameliorated, or prevented by the ceDNA vectors of the present invention. One or more anti-inflammatory factors may be expressed by intraocular (e.g., vitreous or anterior chamber) administration of a ceDNA vector disclosed herein. In other embodiments, eye diseases or disorders characterized by retinal degeneration (e.g., retinitis pigmentosa) may be treated, ameliorated, or prevented by the ceDNA vectors of the present invention. Intraocular (e.g., vitreous) administration of a ceDNA vector disclosed herein encoding one or more neurotrophic factors may be used to treat such retinal degeneration-based diseases. In some embodiments, diseases or disorders involving both angiogenesis and retinal degeneration (e.g., age-related macular degeneration) may be treated with the ceDNA vectors of the present invention. Age-related macular degeneration may be treated by administering one or more neurotrophic factors intraocularly and / or one or more anti-angiogenic factors intraocularly or periocularly (e.g., in the subtenon's area) of a ceDNA vector disclosed herein encoding one or more anti-angiogenic factors. Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment of glaucoma involves the administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the ceDNA vectors disclosed herein.Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors can thus be delivered intraocularly, optionally intravitreally, using the ceDNA vectors disclosed herein.

[0308] In other embodiments, the ceDNA vectors disclosed herein can be used to treat seizures, e.g., to reduce the symptoms, incidence, or severity of seizures. The effectiveness of therapeutic treatment of seizures can be evaluated behaviorally (e.g., rocking, eye or mouth tics) and / or by electrographic means (most seizures have significant electrographic abnormalities). Thus, the ceDNA vectors disclosed herein can also be used to treat epilepsy marked by multiple seizures over time. In a representative embodiment, somatostatin (or an active fragment thereof) is administered to the brain using the ceDNA vectors disclosed herein to treat pituitary tumors. In accordance with this embodiment, the ceDNA vectors disclosed herein that encode somatostatin (or an active fragment thereof) are administered by microinjection into the pituitary gland. Similarly, such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary gland). The nucleic acid (e.g., GenBank Accession No. J00306) and amino acid (e.g., GenBank Accession No. P01166, containing the processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art. In certain embodiments, the ceDNA vector can encode a transgene that includes a secretion signal as described in U.S. Patent No. 7,071,172.

[0309] Another aspect of the present invention relates to the use of the ceDNA vectors described herein to produce antisense RNA, RNAi, or other functional RNA (e.g., ribozymes) for systemic delivery to a subject in vivo. Thus, in some embodiments, ceDNA vectors may contain transgenes encoding antisense nucleic acids, ribozymes (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that affect spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNAs (RNAi) that mediate gene silencing (see Sharp et al., (2000) Science 287:2431) or other non-translated RNAs, such as "guide" RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like.

[0310] In some embodiments, the ceDNA vector may also further comprise a transgene encoding a reporter polypeptide (e.g., an enzyme such as green fluorescent protein or alkaline phosphatase). In some embodiments, the transgene encoding a reporter protein useful for experimental or diagnostic purposes is selected from any of β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. In some aspects, the ceDNA vectors containing transgenes encoding reporter polypeptides can be used for diagnostic purposes or as markers of the activity of the ceDNA vector in the subject to which they are administered.

[0311] In some embodiments, the ceDNA vector may contain a transgene or heterologous nucleotide sequence that shares homology with the host chromosome and recombines with a locus on the host chromosome. This approach can be used to correct genetic defects in the host cell.

[0312] In some embodiments, the ceDNA vector can include a transgene that can be used to express an immunogenic polypeptide in a subject, for example, for vaccination. The transgene can code for any immunogen of interest known in the art, including, but not limited to, immunogens from human immunodeficiency virus, influenza virus, gag protein, tumor antigen, cancer antigen, bacterial antigen, viral antigen, etc.

[0313] XI. Administration In certain embodiments, more than one administration (e.g., 2, 3, 4 or more administrations) may be used to achieve a desired level of gene expression over variously spaced periods (e.g., daily, weekly, monthly, yearly, etc.).

[0314] Exemplary modes of administration of the ceDNA vectors disclosed herein include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intradermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including administration to the skeleton, diaphragm, and / or myocardium], intrapleural, intracerebral, and intra-arterial), topical (e.g., to skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., into the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0315] Administration of the ceDNA vector can be to any site of the subject, including, but not limited to, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. Administration of the ceDNA vector can also be to a tumor (e.g., in or near a tumor or lymph node). The most suitable route in any given case depends on the nature and severity of the condition to be treated, ameliorated, and / or prevented, and the nature of the particular ceDNA vector being used. Additionally, ceDNA allows for the administration of multiple transgenes in a single vector or in two or more ceDNA vectors (e.g., ceDNA cocktail).

[0316] Administration of the ceDNA vectors disclosed herein to skeletal muscles in accordance with the present invention includes, but is not limited to, administration to skeletal muscles of the limbs (e.g., upper arms, lower arms, upper legs, and / or lower legs), lower back, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. The ceDNA vectors disclosed herein can be delivered to skeletal muscles by intravenous administration, intraarterial administration, intraperitoneal, limb perfusion (optionally isolated limb perfusion of the legs and / or arms, see, e.g., Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In certain embodiments, the ceDNA vectors disclosed herein are administered to a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intraarterial administration). In embodiments, the ceDNA vectors disclosed herein can be administered without the use of "hydrodynamic techniques".

[0317] Administration of the ceDNA vector disclosed herein to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. The ceDNA vector described herein can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion. Administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to the smooth muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment, administration can be to endothelial cells present in, near, and / or on the smooth muscle.

[0318] In some embodiments, a ceDNA vector according to the present invention is administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., to treat, ameliorate, and / or prevent muscular dystrophy or cardiac disease (e.g., PAD or congestive heart failure)).

[0319] A. Ex Vivo Treatment In some embodiments, cells are removed from a subject, ceDNA vector is introduced therein, and then the cells are returned to the subject.Methods for removing cells from a subject and then returning them to the subject for ex vivo treatment are known in the art (see, for example, U.S. Patent No. 5,399,346, the disclosure of which is incorporated herein by reference in its entirety).Alternatively, ceDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and these cells are administered to a subject in need thereof.

[0320] The cells transduced with the ceDNA vector are preferably combined with a pharmaceutical carrier and administered to the subject in a "therapeutically effective amount". Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0321] In some embodiments, a ceDNA vector can encode a transgene (sometimes called a heterologous nucleotide sequence), which is any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo. In contrast to the use of ceDNA vectors in, for example, therapeutic methods discussed herein, in some embodiments, a ceDNA vector may be introduced into a cultured cell and the expressed gene product isolated therefrom, for example, for the production of an antigen or vaccine.

[0322] ceDNA vectors can be used for both veterinary and medical purposes.Suitable subjects for the above ex vivo gene delivery method include birds (e.g. chicken, duck, goose, quail, turkey, and pheasant) and mammals (e.g. human, cow, sheep, goat, horse, cat, dog, and lagomorph), with mammals being preferred.Human subjects are most preferred.Human subjects include neonates, infants, juveniles, and adults.

[0323] One aspect of the technology described herein relates to the method of delivering transgene to cells.Typically, for in vitro method, ceDNA vector can be introduced using the method disclosed herein as well as other methods known in the art.The ceDNA vector disclosed herein is preferably administered to cells in a biologically effective amount.When ceDNA vector is administered to cells in vivo (e.g., to a subject), the biologically effective amount of ceDNA vector is sufficient to cause transduction and expression of transgene in target cells.

[0324] B. Dose Range In vivo and / or in vitro assays can be optionally used to help identify the optimal dosage range for use.The exact dosage to be used in the formulation also depends on the route of administration and the severity of the condition, and should be determined according to the judgment of the skilled artisan and the circumstances of each subject.Effective dosages can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0325] The ceDNA vector is administered in an amount sufficient to transfect the cells of the desired tissue and to provide sufficient levels of gene transfer and expression without excessive side effects.Conventional pharmacologic acceptable administration routes include, but are not limited to, those described above in the "administration" section, such as direct delivery to selected organs (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes.Administration routes can be combined if desired.

[0326] The dosage of the amount of ceDNA vector required to achieve a particular "therapeutic effect" will vary based on several factors, including, but not limited to, the route of nucleic acid administration, the level of gene or RNA expression required to achieve the therapeutic effect, the particular disease or disorder being treated, and the stability of the gene(s), RNA product(s), or resulting expressed protein(s). One of skill in the art can readily determine the ceDNA vector dosage range for treating a patient with a particular disease or disorder based on the aforementioned factors as well as other factors well known in the art.

[0327] Dosage regime can be adjusted to provide optimal therapeutic response.For example, oligonucleotide can be repeatedly administered, for example, several doses can be administered every day, or dose can be proportionally reduced as indicated by the exigencies of therapeutic situation.Those skilled in the art can easily determine the appropriate dose and schedule of administration of subject oligonucleotide, regardless of whether oligonucleotide is administered to cell or to subject.

[0328] A "therapeutically effective amount" falls within a relatively broad range that can be determined through clinical trials and depends on the particular application (neuronal cells require very small amounts, while systemic injection requires large amounts). For example, for direct in vivo injection into skeletal or cardiac muscle of a human subject, a therapeutically effective amount would be approximately about 1 μg to about 100 g of ceDNA vector. When exosomes or microparticles are used to deliver the ceDNA vector, the therapeutically effective amount can be determined empirically, but is expected to deliver 1 μg to about 100 g of vector.

[0329] The formulation of pharma- ceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for using the particular compositions described herein in various treatment regimens.

[0330] For in vitro transfection, cells (1 × 10 6 An effective amount of ceDNA vector delivered to a cell will be approximately 0.1-100 μg of ceDNA vector, preferably 1-20 μg, more preferably 1-15 μg or 8-10 μg. Larger ceDNA vectors will require larger doses. When exosomes or microparticles are used, the effective in vitro dose is determined empirically, but is generally intended to deliver the same amount of ceDNA vector.

[0331] Treatment may require administration of a single dose or multiple doses. In some embodiments, multiple doses may be administered to the subject, and in fact multiple doses may be administered as needed, since the ceDNA vector does not induce an anti-capsid host immune response due to the absence of viral capsid. As such, one of skill in the art can determine the appropriate number of doses. The number of doses administered may be, for example, approximately 1-100, preferably 2-20 doses.

[0332] Without being bound by any particular theory, the lack of typical anti-viral immune responses elicited by administration of the ceDNA vectors described by the present disclosure (i.e., the absence of capsid components) allows the ceDNA vector to be administered to the host on multiple occasions. In some embodiments, the number of times the heterologous nucleic acid is delivered to the subject is in the range of 2-10 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the ceDNA vector is delivered to the subject more than 10 times.

[0333] In some embodiments, the dose of the ceDNA vector is administered to the subject only once per calendar day (e.g., 24 hours). In some embodiments, the dose of the ceDNA vector is administered to the subject only once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of the ceDNA vector is administered to the subject only once per calendar week (e.g., 7 calendar days). In some embodiments, the dose of the ceDNA vector is administered to the subject only once every 2 weeks (e.g., once...

Claims

[Claim 1] A non-viral capsid-free DNA vector (ceDNA vector) having covalently closed ends, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site.

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