Scalable, high-purity, cell-free synthesis of closed-end DNA vectors

Abstract

The present disclosure provides a method for scalable and high-purity cell-free synthesis of DNA vectors, particularly closed-end DNA vectors (e.g., ceDNA vectors) with a linear and continuous structure for transgene delivery and expression. The cell-free synthesis involves digesting a double-stranded DNA construct with at least one restriction endonuclease that can cleave the construct at a cleavage site that is different from the recognition site to release an insert with a unique overhang that regulates the high specificity of the subsequent ligation reaction. The insert is then ligated with an inverted terminal repeat (ITR) oligonucleotide to form a closed-end DNA vector. The corresponding DNA vectors and related products prepared by these methods, as well as other bases associated with the methods and intermediate vectors and constructs, are also provided in the present disclosure.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 293,337, filed December 23, 2021. The entire contents of the aforementioned application are expressly incorporated herein by reference.

[0002] The present disclosure relates to the field of gene therapy, including high-throughput and high-purity production of non-viral vectors for the purpose of expressing transgenes in subjects or cells.For example, the present disclosure provides a cell-free method for synthesizing non-viral DNA vectors.The present disclosure also relates to the nucleic acid constructs produced thereby and their methods of use. [Background technology]

[0003] 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 aberrant regulation 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 repair genetic material to the patient, or by altering or silencing the defective gene with repair genetic material to the patient, resulting in therapeutic expression of the genetic material in the patient.

[0004] The basis of gene therapy is to provide a transcription cassette with an active gene product (sometimes referred to as a transgene), which may, for example, produce a positive gain-of-function effect, a negative loss-of-function effect, or another result. 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 repair genes in target cells of patients can be carried out through many methods, including the use of engineered viruses and viral gene delivery vectors. Among the many virus-derived vectors available (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated viruses (rAAV) have gained popularity as a versatile vector in gene therapy.

[0005] Adeno-associated viruses (AAV) belong to the family Parvoviridae and more specifically constitute the genus Dependoparvovirus. Vectors derived from AAV (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for delivery of 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, e.g., 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 as episomes, thus limiting the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively weakly immunogenic and therefore do not elicit a significant immune response (see ii), thus resulting in persistence of vector DNA and long-term expression of potentially therapeutic transgenes.

[0006] However, there are several significant drawbacks to 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), and as a result, the use of AAV vectors is limited to protein-coding capacities 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, potential subjects for rAAV gene therapy need to be screened for the presence of neutralizing antibodies that would clear the vector from the patient. A third drawback concerns capsid immunogenicity, which prevents re-administration to patients who were not excluded from 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 to 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.

[0007] Additionally, conventional AAV virions with capsids are produced by introducing a plasmid containing the AAV genome, rep gene, and cap gene (Grimm et al., 1998). However, such encapsidated AAV viral vectors transduce certain cell and tissue types inefficiently, and the capsids have also been found to induce immune responses.

[0008] Thus, the use of adeno-associated virus (AAV) vectors for gene therapy is limited due to a single administration to the patient (due to the patient immune response), the limited range of transgene genetic material suitable for delivery in AAV vectors due to minimal viral packaging capacity (approximately 4.5 kb), and the slow AAV-mediated gene expression.

[0009] Closed-end DNA vectors have been developed that can deliver one or more desired transgenes in vivo for therapeutic or other purposes and avoid the above-mentioned drawbacks of AAV and other viral vector systems. However, methods to produce such ceDNA vectors have relied on traditional bacterial or insect cell production methods. Such methods can result in contaminants (e.g., nucleic acid contaminants) from the cells used to produce the vector (e.g., Sf9 cells), which can be inconvenient or expensive to remove and can have undesirable side effects when included in a ceDNA therapeutic formulation. Thus, there is a need in the field of technologies that allow for the generation of recombinant vectors used in methods to control gene expression with minimal off-target effects, such as those introduced by such contaminants or other artifacts of purification methods. It would also be advantageous for this technology to be suitable for and support large-scale production of recombinant vectors. The methods provided herein address these unmet needs. Summary of the Invention

[0010] Traditional methods for producing viruses and virus-derived DNA typically use eukaryotic cells, such as mammalian or insect cells. One commonly used insect cell line is Sf9. However, not only do these cells contain both enzymes and other proteins that can adversely affect the replicated DNA, but the process of purifying the desired DNA from cell lysates introduces cellular nucleic acids whose presence can make the purification of the desired DNA product more difficult. Furthermore, such impurities or contaminants can have a range of harmful and / or undesirable effects in the subject to whom the desired DNA is administered. Additionally, such traditional cell-based production methods can be problematic with regard to the amount of DNA vector product produced, and it is not uncommon that significant manipulation of the cell line itself or the production technology is required to produce the desired yield.

[0011] The technical solutions to these vector production challenges provided herein relate to a cell-free synthetic production method that can easily produce closed circular hairpin loop-containing DNA vectors, such as, but not limited to, closed-end DNA vectors (ceDNA vectors), with higher purity than by traditional means using eukaryotic cells (see, for example, FIG. 13).

[0012] Thus, there is provided a method of producing a closed-ended DNA (ceDNA) vector, the method comprising contacting a double-stranded DNA construct having a sense strand and an antisense strand with at least a first restriction endonuclease and at least a second restriction endonuclease, the construct comprising a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette, the first restriction endonuclease being capable of cleaving the double-stranded DNA construct at the first cleavage site, and the second restriction endonuclease being capable of cleaving the double-stranded DNA construct at the second cleavage site. Provided herein is a method comprising contacting the double-stranded DNA construct with a first restriction endonuclease and a second restriction endonuclease, wherein the restriction endonuclease is capable of cleaving the double-stranded DNA construct at a second cleavage site, releasing an insert having a first end comprising a first single-stranded overhang and a second end comprising a second single-stranded overhang; ligating the first end to a first oligonucleotide comprising one or more hairpin structures; and ligating the second end to a second oligonucleotide comprising one or more hairpin structures, thereby producing a ceDNA vector.

[0013] In one embodiment, the first and / or second oligonucleotide comprises an inverted terminal repeat (ITR). In one embodiment, the first oligonucleotide and the second oligonucleotide are different. In another embodiment, the first oligonucleotide and the second oligonucleotide are the same. In one embodiment, each of the oligonucleotides independently comprises one, two, three, four, or more stem-loop regions. In another embodiment, each of the oligonucleotides independently comprises two or three stem-loop regions.

[0014] In one embodiment, the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases. In another embodiment, the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease.

[0015] In one embodiment, the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site are separate and distinct from each other, both sites being located upstream of the transgene expression cassette. In another embodiment, the first cleavage site is about 1 to about 22 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct. In another embodiment, the first cleavage site is about 1 to about 8 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct.

[0016] In one embodiment, the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site are separate and distinct from one another, both sites being located upstream of the transgene expression cassette. In another embodiment, the second cleavage site is about 1 to about 22 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct (e.g., about 2 to about 22, about 2 to about 20, about 5 to about 22, about 5 to about 20, about 10 to about 22, about 10 to about 20, about 15 to about 22, about 15 to about 20, about 2 to about 15, about 5 to about 15, about 10 to about 15, about 2 to about 10, about 5 to about 10, about 2 to about 10, about 2 to about 10, about 2 to about 10 nucleotides away). In another embodiment, the second cleavage site is about 1 to about 8 nucleotides away from a second non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct.

[0017] In one embodiment, the first non-palindromic restriction endonuclease recognition site and the second non-palindromic restriction endonuclease recognition site are each double-stranded polynucleotides having different 5' to 3' nucleotide sequences in each of the sense and antisense strands.

[0018] In one embodiment, one or both of the single-stranded overhangs at the ends of the insert are 5' overhangs, hi another embodiment, one or both of the single-stranded overhangs at the ends of the insert are 3' overhangs.

[0019] In one embodiment, the first oligonucleotide comprising one or more hairpin structures and the second oligonucleotide comprising one or more hairpin structures are each a single-stranded oligonucleotide that self-anneals to form a three-dimensional structure. In one embodiment, the three-dimensional structure is a T-shaped or Y-shaped stem-loop structure. In another embodiment, the first oligonucleotide comprising one or more hairpin structures and the second oligonucleotide comprising one or more hairpin structures each self-anneals to further form a single-stranded overhang at either the 5' end or the 3' end of each oligonucleotide.

[0020] In one embodiment, the first oligonucleotide and the second oligonucleotide each self-anneal to further form a single-stranded overhang at the 5' end of each oligonucleotide. In another embodiment, the first oligonucleotide and the second oligonucleotide each self-anneal to further form a single-stranded overhang at the 3' end of each oligonucleotide. In one embodiment, the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each about 1 to about 12 nucleotides in length, about 1 to about 8 nucleotides in length, about 2 to about 6 nucleotides in length, or about 3, about 4, about 5, or about 6 nucleotides in length.

[0021] In one embodiment, the 5' end of each oligonucleotide is phosphorylated.

[0022] In one embodiment, the 5' to 3' nucleotide sequences of the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are non-complementary to each other. In another embodiment, the 5' to 3' nucleotide sequences of the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are the same. In another embodiment, the first oligonucleotide and the second oligonucleotide have the same nucleotide sequence. In another embodiment, the single-stranded overhangs at each end of the insert comprise the same 5' to 3' nucleotide sequence. In another embodiment, the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each complementary to both of the single-stranded overhangs at the ends of the insert. In another embodiment, the 5' to 3' nucleotide sequences of the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are different. In another embodiment, the first oligonucleotide and the second oligonucleotide comprise different nucleotide sequences. In another embodiment, the 5' to 3' nucleotide sequences of the single-stranded overhangs at each end of the insert are different.

[0023] In one embodiment, the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each complementary to only one of the single-stranded overhangs at the ends of the insert.

[0024] In one embodiment, the single-stranded overhang of the first oligonucleotide and / or the single-stranded overhang of the second oligonucleotide comprises a 5' to 3' nucleotide sequence selected from the group consisting of CTCT, CTCA, CACT, CTC, and GCT, hi another embodiment, one or both of the first and second oligonucleotides are synthetic.

[0025] In one embodiment, the first oligonucleotide and the second oligonucleotide are each about 40 nucleotides to about 75 nucleotides in length, or about 45 to about 65 nucleotides in length. In another embodiment, the first oligonucleotide and the second oligonucleotide each independently comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In another embodiment, the first oligonucleotide and the second oligonucleotide each independently comprise a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In another embodiment, the first oligonucleotide and the second oligonucleotide each independently comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0026] In one embodiment, each hairpin structure and / or each T-shaped or Y-shaped stem loop structure of the first oligonucleotide and each hairpin structure and / or each T-shaped or Y-shaped stem loop structure of the second oligonucleotide comprises a stem region that is at least about 4 base pairs in length, about 4 to about 20 base pairs in length, about 4 to about 15 base pairs in length, about 4 to about 6 base pairs in length, or about 6 to about 8 base pairs in length. In another embodiment, the stem region length does not include any single-stranded overhangs.

[0027] In one embodiment, at least one or both of the restriction endonucleases are Type IIS restriction endonucleases. In another embodiment, the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease. In another embodiment, the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases. In another embodiment, the Type IIS restriction endonuclease is selected from the group consisting of AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, Bsr DI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers thereof. In another embodiment, each of the first and second restriction endonucleases is independently selected from the group consisting of AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, The type IIS restriction endonuclease is selected from the group consisting of BsrDI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers thereof.In further embodiments, the at least one Type IIS restriction endonuclease is selected from the group consisting of BbsI, BsaI, Esp3I, and SapI, and isoschizomers thereof. In further embodiments, the at least one Type IIS restriction endonuclease is BsaI or an isoschizomer thereof.

[0028] In one embodiment, after ligating, the first non-palindromic restriction endonuclease recognition site and the second non-palindromic restriction endonuclease recognition site are not regenerated in the resulting ceDNA vector.

[0029] In one embodiment, the double-stranded DNA construct further comprises at least a first partial ITR and a second partial ITR, each flanking the transgene expression cassette. In another embodiment, the first partial ITR is upstream of the transgene expression cassette and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site. In another embodiment, the second partial ITR is downstream of the transgene expression cassette and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site. In another embodiment, the first cleavage site is adjacent to the first partial ITR and the second cleavage site is adjacent to the second partial ITR. In another embodiment, the double-stranded DNA construct further comprises a first spacer between the first partial ITR and the transgene expression cassette. In another embodiment, the double-stranded DNA construct further comprises a second spacer between the second partial ITR and the transgene expression cassette. In another embodiment, the double stranded DNA construct is selected from the group consisting of a bacmid, a plasmid, a minicircle, or a linear double stranded DNA molecule.

[0030] In one embodiment, the resulting ceDNA vector comprises a transgene expression cassette and at least a first ITR and a second ITR, each flanking the transgene expression cassette. In another embodiment, the first ITR is upstream of the transgene expression cassette. In another embodiment, the second ITR is downstream of the transgene expression cassette. In another embodiment, the first ITR comprises a nucleotide sequence from a first oligonucleotide and a first partial ITR. In another embodiment, the second ITR comprises a nucleotide sequence from a second oligonucleotide and a second partial ITR. In another embodiment, the first ITR lacks a first non-palindromic restriction endonuclease recognition site. In another embodiment, the second ITR lacks a second non-palindromic restriction endonuclease recognition site.

[0031] In one embodiment, the first ITR and the second ITR each comprise a hairpin structure and / or a T-shaped or Y-shaped stem-loop structure. In another embodiment, the first ITR and the second ITR each comprise a T-shaped or Y-shaped stem-loop structure. In another embodiment, the T-shaped or Y-shaped stem-loop structure comprises a stem comprising an A-A' and D-D' stem region and two B-B' and C-C' loops. In another embodiment, one or both of the first ITR and the second ITR are adeno-associated virus (AAV) ITRs or ITRs derived from AAV. In another embodiment, one or both of the first ITR and the second ITR are wild-type ITRs. In another embodiment, both the first ITR and the second ITR are wild-type ITRs. In another embodiment, one or both of the first ITR and the second ITR are modified ITRs. In another embodiment, the first ITR and the second ITR are symmetrical or substantially symmetrical to each other. In another embodiment, the first ITR and the second ITR are asymmetric ITRs.

[0032] In one embodiment, one or both of the first and second ITRs comprise one or more modifications selected from the group consisting of additions, deletions, truncations, and point mutations. In another embodiment, the one or more modifications are located in the A-A' stem region, the B-B' loop, the C-C' loop, and / or the D-D' stem region of one or both of the first and second ITRs. In another embodiment, the one or more modifications are located in the B-B' loop and / or the C-C' loop of one or both of the first and second ITRs. In another embodiment, the B-B' loop and the C-C' loop of one of the first and second ITRs are truncated.

[0033] In one embodiment, the transgene expression cassette further comprises a first spacer between the first ITR and the transgene expression cassette. In another embodiment, the transgene expression cassette further comprises a first spacer between the second ITR and the transgene expression cassette. In another embodiment, the transgene expression cassette further comprises a first spacer between the first ITR and the transgene expression cassette and a second spacer between the second ITR and the transgene expression cassette.

[0034] In one embodiment, the transgene expression cassette comprises a transgene. In another embodiment, the transgene encodes a therapeutic protein. In another embodiment, the therapeutic protein is selected from the group consisting of an enzyme, a clotting factor or cofactor, an antibody or antigen-binding fragment thereof, an antigen, a gene-editing protein, and a cytotoxic protein.

[0035] In one embodiment, the transgene expression cassette further comprises a genetic element selected from the group consisting of a promoter, an enhancer, an intron, a posttranscriptional regulatory element, and a polyadenylation signal, hi another embodiment, the posttranscriptional regulatory element comprises a WHP posttranscriptional regulatory element (WPRE).

[0036] In one embodiment, the ligating is performed by a ligase or AAV Rep protein, hi another embodiment, the ligase is T4 ligase.

[0037] In one embodiment, the method further comprises isolating or purifying the resulting ceDNA vector. In another embodiment, the method further comprises isolating or purifying the insert prior to ligating. In another embodiment, the method does not include isolating or purifying the insert prior to ligating. In another embodiment, the contacting and ligating steps are performed in a single reaction vessel.

[0038] In one embodiment, the resulting ceDNA vector comprises at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the monomeric species of the ceDNA vector.

[0039] In one embodiment, provided herein is a closed-end DNA (ceDNA) vector produced by any of the methods disclosed herein. In another embodiment, provided herein is a pharmaceutical composition comprising a closed-end DNA (ceDNA) vector produced by any of the methods disclosed herein. In another embodiment, provided herein is a lipid nanoparticle composition comprising a closed-end DNA (ceDNA) vector produced by any of the methods disclosed herein. In another embodiment, provided herein is an isolated host cell comprising a closed-end DNA (ceDNA) vector produced by any of the methods disclosed herein. In another embodiment, provided herein is a transgenic animal comprising a closed-end DNA (ceDNA) vector produced by any of the methods disclosed herein.

[0040] In one embodiment, provided herein is a method of treating a disorder, disease, or condition in a subject (e.g., a genetic disorder, disease, or condition), the method comprising administering to the subject a therapeutically effective amount of a closed-ended DNA (ceDNA) vector produced by any of the methods disclosed herein, or a pharmaceutical composition comprising a ceDNA vector produced by any of the methods disclosed herein, or a lipid nanoparticle composition comprising a ceDNA vector produced by any of the methods disclosed herein.

[0041] In one embodiment, provided herein is a method of delivering a therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of a closed-end DNA (ceDNA) vector produced by any of the methods disclosed herein, or a pharmaceutical composition comprising a ceDNA vector produced by any of the methods disclosed herein, or a lipid nanoparticle composition comprising a ceDNA vector produced by any of the methods disclosed herein.

[0042] In another embodiment, the therapeutic protein is selected from the group consisting of an enzyme, a clotting factor or cofactor, an antibody or antigen-binding fragment thereof, an antigen, a gene-editing protein, and a cytotoxic protein.

[0043] In another embodiment, provided herein is an inverted terminal repeat (ITR) nucleotide sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, as set forth below.

[0044] [Table 12]

[0045] In another embodiment, provided herein is an inverted terminal repeat (ITR) nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In another embodiment, provided herein is an inverted terminal repeat (ITR) nucleotide sequence comprising a sequence that is at least 95%, at least 96%, at least 975, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In another embodiment, provided herein is an inverted terminal repeat (ITR) nucleotide sequence consisting of a sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0046] In another embodiment, the ITR nucleotide sequence further comprises a spacer selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, as shown below.

[0047] [Table 13]

[0048] In another embodiment, the ITR nucleotide sequence further comprises a spacer sequence comprising a sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40. In another embodiment, the ITR nucleotide sequence further comprises a spacer sequence comprising a sequence that is at least 95%, at least 96%, at least 975, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, as set forth below. In another embodiment, the ITR nucleotide sequence further comprises a spacer sequence comprising a sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, as set forth below.

[0049] In another embodiment, provided herein is a closed-end DNA (ceDNA) vector comprising a transgene expression cassette and at least a first inverted terminal repeat (ITR) and a second ITR flanking the transgene expression cassette, wherein the first ITR and the second ITR each comprise a nucleotide sequence selected from the group consisting of ITR nucleotide sequences disclosed herein. In another embodiment, the vector comprises double-stranded DNA.

[0050] In another embodiment, provided herein is a pharmaceutical composition comprising a ceDNA vector comprising any of the ITR sequences disclosed herein and at least one pharma- ceutically acceptable excipient.In another embodiment, provided herein is a lipid nanoparticle composition comprising a ceDNA vector comprising any of the ITR sequences disclosed herein.In another embodiment, provided herein is an isolated host cell comprising a ceDNA vector comprising any of the ITR sequences disclosed herein.In another embodiment, provided herein is a transgenic animal comprising a ceDNA vector comprising any of the ITR sequences disclosed herein.

[0051] In embodiments, provided herein is a method of treating a disorder, disease, or condition in a subject (e.g., a genetic disorder, disease, or condition), the method comprising administering to the subject a therapeutically effective amount of a ceDNA vector comprising any of the ITR sequences disclosed herein, or a pharmaceutical composition comprising a ceDNA vector comprising any of the ITR sequences disclosed herein, or a lipid nanoparticle composition comprising a ceDNA vector comprising any of the ITR sequences disclosed herein.

[0052] In another embodiment, provided herein is a method of delivering a therapeutic protein to a subject, the method comprising administering to the subject a therapeutically effective amount of a ceDNA vector comprising any of the ITR sequences disclosed herein, or a pharmaceutical composition comprising a ceDNA vector comprising any of the ITR sequences disclosed herein, or a lipid nanoparticle composition comprising a ceDNA vector comprising any of the ITR sequences disclosed herein. In another embodiment, the therapeutic protein is selected from the group consisting of an enzyme, an antibody or an antigen-binding fragment thereof, an antigen, a gene editing protein, and a cytotoxic protein.

[0053] In one embodiment, a DNA vector for use in synthetic production of closed-end DNA vectors (ceDNA) is provided herein, the DNA vector comprising a multiple cloning site capable of accepting a transgene, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the multiple cloning site, a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the multiple cloning site, and a first partial ITR and a second partial ITR, each flanking the multiple cloning site. In another embodiment, the first partial ITR is upstream of the multiple cloning site and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site. In another embodiment, the second partial ITR is downstream of the multiple cloning site and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site.

[0054] In another embodiment, the DNA vector comprises one or more spacers. In another embodiment, the DNA vector comprises an origin of replication and a selectable marker gene. In another embodiment, the multiple cloning site can accommodate a transgene and one or more additional genetic elements selected from the group consisting of a promoter, an enhancer, an intron, a post-transcriptional regulatory element, and a polyadenylation signal.

[0055] In one embodiment, the first non-palindromic restriction endonuclease recognition site is specific for a first restriction endonuclease and the second non-palindromic restriction endonuclease recognition site is specific for at least a second restriction endonuclease. In another embodiment, the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease. In another embodiment, the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases.

[0056] In one embodiment, at least one of the restriction endonucleases is a Type IIS restriction endonuclease. In another embodiment, each of the first and second restriction endonucleases is a Type IIS restriction endonuclease. In another embodiment, the Type IIS restriction endonuclease is AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, Bsr DI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers thereof. In another embodiment, each Type IIS restriction endonuclease is independently selected from AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, In another embodiment, the Type IIS restriction endonuclease is selected from the group consisting of BbsI, BsaI, Esp3I, and isoschizomers thereof.

[0057] In one embodiment, the DNA vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In one embodiment, the DNA vector comprises a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In one embodiment, the DNA vector consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0058] In one embodiment, provided herein is a kit for preparing a closed-end DNA (ceDNA) vector comprising a transgene, the kit comprising any DNA vector disclosed herein, at least one restriction endonuclease capable of cleaving the DNA vector at a multiple cloning site to enable the multiple cloning site to accept a transgene, at least one restriction endonuclease capable of cleaving at a first cleavage site and a second cleavage site, and a ligase. In another embodiment, the kit further comprises at least one oligonucleotide comprising one or more hairpin structures.

[0059] In one embodiment, provided herein is a double-stranded circular DNA construct engineered to facilitate preparation of a closed-ended DNA (ceDNA) vector comprising a transgene expression cassette, the double-stranded circular DNA construct comprising a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette, and a first partial ITR and a second partial ITR, each flanking the transgene expression cassette.

[0060] In one embodiment, a first partial ITR is upstream of the transgene expression cassette and downstream of a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site. In another embodiment, a second partial ITR is downstream of the transgene expression cassette and upstream of a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site. In another embodiment, the first non-palindromic restriction endonuclease recognition site is specific for a first restriction endonuclease and the second non-palindromic restriction endonuclease recognition site is specific for at least a second restriction endonuclease.

[0061] In one embodiment, the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease. In another embodiment, the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases.

[0062] In one embodiment, at least one of the restriction endonucleases is a Type IIS restriction endonuclease. In another embodiment, each of the first and second restriction endonucleases is a Type IIS restriction endonuclease. In another embodiment, the Type IIS restriction endonuclease is AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, Bsr DI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers thereof. In another embodiment, each Type IIS restriction endonuclease is independently selected from AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, In another embodiment, the at least one Type IIS restriction endonuclease is selected from the group consisting of BbsI, BsaI, Esp3I, and isoschizomers thereof.

[0063] In one embodiment, provided herein is a kit for preparing a closed-end DNA (ceDNA) vector comprising a transgene expression cassette, the kit comprising any of the double-stranded DNA constructs disclosed herein, at least one restriction endonuclease capable of cleaving the double-stranded DNA construct at a first cleavage site and a second cleavage site, a ligase, and instructions for use. In another embodiment, the kit further comprises at least one oligonucleotide comprising one or more hairpin structures.

[0064] In one embodiment, provided herein is a method of producing a double-stranded DNA construct from a plasmid template via rolling circle amplification, comprising: contacting the plasmid template with a thermostable polymerase having strand displacement activity, wherein the ratio of plasmid template concentration (in ng / μl) to polymerase concentration (in U / μl) is greater than about 1; contacting the plasmid template with oligonucleotide primers and dNTPs; and incubating the plasmid template, polymerase, oligonucleotide primers, and dNTPs at a temperature of about 40° C. or less for a period of at least about 5 hours, thereby producing a double-stranded DNA construct.

[0065] In one embodiment, the ratio of plasmid template concentration (in ng / μl) to polymerase concentration (in U / μl) is greater than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0066] In one embodiment, the plasmid template concentration is about 0.01 ng / μl, about 0.05 ng / μl, about 0.1 ng / μl, about 0.15 ng / μl, about 0.2 ng / μl, about 0.21 ng / μl, about 0.22 ng / μl, about 0.23 ng / μl, about 0.24 ng / μl, about 0.2 ng / μl, about 0.26 ng / μl, about 0.27 ng / μl, about 0.28 ng / μl, about 0.29 ng / μl, about 0.3 ng / μl, about 0.35 ng / μl, about 0.4 ng / μl, about 0.45 ng / μl, about 0.5 ng / μl, about 0.6 ng / μl, about 0.7 ng / μl, about 0.8 ng / μl, about 0.9 ng / μl, or about 1.0 ng / μl.

[0067] In one embodiment, the polymerase concentration is about 0.01 U / μl, about 0.02 U / μl, about 0.03 U / μl, about 0.04 U / μl, about 0.05 U / μl, about 0.06 U / μl, about 0.07 U / μl, about 0.08 U / μl, about 0.09 U / μl, about 0.1 U / μl, about 0.15 U / μl, about 0.2 U / μl, about 0.25 U / μl, about 0.3 U / μl, about 0.35 U / μl, about 0.4 U / μl, or about 0.45 U / μl.

[0068] In one embodiment, the temperature in step (c) is about 40°C, about 39°C, about 38°C, about 37°C, about 36°C, about 35°C, about 34°C, about 33°C, about 32°C, about 31°C, about 30°C, about 29°C, about 28°C, about 27°C, about 26°C, about 25°C, about 24°C, about 23°C, about 22°C, or less than about 21°C.

[0069] In one embodiment, the period is at least about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours.

[0070] In one embodiment, the period is less than about 6 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours.

[0071] In another embodiment, the plasmid template concentration is about 0.25 ng / μl, the temperature is about 30° C., the polymerase concentration is about 0.05 U / μl, and the duration is about 18-26 hours. In another embodiment, the oligonucleotide primer concentration is less than about 50 μM, at least about 10 μM, or at least about 10 μM and less than about 50 μM. 171. In another embodiment, the dNTP concentration is about 4 mM.

[0072] In one embodiment, the thermostable polymerase is Phi29 DNA polymerase or a derivative or variant thereof. In another embodiment, the thermostable polymerase is EquiPhi29™.

[0073] In one embodiment, the method is carried out in a total reaction volume of at least about 100 μl. In another embodiment, the method is carried out in a total reaction volume of at least about 100 μl, about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 100 ml, about 150 ... The method is carried out in a total reaction volume of about 1, about 95 ml, about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml, about 800 ml, about 900 ml, about 1 L, about 2 L, about 3 L, 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, or about 1000 L. In another embodiment, the method is carried out in a reaction vessel having a capacity at least twice the total reaction volume.

[0074] In one embodiment, the oligonucleotide primer hybridizes to a backbone sequence in a plasmid template, hi another embodiment, the oligonucleotide primer is a universal primer.

[0075] In one embodiment, provided herein is a double-stranded DNA construct produced by any of the methods disclosed herein.

[0076] In one embodiment, provided herein is a method for producing a closed-end DNA (ceDNA) vector, comprising: producing a double-stranded DNA construct using any of the methods disclosed herein; and performing any of the methods disclosed herein to produce a ceDNA vector from the double-stranded DNA construct. In another embodiment, provided herein is a ceDNA vector produced by any of the above methods, a pharmaceutical composition comprising a ceDNA vector produced by any of the above methods and at least one pharma- ceutically acceptable excipient, and a lipid nanoparticle composition comprising a vector produced by any of the above methods.

[0077] In another embodiment, provided herein is a method of preparing a closed-end DNA (ceDNA) vector, the method comprising contacting a double-stranded DNA construct with at least one restriction endonuclease, wherein the construct comprises a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette, wherein the at least one restriction endonuclease is capable of cleaving the construct at the first cleavage site and the second cleavage site to release an insert having single-stranded overhangs at 5' and 3' ends of the insert; and ligating the 5' and 3' ends of the insert to a first inverted terminal repeat (ITR) oligonucleotide and a second ITR oligonucleotide to form a ceDNA vector.

[0078] These and other aspects of the disclosure are described in further detail below. [Brief description of the drawings]

[0079] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0080]

[0013] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to the exemplary embodiments of the present disclosure, which are depicted in the accompanying drawings. The accompanying drawings, however, depict only typical embodiments of the present disclosure and therefore should not be considered as limiting in scope, since the present disclosure may admit of other equally effective embodiments. [Figure 1A] FIG. 1 shows a schematic diagram of non-limiting exemplary ceDNA vectors having symmetric or substantially symmetric inverted terminal repeats (ITRs), or asymmetric ITRs, at each end of the vector flanking a transgene expression cassette that contains a transgene and one or more regulatory sequences that enable and / or control expression of the transgene. [Figure 1B] FIG. 1 shows a schematic diagram of non-limiting exemplary ceDNA vectors having symmetric or substantially symmetric inverted terminal repeats (ITRs), or asymmetric ITRs, at each end of the vector flanking a transgene expression cassette that contains a transgene and one or more regulatory sequences that enable and / or control expression of the transgene. [Diagram 2] 1 is a map of an exemplary basic vector plasmid 11. [Diagram 3] 1 is a map of an exemplary construct, construct 1, generated by subcloning a Factor VIII (VIII) expression transgene into plasmid 11. [Figure 4] FIG. 1 is a schematic diagram of an exemplary cell-free synthesis of ceDNA using a single ITR oligonucleotide and the restriction endonuclease BsaI. [Diagram 5]FIG. 1 is a schematic diagram comparing the activity of conventional restriction endonucleases such as EcoRI and Type IIS restriction endonucleases, in particular how the enzymes recognize and cleave nucleotide sequences on substrate DNA and the properties of the overhangs generated from cleavage. [Figure 6] FIG. 1 is a schematic illustrating the mechanism of BsaI recognizing and cleaving double-stranded DNA constructs, such that only fragments carrying a transgene expression cassette and with the appropriate overhangs will ligate to ITR oligo 1 with a complementary overhang. [Figure 7A] FIG. 1 is a schematic diagram of exemplary ITR oligonucleotides that self-anneal to form a three-dimensional stem-loop structure, with stem lengths of 7 bp or 3 bp. [Figure 7B] 13 is an agarose gel image analyzing ligation reactions using plasmid 20 insert and ITR oligonucleotides with different stem lengths: 14 bp, 7 bp, 5 bp, and 3 bp. [Figure 7C] 1 is a map of an exemplary base vector plasmid 20. [Figure 8] FIG. 1 is a schematic illustrating the mechanism for generating ceDNA vectors with asymmetric ITRs. [Figure 9A-9C] Figure 9A is an agarose gel image analyzing multiple ligation reactions using different combinations of base vector, ITR oligonucleotides, and type IIS restriction endonucleases. Figures 9B and 9C are agarose gel images confirming ligation specificity at both the 5' and 3' ends of the insert using labeled ITR oligonucleotides. [Figure 10] Schematic diagram showing the stem-loop structure of the wild-type ITR of AAV serotype 2 (AAV2), with identification of the A-A' and D-D' stem regions and the B-B' and C-C' loops (SEQ ID NO: 52 of International Patent Application Publication No. WO2019 / 143885, the entire contents of which are expressly incorporated herein by reference). [Figure 11]13 is an agarose gel image analyzing uncut construct 1 (lane 1), the digestion / ligation reaction of construct 1 (lane 2), and the reaction mixture after exonuclease digestion (lane 3). [Figure 12] Illustrated are (i) the predicted sizes of FVIII-ceDNA produced from construct 1 as uncut or BglII-cut closed-ended vector, as open-stranded DNA, or as closed-ended vector containing one or more nicks or gaps, and (ii) denaturing gel analysis of uncut or BglII-cut FVIII-ceDNA. [Figure 13] Chromatogram of FVIII-ceDNA drug substance eluting as a single sharp peak from an ion exchange chromatography column. [Figure 14] FIG. 1 is a schematic illustrating DNA sequence analysis comparing the DNA sequence of FVIII-ceDNA at the 5' and 3' ligation junction with the DNA sequences of ITR oligo 1 and Construct 1, thereby revealing a unique junction sequence that is present only in the FVIII-ceDNA ligation product, but not in ITR oligo 1 and Construct 1. [Figure 15] 1 is an agarose gel image showing small- and medium-scale ceDNA produced using the cell-free synthesis method described herein. [Figure 16A] 1 is a graph showing the synthesis of increasing dose levels and in vivo FVIII expression levels of FVIII-ceDNA produced in Sf9 in a hydrodynamic tail vein injection study in mice. [Figure 16B] FIG. 13 is a graph showing in vivo FVIII expression levels of synthetic and Sf9-produced FVIII-ceDNA formulated in lipid nanoparticle compositions in a 42-day intravenous injection study in mice. [Figure 17] Schematic diagram of rolling circle plasmid amplification using primer-driven multiple strand displacement, both with (bottom) and without (top) an optional intermediate digestion step, followed by enzymatic conversion of the amplified products to ceDNA. [Figure 18]1 illustrates the evaluation of the effect of primer concentration on amplified plasmid product quality and DNA yield using agarose gel analysis of DNA banding profiles of E. coli plasmid template and amplified plasmid. Lanes from left to right: (1) size marker, (2) plasmid, no BsaI digestion, (3) plasmid, BsaI digestion, (4) amplified plasmid, BsaI digestion, 500 μM primer, (5) amplified plasmid, BsaI digestion, 100 μM primer, (6) amplified plasmid, BsaI digestion, 50 μM primer, (7) amplified plasmid, BsaI digestion, 10 μM primer, (8) amplified plasmid, BsaI digestion, 5 μM primer, (9) amplified plasmid, BsaI digestion, 1 μM primer, (10) amplified plasmid, BsaI digestion, 0 μM primer. [Figure 19A] Illustrates the effect of temperature and polymerase amount on amplified product quality. Shows agarose gel analysis of DNA banding and product quality for BsaI-treated amplified plasmid at different amplification temperatures, amounts of polymerase enzyme, and lengths of amplification time. For each amplification reaction temperature (from left to right: 40° C., 37° C., 33° C., 30° C., and 20° C.), results are shown for 50 units of EquiPhi29™ (top) and 5 units of EquiPhi39™ (bottom). Additionally, for each reaction temperature, four lanes show results for amplification reaction times of 3 hours, 12 hours, 24 hours, and 36 hours, from left to right. The leftmost lane contains size markers. [Figure 19B] FIG. 1 illustrates the effect of temperature and polymerase amount on amplified product quality. Shown is an agarose gel comparing the results of amplification at 30° C. with different amounts of wild-type Phi29 polymerase enzyme (left) and engineered EquiPhi29™ polymerase enzyme (right). Lanes, from left to right for each gel: (1) size marker, (2) 2 units of enzyme, (3) 5 units of enzyme. [Figure 20A]Illustrated is an agarose gel comparison of E. coli plasmid digested with BsaI, plasmid amplified at 30° C., and plasmid amplified at 45° C. (left), and the corresponding ceDNA vectors produced from both the E. coli plasmid and each amplified plasmid (right). Lanes, from left to right: (1) size marker, (2) plasmid digested with BsaI, (3) plasmid amplified at 30° C. digested with BsaI, (4) plasmid amplified at 45° C. digested with BsaI, (5) size marker, (6) purified ceDNA produced from the plasmid, (7) purified ceDNA produced from the plasmid amplified at 30° C., (8) purified ceDNA produced from the plasmid amplified at 45° C., (9) size marker. [Figure 20B] Illustrates an agarose gel comparison of ceDNA produced from five different amplified plasmid constructs. Expected ceDNA product sizes, from left to right, are: 2.9 kb (construct 1), 3.8 kb (construct 2), 5.9 kb (construct 3), 4.7 kb (construct 4), and 6.2 kb (construct 5). The leftmost lane contains size markers. [Figure 21A] Illustrates scaled plasmid amplification with increasing reaction volumes. Agarose gel analysis of amplified plasmid products digested with BsaI is shown with quantified amplified DNA yield. Lanes, from left to right: (1) 100 μl volume, 1.5 mL tube: 25 ng plasmid input, 80 μg amplified DNA yield; (2) 1 mL volume, 1.5 mL tube: 250 ng plasmid input, 800 μg amplified DNA yield; (3) 25 mL volume, 50 mL tube: 6.3 ng plasmid input, 20 mg amplified DNA yield; (4) size markers. [Figure 21B] Scaled plasmid amplification with increasing reaction volumes is illustrated. Fragment chromatogram and agarose gel analysis of 20 mg of ceDNA product from a 25 mL reaction volume is illustrated (corresponding to lane 3 in FIG. 21A). [Figure 22]Illustrated is an agarose gel analysis comparing plasmid amplification using different amounts of polymerase enzyme, reaction temperatures, and reaction time lengths. Results of BsaI digestion of crude reactions (left) and Zymo-purified ceDNA (right) are shown. Initial process: 0.25 ng / μl plasmid template, 10 μM annealing primer, 45°C, 0.5 U / μl EquiPhi29™, 5 mM dNTPs, 3 hours. Updated process: 0.25 ng / μl plasmid template, 25 μM annealing primer, 30°C, 0.05 U / μl EquiPhi29™, 4 mM dNTPs, 18-26 hours. Lanes, from left to right: (1) size marker, (2) plasmid digested with BsaI, (3) crude amplified plasmid digested with BsaI, updated process, (4) crude amplified plasmid digested with BsaI, initial process, (5) size marker, (6) purified ceDNA produced from the plasmid, (7) purified ceDNA produced from the amplified plasmid, updated process, (8) purified plasmid produced from the amplified plasmid, initial process, (9) size marker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] The present disclosure describes the development of a new approach for faster and more cost-effective plasmid or DNA production using cell-free DNA amplification methods. The methods and compositions provided herein are based, at least in part, on the discovery of cell-free synthetic production methods useful for generating DNA vectors, including closed-end DNA (ceDNA) vectors, with higher purity and yield compared to DNA vectors produced in insect cell lines, such as, but not limited to, Sf9 cell lines, and / or the production process is simplified or made more efficient or cost-effective compared to traditional cell-based production methods. For example, in one embodiment, high specificity in the restriction endonuclease digestion and ligation reactions allows both reactions to be performed simultaneously in a single reaction vessel. In some embodiments, high specificity in the digestion and ligation reactions further eliminates the need for purification steps between the two reactions. This high specificity in the restriction endonuclease digestion and ligation reactions is facilitated by the design in the closed-end vectors, i.e., inverted terminal repeat (ITR) oligonucleotides and base materials that create the double-stranded DNA constructs. Specifically, the ITR oligonucleotides and double-stranded DNA constructs contain nucleotide sequences that exploit the inherent activity of the restriction endonucleases used in these cell-free synthesis methods in recognizing, binding to, and cleaving DNA.

[0082] The result of the work described in embodiments herein is a scalable, robust, cell-free, enzymatic method that generated large quantities of fully synthetic closed-ended DNA molecules, representing a significant advantage over cell-based production methods.

[0083] Furthermore, exploiting the inherent activity of the restriction endonucleases used in these cell-free synthesis methods allows for the directionality of the ligation reaction utilizing more than one ITR oligonucleotide to be controlled, thereby enabling the preparation of ceDNA with asymmetric ITRs.

[0084] Another significant advantage provided by the cell-free synthesis methods provided herein over cell-based production methods, in addition to higher yields, is that the methods described herein are easily scalable to small scale reactions (approximately 1 mL) and at least to moderate sizes (>40 mL), without compromising purity.

[0085] A vector synthesized as described herein can express any desired transgene, e.g., a transgene for treating or curing a given disease. One of skill in the art will readily recognize that any transgene used in conventional gene therapy methods using conventional recombinant vectors can be adapted for expression by a ceDNA vector generated, e.g., by the synthetic methods described herein.

[0086] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. It is to be understood that this disclosure is not limited to the specific methodology, protocols, and reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the disclosure, 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 Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6 thEdition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013); Knipe, DM and Howley, PM (eds.), 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, NY, 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 047150338 Strobe, (ed.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737).

[0087] As used herein, the terms "cell-free," "cell-free synthesis," "cell-free production," "synthetic closed-end DNA vector production," and "synthetic production," as well as all other related counterparts, are used interchangeably and refer to the production of one or more molecules in a manner that does not involve replication or other propagation of the molecule by or within a cell, or by use of a cell extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants (e.g., cellular proteins or cellular nucleic acids) and also avoids undesired cell-specific modifications of the molecule during the production process (e.g., methylation or glycosylation or other post-translational modifications).

[0088] As used herein, the terms "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 vectors disclosed herein.

[0089] As used herein, the terms "transgene expression cassette," "expression cassette," "transcription cassette," and "gene expression unit" 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 regulatory genetic elements, including cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, one or more polyadenylation signals, and one or more post-transcriptional regulatory elements, such as a WHP post-transcriptional regulatory element (WPRE).

[0090] The terms "polynucleotide" and "nucleic acid", as used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides, also known as "oligomers" or "oligos", can be isolated from genes or chemically synthesized by methods known in the art. As used herein, an "inverted terminal repeat oligonucleotide" or "ITR oligonucleotide" refers to a single-stranded oligonucleotide containing at least a partial sequence of a complete ITR as defined herein, and can self-anneal to form a three-dimensional configuration of an ITR having a hairpin structure or a T-shaped or Y-shaped stem-loop structure. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides, as applicable to the embodiments being described.

[0091] The term "nucleic acid construct" as used herein refers to a single- or double-stranded nucleic acid molecule (e.g., a DNA construct) that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner that would not otherwise occur in nature, or that is synthetic.

[0092] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., a single-stranded DNA) contains a sequence of nucleotides that allows it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, to "anneal" or "hybridize" (i.e., the nucleic acid specifically binds to a complementary nucleic acid) to another nucleic acid (e.g., another single-stranded DNA) in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C).

[0093] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. In some embodiments, the transgene in the defined transgene expression cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from an enzyme, a clotting factor or cofactor, an antibody or antigen-binding fragment thereof, an antigen, a gene editing protein, and a cytotoxic protein.

[0094] A DNA sequence, such as a transgene, that "encodes" a particular RNA or protein gene product is a DNA nucleic acid sequence that is transcribed into a particular RNA and / or protein. The DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or the DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA-targeting RNA, also called "non-coding" RNA or "ncRNA").

[0095] As used herein, the term "gene delivery" refers to the process by which foreign DNA is introduced into a host cell for gene therapy applications.

[0096] As used herein, the term "terminal repeat" or "TR" may include any viral terminal repeat or synthetic sequence that includes at least one minimally necessary origin of replication and a region that includes a palindromic hairpin structure. The Rep-binding sequence ("Rep-binding sequence, RBS") (also referred to as RBE (Rep-binding element)) and terminal resolution site ("TRS") together constitute the "minimally necessary origin of replication", and thus a TR includes 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 viruses, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found in the present disclosure 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 configuration. The ITRs may be AAV ITRs or non-AAV ITRs or may be derived from AAV ITRs or non-AAV ITRs. For example, the ITRs may be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as the origin of SV40 replication, may be used as an ITR, which may be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses is composed of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates.Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses capable of replication in vertebrate hosts, including, but not limited to, human, primate, bovine, canine, equine, and ovine species. For convenience herein, an ITR located 5' to (upstream of) an expression cassette in a ceDNA vector is referred to as the "5'ITR" or "left ITR" and an ITR located 3' to (downstream of) an expression cassette in a ceDNA vector is referred to as the "3'ITR" or "right ITR."

[0097] "Wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in an AAV or other depend virus that retains, for example, Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring canonical sequence due to degeneracy of the genetic code or drift, and thus WT-ITR sequences encompassed for use herein include WT-ITR sequences as a result of naturally occurring changes (e.g., replication errors) that occur during the production process.

[0098] As used herein, the term "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR" refers to a pair of WT-ITRs in a single ceDNA genome or ceDNA vector, both of which are wild-type ITRs with reverse complement sequences over their entire length. For example, an ITR can be considered to be a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, as long as the changes do not affect the properties and overall three-dimensional structure of the sequence. In some embodiments, the deviating nucleotides represent conservative sequence changes. As a non-limiting example, the sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., as measured using BLAST with default settings) to the canonical sequence and has a symmetric three-dimensional spatial organization with respect to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and a terminal separation site (trs) that pairs with an appropriate Rep protein. Optionally, other functions can be tested, including transgene expression under permissive conditions.

[0099] As used herein, the terms "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably herein and refer to an ITR having a mutation in at least one or more nucleotides compared to the WT-ITR from the same serotype. The mutation may result in a change in one or more of the A-A' and D-D' stem regions and the B-B' and C-C' loops in the ITR (see FIG. 10) and may result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of the WT-ITR of the same serotype.

[0100] As used herein, the term "asymmetric ITR", also referred to as "asymmetric ITR pair", refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are not reverse complements over their entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetric three-dimensional spatial configuration with respect to their cognate ITRs, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair differs in overall geometric structure, i.e., the configuration of their A-A' and D-D' stem regions and B-B' and C-C' loops in three-dimensional space (e.g., one ITR may have a shorter C-C' loop and / or a shorter B-B' loop compared to the cognate ITR). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV ITR, but rather the two ITRs are modified ITRs that have different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' loop, and the other ITR can have a different modification (e.g., a single loop, or a short B-B' loop, etc.) such that they have a different three-dimensional spatial organization compared to their cognate asymmetric mod-ITR.

[0101] As used herein, the term "symmetric ITR" refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are mutated or modified relative to the wild-type Depend virus ITR sequence and are reverse complements over their entire length. Neither ITR is a wild-type ITR (i.e., they are modified ITRs, also referred to as mutant ITRs), and may differ in sequence from the wild-type ITR by nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, 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".

[0102] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs in a single ceDNA genome or ceDNA vector, both of which have reverse complement sequences over their entire length. For example, modified ITRs can be considered substantially symmetric even if there are some nucleotide sequences that deviate from the reverse complement sequence, as long as the changes do not affect the properties and overall shape. As a non-limiting example, the sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) to the canonical sequence and have a symmetric three-dimensional spatial organization to their cognate modified ITRs, such that their three-dimensional structures have the same shape in geometric space. In other words, substantially symmetric modified ITR pairs have the same A-A' and D-D' stem regions and B-B' and C-C' loops organized in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair may have different reverse complement nucleotide sequences but still have the same symmetrical three-dimensional spatial configuration. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., the 5'ITR) of a mod-ITR pair may be from one serotype and the other ITR (e.g., the 3'ITR) may be from a different serotype, but both may have the same corresponding mutations (e.g., if the 5'ITR has a deletion in the C region, the cognate modified 3'ITR of the different serotype has a deletion in the corresponding position of the C' region), so that the modified ITR pair has the same symmetrical three-dimensional spatial configuration. In such an embodiment, each ITR of the modified ITR pair may be derived from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected in the corresponding position of the cognate ITR of the different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs), so long as the differences in nucleotide sequence between the ITRs do not affect the properties or overall shape, and they have substantially the same shape in three-dimensional space.As non-limiting examples, mod-ITRs have at least 95%, 96%, 97%, 98%, or 99% sequence identity to a canonical mod-ITR as determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and have a symmetric three-dimensional spatial organization such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric mod-ITR pair has the same A-A' and D-D' stem regions and B-B' and C-C' loops in three-dimensional space. For example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR has a corresponding deletion of the C-C' loop and has a similar three-dimensional structure of the remaining AA stem region and B-B' loop that are the same shape in geometric space as its cognate mod-ITR.

[0103] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is flanked by A and C. Similarly for the sequence AxBxC. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous or immediately adjacent to the flanked sequence. In one embodiment, the term flanking refers to the terminal repeats at each end of a linear double-stranded ceDNA vector.

[0104] 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 viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0105] 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, a ceDNA spacer region maintains two functional elements at a desired distance for optimal functionality. In some embodiments, a ceDNA spacer region provides or increases the genetic stability of a ceDNA genome, for example, in a plasmid or baculovirus. In some embodiments, a ceDNA spacer region facilitates easy genetic manipulation of a ceDNA genome by providing a convenient location for a cloning site or the like. 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 a ceDNA genome to separate cis-acting elements, for example, inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc., between the terminal separation site and the upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between a polyadenylation signal sequence and the 3' terminal separation site.

[0106] 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 Rep 78 or AAV Rep 68) that, upon binding by the Rep protein, allows the Rep protein to perform 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, SEQ ID NO: 60 of International Patent Application Publication No. WO2019 / 143885), the RBS sequence identified in AAV2. Any known RBS sequence may be used in the embodiments of the present disclosure, 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 (see SEQ ID NO: 60 in WO2019 / 143885), and thus the two known AAV Rep proteins bind directly to the double-stranded oligonucleotide and stably assemble. In addition, the soluble aggregated conformers (i.e., an indefinite number of interrelated Rep proteins) dissociate and bind to oligonucleotides containing the Rep binding site. 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.

[0107] As used herein, the terms "terminal separation 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 a cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. 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, 5'-GGTTGA-3' (SEQ ID NO: 61 in WO2019 / 143885), a hexanucleotide sequence identified in AAV2. Any known TRS sequence can be used in embodiments of the present disclosure, including other known AAV TRS sequences, other naturally known or synthetic TRS sequences such as AGTT, GGTTGG (SEQ ID NO: 63 of WO2019 / 143885), AGTTGG (SEQ ID NO: 64 of WO2019 / 143885), AGTTGA (SEQ ID NO: 65 of WO2019 / 143885), and other motifs such as RRTTRR (SEQ ID NO: 66 of WO2019 / 143885).

[0108] As used herein, the term "ceDNA-plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0109] 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 acting as a shuttle vector for the baculovirus.

[0110] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0111] 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.

[0112] As used herein, the term "closed-ended DNA vector" refers to a capsid-free DNA vector having at least one covalently closed end and at least a portion of the vector having an intramolecular double-stranded structure.

[0113] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector that contains at least one terminal covalently closed end. In some embodiments, the ceDNA contains two covalently closed ends.

[0114] 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.

[0115] 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 renders 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 may include, but are not limited to, restriction endonucleases (whether genomic or extrachromosomal elements) 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 may participate as a factor in another synthetic biological circuit, thereby expanding the scope and complexity of the responsiveness of the biological circuit system.

[0116] Transcriptional regulators refer to transcriptional activators and repressors, including inducible and repressor proteins, that activate or repress 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 a transcriptional promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to a transcriptional promoter and sterically hinder transcription initiation 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.

[0117] As used herein, "carrier" or "excipient" 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-ceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0118] 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 aspects 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 that are 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 the introduction of programmable synthetic biological circuits into a non-cellular system, such as a cell or cell system-free medium, such as a cell extract.

[0119] 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 the promoter itself. 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" boxes 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. A promoter sequence can be bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background.

[0120] 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 start site of the gene they regulate or downstream of the start site of the gene. Enhancers can be located within intronic or exonic regions of unrelated genes.

[0121] 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 can be 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.

[0122] A promoter may be one that is naturally associated with a gene or sequence, which may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons 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.

[0123] 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 isolated from any other prokaryotic, viral, or eukaryotic cell, or 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 of enhancers 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). 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.

[0124] As described herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducer agent. As defined herein, an "inducer" or "inducer agent" can be an endogenous or, usually exogenous, compound or protein 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 inducer 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 the inducible promoter. In some embodiments, an inducible promoter is induced in the absence of a certain agent, such as a repressor. 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.

[0125] The term "promoter" as contemplated herein includes a promoter set, which refers to a system comprising one or more promoters (or promoter sequences) as defined herein and one or more enhancers (or enhancer sequences) as defined herein. As used herein, the term "promoter set" includes sequences in which the promoter and enhancer elements or sequences are separated by a spacer region or sequence that is about 1-50 nucleotides in length (e.g., about 2, 5, 7, 8, 10, 11, 12, 13, 15, 17, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 35, 37, 38, 40, 42, 43, 45, 47, 48, or 50 nucleotides in length).

[0126] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0127] "Operably linked" refers to a juxtaposition in which the components so described are in a relationship that permits them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. An "expression cassette" includes a heterologous DNA sequence operably linked to a promoter or other regulatory sequence sufficient to direct the transcription of a transgene in a ceDNA vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0128] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with a ceDNA vector according to the present disclosure 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. In addition, 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, Latin American, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject has already undergone treatment. In some embodiments, the subject is an embryo, fetus, neonatal, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a human neonatal, a human infant, a human child, a human adolescent, or a human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or a non-human primate embryo, hi some embodiments, the subject is a human embryo.

[0129] As used herein, the term "host cell" includes any cell type that is amenable to transformation, transfection, transduction, etc. with a nucleic acid construct or ceDNA expression vector of the present disclosure. By way of non-limiting example, host cells include isolated primary cells, pluripotent stem cells, CD34 + The host cell may be a human cell, an induced pluripotent stem cell, or any of several immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ, or organism.

[0130] The term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that is not normally found and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that is found in relatively low amounts and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to cause ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is natural to a biological system or cell.

[0131] The term "sequence identity" refers to the relatedness between two nucleotide sequences. For the purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are gap open penalty 10, gap extension penalty 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage, calculated as follows: (identical deoxyribonucleotides x 100) / length of alignment-total number of gaps in alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0132] The term "homology" or "homology" as used herein is defined as the percentage of nucleotide residues in the homology arms that are identical to the nucleotide residues of the corresponding sequence on the target chromosome, after aligning the sequences as necessary and introducing gaps to achieve the maximum sequence identity percentage. Alignment for the purpose of determining the nucleotide sequence homology percentage can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of an arm of homology is considered "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., a genomic sequence) of the host cell.

[0133] The term "heterologous" as used herein refers to a nucleotide or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence that encodes a chimeric polypeptide. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleotide sequence that encodes a fusion variant polypeptide.

[0134] A "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert," can be attached to effect replication of the attached segment in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin and / or final form, but for purposes of this disclosure, "vector," as the term is used herein, generally refers to a ceDNA vector. The term "vector" encompasses any genetic element that can replicate and transfer genetic sequences to a cell when associated with the appropriate control elements. In some embodiments, a vector can be an expression vector or a recombinant vector.

[0135] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, so that it can be maintained in two organisms, for example, human cells for expression, and prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0136] "Recombinant vector" refers to a vector that contains a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in a high copy number of extrachromosomal DNA in a subject, thereby eliminating the potential effects of chromosomal integration.

[0137] The phrase "genetic disease" as used herein refers to a disease that is caused, directly or indirectly, partially or completely, by one or more abnormalities in the genome, particularly a condition that is present from birth. The abnormality may be a mutation, an insertion, or a deletion. The abnormality may affect the coding sequence of the gene or its regulatory sequence. The genetic disease may be, but is not limited to, DMD, hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease.

[0138] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the method or composition, but are open to the inclusion of unspecified elements, whether essential or not.

[0139] 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 characteristics of the embodiment. The use of "comprises" indicates inclusion rather than limitation.

[0140] 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.

[0141] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the present disclosure.

[0142] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will be apparent to one of ordinary skill in the art upon reading this disclosure, and so forth. Similarly, the word "or" is intended to include "and" unless the context clearly dictates 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 denote a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0143] 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 instances by the term "about". When used in relation to percentages, the term "about" can mean ±1%. The present disclosure is further illustrated by the following examples, but the scope of the present disclosure should not be limited thereto.

[0144] Grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed herein to include the modified group, thus satisfying the description of all Markush groups used in the appended claims.

[0145] In some embodiments of any of the aspects, the disclosure described herein does not pertain to human cloning processes, processes for correcting the genetic identity of human germ lines, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, and processes for correcting the genetic identity of animals resulting from such processes.

[0146] Other terms are defined herein within the description of various aspects of the disclosure.

[0147] All patents and other publications, including literature references, issued patents, published patent applications, unpublished patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0148] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Certain embodiments and examples of the present disclosure are described herein for illustrative purposes, however, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as appropriate to provide further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, some changes can be made to protein structures without affecting the type or amount of biological or chemical action, due to considerations of biological functional equivalence. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0149] Particular elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages, to be within the scope of the present disclosure.

[0150] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It is to be understood that the present disclosure is not limited to the specific methodology, protocols, and reagents described herein, and as such may vary. The terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0151] II. Cell-free synthesis of DNA vectors The technology described herein is generally directed to methods for producing DNA vectors in the absence of cells or cell lines. Thus, the resulting vectors have fewer impurities than comparable vectors made using traditional cell-based production methodologies, which may translate into better in vivo expression that is sustained for a longer duration after administration (see, e.g., FIG. 16B). As exemplified herein, this cell-free synthesis is also scalable from small-scale reactions (~1 mL) to large-scale (>40, 100, 200, 500, 1,000 mL) without further compromising purity (see, e.g., FIG. 15), thus allowing vectors to be prepared in large quantities for therapeutic use. Furthermore, cell-based methods can take several weeks for vectors to be produced, whereas the cell-free methods described herein produce vectors in less than a week, such as 2-4 days, depending on scale.

[0152] According to some embodiments, the cell-free methods described herein involve >1000-fold rolling circle and multiple strand displacement (MSD) amplification of a DNA plasmid template, followed by conversion of the resulting products into ceDNA molecules using type II endonuclease, ligase, ITR oligos, and exonuclease enzymes.

[0153] According to one aspect, the disclosure provides a method for producing a closed-ended DNA (ceDNA) vector, the method comprising: (a) contacting a double-stranded DNA construct having a sense strand and an antisense strand with at least a first restriction endonuclease and at least a second restriction endonuclease, the construct comprising a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette, wherein the first restriction endonuclease cleaves the double-stranded DNA construct at the first cleavage site. and a second restriction endonuclease is capable of cleaving the double-stranded DNA construct at a second cleavage site, wherein contacting the double-stranded DNA construct with the first restriction endonuclease and the second restriction endonuclease releases an insert having a first end comprising a first single-stranded overhang and a second end comprising a second single-stranded overhang; (b) ligating the first end to a first oligonucleotide comprising one or more hairpin structures; and (c) ligating the second end to a second oligonucleotide comprising one or more hairpin structures, thereby producing a ceDNA vector.

[0154] According to some embodiments, the first oligonucleotide comprises an inverted terminal repeat (ITR). According to further embodiments, the second oligonucleotide comprises an ITR. According to other embodiments, the first oligonucleotide and the second oligonucleotide are different. According to some other embodiments, the first oligonucleotide and the second oligonucleotide are the same.

[0155] An exemplary embodiment of the cell-free synthesis method for preparing ceDNA vectors is illustrated in Figure 4. Briefly, a transgene expression cassette (shaded) is excised from a double-stranded DNA construct using at least one restriction endonuclease, and the insert is subsequently ligated with an inverted terminal repeat (ITR) oligonucleotide to form ceDNA. The ITR oligonucleotide is a single-stranded oligonucleotide that self-anneals to form an ITR-like three-dimensional structure. The restriction endonucleases used in the methods described herein, such as but not limited to type IIS restriction endonucleases, cut DNA at different sites rather than within the recognition site. These restriction endonucleases used in the cell-free synthesis methods disclosed herein also recognize non-palindromic nucleotide sequences, such that the recognition sequence for the enzyme (which is also the binding site) is encoded only on one strand (see, for example, Figure 5). Thus, cleavage by this class of restriction endonucleases is directional and occurs either upstream or downstream of the recognition site, but not within the recognition site itself, unlike other restriction endonucleases most frequently used in molecular biology, such as EcoRI (see FIG. 5). The strand encoding the recognition sequence determines which side of the sequence (i.e., downstream or upstream) is cleaved. In summary, the intrinsic activity of the restriction endonucleases used in the methods described herein allows any sequence within a given distance from a specific recognition site to be cleaved by the restriction endonuclease, thereby allowing any overhang sequence to be generated. Digestion by a special restriction endonuclease generates sticky overhangs at both the 5' and 3' ends of the excised insert that are compatible with the overhang of the ITR oligonucleotide. In other words, the design of the ITR oligonucleotide and the insert overhang drives high specificity of the ligation process, such that the ITR oligonucleotide overhang and the insert overhang are compatible with each other. Once ligated, the desired ceDNA product is not susceptible to digestion by the restriction endonuclease because the recognition site is not regenerated.However, in the situation where the excised insert and plasmid fragment are religated into the original construct, the recognition site is regenerated, thus allowing the construct to be excised.

[0156] Due to the inherent activity of the restriction endonucleases used in the cell-free synthesis methods described herein, the generation of unique overhangs on the insert ends, and the design of unique overhangs on the ITR oligonucleotides that drive high specificity of the subcloning process, digestion and ligation can occur in a single reaction vessel without the need to purify the digestion products prior to ligation. As shown in Figure 4, digestion / ligation is followed by treatment with an exonuclease to degrade open-ended DNA fragments and intermediates. In some embodiments, restriction endonuclease digestion, ligation, and exonuclease degradation can occur in a single reaction vessel and all reactions can occur simultaneously.

[0157] In addition, the inherent activity of the restriction endonucleases used in the cell-free synthesis methods described herein allows for the directionality of ligation reactions utilizing more than one ITR oligonucleotide, thereby enabling the preparation of ceDNA with asymmetric ITRs.

[0158] General cell-free synthesis production method Disclosed herein is a process for the synthesis of closed-ended DNA vectors that does not require the use of any microbiological steps. In some embodiments, the process allows for the synthesis of closed-ended DNA vectors in a system that uses an enzymatic cleavage step using a restriction endonuclease and a ligation step to generate closed-ended DNA vectors. In nearly all embodiments, the synthesis system for DNA vector production is a cell-free system.

[0159] It will be understood by those skilled in the art that one or more of the enzymes or oligonucleotide components used in the synthetic production method can be produced from cells and used in the method of the present disclosure in purified form.Thus, in some embodiments, the steps in the synthetic production method itself are cell-free.However, base materials such as double-stranded DNA constructs and ITR oligonucleotides, and enzymes such as restriction endonucleases and ligases can be produced using cell-based methods and techniques.

[0160] In one embodiment, the restriction endonuclease and / or ligation competent proteins can be expressed or provided from an expression vector in a cell, e.g., a bacterial cell. In one embodiment, there can be a cell, such as a bacterial cell, that contains an expression vector expressing one or more of the restriction endonuclease or ligase enzymes. Thus, while the methods disclosed herein are primarily directed to cell-free synthetic methods for producing the DNA vectors disclosed herein, in one embodiment, synthetic production methods are also encompassed in which a cell, e.g., a bacterial cell, rather than an insect cell, is present and can be used to express one or more of the enzymes required in the method. In such an embodiment, the cell expressing the restriction endonuclease and / or ligation competent protein is not an insect cell. In all embodiments in which a cell is present and expresses one or more restriction endonuclease or ligation competent proteins, the cell does not replicate the closed-end DNA vector. In other words, the intracellular machinery of the cell does not replicate or is not involved in replicating the DNA vector.

[0161] In some embodiments, the synthesis of the DNA vectors (e.g., ceDNA vectors) described herein is carried out in an in vitro cell-free process starting with either a double-stranded DNA construct or one or more oligonucleotides. The double-stranded DNA construct or one or more oligonucleotides are cleaved with a restriction endonuclease and ligated to form a DNA molecule. In some embodiments, the oligonucleotides can be chemically synthesized, thus avoiding the use of large starting templates that code for the entire desired sequence, which typically must be propagated in bacteria. Once the desired DNA sequence is synthesized, it can be cleaved and ligated with other oligonucleotides as disclosed herein. The use of multiple oligonucleotides in the generation of closed-end DNA vectors using the methods disclosed herein allows for a modular approach to DNA vector generation, allowing for the adjustment and / or specific selection of terminal repeats, e.g., ITRs, and spacing of terminal repeats, as well as the selection of heterologous nucleic acid sequences within the synthetically produced closed-end DNA vector.

[0162] Cell-free synthetic production of DNA vectors Certain methods for the production of ceDNA vectors containing various ITR configurations using cell-based methods are described in Example 1 of International Patent Application Publication Nos. WO2019 / 051255 and WO2019 / 113310, the contents of which are incorporated herein by reference in their entireties.

[0163] In contrast, the methods provided herein relate to synthetic production methods, e.g., in some embodiments, cell-free production methods, also referred to herein as "synthetic closed-end DNA vector production" or "synthetic production."

[0164] In one aspect, the closed-end DNA vector is generated by excising a transgene expression cassette from a double-stranded DNA construct, followed by ligating the ends of the insert to a first oligonucleotide comprising one or more hairpin structures and a second oligonucleotide comprising one or more hairpin structures to form a ceDNA. In some embodiments, each of the oligonucleotides independently comprises one, two, three, four, or more stem-loop regions. In some embodiments, each of the oligonucleotides independently comprises two or three stem-loop regions. In some embodiments, the first oligonucleotide comprising one or more hairpin structures and the second oligonucleotide comprising one or more hairpin structures are each single-stranded oligonucleotides that self-anneal to form a three-dimensional structure. In further embodiments, the three-dimensional structure is a T-shaped or Y-shaped stem-loop structure.

[0165] In another aspect, the closed-end DNA vector is generated by excising the transgene expression cassette from a double-stranded DNA construct, followed by ligating the ends of the insert to ITR oligonucleotides to form ceDNA. Ligation can be accomplished by a ligase (e.g., T4 ligase) or AAV Rep protein. In one embodiment, the reaction mixture is not purified prior to ligation. In such an embodiment, excision (e.g., by one or more restriction endonucleases) of the transgene expression cassette and ligation are performed simultaneously in a single reaction vessel. In an alternative embodiment, the reaction mixture is purified prior to ligation.

[0166] The resulting closed DNA vectors prepared by the cell-free synthesis methods described herein comprise at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the monomeric species of the vector. The resulting closed DNA vectors prepared by the cell-free synthesis methods described herein comprise less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the submonomer species of the vector.

[0167] As used herein, the term "submonomer species" is meant to refer to a complex of nucleic acid that is generally smaller than the monomeric form of a therapeutic nucleic acid defined herein, such as a ceDNA genome, a ceDNA vector, an AAV genome, or an AAV vector, as determined, for example, by ion exchange chromatography (IEX). The terms "submonomer species" and "submonomer DNA" also encompass dimers formed by two submonomer units, and multimers formed by three or more submonomer units. The formation of dimers and multimers is unstable and may therefore be transient, and dimers and multimers may eventually degrade to their submonomer forms. The amount and concentration of submonomer DNA may be quantified using capillary electrophoresis, such as, for example, using ion exchange high-performance liquid chromatography (IEX-HPLC) for peak quantification, and / or using a chip-based capillary electrophoresis machine such as a Bioanalyzer, and may be expressed in mass units (e.g., μg, ng, pg) or mass / volume units.

[0168] In one embodiment, the double-stranded DNA construct is selected from a bacmid, a plasmid, a minicircle, or a linear double-stranded DNA molecule. In such an embodiment, the double-stranded DNA construct comprises at least, in order from 5' to 3', a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette. In one embodiment, the first restriction endonuclease is capable of cleaving the double-stranded DNA construct at the first cleavage site, and the second restriction endonuclease is capable of cleaving the double-stranded DNA construct at the second cleavage site, and contacting the double-stranded DNA construct with the first restriction endonuclease and the second restriction endonuclease releases an insert having a first end comprising a first single-stranded overhang and a second end comprising a second single-stranded overhang. In further embodiments, the first and second restriction endonucleases are different restriction endonucleases. In other embodiments, the first and second restriction endonucleases are the same restriction endonucleases. In another embodiment, the double-stranded DNA construct comprises at least, in the order of 5' to 3' direction, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, a first partial ITR, a transgene expression cassette, a second partial ITR, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette. The double-stranded DNA construct is contacted with at least one restriction endonuclease capable of cleaving the construct at the first and second cleavage sites to release an insert having single-stranded overhangs at the 5' and 3' ends (i.e., sticky ends) of the insert. The ends of these inserts are then ligated to a first inverted terminal repeat (ITR) oligonucleotide and a second ITR oligonucleotide to form the ceDNA vector.In one embodiment, one or both of the single-stranded overhangs at the 5' and 3' ends of the insert are 5' overhangs. In one embodiment, one or both of the single-stranded overhangs at the 5' and 3' ends of the insert are 3' overhangs. In one embodiment, these overhangs are about 1 to about 30 nucleotides in length, for example, about 1 to about 25 nucleotides, or about 1 to about 20 nucleotides, or about 1 to about 18 nucleotides, or about 1 to about 15 nucleotides, or about 1 to about 12 nucleotides, or about 1 to about 10 nucleotides, or about 1 to about 8 nucleotides, or about 2 to about 8 nucleotides, or about 2 to about 7 nucleotides, or about 2 to about 6 nucleotides, or about 1 nucleotide, or about 2 nucleotides, or about 3 nucleotides, or about 4 nucleotides, or about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides in length.

[0169] Use of restriction endonucleases which recognize non-palindromic nucleotide sequences and have cleavage sites distinct from their recognition and binding sites Of note, the restriction endonucleases used in the synthesis methods provided herein recognize non-palindromic nucleotide sequences. As used herein, the term "non-palindromic" refers to having a different 5'→3' nucleotide sequence between the sense and antisense strands when referring to a double-stranded polynucleotide or oligonucleotide, whereas the term "palindromic" refers to having an identical 5'→3' nucleotide sequence between the sense and antisense strands when referring to a double-stranded polynucleotide or oligonucleotide.

[0170] Thus, as illustrated in Figure 5, such restriction endonucleases recognize double-stranded polynucleotides or oligonucleotides that have different 5'→3' nucleotide sequences between the sense and antisense strands. This means that the recognition sequence for the restriction endonuclease is encoded on only one of the strands, such as 5'-GGTCTC-3' in the example illustrated in Figure 5. Another distinguishing feature for the restriction endonucleases used in the synthetic methods described herein is that the enzymes cleave DNA at cleavage sites that are either upstream or downstream of the recognition sequence, but not within the recognition site itself. The strand that encodes the recognition site determines which side of the recognition sequence (i.e., downstream or upstream) is cleaved.

[0171] In certain embodiments, the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site are separate and distinct from each other located upstream of the transgene expression cassette. The first cleavage site is about 1 to 35 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct, for example, about 1 to about 22 nucleotides away, or about 1 to about 20 nucleotides away, or about 1 to about 15 nucleotides away, or about 1 to about 12 nucleotides away, or about 1 to about 10 nucleotides away, or about 1 to about 8 nucleotides away, or about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct.

[0172] In certain embodiments, the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site are separate and distinct from each other located downstream of the expression cassette. The second cleavage site is about 1 to 35 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct, for example, about 1 to about 22 nucleotides away, or about 1 to about 20 nucleotides away, or about 1 to about 15 nucleotides away, or about 1 to about 12 nucleotides away, or about 1 to about 10 nucleotides away, or about 1 to about 8 nucleotides away, or about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct.

[0173] A single restriction endonuclease can target both the first and second non-palindromic restriction endonuclease recognition sites and their corresponding cleavage sites. Alternatively, two different restriction endonucleases target both the first and second non-palindromic restriction endonuclease recognition sites and their corresponding cleavage sites.

[0174] Type IIS restriction endonucleases In one embodiment, the restriction endonucleases used in the synthetic methods provided herein that recognize non-palindromic nucleotide sequences and cleave DNA outside of the recognition site are type IIS restriction endonucleases. Non-limiting examples of type IIS restriction endonucleases include AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrD ... I, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers of any of the above. Isoschizomers are pairs of restriction endonucleases that are specific for the same recognition sequence. For example, BcoDI and BsmAI are isoschizomers of each other, both specific for the recognition sequence of 5'-GTCTC-3'. In one embodiment, the type IIS endonuclease is selected from BbsI, BsaI, Esp3I, and SapI, and isoschizomers thereof. In one embodiment, the Type IIS endonuclease is BbsI or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is BsaI or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is BbsI or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is Esp3I or an isoschizomer thereof. In one embodiment, the Type IIS endonuclease is SapI or an isoschizomer thereof.

[0175] ITR oligonucleotides The first and second ITR oligonucleotides to which the 5' and 3' ends of the insert are ligated in the cell-free synthesis method disclosed herein are each a single-stranded oligonucleotide that self-anneals to form a three-dimensional structure, such as an ITR three-dimensional structure such as a hairpin structure or a T-shaped or Y-shaped stem-loop structure. In one embodiment, one or both of the ITR oligonucleotides are synthetic or are synthesized.

[0176] In some embodiments, in addition to the three-dimensional ITR structure, the ITR oligonucleotides each self-anneal to further form a single-stranded overhang at either the 5' or 3' end of the oligonucleotide. In such embodiments, the single-stranded overhang of the insert is ligated to the single-stranded overhang of the ITR oligonucleotide. The ITR oligonucleotide overhang is about 1 to about 30 nucleotides in length, for example, about 1 to about 25 nucleotides, or about 1 to about 20 nucleotides, or about 1 to about 18 nucleotides, or about 1 to about 15 nucleotides, or about 1 to about 12 nucleotides, or about 1 to about 10 nucleotides, or about 1 to about 8 nucleotides, or about 2 to about 8 nucleotides, or about 2 to about 7 nucleotides, or about 2 to about 6 nucleotides, or about 1 nucleotide, or about 2 nucleotides, or about 3 nucleotides, or about 4 nucleotides, or about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides in length. In certain embodiments, the ITR oligonucleotide overhang comprises the 5' to 3' nucleotide sequence of CTCT, CTCA, CACT, CTC, or GCT.

[0177] The overhangs of the first ITR oligonucleotide and the second ITR oligonucleotide comprise 5'→3' nucleotide sequences that are non-complementary to each other. In one embodiment, the overhangs of the first ITR oligonucleotide and the second ITR oligonucleotide comprise or have the same sequence, i.e., the same 5'→3' nucleotide sequence, and in a further embodiment, the first ITR oligonucleotide and the second ITR oligonucleotide are the same oligonucleotide. In such an embodiment, the overhangs at the 5' and 3' ends of the insert comprise or have the same 5'→3' nucleotide sequence. The overhangs of the ITR oligonucleotide are complementary to either and both of the overhangs of the insert. Such an embodiment, in which a single ITR oligonucleotide is used in the cell-free synthesis of ceDNA, is useful for preparing ceDNA with symmetric ITRs as defined herein.

[0178] In an alternative embodiment, the overhangs of the first ITR oligonucleotide and the second ITR oligonucleotide comprise or have different sequences, i.e., different 5'→3' nucleotide sequences, and thus the first ITR oligonucleotide and the second ITR oligonucleotide are different oligonucleotides. In such an embodiment, the overhangs at the 5' and 3' ends of the insert comprise or have different 5'→3' nucleotide sequences. Each of the overhangs of the ITR oligonucleotides is complementary to only one of the overhangs of the insert.

[0179] With respect to the length of the ITR oligonucleotides, in some embodiments, the ITR oligonucleotides are each about 40 nucleotides to about 75 nucleotides in length, e.g., about 40 nucleotides to about 72 nucleotides, or about 40 nucleotides to about 70 nucleotides, or about 40 nucleotides to about 68 nucleotides, or about 40 nucleotides to about 65 nucleotides, or about 40 nucleotides to about 75 nucleotides, or about 45 nucleotides to about 72 nucleotides, or about 45 nucleotides to about 70 nucleotides, or about 45 nucleotides to about 68 nucleotides, or about 45 nucleotides to about 65 nucleotides. In one embodiment, the first ITR oligonucleotide and the second ITR oligonucleotide each and independently comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 (see Table 1).

[0180] In one embodiment, the first ITR oligonucleotide and the second ITR oligonucleotide each and independently comprise a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 (see Table 1). In one embodiment, the first ITR oligonucleotide and the second ITR oligonucleotide each and independently consist of a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 (see Table 1).

[0181] [Table 1]

[0182] To enhance ligation efficiency, the 5' ends of ITR oligonucleotides, including but not limited to, the ITR oligonucleotides of Table 1, can be phosphorylated.

[0183] In one embodiment, the first ITR and the second ITR each comprise a hairpin structure and / or a T-shaped or Y-shaped stem-loop structure. In another embodiment, the first ITR and the second ITR each comprise a T-shaped or Y-shaped stem-loop structure. In another embodiment, the T-shaped or Y-shaped stem-loop structure comprises a stem comprising an A-A' and D-D' stem region and two B-B' and C-C' loops. In another embodiment, one or both of the first ITR and the second ITR are adeno-associated virus (AAV) ITRs or ITRs derived from AAV. In another embodiment, one or both of the first ITR and the second ITR are wild-type ITRs. In another embodiment, both the first ITR and the second ITR are wild-type ITRs. In another embodiment, one or both of the first ITR and the second ITR are modified ITRs. In another embodiment, the first ITR and the second ITR are symmetrical or substantially symmetrical to each other. In another embodiment, the first ITR and the second ITR are asymmetric ITRs.

[0184] In some embodiments, the T-shaped or Y-shaped stem-loop structure (e.g., single stem+2 loop or single stem+2 loop) of the first ITR oligonucleotide and the second ITR oligonucleotide is at least about 4 base pairs (nucleotides) long, e.g., from about 4 base pairs to about 30 base pairs, or from 4 base pairs to about 25 base pairs, or from 4 base pairs to about 22 base pairs, or from 4 base pairs to about 20 base pairs, or from 4 base pairs to about 18 base pairs, or from 4 base pairs to about 15 base pairs, or from 4 base pairs to about 12 base pairs, or from 4 base pairs to about 10 base pairs. or a stem region that is about 4 base pairs, or 4 base pairs to about 8 base pairs, or 4 base pairs to about 7 base pairs, or 4 base pairs to about 6 base pairs, or 6 base pairs to about 8 base pairs, or about 4 base pairs, or about 5 base pairs, or about 6 base pairs, or about 7 base pairs, or about 8 base pairs, or about 9 base pairs, or about 10 base pairs, or about 11 base pairs, or about 12 base pairs, or about 13 base pairs, or about 14 base pairs, or about 15 base pairs (i.e., the A-A' stem region, the D-D' stem region, or both the A-A' and D-D' stem regions shown in FIG. 10). In one embodiment, this stem region length does not include the overhang length.

[0185] Partial ITR and spacer In some embodiments, the ITR oligonucleotides form the complete ITR of the ceDNA product at both ends. In other embodiments, the ITR oligonucleotides partially form the ITR of the ceDNA, but the remaining continuous sequence of the ITR is found in the partial ITR on the insert. Thus, in some embodiments, the double-stranded DNA construct from which the transgene expression cassette is excised in the cell-free synthesis method disclosed herein further comprises at least a first partial ITR and a second partial ITR, each flanking the transgene expression cassette. In one embodiment, the first partial ITR is upstream of the transgene expression cassette and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site. The second partial ITR is downstream of the transgene expression cassette and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site. In one embodiment, the first cleavage site is adjacent to the first partial ITR and the second cleavage site is adjacent to the second partial ITR (i.e., there is no spacer between the cleavage site and the partial ITR).

[0186] In certain embodiments, the double-stranded DNA construct or the excised insert further comprises one or more spacer regions. In one embodiment, the double-stranded DNA construct or insert further comprises a first spacer between the first partial ITR and the transgene expression cassette. In another embodiment, the double-stranded DNA construct further comprises a second spacer between the second partial ITR and the transgene expression cassette. Each spacer region or sequence is about 1-50 nucleotides in length, for example, about 2, 5, 7, 8, 10, 11, 12, 13, 15, 17, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 35, 37, 38, 40, 42, 43, 45, 47, 48, or 50 nucleotides. In some embodiments, the spacer between the first (or left) partial ITR or the second (or right) partial ITR and the transgene expression cassette is selected from spacers comprising the sequences shown in Table 2. According to some embodiments, the spacer comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40. According to some embodiments, the spacer consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40.

[0187] [Table 2]

[0188] In some aspects, the disclosure provides a method of producing a double-stranded DNA construct from a plasmid template via rolling circle amplification, comprising: (a) contacting the plasmid template with a thermostable polymerase having strand displacement activity, wherein the ratio of plasmid template concentration (in ng / μl) to polymerase concentration (in U / μl) is greater than about 1; (b) contacting the plasmid template with oligonucleotide primers and dNTPs; and (c) incubating the plasmid template, polymerase, oligonucleotide primers, and dNTPs at a temperature of about 40° C. or less for a period of at least about 5 hours, thereby producing a double-stranded DNA construct. In some embodiments, the ratio of plasmid template concentration (in ng / μl) to polymerase concentration (in U / μl) is greater than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20. In some embodiments, the plasmid template concentration is about 0.01ng / μl, about 0.05ng / μl, about 0.1ng / μl, about 0.15ng / μl, about 0.2 ng / μl, about 0.21ng / μl, about 0.22ng / μl, about 0.23ng / μl, about 0.24ng / μl, about 0.2ng / μl, about 0.26ng / μl , about 0.27ng / μl, about 0.28ng / μl, about 0.29ng / μl, about 0.3ng / μl, about 0.35ng / μl, about 0.4ng / μl, about 0.45 ng / μl, about 0.5ng / μl, about 0.6ng / μl, about 0.7ng / μl, about 0.8ng / μl, about 0.9ng / μl, or about 1.0ng / μl. In other embodiments, the polymerase concentration is about 0.01 U / μl, about 0.02U / μl, about 0.03U / μl, about 0.04U / μl, about 0.05U / μl, about 0.06U / μl, about 0.07U / μl, about 0.08U / μl, about 0.09U / μl, about 0.1U / μl, about 0.15U / μl, about 0.2U / μl, about 0.25U / μl, about 0.3U / μl, about 0.35U / μl, about 0.4U / μl, or about 0.45U / μl.In further embodiments, the temperature in step (c) is less than about 40°C, about 39°C, about 38°C, about 37°C, about 36°C, about 35°C, about 34°C, about 33°C, about 32°C, about 31°C, about 30°C, about 29°C, about 28°C, about 27°C, about 26°C, about 25°C, about 24°C, about 23°C, about 22°C, or about 21°C. In some embodiments, the period is at least about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours. In some embodiments, the time period is less than about 6 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours. In some embodiments, the plasmid template concentration is about 0.25 ng / μl, the temperature is about 30° C., the polymerase concentration is about 0.05 U / μl, and the time period is about 18-26 hours. In further embodiments, the oligonucleotide primer concentration is less than about 50 μM. In other embodiments, the oligonucleotide primer concentration is at least about 10 μM. In further embodiments, the oligonucleotide primer concentration is at least about 10 μM and less than about 50 μM. In further embodiments, the thermostable polymerase is Phi29 DNA polymerase or a derivative or variant thereof. In other further embodiments, the thermostable polymerase is EQUIPHI29™. In other embodiments, the method is performed in a total reaction volume of at least about 100 μl.In other further embodiments, the method comprises at least about 100 μl, about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 100 ml ...15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 6 In some embodiments, the reaction is carried out in a total reaction volume of about 0 ml, about 95 ml, about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml, about 800 ml, about 900 ml, about 1 L, about 2 L, about 3 L, 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, or about 1000 L.

[0189] In some embodiments, the method is carried out in a reaction vessel having a volume of at least twice the total reaction volume.In some embodiments, the oligonucleotide primer hybridizes to the backbone sequence in the plasmid template.In some embodiments, the oligonucleotide primer is a universal primer.

[0190] In some embodiments, the dNTP concentration is about 4 mM.

[0191] Isolation and purification Methods for generating and isolating exemplary closed-end DNA vectors, ceDNA vectors, are described herein. For example, the closed-end DNA vectors, e.g., ceDNA vectors, produced by the synthesis methods described herein can be harvested or collected at an appropriate time point after the final ligation reaction and optimized to achieve high-yield production of ceDNA vectors. The closed-end DNA vectors, e.g., ceDNA vectors, can be purified by any means known to those skilled in the art for purifying DNA. In one embodiment, the ceDNA vectors are purified as DNA molecules. In general, any art-known nucleic acid purification method can be employed, as well as commercially available DNA extraction kits.

[0192] Alternatively, purification can be carried out by subjecting the reaction mixture to chromatographic separation.As one non-limiting example, this process can be carried out by loading the reaction mixture onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acid, then eluting (e.g., with 1.2M NaCl solution), and carrying out further chromatographic purification on a gel filtration column (e.g., 6 Fast Flow GE).The DNA vector, e.g., ceDNA vector, is then recovered, e.g., by precipitation.

[0193] The presence of a ceDNA vector can be confirmed by digesting vector DNA isolated from a cell with a restriction enzyme that has a single recognition site on the DNA vector and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic linear and continuous DNA compared to linear and discontinuous DNA.

[0194] In some embodiments, the closed-end DNA vectors produced by the synthetic production methods disclosed herein can be delivered to target cells in vitro or in vivo by various suitable methods as discussed herein. The vector alone can be applied or injected. The vector can be delivered to cells without the aid of transfection reagents or other physical means. Alternatively, the vector can be delivered using transfection reagents or other physical means that facilitate DNA entry into cells, such as liposomes, alcohol, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, microinjection, etc.

[0195] Circular DNA vectors produced using synthetic production methods Provided herein are various methods for in vitro production of DNA molecules and closed-end DNA vectors.In some embodiments, the closed-end DNA vector is the ceDNA vector described herein.In alternative embodiments, the closed-end DNA vector is, for example, a dumbbell DNA vector or a dogbone DNA vector (see, for example, WO2010 / 0086626, the entirety of which is incorporated herein by reference).

[0196] III. Common ceDNA Vectors In some embodiments, the closed-end DNA vector produced using the synthesis process described herein is a ceDNA vector, including a ceDNA vector capable of expressing a transgene. The ceDNA vector described herein is not limited by size, thereby allowing, for example, the expression of all components required for the expression of a transgene from a single vector. 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 vector has covalently closed ends and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), for example, at 37° C. for 1 hour or more.

[0197] In general, a ceDNA vector produced using the synthetic process described herein comprises a transgene expression cassette, a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette. In some embodiments, a ceDNA vector produced using the synthetic process described herein comprises, in a 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., a transgene expression cassette described herein), and a second AAV ITR. The ITRs are selected from either: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR); (ii) two modified ITRs (e.g., an asymmetric modified ITR), where the mod-ITR pair have different three-dimensional spatial configurations relative to each other; or (iii) a symmetric or substantially symmetric WT-WT ITR pair, where each WT-ITR has the same three-dimensional spatial configuration; or (iv) a symmetric or substantially symmetric modified ITR pair, where each mod-ITR has the same three-dimensional spatial configuration.

[0198] Methods and compositions comprising ceDNA vectors produced using the synthesis process described herein are included herein, and may further include delivery systems, such as, but not limited to, liposomal nanoparticle delivery systems. Non-limiting exemplary liposomal nanoparticle systems included for use are disclosed herein. In some aspects, the present disclosure provides lipid nanoparticles comprising ceDNA and ionized lipids. For example, lipid nanoparticle formulations made and loaded with ceDNA obtained by the process are disclosed in International Patent Application Publication No. WO2019 / 051289, the entirety of which is incorporated herein by reference.

[0199] The ceDNA vectors produced using the synthetic process described herein are free of packaging constraints imposed by the limited space within the viral capsid, which allows for the insertion of control elements, such as regulatory switches as disclosed herein, large transgenes, multiple transgenes, etc.

[0200] 1A-1G of International Patent Application Publication No. WO2019 / 143885 show schematic diagrams of non-limiting exemplary ceDNA vectors or sequences of corresponding ceDNA plasmids. A ceDNA vector does not contain a capsid and can be obtained from a plasmid encoding a first ITR, an expression cassette containing a transgene, and a second ITR in that order. The expression cassette can include one or more regulatory sequences that enable and / or control the expression of the transgene, for example, the expression cassette can include one or more of an enhancer / promoter, an ORF reporter such as luciferase or a transgene, a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA), in that order.

[0201] 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 for the transgene. In some embodiments, the ceDNA vector may include additional components for regulating expression of the transgene, such as a regulatory switch, described herein in the section entitled "Regulatory Switches," for controlling and regulating 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.

[0202] The 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 of about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-50,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-75,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-10,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 1000-10,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-5,000 nucleotides in length. ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and therefore allow the delivery of large expression cassettes to result in efficient transgenes. In some embodiments, ceDNA vectors lack prokaryotic cell-specific methylation.

[0203] A ceDNA expression cassette may contain, for example, an expressible exogenous sequence (e.g., an open reading frame) or transgene that encodes a protein that is absent, inactive, or insufficiently active in a recipient subject, or a gene that encodes a protein with a desired biological or therapeutic effect. A transgene may encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, an expression cassette may contain any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect where overexpression is considered within the scope of this disclosure.

[0204] The expression cassette may include any transgene useful for treating a disease or disorder of a subject. The ceDNA vectors produced using the synthetic process described herein can be used to deliver and express any gene of interest to a subject, including, but not limited to, exogenous genes and nucleotide sequences, including nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miR, etc.), as well as viral sequences in the genome of a subject, such as HIV viral sequences. Preferably, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or immunogenic polypeptides. In certain embodiments, the ceDNA vectors are useful for expressing any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAi's, antisense oligonucleotides, antisense polynucleotides, or RNAs (coding or non-coding, e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)), antibodies, fusion proteins, or any combination thereof.

[0205] Expression cassettes may also encode polypeptides, sense or antisense oligonucleotides, or RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). Expression cassettes may also include exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0206] The sequences provided in the expression cassette, the expression construct of the ceDNA vector described herein, can be codon optimized for the target 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, e.g., mouse or human. Different species exhibit a particular bias for certain codons of certain amino acids. 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., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another public database.

[0207] In some embodiments, the transgene expressed by the ceDNA vector is a therapeutic gene, hi some embodiments, the therapeutic gene is an antibody, or an antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment.

[0208] In particular, a therapeutic gene is one or more therapeutic agents including, but not limited to, proteins, polypeptides, peptides, enzymes, 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 therapeutic genes are described herein in the section entitled "Methods of Treatment."

[0209] There are many structural features of ceDNA vectors that are different from plasmid-based expression vectors. The ceDNA vectors produced by the synthetic methods herein may have one or more of the following features: lack of original (i.e., uninserted) bacterial DNA, lack of a prokaryotic replication origin, are self-contained (i.e., do not require any sequences other than the two ITRs containing Rep binding and terminal separation sites (RBS and TRS) and no 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 associated with production in a given cell type and 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, in 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.

[0210] The ceDNA vectors produced by the synthesis methods provided herein preferably have a linear, continuous structure, as determined by restriction enzyme digestion assays, rather than a discontinuous structure (see, e.g., Figures 11 and 12). A linear, continuous structure is believed to be more stable against attack by cellular endonucleases, while at the same time less likely to recombine and cause mutagenesis. Thus, a linear, continuous structure ceDNA vector 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, may be produced without DNA base methylation of prokaryotic cell types. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (specifically 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.

[0211] The use of the ceDNA vectors described herein has several advantages over plasmid-based expression vectors, including, but not limited to, the following: 1) plasmids contain bacterial DNA sequences and are subject to prokaryote-specific methylation (e.g., methylation of 6-methyladenosine and 5-methylcytosine), whereas capsid-free AAV vector sequences are of eukaryotic origin and are not subject to prokaryote-specific methylation; as a result, capsid-free AAV vectors are less likely to induce inflammatory 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 avoid degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements (i.e., ITRs), which can be designed to confer resistance to nucleases and target and deliver to the nucleus. The minimal defining elements essential for ITR function are the Rep binding site (for AAV2, SEQ ID NO: 60 in International Patent Application Publication No. WO2019 / 143885), and the terminal separation site (TRS, for AAV2 5'-AGTTGG-3' (SEQ ID NO: 64 in WO2019 / 143885)) plus a variable palindrome sequence allowing hairpin formation; and 4). ceDNA vectors do not have the over-representation of CpG dinucleotides often found in prokaryotic-derived plasmids that are reported to bind members of the Toll-like family of receptors and induce 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.

[0212] Unique Junction Array The ceDNA vector prepared using the cell-free synthesis method described herein contains nucleotide sequences at the ligation junction between the insert and the ITR oligonucleotide on both ends that are unique to ceDNA. Such junction sequences are not found in the ITR oligonucleotides or in the double-stranded DNA construct from which the transgene expression cassette is excised. The presence of such junction sequences, as revealed in DNA sequencing analysis (see, for example, Figure 14), is useful for confirming the success of the ligation reaction.

[0213] Thus, another aspect of the present disclosure is directed to ITR nucleotide sequences found in ceDNA vectors, ceDNA vectors comprising these ITR nucleotide sequences, as well as compositions, host cells, and transgenic animals comprising such ceDNA vectors. A non-exhaustive list of examples of such ITR nucleotide sequences is provided in Table 3 below.

[0214] [Table 3]

[0215] In some embodiments, the ITR nucleotide sequences provided herein further comprise a spacer sequence. In one embodiment, the spacer is selected from any one of the spacer sequences provided in Table 2. In one embodiment, any one of the ITR nucleotide sequences in Table 3 may further comprise any one of the spacer sequences provided in Table 2.

[0216] IV.ITR As disclosed herein, a ceDNA vector contains a transgene or heterologous nucleic acid sequence located between at least two inverted terminal repeats (ITRs), which may be an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. The ceDNA vector disclosed herein may include an ITR sequence selected from any of: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration, and the method of the present disclosure may further include a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system.

[0217] In some embodiments, the ITR sequences may be derived from viruses of the family Parvoviridae, which includes two subfamilies, Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. The Parvovirinae subfamily (referred to as parvoviruses) includes the genus Dependovirus, whose members require co-infection with a helper virus, such as an adenovirus or a herpesvirus, for productive infection under most conditions. The Dependovirus genus includes the 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 family Parvoviridae are described generally in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[0218] WT-ITR is exemplified in the specification, and the example herein is the AAV2 WT-ITR, however, 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, the 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 from Mouse (MVM) (GenBank Accession No. NC001510), goose parvovirus (GenBank Accession No. NC001701), snake parvovirus 1 (GenBank Accession No. NC006148). In some embodiments, the 5'WT-ITR can be derived from one serotype and the 3'WT-ITR can be derived from a different serotype, as discussed herein.

[0219] Those skilled in the art know that ITR sequences have the general structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure (see, e.g., FIG. 10), and each WT-ITR is formed by two loops (B-B' and C-C', both of which contain palindromic double-stranded DNA sequences) and a stem (A-A', which also contains a palindromic double-stranded DNA sequence), as well as a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). See, for example, the structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6) and described in Grimm et al., J. Virology, 2006, 80(1); 426-439, Yan et al., J. Virology, 2005; 364-379, Duan et al., Virology 1999; 261; 8-14. Based on the exemplary AAV2 ITR sequences provided herein, one of skill in the art can easily determine the WT-ITR sequence from any AAV serotype for use in a ceDNA vector or ceDNA-plasmid. See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1-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%).

[0220] Symmetric or substantially symmetric ITR pairs In some embodiments, the ceDNA vector described herein 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 described herein), and a second AAV ITR, where the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other, i.e., the ceDNA vector may comprise ITRs with a symmetric three-dimensional spatial organization, whereby their structures are the same shape in geometric space or have the same A-A' stem, C-C' and B-B' loops in three-dimensional space. In such embodiments, the symmetric ITR pair or the substantially symmetric ITR pair may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have one or more modifications from the wild-type ITR and have the same sequence that is the reverse complement (inversion) of each other. In alternative embodiments, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetric three-dimensional shapes.

[0221] Wild type ITR (WT-ITR) In some embodiments, the symmetric or substantially symmetric ITRs are wild-type (WT-ITR) as described herein. That is, both ITRs have a wild-type sequence, but are not necessarily WT-ITRs of the same AAV serotype. That is, in some embodiments, one WT-ITR can be from one AAV serotype and the other WT-ITR can be from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, that is, they can have one or more conservative nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0222] Thus, as disclosed herein, a ceDNA vector contains a transgene or heterologous nucleic acid sequence located between at least two adjacent wild-type inverted terminal repeats (WT-ITRs), which are reverse complements (inverted) of each other, or alternatively are substantially symmetrical to each other. That is, the WT-ITR pair has a symmetrical three-dimensional spatial configuration. In some embodiments, the wild-type ITR sequence (e.g., AAV WT-ITR) comprises a functional Rep binding site (RBS, e.g., for AAV2, SEQ ID NO: 60 in International Patent Publication No. WO2019 / 143885, incorporated herein by reference in its entirety), and a functional terminal separation site (TRS, e.g., 5'-AGTT-3', SEQ ID NO: 62 in WO2019 / 143885, incorporated herein by reference in its entirety).

[0223] In one aspect, the ceDNA vector can be obtained from a double-stranded DNA construct encoding a transgene operably positioned between at least two WT-ITRs (e.g., AAV WT-ITRs). That is, both ITRs have wild-type sequences, but not necessarily WT-ITRs of the same AAV serotype. That is, in some embodiments, one WT-ITR can be derived from one AAV serotype and the other WT-ITR can be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they can have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial configuration. In some embodiments, the 5'WT-ITR is derived from one AAV serotype and the 3'WT-ITR is derived from the same or different AAV serotype. In some embodiments, the 5'WT-ITR and the 3'WT-ITR are mirror images of each other, i.e., they are symmetrical. In some embodiments, the 5'WT-ITR and the 3'WT-ITR are derived from the same AAV serotype.

[0224] WT-ITR is well known. In one embodiment, the two ITRs are from the same AAV2 serotype. In certain embodiments, WT from other serotypes can be used. There are several serotypes that are homologous, such as AAV2, AAV4, AAV6, AAV8. In one embodiment, closely homologous ITRs (e.g., ITRs with similar loop structures) can be used. In another embodiment, more diverse AAV WT ITRs can be used, such as AAV2 and AAV5, and in yet another embodiment, an ITR that is substantially WT can be used, i.e., it has the basic loop structure of WT, but has some conservative nucleotide changes that do not change or affect the properties. When using WT-ITR from the same virus serotype, one or more regulatory sequences can be further used. In certain embodiments, the regulatory sequence is a regulatory switch that allows the activity of ceDNA to be adjusted.

[0225] In some embodiments, one aspect of the technology described herein relates to synthetically produced ceDNA vectors, which comprise at least one heterologous nucleotide sequence operably positioned between two wild-type inverted terminal repeats (WT-ITRs), where the WT-ITRs can be from the same serotype, different serotypes, or can be substantially symmetrical with respect to each other (i.e., have a symmetrical three-dimensional spatial organization such that their structures are the same shape in geometric space, or have the same A, C-C', and B-B' loops in three-dimensional space). In some embodiments, the symmetric WT-ITRs comprise functional terminal separation sites and Rep binding sites. In some embodiments, the heterologous nucleic acid sequence encodes a transgene and the vector is not in a viral capsid.

[0226] In some embodiments, the WT-ITRs are the same but are reverse complements of each other. For example, the sequence AACG of the 5'ITR can be CGTT (i.e., the reverse complement) of the 3'ITR of the corresponding site. In one example, the 5'WT-ITR sense strand comprises a sequence of ATCGATCG and the corresponding 3'WT-ITR sense strand comprises CGATCGAT (i.e., the reverse complement of ATCGATCG). In some embodiments, the WT-ITR ceDNA further comprises a terminal separation site and a replication protein binding site (sometimes referred to as a replication protein binding site), e.g., a Rep binding site (RBS).

[0227] Exemplary WT-ITR sequences for use in ceDNA vectors containing WT-ITRs are shown in Table 2 of International Patent Application Publication No. WO2019 / 143885, which shows a pair of WT-ITRs (5'WT-ITR and 3'WT-ITR).

[0228] As an illustrative example, the disclosure provides a synthetically produced ceDNA vector comprising a promoter, with or without a regulatory switch, operably linked to a transgene (e.g., a gene editing sequence), where the ceDNA lacks a capsid protein, (a) is produced from a ceDNA-plasmid (see, e.g., Figures 1F-1G of WO2019 / 143885) encoding WT-ITRs, each WT-ITR having the same number of intramolecularly duplexed base pairs in its hairpin secondary configuration (preferably excluding any AAA or TTT terminal loop deletions in this configuration compared to these reference sequences), and (b) is identified as ceDNA using an assay for identification of ceDNA by agarose gel electrophoresis under native gel and denaturing conditions.

[0229] In some embodiments, the adjacent WT-ITRs are substantially symmetrical to each other. In this embodiment, the 5'WT-ITR can be derived from one serotype of AAV and the 3'WT-ITR can be derived from a different serotype of AAV, such that the WT-ITRs are not identical reverse complements. For example, the 5'WT-ITR can be derived from AAV2 and the 3'WT-ITR can be derived from a different serotype (e.g., AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12). In some embodiments, the WT-ITRs may be selected from two different parvoviruses selected from any of 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. In some embodiments, such a combination of WT ITRs is a combination of WT-ITRs from AAV2 and AAV6. In one embodiment, the substantially symmetric WT-ITRs are at least 90% identical, at least 95% identical, at least 96%...97%...98%...99%...99.5%, and all percentages in between, identical when one is inverted relative to the other ITR, and have the same symmetric three-dimensional spatial organization. In some embodiments, the WT-ITR pair is substantially symmetric because they have a symmetric three-dimensional spatial organization, e.g., the same three-dimensional organization of the A-A' and D-D' stem regions, and the B-B' and C-C' stem regions. In one embodiment, a substantially symmetric WT-ITR pair is inverted relative to the other and is at least 95% identical, at least 96%...97%...98%...99%....99.5% identical, and all points in between, where one WT-ITR retains the Rep binding site (RBS) (SEQ ID NO: 60 of International Patent Application Publication No. WO2019 / 143885 and the terminal separation site (trs).In some embodiments, the substantially symmetric WT-ITR pair is inverted relative to each other and is at least 95% identical, at least 96%...97%...98%...99%....99.5% identical and all points in between, where one WT-ITR retains a Rep binding site (RBS) (SEQ ID NO: 60 of WO2019 / 143885) and a terminal separation site (trs) in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. Homology can be determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool), BLASTN with default settings.

[0230] 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 separation sites (trs).

[0231] Table 1 of International Patent Application Publication No. WO2019 / 143885 provides a non-exhaustive list of exemplary combinations of WT-ITRs. Table 2 of WO2019 / 143385 provides sequences of a non-exhaustive list of exemplary WT-ITRs from different AAV serotypes.

[0232] In some embodiments, the nucleotide sequence of the WT-ITR sequence may be modified (e.g., by modifying 1, 2, 3, 4, 5 or more nucleotides, or any range therein), whereby the modification is a substitution of a complementary nucleotide, e.g., G for C and vice versa, T for A and vice versa.

[0233] In certain embodiments of the present disclosure, the synthetically produced ceDNA vector does not have a WT-ITR consisting of a nucleotide sequence selected from any of SEQ ID NOs: 1, 2, 5-14 of WO2019 / 143885. In alternative embodiments of the present disclosure, when the ceDNA vector has a WT-ITR comprising a nucleotide sequence selected from any of SEQ ID NOs: 1, 2, 5-14 of WO2019 / 143885, the adjacent ITR is also WT, and the ceDNA comprises a regulatory switch, e.g., as disclosed herein and in International Patent Publication No. WO2019 / 051255 (see, e.g., Table 11 of WO2019 / 051255, which is incorporated herein by reference in its entirety). In some embodiments, the ceDNA vector comprises a regulatory switch as disclosed herein and the selected WT-ITR having a nucleotide sequence selected from any of the group consisting of SEQ ID NOs: 1, 2, 5-14 of WO2019 / 143885.

[0234] The ceDNA vectors described herein may include a WT-ITR structure that retains operable RBE, trs, and RBE' portions. Using the wild-type ITR for illustrative purposes, Figures 2A and 2B of WO2019 / 143885 show one possible mechanism for the operation of the trs site within the wild-type ITR structure portion of the ceDNA vector. In some embodiments, the ceDNA vector contains one or more functional WT-ITR polynucleotide sequences that include a Rep-binding site (for AAV2 (SEQ ID NO: 60 in WO2019 / 143885)) and a terminal separation site (TRS, 5'-AGTT (SEQ ID NO: 62 in WO2019 / 143885)). In some embodiments, at least one WT-ITR is functional. In alternative embodiments where the ceDNA vector includes two WT-ITRs that are substantially symmetrical to each other, at least one WT-ITR is functional and at least one WT-ITR is non-functional.

[0235] Common modified ITRs (mod-ITRs) of ceDNA vectors containing asymmetric or symmetric ITR pairs As discussed herein, synthetically produced ceDNA vectors may contain symmetric or asymmetric ITR pairs. In either case, one or both ITRs may be modified ITRs, the difference being that in the first case (i.e., symmetric mod-ITR), the mod-ITRs have the same three-dimensional spatial organization (i.e., have the same A-A' and D-D' stem regions, and B-B' and C-C' loop organization), whereas in the second case (i.e., asymmetric mod-ITR), the mod-ITRs have a different three-dimensional spatial organization (i.e., have a different organization of A-A' and D-D' stem regions, and B-B' and C-C' loops).

[0236] In some embodiments, the modified ITR is an ITR that is modified by deletion, insertion, and / or substitution compared to a wild-type ITR sequence (e.g., AAV ITR). In some embodiments, at least one of the ITRs in the ceDNA vector has a functional Rep binding site (RBS; e.g., SEQ ID NO: 60 in WO2019 / 143885) and a functional terminal separation site (TRS; e.g., 5'-AGTT-3', SEQ ID NO: 62 in WO2019 / 143885). In one embodiment, at least one of the ITRs is a non-functional ITR. In one embodiment, each of the different or modified ITRs is not a wild-type ITR from a different serotype.

[0237] Although certain alterations and mutations of ITRs are described in detail herein, in the context of ITRs, "altered" or "mutated" or "modified" refers to nucleotides that have been inserted, deleted, and / or substituted relative to the wild-type, reference, or original ITR sequence. An altered or mutant ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated 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 manipulated by the hand of man and is different from the aspect it occurs in nature.

[0238] In some embodiments, the mod-ITR may 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 include AAV base sequences. In yet another embodiment, the synthetic ITR preserves the ITR structure described above, but has little or no AAV source sequence. In some aspects, the synthetic ITR may 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 by mutant Rep.

[0239] Those skilled in the art can determine the corresponding sequences in other serotypes by known means. For example, determine whether there are changes in the A, A', B, B', C, C', or D-D' regions to determine the corresponding regions in another serotype. BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs can be used in default settings to determine the corresponding sequences. The present disclosure further provides a collection and a plurality of ceDNA vectors comprising mod-ITRs from a combination of different AAV serotypes. That is, one mod-ITR can be from one AAV serotype and the other mod-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).

[0240] Any parvovirus ITR can be used for modification as an ITR or as a basic ITR. Preferably, the parvovirus is a dependovirus. More preferably, it is AAV. The serotype selected can be based on the tissue tropism of the serotype. AAV2 has broad tissue tropism, AAV1 preferentially targets neurons and skeletal muscle, and AAV5 preferentially 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.

[0241] 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.

[0242] Table 3 of International Patent Application Publication No. WO2019 / 143885 provides exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in a region of a modified ITR, where X provides non-exhaustive examples of modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. In some embodiments, any modification (e.g., deletions, insertions, and / or substitutions) 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' loop, or a single B-B' loop), 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' loop and / or a truncated B-B' loop), at least the single loop, or at least one of the loops of the two-loop 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.

[0243] In some embodiments, mod-ITRs for use in synthetically produced ceDNA vectors comprising asymmetric or symmetric mod-ITR pairs disclosed herein may comprise any one of the combinations of modifications shown in Table 3 of WO2019 / 143885, 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 3, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any 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 3 of WO2019 / 143885, as well as at least one 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 3 of WO2019 / 143885, as well as a modification (e.g., a deletion, insertion, and / or substitution) of at least one nucleotide in the A' region.In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 3 of WO2019 / 143885, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A and / or A' region. In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 3 of WO2019 / 143885, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the D region.

[0244] In one embodiment, the nucleotide sequence of the 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 shown in SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187 of WO2019 / 143885, or in Figures 7A-7B of International Patent Application Publication No. WO2019 / 113310, which are incorporated by reference in their entirety (e.g., SEQ ID NOs: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 of WO2019 / 113310). In some embodiments, the ITRs may 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 can 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 one of the modified ITRs of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or to the A-A' stem region and the RBE-containing sections of the C-C' and B-B' loops as set forth in SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187 of WO 2019 / 143885, or Tables 2-9 of International Patent Application Publication No. WO 2019 / 051255 (i.e., SEQ ID NOs: 110-112, 115-190, 200-468).

[0245] In some embodiments, the modified ITR may include, for example, removal or deletion of all of a particular arm, e.g., all or part of the A-A' stem region, or all or part of the B-B' loop, or all or part of the C-C' loop, or alternatively, removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form a loop (see, e.g., ITR-21 in Figure 7A of WO2019 / 113310), 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 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 removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm (see, for example, ITR-1 in Figure 3B or ITR-45 in Figure 7A of WO2019 / 113310). 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' loop. Any combination of base pair removal is envisioned, for example, six base pairs in the C-C' loop and two base pairs in the B-B' loop may be removed.

[0246] 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 WT ITR sequence. In some embodiments, the modified ITR has a 2-20 nucleotide deletion relative to the full-length wild-type ITR sequence.

[0247] In some embodiments, the modified ITRs do not contain any nucleotide deletions in the RBE-containing portions of the A or A' stem regions so as not to interfere with DNA replication (e.g., binding to the RBE by Rep proteins or nicking at the terminal separation sites). 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.

[0248] In some embodiments, the synthetically produced ceDNA vector comprising a symmetric or asymmetric ITR pair comprises a regulatory switch as disclosed herein and at least one selected modified ITR having a nucleotide sequence selected from any of the group consisting of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187 of International Patent Application Publication No. WO2019 / 143885.

[0249] In another embodiment, the structure of the structural element may be modified. For example, the structural element may have a change in stem height and / or the number of nucleotides in the loop. For example, the stem height 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 stem height may have about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0250] 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.

[0251] 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.

[0252] The synthetically produced 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 operable RBE, trs, and RBE' portions. In some embodiments, such ITRs (wild-type or modified ITRs) are functional. In alternative embodiments where the ceDNA vector contains two modified ITRs that are different or asymmetric with respect to each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0253] In some embodiments, the synthetically produced ceDNA vector does not have modified ITRs selected from any sequence consisting of, or consisting essentially of, SEQ ID NOs: 500-529 as disclosed in International Patent Application Publication No. WO2019 / 143885. In some embodiments, the ceDNA vector does not have ITRs selected from any sequence selected from SEQ ID NOs: 500-529 of WO2019 / 143885.

[0254] In some embodiments, a modified ITR (e.g., left or right ITR) of a synthetically produced ceDNA vector described herein has a modification in a loop, truncated loop, or spacer. Exemplary sequences of ITRs with modifications in a loop, truncated loop, or spacer are set forth in Table 2 of International Patent Application Publication No. WO2019 / 051255 (i.e., SEQ ID NOs: 135-190, 200-233 of WO2019 / 143885), Table 3 of WO2019 / 051255 (e.g., SEQ ID NOs: 234-263 of WO2019 / 143885), Table 4 of WO2019 / 051255 (e.g., SEQ ID NOs: 264-293 of WO2019 / 143885), Table 5 of WO2019 / 051255 (e.g., SEQ ID NOs: 270-293 of WO2019 / 143885), Table 6 of WO2019 / 051255 (e.g., SEQ ID NOs: 290-300 of WO2019 / 143885), Table 7 of WO2019 / 051255 (e.g., SEQ ID NOs: 300-310 of WO2019 / 143885), Table 8 of WO2019 / 051255 (e.g., SEQ ID NOs: 310-320 of WO2019 / 143885), Table 9 of WO2019 / 051255 (e.g., SEQ ID NOs: 320-330 of WO2019 / 143885), Table 10 of W WO2019 / 143885), Table 6 (e.g., SEQ ID NOs: 319-468 of WO2019 / 143885), and Tables 7-9 of WO2019 / 051255 (e.g., SEQ ID NOs: 101-110, 111-112, 115-134 of WO2019 / 143885), or Tables 10A or 10B of WO2019 / 051255 (e.g., SEQ ID NOs: 9, 100, 469-483, 484-499 of WO2019 / 143885).

[0255] In some embodiments, modified ITRs for use in synthetically produced ceDNA vectors comprising asymmetric ITR pairs or symmetric mod-ITR pairs are selected from any of those shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, and 10A-10B of WO2019 / 051255, or combinations thereof.

[0256] Additional exemplary modified ITRs for use in synthetically produced ceDNA vectors containing asymmetric or symmetric mod-ITR pairs in each of the above classes are shown in Tables 4A and 4B of WO2019 / 143885.

[0257] In one embodiment, the synthetically produced 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 (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other. That is, they have different three-dimensional spatial configurations from each other. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutant or modified ITR, or vice versa, where the first ITR can be a mutant or modified ITR and the second ITR can be a wild-type ITR. In some embodiments, the first ITR and the second ITR are both mod-ITRs, but have different sequences or have different modifications, and thus are not the same modified ITRs, but have different three-dimensional spatial configurations. In other words, a ceDNA vector with asymmetric ITRs contains ITRs in which any change in one ITR relative to the WT-ITR is not reflected in the other ITR, or alternatively, the asymmetric ITRs may have different sequences and different three-dimensional shapes relative to each other if they have a modified asymmetric ITR pair. Exemplary asymmetric ITRs in ceDNA vectors for use in generating ceDNA-plasmids are shown in Tables 4A and 4B of WO2019 / 143885.

[0258] In alternative embodiments, the synthetically produced ceDNA vector comprises two symmetric mod-ITRs. That is, both ITRs have the same sequence but are the reverse complements of each other. In some embodiments, the symmetric mod-ITR pair comprises at least one or any combination of deletions, insertions, or substitutions compared to the wild-type ITR sequence from the same AAV serotype. The additions, deletions, or substitutions of the symmetric ITRs are the same but are the reverse complements of each other. For example, an insertion of three nucleotides into the C region of the 5'ITR is reflected by an insertion of three reverse complement nucleotides into the corresponding section of the C' region of the 3'ITR.

[0259] In alternative embodiments, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair may have different sequences but have corresponding or the same symmetrical three-dimensional shapes. For example, one modified ITR may be derived from one serotype and the other modified ITR may be derived from a different serotype, but they have the same mutation (e.g., nucleotide insertion, deletion, or substitution) in the same region. In other words, for illustrative purposes only, the 5'mod-ITR may be derived from AAV2 and have a deletion in the C region, and the 3'mod-ITR may be derived from AAV5 and have a corresponding deletion in the C' region. Provided that the 5'mod-ITR and the 3'mod-ITR have the same or symmetrical three-dimensional spatial configuration, they are encompassed in the use of modified ITR pair herein.

[0260] In some embodiments, a substantially symmetric mod-ITR pair has the same A-A' stem region, C-C' and B-B' loops in three-dimensional space. For example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR has a corresponding deletion of the C-C' loop and has a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in geometric space of the cognate mod-ITR. By way of example only, substantially symmetric ITRs can have a symmetric spatial organization such that their structures are the same shape in geometric space. This can occur, for example, when a GC pair is modified, for example, to a CG pair, or vice versa, or when an AT pair is modified to a TA pair, or vice versa. In some embodiments, such modified ITRs are substantially symmetric because the modified ITR pair has symmetric stereochemistry.

[0261] Table 5 of International Patent Application Publication No. WO2019 / 143885 provides exemplary symmetrically modified ITR pairs (i.e., a left-modified ITR and a symmetrically right-modified ITR).

[0262] In some embodiments, a ceDNA vector comprising an asymmetric ITR pair may comprise an ITR having an ITR sequence or ITR subsequence set forth in any one or more of Tables 4A-4B of WO2019 / 143885, or a modification corresponding to any of the modifications in the sequences shown in Figures 7A and 7B of International Patent Application Publication No. WO2019 / 113310, or disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9, or 10A-10B of International Patent Application Publication No. WO2019 / 051255.

[0263] V. Exemplary ceDNA Vectors As mentioned above, the present disclosure relates to synthetically produced recombinant ceDNA expression vectors and ceDNA vectors encoding a transgene comprising any one of the above-mentioned asymmetric, symmetric, or substantially symmetric ITR pairs. In certain embodiments, the present disclosure relates to synthetically produced recombinant ceDNA vectors having adjacent ITRs and a transgene, the ITRs being asymmetric, symmetric, or substantially symmetric with respect to each other as defined herein, and the ceDNA further comprising a nucleotide sequence of interest (e.g., an expression cassette comprising the nucleic acid of the transgene) located between the adjacent ITRs, the nucleic acid molecule lacking a viral capsid protein coding sequence.

[0264] The synthetically produced ceDNA expression vector may be any ceDNA vector that can be conveniently subjected to recombinant DNA procedures, including the nucleotide sequence described herein, when at least one ITR is modified. The synthetically produced ceDNA vector of the present disclosure is compatible with the host cell into which the ceDNA vector is introduced. In certain embodiments, the synthetically produced ceDNA vector may be linear. In certain embodiments, the synthetically produced ceDNA vector may exist as an extrachromosomal entity. In certain embodiments, the synthetically produced ceDNA vector of the present disclosure may contain elements that allow the integration of donor sequences into the genome of the host cell.

[0265] 1A-1B provided herein, and further referring to FIG. 1A-1G of International Patent Application Publication No. WO2019 / 143885, schematic diagrams of the functional components of two non-limiting examples of ceDNAs with asymmetric ITRs or symmetric or substantially symmetric ITRs are shown. In some embodiments, the expressible transgene cassette optionally includes an enhancer / promoter, one or more homology arms, donor sequences, post-transcriptional regulatory elements (e.g., WPRE, e.g., SEQ ID NO: 67 of WO2019 / 143885), and polyadenylation and termination signals (e.g., BGH polyA, e.g., SEQ ID NO: 68 of WO2019 / 143885).

[0266] Regulatory elements The ceDNA vectors described herein and produced using the synthesis process described herein may contain asymmetric or symmetric ITR pairs as defined herein, and may further contain a particular 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, the ITRs may act as promoters of the transgene. In some embodiments, the ceDNA vectors contain additional components for regulating the expression of the transgene, such as a regulatory switch as described herein, to regulate the expression of the transgene, or a kill switch that may kill a cell containing the ceDNA vector. Regulatory elements, including regulatory switches, that may be used in the present disclosure are more fully discussed in International Patent Application Publication No. WO2019 / 051255.

[0267] In some embodiments, the second nucleotide sequence comprises a regulatory sequence and a nucleotide sequence encoding a nuclease. In certain embodiments, the gene regulatory sequence is operably linked to the nucleotide sequence encoding the nuclease. In certain embodiments, the regulatory sequence is suitable for controlling the expression of the nuclease in the host cell. In certain embodiments, the regulatory sequence comprises a suitable promoter sequence capable of directing the transcription of a gene operably linked to a promoter sequence, such as a nucleotide sequence encoding a nuclease of the present disclosure. In certain embodiments, the second nucleotide sequence comprises an intron sequence linked to the 5' end of the nucleotide sequence encoding the nuclease. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the efficacy of the promoter. In certain embodiments, the regulatory sequence comprises an enhancer and a promoter, and the second nucleotide sequence comprises an intron sequence upstream of the nucleotide sequence encoding the nuclease, the intron comprising one or more nuclease cleavage sites, and the promoter operably linked to the nucleotide sequence encoding the nuclease.

[0268] The ceDNA vectors produced using the synthesis process described herein may further include a specific combination of cis-regulatory elements such as the WHP post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67 of WO2019 / 143885) and BGH polyA (SEQ ID NO: 68 of WO2019 / 143885). Suitable expression cassettes for use in expression constructs are not limited by packaging constraints imposed by viral capsids.

[0269] Promoter Those skilled in the art will understand that the promoters used in the synthetically produced ceDNA vectors of the present disclosure should be adjusted appropriately to the specific sequences they promote.For example, guide RNAs may not require a promoter at all, since their function is to form a duplex with a specific target sequence on native DNA to generate a recombination event.In contrast, the nucleases encoded by ceDNA vectors benefit from a promoter, and therefore can be expressed efficiently and optionally in a regulatable manner from the vector.

[0270] The expression cassettes of the present disclosure contain promoters that can affect overall expression levels as well as cell specificity. 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: 72 of WO2019 / 143885). The CAG promoter contains (i) a cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of the chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette may contain an alpha-1-antitrypsin (AAT) promoter (SEQ ID NO: 73 or SEQ ID NO: 74 of WO2019 / 143885), a liver-specific (LP1) promoter (SEQ ID NO: 75 or SEQ ID NO: 76 of WO2019 / 143885), or a human elongation factor-1 alpha (EF1a) promoter (e.g., SEQ ID NO: 77 or SEQ ID NO: 78 of WO2019 / 143885). In some embodiments, the expression cassette comprises one or more constitutive promoters, such as the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with a RSV enhancer), or the cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer, e.g., SEQ ID NO: 79 of WO2019 / 143885). Alternatively, an inducible promoter, the transgene's native promoter, a tissue-specific promoter, or a variety of promoters known in the art can be used.

[0271] Suitable promoters, including those mentioned above, can be derived from viruses and therefore can be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6, e.g., SEQ ID NO: 80 in WO2019 / 143885) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the 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: 81 or SEQ ID NO: 155 of WO2019 / 143885), CAG promoter, human alpha 1-antitrypsin (HAAT) promoter (e.g., SEQ ID NO: 82 of WO2019 / 143885), and the like. In certain embodiments, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites. In certain embodiments, the DNA containing the nuclease cleavage sites is foreign to the promoter DNA.

[0272] In one embodiment, the promoter used is the native promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of each gene encoding the 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: 79 and SEQ ID NO: 83 of WO2019 / 143885).

[0273] Non-limiting examples of suitable promoters or promoter sets for use according to the present disclosure include, for example, the CAG promoter (SEQ ID NO: 72 of WO2019 / 143885), the HAAT promoter (SEQ ID NO: 82 of WO2019 / 143885), the human EF1-alpha promoter (SEQ ID NO: 77 of WO2019 / 143885), or the EF1a promoter (SEQ ID NO: 78 of WO2019 / 143885), the IE2 promoter (e.g., SEQ ID NO: 84 of WO2019 / 143885), and the rat EF1-alpha promoter (SEQ ID NO: 85 of WO2019 / 143885), or the 1E1 promoter fragment (SEQ ID NO: 125 of WO2019 / 143885).

[0274] Polyadenylation sequence A sequence encoding a polyadenylation sequence may be included in the ceDNA vector to stabilize the mRNA expressed from the synthetically produced ceDNA vector and to aid in nuclear transport and translation. In one embodiment, the synthetically produced ceDNA vector does not include a polyadenylation sequence. In other embodiments, the vector includes 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 includes about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0275] 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: 68 of WO2019 / 143885) or viral SV40pA (e.g., SEQ ID NO: 86 of WO2019 / 143885), or a synthetic sequence (e.g., SEQ ID NO: 87 of WO2019 / 143885). 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.

[0276] 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: 67 of WO2019 / 143885) is used to increase expression of the transgene. Other post-transcriptional processing elements can be used, such as 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, such as SEQ ID NO: 88 and SEQ ID NO: 89 of WO2019 / 143885.

[0277] nuclear localization sequence In some embodiments, the vector encoding the RNA-guided endonuclease comprises one or more nuclear localization sequences (NLSs), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the one or more NLSs are located at or near the amino terminus, at or near the carboxy terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and / or one or more NLSs at the carboxy terminus). When multiple NLSs are present, each can be selected independently from the other NLSs, such that a single NLS is present in multiple copies and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs are shown in Table 6 of International Patent Application Publication No. WO2019 / 143885.

[0278] Additional components of ceDNA vectors The ceDNA vectors produced using the synthesis process described herein may contain nucleotides encoding other components for gene expression. For example, to select for a specific gene targeting event, a protective shRNA can be embedded in a microRNA and inserted into a recombinant ceDNA vector designed for site-specific integration into a highly active locus, such as the albumin locus. Such an embodiment may provide a system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background, such as described in Nygaard et al., A universal system to select gene-modified hepatocytes in vivo, Gene Therapy, June 8, 2016. The ceDNA vectors of the present disclosure may contain one or more selectable markers that allow for the selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, NeoR, etc. In certain embodiments, a positive selection marker is incorporated into the donor sequence, such as NeoR. A negative selection marker can be incorporated downstream of the donor sequence, for example, a nucleic acid sequence encoding the negative selection marker, HSV-tk, can be incorporated into the nucleic acid construct downstream of the donor sequence.

[0279] In embodiments, the ceDNA vectors produced using the synthesis process described herein can be used for gene editing, for example, as disclosed in International Patent Application Publication No. WO2019 / 113310, and can include one or more of a 5' homology arm, a 3' homology arm, a polyadenylation site upstream, and a 5' homology arm adjacent. Exemplary homology arms are 5' and 3' albumin homology arms (SEQ ID NOs: 151 and 152 of WO2019 / 143885) or CCR5 5' and 3' homology arms (e.g., SEQ ID NOs: 153, 154 of WO2019 / 143885).

[0280] 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 ceDNA vectors produced using the synthetic processes described herein to control the output of transgene expression from the ceDNA vector. In some embodiments, the ceDNA vector includes a regulatory switch that serves to fine-tune the expression of the 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. Exemplary regulatory switches encompassed for use in ceDNA vectors can be used to regulate expression of a transgene and are discussed more fully in International Patent Application Publication No. WO2019 / 113310.

[0281] (i) Binary Adjustment Switch In some embodiments, the ceDNA vector produced using the synthesis process described herein comprises a regulatory switch that can help to controllably regulate the expression of the transgene. For example, the expression cassette located between the ITRs of the ceDNA vector can additionally comprise a regulatory region, such as a promoter, a cis-element, a repressor, an enhancer, etc., operably linked to the gene of interest, which is regulated by one or more cofactors or exogenous drugs. By way of example only, the regulatory region can be regulated by a small molecule switch or an inducible or repressible promoter. A non-limiting example of an inducible promoter is a hormone-inducible or metal-inducible promoter. Other exemplary inducible promoter / enhancer elements include, but are not limited to, the RU486-inducible promoter, the ecdysone-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

[0282] (ii) Small molecule regulatory switches A variety of small molecule-based regulatory switches known in the art can be combined with synthetically produced ceDNA vectors known in the art and disclosed herein to form ceDNA vectors controlled by regulatory switches. In some embodiments, the regulatory switch is an artificial promoter that controls the expression of an operably linked transgene, as disclosed in Taylor, et al., BMC Biotechnology 10(2010):15, along with an orthogonal ligand / nuclear receptor pair, such as retinoid receptor variants / LG335 and GRQCIMFI, engineered steroid receptors, such as modified progesterone receptors with C-terminal truncations that cannot bind progesterone but bind RU486 (mifepristone) (U.S. Pat. No. 5,364,791), ecdysone receptors from Drosophila and their ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517, or Sando R 3 rd ;Nat Methods. 2013,10(11):1085-8, or any one or combination of switches controlled by the antibiotic trimethoprim (TMP). 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.

[0283] (iii) "Passcode" adjustment switch In some embodiments, the regulatory switch can be a "passcode switch" or "passcode circuit." A passcode switch allows fine tuning of the control of transgene expression from a synthetically produced ceDNA vector when certain conditions occur, i.e., a combination of conditions must be present for transgene expression and / or repression to occur. For example, at least conditions A and B must occur for transgene expression to occur. A passcode regulatory switch can be any number of conditions, e.g., 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 transgene expression to occur. In some embodiments, at least 2 conditions (e.g., A, B conditions) must occur, and in some embodiments, at least 3 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 can 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. For example, if the transgene edits a defective EPO gene, Condition A is the presence of Chronic Kidney Disease (CKD), Condition B occurs when the subject has hypoxia in the kidney, and Condition C is a failure of Erythropoietin-producing cell (EPC) mobilization in the kidney or, alternatively, a failure of HIF-2 activation. Once oxygen levels increase or a desired EPO level is reached, the transgene is turned off again and turned back on until the three conditions occur.

[0284] In some embodiments, the passcode regulatory switches or "passcode circuits" included for use in synthetically produced ceDNA vectors contain hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biological containment conditions. In contrast to dead-man switches, which induce 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.

[0285] 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 of skill in the art disclosed herein, can be used in the passcode regulatory switches disclosed herein. Regulatory switches encompassed for use are also described in the review article Kis et al., JR Soc Interface. 12:20141000 (2015) and summarized in Table 1 of Kis.

[0286] (iv) a nucleic acid-based regulatory switch for controlling transgene expression In some embodiments, the regulatory switch for controlling the transgene expressed by the synthetically produced ceDNA vector 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 US Patent Application Publication Nos. 2009 / 0305253, 2008 / 0269258, 2017 / 0204477, International Patent Application Publication No. WO2018026762(A1), US Patent No. 9,222,093, and the review by Villa JK et al., Microbiol Spectr. 2018 May; 6(3). Metabolite-responsive transcriptional biosensors, such as those disclosed in International Patent Application Publication Nos. WO2018 / 075486 and WO2017 / 147585, are also included. Other mechanisms known in the art that can be used include silencing transgenes by siRNA or RNAi molecules (e.g., miR, shRNA).For example, ceDNA vectors can contain regulatory switches that code for RNAi molecules that are complementary to the transgenes expressed by ceDNA vectors.If such RNAi is expressed even when expressed by ceDNA vectors, the transgene will be silenced by complementary RNAi molecules, and if RNAi is not expressed when transgenes are expressed by ceDNA vectors, the transgene will not be silenced by RNAi.

[0287] In some embodiments, the regulatory switch is a tissue-specific self-inactivating regulatory switch, e.g., as disclosed in U.S. Patent Application Publication No. 2002 / 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 U.S. Patent Application Publication No. 2014 / 0127162 and U.S. Patent No. 8,324,436, which are incorporated by reference in their entireties.

[0288] (v) Post-transcriptional and post-translational regulatory switches. In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the synthetically produced ceDNA vector is a post-transcriptional modification system. For example, such a regulatory switch can be an aptazyme riboswitch sensitive to tetracycline or theophylline, as disclosed in US Patent Application Publication No. 2018 / 0119156, UK Patent Application Publication No. GB2011 / 07768, International Patent Application Publication No. WO2001 / 064956(A3), European Patent No. 2707487, and Beilstein 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 a person skilled in the art can code both the transgene and the inhibitory siRNA that contains a ligand-sensitive (off-switch) aptamer, the end result of which is a ligand-sensitive on-switch.

[0289] (vi) Other exemplary adjustment switches Any known regulatory switch can be used in the synthetically produced ceDNA vector to control gene expression of the transgene expressed by the ceDNA vector, including those triggered by environmental changes. Further 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- or ultrasound-regulated on / off switches (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 International Patent Application Publication No. WO1999 / 025385(A1). In some embodiments, the regulatory switch is controlled by an implantable system, e.g., as disclosed in U.S. Patent No. 7,840,263, U.S. Patent Application Publication No. 2007 / 0190028(A1), 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.

[0290] In some embodiments, regulatory switches contemplated for use in synthetically produced ceDNA vectors are hypoxia-mediated or stress-activated switches, such as those disclosed in International Patent Application Publication No. WO1999060142(A2), U.S. Pat. Nos. 5,834,306, 6,218,179, 6,709,858, U.S. Pat. Appl. Publication No. 2015 / 0322410, Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD, and NRSE elements, and conditionally inducible silencing elements, including the hypoxia response element (HRE), inflammatory response element (IRE), and shear-stress activated element (SSAE) disclosed in U.S. Pat. No. 9,394,526. Such embodiments are useful for turning on expression of a transgene from a ceDNA vector after ischemia or in ischemic tissues and / or tumors.

[0291] (vii) Kill Switch Another embodiment of the present disclosure relates to synthetically produced ceDNA vectors that include a kill switch. The kill switch disclosed herein can cause cells containing 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 synthetically produced ceDNA vector of the present disclosure 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 where apoptosis is desired (e.g., cancer cells). In all aspects, the "kill switch" disclosed herein is designed to provide rapid and robust cell killing of cells containing 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 containing 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 synthetically produced ceDNA vector from the subject or ensure that the encoded transgene is not expressed.

[0292] VI. Related Basic Vectors, Constructs, and Kits Further aspects of the present disclosure are directed to base vectors and double-stranded DNA constructs engineered to facilitate cell-free synthesis of DNA vectors, such as closed-ended DNA (ceDNA) vectors.

[0293] Basic Vector The basic vectors provided herein are vectors that do not carry any transgene or heterologous nucleic acid. Instead, these basic vectors contain a multiple cloning site that can accept a transgene. Additionally, the basic vectors contain non-palindromic restriction endonuclease recognition sites and their corresponding cleavage sites adjacent to the multiple cloning sites. The basic vectors also further contain partial ITRs adjacent to the multiple cloning sites. Thus, in one embodiment, and in all embodiments of any of the above disclosed herein, including the embodiment of the restriction endonuclease whose recognition site is specific, the basic vector contains, in the order 5'→3': a first non-palindromic restriction endonuclease recognition site and corresponding first cleavage site, a first partial ITR, a multiple cloning site, a second partial ITR, and a second non-palindromic restriction endonuclease recognition site and corresponding second cleavage site. In some embodiments, the basic vector further comprises an origin of replication and a selectable marker gene. In some embodiments, the base vector further comprises one or more spacer regions, such as a spacer between the first partial ITR and the transgene expression cassette, a spacer between the second partial ITR and the transgene expression cassette, and / or a spacer within the multiple cloning site (see, e.g., FIG. 2 showing an exemplary base vector plasmid 11).

[0294] Exemplary base vectors include plasmid 17 (SEQ ID NO:9), plasmid 20 (SEQ ID NO:10), plasmid 18 (SEQ ID NO:11), plasmid 15 (SEQ ID NO:12), plasmid 1 (SEQ ID NO:13), plasmid 11 (SEQ ID NO:14), plasmid 8 (SEQ ID NO:15), plasmid 7 (SEQ ID NO:16), and plasmid 6 (SEQ ID NO:17), as shown below.

[0295] Plasmid 17 (SEQ ID NO:9)

[0296] According to some embodiments, the base vector is 85% identical to SEQ ID NO:9. According to some embodiments, the base vector is 90% identical to SEQ ID NO:9. According to some embodiments, the base vector is 95% identical to SEQ ID NO:9. According to some embodiments, the base vector is 96% identical to SEQ ID NO:9. According to some embodiments, the base vector is 97% identical to SEQ ID NO:9. According to some embodiments, the base vector is 98% identical to SEQ ID NO:9. According to some embodiments, the base vector is 99% identical to SEQ ID NO:9. According to some embodiments, the base vector consists of SEQ ID NO:9.

[0297] Plasmid 20 (SEQ ID NO: 10)

[0298] According to some embodiments, the base vector is 85% identical to SEQ ID NO:10. According to some embodiments, the base vector is 90% identical to SEQ ID NO:10. According to some embodiments, the base vector is 95% identical to SEQ ID NO:10. According to some embodiments, the base vector is 96% identical to SEQ ID NO:10. According to some embodiments, the base vector is 97% identical to SEQ ID NO:10. According to some embodiments, the base vector is 98% identical to SEQ ID NO:10. According to some embodiments, the base vector is 99% identical to SEQ ID NO:10. According to some embodiments, the base vector consists of SEQ ID NO:10.

[0299] Plasmid 18 (SEQ ID NO:11)

[0300] According to some embodiments, the base vector is 85% identical to SEQ ID NO:11. According to some embodiments, the base vector is 90% identical to SEQ ID NO:11. According to some embodiments, the base vector is 95% identical to SEQ ID NO:11. According to some embodiments, the base vector is 96% identical to SEQ ID NO:11. According to some embodiments, the base vector is 97% identical to SEQ ID NO:11. According to some embodiments, the base vector is 98% identical to SEQ ID NO:11. According to some embodiments, the base vector is 99% identical to SEQ ID NO:11. According to some embodiments, the base vector consists of SEQ ID NO:11.

[0301] Plasmid 15 (SEQ ID NO: 12)

[0302] According to some embodiments, the base vector is 85% identical to SEQ ID NO:12. According to some embodiments, the base vector is 90% identical to SEQ ID NO:12. According to some embodiments, the base vector is 95% identical to SEQ ID NO:12. According to some embodiments, the base vector is 96% identical to SEQ ID NO:12. According to some embodiments, the base vector is 97% identical to SEQ ID NO:12. According to some embodiments, the base vector is 98% identical to SEQ ID NO:12. According to some embodiments, the base vector is 99% identical to SEQ ID NO:12. According to some embodiments, the base vector consists of SEQ ID NO:12.

[0303] Plasmid 1 (SEQ ID NO: 13)

[0304] According to some embodiments, the base vector is 85% identical to SEQ ID NO:13. According to some embodiments, the base vector is 90% identical to SEQ ID NO:13. According to some embodiments, the base vector is 95% identical to SEQ ID NO:13. According to some embodiments, the base vector is 96% identical to SEQ ID NO:13. According to some embodiments, the base vector is 97% identical to SEQ ID NO:13. According to some embodiments, the base vector is 98% identical to SEQ ID NO:13. According to some embodiments, the base vector is 99% identical to SEQ ID NO:13. According to some embodiments, the base vector consists of SEQ ID NO:13.

[0305] Plasmid 11 (SEQ ID NO: 14)

[0306] According to some embodiments, the base vector is 85% identical to SEQ ID NO:14. According to some embodiments, the base vector is 90% identical to SEQ ID NO:14. According to some embodiments, the base vector is 95% identical to SEQ ID NO:14. According to some embodiments, the base vector is 96% identical to SEQ ID NO:14. According to some embodiments, the base vector is 97% identical to SEQ ID NO:14. According to some embodiments, the base vector is 98% identical to SEQ ID NO:14. According to some embodiments, the base vector is 99% identical to SEQ ID NO:14. According to some embodiments, the base vector consists of SEQ ID NO:14.

[0307] Plasmid 8 (SEQ ID NO: 15)

[0308] According to some embodiments, the base vector is 85% identical to SEQ ID NO: 15. According to some embodiments, the base vector is 90% identical to SEQ ID NO: 15. According to some embodiments, the base vector is 95% identical to SEQ ID NO: 15. According to some embodiments, the base vector is 96% identical to SEQ ID NO: 15. According to some embodiments, the base vector is 97% identical to SEQ ID NO: 15. According to some embodiments, the base vector is 98% identical to SEQ ID NO: 15. According to some embodiments, the base vector is 99% identical to SEQ ID NO: 15. According to some embodiments, the base vector consists of SEQ ID NO: 15.

[0309] Plasmid 7 (SEQ ID NO: 16)

[0310] According to some embodiments, the base vector is 85% identical to SEQ ID NO: 16. According to some embodiments, the base vector is 90% identical to SEQ ID NO: 16. According to some embodiments, the base vector is 95% identical to SEQ ID NO: 16. According to some embodiments, the base vector is 96% identical to SEQ ID NO: 16. According to some embodiments, the base vector is 97% identical to SEQ ID NO: 16. According to some embodiments, the base vector is 98% identical to SEQ ID NO: 16. According to some embodiments, the base vector is 99% identical to SEQ ID NO: 16. According to some embodiments, the base vector consists of SEQ ID NO: 16.

[0311] Plasmid 6 (SEQ ID NO: 17)

[0312] According to some embodiments, the base vector is 85% identical to SEQ ID NO:17. According to some embodiments, the base vector is 90% identical to SEQ ID NO:17. According to some embodiments, the base vector is 95% identical to SEQ ID NO:17. According to some embodiments, the base vector is 96% identical to SEQ ID NO:17. According to some embodiments, the base vector is 97% identical to SEQ ID NO:17. According to some embodiments, the base vector is 98% identical to SEQ ID NO:17. According to some embodiments, the base vector is 99% identical to SEQ ID NO:17. According to some embodiments, the base vector consists of SEQ ID NO:17.

[0313] Double-stranded DNA constructs As disclosed herein, cell-free synthesis of DNA vectors involves excising a transgene expression cassette from a double-stranded DNA construct. The double-stranded DNA construct can be prepared, for example, by subcloning a transgene expression cassette into a base vector. Thus, in one embodiment, and in all embodiments of any of the above disclosed herein, including those in which the recognition site is specific for the restriction endonuclease, the double-stranded DNA construct contains, in the order 5'→3': a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site, a first partial ITR, a transgene expression cassette, a second partial ITR, and a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site. In some embodiments, the double-stranded DNA construct further comprises an origin of replication and a selectable marker gene. In some embodiments, the base vector further comprises one or more spacer regions, such as a spacer between the first partial ITR and the transgene expression cassette and a spacer between the second partial ITR and the transgene expression cassette (see, e.g., FIG. 3 showing an exemplary double-stranded DNA construct, construct 1, carrying a FVIII-expressing transgene expression cassette).

[0314] kit In one embodiment, a kit for preparing a DNA vector, such as a closed-end DNA (ceDNA) vector, comprises the basic vector described above, at least one restriction endonuclease capable of cleaving the DNA vector at the multiple cloning site to enable the multiple cloning site to accept an introduced gene (e.g., BsaI for plasmid 11, see FIG. 2), at least one restriction endonuclease capable of cleaving at a first cleavage site and a second cleavage site (e.g., NotI and XbaI for plasmid 11, see FIG. 2), and a ligase.

[0315] In another embodiment, a kit for preparing a DNA vector, such as a closed-end DNA (ceDNA) vector, comprises a double-stranded DNA construct as described above, at least one restriction endonuclease capable of cleaving the double-stranded DNA construct at a first cleavage site and a second cleavage site (e.g., BsaI for construct 1, see FIG. 3), and a ligase.

[0316] Further embodiments of the above kits may comprise one or more ITR oligonucleotides as defined herein.

[0317] VII. Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided, comprising a closed-end DNA vector, e.g., a ceDNA vector, produced using the synthetic process described herein, and a pharma- ceutically acceptable carrier or diluent.

[0318] The closed-end DNA vectors, including the ceDNA vectors produced using the synthetic process described herein, can be incorporated into pharmaceutical compositions suitable for administration to a subject for in vivo delivery to a cell, tissue, or organ of the subject. Typically, the pharmaceutical composition comprises the ceDNA vectors disclosed herein and a pharma- ceutically acceptable carrier. For example, the closed-end DNA vectors, including the ceDNA vectors produced using the synthetic process described herein, can be incorporated into pharmaceutical compositions suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high pressure intravenous or intra-arterial infusion, as well as intracellular injection, such as 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 concentrations of synthetically produced closed-end DNA vectors, such as ceDNA vector concentrations. Sterile injections can be prepared by incorporating a required amount of synthetically produced closed-end DNA vectors, such as ceDNA vector compounds, in a suitable buffer with one or a combination of the above-listed components, as needed, followed by filter sterilization containing the ceDNA vector, and can be formulated to deliver the transgene in the nucleic acid to the recipient's cells, resulting in the therapeutic expression of the transgene or donor sequence therein. The composition can also include a pharmaceutically acceptable carrier.

[0319] Pharmaceutically active compositions comprising closed-ended DNA vectors, including ceDNA vectors, produced using the synthetic processes described herein can be formulated to deliver transgenes for a variety of purposes to cells, e.g., cells of a subject.

[0320] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under the conditions of manufacture and storage.Compositions can be formulated as solution, microemulsion, dispersion, liposome, or other ordered structures suitable for high concentration synthetically produced closed-end DNA vectors, such as ceDNA vectors.Sterile injectable solutions can be prepared by incorporating the required amount of synthetically produced closed-end DNA vectors, such as ceDNA vector compounds in a suitable buffer with one or a combination of the components listed above, followed by filtration sterilization.

[0321] As disclosed herein, closed-end DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intra-thecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastitial, anterior chamber, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intra-arterial infusion is also contemplated, as well as intracellular injections such as intranuclear microinjection or intracytoplasmic injection.

[0322] In some aspects, the methods provided herein include delivering one or more closed-end DNA vectors, including ceDNA vectors, produced using the synthetic process described herein to a host cell. Also provided herein are cells produced by such methods, and organisms (such as animals, plants, or fungi) that contain or are produced from such cells. Nucleic acid delivery methods may include lipofection, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycations, or lipid:nucleic acid conjugates, naked DNA, and drug-enhanced DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0323] Various techniques and methods for delivering nucleic acid to cells are known in the art.For example, the closed-end DNA vectors, including the ceDNA vectors produced using the synthesis process described herein, can be formulated into lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core-shell nanoparticles.Typically, LNPs are composed of nucleic acid (e.g., ceDNA) molecules, 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 conjugates), and optionally sterols (e.g., cholesterol).

[0324] Another method for delivering closed-end DNA vectors, including ceDNA vectors produced using the synthesis process described herein, to cells is by binding nucleic acid with a ligand that is internalized by cells.For example, the ligand can bind to a receptor on the cell surface and be internalized via plasma membrane invagination.The ligand can be covalently linked to the nucleotide in the nucleic acid. Exemplary conjugates for delivering nucleic acids into cells are described, for example, in International Patent Application Publication Nos. WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515, and WO2017 / 177326.

[0325] Nucleic acids and closed-end DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, 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 England Biolabs), CHARIOT™ Protein Delivery Reagent (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMD Millipore), 293fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific), LIPOFECTAMINE™ (Thermo Fisher Scientific), LIPOFECTIN™ (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific), CELLFECTIN ... Scientific), LIPOFECTACE(TM), FUGENE(TM)(Roche, Basel, Switzerland), FUGENE(TM) HD(Roche), TRANSFECTAM(TM)(Transfectam, Promega, Madison, Wis.), TFX-10(TM)(Pr omega), TFX-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™ (Gene Therapy Systems, San Diego, Calif.), DHARMAFECT 1™ (Dharmacon, Lafayette, Colo.), 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.

[0326] The closed-end DNA vectors, including ceDNA vectors, produced using the synthetic process described herein can also be administered directly to an organism for in vivo cell transduction. Administration is by any of the routes normally used to ultimately bring molecules into contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known by those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route may often provide a more immediate and more effective response than another route.

[0327] Methods for the introduction of closed-ended DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, can be delivered to hematopoietic stem cells, for example, by the methods described in U.S. Pat. No. 5,928,638.

[0328] The closed-end DNA vectors, including ceDNA vectors produced using the synthetic process described herein, can be added to liposomes for delivery to cells or target organs of a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug 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). Liposomal compositions for such delivery are composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also include other lipids. Exemplary liposomes and liposomal formulations are disclosed in International Patent Application Publication Nos. WO2019 / 051289 and WO2019 / 113310, see, for example, the section entitled "Pharmaceutical Formulations."

[0329] A variety of delivery methods known in the art or modifications thereof can be used to deliver closed-end DNA vectors, including ceDNA vectors produced in vitro or in vivo using the synthesis process described herein. For example, in some embodiments, ceDNA vectors are delivered by creating a temporary penetration in the cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy, thereby facilitating DNA entry into the targeted cell. For example, ceDNA vectors can be delivered by temporarily disrupting the cell membrane by squeezing the cell through a size-restricted channel or by other means known in the art. In some cases, ceDNA vectors are directly injected as naked DNA alone into skin, thyroid, cardiac, skeletal muscle, or liver cells. 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 can be accelerated to high velocities by pressurized gas and penetrate into the target tissue cells.

[0330] Specifically contemplated herein are compositions comprising closed-end DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, and a pharma- ceutically acceptable carrier. In some embodiments, the ceDNA vectors are formulated in lipid delivery systems, such as liposomes, as described herein. In some embodiments, such compositions are administered by any route desired by a skilled practitioner. The compositions may be administered to a subject by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or combinations thereof. For veterinary use, the compositions may be administered in a suitably acceptable formulation according to normal veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The compositions may be administered by conventional syringes, needle-free injection devices, "microprojectile bombardment gene guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0331] In some cases, closed-end DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, are delivered by hydrodynamic injection, a simple and highly efficient method for direct intracellular delivery of any water-soluble compound and particle to skeletal muscles throughout the viscera and limbs.

[0332] In some cases, closed-end DNA vectors, including ceDNA vectors produced using the synthesis process described herein, are delivered by ultrasound, creating 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 closed-end DNA play a major role in the efficiency of this system.In some cases, closed-end DNA vectors, including ceDNA vectors produced using the synthesis process described herein, are delivered by magnetofection, using a magnetic field to concentrate particles containing nucleic acids in target cells.

[0333] 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 belonging to cationic polymers. The cationic lipids used in the 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.

[0334] Exosomes In some embodiments, closed-end DNA vectors, including ceDNA vectors produced using the synthetic process described herein, are delivered by being encapsulated in exosomes. Exosomes are small membrane vesicles of plasma membrane invagination 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 cell that produced the exosomes, and exhibit membrane proteins from the parent cell on their surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MC), and dendritic cells (DC). 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 present disclosure.

[0335] Microparticles / Nanoparticles In some embodiments, the closed-end DNA vectors, including the ceDNA vectors produced using the synthesis process described herein, are delivered by lipid nanoparticles. In general, 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. Pharmaceuticals 5(3):498-507.

[0336] In some embodiments, the lipid nanoparticles have an average diameter of about 10 to about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter that is less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of about 10 to about 300 nm. In some embodiments, the lipid nanoparticles have a diameter that is less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of about 25 to 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.

[0337] Various lipid nanoparticles known in the art can be used to deliver closed-end DNA vectors, including the ceDNA vectors produced using the synthesis process described 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.

[0338] In some embodiments, the closed-end DNA vectors, including ceDNA vectors, produced using the synthesis process described herein are delivered by gold nanoparticles. In general, nucleic acids can be covalently bound to gold nanoparticles or non-covalently bound to gold nanoparticles (e.g., bound by charge-charge interactions), 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 conjugates are produced using the methods described, for example, in U.S. Patent No. 6,812,334.

[0339] Conjugates In some embodiments, as disclosed herein, closed-end DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, are conjugated (e.g., covalently linked to agents that increase cellular uptake). An "agent that increases cellular uptake" is a molecule that promotes 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 are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[0340] In some embodiments, as disclosed herein, closed-end DNA vectors, including ceDNA vectors produced using the synthesis process described herein, are 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 a polymer is known in the art, for example, as described in International Patent Application Publication Nos. WO2000 / 34343 and 2008 / 022309. In some embodiments, the ceDNA vectors disclosed herein are conjugated to a poly(amide) polymer, for example, as described by U.S. Patent No. 8,987,377. In some embodiments, the nucleic acids described by this disclosure are conjugated to a folic acid molecule, as described in U.S. Patent No. 8,507,455.

[0341] In some embodiments, as disclosed herein, closed-ended DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, are conjugated to carbohydrates, e.g., as described in U.S. Pat. No. 8,450,467.

[0342] Nanocapsules Alternatively, as disclosed herein, nanocapsule formulations of closed-end DNA vectors, including ceDNA vectors produced using the synthesis process described 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 that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0343] Liposomes The closed-end DNA vectors, including ceDNA vectors produced using the synthetic process described herein, can be added to liposomes for delivery to cells or target organs of interest. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug 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.

[0344] The formation and use of liposomes is generally known to those skilled in the art. Liposomes with improved serum stability and circulation half-life have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0345] Exemplary Liposome and Lipid Nanoparticle (LNP) Compositions The closed-end DNA vectors, including ceDNA vectors, produced using the synthetic process described herein can be added to liposomes for delivery to cells, for example, cells requiring expression of a transgene. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug 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.

[0346] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application Publication Nos. WO2019 / 051289 and WO2019 / 113310, and International Patent Application No. PCT / US2021 / 04043, filed July 16, 2021, and are contemplated for use in the methods and compositions disclosed herein.

[0347] 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 hydrophilic and hydrophobic properties to the compounds, 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.

[0348] In some aspects, the present disclosure provides liposomal formulations that will deliver APIs with extended or controlled release profiles over a period of hours to weeks. In some related aspects, the liposomal formulations may include aqueous chambers bounded by a lipid bilayer. In other related aspects, the liposomal formulations encapsulate APIs with components that undergo a physical transition at elevated temperatures that release the API over a period of hours to weeks.

[0349] In some embodiments, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein, hi some embodiments, the liposomal formulation comprises an optisome.

[0350] In some aspects, the present disclosure provides a lipid composition comprising N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine), PEG (polyethylene glycol), DSPE (distearoyl-sn-glycero-phosphoethanolamine), DSPC (distearoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPG (dipalmitoylphosphatidylglycerol), EPC (egg phosphatidylcholine), DOPS ... The present invention provides a liposome formulation comprising one or more lipids selected from dioleoyl-sn-glycero-phosphoethanolamine (DSPG), ...

[0351] 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 lipid containing a phosphatidylcholine functional group, cholesterol, 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 aspects, the present disclosure provides a liposome formulation comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[0352] In some embodiments, the present disclosure provides liposomal formulations 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 liposomal formulations 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.

[0353] In some aspects, the present disclosure provides liposome formulations further comprising one or more pharmaceutical excipients, such as sucrose and / or glycine.

[0354] In some embodiments, the present disclosure provides liposomal formulations that are either unilamellar or multilamellar in structure. In some embodiments, the present disclosure provides liposomal formulations that include multivesicular particles and / or foam-based particles. In some embodiments, the present disclosure provides liposomal formulations that are larger in size relative to typical nanoparticles, about 150-250 nm in size. In some embodiments, the liposomal formulation is a lyophilized powder.

[0355] In some aspects, 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, sending the API into the liposome. In some aspects, the present disclosure provides liposomal formulations with 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 aspects, 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, are utilized.

[0356] In some aspects, the present disclosure provides lipid nanoparticles comprising a DNA vector, including a ceDNA vector produced using the synthesis process described herein, and an ionized lipid. For example, lipid nanoparticle formulations made and loaded with ceDNA obtained by the process disclosed in International Patent Application Publication No. WO2019 / 051289. This can be achieved by high energy mixing of ethanol lipids and aqueous ceDNA at low pH, which protonates the ionized lipids and provides favorable energetics for ceDNA / lipid association and nucleation of the particles. The particles can be further stabilized by aqueous dilution and removal of organic solvent. The particles can be concentrated to a desired level.

[0357] Typically, lipid particles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 30:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of lipid and ceDNA can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Typically, the total lipid content of the lipid particle formulation can range from about 5 mg / ml to about 30 mg / mL.

[0358] Ionizable lipids are typically used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and fusogenicity.Generally, ionizable lipids are lipids that contain at least one amino group that is positively charged or protonated under acidic conditions, such as pH 6.5 or less.Ionizable lipids are also referred to herein as cationic lipids.

[0359] Exemplary ionizable lipids are described in International Patent Application Publication Nos. 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, 2017 / 075531, 2017 / 117528, 2011 / 022460, 2013 / 148541, 2013 / 116126, 201 1 / 153120, 2012 / 044638, 2012 / 054365, 2011 / 090965, 2013 / No. 016058, No. 2012 / 162210, No. 2008 / 042973, No. 2010 / 129709, No. 2010 / 14 No. 4740, No. 2012 / 099755, No. 2013 / 049328, No. 2013 / 086322, No. 2013 / 0863 No. 73, No. 2011 / 071860, No. 2009 / 132131, No. 2010 / 048536, No. 2010 / 088537 , 2010 / 054401, 2010 / 054406, 2010 / 054405, 2010 / 054384, No. 2012 / 016184, No. 2009 / 086558, No. 2010 / 042877, No. 2011 / 000106, No. 2 011 / 000107, 2005 / 120152, 2011 / 141705, 2013 / 126803, 200 6 / 007712, 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / 1 Nos. 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, and U.S. Patent Application Publication Nos. 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, and 2013 / 0178541;Same No. 2013 / 0303587, No. 2015 / 0141678, No. 2015 / 0239926, No. 2016 / 0376224, No. 2017 / 0119904, No. 2012 / 01 No. 49894, No. 2015 / 0057373, No. 2013 / 0090372, No. 2013 / 0274523, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 0023673, 2012 / 0128760, 2010 / 0324120, 2014 / 0200257, 2015 / 0203446, 2018 / 0005 No. 363, No. 2014 / 0308304, No. 2013 / 0338210, No. 2012 / 0101148, No. 2012 / 0027796, No. 2012 / 0058144, No. 20 13 / 0323269, 2011 / 0117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 008378 No. 0, No. 2013 / 0123338, No. 2015 / 0064242, No. 2006 / 0051405, No. 2013 / 0065939, No. 2006 / 0008910, No. 2003 2010 / 0130588, 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920.

[0360] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), having the following structure:

[0361] [ka] may be selected from the group consisting of:

[0362] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533.

[0363] In some embodiments, the ionizable lipid is the lipid ATX-002, which is described in International Patent Application Publication No. WO2015 / 074085.

[0364] In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32), described in International Patent Application Publication No. WO2012 / 040184.

[0365] In some embodiments, the ionizable lipid is compound 6 or compound 22 described in WO2015 / 199952.

[0366] Without limitation, the ionized lipids may comprise 20-90% (mol) of the total lipids present in the lipid nanoparticles. For example, the ionized lipid molar content may be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the ionized lipids comprise about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticles.

[0367] In some aspects, the lipid nanoparticles may further comprise non-cationic lipids. Non-ionic lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, non-cationic lipids may be neutral uncharged, zwitterionic, or anionic lipids. Non-cationic lipids are typically used to enhance membrane fusogenicity.

[0368] Exemplary non-cationic lipids contemplated for use in the methods and compositions comprising DNA vectors, including ceDNA vectors, produced using the synthetic processes described herein are described in International Patent Application Publication Nos. WO2019 / 051289 and WO2019 / 113310.

[0369] Exemplary non-cationic lipids are described in International Patent Application Publication No. WO2017 / 099823 and U.S. Patent Application Publication No. US2018 / 0028664.

[0370] The non-cationic lipid may comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In various embodiments, the molar ratio of ionized lipid to neutral lipid ranges from about 2:1 to about 8:1.

[0371] In some embodiments, the lipid nanoparticles are completely free of phospholipids. In some aspects, the lipid nanoparticles may further comprise components such as sterols to provide membrane integrity.

[0372] One exemplary sterol that can be used in the lipid nanoparticles is cholesterol and its derivatives. Exemplary cholesterol derivatives are described in International Patent Application Publication No. 2009 / 127060 and US Patent Application Publication No. 2010 / 0130588.

[0373] Components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipids present in the lipid nanoparticles, in some embodiments, such components comprise 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticles.

[0374] In some aspects, the lipid nanoparticles may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit lipid nanoparticle aggregation and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as (methoxypolyethylene glycol)-conjugated lipid. Exemplary PEG-lipid conjugates include PEG-diacylglycerol (DAG) (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl- Methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Patent Nos. 5,885,613, 6,287,591, and U.S. Patent Application Publication Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, and 2017 / 0119904.

[0375] In some embodiments, the PEG-lipid is a compound disclosed in U.S. Patent Application Publication No. 2018 / 0028664.

[0376] In some embodiments, the PEG-lipids are disclosed in U.S. Patent Application Publication No. 2015 / 0376115 or 2016 / 0376224.

[0377] The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be, for example, PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG -DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid may be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0378] Lipids conjugated with molecules other than PEG can also be used instead of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used instead of or in addition to PEG-lipids. Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in International Patent Application Publication Nos. 1996 / 010392, 1998 / 051278, 2002 / 087541, 2005 / 026372, 2008 / 030363, 2009 / 026372, 2010 / 026373, 2011 / 026374, 2012 / 026375, 2013 / 026376, 2014 / 026377, 2015 / 026378, 2016 / 026379, 2017 / 026379, 2010 / 026379, 2010 / 026379, 2011 / 026379, 2012 / 026379, 2013 / 026379, 2014 / 026379, 2015 / 026379, 2016 / 026379, 2015 / 026379, 2016 / 026379, 2017 / 026379, 2018 / 026379, 2015 / 026 147438, 2009 / 086558, 2012 / 000104, 2017 / 117528, 2017 / 099823, 2 015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 006282, U.S. Patent Application Publication Nos. 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0028664, 2017 / 0028665, 2019 / 0028666, 2020 / 0028667, 2021 / 0028668, 2022 / 0028669, 2023 / 0028669, 2024 / 0028669, 2025 / 0028669, 2026 / 0028669, 2027 / 0028669, 2028 / 0028669, 2029 / 0028669, 2030 ... Nos. 0376224, 2016 / 0317458, 2013 / 0303587, 2013 / 0303587, and 2011 / 0123453, as well as U.S. Patent Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559.

[0379] In some embodiments, the one or more additional compounds may be therapeutic agents. The therapeutic agents may be selected from any class suitable for therapeutic purposes. In other words, the therapeutic agents may be selected according to the desired therapeutic purpose and biological effect. For example, if the ceDNA in the LNPs is useful for treating cancer, the additional compound may be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if the LNPs containing ceDNA are useful for treating an infectious disease, the additional compound may be an anti-microbial agent (e.g., an antibiotic or an anti-viral compound). In yet another example, if the LNPs containing ceDNA are useful for treating an immune disease or disorder, the additional compound may be a compound that modulates an immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as ceDNAs encoding different proteins or different compounds (e.g., therapeutic agents), may be used in the compositions and methods of the present disclosure.

[0380] In some embodiments, the additional compound is an immunomodulatory agent, e.g., the additional compound is an immunosuppressant, hi some embodiments, the additional compound is an immunostimulant.

[0381] Also provided herein is a pharmaceutical composition comprising a synthetically produced ceDNA vector encapsulated in a lipid nanoparticle and a pharma- ceutically acceptable carrier or excipient.

[0382] In some aspects, the present disclosure provides lipid nanoparticle formulations further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, Tris, trehalose, and / or glycine.

[0383] The closed-end DNA vectors, including the ceDNA vectors produced using the synthesis process described herein, can be complexed with the lipid portion of the particle or encapsulated in the lipid portion of the lipid nanoparticle. In some embodiments, the DNA vectors, including the ceDNA vectors produced using the synthesis process described herein, can be fully encapsulated in the lipid portion of the lipid nanoparticle, thereby protecting it from degradation by nucleases in aqueous solution, for example. In some embodiments, the DNA vectors, including the ceDNA vectors produced using the synthesis process described herein in lipid nanoparticles, are not substantially degraded after exposure of the lipid nanoparticles to nucleases at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments, the ceDNA in the lipid nanoparticles is not substantially degraded after incubation of the particles in serum at 37°C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0384] In certain embodiments, the lipid nanoparticles are substantially non-toxic to a subject, e.g., a mammal, such as a human. In some aspects, the lipid nanoparticle formulation is a lyophilized powder.

[0385] In some embodiments, lipid nanoparticles are solid core particles with at least one lipid bilayer. In other embodiments, lipid nanoparticles have a non-bilayer structure, i.e., non-lamellar (i.e., non-bilayer) morphology. Without limitation, non-bilayer morphology can include, for example, three-dimensional tube, rod, cubic symmetry, etc. For example, lipid nanoparticle morphology (lamellar vs. non-lamellar) can be easily evaluated and characterized using Cryo-TEM analysis, for example, as described in US Patent Application Publication No. 2010 / 0130588.

[0386] In some further embodiments, lipid nanoparticles having a non-lamellar morphology are electron dense. In some aspects, the present disclosure provides lipid nanoparticles that are either unilamellar or multilamellar structures. In some aspects, the present disclosure provides lipid nanoparticles that include multivesicular particles and / or effervescent base particles.

[0387] By controlling the composition and concentration of lipid components, the rate at which lipid conjugates exchange out of lipid particles, which in turn controls the rate at which lipid nanoparticles become fusogenic, can be controlled.In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which lipid nanoparticles become fusogenic.Other methods that can be used to control the rate at which lipid nanoparticles become fusogenic will be clear to those skilled in the art based on this disclosure.It will also be clear that by controlling the composition and concentration of lipid conjugates, lipid particle size can be controlled.

[0388] The pKa of formulated cationic lipids can be correlated with the efficacy of LNPs for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010). A preferred range of pKa is from about 5 to about 7. The pKa of cationic lipids can be determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0389] VIII. Methods for delivering closed-ended DNA vectors In some embodiments, the closed-end DNA vectors, including the ceDNA vectors produced using the synthetic process described herein, can be delivered to target cells in vitro or in vivo by various suitable methods. Only the closed-end DNA vectors, including the ceDNA vectors produced using the synthetic process described herein, can be applied or injected. The closed-end DNA vectors, including the ceDNA vectors produced using the synthetic process described herein, can be delivered to cells without the aid of transfection reagents or other physical means. Alternatively, the closed-end DNA, including the ceDNA vectors produced using the synthetic process described herein, can be delivered using any art-known transfection reagent or other art-known physical means that facilitate DNA entry into cells, such as liposomes, alcohol, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, microinjection, electroporation, etc.

[0390] In another embodiment, the closed-end DNA vectors, including the ceDNA vectors produced using the synthetic process described herein, are administered to the CNS (e.g., brain or eye). For example, the ceDNA vectors 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 lobes, temporal lobes, parietal lobes, and frontal lobes, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. The ceDNA vectors may also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve. The ceDNA vectors may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture). The ceDNA vectors may also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0391] In some embodiments, closed-ended DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, can be administered to the desired region 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, and intramuscular delivery with retrograde delivery to motor neurons.

[0392] In some embodiments, closed-end DNA vectors, including ceDNA vectors produced using the synthetic processes described herein, are administered in a liquid formulation by direct injection (e.g., stereotactic injection) into the desired region or compartment of the CNS. In other embodiments, for example, synthetically produced ceDNA vectors 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, for example, ceDNA vectors can be administered as solid sustained release formulations (see, for example, U.S. Patent No. 7,201,898). In yet additional embodiments, for example, synthetically produced ceDNA vectors 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.). As an example, for example, synthetically produced ceDNA vectors can be delivered to muscle tissue and from there translocate into neurons.

[0393] IX. Additional Uses of ceDNA Vectors Compositions produced using the synthetic processes described herein, and closed-end DNA vectors, including ceDNA vectors, can be used to express target genes or transgenes for a variety of purposes. In some embodiments, the resulting 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 resulting 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 resulting transgene can be introduced (e.g., expressed) in a subject in an amount sufficient to treat a disease associated with reduced expression, lack of expression, or dysfunction of a gene. In some embodiments, the resulting transgene can be expressed in 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 (whose expression the resulting transgene suppresses or otherwise reduces). In yet other embodiments, the resulting transgene replaces or complements a defective copy of the native gene. It will be understood by those skilled in the art that the transgene may not itself be the open reading frame of a transcribed gene, instead it may be the promoter or repressor region of a target gene, and the ceDNA vector may modify such regions to so regulate expression of the gene of interest.

[0394] In some embodiments, the transgene encodes a protein or functional RNA intended to be used to generate an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating or preventing a disease state in a mammalian subject. The transgene may be introduced (e.g., expressed) into a patient in an amount sufficient to treat a disease associated with reduced expression, lack of expression, or dysfunction of the gene.

[0395] X. Method of Use Synthetically produced closed-end DNA vectors, such as ceDNA vectors as disclosed herein, can also be used in methods for delivering a nucleotide sequence of interest (e.g., a transgene) to a target cell (e.g., a host cell). This method can be specifically for delivering a transgene to a cell of a subject in need thereof and for treating a disease of interest. The present disclosure allows for in vivo expression of a transgene, such as a protein, an antibody, a nucleic acid such as miRNA, etc., encoded in the ceDNA vector in the cell of a subject, such that a therapeutic effect of expression of the transgene occurs. These results are seen in both in vivo and in vitro forms of closed-end DNA vector (e.g., ceDNA vector) delivery.

[0396] In addition, the present disclosure provides a method for the delivery of gene editing molecules in cells of a subject in need thereof, comprising multiple administrations of synthetically produced closed-end DNA vectors (e.g., ceDNA vectors) of the present disclosure that contain a nucleic acid or transgene of interest. Because the ceDNA vectors of the present disclosure do not induce immune responses as typically observed against encapsidated viral vectors, such multiple administration strategies will be more successful in ceDNA-based systems.

[0397] Synthetically produced closed-end DNA vector (e.g., ceDNA vector) nucleic acid is administered in an amount sufficient to transfect cells of desired tissue and to provide sufficient levels of gene transfer and expression without undue 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.

[0398] Delivery of closed-end DNA vectors (e.g., ceDNA vectors) is not limited to delivery gene replacement. For example, the synthetically produced closed-end DNA vectors (e.g., ceDNA vectors) described herein may be used with other delivery systems provided to provide par...

Claims

1. 1. A method for producing a closed-ended DNA (ceDNA) vector, said method comprising: (a) contacting a double-stranded DNA construct having a sense strand and an antisense strand with at least a first restriction endonuclease and at least a second restriction endonuclease; The double-stranded DNA construct a transgene expression cassette; a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette; said first restriction endonuclease is capable of cleaving said double-stranded DNA construct at said first cleavage site, and said second restriction endonuclease is capable of cleaving said double-stranded DNA construct at said second cleavage site; contacting the double-stranded DNA construct with the first restriction endonuclease and the second restriction endonuclease to release an insert having a first end comprising a first single-stranded overhang and a second end comprising a second single-stranded overhang; (b) ligating the first end including the first single-stranded overhang to a first oligonucleotide including one or more hairpin structures; (c) ligating the second end including the second single-stranded overhang to a second oligonucleotide including one or more hairpin structures; thereby producing the ceDNA vector.

2. 2. The method of claim 1, wherein the first oligonucleotide comprises an inverted terminal repeat (ITR) and / or the second oligonucleotide comprises an ITR.

3. the first oligonucleotide and the second oligonucleotide are different; the first oligonucleotide and the second oligonucleotide are the same; the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases; the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease; each of said oligonucleotides independently comprises one, two, three, four, or more stem-loop regions; and / or 2. The method of claim 1, wherein each of the oligonucleotides independently comprises two or three stem-loop regions.

4. the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site are separate and distinct from one another, and both sites are located upstream of the transgene expression cassette; the first cleavage site is about 1 to about 22 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense strand and the antisense strand of the construct; the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site are separate and distinct from each other, and both sites are located downstream of the expression cassette; the second cleavage site is about 1 to about 22 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct; and / or 2. The method of claim 1, wherein the first non-palindromic restriction endonuclease recognition site and the second non-palindromic restriction endonuclease recognition site are each double-stranded polynucleotides having different 5' to 3' nucleotide sequences in each of the sense strand and the antisense strand.

5. the first cleavage site is about 1 to about 8 nucleotides away from the first non-palindromic restriction endonuclease recognition site in at least one of the sense strand and the antisense strand of the construct; and / or 5. The method of claim 4, wherein the second cleavage site is about 1 to about 8 nucleotides away from the second non-palindromic restriction endonuclease recognition site in at least one of the sense and antisense strands of the construct.

6. one or both of the single-stranded overhangs at the ends of the insert is a 5' overhang; one or both of the single-stranded overhangs at the ends of the insert is a 3' overhang; and / or The method of claim 1, wherein the 5' end of each oligonucleotide is phosphorylated.

7. the first oligonucleotide comprising one or more hairpin structures and the second oligonucleotide comprising one or more hairpin structures are each single-stranded oligonucleotides that self-anneal to form a three-dimensional structure; and / or 2. The method of claim 1, wherein the first oligonucleotide comprising one or more hairpin structures and the second oligonucleotide comprising one or more hairpin structures each self-anneal to further form a single-stranded overhang at either the 5' end or the 3' end of each oligonucleotide.

8. The method of claim 7, wherein the three-dimensional structure is a T-shaped or Y-shaped stem-loop structure.

9. the first oligonucleotide and the second oligonucleotide each self-anneal to further form a single-stranded overhang at the 5' end of each oligonucleotide; and / or 8. The method of claim 7, wherein the first oligonucleotide and the second oligonucleotide each self-anneal to further form a single-stranded overhang at the 3' end of each oligonucleotide.

10. the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each about 1 to about 12 nucleotides in length; the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each about 1 to about 8 nucleotides in length; the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each about 2 to about 6 nucleotides in length; the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each about 3, about 4, about 5, or about 6 nucleotides in length; and / or 8. The method of claim 7, wherein the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each 3 or 4 nucleotides in length.

11. the nucleotide sequences in the 5' to 3' direction of the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are non-complementary to each other; the nucleotide sequences in the 5' to 3' direction of the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are the same; the first oligonucleotide and the second oligonucleotide have the same nucleotide sequence; the single-stranded overhangs at each end of the insert comprise the same 5' to 3' nucleotide sequence; the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each complementary to both of the single-stranded overhangs at the ends of the insert; and / or 11. The method of claim 10, wherein the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide differ in 5' to 3' nucleotide sequence.

12. the first oligonucleotide and the second oligonucleotide comprise different nucleotide sequences; the nucleotide sequence of the single-stranded overhang at each end of the insert differs in the 5' to 3' direction; 2. The method of claim 1, wherein the single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide are each complementary to only one of the single-stranded overhangs at the ends of the insert.

13. one or both of the first oligonucleotide and the second oligonucleotide are synthetic; the first oligonucleotide and the second oligonucleotide are each about 40 nucleotides to about 75 nucleotides in length; the first oligonucleotide and the second oligonucleotide are each about 45 nucleotides to about 65 nucleotides in length; and / or 2. The method of claim 1, wherein the first oligonucleotide and the second oligonucleotide each independently comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

14. each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the first oligonucleotide and each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the second oligonucleotide comprises a stem region that is at least about 4 base pairs in length; each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the first oligonucleotide and each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the second oligonucleotide comprises a stem region that is about 4 base pairs to about 20 base pairs in length; each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the first oligonucleotide and each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the second oligonucleotide comprises a stem region that is about 4 base pairs to about 15 base pairs in length; each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the first oligonucleotide and each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the second oligonucleotide comprises a stem region that is about 4 base pairs to about 6 base pairs in length; each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the first oligonucleotide and each hairpin structure and / or each T-shaped or Y-shaped stem-loop structure of the second oligonucleotide comprises a stem region that is from about 6 base pairs to about 8 base pairs in length; and / or The method of claim 1 , wherein the stem region length does not include any single-stranded overhangs.

15. at least one of the restriction endonucleases is a Type IIS restriction endonuclease; the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease; the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases; each of the first restriction endonuclease and the second restriction endonuclease is a Type IIS restriction endonuclease; each of the first restriction endonuclease and the second restriction endonuclease independently selected from AcuI, AlwI, Alw26I, BasI, Bbsl, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, Bsr a type IIS restriction endonuclease selected from the group consisting of: DI, BsrI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers thereof; the at least one Type IIS restriction endonuclease is selected from the group consisting of Bbsl, BsaI, Esp3I, and SapI, and isoschizomers thereof; the at least one Type IIS restriction endonuclease is BsaI or an isoschizomer thereof; and / or 2. The method of claim 1, wherein the at least one Type IIS restriction endonuclease is Esp3I or an isoschizomer thereof.

16. 2. The method of claim 1, wherein after the ligation, the first non-palindromic restriction endonuclease recognition site and the second non-palindromic restriction endonuclease recognition site are not reproduced in the resulting ceDNA vector.

17. 2. The method of claim 1, wherein the double-stranded DNA construct further comprises at least a first partial ITR and a second partial ITR, each flanking the transgene expression cassette.

18. the first partial ITR is upstream of the transgene expression cassette and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site; the second partial ITR is downstream of the transgene expression cassette and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site; the first cleavage site is adjacent to the first partial ITR and the second cleavage site is adjacent to the second partial ITR; the double-stranded DNA construct further comprises a first spacer between the first partial ITR and the transgene expression cassette; and / or 18. The method of claim 17, wherein the double-stranded DNA construct further comprises a second spacer between the second partial ITR and the transgene expression cassette.

19. 2. The method of claim 1, wherein the double-stranded DNA construct is selected from the group consisting of a bacmid, a plasmid, a minicircle, or a linear double-stranded DNA molecule.

20. 2. The method of claim 1, wherein the resulting ceDNA vector comprises the transgene expression cassette and at least a first ITR and a second ITR, each flanking the transgene expression cassette.

21. the first ITR is upstream of the transgene expression cassette; the second ITR is downstream of the transgene expression cassette; the first ITR comprises a nucleotide sequence from the first oligonucleotide and the first partial ITR; the second ITR comprises a nucleotide sequence from the second oligonucleotide and the second partial ITR; the first ITR lacks the first non-palindromic restriction endonuclease recognition site; the second ITR lacks the second non-palindromic restriction endonuclease recognition site; the first ITR and the second ITR each comprise a hairpin structure and / or a T-shaped or Y-shaped stem-loop structure; and / or 21. The method of claim 20, wherein the T-shaped or Y-shaped stem-loop structure comprises a stem comprising an AA' and a DD' stem region and two BB' and CC' loops.

22. One or both of the first ITR and the second ITR is selected from the group consisting of an adeno-associated virus (AAV) ITR and an ITR derived from an AAV. one of the first ITR or the second ITR is a wild-type ITR; Both the first ITR and the second ITR are wild-type ITRs. one or both of the first ITR and the second ITR is a modified ITR; the first ITR and the second ITR are symmetrical or substantially symmetrical to each other; the first ITR and the second ITR are asymmetric ITRs; and / or 21. The method of claim 20, wherein one or both of the first ITR and the second ITR comprises one or more modifications selected from the group consisting of additions, deletions, truncations, and point mutations.

23. the one or more modifications are located in the A-A' stem region, the B-B' loop, the C-C' loop, and / or the D-D' stem region of one or both of the first ITR and the second ITR; the one or more modifications are located in the BB' loop and / or the CC' loop of one or both of the first and second ITRs; and / or 23. The method of claim 22, wherein the BB' loop and the CC' loop of one of the first ITR and the second ITR are truncated.

24. the transgene expression cassette further comprises a first spacer between the first ITR and the transgene expression cassette. the transgene expression cassette further comprises a first spacer between the second ITR and the transgene expression cassette; and / or 21. The method of claim 20, wherein the transgene expression cassette further comprises a first spacer between the first ITR and the transgene expression cassette, and a second spacer between the second ITR and the transgene expression cassette.

25. The method of claim 1 , wherein the transgene expression cassette comprises a transgene.

26. 26. The method of claim 25, wherein the transgene encodes a therapeutic protein.

27. 27. The method of claim 26, wherein the therapeutic protein is selected from the group consisting of an enzyme, a clotting factor or cofactor, an antibody or antigen-binding fragment thereof, an antigen, a gene-editing protein, and a cytotoxic protein.

28. 2. The method of claim 1, wherein the transgene expression cassette further comprises a genetic element selected from the group consisting of a promoter, an enhancer, an intron, a post-transcriptional regulatory element, and a polyadenylation signal.

29. 10. The method of claim 1, wherein the ligating is performed by a ligase or an AAV Rep protein.

30. 30. The method of claim 29, wherein the ligase is T4 ligase.

31. the method further comprising isolating or purifying the resulting ceDNA vector. the method further comprising isolating or purifying the insert prior to said ligating. the method does not include isolating or purifying the insert prior to the ligating; and / or 10. The method of claim 1, wherein steps (a), (b), and (c) are carried out in a single reaction vessel.

32. 2. The method of claim 1, wherein the resulting ceDNA vector comprises at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the monomer species of the ceDNA vector.

33. 1. A DNA vector for use in the synthetic production of closed-ended DNA vectors (ceDNA), comprising: a multiple cloning site capable of accepting a transgene; a first non-palindromic restriction endonuclease recognition site and corresponding first cleavage site upstream of said multiple cloning site; a second non-palindromic restriction endonuclease recognition site and corresponding second cleavage site downstream of the multiple cloning site; a first partial ITR and a second partial ITR, each flanking the multiple cloning site.

34. the first partial ITR is upstream of the multiple cloning site and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site; the second partial ITR is downstream of the multiple cloning site and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site; the multiple cloning site is capable of accepting a transgene and one or more additional genetic elements selected from the group consisting of a promoter, an enhancer, an intron, a post-transcriptional regulatory element, and a polyadenylation signal; 34. The DNA vector of claim 33, wherein the first non-palindromic restriction endonuclease recognition site is specific for a first restriction endonuclease and the second non-palindromic restriction endonuclease recognition site is specific for at least a second restriction endonuclease.

35. 34. The DNA vector of claim 33, further comprising one or more spacers and / or further comprising an origin of replication and a selectable marker gene.

36. the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease; the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases; at least one of the restriction endonucleases is a Type IIS restriction endonuclease; each of the first restriction endonuclease and the second restriction endonuclease is a Type IIS restriction endonuclease; and / or 34. The DNA vector of claim 33, wherein the DNA vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:

17.

37. Each type IIS restriction endonuclease independently binds AcuI, AlwI, Alw26I, BasI, BbsI, BbvI, BceAI, BcgI, BCiVI, BcoDI, BruAI, BmrI, BpiI, BpuEI, BsaI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, Bs and / or selected from the group consisting of rI, BtgZI, BtsCI, BtsI, MutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, LguI, MboII, MlyI, MmeI, MnlI, Mva1269I, NmeAIII, PaqCI, PleI, SapI, SfaNI, and isoschizomers thereof; and / or 37. The DNA vector of claim 36, wherein each Type IIS restriction endonuclease is selected from the group consisting of Bbsl, BsaI, Esp3I, and SapI, and isoschizomers thereof.

38. 1. A kit for preparing a closed-ended DNA (ceDNA) vector containing a transgene, the kit comprising: A DNA vector according to claim 33; at least one restriction endonuclease capable of cleaving the DNA vector at the multiple cloning site to enable the multiple cloning site to accept a transgene; at least one restriction endonuclease capable of cleaving at said first cleavage site and said second cleavage site; Ligase and A kit including instructions for use.

39. 39. The kit of claim 38, further comprising at least one oligonucleotide comprising one or more hairpin structures.

40. 1. A double-stranded circular DNA construct engineered to facilitate the preparation of a closed-ended DNA (ceDNA) vector containing a transgene expression cassette, the double-stranded circular DNA construct comprising: a transgene expression cassette; a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette; a first partial ITR and a second partial ITR, each flanking the transgene expression cassette.

41. the first partial ITR is upstream of the transgene expression cassette and downstream of the first non-palindromic restriction endonuclease recognition site and the corresponding first cleavage site; the second partial ITR is downstream of the transgene expression cassette and upstream of the second non-palindromic restriction endonuclease recognition site and the corresponding second cleavage site; and / or 41. The double-stranded circular DNA construct of claim 40, wherein the first non-palindromic restriction endonuclease recognition site is specific for a first restriction endonuclease and the second non-palindromic restriction endonuclease recognition site is specific for at least a second restriction endonuclease.

42. the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease; or 42. The double-stranded circular DNA construct of claim 41, wherein the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases.

43. at least one of the restriction endonucleases is a Type IIS restriction endonuclease; or 41. The double-stranded circular DNA construct of claim 40, wherein each of the first restriction endonuclease and the second restriction endonuclease is a Type IIS restriction endonuclease.

44. 1. A kit for preparing a closed-ended DNA (ceDNA) vector containing a transgene expression cassette, the kit comprising: The double-stranded DNA construct of claim 40; and at least one restriction endonuclease capable of cleaving the double-stranded DNA construct at the first cleavage site and the second cleavage site; Ligase and A kit including instructions for use.

45. 45. The kit of claim 44, further comprising at least one oligonucleotide comprising one or more hairpin structures.

46. 1. A method for producing a double-stranded DNA construct from a plasmid template via rolling circle amplification, comprising: (a) contacting the plasmid template with a thermostable polymerase having strand displacement activity, wherein the ratio of plasmid template concentration (in ng / μl) to polymerase concentration (in U / μl) is greater than about 1; (b) contacting the plasmid template with an oligonucleotide primer and dNTPs; (c) incubating the plasmid template, the polymerase, the oligonucleotide primers, and the dNTPs at a temperature of about 40° C. or less for a period of at least about 5 hours; thereby producing a double-stranded DNA construct.

47. the ratio of plasmid template concentration (in ng / μl) to polymerase concentration (in U / μl) is greater than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20; The plasmid template concentration is about 0.01 ng / μl, about 0.05 ng / μl, about 0.1 ng / μl, about 0.15 ng / μl, about 0.2 ng / μl, about 0.21ng / μl, about 0.22ng / μl, about 0.23ng / μl, about 0.24ng / μl, about 0.2ng / μl, about 0.26ng / μl, about 0.2 7ng / μl, about 0.28ng / μl, about 0.29ng / μl, about 0.3ng / μl, about 0.35ng / μl, about 0.4ng / μl, about 0.45ng / μl μl, about 0.5ng / μl, about 0.6ng / μl, about 0.7ng / μl, about 0.8ng / μl, about 0.9ng / μl, or about 1.0ng / μl, The polymerase concentration is about 0.01U / μl, about 0.02U / μl, about 0.03U / μl, about 0.04U / μl, about 0.05U / μl, about 0.06U / μl, about 0.07U / μl, about 0.08U / μl , about 0.09U / μl, about 0.1U / μl, about 0.15U / μl, about 0.2U / μl, about 0.25U / μl, about 0.3U / μl, about 0.35U / μl, about 0.4U / μl, or about 0.45U / μl, the temperature in step (c) is less than about 40°C, about 39°C, about 38°C, about 37°C, about 36°C, about 35°C, about 34°C, about 33°C, about 32°C, about 31°C, about 30°C, about 29°C, about 28°C, about 27°C, about 26°C, about 25°C, about 24°C, about 23°C, about 22°C, or about 21°C; the period of time is at least about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours; the period of time is less than about 6 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 21 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, or about 40 hours; the oligonucleotide primer concentration is less than about 50 μM; the oligonucleotide primer concentration is at least about 10 μM; The thermostable polymerase is Phi29 DNA polymerase or a derivative or variant thereof. The method is carried out in a total reaction volume of at least about 100 μl. The method may include at least about 100 μl, about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml. , about 9 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml , about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml, about 800 ml, about 900 ml, about 1 L, about 2 L, about 3 L, 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, or about 1000 L total reaction volume; and / or 47. The method of claim 46, wherein the concentration of the dNTP is about 4 mM.

48. the oligonucleotide primers hybridize to backbone sequences in the plasmid template; and / or 47. The method of claim 46, wherein the oligonucleotide primer is a universal primer.

49. 1. A method for preparing a closed-ended DNA (ceDNA) vector, said method comprising: (a) contacting the double-stranded DNA construct with at least one restriction endonuclease; the restriction endonuclease is a Type IIS restriction endonuclease; The construct comprises: a transgene expression cassette; a first non-palindromic restriction endonuclease recognition site and a corresponding first cleavage site upstream of the transgene expression cassette; a second non-palindromic restriction endonuclease recognition site and a corresponding second cleavage site downstream of the transgene expression cassette; contacting the construct with a Type IIS restriction endonuclease capable of cleaving the construct at the first cleavage site and the second cleavage site to release the insert having single-stranded overhangs at the 5' and 3' ends of the insert that match the overhangs of a first inverted terminal repeat (ITR) oligonucleotide and a second inverted terminal repeat (ITR) oligonucleotide; (b) ligating the 5' and 3' ends of the insert to the first inverted terminal repeat (ITR) oligonucleotide and the second ITR oligonucleotide to form the ceDNA vector.

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