Synthetic generation of circular DNA vectors

Abstract

Provided herein is an improved method for producing therapeutic circular DNA vectors, pharmaceutical compositions produced by such methods, and methods for using the pharmaceutical compositions.The present invention is based at least in part on a cell-free manufacturing process that includes a restriction digestion and ligation scheme, for example, a restriction digestion process that includes a type IIS restriction enzyme.The method provided herein is suitable for large-scale production of high-purity compositions of therapeutic circular DNA vectors.

Description

[Technical field]

[0001] cross reference This international application claims priority to U.S. Provisional Patent Application No. 63 / 248,801.

[0002] In general, the present invention relates to synthetic circular DNA vectors. [Background technology]

[0003] Gene therapy has emerged as a promising approach to treat a wide variety of diseases or disorders in human patients. Recombinant adeno-associated virus (rAAV) vectors have an established track record of highly efficient gene transfer in human patients and various model systems. The genome of rAAV vectors is advantageous due to its ability to persist in vivo as circular episomes throughout the lifespan of target cells. On the other hand, rAAV-based vectors have significant drawbacks, such as limited maximum payload, immunogenicity, and manufacturing inefficiencies.

[0004] To address some of these challenges in rAAV technology, non-viral alternatives have gained momentum in recent years. However, the development of a scalable non-viral gene therapy platform with the efficacy and persistence of rAAV has proven difficult to achieve. For example, traditional bacterial plasmid DNA vectors are versatile tools in gene delivery, but are limited by their bacterial origin. Bacterial elements of plasmid DNA vectors, such as antibiotic resistance genes, origins of replication, and impurities from bacterial hosts, such as endotoxins, bacterial genomic DNA and RNA, and host cell proteins, can lead to immunogenicity and loss of gene expression due to transcriptional silencing.

[0005] Although minor improvements in plasmid DNA vectors have been achieved by removing bacterial elements through site-specific recombination, such processes still rely on production in bacterial host cells, which inherently poses the risk of unacceptable impurity profiles in the resulting pharmaceutical composition. Synthetic DNA vectors are produced in cell-free conditions, avoiding these risks, but their scalability is currently limited by inefficient manufacturing processes, where gel purification steps and multiple restriction enzymes are often required.

[0006] Thus, there is a need in the art for controllable, scalable methods for producing non-viral DNA vectors with high purity and efficiency. Summary of the Invention

[0007] Provided herein are improved cell-free methods for producing therapeutic circular DNA vectors, pharmaceutical compositions produced by such methods, and methods of using the pharmaceutical compositions. The present invention is based at least in part on the development of cell-free manufacturing processes that include restriction digestion and ligation schemes, such as a restriction digestion process that includes a type IIS restriction enzyme. Furthermore, applicants have identified conditions (e.g., DNA and ligase concentrations), step sequences, and highly efficient overhang compositions that result in dramatic improvements in synthetic DNA vector manufacturing efficiency. The methods and compositions provided herein are suitable for large-scale production of high-purity compositions of therapeutic circular DNA vectors.

[0008] In one aspect, provided herein is a method for generating a therapeutic circular DNA vector, comprising the steps of: (a) providing a sample comprising a template DNA vector (e.g., a plasmid DNA vector) comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification (e.g., Phi29-mediated rolling circle amplification) to generate linear concatemers; (c) isolating a first site and a second site for each unit of the linear concatemers. (c) digesting the linear concatemer with a type IIS restriction enzyme that cuts a site, wherein the first and second sites flank a therapeutic sequence and form self-complementary overhangs, and wherein digestion produces a linear therapeutic fragment and a linear backbone fragment, wherein the linear therapeutic fragment comprises the therapeutic sequence and the linear backbone fragment comprises the backbone sequence or a portion thereof; and (d) contacting the linear backbone fragment and the linear therapeutic fragment with a ligase to produce a circular backbone and a therapeutic circular DNA vector lacking a type IIS restriction site. In some embodiments, the linear backbone fragment of (c) comprises a type IIS restriction site and the circular backbone of (d) comprises a type IIS restriction site, and the type IIS restriction enzyme cuts the circular backbone and does not cut the therapeutic circular DNA vector.

[0009] In another aspect, provided herein is a method for generating a therapeutic circular DNA vector, comprising the steps of: (a) providing a sample comprising a template DNA vector (e.g., a plasmid DNA vector) comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) amplifying the template DNA vector with one or more restriction enzymes that cleave at least a first site, a second site, and a third site per unit of the linear concatemers. digesting the linear concatemer, where (i) the first and second sites flank the therapeutic sequence and form self-complementary overhangs, and (ii) the third site is within the scaffold sequence and forms a non-complementary overhang to the first or second site, such that digestion produces a linear therapeutic fragment that contains the therapeutic sequence and at least two linear scaffold fragments, each containing a portion of the scaffold sequence; and (d) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0010] In some embodiments of any of the above aspects, the method further comprises diluting the DNA between step (c) and step (d). In some embodiments, the DNA concentration at the start of step (d) is greater than or equal to 20 μg / mL but less than 160 μg / mL. In some embodiments, the DNA concentration at the start of step (d) is about 40 μg / mL. In some embodiments, the DNA concentration at the start of step (d) is about 80 μg / mL. In some embodiments, the ligase concentration in step (d) is about 10 to about 20 U of ligase per μg of DNA. In some embodiments, the ligase is T4 ligase. In some embodiments, no temperature increase is performed immediately after step (d).

[0011] In some embodiments, the linear concatemers are digested with a single restriction enzyme that cuts at the first site, the second site, and the third site (e.g., step (b) requires a single (i.e., only one) restriction enzyme (e.g., a Type IIS restriction enzyme, e.g., BsaI)). In some embodiments, the one or more restriction enzymes cut the linear concatemers at a fourth site per unit, where the fourth site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site, and digestion generates at least three linear scaffold fragments, each of which comprises a portion of the scaffold sequence. In some embodiments, step (b) requires a single restriction enzyme (e.g., a Type IIS restriction enzyme, e.g., BsaI) that cuts the linear concatemer unit-by-unit at a fourth site, where the fourth site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site, such that digestion generates at least three linear scaffold fragments, each of which contains a portion of the scaffold sequence.

[0012] In some embodiments, a restriction enzyme inactivation step does not precede step (d) (e.g., heat inactivation of the restriction enzyme does not precede step (d)). In some embodiments, no temperature increase is performed between steps (c) and (d). In some embodiments, the temperature is reduced between steps (c) and (d). In some embodiments, no temperature increase is performed immediately after step (d). In some embodiments where heat inactivation is not performed, the reaction is performed in a disposable vessel that is not suitable for high temperatures. In some embodiments, steps (c) and (d) are performed simultaneously.

[0013] In some embodiments, the method further comprises increasing the temperature of the solution containing the therapeutic circular DNA vector to about 65°C.

[0014] In some embodiments, the method further comprises (e) contacting the therapeutic circular DNA vector with a topoisomerase or helicase. In some embodiments, step (e) is carried out at about 37° C.

[0015] In some embodiments, the method further comprises (f) contacting the linear backbone fragment with an exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease). In some embodiments, step (f) is carried out at about 37°C.

[0016] In some embodiments, the method further comprises (e) contacting the therapeutic circular DNA vector with a topoisomerase or helicase, and (f) contacting the linear backbone fragment with an exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease), where no enzyme inactivation step is performed between steps (e) and (f). In some embodiments, contacting the therapeutic circular DNA vector with the topoisomerase or helicase is performed prior to contacting the linear backbone fragment with the exonuclease (e.g., a terminal exonuclease). In other embodiments, contacting the therapeutic circular DNA vector with the topoisomerase or helicase is performed after contacting the linear backbone fragment with the exonuclease (e.g., a terminal exonuclease).

[0017] In some embodiments of any of the above methods, the restriction enzyme is provided at a concentration of about 0.5 U / μg to about 20 U / μg, e.g., about 1 U / μg DNA to about 10 U / μg DNA, e.g., about 2 U / μg DNA to about 5 U / μg DNA, e.g., about 2.5 U / μg DNA. For example, restriction enzymes are approximately 0.5U / μg DNA, 1.0U / μg DNA, 1.5U / μg DNA, 2.0U / μg DNA, 2.5U / μg DNA, 3.0U / μg DNA, 3.5U / μg DNA, 4.0U / μg DNA, 4.5U / μg DNA, 5.0U / μg DNA, 5.5U / μg DNA, 6.0U / μg DNA, 6.5U / μg DNA, 7.0U / μg DNA, 7.5U / μg DNA, 8.0U / μg DNA, 8.5U / μg DNA, 9.0U / μg DNA, 9.5U / μg DNA, 10.0U / μg DNA, 11U / μg DNA, 12U / μg DNA, 13U / μg DNA, 14U / μg DNA, 15U / μg DNA, 16U / μg DNA, 17U / μg The restriction enzyme may be provided at a concentration of about 0.5 U / μg to about 2.5 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, or 20 U / μg DNA. In some embodiments, the restriction enzyme is provided at a concentration of about 0.5 U / μg to about 2.5 U / μg DNA.

[0018] In some embodiments, the restriction enzyme is provided at a concentration of about 2.5 U / μg.

[0019] In some embodiments, digestion (e.g., step (c)) comprises incubation for 1 to 12 hours, e.g., about 1 hour. In some embodiments, digestion (e.g., step (c)) comprises incubation for 1 hour or less.

[0020] In some embodiments, the ligase has a concentration of 50 U per μg of DNA (U / μg) or less (e.g., 40 U / μg DNA or less, 30 U / μg DNA or less, 25 U / μg DNA or less, 20 U / μg DNA or less, 15 U / μg DNA or less, 10 U / μg DNA or less, 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 20 U / μg DNA; e.g., 0.1 U / μg DNA to 30 U / μg DNA, 0.1 U / μg DNA to 20 U / μg DNA, 0.2 U / μg DNA to 15 U / μg DNA, 0.5 U / μg DNA to 12 U / μg DNA, or 1 U / μg DNA). DNA~10U / μg DNA; For example, 0.1U / μg DNA~0.5U / μg DNA, 0.5U / μg DNA~1.0U / μg DNA, 1.0U / μg DNA~2.0U / μg DNA, 2.0U / μg DNA~3.0U / μg DNA, 3.0U / μg DNA~4.0U / μg DNA, 4.0U / μg DNA~5.0U / μg DNA, 5.0~6.0U / μg DNA, 6.0U / μg DNA~7.0U / μg DNA, 7.0U / μg DNA~8.0U / μg DNA, 8.0U / μg DNA~9.0U / μg DNA, 9.0U / μg DNA~11U / μg DNA, 11U / μg DNA~12U / μg DNA, 12U / μg DNA~15U / μg DNA, 15U / μg The ligase may be provided at a concentration of 20 U / μg DNA, 20 U / μg DNA to 25 U / μg DNA, 25 U / μg DNA to 30 U / μg DNA, 30 U / μg DNA to 35 U / μg DNA, 35 U / μg DNA to 40 U / μg DNA, or 40 U / μg DNA to 50 U / μg DNA. In some embodiments, the ligase is provided at a concentration of 20 U / μg DNA or less. In some embodiments, the ligase is provided at a concentration of about 10 U / μg DNA. In some embodiments, the ligase is T4 ligase.

[0021] In some embodiments, the topoisomerase is at 10 U per μg of DNA (U / μg) or less (e.g., 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 10 U / μg DNA, e.g., 0.5 U / μg DNA to 8 U / μg DNA, or 1 U / μg DNA to 5 U / μg DNA; e.g., 0.1 U / μg DNA to 0.5 U / μg DNA, 0.5 U / μg DNA to 1.0 U / μg DNA, 1.0 U / μg DNA to 2.0 U / μg DNA, 2.0 U / μg DNA to 3.0 U / μg DNA, 3.0 U / μg DNA to 4.0 U / μg DNA, 4.0 U / μg DNA, The topoisomerase is provided at concentrations of: 5.0U / μg DNA, 5.0-6.0U / μg DNA, 6.0U / μg DNA-7.0U / μg DNA, 7.0U / μg DNA-8.0U / μg DNA, 8.0U / μg DNA-9.0U / μg DNA, or 9.0U / μg DNA-10U / μg DNA.

[0022] In some embodiments, the topoisomerase is a type II topoisomerase. In some embodiments, the topoisomerase is a gyrase. In some embodiments, the topoisomerase is a topoisomerase IV.

[0023] In some embodiments, the exonuclease (e.g., a terminal exonuclease, e.g., a T5 exonuclease) is provided at a concentration of about 0.5 U / μg to about 20 U / μg, e.g., about 0.5 U / μg to about 10 U / μg, e.g., about 1 U / μg to about 10 U / μg, e.g., about 2 U / μg to about 5 U / μg, e.g., about 2.5 U / μg. For example, the exonuclease (e.g., a terminal exonuclease) is provided at a concentration of about 0.5 U / μg, 1.0 U / μg, 1.5 U / μg, 2.0 U / μg, 2.5 U / μg, 3.0 U / μg, 3.5 U / μg, 4.0 U / μg, 4.5 U / μg, 5.0 U / μg, 5.5 U / μg, 6.0 U / μg, 6.5 U / μg, 7. .0U / μg, 7.5U / μg, 8.0U / μg, 8.5U / μg, 9.0U / μg, 9.5U / μg, 10.0U / μg, 11U / μg, 12U / μg , 13U / μg, 14U / μg, 15U / μg, 16U / μg, 17U / μg, 18U / μg, 19U / μg, or 20U / μg.

[0024] In some embodiments, step (f) is performed two or more times (e.g., two, three, or four times). In some embodiments, step (f) comprises incubation for 1 hour to 12 hours. In some embodiments, step (f) comprises incubation for 1 hour to 18 hours. In some embodiments, step (f) comprises incubation for 3 hours to 18 hours. In some embodiments, the exonuclease is a terminal exonuclease (e.g., T5 exonuclease).

[0025] In some embodiments of any of the above methods, the methods further include (g) passing the therapeutic circular DNA vector through a column (e.g., a capture column), and / or (h) precipitating the therapeutic circular DNA vector with isopropyl alcohol.

[0026] In some embodiments, step (b) is performed using site-specific primers. In other embodiments, step (b) is performed using random primers.

[0027] In some embodiments, the amount of therapeutic circular DNA vector generated is at least 5-fold the amount of template DNA vector (e.g., plasmid DNA vector) in the sample in step (a).

[0028] In some embodiments, no DNA purification or gel extraction step is performed prior to step (d).

[0029] In some embodiments, the amount of therapeutic circular DNA in the solution in step (d) is at least 2.0% by weight of the amount of linear concatemers in step (b) (e.g., at least 3.0% by weight, at least 4.0% by weight, at least 5.0% by weight, at least 6.0% by weight, at least 7.0% by weight, at least 8.0% by weight, at least 9.0% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, or at least 50% by weight) of the amount of linear concatemers in step (b).

[0030] In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 1.0 mg (e.g., 1.0 mg to 10 mg, 2.0 mg to 10 mg, 3.0 mg to 10 mg, 4.0 mg to 10 mg, or 5.0 mg to 10 mg; e.g., 1.0 mg to 2.5 mg, 2.5 mg to 5.0 mg, 5.0 mg to 7.5 mg, or 7.5 mg to 10 mg). In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 2.0 mg. For example, in some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 5.0 mg.

[0031] In some embodiments, the concentration of the therapeutic circular DNA in the solution after step (d) is 1.0 μg / mL to 1.0 mg / mL without any purification or concentration being performed (e.g., 5.0 μg / mL to 100 μg / mL or 10 μg / mL to 50 μg / mL without any purification or concentration being performed, e.g., 1.0 μg / mL to 10 μg / mL, 5.0 μg / mL to 10 μg / mL, 10 μg / mL to 50 μg / mL, 50 μg / mL to 100 μg / mL, or more without any purification or concentration being performed).

[0032] In some embodiments, the volume of the solution in step (d) is at least 5 liters (e.g., between 5 liters and 200 liters, e.g., between 7 liters and 100 liters, between 10 liters and 80 liters, between 15 liters and 75 liters, or between 20 liters and 70 liters, e.g., at least 1.0 liter, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, or at least 100 liters).

[0033] In some embodiments, step (b) is performed in a reaction vessel having a volume of at least 0.5 liters (e.g., at least 1.0 liters, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 150 liters, or at least 200 liters). In some embodiments, step (b) is performed in a reaction vessel having a volume of at least 5 liters. Additionally or alternatively, steps (c) and (d) are performed in a reaction vessel having a volume of at least 0.5 liters (e.g., at least 1.0 liters, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 150 liters, or at least 200 liters). For example, in some embodiments, steps (c) and (d) are performed in a reaction vessel having a volume of at least 5 liters. In some embodiments, each of steps (b)-(d) is performed in a reaction vessel having a volume of at least 0.5 liters (e.g., at least 1.0 liters, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 150 liters, or at least 200 liters). In some embodiments, each of steps (b)-(d) is performed in a reaction vessel having a volume of at least 5 liters.

[0034] In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 20% of the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a) (e.g., at least 50%, at least 75%, at least 100%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a), e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a). In certain embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 5-fold the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a). In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 10-fold the amount of template DNA vector (eg, plasmid DNA vector) provided in step (a).

[0035] In another aspect, provided herein is a method of removing a backbone sequence from a DNA molecule to generate a therapeutic circular DNA vector, the DNA molecule comprising a backbone sequence and a therapeutic sequence, the method comprising: (a) digesting the DNA molecule with a type IIS restriction enzyme that cleaves a first site and a second site for each unit of a linear concatemer, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs, wherein digestion generates a linear therapeutic fragment and a linear backbone fragment, the linear therapeutic fragment comprising the therapeutic sequence, and the linear backbone fragment comprising at least a portion of the backbone sequence and a type IIS restriction site; and (b) contacting the linear backbone fragment and the linear therapeutic fragment with a ligase to generate a circular backbone that comprises the type IIS restriction site and a therapeutic circular DNA vector lacking a type IIS restriction site.

[0036] In another aspect, the method includes providing a sample comprising a template DNA vector (e.g., a plasmid DNA vector) comprising a therapeutic sequence, and amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers. The linear concatemers are digested with a restriction enzyme that cuts at least two sites in the linear concatemers per unit of the template DNA vector to generate linearized fragments of the DNA vector. The method further includes self-ligating the linearized fragments of the DNA vector comprising the therapeutic sequence to generate a therapeutic circular DNA vector. In some embodiments, the digestion and self-ligation are performed simultaneously. The sample can then be treated with a topoisomerase or helicase. In some embodiments, the method further includes digesting the sample with an exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease).

[0037] In another aspect, the method includes providing a sample containing a template DNA vector (e.g., a plasmid DNA vector) containing a therapeutic sequence, and amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers. The method further includes digesting the linear concatemers with a restriction enzyme to generate linearized fragments of the DNA vector. The linear concatemers have multiple copies of the template DNA vector, each copy having a unit length, and the linear concatemers have a unit length of multiple vectors. The restriction enzyme cuts at least two sites in the linear concatemers for each unit of the template DNA vector. The method further includes self-ligating the linearized fragments of the DNA vector to generate a therapeutic circular DNA vector. The method also includes digesting the sample with an exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease). In some embodiments, the method further includes treating the sample with a topoisomerase or a helicase. In some embodiments, the digestion and self-ligation are performed simultaneously.

[0038] In another aspect, the method includes providing a sample containing a template DNA vector (e.g., a plasmid DNA vector) containing a therapeutic sequence, and amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers. The method further includes digesting the linear concatemers with a restriction enzyme to generate linearized fragments of the DNA vector. The restriction enzyme cuts at least two sites in the linear concatemers per unit of the template DNA vector. The method further includes self-ligating the linearized fragments of the DNA vector to generate a therapeutic circular DNA vector. The method may further include treating the sample with a topoisomerase or helicase, and digesting the sample with an exonuclease (e.g., a terminal exonuclease). In some embodiments, the digestion and self-ligation are performed simultaneously (in the same reaction conditions).

[0039] In another aspect, the present invention provides a method for removing a backbone sequence from a DNA molecule to generate a therapeutic circular DNA vector. The DNA molecule includes a backbone sequence and a therapeutic sequence. The method includes the steps of (a) digesting the DNA molecule with one or more restriction enzymes that cleave at least a first site, a second site, and a third site per unit of the DNA molecule, where (i) the first and second sites are adjacent to the therapeutic sequence and form self-complementary overhangs, and (ii) the third site is within the backbone sequence and forms a non-complementary overhang to the first or second site, and digestion produces a linear therapeutic fragment that includes the therapeutic sequence and at least two linear backbone fragments, each of which includes a portion of the backbone sequence; and (b) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0040] In some embodiments, the linear concatemer is digested with a single restriction enzyme that cleaves at a first site, a second site, and a third site. In some embodiments, one or more restriction enzymes cleave a fourth site in the DNA molecule, where the fourth site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site, and digestion produces at least three linear scaffold fragments, each of which comprises a portion of the scaffold sequence. In some embodiments, a single restriction enzyme cleaves a fourth site in the DNA molecule, where the fourth site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site, and digestion produces at least three linear scaffold fragments, each of which comprises a portion of the scaffold sequence.

[0041] In some embodiments, the DNA molecule is a concatemer generated by amplification of a template DNA vector. In some embodiments, the DNA molecule is a template DNA vector. In some embodiments, the template DNA vector is a plasmid DNA vector.

[0042] In some embodiments, the single restriction enzyme is a Type IIS restriction enzyme, for example, BsaI.

[0043] In some embodiments, a restriction enzyme inactivation step does not precede step (b).

[0044] In some embodiments, no temperature increase is performed between steps (a) and (b).

[0045] In some embodiments, steps (a) and (b) are performed simultaneously.

[0046] In some embodiments, the method further comprises increasing the temperature of the solution containing the therapeutic circular DNA vector to about 65°C.

[0047] In some embodiments, the method further comprises (c) contacting the therapeutic circular DNA vector with a topoisomerase or helicase. In some embodiments, step (c) is performed at about 37° C. In some embodiments, the method further comprises (d) contacting the linear backbone fragment with an exonuclease (e.g., a terminal exonuclease). In some embodiments, step (d) is performed at about 37° C.

[0048] In some embodiments, the method further comprises (c) contacting the therapeutic circular DNA vector with a topoisomerase or helicase, and (d) contacting the linear backbone fragment with an exonuclease (e.g., a terminal exonuclease), wherein no enzyme inactivation step is performed between steps (c) and (d). In some embodiments, step (c) is performed prior to step (d).

[0049] In some embodiments, the restriction enzyme is provided at a concentration of about 0.5 U / μg to about 20 U / μg, e.g., about 1 U / μg DNA to about 10 U / μg DNA, e.g., about 2 U / μg DNA to about 5 U / μg DNA, e.g., about 2.5 U / μg DNA. For example, restriction enzymes are approximately 0.5U / μg DNA, 1.0U / μg DNA, 1.5U / μg DNA, 2.0U / μg DNA, 2.5U / μg DNA, 3.0U / μg DNA, 3.5U / μg DNA, 4.0U / μg DNA, 4.5U / μg DNA, 5.0U / μg DNA, 5.5U / μg DNA, 6.0U / μg DNA, 6.5U / μg DNA, 7.0U / μg DNA, 7.5U / μg DNA, 8.0U / μg DNA, 8.5U / μg DNA, 9.0U / μg DNA, 9.5U / μg DNA, 10.0U / μg DNA, 11U / μg DNA, 12U / μg DNA, 13U / μg DNA, 14U / μg DNA, 15U / μg DNA, 16U / μg DNA, 17U / μg The restriction enzyme may be provided at a concentration of about 2.5 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, or 20 U / μg DNA. In some embodiments, the restriction enzyme is provided at a concentration of about 2.5 U / μg DNA.

[0050] In some embodiments, step (a) comprises incubation for 1 to 12 hours (eg, about 1 hour).

[0051] In some embodiments, the ligase has a ligase activity of 20 U per μg of DNA (U / μg) or less (e.g., 15 U / μg DNA or less, 10 U / μg DNA or less, 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 20 U / μg DNA; e.g., 0.2 U / μg DNA to 15 U / μg DNA, 0.5 U / μg DNA to 12 U / μg DNA, or 1 U / μg DNA to 10 U / μg DNA; e.g., 0.1 U / μg DNA to 0.5 U / μg DNA, 0.5 U / μg DNA to 1.0 U / μg DNA, 1.0 U / μg DNA to 2.0 U / μg DNA, 2.0 U / μg DNA, The ligase may be provided at a concentration of from about 3.0 U / μg DNA, 3.0 U / μg DNA to 4.0 U / μg DNA, 4.0 U / μg DNA to 5.0 U / μg DNA, 5.0 to 6.0 U / μg DNA, 6.0 U / μg DNA to 7.0 U / μg DNA, 7.0 U / μg DNA to 8.0 U / μg DNA, 8.0 U / μg DNA to 9.0 U / μg DNA, 9.0 U / μg DNA to 11 U / μg DNA, 11 U / μg DNA to 12 U / μg DNA, 12 U / μg DNA to 15 U / μg DNA, or 15 U / μg DNA to 20 U / μg DNA. In some embodiments, the ligase is at a concentration of about 10 U / μg DNA. In some embodiments, the ligase is T4 ligase.

[0052] In some embodiments, the topoisomerase is at 10 U per μg of DNA (U / μg) or less (e.g., 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 10 U / μg DNA, e.g., 0.5 U / μg DNA to 8 U / μg DNA, or 1 U / μg DNA to 5 U / μg DNA; e.g., 0.1 U / μg DNA to 0.5 U / μg DNA, 0.5 U / μg DNA to 1.0 U / μg DNA, 1.0 U / μg DNA to 2.0 U / μg DNA, 2.0 U / μg DNA to 3.0 U / μg DNA, 3.0 U / μg DNA to 4.0 U / μg DNA, 4.0 U / μg DNA, The topoisomerase is provided at concentrations of: 5.0U / μg DNA, 5.0-6.0U / μg DNA, 6.0U / μg DNA-7.0U / μg DNA, 7.0U / μg DNA-8.0U / μg DNA, 8.0U / μg DNA-9.0U / μg DNA, or 9.0U / μg DNA-10U / μg DNA.

[0053] In some embodiments, the topoisomerase is a type II topoisomerase. In some embodiments, the topoisomerase is gyrase or topoisomerase IV. In some embodiments, the exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease) is provided at a concentration of about 0.5 U / μg to about 20 U / μg, such as about 0.5 U / μg to about 10 U / μg, such as about 1 U / μg to about 10 U / μg, such as about 2 U / μg to about 5 U / μg, for example, about 2.5 U / μg. For example, exonucleases (e.g., terminal exonucleases) may be at about 0.5 U / μg, 1.0 U / μg, 1.5 U / μg, 2.0 U / μg, 2.5 U / μg, 3.0 U / μg, 3.5 U / μg, 4.0 U / μg, 4.5 U / μg, 5.0 U / μg, 5.5 U / μg, 6.0 U / μg, 6.5 U / μg, 7. .0U / μg, 7.5U / μg, 8.0U / μg, 8.5U / μg, 9.0U / μg, 9.5U / μg, 10.0U / μg, 11U / μg, 12U / μg , 13U / μg, 14U / μg, 15U / μg, 16U / μg, 17U / μg, 18U / μg, 19U / μg, or 20U / μg.

[0054] In some embodiments, step (d) is performed more than once. In some embodiments, step (d) comprises incubation for 1 hour to 12 hours. In some embodiments, the exonuclease is a terminal exonuclease. In some embodiments, the terminal exonuclease is T5 exonuclease. In some embodiments, the method further comprises (e) passing the therapeutic circular DNA vector through a column (e.g., a capture column) and / or (f) precipitating the therapeutic circular DNA vector with isopropyl alcohol.

[0055] In some of any of the preceding embodiments, the therapeutic circular DNA vector is generated without a gel extraction step (e.g., an intermediate gel extraction step).

[0056] In another aspect, a method of generating a therapeutic circular supercoiled DNA vector includes: (a) providing a sample comprising a template DNA vector comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) digesting the linear concatemers with a Type IIS restriction enzyme that cleaves a first site and a second site per unit of the linear concatemer, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs, wherein digestion generates a linear therapeutic fragment and a linear backbone fragment, the linear therapeutic fragment comprising the therapeutic sequence, and the linear therapeutic fragment comprising the therapeutic sequence. The method includes: (a) digesting the linear backbone fragment, wherein the linear backbone fragment comprises at least a portion of the backbone sequence; (b) diluting the linear therapeutic fragment and the linear backbone fragment to a cumulative DNA concentration of 20 μg / mL to 160 μg / mL; (c) contacting the diluted linear backbone fragment and the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector lacking a circular backbone and a type IIS restriction site; (d) contacting the therapeutic circular DNA vector with a gyrase at a concentration of about 1.5 U per μg of DNA to generate a mixture of therapeutic supercoiled circular DNA vector and linear backbone fragment; and (e) digesting the linear backbone fragment with an exonuclease after step (f).

[0057] In another aspect, a method of generating a therapeutic circular supercoiled DNA vector includes: (a) providing a sample comprising a template DNA vector comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) digesting the linear concatemers with a Type IIS restriction enzyme that cleaves a first site and a second site per unit of the linear concatemer, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs, wherein digestion generates a linear therapeutic fragment and a linear backbone fragment, and the linear therapeutic fragments are digested with a Type IIS restriction enzyme that cleaves a first site and a second site per unit of the linear concatemer, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs ... The method includes: (a) digesting a linear therapeutic fragment, the linear therapeutic fragment comprising a therapeutic sequence and the linear backbone fragment comprising at least a portion of the backbone sequence; (d) diluting the linear therapeutic fragment and the linear backbone fragment to a cumulative DNA concentration of 20 μg / mL to 160 μg / mL; (e) contacting the diluted linear backbone fragment and the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector lacking a circular backbone and a type IIS restriction site; (f) digesting the linear backbone fragment with an exonuclease; and (g) supercoiling the therapeutic circular DNA vector after step (f) with gyrase at a concentration of less than 1.5 U per μg of DNA. In some embodiments, the ligase in step (e) is at a concentration of 10-20 U of ligase per μg of DNA. In some embodiments, the diluted cumulative DNA concentration in step (d) is about 10% to about 80% of the cumulative DNA concentration immediately after step (c). In some embodiments, the cumulative DNA concentration immediately after step (c) is 100 μg / mL to 300 μg / mL. In some embodiments, the first or second cleavage site adjacent to the therapeutic sequence comprises AAAA or AACC.

[0058] In another aspect, a method for large-scale production of therapeutic circular DNA vectors includes: (a) providing a sample of a template DNA vector (e.g., a plasmid DNA vector) comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector in a reaction volume of at least 1.0 liter (e.g., at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 150 liters, or at least 200 liters) using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) amplifying the template DNA vector in a reaction volume of at least 1.0 liter (e.g., at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 150 liters, or at least 200 liters) using polymerase-mediated rolling circle amplification to generate linear concatemers; and (d) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0059] In some embodiments, the amount of template DNA vector provided in step (a) is at least 0.5 mg, at least 0.75 mg, or at least 1.0 mg (e.g., 1.0 mg to 10 mg, 2.0 mg to 10 mg, 3.0 mg to 10 mg, 4.0 mg to 10 mg, or 5.0 mg to 10 mg; e.g., 1.0 mg to 2.5 mg, 2.5 mg to 5.0 mg, 5.0 mg to 7.5 mg, or 7.5 mg to 10 mg). In some embodiments, the amount of template DNA vector provided in step (a) is at least 5.0 mg. For example, in some embodiments, the amount of template DNA vector provided in step (a) is at least 10.0 mg.

[0060] In some embodiments, step (b) produces at least 100 mg (e.g., 100 mg to 10 g, 500 mg to 5 g, or 1 g to 3 g; e.g., at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, at least 1 g, at least 2 g, or at least 3 g; e.g., 200 mg to 10 g, 300 mg to 10 g, 400 mg to 10 g, 500 mg to 10 g, or 1 g to 10 g) of linear concatemers. In some embodiments, step (b) produces a solution containing 0.5 g to 2 g of DNA per liter of reaction volume (e.g., about 1 g of DNA per liter of reaction volume).

[0061] In some embodiments, step (d) produces at least 1.0 mg (e.g., 1.0 mg to 10 mg, 2.0 mg to 10 mg, 3.0 mg to 10 mg, 4.0 mg to 10 mg, or 5.0 mg to 10 mg; e.g., 1.0 mg to 2.5 mg, 2.5 mg to 5.0 mg, 5.0 mg to 7.5 mg, or 7.5 mg to 10 mg) of therapeutic circular DNA vector. In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 2.0 mg (e.g., as in large scale production). For example, in some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 5.0 mg.

[0062] In some embodiments, steps (c) and (d) are performed simultaneously. In some embodiments, no DNA purification is performed between steps (b), (c), and (d), or between steps (b), (c), and (d).

[0063] In some embodiments, the amount of therapeutic circular DNA in the solution in step (d) is at least 2.0% by weight of the amount of linear concatemers in step (b) (e.g., at least 3.0% by weight, at least 4.0% by weight, at least 5.0% by weight, at least 6.0% by weight, at least 7.0% by weight, at least 8.0% by weight, at least 9.0% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, or at least 50% by weight) of the amount of linear concatemers in step (b).

[0064] In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 20% of the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a) (e.g., at least 50%, at least 75%, at least 100%, or at least 150% of the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a); e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a). In certain embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 5-fold the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a). In some embodiments, the amount of therapeutic circular DNA produced in step (d) is at least 10-fold the amount of the template DNA vector (e.g., a plasmid DNA vector) provided in step (a).

[0065] In some embodiments, the DNA concentration at the start of step (d) is greater than or equal to 20 μg / mL but less than 160 μg / mL. In some embodiments, the DNA concentration at the start of step (d) is between about 40 μg / mL and about 80 μg / mL. In some embodiments, the DNA concentration at the start of step (d) is about 40 μg / mL. In some embodiments, the DNA concentration at the start of step (d) is about 80 μg / mL. In some embodiments, the ligase concentration (e.g., T4 ligase concentration) in step (d) is between about 10 and about 20 U of ligase per μg of DNA. In some embodiments, no temperature increase is performed immediately after step (d).

[0066] In another aspect, a method of generating a therapeutic circular DNA vector is provided, the method comprising: (a) providing a solution containing DNA molecules, each DNA molecule containing a backbone sequence and a therapeutic sequence; (b) adding a type IIS restriction enzyme to the solution to digest the DNA molecules, thereby separating the backbone sequence from the therapeutic sequence; (c) adding a ligase to the solution to generate a reaction in a mixture containing (i) the ligase; (ii) the type IIS restriction enzyme; (iii) therapeutic circular DNA vectors, each containing a single therapeutic sequence, each lacking a type IIS recognition site; and (iv) by-products, each by-product containing one or more type IIS restriction sites, wherein the ratio of the therapeutic circular DNA vectors to the by-products containing one or more type IIS restriction sites increases as the reaction progresses. In some embodiments, some or all of the by-products include one, two, three, four, or more backbone sequences (e.g., circular DNA with two or more backbone sequences linked via type IIS restriction sites and / or linear DNA with two or more backbone sequences linked via type IIS restriction sites). In some embodiments, some or all of the by-products further include two, three, four, or more therapeutic sequences (e.g., circular DNA with two or more copies of a therapeutic sequence linked via type IIS restriction sites and / or linear DNA with two or more copies of a therapeutic sequence linked via type IIS restriction sites). In some embodiments, some or all of the by-products are circular. In some embodiments, the DNA molecule of (a) is a concatemer.

[0067] In some embodiments, the method further comprises amplifying a template DNA vector (e.g., a plasmid DNA vector) using rolling circle amplification prior to step (a) to generate concatemers.

[0068] In some embodiments, the Type IIS restriction enzyme is BsaI.

[0069] In some embodiments, a restriction enzyme inactivation step does not precede step (c). In some embodiments, no temperature increase is performed between steps (b) and (c). In some embodiments, the method further comprises increasing the temperature of the solution containing the therapeutic circular DNA vector to about 65° C. In some embodiments, the method further comprises (d) contacting the therapeutic circular DNA vector with a topoisomerase or helicase. In some embodiments, step (d) is performed at about 37° C. In some embodiments, the method additionally or alternatively comprises (e) contacting the linear by-product with an exonuclease. In some embodiments, step (e) is performed at about 37° C.

[0070] In some embodiments, the method further comprises (d) contacting the therapeutic circular DNA vector with a topoisomerase or helicase, and (e) contacting the linear by-product with an exonuclease, wherein an enzyme inactivation step is not performed between steps (d) and (e). In some embodiments, step (d) is performed prior to step (e).

[0071] In some embodiments, the restriction enzyme is provided at a concentration of about 0.5 U / μg to about 20 U / μg, e.g., about 1 U / μg DNA to about 10 U / μg DNA, e.g., about 2 U / μg DNA to about 5 U / μg DNA, e.g., about 2.5 U / μg DNA. For example, restriction enzymes are approximately 0.5U / μg DNA, 1.0U / μg DNA, 1.5U / μg DNA, 2.0U / μg DNA, 2.5U / μg DNA, 3.0U / μg DNA, 3.5U / μg DNA, 4.0U / μg DNA, 4.5U / μg DNA, 5.0U / μg DNA, 5.5U / μg DNA, 6.0U / μg DNA, 6.5U / μg DNA, 7.0U / μg DNA, 7.5U / μg DNA, 8.0U / μg DNA, 8.5U / μg DNA, 9.0U / μg DNA, 9.5U / μg DNA, 10.0U / μg DNA, 11U / μg DNA, 12U / μg DNA, 13U / μg DNA, 14U / μg DNA, 15U / μg DNA, 16U / μg DNA, 17U / μg The restriction enzyme may be provided at a concentration of about 100 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, or 20 U / μg DNA. In some embodiments, the restriction enzyme is provided at a concentration of about 2.5 U / μg. In some embodiments, the restriction enzyme is provided at a concentration of about 0.5 U / μg to about 2.5 U / μg.

[0072] In some embodiments, the digestion (eg, step (b)) comprises an incubation period of 1 to 12 hours, for example, about 1 hour.

[0073] In some embodiments, the ligase has a concentration of 50 U per μg of DNA (U / μg) or less (e.g., 40 U / μg DNA or less, 30 U / μg DNA or less, 25 U / μg DNA or less, 20 U / μg DNA or less, 15 U / μg DNA or less, 10 U / μg DNA or less, 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 20 U / μg DNA; e.g., 0.1 U / μg DNA to 30 U / μg DNA, 0.1 U / μg DNA to 20 U / μg DNA, 0.2 U / μg DNA to 15 U / μg DNA, 0.5 U / μg DNA to 12 U / μg DNA, or 1 U / μg DNA). DNA~10U / μg DNA; For example, 0.1U / μg DNA~0.5U / μg DNA, 0.5U / μg DNA~1.0U / μg DNA, 1.0U / μg DNA~2.0U / μg DNA, 2.0U / μg DNA~3.0U / μg DNA, 3.0U / μg DNA~4.0U / μg DNA, 4.0U / μg DNA~5.0U / μg DNA, 5.0~6.0U / μg DNA, 6.0U / μg DNA~7.0U / μg DNA, 7.0U / μg DNA~8.0U / μg DNA, 8.0U / μg DNA~9.0U / μg DNA, 9.0U / μg DNA~11U / μg DNA, 11U / μg DNA~12U / μg DNA, 12U / μg DNA~15U / μg DNA, 15U / μg The ligase is provided at a concentration of 20 U / μg DNA, 20 U / μg DNA to 25 U / μg DNA, 25 U / μg DNA to 30 U / μg DNA, 30 U / μg DNA to 35 U / μg DNA, 35 U / μg DNA to 40 U / μg DNA, or 40 U / μg DNA to 50 U / μg DNA. In some embodiments, the ligase is provided at a concentration of 20 U / μg DNA or less, e.g., about 10 U / μg DNA. In some embodiments, the ligase is T4 ligase.

[0074] In some embodiments, the topoisomerase is at 10 U per μg of DNA (U / μg) or less (e.g., 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 10 U / μg DNA, e.g., 0.5 U / μg DNA to 8 U / μg DNA, or 1 U / μg DNA to 5 U / μg DNA; e.g., 0.1 U / μg DNA to 0.5 U / μg DNA, 0.5 U / μg DNA to 1.0 U / μg DNA, 1.0 U / μg DNA to 2.0 U / μg DNA, 2.0 U / μg DNA to 3.0 U / μg DNA, 3.0 U / μg DNA to 4.0 U / μg DNA, 4.0 U / μg DNA, The topoisomerase is provided at concentrations of: 5.0U / μg DNA, 5.0-6.0U / μg DNA, 6.0U / μg DNA-7.0U / μg DNA, 7.0U / μg DNA-8.0U / μg DNA, 8.0U / μg DNA-9.0U / μg DNA, or 9.0U / μg DNA-10U / μg DNA.

[0075] In some embodiments, the topoisomerase is a type II topoisomerase. In some embodiments, the topoisomerase is a gyrase. In some embodiments, the topoisomerase is a topoisomerase IV.

[0076] In some embodiments, the exonuclease (e.g., a terminal exonuclease, e.g., a T5 exonuclease) is provided at a concentration of about 0.5 U / μg to about 20 U / μg, e.g., about 0.5 U / μg to about 10 U / μg, e.g., about 1 U / μg to about 10 U / μg, e.g., about 2 U / μg to about 5 U / μg, e.g., about 2.5 U / μg. For example, the exonuclease (e.g., a terminal exonuclease) is provided at a concentration of about 0.5 U / μg, 1.0 U / μg, 1.5 U / μg, 2.0 U / μg, 2.5 U / μg, 3.0 U / μg, 3.5 U / μg, 4.0 U / μg, 4.5 U / μg, 5.0 U / μg, 5.5 U / μg, 6.0 U / μg, 6.5 U / μg, 7. .0U / μg, 7.5U / μg, 8.0U / μg, 8.5U / μg, 9.0U / μg, 9.5U / μg, 10.0U / μg, 11U / μg, 12U / μg , 13U / μg, 14U / μg, 15U / μg, 16U / μg, 17U / μg, 18U / μg, 19U / μg, or 20U / μg.

[0077] In some embodiments, step (e) is performed two or more times (e.g., two, three, or four times). In some embodiments, step (e) comprises incubation for 1 hour to 12 hours. In some embodiments, the exonuclease is a terminal exonuclease (e.g., T5 exonuclease).

[0078] In some embodiments of any of the above methods, the method further comprises: (f) passing the therapeutic circular DNA vector through a column (e.g., a capture column or an anion exchange column); and / or (g) precipitating the therapeutic circular DNA vector with isopropyl alcohol.

[0079] In some embodiments, amplification is performed using site-specific primers, while in other embodiments, amplification is performed using random primers.

[0080] In some embodiments, no intermediate gel extraction step is performed prior to step (c).In some embodiments, no intermediate DNA purification is performed prior to step (c).

[0081] In some embodiments, the amount of therapeutic circular DNA in the solution in step (c) is at least 2.0% by weight of the amount of DNA molecules in step (a) (e.g., at least 3.0% by weight, at least 4.0% by weight, at least 5.0% by weight, at least 6.0% by weight, at least 7.0% by weight, at least 8.0% by weight, at least 9.0% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, or at least 50% by weight of the DNA molecules in step (a).

[0082] In some embodiments, the amount of therapeutic circular DNA produced in step (c) is at least 1.0 mg (e.g., 1.0 mg to 10 mg, 2.0 mg to 10 mg, 3.0 mg to 10 mg, 4.0 mg to 10 mg, or 5.0 mg to 10 mg; e.g., 1.0 mg to 2.5 mg, 2.5 mg to 5.0 mg, 5.0 mg to 7.5 mg, or 7.5 mg to 10 mg). In some embodiments, the amount of therapeutic circular DNA produced in step (c) is at least 2.0 mg (e.g., as in large-scale production). For example, in some embodiments, the amount of therapeutic circular DNA produced in step (c) is at least 5.0 mg.

[0083] In some embodiments, the concentration of the therapeutic circular DNA in the solution after step (c) is 1.0 μg / mL to 1.0 mg / mL without any purification or concentration being performed (e.g., 5.0 μg / mL to 100 μg / mL or 10 μg / mL to 50 μg / mL without any purification or concentration being performed, e.g., 1.0 μg / mL to 10 μg / mL, 5.0 μg / mL to 10 μg / mL, 10 μg / mL to 50 μg / mL, 50 μg / mL to 100 μg / mL, or more without any purification or concentration being performed). In some embodiments, the volume of the solution in step (c) is at least 5 liters (e.g., between 5 liters and 200 liters, e.g., between 7 liters and 100 liters, between 10 liters and 80 liters, between 15 liters and 75 liters, or between 20 liters and 70 liters, e.g., at least 1.0 liter, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, or at least 100 liters).

[0084] In some embodiments, steps (b) and (c) are carried out in a reaction vessel having a volume of at least 0.5 liters (e.g., at least 1.0 liters, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 150 liters, or at least 200 liters). In some embodiments, steps (b) and (c) are carried out in a reaction vessel having a volume of at least 5 liters (e.g., from 5 liters to 200 liters, e.g., from 7 liters to 100 liters, from 10 liters to 80 liters, from 15 liters to 75 liters, or from 20 liters to 70 liters, e.g., at least 1.0 liters, at least 2.0 liters, at least 5.0 liters, at least 10 liters, at least 20 liters, at least 50 liters, or at least 100 liters).

[0085] In another aspect, a method of generating a therapeutic circular DNA vector is provided, the method comprising: (a) providing a mixture of DNA including a plurality of linear therapeutic DNA fragments and a plurality of linear backbone DNA fragments, each of the linear therapeutic DNA fragments including a therapeutic sequence and a self-complementary end, the plurality of linear therapeutic DNA fragments and the linear backbone DNA fragments being at a cumulative DNA concentration of 20 μg / mL to 160 μg / mL; and (b) performing a ligation reaction by contacting the mixture of DNA with a ligase at a concentration of 10 to 20 U of ligase per μg of DNA to generate a therapeutic circular DNA vector. In some embodiments, the mixture of DNA is generated by a type IIS restriction digestion reaction, where the type IIS restriction enzyme cleaves the linear therapeutic DNA fragments from the linear backbone DNA fragments, and the self-complementary ends are type IIS overhangs.

[0086] In another aspect, a method of generating a therapeutic circular DNA vector is provided, the method comprising: (a) generating a mixture of DNA comprising a plurality of linear therapeutic DNA fragments and a plurality of linear backbone DNA fragments by a type IIS restriction digestion reaction, where a type IIS restriction enzyme cleaves the linear therapeutic DNA fragments from the linear backbone DNA fragments, each linear therapeutic DNA fragment comprising a therapeutic sequence and a self-complementary type IIS overhang, the plurality of linear therapeutic DNA fragments and the linear backbone DNA fragments being at a cumulative DNA concentration of 20 μg / mL to 160 μg / mL; and (b) performing a ligation reaction by contacting the mixture of DNA with a ligase at a concentration of 10-20 U of ligase per μg of DNA to generate a therapeutic circular DNA vector.

[0087] In some embodiments of either of the two aspects immediately preceding, the cumulative DNA concentration in step (a) is achieved by adjusting (e.g., diluting) the cumulative DNA concentration immediately after type IIS restriction digestion. In some embodiments, the cumulative DNA concentration is diluted immediately after type IIS restriction digestion to achieve the cumulative DNA concentration in step (a). In some embodiments, the cumulative DNA concentration immediately after type IIS restriction digestion is 100 μg / mL to 300 μg / mL. In some embodiments, the cumulative DNA concentration in step (a) is diluted to about 10% to about 80% of the cumulative DNA concentration immediately after type IIS restriction digestion. In some embodiments, the cumulative DNA concentration in step (a) is about 40 μg / mL to about 80 μg / mL. In some embodiments, the type IIS restriction enzyme in the type IIS restriction digestion reaction is at a concentration of about 0.5 to about 2.5 U per μg of DNA. In some embodiments, the ligase (e.g., T4 ligase) is at a concentration of about 10 U / ug. In some embodiments, the ligation reaction is carried out for at least 5 hours, for example, 18-24 hours.

[0088] In some embodiments, the Type IIS restriction enzyme (e.g., BsaI) in the Type IIS restriction digestion reaction is at a concentration of about 0.5 to about 2.5 U per μg of DNA. In some embodiments, the Type IIS restriction digestion reaction is carried out for 2 hours or less, for example, 10 minutes to 1 hour.

[0089] In some embodiments, the Type IIS overhangs each comprise four bases. In some embodiments, only two of the four bases are A or T. In some embodiments, the Type IIS overhangs comprise AAAA or AACC.

[0090] In some embodiments, the method further comprises (c) contacting the therapeutic circular DNA vector with a topoisomerase or helicase, and / or (d) contacting the linear backbone fragment with an exonuclease.

[0091] In some embodiments, the method further comprises (c) contacting the therapeutic circular DNA vector with a topoisomerase or helicase, and (d) contacting the linear backbone fragment with an exonuclease (e.g., a terminal exonuclease). In some embodiments, no enzyme inactivation step is performed between steps (c) and (d). In some embodiments, step (c) is performed before step (d). In other embodiments, step (d) is performed before step (c).

[0092] In some embodiments, the topoisomerase (e.g., gyrase) is provided at a concentration of 10 U of topoisomerase per μg of DNA (U / μg) or less. In some embodiments, the topoisomerase is a type II topoisomerase. In some embodiments, the topoisomerase is gyrase or topoisomerase IV.

[0093] In some embodiments, the exonuclease (e.g., T5 exonuclease) is provided at a concentration of about 0.5 U / μg to about 20 U / μg. In some embodiments, step (d) is performed two or more times. In some embodiments, step (d) comprises incubation for 1 hour to 18 hours. In some embodiments, step (d) comprises incubation for 3 hours to 18 hours.

[0094] In some embodiments, the method further comprises (e) passing the therapeutic circular DNA vector through a column, and / or (f) precipitating the therapeutic circular DNA vector with isopropyl alcohol.

[0095] In some embodiments, the amount of therapeutic circular DNA produced in step (b) is at least 1.0 mg. In some embodiments, the concentration of therapeutic circular DNA in the solution after step (b) is at least 5 μg / mL without any purification or concentration being performed. In some embodiments, the volume of the solution in step (d) is at least 5 liters. In some embodiments, step (b) is performed in a reaction vessel having a volume of at least 1 liter.

[0096] In some embodiments, the mixture of DNA is the product of in vitro amplification.

[0097] In some embodiments, the in vitro amplification is polymerase-mediated rolling circle amplification.

[0098] In some embodiments, the method does not include a gel extraction step.

[0099] In some embodiments, the DNA mixture contains only one linear backbone DNA fragment (eg, restriction digest produces a single fragment with a plasmid backbone).

[0100] In another aspect, a method of generating a therapeutic circular supercoiled DNA vector is provided, the method comprising: (a) providing a sample comprising a therapeutic circular DNA vector in a relaxed circular form, wherein the therapeutic circular DNA vector comprises a therapeutic sequence; (b) contacting the sample with gyrase, wherein the concentration of the gyrase is about 1.5 U per mg of therapeutic circular DNA vector, thereby generating a therapeutic supercoiled circular DNA vector composition. In some embodiments, the sample of (a) further comprises a linear DNA by-product, wherein the method further comprises, after (b), contacting the therapeutic supercoiled circular DNA vector composition with an exonuclease under conditions suitable for digesting the linear DNA by-product.

[0101] In another aspect, the invention includes a method of generating a therapeutic supercoiled circular DNA vector, the method comprising: (a) providing a sample containing a therapeutic circular DNA vector in a relaxed circular form and a linear DNA by-product, where the therapeutic circular DNA vector contains a therapeutic sequence; (b) contacting the sample with an exonuclease under conditions suitable for digesting the linear DNA by-product to form a digested sample; and (c) contacting the digested sample with gyrase, where the concentration of gyrase is greater than 0.1 U per mg of therapeutic circular DNA vector and less than 1.5 U per mg of therapeutic circular DNA vector, thereby generating a therapeutic supercoiled circular DNA vector.

[0102] In some embodiments of any of the above aspects, the exonuclease is T5 exonuclease and / or the ligase is T4 ligase. In some embodiments, the method includes, prior to step (a), contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector. In some embodiments, the method includes, prior to contacting the linear therapeutic fragment with the ligase, digesting the linear concatemers comprising the therapeutic sequence with a restriction enzyme to cleave a first site and a second site per unit of the linear concatemer, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs, and digestion generates a linear therapeutic fragment and a linear DNA by-product. In some embodiments, the therapeutic supercoiled circular DNA vector is present in a composition of therapeutic circular DNA vectors, wherein at least 70% of the therapeutic circular DNA vector is supercoiled (e.g., at least 80% of the therapeutic circular DNA vector is supercoiled).

[0103] In some embodiments of any of the above aspects, the therapeutic circular DNA vector is formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises 1.0% or less by weight (e.g., 0.5% or less by weight) of residual protein or backbone sequence relative to the amount of the therapeutic circular DNA vector. In some embodiments, the therapeutic sequence is greater than 5 kb. In some embodiments, the therapeutic sequence is between 5 kb and 15 kb. In some embodiments, the therapeutic sequence is between 5 kb and 10 kb. In some embodiments, the therapeutic sequence is between 10 kb and 15 kb. In some embodiments, the therapeutic sequence comprises two or more transcription units. In some embodiments, the therapeutic sequence encodes one or more therapeutic proteins. In some embodiments, the one or more therapeutic proteins are multimeric proteins. In some embodiments, the therapeutic sequence encodes a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an RNA molecule. In some embodiments, the RNA molecule is a self-replicating RNA molecule, a short hairpin RNA, or a microRNA.

[0104] In some embodiments of any of the above aspects, the method further comprises formulating the therapeutic circular DNA vector in a pharma- ceutically acceptable carrier to produce a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises at least 1.0 mg of the therapeutic circular DNA vector in a pharma- ceutically acceptable carrier. In some embodiments, the therapeutic circular DNA vector in the pharmaceutical composition is at least 70% supercoiled monomer (e.g., by densitometric analysis of gel electrophoresis). In some embodiments, the therapeutic circular DNA vector in the pharmaceutical composition is at least 80% supercoiled monomer (e.g., by densitometric analysis of gel electrophoresis). In some embodiments, the pharmaceutical composition comprises less than 1.0% protein by weight, less than 1.0% RNA by weight, and less than 0.5 EU / mg endotoxin.

[0105] In another aspect, provided herein is a composition (e.g., a pharmaceutical composition) produced by the method of any of the preceding embodiments of any of the above aspects.

[0106] In another aspect, provided herein is a method of expressing a therapeutic sequence in an individual, comprising administering to the individual a pharmaceutical composition produced by the method of any of the preceding embodiments of any of the above aspects. The therapeutic sequence of any of the therapeutic circular DNA vectors or pharmaceutical compositions thereof described herein can be expressed in skin, skeletal muscle, tumors (including, for example, melanoma), eyes, or lungs by electrical transfer in vivo.

[0107] In another aspect, provided herein is a method of treating a disease or disorder in an individual in need thereof, comprising administering to said individual a pharmaceutical composition produced by the method of any of the preceding embodiments of any of the above aspects. In some embodiments, the method comprises in vivo electrical introduction of a therapeutic circular DNA vector into the skin, skeletal muscle, tumor (including, for example, melanoma), eye, or lung of the individual.

[0108] In another aspect, a therapeutic circular DNA vector is provided that comprises a therapeutic sequence having a 3' end and a 5' end, the 3' end of the therapeutic sequence being linked to the 5' end of the therapeutic sequence by a four base pair sequence comprising at least two consecutive adenines (A). In some embodiments, the four base pair sequence consists of AAAA. In some embodiments, the therapeutic circular DNA vector comprises (e.g., consists of) a nucleic acid sequence having 85% sequence identity to SEQ ID NO:1. In some embodiments, the therapeutic circular DNA vector comprises SEQ ID NO:1. In some embodiments, only two consecutive bases of the four base pair sequence are AA. In some embodiments, the four base pair sequence consists of AACC. In some embodiments, the therapeutic circular DNA vector comprises a nucleic acid sequence having at least 85% sequence identity (e.g., 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 100% sequence identity) to SEQ ID NO: 3. In some embodiments, the therapeutic circular DNA vector comprises or consists of SEQ ID NO: 3.

[0109] In another aspect, the invention provides a pharmaceutical composition comprising the therapeutic circular DNA vector of the immediately preceding aspect. In some embodiments, the pharmaceutical composition comprises at least 1.0 mg of the therapeutic circular DNA vector in a pharma- ceutically acceptable carrier. In some embodiments, the therapeutic circular DNA vector is at least 70% supercoiled monomers. In some embodiments, the pharmaceutical composition comprises no more than 1.0% residual protein or backbone sequence. In some embodiments, the pharmaceutical composition comprises less than 1.0% protein by weight, less than 1.0% RNA by weight, and less than 5 EU / mg endotoxin.

[0110] In another aspect, the invention includes a method of expressing a therapeutic sequence in an individual (e.g., a human), comprising administering to the individual a pharmaceutical composition of any embodiment of the immediately preceding aspect, hi some embodiments, the method comprises delivering a therapeutic circular DNA vector to the eye of the individual by in vivo electrotransfer.

[0111] In another aspect, the invention includes a method of treating an ocular disease or disorder in an individual (e.g., a human) in need thereof, comprising administering to the individual a pharmaceutical composition of any embodiment of the immediately preceding aspect. In some embodiments, the method comprises delivering a therapeutic circular DNA vector to the eye of the individual by in vivo electrotransfer. In another aspect, provided herein is a kit comprising any of the therapeutic circular DNA vectors or compositions (e.g., pharmaceutical compositions) thereof described herein (or generated by the methods described herein) and instructions for expressing the therapeutic circular DNA vector in a cell or culture of cells using electroporation (e.g., in vitro or ex vivo electroporation) or electrotransfer (e.g., in vivo electrotransfer).

[0112] In another aspect, provided herein are cells (e.g., mammalian cells) that express any of the therapeutic circular DNA vectors described herein (or generated by the methods described herein). In some embodiments, the cells have been electrotransduced with the vector by electroporation (e.g., in vitro or ex vivo electroporation). [Brief description of the drawings]

[0113] [Figure 1]A-E show schematic diagrams illustrating the sequence of reaction steps described herein in which two restriction enzymes are used in a cell-free process to generate c3DNA. A shows a plasmid DNA vector with a therapeutic sequence (solid) and a backbone sequence (hatched). The backbone sequence has two PvuII restriction sites within the sequence. Two EcoRI restriction sites flank the backbone and therapeutic sequences. B shows the product of reaction of the plasmid DNA vector or its amplified concatemer with EcoRI. A linear therapeutic fragment (solid) and a linear backbone fragment (hatched) are generated for each unit of the plasmid DNA or linear concatemer. C shows the circularization products of reaction of the linear fragments with ligase, i.e., a therapeutic circular DNA vector and a circularized backbone. D shows the product of the circularization product of C with PvuII. The circularized backbone can be linearized and subsequently digested with an exonuclease. E shows a therapeutic supercoiled circular DNA vector obtained by reaction of the therapeutic circular DNA vector with a topoisomerase such as gyrase. [Figure 2A] 2B shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts two sites within the plasmid DNA vector. A plasmid DNA vector is shown containing a therapeutic sequence (the "C3 region"; its nucleotide base is shown as N in FIG. 2B for illustration purposes) and a backbone sequence (which includes an origin of replication and two BsaI recognition sites, the BsaI recognition sites being distal to the therapeutic sequence relative to the corresponding BsaI overhangs (cutting sites)). [Figure 2B] Figure 2A shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts two sites in the plasmid DNA vector. The linear sequence corresponding to Figure 2A is shown. [Figure 2C] Figure 2B shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts two sites within the plasmid DNA vector. Figure 2C shows the therapeutic circular DNA vector resulting from the plasmid in Figure 2A. The therapeutic circular DNA does not contain an origin of replication, only the four base pair BsaI overhangs remain. [Figure 2D] Figure 2C shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts two sites in the plasmid DNA vector.The linear sequence corresponding to Figure 2C is shown. [Figure 2E] 2A shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts two sites in the plasmid DNA vector. 2B shows the circularized backbone resulting from the plasmid of FIG. 2A, containing BsaI recognition sites separated by BsaI cutting sites (overhangs). [Figure 2F] Figure 2B shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts two sites in the plasmid DNA vector. The corresponding linear sequence is shown in Figure 2F. [Diagram 3] A-D show schematic diagrams illustrating the sequence of reaction steps described herein in which a single restriction enzyme is used in a cell-free process to generate c3DNA. A shows a plasmid DNA vector with a therapeutic sequence (solid) and a backbone sequence (hatched). The plasmid DNA vector contains four BsaI restriction sites, two in the backbone sequence and two flanking the backbone and therapeutic sequences. B shows the product of reaction of the plasmid DNA vector or its amplified concatemer with BsaI. For each unit of the plasmid DNA or linear concatemer, a linear therapeutic fragment (solid) and three linear backbone fragments (hatched) are generated. C shows the product of reaction of the linear fragments with ligase, i.e., a therapeutic circular DNA vector and a linear backbone fragment that is digestible with an exonuclease. D shows a therapeutic supercoiled circular DNA vector obtained by reaction of the therapeutic circular DNA vector with a topoisomerase such as gyrase. [Figure 4]A-C show diagrams depicting three DNA vectors generated using the methods described herein. A is a single transcription unit (TU) DNA vector (1103) with a CMV promoter (PCMV), coding sequence, and polyA tail arranged in a 5'→3' direction. B is a composite TU DNA vector (1147) with a first TU with a first promoter, a first coding sequence, and a first polyA tail; a second TU with a second promoter, a second coding sequence, and a second polyA tail; a third TU with a third promoter, a third coding sequence, and a third polyA tail; and a fourth TU with a fourth promoter, a fourth coding sequence, and a fourth polyA tail, arranged in a 5'→3' direction. C is a single TU DNA vector (1258) with three coding sequences under a single promoter control followed by a polyA tail. [Figure 5A] 1 shows a photograph of an electrophoretic gel showing bands corresponding to DNA fragments after digestion, with actual and theoretical bands shown, respectively. [Figure 5B] 1 shows a photograph of an electrophoretic gel showing bands corresponding to DNA fragments after digestion, with actual and theoretical bands shown, respectively. [Figure 5C] 1 shows a photograph of an electrophoretic gel showing bands corresponding to the digested DNA fragments, and the band pattern after ligation. [Figure 5D] 1 shows a photograph of an electrophoretic gel showing bands corresponding to digested DNA fragments, and the band pattern after exonuclease digestion. [Figure 6A] 1 shows a schematic diagram illustrating two variations of the reaction step process described herein: An initial BsaI digestion is followed by heat inactivation before ligation (condition 1). [Figure 6B] 1 shows a schematic diagram illustrating two variations of the reaction step process described herein: A streamlined process (condition 2) is shown, combining BsaI digestion with ligation. [Figure 7A]7B shows a map illustrating the process of removing the backbone from a plasmid DNA vector using BsaI, which cuts five sites in the plasmid DNA vector. 7C shows a plasmid DNA vector containing the therapeutic sequence (C3 region; its nucleotide base is shown as N in FIG. 7B for illustration purposes) and the backbone sequence (containing the origin of replication and resistance genes). [Figure 7B] Figure 7A shows a map illustrating the process of backbone removal from a plasmid DNA vector using BsaI, which cuts at five sites within the plasmid DNA vector. The linear sequence corresponding to Figure 7A is shown. [Figure 8] A and B show gels showing the theoretical (A) or actual (B) bands following exonuclease digestion of the three constructs shown in Figures 4A-4C. Lanes 1-3 contain products from condition 1, while lanes 4-6 contain products from condition 2. Lanes 1 and 4 contain construct 1103 (1431 bp), lanes 2 and 5 contain construct 1147 (6293 bp), and lanes 3 and 6 contain construct 1258 (5065 bp). [Figure 9] A photograph of an electrophoresis gel showing recovery of the 12.75 kb C3 DNA construct after T5 exonuclease digestion is shown. [Figure 10] 1 shows a photograph of an electrophoretic gel illustrating a ligation reaction in which a linear fragment containing a reporter gene sequence self-ligated to form a closed circular DNA vector. [Figure 11] Photograph of an electrophoretic gel showing a ligation reaction in which a linear fragment containing a reporter gene sequence self-ligated to form a closed circular DNA vector. Lanes 1-3 show 20 μg / mL DNA treated with 100 U / μg ligase, 20 U / μg ligase, and 5 U / μg ligase, respectively. Lanes 4-6 show 40 μg / mL DNA treated with 100 U / μg ligase, 20 U / μg ligase, and 5 U / μg ligase, respectively. Lanes 7-9 show 100 μg / mL DNA treated with 100 U / μg ligase, 20 U / μg ligase, and 5 U / μg ligase, respectively. [Figure 12]Photograph of a gel showing the results of a time course of T5 exonuclease digestion from 0 hours (lane 2) to 2 hours (lane 6) or overnight (lane 11). [Figure 13] Electrophoretic gel photograph showing the banding profile of C3DNA after ligation with various ligase enzymes and DNA concentrations during ligation. The black box identifies the band of the C3DNA vector of interest. The lane numbering corresponds to the sample number identified in Table 3. [Figure 14] Photograph of an electrophoretic gel showing the banding profile of C3DNA after various times of ligation with T4 ligase. White boxes indicate the C3DNA bands of interest. Lane 1 is the control sample after BsaI treatment, lanes 2-5 show sample 1 from Table 3 at t=0 (lane 2), t=2 hours (lane 3), t=5 hours (lane 4), and t=21.5 hours (lane 5), lanes 6-9 show sample 2 from Table 3 at t=0 (lane 6), t=2 hours (lane 7), t=5 hours (lane 8), and t=21.5 hours (lane 9), and lanes 10-13 show sample 2 from Table 3 at t=0 (lane 10), t=2 hours (lane 11), t=5 hours (lane 12), and t=21.5 hours (lane 13). [Figure 15]Photograph of an electrophoretic gel showing the banding profile of C3 DNA after various times of ligation with T3 ligase (lanes 2-9) and T7 ligase (lanes 10-17). The white boxes indicate the C3 DNA bands of interest. Lane 1 is a control sample after BsaI treatment, lanes 2 to 5 show sample 4 from Table 3 at t=0 (lane 2), t=2 hours (lane 3), t=5 hours (lane 4), and t=21.5 hours (lane 5), lanes 6 to 9 show sample 5 from Table 3 at t=0 (lane 6), t=2 hours (lane 7), t=5 hours (lane 8), and t=21.5 hours (lane 9), lanes 10 to 13 show sample 6 from Table 3 at t=0 (lane 10), t=2 hours (lane 11), t=5 hours (lane 12), and t=21.5 hours (lane 13), and lanes 14 to 17 show sample 7 from Table 3 at t=0 (lane 14), t=2 hours (lane 15), t=5 hours (lane 16), and t=21.5 hours (lane 17). [Figure 16] 1 shows a graph depicting the ligation kinetics for samples 1 to 7 in Table 3 as the decrease in linear DNA over time. [Figure 17] Figure 1 shows a photograph of an electrophoretic gel showing the banding patterns of various C3DNA construct preparations after ligation and before heat killing. Lanes correspond to sample numbers in Table 4. White boxes indicate the C3DNA monomer bands of interest for each construct size. [Figure 18] A and B are photographs of electrophoretic gels showing the banding patterns of various C3DNA construct preparations after supercoiling by gyrase treatment. Lanes correspond to sample numbers in Table 4. White boxes indicate the C3DNA monomer bands of interest for each construct size. [Figure 19] A and B are photographs of electrophoretic gels showing the banding patterns of various C3DNA construct preparations after exonuclease digestion. Lanes correspond to sample numbers in Table 4. White boxes indicate the C3DNA monomer bands of interest for each construct size. [Figure 20] 1 shows a graphical quantification of C3DNA monomer yields of purified C3DNA construct preparations. The left bar in each sample represents the heat-killed sample. [Figure 21] 1 is a photograph of an electrophoretic gel showing the band pattern of C3 DNA produced by supercoiling and exonuclease digestion under the conditions specified in Table 6, before downstream column purification. Lane numbers correspond to sample numbers in Table 6. [Figure 22] 22 is a graph showing relative quantification of C3 DNA monomer yields for the samples shown in FIG. 21. [Diagram 23] 6 is a photograph of an electrophoresis gel showing the banding pattern of C3 DNA produced by downstream purification under the conditions specified in Table 6, except for the sample with a DNA concentration of 160 ug / mL. [Figure 24] FIG. 24 is a graph showing relative quantification of C3 DNA monomer yields for the samples shown in FIG. 23. [Diagram 25] Photographs of electrophoretic gels showing the banding patterns of C3 DNA produced under various gyrase concentrations specified in Table 8 are shown. [Figure 26] Electrophoretic gel photographs showing the band patterns of different sizes of C3 DNA generated by BsaI digestion steps at various times using different restriction treatments (overhang sequences and number of cleavage sites). Sample numbers are shown in Table 9. [Figure 27] A and B show photographs of electrophoretic gels showing the band patterns of different sizes of C3 DNA generated by ligation steps at various times performed as part of the same experiment using different restriction procedures (overhang sequences and number of cleavage sites). A shows the 1, 3, and 18 hour time points. B shows the 3, 18, and 24 hour time points. Sample numbers are shown in Table 9. White arrows indicate the bands of interest. White boxes indicate by-product bands. [Figure 28] A shows a photograph of an electrophoretic gel showing the post-exonuclease banding patterns of different sizes of C3DNA generated using different restriction treatments (overhang sequences and number of cleavage sites) as shown in Table 9. B shows a graph showing the post-exonuclease C3DNA concentration in each sample listed in Table 9, as quantified by Qubit. [Figure 29]Graph showing DNA concentration of by-product DNA over time of exonuclease treatment. Samples were run in duplicate (A and B). [Diagram 30] Schematic diagram showing the two constructs tested in Example 11. Each construct was generated with a different restriction procedure (combining either AAAA or AACC overhangs with either a one-fragment or four-fragment backbone). [Diagram 31] Figure 1 shows the plasmid map of the 8.7 kb construct with AAAA overhangs exemplified in Example 11. The BsaI recognition site (GGTCTC) is indicated by a black arrow near the BsaI cleavage site (cohesive ends outlined in black). For cleavage sites adjacent to a therapeutic sequence, either the therapeutic sequence or the backbone sequence is noted on either side of the cleavage site. [Diagram 32] Figure 1 shows the plasmid map of the 8.7 kb construct with AACC overhangs exemplified in Example 11. The BsaI recognition site (GGTCTC) is indicated by a black arrow near the BsaI cleavage site (cohesive ends outlined in black). Either the therapeutic or backbone sequence is indicated on either side of each cleavage site. [Diagram 33] 1 shows a photograph of an electrophoretic gel showing the 8.7 kb C3 DNA banding pattern after the ligation step for the samples identified in Table 14. The white box indicates the C3 DNA monomer band of interest. [Diagram 34] Photographs of electrophoretic gels showing the 8.7 kb C3 DNA band patterns after the supercoiling step under conditions 1, 2, and 4. The white box indicates the desired C3 DNA monomer band. [Diagram 35] A photograph of an electrophoretic gel showing the 8.7 kb C3 DNA band pattern after the exonuclease digestion step under condition 3 is shown. The white box indicates the desired C3 DNA monomer band. [Diagram 36]Photographs of electrophoretic gels showing the 8.7 kb C3 DNA band patterns after the exonuclease digestion steps under conditions 1, 2, and 4, and after the supercoiling step under condition 3. The white boxes indicate the desired C3 DNA monomer band. [Figure 37] 1 shows a photograph of an electrophoretic gel showing the 10.3 kb C3 DNA banding pattern after the ligation step for the samples identified in Table 16. The white box indicates the C3 DNA monomer band of interest. [Figure 38] Photographs of electrophoretic gels showing the 10.3 kb C3 DNA band patterns after the supercoiling step under conditions 1, 2, and 4. The white box indicates the desired C3 DNA monomer band. [Figure 39] Photographs of electrophoretic gels showing the 10.3 kb C3 DNA band patterns after the exonuclease digestion steps under conditions 1, 2, and 4, and after the supercoiling step under condition 3. The white boxes indicate the desired C3 DNA monomer band. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] The present invention features an improved method for generating non-viral DNA vectors, e.g., therapeutic circular DNA vectors. The invention is based in part on the development of a cell-free process for synthetically generating circular DNA by rolling circle amplification and ligation-mediated circularization (as opposed to, e.g., bacterial expression and / or site-specific recombination). The method of the present invention allows for improved scalability and manufacturing efficiency in the generation of non-viral circular DNA vectors and can reduce the risks associated with bacterial processing. The present invention allows for the generation of circular DNA vectors with therapeutic sequences that can be used to treat diseases or disorders, e.g., by transfecting target cells.

[0115] The methods disclosed herein may result in enhanced purity and yield of the desired product compared to conventional methods. In particular, the use of steps including treatment with certain restriction enzymes (e.g., type IIS restriction enzymes), exonucleases, such as terminal exonucleases (e.g., T5 exonuclease), and / or helicases or topoisomerases (e.g., type II topoisomerases, e.g., gyrase), results in products with enhanced yield and purity by reducing and / or degrading impurities such as bacterial sequences. In certain embodiments, the methods of the present invention streamline the manufacturing process by simultaneously performing restriction digestion and ligation by using type IIS restriction enzymes.

[0116] The therapeutic circular DNA vector and its pharmaceutical composition produced by the method of the present invention show several advantageous properties. For example, by removing or reducing the repression site of bacterial plasmid DNA sequence, such as RNAPII, the transcriptional silencing of the therapeutic circular DNA vector can be reduced or eliminated, which leads to the persistence of the therapeutic sequence in an individual. In a specific embodiment of the present invention, immunogenic components (e.g., bacterial endotoxins, DNA or RNA, or bacterial signatures, such as CpG motifs) are not present in the therapeutic circular DNA vector of the present invention, and therefore the risk of stimulating host immune response is reduced compared to conventional DNA vectors such as plasmid DNA vectors.

[0117] Thus, the methods described herein generate DNA vectors that are substantially devoid of bacterial plasmid DNA sequences (e.g., repression sites of RNAPII, origins of replication, and / or resistance genes) and other bacterial signatures (e.g., immunogenic CpG motifs) and / or can be amplified and synthesized entirely in vitro (e.g., no bacterial replication is required, no bacterial origins of replication and no bacterial resistance genes are required, no recombination sites are required). These methods and steps are described in more detail below.

[0118] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and as understood by reference to published textbooks which provide those skilled in the art with a general guide to many of the terms used in this application. In the event of any discrepancy between a definition set forth herein and a definition in a referenced publication, the definition provided herein shall control.

[0119] As used herein, the term "circular DNA vector" refers to a nucleic acid molecule in a circular form. Such a circular form can be amplified into concatemers, usually by rolling circle amplification. A linear double-stranded nucleic acid in which the strands are linked at the ends (e.g., a backbone covalently linked by a hairpin loop or other structure) is not a circular vector as used herein. The term "circular DNA vector" is used interchangeably herein with the terms "covalently closed circular DNA vector" and "C3DNA". Those skilled in the art will understand that such a circular vector encompasses a covalently closed vector that is supercoiled and has a complex DNA topology, as described herein. In certain embodiments, the circular DNA vector is supercoiled (e.g., monomeric supercoiled). In certain instances, the circular DNA vector lacks a bacterial origin of replication (e.g., if the circular DNA vector encodes a self-replicating RNA molecule, the circular DNA vector lacks a bacterial origin of replication and encodes an RNA origin of replication).

[0120] As used herein, a "cell-free" method for generating a circular DNA vector refers to a method in which every step of the method, from providing a template DNA vector (e.g., a plasmid DNA vector) to generating a therapeutic circular DNA vector, is facilitated without relying on the containment of any DNA in a host cell, such as a bacterial (e.g., E. coli) host cell. For example, the cell-free method is carried out in a suitable solution (e.g., a buffer) in one or more synthetic vessels (e.g., glass or plastic tubes, bioreactors, vessels, tanks, or other suitable vessels), to which enzymes and other agents may be added to facilitate the amplification, modification, and isolation of DNA. The cell-free production method may use template DNA generated within a cell.

[0121] As used herein, the term "therapeutic sequence" refers to a portion of a DNA molecule (e.g., a plasmid DNA vector or a concatemer thereof) that contains any genetic material necessary for transcription of one or more therapeutic moieties in a target cell, and may include one or more coding sequences, promoters, terminators, introns, and / or other regulatory elements. The therapeutic moiety may be a therapeutic protein (e.g., a replacement protein (e.g., a protein that replaces a defective protein in a target cell) or an endogenous protein (e.g., a regulatory protein such as a cytokine)) and / or a therapeutic nucleic acid (e.g., one or more microRNAs). In a DNA vector with two or more transcription units, the therapeutic sequence includes multiple transcription units. The therapeutic sequence may include one or more genes (e.g., heterologous genes or transgenes) that are administered for therapeutic purposes.

[0122] As used herein, the term "protein" refers to multiple amino acids bound together by peptide bonds (i.e., as a primary structure) and includes non-covalently associated multimeric (e.g., dimeric, trimeric, etc.) proteins (e.g., proteins having quaternary structure). Thus, the term "protein" includes peptides (e.g., polypeptides), natural proteins, recombinant proteins, and fragments thereof. In some embodiments, a protein has a primary structure and no secondary, tertiary, or quaternary structure under physiological conditions. In some embodiments, a protein has a primary structure and a secondary structure and no tertiary or quaternary structure under physiological conditions. In certain embodiments, a protein has a primary structure, a secondary structure, and a tertiary structure, but no quaternary structure under physiological conditions (e.g., a monomeric protein having one or more folded alpha helices and / or beta sheets). In some embodiments, any of the proteins described herein has a length of at least 25 amino acids (e.g., between 50 amino acids and 1000 amino acids).

[0123] The term "therapeutic gene" refers to a transgene that is administered (e.g., as part of a DNA vector or a self-replicating RNA molecule). A therapeutic gene can be a mammalian gene that encodes a therapeutic protein.

[0124] As used herein, the term "therapeutic protein" refers to a protein that can treat a disease or disorder in a subject. In some embodiments, a therapeutic protein is a therapeutic replacement protein that is administered to replace a defective (e.g., mutant) protein in a subject. In some embodiments, a therapeutic protein is identical or functionally equivalent to a native protein (e.g., a cytokine, chemokine, or growth factor) that is not defective in the subject. In some embodiments, a therapeutic protein is an antigen. In some embodiments, a therapeutic protein is an antigen-binding protein.

[0125] As used herein, the term "therapeutic replacement protein" refers to a protein that is structurally similar (e.g., structurally identical) to a protein that is endogenously expressed by a normal (e.g., healthy) individual. Therapeutic replacement proteins can be administered to individuals suffering from a disorder associated with a dysfunction (or lack) of the protein to be replaced. In some embodiments, the therapeutic replacement protein corrects a protein abnormality resulting from a mutation (e.g., a point mutation, an insertion mutation, a deletion mutation, or a splice variant mutation) in the gene encoding the protein. Therapeutic replacement proteins do not include non-endogenous proteins, such as proteins associated with pathogens (e.g., as part of a vaccine). Therapeutic replacement proteins can include enzymes, growth factors, hormones, interleukins, interferons, cytokines, anti-apoptotic factors, anti-diabetic factors, clotting factors, anti-tumor factors, liver-secreted proteins, or neuroprotective factors. In some examples, the therapeutic replacement protein is monogenic.

[0126] As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid that binds (e.g., hybridizes) to a molecule (e.g., a protein or nucleic acid) in a subject to produce its therapeutic effect (i.e., without necessarily being transcribed or translated). Therapeutic nucleic acids can be DNA or RNA, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), CRISPR molecules (e.g., guide RNA (gRNA)), oligonucleotides (e.g., antisense oligonucleotides), aptamers, or DNA vaccines. In some embodiments, therapeutic nucleic acids can be non-inflammatory or non-immunogenic therapeutic nucleic acids. In other embodiments, therapeutic nucleic acids can be recognized by the immune system (e.g., adaptive immune system) and induce an immune response (e.g., innate immune response). Such therapeutic nucleic acids include Toll-like receptor (TLR) agonists.

[0127] As used herein, the term "Type IIS restriction enzyme" refers to an enzyme that recognizes a recognition site on a DNA molecule and cleaves the DNA molecule at a cleavage site that is outside the recognition site, thereby generating an overhang (sticky end) having a sequence unrelated to the recognition site. IIS type restriction enzymes include natural type IIS restriction enzymes (e.g., BsaI, AcuI, AlwI, BaeI, BbsI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, BtsIMutI, CspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MlyI, MmeI, MnII, NmeAIII, PaqCI, PleI, SapI, and SfaNI) and (e.g., Lippow et al. al. Nucleic Acids Res. 2009, 37(9):3061-3073, which is incorporated by reference in its entirety.

[0128] As used herein, the term "backbone sequence" refers to a portion of the plasmid DNA outside the therapeutic sequence, including one or more bacterial origins of replication or fragments thereof, one or more drug resistance genes or fragments thereof, one or more recombination sites, or any combination thereof. In some embodiments, the backbone sequence includes one or more bacterial origins of replication. The backbone sequence includes a truncated plasmid backbone of 20 base pairs or more (e.g., 31-40, e.g., 38 base pairs), which may include, for example, a functional origin of replication.

[0129] As used herein, the term "recombination site" refers to a nucleic acid sequence that is a product of site-specific recombination, comprising a first sequence corresponding to a portion of a first recombinase attachment site and a second sequence corresponding to a portion of a second recombinase attachment site. An example of a hybrid recombination site is attR, which is a product of site-specific recombination and comprises a first sequence corresponding to a portion of attP and a second sequence corresponding to a portion of attB. Alternatively, a recombination site can be generated by Cre / Lox recombination. Thus, a vector generated by Cre / Lox recombination (e.g., a vector containing a LoxP site) comprises a recombination site as used herein. Other site-specific recombination events that generate recombination sites involve, for example, lambda integrase, FLP recombinase, and Kw recombinase. A nucleic acid sequence generated by a non-site-specific recombination event (e.g., intermolecular recombination mediated by ITRs) is not a recombination site as defined herein.

[0130] As used herein, the terms "adjacent," "adjacent," and "adjacent" refer to a pair of regions or positions on a nucleic acid molecule (e.g., a plasmid DNA vector) that are outside the reference region of the nucleic acid molecule. In some embodiments, a pair of regions or positions on a nucleic acid that are adjacent to a reference region are adjacent (i.e., contiguous) to the reference region (i.e., there are no intervening bases between the reference and adjacent positions). In other embodiments, a pair of regions or positions on a nucleic acid molecule that are adjacent to a reference region are separated from the reference region by one or more intervening bases (e.g., up to 1,000 intervening bases). For example, if a first restriction site is 200 bases upstream of a therapeutic sequence and a second restriction site is 100 bases downstream of the therapeutic sequence, the first and second restriction sites are said to be adjacent to the therapeutic sequence.

[0131] In some embodiments, all intervening sequences between the flanking region or position and the reference region are devoid of bacterial sequences. Thus, the circular DNA vector generated by self-ligating the therapeutic sequence excised from the plasmid DNA vector at the restriction site adjacent to the therapeutic sequence is free of bacterial sequences. For example, in such an embodiment, a type IIS restriction enzyme that cuts at a site adjacent to the therapeutic sequence can generate a therapeutic circular DNA vector with a sequence between the 5' and 3' ends of the therapeutic sequence, but this region does not have bacterial sequences (e.g., bacterial origin of replication or drug resistance genes). Such intervening sequences may be artifacts of sticky end ligation, e.g., they correspond to overhanging bases generated by type IIS restriction enzymes.

[0132] As used herein, steps are performed "concurrently" if they overlap completely or partially. Thus, restriction enzyme digestion and ligation are performed simultaneously in any of the following scenarios: (i) the restriction enzyme acts on the DNA simultaneously with the ligase and both enzymes are inactivated simultaneously; (ii) the restriction enzyme acts on the DNA simultaneously with the ligase and the enzymes are inactivated at different times; (iii) the restriction enzyme acts on the DNA before the ligase and both enzymes are inactivated simultaneously; or (iv) the restriction enzyme acts on the DNA before the ligase, the ligase acts on the DNA before the restriction enzyme is inactivated, and the restriction enzyme is inactivated before the ligase is inactivated.

[0133] As used herein, a step is said to follow "immediately" after a previous step if there is no intervening functional step, such as purification (e.g., purification that reduces DNA yield, e.g., gel purification or column purification), enzymatic reaction, or enzyme inactivation step (e.g., heat inactivation step, also referred to as heat killing step). It will be understood that when going from one step to the immediately following step, a transition of conditions, such as an increase or decrease in temperature and / or an increase or decrease in reagent concentration, may occur. Regardless of whether such transition of conditions occurs instantly or gradually (e.g., over a period of seconds or minutes), a subsequent step is still said to follow "immediately" after a previous step if there is no intervening functional step. For example, a heat inactivation step at 65°C after ligation may be immediately followed by a supercoiling step at 37°C after a 2 hour cooling period during which the temperature is reduced from 65°C to 37°C.

[0134] As used herein, "large-scale production" refers to the production of at least 2 mg of therapeutic circular DNA vector per batch. Large-scale production allows for one or more administrations of a therapeutically effective amount of therapeutic circular DNA vector.

[0135] As used herein, the term "self-replicating RNA molecule" refers to a self-replicating genetic element that contains RNA that replicates autonomously from a single origin of replication. The terms "self-replicating RNA", "replicon RNA" and "self-amplifying replicon RNA" are used interchangeably herein.

[0136] As used herein, the term "operably linked" refers to an arrangement of elements in which the components so described are positioned to perform their normal functions. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to one or more heterologous genes if it affects the transcription of the gene(s). Furthermore, regulatory elements operably linked to a coding sequence can affect the expression of the coding sequence. Regulatory elements need not be contiguous with the coding sequence, so long as they function to induce expression of the coding sequence. Thus, for example, non-translated but transcribed intervening sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0137] As used herein, the term "isolated" means artificially produced and not integrated into the genome of a natural host. For example, an isolated nucleic acid vector encompasses a nucleic acid vector that is encapsulated in a lipid envelope (e.g., a liposome) or a polymer matrix. In some embodiments, the term "isolated" refers to a DNA vector that is (i) amplified in vitro (e.g., in a cell-free environment), for example, by rolling circle amplification or polymerase chain reaction (PCR); (ii) recombinantly produced by molecular cloning; (iii) purified, for example, by restriction endonuclease cleavage and gel electrophoretic fractionation, or column chromatography; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid vector is one that can be readily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for which the 5' and 3' restriction sites are known or for which the polymerase chain reaction (PCR) primer sequences are disclosed is considered to be isolated, while a nucleic acid sequence that exists in its natural state in its natural host is not considered to be isolated. An isolated nucleic acid vector may, but need not be, substantially purified.

[0138] As used herein, the term "naked" refers to a nucleic acid molecule (e.g., a circular DNA vector) that is not encapsulated in a lipid envelope (e.g., a liposome) or a polymer matrix and is not physically associated (e.g., not covalently or non-covalently bound) with a three-dimensional structure (e.g., a particulate structure) upon administration to an individual. In some examples of the present invention, the pharmaceutical composition comprises a naked circular DNA vector.

[0139] As used herein, a "vector" refers to a nucleic acid molecule capable of delivering a therapeutic sequence linked thereto to a target cell, where the therapeutic sequence can then be transcribed, replicated, processed, and / or expressed within the target cell. Once the target or host cell has processed the therapeutic sequence of the vector, the therapeutic sequence is no longer considered a vector. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop containing a bacterial backbone into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome.

[0140] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors").

[0141] As used herein, the terms "individual" and "subject" are used interchangeably and include any mammalian subject in need of treatment or prevention, for example, with a therapeutic circular DNA vector or pharmaceutical composition thereof described herein. In some embodiments, the individual or subject is a human. In other embodiments, the individual or subject is a non-human mammalian subject, such as a non-human primate (e.g., monkey), mouse, pig, rabbit, cat, or dog. The individual or subject may be male or female.

[0142] As used herein, an "effective amount" or "effective dose" of a therapeutic circular DNA vector or pharmaceutical composition thereof refers to an amount sufficient to achieve a desired biological, pharmacological, or therapeutic effect when administered to an individual, for example, according to a selected administration form, route, and / or schedule. As will be appreciated by those skilled in the art, the effective absolute amount of a particular composition may vary depending on factors such as the desired biological or pharmacological endpoint, the agent being delivered, the target tissue, and the like. Furthermore, those skilled in the art will understand that an "effective amount" may be contacted with a cell or administered to a subject using a single administration or repeated administrations. An effective amount of a composition for treating a disease may slow or stop the progression of the disease, or increase partial or complete responses compared to a reference population, for example, an untreated or placebo population, or a population receiving standard treatment.

[0143] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to a clinical intervention to alter the natural course of the individual being treated, which may be performed for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or alleviation of the pathology, and improvement of prognosis. In some embodiments, the therapeutic circular DNA vector of the present invention is used to delay the onset of disease or to slow the progression of disease.

[0144] The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a polynucleotide, or an amino acid product or protein, in a biological sample (e.g., retina). "Expression" generally refers to the process by which genetically encoded information is converted into structures present and functional within a cell.

[0145] Thus, according to the present invention, "expression" can refer to transcription into a polynucleotide, translation into a protein, or post-translational modification of a protein. A transcribed polynucleotide, a translated protein, or a post-translationally modified fragment of a protein is also considered to be expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a protein, for example, by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a protein, and those that are further transcribed into RNA but not translated into a protein (e.g., transfer RNA and ribosomal RNA).

[0146] As used herein, the term "expression persistence" refers to the duration during which a therapeutic sequence or a functional portion thereof (e.g., one or more coding sequences of a therapeutic DNA vector) is expressible in a transfected cell ("intracellular persistence") or in any descendants of the transfected cell ("intergenerational persistence"). A therapeutic sequence or a functional portion thereof may be expressible if it is not silenced, for example, by DNA methylation and / or histone methylation and compaction. Expression persistence may be assessed by detecting or quantifying (i) the mRNA transcribed from the therapeutic sequence in the target cell or its descendants (e.g., by qPCR, RNA-seq, or any other suitable method), and (ii) the protein translated from the therapeutic sequence in the target cell or its descendants (e.g., by Western blot, ELISA, or any other suitable method). In some examples, persistence of expression can be assessed by detecting or quantifying the therapeutic DNA in the target cell or its progeny (e.g., by episomal DNA copy number analysis of the presence of the therapeutic circular DNA vector in the target cell) in conjunction with one or both of (i) the mRNA transcribed from the therapeutic sequence in the target cell or its progeny, and (ii) the protein translated from the therapeutic sequence in the target cell or its progeny. The persistence of expression of the therapeutic sequence or a functional portion thereof can be quantified in comparison to a reference vector, such as a control vector produced in bacteria (e.g., a circular vector (e.g., a plasmid) produced in bacteria or having one or more bacterial signatures that are not present in the vector of the present invention), using any method known in the art for characterizing gene expression. The persistence of expression can be quantified at any given time point after administration of the vector. For example, in some embodiments, expression of the therapeutic circular DNA vector of the present invention persists for at least two weeks after administration if it is detectable in the target cell or its progeny two weeks after administration of the therapeutic circular DNA vector.In some embodiments, expression of a gene is "sustained" in a target cell if it is detectable in the target cell for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration. In some embodiments, expression of a therapeutic sequence is said to be sustained for a given period of time after administration if any remaining portion of the original expression level (e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, or at least 100%) is detectable after the given period of time (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration).

[0147] As used herein, "intracellular persistence" refers to the duration that a therapeutic sequence or a functional portion thereof (e.g., one or more coding sequences of a therapeutic DNA vector) is expressible in a transfected cell (e.g., a target cell, e.g., a postmitotic or quiescent cell). Intracellular persistence can be assessed by detecting or quantifying (i) the mRNA transcribed from the therapeutic sequence in the target cell, and (ii) the protein translated from the therapeutic sequence in the target cell. In some examples, intracellular persistence is assessed by detecting or quantifying the therapeutic DNA in the target cell (e.g., the presence of a therapeutic circular DNA vector in the target cell) in conjunction with one or both of (i) the mRNA transcribed from the therapeutic sequence in the target cell, and (ii) the protein translated from the therapeutic sequence in the target cell. In some embodiments, the therapeutic circular DNA vector of the present invention exhibits improved intracellular persistence compared to a reference vector (e.g., a plasmid DNA vector).

[0148] As used herein, "transgenerational persistence" refers to the duration during which a therapeutic sequence or a functional portion thereof (e.g., one or more coding sequences of a therapeutic DNA vector) can be expressed in the progeny of a cell transfected with the gene (e.g., the progeny of a target cell, e.g., the first, second, third, or fourth generation progeny of a cell transfected with the gene by a therapeutic circular DNA vector). Transgenerational persistence accounts for any dilution of a gene through cell division and can therefore be useful to measure the persistence of a vector over time in a dividing tissue. In some embodiments, the therapeutic circular DNA vector of the present invention exhibits improved transgenerational persistence compared to a reference vector (e.g., a plasmid DNA vector). Transgenerational persistence can be assessed by detecting or quantifying (i) the mRNA transcribed from the therapeutic sequence in the progeny of the target cell, and (ii) the protein translated from the therapeutic sequence in the progeny of the target cell. In some instances, intracellular persistence is assessed by detecting or quantifying the therapeutic DNA in the progeny of the target cell (e.g., the presence of the therapeutic circular DNA vector in the progeny of the target cell) in conjunction with one or both of (i) the mRNA transcribed from the therapeutic sequence in the progeny of the target cell, and (ii) the protein translated from the therapeutic sequence in the progeny of the target cell. In some embodiments, the therapeutic circular DNA vectors of the present invention exhibit improved transgenerational persistence compared to a reference vector (e.g., a plasmid DNA vector).

[0149] The term "pharmaceutical acceptable" means safe for administration to a mammal, such as a human. In some embodiments, a pharmaceutical acceptable composition is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0150] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the vector or composition of the present invention is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, PA., 23. rd It is described in the 2020 edition.

[0151] The terms "a" and "an" mean "one or more of." For example, "a gene" is understood to represent one or more of such genes. Thus, the terms "a" and "an," "one or more of a (or an)," and "at least one of a (or an)" are used interchangeably herein.

[0152] As used herein, the term "about" refers to a value within ±10% variation from a reference value, unless otherwise specified.

[0153] In the event of any discrepancies in definitions among various sources or references, the definitions provided herein shall prevail.

[0154] II. Methods for generating therapeutic circular DNA vectors The methods provided herein include cell-free synthesis of therapeutic circular DNA vectors as an alternative to traditional production methods based on synthesis by bacterial cells. Because amplification of bacterial plasmid DNA vectors in cell-free conditions using polymerases is possible, circular DNA vectors can be isolated from bacterial components of cloned plasmids, with the isolated product vector being substantially free of bacterial signatures. Thus, cell-free synthesis minimizes the risk of bacterial impurities and provides a purer composition of the resulting circular DNA vector (i.e., synthetic circular DNA vector) compared to bacterial-derived vectors (i.e., non-synthetic circular DNA vectors). The methods of the present invention are amenable to scale-up and provide improved manufacturing efficiency. No gel extraction step is required. Thus, in some embodiments, gel purification (e.g., agarose gel purification) as part of the production process is not performed (e.g., gel electrophoresis can be performed in parallel for analytical purposes). In some embodiments, streamlined restriction digestion schemes are provided. The therapeutic circular DNA vectors generated using such cell-free processes are referred to herein as "synthetic" vectors, reflecting the lack of bacterial cells in their generation from templates.

[0155] In one aspect, the method includes providing a sample including a template DNA molecule (e.g., a template DNA vector (e.g., a plasmid DNA vector)) that includes a therapeutic gene sequence, and amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers. The linear concatemers are digested with a restriction enzyme that cuts at least two sites in the linear concatemers per unit of the bacterial plasmid DNA vector to generate linearized fragments of the DNA vector. The method of the invention further includes self-ligating the linearized fragments of the DNA vector that include the therapeutic sequence to generate a therapeutic circular DNA vector. The method also includes treating the sample with a topoisomerase or helicase. In some embodiments, the method further includes digesting the sample with an exonuclease (e.g., a terminal exonuclease). In some embodiments, the digesting and self-ligating are performed simultaneously.

[0156] In one aspect, the method includes providing a sample including a template DNA molecule (e.g., a template DNA vector (e.g., a plasmid DNA vector)) that includes a therapeutic sequence, and amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers. The method further includes digesting the linear concatemers with a restriction enzyme (e.g., a type IIS restriction enzyme, e.g., BsaI) to generate linearized fragments of the DNA vector. The linear concatemers have multiple copies of the template DNA vector, each copy having a unit length, and the linear concatemers have a unit length of multiple vectors. The restriction enzyme cuts at least two sites (e.g., only two sites, or more than two sites (e.g., three, four, five, or more sites)) of the linear concatemers per unit of bacterial plasmid DNA vector. The method includes self-ligating the linearized fragments of the DNA vector to generate closed circular DNA vectors (e.g., C 3In some embodiments, the method further comprises generating a self-ligated DNA. The method also comprises digesting the sample with an exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease). In some embodiments, the method further comprises treating the sample with a topoisomerase (e.g., gyrase) or a helicase. In some embodiments, the digesting and self-ligating are performed simultaneously.

[0157] In another aspect, the method includes providing a sample containing a template DNA molecule (e.g., a template DNA vector (e.g., a plasmid DNA vector)) that includes a therapeutic sequence, and amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers. The method further includes digesting the linear concatemers with a restriction enzyme (e.g., a type IIS restriction enzyme, e.g., BsaI) to generate linearized fragments of the DNA vector. The restriction enzyme cuts at least two sites (e.g., only two sites, or more than two sites (e.g., three, four, five, or more sites)) of the linear concatemers per unit of the template DNA vector. The method includes self-ligating the linearized fragments of the DNA vector to generate closed circular DNA vectors (e.g., C 3 The method may further include treating the sample with a topoisomerase (e.g., gyrase) or a helicase. The method may also include digesting the sample with an exonuclease (e.g., a terminal exonuclease, e.g., T5 exonuclease). In some embodiments, the digestion and self-ligation are performed simultaneously (in the same reaction conditions).

[0158] In some embodiments, the method utilizes a single restriction enzyme to generate an overhang so that the restriction digestion step can be integrated with the ligation step (e.g., the restriction digestion step can overlap with the ligation step or can be performed simultaneously with the ligation step). For example, some embodiments of such a method include: (a) providing a sample containing a template DNA vector containing a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) digesting the linear concatemers with a restriction enzyme (e.g., a type IIS restriction enzyme, e.g., BsaI) that cuts at least a first site, a second site, and a third site for each unit of the linear concatemer, thereby generating a linear therapeutic fragment containing the therapeutic sequence and at least two linear backbone fragments each containing a portion of the backbone sequence; and (d) contacting the linear therapeutic fragments with a ligase to generate a therapeutic circular DNA vector in solution. The first and second sites flank the therapeutic sequence and form self-complementary overhangs, and the third site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site.

[0159] Alternatively, a type IIS restriction enzyme can be used to cleave a template DNA molecule at two sites, thereby generating a single backbone and therapeutic fragment. By designing the template such that a type IIS recognition site (e.g., GGTCTC in embodiments calling for BsaI) is present in the backbone fragment but absent in the therapeutic fragment, self-ligation of the backbone fragment reconstitutes a type IIS restriction site on the circularized backbone, while self-ligation of the therapeutic fragment generates a therapeutic DNA vector lacking a type IIS restriction site. Thus, the backbone is subject to further digestion, but the therapeutic DNA vector is not cleaved.

[0160] In some embodiments, the restriction enzyme cleaves the linear concatemer unit-by-unit at a fourth site, where the fourth site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site, and digestion generates at least three linear scaffold fragments, each of which contains a portion of the scaffold sequence.

[0161] In some embodiments, a restriction enzyme inactivation step does not precede step (d). For example, no heat inactivation of the restriction enzyme (e.g., type IIS restriction enzyme, e.g., BsaI) is performed prior to ligation. This allows for streamlined DNA production, enabled by the overhang design, which allows the restriction digestion and ligation steps to be performed simultaneously, thereby eliminating the need to inactivate the restriction enzyme in step (c). This also allows for the use of disposable containers that are not suitable for elevated temperatures.

[0162] After step (d), the temperature of the solution containing the therapeutic circular DNA vector may be raised to about 65° C. to inactivate the enzymes (e.g., restriction enzymes and / or ligases). Alternatively, no heat inactivation is performed after (e.g., immediately after) restriction enzyme digestion and / or ligation.

[0163] In another aspect, the present invention provides a method for removing a backbone sequence from a DNA molecule (e.g., a template DNA vector) to generate a therapeutic circular DNA vector. The DNA molecule (e.g., the template DNA vector) comprises a backbone sequence and a therapeutic sequence. The method includes the steps of (a) digesting the DNA molecule with one or more restriction enzymes (e.g., type IIS restriction enzymes) that cleave at least a first site and a second site in the DNA molecule, where (i) the first and second sites are adjacent to the therapeutic sequence and form self-complementary overhangs, and (ii) the recognition site is within the backbone sequence, and digestion produces a linear therapeutic fragment that includes the therapeutic sequence, and a linear backbone fragment that includes the backbone sequence and the recognition site (e.g., type IIS recognition site); and (b) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0164] In another aspect, the present invention provides a method for removing a backbone sequence from a DNA molecule (e.g., a template DNA vector) to generate a therapeutic circular DNA vector. The DNA molecule (e.g., a template DNA vector) comprises a backbone sequence and a therapeutic sequence. The method includes the steps of (a) digesting the DNA molecule with one or more restriction enzymes that cleave at least a first site, a second site, and a third site per unit of the DNA molecule, where (i) the first and second sites are adjacent to the therapeutic sequence and form self-complementary overhangs, and (ii) the third site is within the backbone sequence and forms a non-complementary overhang at the first or second site, and digestion generates a linear therapeutic fragment that includes the therapeutic sequence and at least two linear backbone fragments, each of which includes a portion of the backbone sequence; and (b) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0165] In some embodiments, the therapeutic circular DNA vector is contacted with a topoisomerase (e.g., gyrase) or helicase. Such reactions may be carried out at about 37° C. Additionally or alternatively, the therapeutic circular DNA vector may be contacted with an exonuclease (e.g., terminal exonuclease) (e.g., in a reaction carried out at about 37° C.). In certain embodiments, the therapeutic circular DNA vector (and its reaction mixture) is contacted with a topoisomerase or helicase, and then the therapeutic circular DNA vector (and its reaction mixture) is contacted with an exonuclease (e.g., terminal exonuclease) without increasing the reaction temperature to inactivate the topoisomerase or helicase. Alternatively, exonuclease digestion is carried out prior to contact with the topoisomerase or helicase.

[0166] In some embodiments, the method includes contacting the therapeutic circular DNA vector with a topoisomerase or helicase and / or terminal exonuclease followed by passing the therapeutic circular DNA vector through a column (e.g., a capture column). In some embodiments, the therapeutic circular DNA vector is then precipitated with isopropyl alcohol. In some embodiments, the method includes amplifying a template vector in vitro using any combination of the steps described in Sections A-G below, digesting the amplified vector with a restriction enzyme, allowing the resulting fragments to self-ligate, and treating the sample with a terminal exonuclease and / or a helicase or topoisomerase.

[0167] A. Templates Generally, the generation of a therapeutic circular DNA vector begins with providing a sample containing a template DNA molecule (e.g., template DNA vector), e.g., a plasmid DNA vector having a therapeutic sequence and a backbone sequence. The backbone sequence can be separated from the therapeutic sequence by designing the template DNA vector to have a restriction site (e.g., a type IIS restriction site, e.g., a BsaI restriction site, e.g., GGTCTC) adjacent to the therapeutic sequence (e.g., within the backbone sequence). The therapeutic sequence can then be self-ligated to generate the therapeutic circular DNA vector. The restriction site can be designed at a position within the backbone sequence to allow removal of the backbone sequence from the product by further restriction and / or exonuclease digestion without performing a yield-reducing step such as gel purification. For example, a type IIS recognition site can be placed distal to its corresponding cleavage site compared to the therapeutic sequence (see FIG. 2A), i.e., the cleavage site is between the recognition site and the therapeutic sequence.

[0168] In some embodiments, the template comprises the following linked in the following order: a first type IIS recognition site, a first type IIS cleavage site corresponding to the first type IIS recognition site, a therapeutic sequence, a second type IIS cleavage site, and a second type IIS recognition site corresponding to the second type IIS cleavage site. The second type IIS recognition site may be linked to the first type IIS recognition site by a scaffold sequence (or a portion thereof, in which case one or both of the first and second type IIS recognition sites are present in the scaffold sequence). In some examples, only two (i.e., no more than two) type IIS recognition sites are present in the template DNA molecule. In some examples, only two BsaI recognition sites are present in the template DNA molecule (e.g., as shown in FIG. 32). Such a design allows both type IIS recognition sites to be positioned in a linear scaffold sequence generated upon type IIS restriction enzyme digestion, and upon religation, the type IIS restriction enzyme may cleave the circularized scaffold sequence. In some embodiments, the therapeutic sequence does not include a type IIS recognition site (e.g., a BsaI recognition site, e.g., GGTCTC). In some embodiments, the therapeutic sequence does not include a restriction enzyme recognition site.

[0169] In some embodiments where the template is a plasmid DNA vector having a therapeutic sequence and a backbone sequence, the plasmid DNA has at least three restriction sites (e.g., at least four restriction sites or at least five restriction sites; e.g., three restriction sites, four restriction sites, or five restriction sites) recognized by the same restriction enzyme (e.g., a type IIS restriction enzyme, e.g., BsaI). Two of the at least three restriction sites flank the therapeutic sequence such that upon restriction digestion with the restriction enzyme (e.g., a type IIS restriction enzyme, e.g., BsaI), linear therapeutic fragments are formed having self-complementary overhangs at their ends (e.g., a single linear therapeutic fragment is formed). At least one remaining restriction site is present within the backbone sequence such that upon digestion of the plasmid DNA vector with the restriction enzyme (e.g., a type II restriction enzyme, e.g., BsaI), at least two linear backbone fragments are generated, each of which comprises a portion of the backbone sequence. At least one remaining restriction site is positioned within the backbone sequence such that upon digestion of the plasmid DNA vector with a restriction enzyme (e.g., a type II restriction enzyme, e.g., BsaI), the overhang generated by the restriction site in the backbone sequence is non-complementary to the overhang generated at the flanking end of the therapeutic sequence.

[0170] In some embodiments where the plasmid DNA has four restriction sites recognized by a type IIS restriction enzyme (e.g., BsaI), the remaining two restriction sites in the backbone sequence are positioned such that upon digestion of the plasmid DNA vector with a type IIS restriction enzyme (e.g., BsaI), three linear backbone fragments are generated, each containing a portion of the backbone sequence, and the overhangs generated by the restriction sites in the backbone sequence are all non-complementary to the overhangs generated at the flanking ends of the therapeutic sequence. In some such embodiments, the two type IIS restriction sites in the backbone sequence generate overhangs that are non-complementary to each other.

[0171] In embodiments in which two different restriction enzymes are used, the template (e.g., a plasmid DNA vector) may have at least three restriction sites (e.g., at least four restriction sites or at least five restriction sites; e.g., three restriction sites, four restriction sites, or five restriction sites), where two of the restriction sites are adjacent to the therapeutic sequence and are recognized by a first restriction enzyme. At least one remaining restriction site is in the backbone sequence and is recognized by a different second restriction enzyme. In some embodiments, the restriction sites adjacent to the therapeutic sequence are EcoRI restriction sites and the first restriction enzyme is EcoRI. In some embodiments, the restriction sites adjacent to the therapeutic sequence are PvuII restriction sites and the first restriction enzyme is PvuII. In some embodiments, the restriction site in the backbone sequence is a PvuII restriction site and the second restriction enzyme is PvuII. In some embodiments, the restriction site in the backbone sequence is an EcoRI restriction site and the second restriction enzyme is EcoRI.

[0172] In some embodiments, the template (e.g., a plasmid DNA vector) has four restriction sites, where two restriction sites flanking the therapeutic sequence are recognized by a first restriction enzyme and the remaining two restriction sites in the backbone sequence are recognized by a second restriction enzyme. In some embodiments, the restriction sites flanking the therapeutic sequence are EcoRI restriction sites and the first restriction enzyme is EcoRI. In some embodiments, the restriction sites flanking the therapeutic sequence are PvuII restriction sites and the first restriction enzyme is PvuII. In some embodiments, the restriction sites in the backbone sequence are PvuII restriction sites and the second restriction enzyme is PvuII. In some embodiments, the restriction sites in the backbone sequence are EcoRI restriction sites and the second restriction enzyme is EcoRI.

[0173] The sample containing template DNA may be a lysate or other preparation of cells or tissues (e.g., mammalian cells or tissues or bacterial cells) containing a template DNA vector (e.g., a bacterial plasmid DNA vector). Double-stranded circular DNA may be harvested from cells using standard DNA extraction / isolation techniques. In some embodiments, to purify the plasmid DNA vector before further processing, linear DNA is specifically degraded, for example, using a DNase that does not destroy the plasmid.

[0174] In other embodiments, the template DNA lacks one or more bacterial elements of a plasmid DNA vector. In some examples of the methods described herein, a synthetic DNA vector described in International Publication No. WO2021 / 055760 (herein incorporated by reference in its entirety) is used as the template DNA. Such a synthetic DNA vector can be amplified using rolling circle amplification and recircularized by restriction digestion and ligation. In embodiments in which a synthetic DNA vector lacking backbone sequences is used as a template, the step involving exonuclease digestion of linear backbone fragments is unnecessary and is omitted from the production method of the present invention.

[0175] B. Amplification In some examples, the cell-free synthesis of circular DNA vectors relies on the effective amplification using a polymerase such as a phage polymerase (e.g., Phi29 polymerase). The polymerase used herein may be, for example, a thermophilic polymerase with high processivity at GC-rich residues. In certain embodiments, the polymerase used to amplify vectors is Phi29 polymerase.

[0176] In some embodiments, the plasmid DNA vector is amplified in vitro in a cell-free preparation by incubating the DNA with a polymerase (e.g., a phage polymerase, e.g., Phi29 DNA polymerase; TempliPhi Kit, GE Healthcare), a primer (e.g., a site-specific or random primer, e.g., a random hexamer primer), and a nucleotide mixture (e.g., dNTPs, e.g., dATP, dCTP, dGTP, and dTTP). The polymerase (e.g., a phage polymerase, e.g., Phi29 polymerase) amplifies the template by rolling circle amplification (e.g., isothermal rolling circle amplification) to generate linear concatemers with multiple copies of the unit length of the template vector (e.g., a plasmid DNA vector). Suitable polymerases include thermophilic polymerases and polymerases characterized by high processivity.

[0177] A suitable polymerase concentration (e.g., Phi29 DNA polymerase concentration) is 10 U / mL to 2,000 U / mL (e.g., 50 U / mL to 1,000 U / mL, 100 U / mL to 500 U / mL, or 150 U / mL to 300 U / mL, e.g., 10 U / mL to 50 U / mL, 50 U / mL to 100 U / mL, 100 U / mL to 150 U / mL, 150 U / mL to 200 U / mL, 200 U / mL to 250 U / mL, 250 U / mL to 300 U / mL, 300 U / mL to 400 U / mL, 400 U / mL to 500 U / mL, 500 U / mL to 750 U / mL, 750 U / mL to 1,000 U / mL, 1,000 U / mL to 1,500 U / mL, or 1,500 U / mL to 2,000 U / mL). In some embodiments, the polymerase (e.g., Phi29 DNA polymerase) concentration is about 200 U / mL.

[0178] The starting concentration of the template DNA vector (e.g., plasmid DNA vector) is 10 ng / mL to 5 mg / mL (e.g., 0.1 μg / mL to 1 mg / mL, 0.2 μg / mL to 0.5 mg / mL, 0.5 μg / mL to 0.1 mg / mL, 1.0 μg / mL to 50 μg / mL, 2.0 μg / mL to 25 μg / mL, 4.0 μg / mL to 10 μg / mL, or about 5.0 μg / mL; e.g., 10 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 500 ng / mL, 500 ng / mL to 1 μg / mL, 1 μg / mL to 2 μg / mL, 2 μg / mL to 3 μg / mL, 3 μg / mL to 4 μg / mL, 4 μg / mL to 5 μg / mL, or about 5 μg / mL; g / mL~5μg / mL, 5μg / mL~6μg / mL, 6μg / mL~7μg / mL, 7μg / mL~8μg / mL, 8μg / mL~9μg / mL, 9μg / mL mL~10μg / mL, 10μg / mL~20μg / mL, 20μg / mL~50μg / mL, 50μg / mL~100μg / mL, 100μg / mL~500 μg / mL, 500 μg / mL to 1 mg / mL, or 1 mg / mL to 5 mg / mL; for example, about 0.5 μg / mL, about 1.0 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, or about 10 μg / mL). In some embodiments, the starting concentration of the template DNA vector (e.g., a plasmid DNA vector) is 1.0 μg / mL to 10 μg / mL (e.g., about 1.0 μg / mL, about 5.0 μg / mL, or about 10 μg / mL). In some embodiments, the starting concentration of the template DNA vector (e.g., a plasmid DNA vector) is 10 μg / mL. In some embodiments, the starting concentration of the template DNA vector (e.g., a plasmid DNA vector) is 1 μg / mL. In some embodiments, the starting concentration of the template DNA vector (e.g., a plasmid DNA vector) is 5 μg / mL.

[0179] The starting concentration of primers (e.g., random or specific primers) may be 0.1 μM to 1.0 mM (e.g., 0.5 μM to 500 μM, 1.0 μM to 250 μM, 2.0 μM to 200 μM, 4 μM to 100 μM, or 5 μM to 50 μM; e.g., 0.1 μM to 0.5 μM, 0.5 μM to 1.0 μM, 1.0 μM to 2.0 μM, 2.0 μM to 5.0 μM, 5.0 μM to 10 μM, 10 μM about 1 μM, about 2 μM, about 5 μM, about 10 μM, about 20 μM, about 25 μM, about 50 μM, about 100 μM, about 200 μM, about 250 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 750 μM, about 800 μM, about 900 μM, or about 1.0 mM).

[0180] In some examples, the starting concentration of the template DNA vector (e.g., plasmid DNA vector) at the start of amplification is 1 μg / mL to 10 μg / mL, and the starting concentration of the primer is 1 μM to 100 μM. In some examples, the starting concentration of the plasmid DNA vector at the start of amplification is about 5 μg / mL, and the starting concentration of the primer is 1 μM to 100 μM (e.g., about 50 μM).

[0181] Any suitable amplification buffer known in the art or described herein may be used in the methods of the present invention.

[0182] In some embodiments, the amplification reaction proceeds for about 1 hour to about 24 hours, for example, about 18 hours. For example, the amplification reaction can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the amplification reaction proceeds for about 18 hours.

[0183] In some embodiments, the amplification reaction is carried out at a temperature of about 25°C to about 42°C (e.g., about 28°C to about 40°C, e.g., about 29°C to about 40°C, e.g., about 30°C). For example, the amplification step can be carried out at about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. In some embodiments, the amplification step is carried out at about 30°C.

[0184] In some examples, the total amount of DNA present after amplification is at least 5 times the amount of template DNA (e.g., a plasmid DNA vector) present at the start of the amplification reaction (e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times the amount of template DNA present at the start of the amplification reaction; e.g., 10 times to 50 times, 10 times to 40 times, 10 times to 30 times, or 10 times to 20 times the amount of template DNA present at the start of the amplification reaction; e.g., 20 times to 50 times, 20 times to 40 times, or 20 times to 30 times the amount of template DNA present at the start of the amplification reaction; e.g., 30 times to 50 times or 40 times to 50 times the amount of template DNA present at the start of the amplification reaction).

[0185] In some embodiments, the total amount of DNA present after amplification is at least 50 times the amount of template DNA (e.g., a plasmid DNA vector) present at the start of the amplification reaction (e.g., between 50 and 300 times, e.g., at least 82 times, e.g., between 82 and 236 times the amount (e.g., by mass) of template DNA present at the start of the amplification reaction.

[0186] In some instances, the restriction digest step is performed immediately after the amplification step (eg, there is no heat inactivation step between the amplification step and the restriction digestion step of the template DNA (eg, a plasmid DNA vector).

[0187] In other examples, a heat inactivation step is performed after amplification (e.g., immediately after amplification). Heat inactivation can be performed by increasing the temperature to at least 50°C, at least 55°C, at least 60°C, at least 65°C, or at least 70°C to inactivate the polymerase (e.g., a phage polymerase, e.g., Phi29 polymerase). In some examples, the temperature is increased to at least 65°C. In some examples, the temperature of post-amplification heat inactivation is about 65°C. In some embodiments, restriction digestion is performed immediately after heat inactivation.

[0188] Certain embodiments of the methods of the invention allow the manufacturing process to proceed from amplification to restriction digestion without an intervening step that may impair yield, such as intermediate purification (e.g., gel purification (e.g., agarose gel extraction) or column purification). Thus, in some embodiments, there is no purification step (e.g., gel purification step (e.g., agarose gel extraction) or column purification step) between amplification and restriction digestion. Additionally or alternatively, in some embodiments, at least 90% of the amplified DNA products proceed to restriction digestion (e.g., 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 of the amplified DNA products proceed to restriction digestion; e.g., 90%-95%, 95%-97%, 97%-98%, 98%-99%, or 99%-100% of the amplified DNA products proceed to restriction digestion).

[0189] C. Restriction Digestion The template DNA vector (e.g., a plasmid DNA vector) and / or its concatemers generated by rolling circle amplification can be digested using restriction enzymes. In some embodiments, one or more restriction enzymes cleave at least two sites (e.g., at least three sites or at least four sites) per unit of the template DNA vector (e.g., a plasmid DNA vector) to generate linear fragments of DNA, some of which contain the therapeutic gene sequence.

[0190] In some embodiments, multiple restriction enzymes are used (e.g., two restriction enzymes are used). In such an example, a first restriction enzyme can be used to cleave the therapeutic gene sequence from the backbone sequence (e.g., by designing a plasmid DNA vector in which first restriction sites flank the therapeutic gene sequence). A second restriction enzyme can be used to cleave the backbone sequence into one or more (e.g., two, three, or more) linear backbone fragments, which can then be degraded using an exonuclease (e.g., T5 exonuclease or Plasmid-Safe). Restriction enzymes suitable for such methods include, for example, EcoRI and PvuII (e.g., EcoRI as the first restriction enzyme and PvuII as the second restriction enzyme).

[0191] In certain embodiments, a single restriction enzyme is used (i.e., this step involves the use of only one type of restriction enzyme). In such an example, a type IIS restriction enzyme is suitable as the single restriction enzyme. Type IIS restriction enzymes can be particularly useful because they recognize a restriction site that exists outside the cleavage site. Thus, after cleavage and ligation, the restriction site is no longer present in the ligated product (e.g., therapeutic circular DNA vector). This allows for simultaneous treatment of DNA fragments with restriction enzyme and ligase.

[0192] In some instances, as described herein, a type IIS restriction site is positioned outside of the therapeutic sequence of a DNA molecule (e.g., a template DNA vector, e.g., a plasmid DNA vector) such that a reaction containing a ligase and a type IIS restriction enzyme drives a reaction that increases the relative concentration of the therapeutic circular DNA vector relative to a by-product having a type IIS restriction site (e.g., a by-product having one or more backbone sequences and a type IIS restriction site).

[0193] In some embodiments, the Type IIS restriction enzyme used in such embodiments is BsaI. Other suitable Type IIS restriction enzymes that can be used with the methods described herein include, for example, AcuI, AlwI, BaeI, BbsI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, BtsIMutI, CspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MlyI, MmeI, MnII, NmeAIII, PaqCI, PleI, SapI, and SfaNI.

[0194] In some embodiments, the restriction enzyme is provided at a concentration of about 0.5 U / μg DNA to about 20 U / μg DNA, such as about 1 U / μg DNA to about 10 U / μg DNA, such as about 2 U / μg DNA to about 5 U / μg DNA, e.g., about 2.5 U / μg DNA. For example, restriction enzymes are approximately 0.5U / μg DNA, 1.0U / μg DNA, 1.5U / μg DNA, 2.0U / μg DNA, 2.5U / μg DNA, 3.0U / μg DNA, 3.5U / μg DNA, 4.0U / μg DNA, 4.5U / μg DNA, 5.0U / μg DNA, 5.5U / μg DNA, 6.0U / μg DNA, 6.5U / μg DNA, 7.0U / μg DNA, 7.5U / μg DNA, 8.0U / μg DNA, 8.5U / μg DNA, 9.0U / μg DNA, 9.5U / μg DNA, 10.0U / μg DNA, 11U / μg DNA, 12U / μg DNA, 13U / μg DNA, 14U / μg DNA, 15U / μg DNA, 16U / μg DNA, 17U / μg The restriction enzyme may be provided at a concentration of about 2.5 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, or 20 U / μg DNA. In some embodiments, the restriction enzyme is BsaI at a concentration of about 2.5 U / μg DNA.

[0195] In some embodiments, the restriction digestion step is about 1 hour to about 24 hours, for example, about 1 hour to about 12 hours. For example, the digestion step can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the digestion step is about 2 hours. In some embodiments, the digestion step is about 1 hour or less, for example, about 30 minutes or less.

[0196] In some embodiments, the total amount of DNA present after restriction digestion is at least 50 times the amount of template DNA (e.g., a plasmid DNA vector) present at the start of the amplification reaction (e.g., between 50 and 300 times, e.g., at least 82 times, e.g., between 82 and 236 times the amount (e.g., by mass) of template DNA present at the start of the amplification reaction.

[0197] The restriction enzyme digestion may be carried out at a reaction temperature of about 30°C to about 42°C (e.g., about 32°C to about 40°C, e.g., about 35°C to about 40°C, e.g., about 37°C). For example, in some examples, the restriction enzyme digestion step is carried out at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. In some embodiments, the restriction enzyme digestion step is carried out at about 37°C.

[0198] Some embodiments of the methods of the invention allow the manufacturing process to proceed from restriction digestion to ligation without intervening steps that may compromise yield, such as purification (e.g., gel purification (e.g., agarose gel extraction) or column purification). Thus, in some embodiments, there is no purification step (e.g., a gel purification step (e.g., agarose gel extraction) or column purification step) between restriction digestion and ligation. Additionally or alternatively, in some embodiments, at least 90% of the total DNA present at or after restriction digestion (including digested linear fragments (e.g., backbone and / or therapeutic sequences) and any undigested or circular DNA) proceeds to ligation (e.g., 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 of the total DNA present at or after restriction digestion proceeds to ligation, e.g., 90%-95%, 95%-97%, 97%-98%, 98%-99%, or 99%-100% of the total DNA present at or after restriction digestion (including digested linear fragments (e.g., backbone and / or therapeutic sequences) and any undigested or circular DNA) proceeds to ligation).

[0199] In some examples, a heat inactivation step is performed after restriction digestion and before ligation (e.g., a restriction digest involving one or more non-type II restriction enzymes, e.g., EcoRI and / or PvuII). Heat inactivation can be performed by increasing the temperature to at least 50°C, at least 55°C, at least 60°C, at least 65°C, or at least 70°C to inactivate the restriction enzyme (e.g., non-type II restriction enzymes, e.g., EcoRI and / or PvuII). In some examples, the temperature is increased to at least 65°C. In some examples, the temperature of post-amplification heat inactivation is about 65°C. In some embodiments, ligation is performed immediately after heat inactivation of the restriction enzyme(s).

[0200] Alternatively, no heat inactivation step is performed (eg, immediately after) ligation (eg, no heat inactivation step is performed throughout the process).

[0201] In some embodiments involving a type II restriction enzyme such as BsaI, inactivation (e.g., heat inactivation) of the restriction enzyme prior to ligation is not required. In some embodiments, ligation is performed immediately after restriction digestion (e.g., there is no heat inactivation step between the restriction digestion step (e.g., restriction digestion with a type II restriction enzyme, e.g., BsaI) and the ligation step). In some embodiments, ligation is performed completely or partially during restriction digestion. For example, the ligation reaction can be initiated at or during the start of the restriction digest (e.g., ligase can be added to the DNA at the same time that the restriction enzyme (e.g., a type II restriction enzyme, e.g., BsaI) is added, or after the restriction enzyme is added (e.g., within 1 minute after the restriction enzyme is added, within 5 minutes after the restriction enzyme is added, within 10 minutes after the restriction enzyme is added, within 30 minutes after the restriction enzyme is added, within 60 minutes after the restriction enzyme is added, within 90 minutes after the restriction enzyme is added, or within 120 minutes after the restriction enzyme is added). Alternatively, the ligation reaction can be initiated after the restriction digest is completed (e.g., 2 hours after the restriction enzyme is added).

[0202] After restriction digestion (eg, immediately after restriction digestion or immediately after heat inactivation, if performed), the reaction temperature can be adjusted to match that of the ligation reaction described above (eg, 25° C.).

[0203] D. Ligation Self-ligation of the linear therapeutic fragment with the therapeutic sequence results in a therapeutic circular DNA vector (e.g., a monomeric therapeutic circular DNA vector). In some embodiments, the self-ligation step includes providing a ligase (e.g., a DNA ligase) to the digested DNA sample to obtain a ligation solution. The ligase can be, for example, T3 ligase, T4 ligase, or T7 ligase. In certain embodiments, T4 ligase is used. The ligation solution can contain additional components, such as ATP (e.g., ribo-ATP) or other buffering agents at suitable concentrations known in the art or described herein. For example, some examples of the methods of the invention include preparing a ligation solution containing ligase with ribo-ATP in CutSmart® or recombinant CutSmart® (rCutSmart®) buffer or an equivalent buffer, where the ribo-ATP is at a concentration of 0.1 to 100 mM (e.g., about 10 mM).

[0204] In some embodiments, the total amount of DNA contacted with the ligase is 90% or more of the total amount of DNA produced at the end of the amplification (e.g., 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 of the total amount of DNA produced at the end of the amplification; e.g., 90%-99%, 91%-99%, 92%-99%, 93%-99%, or more of the total amount of DNA produced at the end of the amplification). %, 94%-99%, 95%-99%, 96%-99%, 97%-99%, 98%-99%, 90%-98%, 91%-98%, 92%-98%, 93%-98%, 94%-98%, 95%-98%, 96%-98%, 97%-98%, 90%-97%, 91%-97%, 92%-97%, 93%-97%, 94%-97%, 95%-97%, 96%-97%, 90%-96%, 91%-96%, 92%-96%, 93%-96%, 94%-96%, 95%-96%, or 90%-95%).

[0205] The ligase (e.g., T4 ligase) may be present in the ligation solution at a concentration of about 0.5 U / μg DNA to about 20 U / μg DNA, such as, for example, about 0.5 U / μg DNA to about 10 U / μg DNA, such as, for example, about 1 U / μg DNA to about 10 U / μg DNA, such as, for example, about 1 U / μg DNA to about 5 U / μg DNA, such as, for example, about 1.5 U / μg DNA, about 2.0 U / μg DNA, or about 2.5 U / μg DNA. For example, a ligase (e.g., T4 ligase) may be approximately 0.5U / μg DNA, 1.0U / μg DNA, 1.5U / μg DNA, 2.0U / μg DNA, 2.5U / μg DNA, 3.0U / μg DNA, 3.5U / μg DNA, 4.0U / μg DNA, 4.5U / μg DNA, 5.0U / μg DNA, 5.5U / μg DNA, 6.0U / μg DNA, 6.5U / μg DNA, 7.0U / μg DNA, 7.5U / μg DNA, 8.0U / μg DNA, 8.5U / μg DNA, 9.0U / μg DNA, 9.5U / μg DNA, 10.0U / μg DNA, 11U / μg DNA, 12U / μg DNA, 13U / μg DNA, 14U / μg DNA, 15U / μg DNA, 16U / μg The antibody may be provided at a concentration of 17 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, or 20 U / μg DNA.In some embodiments, the ligase has a concentration of 50 U per μg of DNA (U / μg) or less (e.g., 40 U / μg DNA or less, 30 U / μg DNA or less, 25 U / μg DNA or less, 20 U / μg DNA or less, 15 U / μg DNA or less, 10 U / μg DNA or less, 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 20 U / μg DNA; e.g., 0.1 U / μg DNA to 30 U / μg DNA, 0.1 U / μg DNA to 20 U / μg DNA, 0.2 U / μg DNA to 15 U / μg DNA, 0.5 U / μg DNA to 12 U / μg DNA, or 1 U / μg DNA). DNA~10U / μg DNA; For example, 0.1U / μg DNA~0.5U / μg DNA, 0.5U / μg DNA~1.0U / μg DNA, 1.0U / μg DNA~2.0U / μg DNA, 2.0U / μg DNA~3.0U / μg DNA, 3.0U / μg DNA~4.0U / μg DNA, 4.0U / μg DNA~5.0U / μg DNA, 5.0~6.0U / μg DNA, 6.0U / μg DNA~7.0U / μg DNA, 7.0U / μg DNA~8.0U / μg DNA, 8.0U / μg DNA~9.0U / μg DNA, 9.0U / μg DNA~11U / μg DNA, 11U / μg DNA~12U / μg DNA, 12U / μg DNA~15U / μg DNA, 15U / μg Ligase concentrations are provided at: 20U / μg DNA, 20U / μg DNA, 25U / μg DNA, 30U / μg DNA, 35U / μg DNA, 40U / μg DNA, or 50U / μg DNA.In some embodiments, the ligase (e.g., T4 ligase) has a concentration of 20 U / μg DNA or less (e.g., 15 U / μg DNA or less, 10 U / μg DNA or less, 5 U / μg DNA or less, 4 U / μg DNA or less, 3 U / μg DNA or less, 2.5 U / μg DNA or less, 2.0 U / μg DNA or less, 1.5 U / μg DNA or less, or 1.0 U / μg DNA or less; e.g., 0.1 U / μg DNA to 20 U / μg DNA; e.g., 0.2 U / μg DNA to 15 U / μg DNA, 0.5 U / μg DNA to 12 U / μg DNA, or 1 U / μg DNA to 10 U / μg DNA; e.g., 0.1 U / μg DNA to 0.5 U / μg DNA, 0.5 U / μg DNA to 1.0 U / μg DNA, 1.0 U / μg DNA to 2.0 U / μg DNA, 2.0 U / μg DNA, DNA~3.0U / μg DNA, 3.0U / μg DNA~4.0U / μg DNA, 4.0U / μg DNA~5.0U / μg DNA, 5.0~6.0U / μg DNA, 6.0U / μg DNA~7.0U / μg DNA, 7.0U / μg DNA~8.0U / μg DNA, 8.0U / μg DNA~9.0U / μg DNA, 9.0U / μg DNA to 11U / μg DNA, 11U / μg DNA to 12U / μg DNA, 12U / μg DNA to 15U / μg DNA, or 15U / μg DNA to 20U / μg DNA).

[0206] In certain embodiments, the linear therapeutic fragment is contacted with T4 ligase at a concentration of between 5.0 U / μg DNA and 15 U / μg DNA (e.g., about 10 U / μg DNA).

[0207] In some embodiments, the ligation step is about 30 minutes to about 24 hours, e.g., about 1 hour to about 12 hours. For example, the ligation step can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the ligation step is about 2 hours. In some embodiments, the ligation step is about 18 to about 24 hours (e.g., about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours).

[0208] Ligation may be carried out at a reaction temperature of about 20° C. to about 42° C. (e.g., about 20° C. to about 37° C., about 22° C. to about 30° C., or about 25° C.). For example, in some examples, the ligation step is carried out at about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C. In some embodiments, the ligation step is carried out at about 25° C.

[0209] A heat inactivation step may be performed to inactivate the ligase after ligation. In processes that require a type II restriction enzyme (e.g., ligation simultaneously with or immediately after restriction digestion), this post-ligation heat inactivation step may inactivate both the type II restriction enzyme (e.g., BsaI) and the ligase (e.g., T4 ligase). Heat inactivation may be performed by increasing the temperature to at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, or at least 80°C. In some examples, the temperature is increased to at least 65°C. In some examples, the temperature of post-amplification heat inactivation is about 65°C. In some embodiments, ligation is performed immediately after heat inactivation of the restriction enzyme(s). Heat inactivation may proceed for 10 minutes to 2 hours (e.g., 30 minutes to 90 minutes, 40 minutes to 60 minutes, or about 45 minutes). In some embodiments, heat inactivation comprises incubation at about 65° C. for about 45 minutes after ligation.

[0210] In some embodiments, the method includes reducing the temperature of the solution after post-ligation heat inactivation to less than 50° C. (e.g., between 20° C. and 40° C., between 25° C. and 37° C., or about 37° C.).

[0211] In other examples, no heat inactivation step is performed after ligation (e.g., immediately after ligation). In some examples, the reaction temperature after ligation (e.g., immediately after ligation) is less than 50° C. or less than 45° C. In some examples, the temperature is maintained within (+ / -) 10° C. of the ligation reaction temperature (e.g., within (+ / -) 8° C., within (+ / -) 5° C., or within (+ / -) 2° C. of the ligation reaction temperature).

[0212] Some embodiments of the methods of the invention allow the manufacturing process to proceed from ligation to supercoiling and / or exonuclease digestion without intervening steps that may compromise yield, such as purification (e.g., gel purification (e.g., agarose gel extraction) or column purification). Thus, in some embodiments, there is no purification step (e.g., a gel purification step (e.g., agarose gel extraction) or column purification step) between ligation and supercoiling and / or exonuclease digestion. Additionally or alternatively, in some embodiments, at least 90% of the total DNA present at or after ligation (including the therapeutic circular DNA, the linear backbone fragment, and any unligated therapeutic fragments) proceeds to supercoiling and / or exonuclease digestion (e.g., 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 of the total DNA present at or after ligation proceeds to supercoiling and / or exonuclease digestion, e.g., 90%-95%, 95%-97%, 97%-98%, 98%-99%, or 99%-100% of the total DNA present at or after ligation proceeds to supercoiling and / or exonuclease digestion).

[0213] E. Supercoiling A cell-free method for producing a supercoiled therapeutic circular DNA vector (and pharmaceutical composition thereof) may include contacting a relaxed circular DNA vector with a topoisomerase or helicase under conditions suitable for supercoiling. In some embodiments, the therapeutic circular DNA vector produced by the methods described herein is positively supercoiled. The methods described herein include any reagents and conditions known in the art or described herein to promote effective supercoiling.

[0214] For example, an exemplary buffer suitable for a supercoiling reaction contains 35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 1 mM ATP, 2 mM DTT, 1.8 mM spermidine, 32% glycerol (w / v), and 100 μg / mL BSA.

[0215] In some embodiments, the total amount of DNA contacted with the topoisomerase or helicase is 90% or more of the total amount of DNA generated at the end of amplification or ligation (e.g., 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 of the total amount of DNA generated at the end of amplification or ligation; e.g., 90% to 99% of the total amount of DNA generated at the end of amplification or ligation). 99%, 91%~99%, 92%~99%, 93%~99%, 94%~99%, 95%~99%, 96%~99%, 97%~99%, 98%~99%, 90%~98%, 91%~98%, 92%~98%, 93%~98%, 94%~98%, 95%~98%, 96%~98%, 9 7%-98%, 90%-97%, 91%-97%, 92%-97%, 93%-97%, 94%-97%, 95%-97%, 96%-97%, 90%-96%, 91%-96%, 92%-96%, 93%-96%, 94%-96%, 95%-96%, or 90%-95%).

[0216] In some embodiments, the topoisomerase is a type II topoisomerase. The type II topoisomerase can be, for example, gyrase or topoisomerase IV.

[0217] In some embodiments, the topoisomerase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) or helicase is provided at a concentration of about 0.5 U / μg DNA to about 20 U / μg DNA, e.g., about 0.5 U / μg DNA to about 10 U / μg DNA, e.g., about 1 U / μg DNA to about 10 U / μg DNA, e.g., about 1 U / μg DNA to about 5 U / μg DNA, e.g., about 1.5 U / μg DNA, about 2.0 U / μg DNA, or about 2.5 U / μg DNA. For example, a topoisomerase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) or helicase may be at about 0.5 U / μg DNA, 1.0 U / μg DNA, 1.5 U / μg DNA, 2.0 U / μg DNA, 2.5 U / μg DNA, 3.0 U / μg DNA, 3.5 U / μg DNA, 4.0 U / μg DNA, 4.5 U / μg DNA, 5.0 U / μg DNA, 5.5 U / μg DNA, 6.0 U / μg DNA, 6.5 U / μg DNA, 7.0 U / μg DNA, 7.5 U / μg DNA, 8.0 U / μg DNA, 8.5 U / μg DNA, 9.0 U / μg DNA, 9.5 U / μg DNA, 10.0 U / μg DNA, 11 U / μg DNA, 12 U / μg DNA, 13 U / μg DNA, 14 U / μg DNA, 15 U / μg DNA, 16 U / μg DNA, 17 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, 20 U / μg DNA, 21 U / μg DNA, 22 U / μg DNA, 23 U / μg DNA, 24 U / μg DNA, 25 U / μg DNA, 26 U / μg DNA, 27 U / μg DNA, 28 U / μg DNA, 29 U / μg DNA, 30 U / μg DNA, 31 U / μg DNA, 32 U / μg DNA, 33 U / μg DNA, 34 U / μg DNA, 35 U / μg DNA, 36 U / μg DNA, 37 U / μg DNA, 38 U / μg DNA, 39 U / μg DNA The antibody may be provided at a concentration of 14 U / μg DNA, 15 U / μg DNA, 16 U / μg DNA, 17 U / μg DNA, 18 U / μg DNA, 19 U / μg DNA, or 20 U / μg DNA.In some embodiments, the topoisomerase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) or helicase is at or below 10 U / μg DNA (e.g., below 5 U / μg DNA, below 4 U / μg DNA, below 3 U / μg DNA, below 2.5 U / μg DNA, below 2.0 U / μg DNA, below 1.5 U / μg DNA, or below 1.0 U / μg DNA; e.g., between 0.1 U / μg DNA and 10 U / μg DNA; e.g., between 0.5 U / μg DNA and 8 U / μg DNA or between 1 U / μg DNA and 5 U / μg DNA; e.g., between 0.1 U / μg DNA and 0.5 U / μg DNA, between 0.5 U / μg DNA and 1.0 U / μg DNA, between 1.0 U / μg DNA and 2.0 U / μg DNA, between 2.0 U / μg DNA and 3.0 U / μg DNA, between 3.0 U / μg DNA, It is provided in concentrations of: 4.0U / μg DNA, 4.0U / μg DNA, 5.0U / μg DNA, 6.0U / μg DNA, 7.0U / μg DNA, 8.0U / μg DNA, 9.0U / μg DNA, or 10U / μg DNA.

[0218] In certain embodiments, the relaxed circular DNA vector is contacted with gyrase at a concentration of 1.0 U / μg DNA to 2.5 U / μg DNA (e.g., about 1.0 U / μg DNA, about 1.5 U / μg DNA, or about 2.0 U / μg DNA). In embodiments in which gyrase is contacted with the DNA after terminal exonuclease digestion (e.g., T5 exonuclease digestion), the gyrase is at a concentration of 0.1 U / μg DNA to 1.5 U / μg DNA (e.g., 0.2 U / μg DNA to 1.5 U / μg DNA, 0.5 U / μg DNA to 1.5 U / μg DNA, 0.5 U / μg DNA to 1.0 U / μg DNA, or 1.0 U / μg DNA to 1.5 U / μg DNA, e.g., about 0.1 U / μg DNA, about 0.2 U / μg DNA, about 0.3 U / μg DNA, about 0.4 U / μg DNA, about 0.5 U / μg DNA, about 1.0 U / μg DNA, or about 1.5 U / μg DNA).

[0219] In some embodiments, the step of contacting the circular DNA vector with a topoisomerase or helicase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) is from about 1 hour to about 24 hours, e.g., from about 1 hour to about 12 hours. For example, the step of contacting the circular DNA vector with a topoisomerase or helicase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) is from about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the step of contacting the circular DNA vector with a topoisomerase or helicase (eg, a type II topoisomerase, such as topoisomerase IV or gyrase) is for about 12 hours.

[0220] In some embodiments, the step of contacting the circular DNA vector with a topoisomerase or helicase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) is carried out at a temperature of about 30°C to about 42°C (e.g., about 32°C to about 40°C, e.g., about 35°C to about 40°C, e.g., about 37°C). For example, the digestion step can be carried out at about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. In some embodiments, the step of contacting the circular DNA vector with a topoisomerase or helicase (e.g., a type II topoisomerase, e.g., topoisomerase IV or gyrase) is carried out at about 37°C.

[0221] F. Terminal exonuclease Any of the methods for cell-free production of therapeutic circular DNA vectors described herein may include a clean-up step in which undesired DNA (e.g., bacterial sequences, linear or nicked DNA by-products, etc.) is enzymatically degraded. In a specific example, linear backbone fragments generated by restriction enzyme digestion can be selectively degraded in a solution containing the circularized therapeutic fragments (e.g., relaxed circular therapeutic DNA vectors or therapeutic supercoiled DNA vectors) using terminal exonucleases under any suitable conditions known in the art or described herein. In some embodiments, the terminal exonuclease is T5 exonuclease.

[0222] The methods described herein include any reagents and conditions known in the art or described herein to promote effective terminal exonuclease activity. For example, an exemplary buffer suitable for a terminal exonuclease reaction can be a potassium acetate buffer (e.g., 10 mM to 100 mM potassium acetate, e.g., about 50 mM potassium acetate).

[0223] In some embodiments, the total amount of DNA contacted with the terminal exonuclease is 90% or more of the total amount of DNA generated at the end of amplification or ligation (e.g., 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 of the total amount of DNA generated at the end of amplification or ligation; e.g., 90% to 99% of the total amount of DNA generated at the end of amplification or ligation). , 91%-99%, 92%-99%, 93%-99%, 94%-99%, 95%-99%, 96%-99%, 97%-99%, 98%-99%, 90%-98%, 91%-98%, 92%-98%, 93%-98%, 94%-98%, 95%-98%, 96%-98%, 97%-98%, 90%-97%, 91%-97%, 92%-97%, 93%-97%, 94%-97%, 95%-97%, 96%-97%, 90%-96%, 91%-96%, 92%-96%, 93%-96%, 94%-96%, 95%-96%, or 90%-95%).

[0224] In some embodiments, the terminal exonuclease (e.g., T5 exonuclease) is provided at a concentration of about 0.5 U / μg to about 20 U / μg, such as about 0.5 U / μg to about 10 U / μg, such as about 1 U / μg to about 10 U / μg, such as about 2 U / μg to about 5 U / μg, such as about 2.5 U / μg. For example, the terminal exonuclease is provided at a concentration of about 0.5 U / μg, 1.0 U / μg, 1.5 U / μg, 2.0 U / μg, 2.5 U / μg, 3.0 U / μg, 3.5 U / μg, 4.0 U / μg, 5.0 U / μg, 6.0 U / μg, 7.0 U / μg, 8.0 U / μg, 9.0 U / μg, 10.0 U / μg, 11.0 U / μg, 12.0 U / μg, 13.0 U / μg, 14.0 U / μg, 15.0 U / μg, 16.0 U / μg, 17.0 U / μg, 18.0 U / μg, 19.0 U / μg, 20.0 U / μg, 21.0 U / μg, 22.0 U / μg, 23.0 U / μg, 24.0 U / μg, 25.0 U / μg, 26.0 U / μg, 27.0 U / μg, 28.0 U / μg, 29.0 U / μg, 30.0 U / μg, 31.0 U / μg, 32.0 U / μg, 33.0 U / μg, 34.0 U / μg, 35.0 U / U / μg, 4.5U / μg, 5.0U / μg, 5.5U / μg, 6.0U / μg, 6.5U / μg, 7.0U / μg, 7.5U / μg, 8.0U / μg, 8.5U / μg, 9.0U / μg, 9.5U / μg , 10.0U / μg, 11U / μg, 12U / μg, 13U / μg, 14U / μg, 15U / μg, 16U / μg, 17U / μg, 18U / μg, 19U / μg, or 20U / μg.

[0225] In some embodiments, the digestion step with the terminal exonuclease is from about 1 hour to about 24 hours, e.g., from about 1 hour to about 12 hours. For example, the digestion step is about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the digestion step is from 2 hours to 12 hours (e.g., from 2 to 5 hours, from 2 to 4 hours, or from 2 to 3 hours).

[0226] In some embodiments, digestion of the sample with a terminal exonuclease is carried out at about 30° C. to about 42° C. (e.g., about 32° C. to about 40° C., e.g., about 35° C. to about 40° C., e.g., about 37° C.). For example, the digestion step can be carried out at about 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., or 42° C. In some embodiments, digestion of the sample with a terminal exonuclease is carried out at about 37° C.

[0227] In some instances, heat inactivation of terminal exonucleases is not performed immediately following exonuclease digestion.

[0228] In some instances, terminal exonuclease digestion is performed after supercoiling (e.g., immediately after supercoiling). Alternatively, terminal exonuclease digestion is performed before supercoiling (e.g., immediately before supercoiling). In some embodiments, terminal exonuclease digestion is performed simultaneously with supercoiling.

[0229] G. Purification / Precipitation In some embodiments of any of the methods described herein, the method further comprises precipitating the therapeutic circular DNA vector, for example, by isopropanol precipitation.

[0230] In some embodiments, prior to precipitation, the solution containing the therapeutic circular DNA vector (e.g., supercoiled circular DNA vector) is sterile filtered, for example through a 0.22 μm filter. The solution may be reconstituted in a buffer containing IPA by methods known in the art and described herein. In some embodiments, the sterile filtrate from section F above is reconstituted in IPA buffer with a final concentration of 760 mM NaCl, 50 mM MOPS, 15% isopropyl alcohol (IPA), and 0.15% Triton X-100 (v / v). The therapeutic circular DNA vector (e.g., supercoiled circular DNA vector) in IPA buffer is added to an equilibrated QIAGEN-tip column (Qiagen Plasmid Kit), the column is washed, and the contents are eluted. 24.5 mL of IPA buffer is added per 35 mL of elution. IPA precipitation may then be performed in which the sample is centrifuged at 15,000 g for 30 minutes at 4°C. The dried pellet can be resuspended in water or the final buffer of interest.

[0231] In some embodiments, the amount of therapeutic circular DNA vector obtained after purification / precipitation (e.g., a single purification / precipitation step, e.g., no more than one purification / precipitation step) is at least twice the number of therapeutic sequences (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times; e.g., 2 times to 1,000 times, 2 times to 500 times, 2 times to 100 times, 2 times to 50 times, 2 times to 40 times, 2 times to 30 times, 2 times to 20 times, or 2 times to 10 times; e.g., 5 times to 1,000 times, 5 times to 500 times, 5 times to 100 times, 5 times to 50 times, 5 times to 40 times, fold, 5x to 30x, 5x to 20x, or 5x to 10x; for example, 10x to 1,000x, 10x to 500x, 10x to 100x, 10x to 50x, 10x to 40x, 10x to 30x, or 10x to 20x; for example, 2x to 5x, 5x to 10x, 10x to 20x, 20x to 30x, 30x to 40x, 40x to 50x, 50x to 60x, 60x to 70x, 70x to 80x, 80x to 90x, 90x to 100x , 100-200-fold, 200-500-fold, or 500-1,000-fold; e.g., about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold) the amount of template DNA vector (e.g., plasmid DNA vector) from which it was generated. In some embodiments, the amount of the resulting therapeutic circular DNA vector after purification / precipitation (e.g., a single purification / precipitation step, e.g., no more than one purification / precipitation step) is at least 3 times the amount of the template DNA vector (e.g., plasmid DNA vector) from which it was generated.Additionally or alternatively, the amount of the resulting therapeutic circular DNA vector after purification / precipitation (e.g., a single purification / precipitation step, e.g., no more than one purification / precipitation step) is at least 1.0 mg (e.g., 1.0 mg to 10 g, 1.0 mg to 5.0 g, 1.0 mg to 1.0 g, 1.0 mg to 500 mg, 1.0 mg to 200 mg, 1.0 mg to 100 mg, 1.0 mg to 50 mg, 1.0 mg to 25 mg, 1.0 mg to 20 mg, 1.0 mg to 15 mg, 1.0 mg to 10 mg, 1.0 mg to 5.0 mg, 2.0 mg to 10 g, 2.0 mg to 5.0 g, 2.0 mg to 1.0 g, 2.0 mg to 500 mg, 2.0 mg to 200 mg, 2.0 mg to 100 mg, 2.0 mg to 2.0 mg mg-50mg, 2.0mg-25mg, 2.0mg-20mg, 2.0mg-15mg, 2.0mg-10mg, 2.0mg-5.0mg, 5.0mg-10g, 5.0mg-5.0g, 5.0mg-1.0g, 5.0mg-500mg, 5.0mg-200mg, 5.0mg-100mg, 5.0mg-50mg, 5.0mg-25mg, 5.0mg-20mg, 5.0mg-15mg, 5.0mg-10mg, 10mg-10g, 10mg-5.0g, 10mg-1.0g, 10mg-500mg, 10mg-200mg, 10mg-100mg, 10mg-50mg, 10mg-25mg, 10mg-20mg, or 10mg-15mg). In some embodiments, the amount of resulting therapeutic circular DNA vector after purification / precipitation (eg, a single purification / precipitation step, eg, no more than one purification / precipitation step) is at least 2.0 mg.

[0232] In some examples, the amount (mass) of therapeutic circular DNA vector produced by the method of the present invention is at least twice the amount (mass) of the template DNA (e.g., plasmid DNA vector) input during amplification in production (e.g., at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the mass of the template DNA (e.g., plasmid DNA vector) input during amplification in production; for example, 2 times to 20 times, 2 times to 15 times, 2 times to 13 times, 3 times to 10 times, or 4 times to 8 times the mass of the template DNA (e.g., plasmid DNA vector) input during amplification in production; for example, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, or about 13 times the mass of the template DNA (e.g., plasmid DNA vector) input during amplification in production).

[0233] IV. Therapeutic Circular DNA Vectors Provided herein is a therapeutic circular DNA vector generated by any of the production methods described herein. In some examples, such therapeutic circular DNA vectors persist as episomes in cells (e.g., in dividing or quiescent cells, e.g., postmitotic cells), for example, in a manner similar to AAV vectors. In any of the embodiments described herein, the therapeutic circular DNA vector can be a non-integrating vector. Provided herein is a therapeutic circular DNA vector can be a naked DNA vector that lacks components unique to viral vectors (e.g., viral proteins) and bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG islands or CpG motifs)), or components additionally or otherwise associated with reduced persistence (e.g., CpG islands or CpG motifs). The therapeutic circular DNA vectors generated as described herein can be characterized by one or more therapeutic sequences and lack plasmid backbone elements (e.g., bacterial elements, e.g., (i) bacterial origins of replication and / or (ii) drug resistance genes) and recombination sites.

[0234] The therapeutic circular DNA vectors provided herein can be naked DNA vectors that lack components unique to viral vectors (e.g., viral proteins) and bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG islands)) or components additionally or otherwise associated with reduced persistence (e.g., CpG islands). For example, in some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of the DNA lacks one or more elements of bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG motifs)) or components additionally or otherwise associated with reduced persistence (e.g., CpG islands). In some embodiments, at least 50% of the DNA (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) lacks CpG methylation. In some embodiments, the vector comprises DNA in which at least 50% of the DNA (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) lacks bacterial methylation signatures, e.g., Dam methylation and Dcm methylation. For example, in some embodiments, the vector comprises DNA in which at least 50% of the GATC sequences (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) are unmethylated (e.g., by Dam methylase). Additionally or alternatively, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or essentially all) of its CCAGG and / or CCTGG sequences are unmethylated (e.g., by Dcm methylase).

[0235] In some embodiments, the therapeutic circular DNA vector is persistent in vivo (e.g., the therapeutic circular DNA vector exhibits improved expression persistence (e.g., intracellular and / or transgenerational persistence) and / or therapeutic persistence compared to a reference vector, e.g., a circular DNA vector, e.g., a plasmid DNA, that is generated in bacteria or has one or more bacterial signatures that are not present in the vectors of the invention). In some embodiments, the expression persistence of the therapeutic circular DNA vector is 5%-50% higher, 50%-100% higher, 1-5-fold, or 5-10-fold (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) higher than the reference vector. In some embodiments, the intracellular persistence of the therapeutic circular DNA vector is 5%-50% higher, 50%-100% higher, 1-5-fold, or 5-10-fold higher (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold, or more) than the reference vector. In some embodiments, the intracellular persistence of the therapeutic circular DNA vector is 5%-50% higher, 50%-100% higher, 1-5-fold, or 5-10-fold higher (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold, or more) than the reference vector. In some embodiments, the therapeutic persistence of the therapeutic circular DNA vector is 5%-50% higher, 50%-100% higher, 1-5-fold, or 5-10-fold higher (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) than the reference vector. In some embodiments, the reference vector is a circular vector or plasmid that (a) has the same therapeutic sequence as the therapeutic circular DNA vector to which it is being compared, and (b) is generated in bacteria and / or has one or more bacterial signatures that are not present in the therapeutic circular DNA vector to which it is being compared, which may include, for example, an antibiotic resistance gene or a bacterial origin of replication.

[0236] In some embodiments, expression of the therapeutic circular DNA vector persists for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration. In certain embodiments, the therapeutic circular DNA vector exhibits intracellular and / or transgenerational persistence for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration. In some embodiments, therapeutic persistence of the therapeutic circular DNA vector persists for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer after administration.

[0237] In some embodiments, expression and / or therapeutic effect of the therapeutic circular DNA vector persists for 1 week to 4 weeks, 1 month to 4 months, or 4 months to 1 year (e.g., at least 1 week, at least 2 weeks, at least 1 month, or longer). In some embodiments, expression levels of the therapeutic circular DNA vector do not show a greater than 90%, greater than 50%, or greater than 10% decrease from levels observed within the first 1, 2, or 3 days for 1 week or more, e.g., 2 weeks, 3 weeks, 5 weeks, 7 weeks, 9 weeks or more, 13 weeks or more, 18 weeks or more, following transfection.

[0238] The therapeutic circular DNA vector can be a monomer, dimer, trimer, tetramer, pentamer, hexamer, etc. In some preferred embodiments, the circular DNA vector is monomeric. In some embodiments, the DNA vector is supercoiled, for example, after treatment with a topoisomerase (e.g., gyrase). In some embodiments, the therapeutic circular DNA vector is a monomeric supercoiled circular DNA molecule. In some embodiments, the therapeutic circular DNA vector is nicked. In some embodiments, the therapeutic circular DNA vector is open circular. In some embodiments, the therapeutic circular DNA vector is double-stranded circular.

[0239] Therapeutic Sequences The therapeutic circular DNA vectors described herein comprise a therapeutic sequence (eg, a therapeutic nucleic acid) that may include one or more protein-coding domains and / or one or more non-protein-coding domains.

[0240] In specific embodiments comprising a therapeutic domain encoding a therapeutic protein, the therapeutic sequence comprises, linked in a 5'→3' direction: a promoter and a single therapeutic protein coding domain (e.g., a single transcription unit); a promoter and two or more therapeutic protein coding domains (e.g., a polycistronic unit); or a first transcription unit and one or more additional transcription units (e.g., multiple transcription units). Any such therapeutic sequence encoding a protein may further comprise non-protein coding domains, such as polyadenylation sites, regulatory elements, enhancers, sequences for labeling DNA (e.g., antibody recognition sequences), PCR amplification sites, sequences defining restriction enzyme sites, site-specific recombinase recognition sites, sequences recognized by proteins that bind and / or modify nucleic acids, linkers, splice sites, pre-mRNA binding domains, regulatory sequences, and / or therapeutic nucleic acids (e.g., sequences encoding microRNAs). The therapeutic protein coding domain may be a domain encoding a full-length protein (e.g., corresponding to a native gene or a naturally occurring variant thereof) or a functional portion thereof, such as a domain encoding a truncated protein (e.g., a minigene).

[0241] In some embodiments, the therapeutic sequence encodes a monomeric protein (e.g., a monomeric protein that has secondary structure under physiological conditions, e.g., a monomeric protein that has secondary and tertiary structure under physiological conditions, e.g., a monomeric protein that has secondary, tertiary, and quaternary structure under physiological conditions). Additionally or alternatively, the therapeutic sequence may encode a multimeric protein (e.g., a dimeric protein (e.g., a homodimeric or heterodimeric protein), a trimeric protein, etc.).

[0242] In some embodiments, the therapeutic sequence encodes an antibody, or a portion, fragment, or variant thereof. Antibodies include fragments capable of binding antigen, such as Fv, single chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody), (Fab')2 (including chemically linked F(ab')2), and nanobodies. Papain digestion of an antibody produces two identical antigen-binding fragments, referred to as "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, the latter name reflecting its ability to crystallize readily. Pepsin treatment produces a F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Antibodies also include chimeric and humanized antibodies. Additionally, for all antibody constructs listed herein, variants with sequences from other organisms are also contemplated. Thus, where a human version of an antibody is disclosed, one of skill in the art would understand how to convert the human sequence-based antibody to mouse, rat, cat, dog, horse, etc. sequences. Antibody fragments also include single chain scFvs of any orientation, tandem di-scFvs, diabodies, tandem tri-sdcFvs, minibodies, nanobodies, etc. In some embodiments, for example, when the antibody is an scFv, a single polynucleotide of the therapeutic gene sequence encodes a single polypeptide comprising a heavy chain and a light chain linked together. Antibody fragments also include nanobodies (e.g., sdAbs, antibodies with a single monomer domain, e.g., a pair of heavy chain variable domains without a light chain). Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.) are known in the art and are contemplated as expression products of the therapeutic gene sequences of the present invention.

[0243] In some examples, the therapeutic sequences each have at least 25 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 500 amino acids, at least 1,000 amino acids, at least 1,500 amino acids, at least 2,000 amino acids, at least 2,500 amino acids, at least 3,000 amino acids, or more (e.g., from 25-5,000 amino acids, 50-5,000 amino acids, 100-5,000 amino acids, 200-5,000 amino acids, 500-5,000 amino acids, 1,000-5,000 amino acids, 1,500-5,000 amino acids, or 2,000-5,000 amino acids; e.g., 25-4,000 amino acids, For example, 25-3,000 amino acids, 50-3,000 amino acids, 100-3,000 amino acids, 200-3,000 amino acids, 500-3,000 amino acids, 1,000-3,000 amino acids, 1,500-3,000 amino acids, or 2,000-3,000 amino acids) in length (e.g., a single protein, two proteins, three proteins, four proteins, or more). In embodiments where such therapeutic sequences encode more than one protein, the therapeutic sequence may be a polycistronic therapeutic sequence or a therapeutic sequence of multiple transcription units.

[0244] In some embodiments, the therapeutic sequence encodes an ocular protein. In certain embodiments, the ocular protein is ABCA4. An exemplary human ABCA4 sequence is provided as NCBI Reference Sequence: NG_009073 or NM_000350.

[0245] In embodiments that include a therapeutic sequence that does not encode a protein, the therapeutic sequence lacks a protein-coding domain (e.g., a therapeutic protein-coding domain). For example, in some embodiments, the therapeutic sequence includes a therapeutic nucleic acid that does not encode a protein, such as a short hairpin RNA (shRNA) coding sequence or an immune-activating therapeutic nucleic acid (e.g., a TLR agonist).

[0246] In some embodiments, the therapeutic sequence is between 0.1 Kb and 100 Kb in length (e.g., the therapeutic gene sequence may be between 0.2 Kb and 90 Kb, 0.5 Kb and 80 Kb, 1.0 Kb and 70 Kb, 1.5 Kb and 60 Kb, 2.0 Kb and 50 Kb, 2.5 Kb and 45 Kb, 3.0 Kb and 40 Kb, 3.5 Kb and 35 Kb, 4.0 Kb and 30 Kb, 4.5 Kb and 25 Kb, 4.6 Kb and 24 Kb, 4.7 Kb and 23 Kb, 4.8 Kb and 22 Kb, 4.9 Kb and 21 Kb, 5.0 Kb and 20 Kb, 5.5 Kb and 18 Kb, 6.0 Kb and 17Kb, 6.5Kb to 16Kb, 7.0Kb to 15Kb, 7.5Kb to 14Kb, 8.0Kb to 13Kb, 8.5Kb to 12.5Kb, 9.0Kb to 12.0Kb, 9.5Kb to 11.5Kb, or 10.0Kb to 11.0Kb in length, for example, 0.1Kb to 0.5Kb, 0.5Kb to 1.0Kb, 1.0Kb to 2.5Kb, 2.5Kb to 4.5Kb, 4.5Kb to 8Kb, 8Kb to 10Kb, 10Kb to 15Kb, 15Kb to 20Kb, or longer in length, for example, 0.1Kb to 0.25Kb, 0.25Kb ~0.5Kb, 0.5Kb~1.0Kb, 1.0Kb~1.5Kb, 1.5Kb~2.0Kb, 2.0Kb~2.5Kb, 2.5Kb~3.0Kb, 3.0Kb~3.5Kb, 3.5Kb~4.0Kb, 4.0Kb~4.5Kb, 4.5Kb~5.0Kb, 5 .0Kb~5.5Kb, 5.5Kb~6.0Kb, 6.0Kb~6.5Kb, 6.5Kb~7.0Kb, 7.0Kb~7.5Kb , 7.5Kb~8.0Kb, 8.0Kb~8.5Kb, 8.5Kb~9.0Kb, 9.0Kb~9.5Kb, 9.5Kb~10K b, 10Kb~10.5Kb, 10.5Kb~11Kb, 11Kb~11.5Kb, 11.5Kb~12Kb, 12Kb~12.5Kb, 12.5Kb~13Kb, 13Kb~13.5Kb, 13.5Kb~14Kb, 14Kb~14.5Kb, 14.5Kb ~15Kb, 15Kb~15.5Kb, 15.5Kb~16Kb, 16Kb~16.5Kb, 16.5Kb~17Kb, 17Kb ~17.5Kb, 17.5Kb~18Kb, 18Kb~18.5Kb, 18.5Kb~19Kb, 19Kb~19.5Kb, 19.The nucleic acid sequence may be 5Kb to 20Kb, 20Kb to 21Kb, 21Kb to 22Kb, 22Kb to 23Kb, 23Kb to 24Kb, 24Kb to 25Kb, or more in length, e.g., about 4.5Kb, about 5.0Kb, about 5.5Kb, about 6.0Kb, about 6.5Kb, about 7.0Kb, about 7.5Kb, about 8.0Kb, about 8.5Kb, about 9.0Kb, about 9.5Kb, about 10Kb, about 11Kb, about 12Kb, about 13Kb, about 14Kb, about 15Kb, about 16Kb, about 17Kb, about 18Kb, about 19Kb, about 20Kb, or more in length). In some embodiments, the therapeutic sequence is at least 10 Kb (e.g., 10 Kb to 15 Kb, 15 Kb to 20 Kb, or 20 Kb to 30 Kb; e.g., 10 Kb to 13 Kb, 10 Kb to 12 Kb, or 10 Kb to 11 Kb; e.g., 10 to 11 Kb, 11 to 12 Kb, 12 to 13 Kb, 13 to 14 Kb, or 14 to 15 Kb). In some embodiments, the therapeutic sequence is at least 1,100 bp in length (e.g., 1,100 bp to 10,000 bp, 1,100 bp to 8,000 bp, or 1,100 bp to 5,000 bp in length). In some embodiments, the therapeutic sequence is at least 2,500 bp in length (e.g., 2,500 bp to 15,000 bp, 2,500 bp to 10,000 bp, or 2,500 bp to 5,000 bp in length; e.g., 2,500 bp to 5,000 bp, 5,000 bp to 7,500 bp, 7,500 bp to 10,000 bp, 10,000 bp to 12,500 bp, or 12,500 bp to 15,000 bp). In some embodiments, the therapeutic sequence is at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 11,000 bp, at least 12,000 bp at least 13,000 bp, at least 14,000 bp, at least 15,000 bp, at least 16,000 bp (e.g., 11,000 bp to 16,000 bp, 12,000 bp to 16,000 bp, 13,000 bp to 16,000 bp, 14,000 bp to 16,000 bp, or 15,000 bp to 16,000 bp). In certain embodiments, the therapeutic sequence is long enough to encode a protein and is not an oligonucleotide therapeutic (e.g., not an antisense, siRNA, shRNA therapeutic, etc.).

[0247] In some embodiments, the 3' end of the therapeutic sequence in the therapeutic circular DNA vector is linked to the 5' end of the therapeutic sequence by a non-bacterial sequence of 30 bp or less (e.g., 3 bp to 24 bp, 4 bp to 18 bp, 5 bp to 12 bp, or 6 bp to 10 bp; e.g., 3 bp to 5 bp, 4 bp to 6 bp, 8 bp to 12 bp, 12 bp to 18 bp, 18 bp to 24 bp, or 24 bp to 30 bp; e.g., 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, or 8 bp). For example, in any of the therapeutic circular DNA vectors generated using Type IIS restriction enzymes described herein, the 3' end of the therapeutic sequence may be linked to the 5' end of the therapeutic sequence by a non-bacterial sequence that corresponds to the sticky end or overhang of the Type IIS restriction enzyme cleavage site (e.g., TTTT, AAAA, or AACC). In some examples, the sticky end or overhang of a Type IIS restriction enzyme cleavage site comprises (or consists of) four bases, where only two of the four bases are A or T (e.g., AACC or TTGG). In some examples, the sticky end or overhang of a Type IIS restriction enzyme cleavage site comprises (or consists of) four bases, where only two of the four bases are A (e.g., AACC). In some examples, the sticky end or overhang of a Type IIS restriction enzyme cleavage site comprises (or consists of) four bases, where only two of the four bases are T (e.g., TTGG).

[0248] In some embodiments, the therapeutic sequence comprises a reporter sequence in addition to a domain encoding a therapeutic protein or a domain not encoding a therapeutic protein. Such reporter genes can be useful, for example, to confirm the expression of the therapeutic gene sequence in specific cells and tissues. Reporter sequences that can be added to the transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When linked to regulatory elements that drive expression, the reporter sequence produces a signal that is detectable by conventional means, including enzymatic, radioactive, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemical staining. For example, if the marker sequence is the LacZ gene, the presence of the vector that produces a signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the vector that produces a signal can be visually measured by color or light production in a luminometer.

[0249] In some embodiments, the therapeutic sequence lacks a reporter sequence.

[0250] The therapeutic circular DNA vector of the present invention may include conventional regulatory elements as part of the therapeutic sequence that regulate or improve transcription, translation, and / or expression in target cells. Suitable regulatory elements are described in International Publication No. WO2021 / 055760, which is incorporated herein by reference in its entirety.

[0251] In some instances, any of the therapeutic circular DNA vectors of the present invention encode a self-replicating RNA molecule. Such self-replicating RNA molecules contain a replicase sequence derived from an alphavirus, characterized by having a positive-stranded replicon that is translated into a replicase (or replicase / transcriptase) after delivery to a target cell. The replicase is translated into a polyprotein that self-cleaves to generate a replication complex that creates a negative-stranded genomic copy of the delivered positive-stranded RNA. These negative-stranded transcripts can themselves be transcribed to generate further copies of the positive-stranded parent RNA, as well as subgenomic transcripts (e.g., regulatory sequences). Translation of the subgenomic transcripts then results in in situ expression of the regulatory protein by the infected cell.

[0252] Non-limiting examples of alphaviruses from which the replicase coding sequences of the invention may be derived include Venezuelan equine encephalitis virus (VEE), Semliki Forest virus (SF), Sindbis virus (SIN), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Everglades virus (EVE), Mucambo virus (MUC), Pixuna virus (PIX), Semliki Forest virus (SF), Middelburg virus (MID), Chikungunya virus (CHIK), O'nyong-nyong virus (ONN), Ross River virus (RR), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAG), Bebaru virus (BEB), Mayaro virus (MAY), Una virus (UNA), Aura virus (AURA), Babanki virus (BAB), Highlands J virus (HJ), and Fort Morgan virus (FM). In a specific example of the invention, the self-replicating RNA molecule comprises VEE replicase or a variant thereof.

[0253] Mutant or wild-type virus sequence can be used.For example, in some examples, the self-replicating RNA comprises the attenuated TC83 mutant of VEE replicase.Other mutations in replicase are contemplated herein, including mutant replicase (e.g. mutant VEE replicase) obtained by in vitro evolution method, for example, as taught by Yingzhong et al., Sci Rep.2019,9:6932 (this methodology is incorporated herein by reference).

[0254] In some examples, the self-replicating RNA molecule comprises (i) a replicase coding sequence (e.g., an RNA sequence encoding an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule) and (ii) a heterologous regulatory gene. The polymerase can be an alphavirus replicase, e.g., an alphavirus replicase that includes one, two, three, or all four alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some examples, the polymerase is a VEE replicase, e.g., a VEE replicase that includes one, two, three, or all four alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4.

[0255] In some examples of the invention, the self-replicating RNA molecule does not encode an alphavirus structural protein (e.g., capsid protein). Such a self-replicating RNA can cause the production of its own genomic RNA copy in a cell, but cannot cause the production of RNA-containing viral particles. The inability to produce these viral particles means that the self-replicating RNA molecule cannot perpetuate itself in an infectious form, unlike wild-type alphaviruses. The alphavirus structural proteins can be replaced with a gene(s) encoding the heterologous regulatory protein(s) of interest, such that the subgenomic transcript encodes the heterologous regulatory protein(s) rather than the structural proteins of the alphavirus viral particles.

[0256] Thus, in some instances, a self-replicating RNA molecule of the invention may have two open reading frames: a first (5') open reading frame that encodes a replicase and a second (3') open reading frame that encodes one or more (e.g., two or three) therapeutic proteins. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, e.g., for encoding additional genes or for encoding accessory polypeptides.

[0257] Suitable self-replicating RNA molecules may have a variety of lengths. In some embodiments of the invention, the length of the self-replicating RNA molecule is 5,000 to 50,000 nucleotides (i.e., 5 kb to 50 kb). In some examples, the self-replicating RNA molecule is 5 kb to 20 kb in length (e.g., 6 kb to 18 kb, 7 kb to 16 kb, 8 kb to 14 kb, or 9 kb to 12 kb in length, e.g., 5 kb to 6 kb, 6 kb to 7 kb, 7 kb to 8 kb, 8 kb to 9 kb, 9 kb to 10 kb, 10 kb to 11 kb, 11 kb to 12 kb, 12 kb to 13 kb, 13 kb to 14 kb, 14 kb to 1 kb). In some embodiments, the length of the nucleic acid is about 5 kb, 15 kb to 16 kb, 16 kb to 18 kb, or 18 kb to 20 kb, e.g., about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 10.5 kb, about 11 kb, about 11.5 kb, about 12 kb, about 12.5 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, or about 20 kb.

[0258] The self-replicating RNA molecule may have a 3' poly A tail. In addition, the self-replicating RNA molecule may contain a poly A polymerase recognition sequence (e.g., AAUAAA).

[0259] In certain embodiments, the RNA according to the invention does not encode a reporter molecule, such as luciferase or a fluorescent protein, such as green fluorescent protein (GFP).

[0260] In some embodiments, the replicase encoded by the self-replicating RNA can be a variant of any of the replicases described herein. In some embodiments, the variant is a functional fragment (e.g., a protein fragment that is functionally similar or functionally equivalent to the protein).

[0261] V. Pharmaceutical Compositions The improved efficiency makes the method of the present invention particularly suitable for scalable production of pharmaceutical compositions containing therapeutic circular DNA vectors.Any of the methods for generating therapeutic circular DNA vectors described herein may be suitable for generating pharmaceutical compositions containing therapeutic circular DNA vectors in a pharma-ceutically acceptable carrier.

[0262] Provided herein is a method for generating a pharmaceutical formulation containing a therapeutic circular DNA vector (e.g., a therapeutic supercoiled circular DNA vector). In some embodiments, such a method includes the following steps: First, a sample is provided containing a plasmid DNA vector having a therapeutic gene sequence and a backbone sequence. The plasmid DNA vector is amplified using polymerase-mediated rolling circle amplification to generate linear concatemers. The linear concatemers are then digested with a restriction enzyme that cuts at least a first site and a second site of the linear concatemer unit-by-unit, where the first and second sites are adjacent to the therapeutic sequence. This digestion generates a linear therapeutic fragment having the therapeutic sequence and a linear backbone fragment having the backbone sequence. The linear therapeutic fragment is then self-ligated to generate a relaxed circular DNA vector, which is then contacted with a topoisomerase or helicase to generate a supercoiled circular DNA vector. In some embodiments, the linear bacterial fragments are digested with a terminal exonuclease.

[0263] In certain examples, a method for generating a pharmaceutical formulation containing a therapeutic circular DNA vector includes the following steps: A sample is provided containing a plasmid DNA vector having a therapeutic sequence and a backbone sequence. The plasmid DNA vector is amplified using polymerase-mediated rolling circle amplification to generate linear concatemers. The linear concatemers are then digested with a restriction enzyme that cuts at least a first site and a second site of the linear concatemers unit-by-unit, where the first and second sites are adjacent to the therapeutic sequence. This digestion generates a linear therapeutic fragment having the therapeutic sequence and a linear backbone fragment having the backbone sequence. The linear therapeutic fragment is then self-ligated to generate a circular DNA vector, and the linear backbone fragment is digested with a terminal nuclease. In some embodiments, the circular DNA vector is contacted with a topoisomerase or helicase to generate a supercoiled circular DNA vector.

[0264] In some examples, a method for generating a pharmaceutical formulation containing a therapeutic circular DNA vector includes the following steps: A sample is provided that includes a plasmid DNA vector that includes a therapeutic sequence and a backbone sequence. The plasmid DNA vector is amplified using polymerase-mediated rolling circle amplification to generate linear concatemers. The linear concatemers are then digested with a restriction enzyme (e.g., a type IIS restriction enzyme, e.g., BsaI) that cuts at least a first site, a second site, and a third site per unit of the linear concatemer. The first and second sites flank the therapeutic sequence and form self-complementary overhangs, and the third site is present in the backbone sequence and forms an overhang that is non-complementary to the first or second site. The digestion generates a linear therapeutic fragment with the therapeutic sequence and at least two linear backbone fragments, each of which includes a portion of the backbone sequence. The linear therapeutic fragments are contacted with a ligase to generate a therapeutic circular DNA vector in solution.

[0265] In some embodiments, the restriction enzyme cleaves the linear concatemer unit-by-unit at a fourth site, where the fourth site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second site, and digestion generates at least three linear scaffold fragments, each of which contains a portion of the scaffold sequence.

[0266] In some embodiments, the therapeutic circular DNA vector is contacted with a topoisomerase or helicase. Such reactions may be carried out at about 37° C. Additionally or alternatively, the therapeutic circular DNA vector may be contacted with a terminal exonuclease (e.g., in a reaction carried out at about 37° C.). In certain embodiments, the therapeutic circular DNA vector is contacted with a topoisomerase or helicase, and then the therapeutic circular DNA vector is contacted with a terminal exonuclease without increasing the reaction temperature to inactivate the topoisomerase or helicase.

[0267] In some embodiments, the method includes contacting the therapeutic circular DNA vector with a topoisomerase or helicase and / or terminal exonuclease, followed by passing the therapeutic circular DNA vector through a column (e.g., a capture column). In some embodiments, the therapeutic circular DNA vector is then precipitated with isopropyl alcohol.

[0268] The above-described methods can produce pharmaceutical formulations containing therapeutic circular DNA vectors (e.g., therapeutic supercoiled circular DNA vectors) in large quantities and with high purity. Thus, the present invention includes any of the pharmaceutical formulations described herein. In some embodiments, the pharmaceutical formulations of the present invention contain at least twice as many therapeutic sequences as the sample of plasmid DNA vector from which the therapeutic sequences are generated. In some embodiments, the pharmaceutical formulations contain at least five times as many therapeutic sequences as the sample of plasmid DNA vector. In some embodiments, the pharmaceutical formulations contain at least ten times as many therapeutic sequences as the sample of plasmid DNA vector.

[0269] In certain embodiments, a pharmaceutical formulation of the invention (e.g., a pharmaceutical composition produced by any of the methods described herein) contains at least twice the number of therapeutic sequences (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold; e.g., 2-fold to 1,000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold, 2-fold to 40-fold, 2-fold to 30-fold, 2-fold to 20-fold, or 2-fold to 10-fold; e.g., 5-fold to 1,000-fold). , 5x to 500x, 5x to 100x, 5x to 50x, 5x to 40x, 5x to 30x, 5x to 20x, or 5x to 10x; for example, 10x to 1,000x, 10x to 500x, 10x to 100x, 10x to 50x, 10x to 40x, 10x to 30x, or 10x to 20x; for example, 2x to 5x, 5x to 10x, 10x to 20x, 20x to 30x, 30x to 40x, 40x to 50x, 50x to 60x, 6 0-70x, 70-80x, 80-90x, 90-100x, 100-200x, 200-500x, or 500-1,000x; for example, about 2-, about 3-, about 4-, about 5-, about 6-, about 7-, about 8-, about 9-, about 10-, about 15-, about 20-, about 25-, about 30-, about 40-, about 50-, about 60-, about 70-, about 80-, about 90-, or about 100-fold the number of therapeutic sequences).

[0270] In some embodiments, a pharmaceutical formulation of the invention (e.g., a pharmaceutical composition produced by any of the methods described herein) contains at least 1.0 mg of a therapeutic circular DNA vector in a pharma- ceutically acceptable carrier (e.g., 1.0 mg to 10 g, 1.0 mg to 5.0 g, 1.0 mg to 1.0 g, 1.0 mg to 500 mg, 1.0 mg to 200 mg, 1.0 mg to 100 mg, 1.0 mg to 50 mg, 1.0 mg to 25 mg, 1.0 mg to 20 mg, 1.0 mg to 15 mg, 1.0 mg to 10 mg, 1.0 mg to 5.0 mg, 2.0 mg to 10 g, 2.0 mg to 5.0 g, 2.0 mg to 1.0 g, 2.0 mg to 500 mg, 2.0 mg to 200 mg, 2.0 mg to 1 00mg, 2.0mg~50mg, 2.0mg~25mg, 2.0mg~20mg, 2.0mg~15mg, 2.0mg~10mg, 2.0mg~5.0mg, 5.0 mg~10g, 5.0mg~5.0g, 5.0mg~1.0g, 5.0mg~500mg, 5.0mg~200mg, 5.0mg~100mg, 5.0mg~50mg , 5.0mg-25mg, 5.0mg-20mg, 5.0mg-15mg, 5.0mg-10mg, 10mg-10g, 10mg-5.0g, 10mg-1.0g, 10mg-500mg, 10mg-200mg, 10mg-100mg, 10mg-50mg, 10mg-25mg, 10mg-20mg, or 10mg-15mg).

[0271] In some embodiments, the pharmaceutical formulations of the present invention (e.g., pharmaceutical compositions produced by any of the methods described herein) contain at least 2.0 mg of therapeutic circular DNA vector in a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical formulations produced by any of the methods described herein contain at least 5.0 mg of therapeutic circular DNA vector in a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical formulations produced by any of the methods described herein contain at least 10.0 mg of therapeutic circular DNA vector in a pharma- ceutically acceptable carrier.

[0272] In some embodiments, the pharmaceutical formulations of the invention (e.g., pharmaceutical compositions produced by the methods described herein (e.g., methods comprising contacting a therapeutic circular DNA vector with a topoisomerase or helicase)) comprise a therapeutic circular DNA vector that is at least 60% supercoiled monomers, at least 70% supercoiled monomers, at least 80% supercoiled monomers, or at least 90% supercoiled monomers (e.g., 60%-80% supercoiled monomers, 60%-90% supercoiled monomers, or at least 90% supercoiled monomers). Supercoiled monomer, 60%~95% supercoiled monomer, 60%~99% supercoiled monomer, 60%~99.5% supercoiled monomer, 60%~99.9% supercoiled monomer, 65%~80% supercoiled monomer, 65%~90% supercoiled monomer, 65%~95% supercoiled monomer, 65%~99% supercoiled monomer, 65%~99.5% supercoiled monomer, 65%~99.9% supercoiled monomer, 70%~80% supercoiled monomer, 70%~90% supercoiled Monomer, 70%~95% supercoiled monomer, 70%~99% supercoiled monomer, 70%~99.5% supercoiled monomer, 70%~99.9% supercoiled monomer, 75%~80% supercoiled monomer, 75%~90% supercoiled monomer, 75%~95% supercoiled monomer, 75%~99% supercoiled monomer, 75%~99.5% supercoiled monomer, 75%~99.9% supercoiled monomer, 80%~85% supercoiled monomer, 80%~90% supercoiled monomer, 80 %~95% supercoiled monomer, 80%~99% supercoiled monomer, 80%~99.5% supercoiled monomer, 80%~99.9% supercoiled monomer, 85%~90% supercoiled monomer, 85%~95% supercoiled monomer, 85%~99% supercoiled monomer, 85%~99.5% supercoiled monomer, 85%~99.9% supercoiled monomer, 90%~95% supercoiled monomer, 90%~99% supercoiled monomer, 90%~99.5% supercoiled monomer, 90%~99.9% supercoiled monomer, 95%-99% supercoiled monomer, 95%-99.5% supercoiled monomer, 95%-99.9% supercoiled monomer, 98%-99% supercoiled monomer, 98%-99.5% supercoiled monomer, or 98%-99.9% supercoiled monomer; e.g., about 60% supercoiled monomer, about 65% supercoiled monomer, about 70% supercoiled monomer, The nucleic acid sequence may contain about 10% supercoiled monomer, about 75% supercoiled monomer, about 80% supercoiled monomer, about 85% supercoiled monomer, about 90% supercoiled monomer, about 95% supercoiled monomer, about 96% supercoiled monomer, about 97% supercoiled monomer, about 98% supercoiled monomer, about 99% supercoiled monomer, or about 99.9% supercoiled monomer). In any of these examples, the supercoiled monomer is calculated using densitometric analysis of gel electrophoresis (e.g., as described in Example 5 below).

[0273] In other embodiments, the pharmaceutical formulations of the invention (e.g., pharmaceutical compositions produced by the methods described herein (e.g., methods in which the therapeutic circular DNA vector is not contacted with a topoisomerase or helicase)) contain a therapeutic circular DNA vector that is not supercoiled (i.e., is a relaxed, circular DNA).

[0274] In some embodiments, the percentage of supercoiled monomer is determined by agarose gel electrophoresis or capillary electrophoresis. Additionally or alternatively, the percentage of supercoiled monomer is determined by anion exchange HPLC.

[0275] In some embodiments, the pharmaceutical formulations of the present invention (e.g., pharmaceutical compositions produced by the methods described herein) are substantially devoid of impurities. For example, in some embodiments, the pharmaceutical formulations have a protein content of less than 1.0% by weight (e.g., a protein content of less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% by weight). In some examples, the protein content is determined by bicinchoninic acid assay. Additionally or alternatively, the protein content is determined by ELISA.

[0276] In some examples, the pharmaceutical formulations of the invention (e.g., pharmaceutical compositions produced by the methods described herein) have an RNA content of less than 1.0% by weight (e.g., an RNA content of less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% by weight). In some embodiments, the RNA content is determined by agarose gel electrophoresis. In some embodiments, the RNA content is determined by quantitative PCR. In some embodiments, the RNA content is determined by a fluorescent assay (e.g., RiboGreen).

[0277] In some embodiments, the pharmaceutical formulations of the invention (e.g., pharmaceutical compositions produced by the methods described herein) have a gDNA content of less than 1.0% by weight (e.g., a gDNA content of less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% by weight). In some embodiments, the gDNA content is determined by agarose gel electrophoresis or capillary electrophoresis. In some embodiments, the gDNA content is determined by quantitative PCR. In some embodiments, the gDNA content is determined by Southern blot.

[0278] In some embodiments, the pharmaceutical formulations of the invention (e.g., pharmaceutical compositions produced by the methods described herein) contain less than 40 EU / mg endotoxin. In some embodiments, the pharmaceutical formulations contain less than 20 EU / mg endotoxin. In some embodiments, the pharmaceutical formulations contain less than 10 EU / mg endotoxin. In some embodiments, the pharmaceutical formulations contain less than 5 EU / mg endotoxin (e.g., less than 4 EU / mg endotoxin, less than 3 EU / mg endotoxin, less than 2 EU / mg endotoxin, less than 1 EU / mg endotoxin, less than 0.5 EU / mg endotoxin), e.g., as measured by Limulus amebocyte extract (LAL) assay.

[0279] The pharmaceutical compositions provided herein may include one or more pharma- ceutically acceptable carriers, such as excipients and / or stabilizers, that are non-toxic to the individual (e.g., human patient) treated at the dosages and concentrations used. In some embodiments, the pharma-ceutically acceptable carrier is a pH-buffered aqueous solution. Examples of pharma-ceutically acceptable carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as Tween, polyethylene glycol (PEG), and Pluronic.

[0280] When the pharmaceutical composition is provided in liquid form, the pharma- ceutically acceptable carrier can be water (e.g., pyrogen-free water), isotonic saline, or an aqueous buffer solution, such as a phosphate or citrate buffer. Injection of the pharmaceutical composition in water or a buffer solution, such as an aqueous buffer containing a sodium salt (e.g., at least 50 mM sodium salt), a calcium salt (e.g., at least 0.01 mM calcium salt), or a potassium salt (e.g., at least 3 mM potassium salt), can be performed. According to certain embodiments, the sodium, calcium, or potassium salts can be present in the form of their halides, such as chlorides, iodides, or bromides, their hydroxides, carbonates, bicarbonates, or sulfates, etc. Examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na2CO2, NaHCO2, and Na2SO4. Examples of potassium salts include, for example, KCl, KI, KBr, K2CO2, KHCO2, and K2SO4. Examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO2, CaSO4, and Ca(OH)2. In addition, organic anions of the above-mentioned cations may be contained in the buffer. According to certain embodiments, the buffer suitable for injection purposes described above may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2), or potassium chloride (KCl), where additional anions may be present. CaCl2 may be exchanged for another salt, such as KCl. In some embodiments, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic, or hypotonic relative to a particular reference medium, i.e., the buffer may have a higher, the same, or lower salt content relative to a particular reference medium, and preferably, such concentrations of the above-mentioned salts that do not cause cell damage due to osmotic or other concentration effects may be used. The reference medium may be a liquid, such as blood, lymph, interstitial fluid, other bodily fluids, or a common buffer solution. Such common buffer solutions or liquids are known to those skilled in the art.Lactated Ringer's solution is an especially preferred liquid base.

[0281] One or more compatible solid or liquid fillers, diluents, or encapsulating materials may be suitable for administration to humans. The components of the pharmaceutical composition according to the invention can be mixed with the nucleic acid vector according to the invention described herein in a manner such that there is no interaction that would substantially reduce the pharmaceutical effectiveness of the (pharmaceutical) composition according to the invention under typical conditions of use. Pharmaceutically acceptable carriers, fillers, and diluents can have sufficiently high purity and sufficiently low toxicity to be suitable for administration to the individual being treated. Some examples of compounds that can be used as pharma- ceutically acceptable carriers, fillers, or components thereof are sugars such as lactose, glucose, trehalose, and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil derived from cocoa; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; or alginic acid.

[0282] The choice of pharma- ceutically acceptable carrier can depend on the way the pharmaceutical composition is to be administered.

[0283] Suitable unit dosage forms for injection include sterile solutions of water, saline, and mixtures thereof. The pH of such solutions can be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Suitable pharma- ceutically acceptable carriers for topical application include those suitable for use in lotions, creams, gels, etc. When the pharmaceutical composition is administered orally, tablets, capsules, etc. are preferred unit dosage forms.

[0284] Additional additives that may be included in the pharmaceutical composition are emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; pharmaceutical carriers; stabilizers; antioxidants; and preservatives.

[0285] The pharmaceutical composition according to the present invention can be provided in liquid or dry (e.g., lyophilized) form. In certain embodiments, the nucleic acid vector of the pharmaceutical composition is provided in lyophilized form. The lyophilized composition comprising the nucleic acid vector of the present invention can be reconstituted before administration in a suitable buffer, preferably based on an aqueous carrier, such as lactate Ringer's solution, Ringer's solution, or phosphate buffer.

[0286] In certain embodiments of the invention, any of the therapeutic circular DNA vectors of the invention may be complexed with one or more cationic or polycationic compounds, such as cationic or polycationic polymers, cationic or polycationic peptides or proteins, such as protamine, cationic or polycationic polysaccharides, and / or cationic or polycationic lipids.

[0287] According to certain embodiments, the therapeutic circular DNA vector of the present invention may be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Thus, in one embodiment, the pharmaceutical composition comprises a liposome, lipoplex, and / or lipid nanoparticle comprising the therapeutic circular DNA vector.

[0288] Lipid-based formulations can be effective delivery systems for nucleic acid vectors due to their biocompatibility and ease of their large-scale manufacture. Cationic lipids have been widely studied as synthetic materials for the delivery of nucleic acids. After mixing, the nucleic acid is condensed by the cationic lipids to form lipid / nucleic acid complexes known as lipoplexes. These lipid complexes can protect the genetic material from the action of nucleases and can deliver the genetic material into cells by interacting with the negatively charged cell membrane. Lipoplexes can be prepared by directly mixing positively charged lipids with negatively charged nucleic acids at physiological pH.

[0289] Conventional liposomes consist of a lipid bilayer that may be composed of cationic, anionic, or neutral phospholipids and cholesterol, which surrounds an aqueous core. Both the lipid bilayer and the aqueous space may incorporate hydrophobic or hydrophilic compounds, respectively. The properties and in vivo behavior of liposomes may be modified by adding hydrophilic polymer coatings, such as polyethylene glycol (PEG), to the liposome surface to provide steric stabilization. Furthermore, liposomes may be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of attached PEG chains.

[0290] Liposomes are colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. Liposomes can exist as spherical vesicles and can range in size from 20 nm to several microns. Cationic lipid-based liposomes can form complexes with negatively charged nucleic acids through electrostatic interactions, resulting in complexes that exhibit the biocompatibility, low toxicity, and feasibility of large-scale manufacturing required for clinical applications in vivo. Liposomes can fuse with the plasma membrane for uptake. Once inside the cell, the liposomes are processed by the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm.

[0291] Cationic liposomes can function as a delivery system for therapeutic circular DNA vectors. Cationic lipids, such as MAP, (1,2-dioleoyl-3-trimethylammonium-propane), and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate), can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions to form nanoparticles, resulting in high transfection efficiency in vitro. In addition, neutral lipid-based nanoliposomes, such as neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes for nucleic acid vector delivery are available.

[0292] Thus, in one embodiment of the present invention, the therapeutic circular DNA vector of the present invention is complexed with cationic lipids and / or neutral lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes, or neutral lipid-based nanoliposomes in the pharmaceutical composition of the present invention.

[0293] In a particular embodiment, the pharmaceutical composition according to the invention comprises a therapeutic circular DNA vector of the invention formulated with a cationic or polycationic compound and / or a polymeric carrier. Thus, in a further embodiment of the invention, the therapeutic circular DNA vector described herein is optionally formulated with a nucleic acid vector to cationic or polycationic compound and / or a polymeric carrier in a ratio selected from the range of about 5:1 (w / w) to about 0.25:1 (w / w), such as about 5:1 (w / w) to about 0.5:1 (w / w), such as about 4:1 (w / w) to about 1:1 (w / w), or about 3:1 (w / w) to about 1:1 (w / w), such as about 3:1 (w / w) to about 2:1 (w / w). The nucleic acid vector is associated with or complexed to the cationic or polycationic compound or polymeric carrier by weight ratio to the polymeric carrier, or optionally with a nitrogen / phosphate (N / P) ratio of the nucleic acid vector to the cationic or polycationic compound and / or polymeric carrier in the range of about 0.1 to 10, e.g., in the range of about 0.3 to 4 or 0.3 to 1, e.g., in the range of about 0.5 to 1 or 0.7 to 1, e.g., in the range of about 0.3 to 0.9 or 0.5 to 0.9. For example, the N / P ratio of the therapeutic circular DNA vector to the one or more polycations is in the range of about 0.1 to 10, including the ranges of about 0.3 to 4, about 0.5 to 2, about 0.7 to 2, and about 0.7 to 1.5.

[0294] The nucleic acid vectors described herein may be associated with vehicles, transfection agents, or complexing agents to increase transfection efficiency and / or expression of the regulated genes according to the present invention.

[0295] In some examples, the therapeutic circular DNA vector according to the invention is complexed with one or more polycations, preferably protamine or oligofectamine. Additional cationic or polycationic compounds that can be used as transfection or complexing agents include cationic polysaccharides, such as chitosan, polybrene, cationic polymers, such as polyethyleneimine (PEI), cationic lipids, such as DOTMA: [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristooxypropyldimethylhydroxyethylammonium bromide, MA P: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, Oligofectamine, or cationic or polycationic polymers, such as modified polyamino acids, such as β-amino acid polymers or reversed polyamides. modified polyethylenes, such as poly(N-ethyl-4-vinylpyridinium bromide) (PVP); modified acrylates, such as poly(dimethylaminoethyl methyl acrylate) (pDMAEMA); modified amidoamines, such as pAMAM (poly(amidoamine)); modified poly-β-aminoesters (PBAE), such as diamine-terminated 1,Examples of the polymer include 4-butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, polyimines such as PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar-based polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, silane-based polymers such as PMOXA-PDMS copolymers, and block polymers consisting of a combination of one or more cationic blocks (e.g., selected from the cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0296] According to a particular embodiment, the pharmaceutical composition of the present invention comprises a nucleic acid vector (e.g., a circular DNA vector) encapsulated in or attached to a polymeric carrier. The polymeric carrier used according to the present invention may be a polymeric carrier formed by disulfide-bridged cationic components. The disulfide-bridged cationic components may be the same or different from each other. The polymeric carrier may contain further components. It is also particularly preferred that the polymeric carrier used according to the present invention comprises a mixture of cationic peptides, proteins or polymers, cross-linked by disulfide bonds as described herein, and optionally further components as described herein. In this context, the disclosure of WO2012 / 013326 is incorporated herein by reference. In this context, the cationic components forming the basis of the polymeric carrier by disulfide bridges are usually selected from any suitable cationic or polycationic peptides, proteins or polymers suitable for this purpose, in particular any cationic or polycationic peptides, proteins or polymers capable of complexing further nucleic acids contained in the nucleic acid vector or composition described herein, thereby preferably condensing the nucleic acid vector. The cationic or polycationic peptides, proteins, or polymers may be linear molecules, although branched cationic or polycationic peptides, proteins, or polymers may also be used.

[0297] All disulfide-bridged cationic or polycationic proteins, peptides or polymers of the polymeric carrier that can be used to complex the therapeutic circular DNA vector according to the invention included as part of the pharmaceutical composition of the present invention may contain at least one SH moiety (e.g. at least one cysteine ​​residue, or any further chemical group that exhibits an SH moiety) that can form a disulfide bond upon condensation with at least one further cationic or polycationic protein, peptide or polymer as the cationic component of the polymeric carrier as referred to herein.

[0298] Such polymeric carriers used for complexing the therapeutic circular DNA vector of the present invention may be formed by disulfide-bridged cationic (or polycationic) components. In particular, such cationic or polycationic peptides or proteins or polymers of the polymeric carrier, which contain at least one SH moiety or are further modified to contain at least one SH moiety, may be selected from proteins, peptides and polymers as complexing agents.

[0299] In other embodiments, the therapeutic circular DNA vector according to the present invention in a suitable buffer may be administered naked, without association with any additional vehicles, transfection agents, or complexing agents.

[0300] VI.How to use Provided herein is a method of inducing expression (e.g., sustained expression) of a therapeutic sequence in a subject in need of such induction (e.g., as part of a gene therapy regimen) by administering any of the therapeutic circular DNA vectors described herein or pharmaceutical compositions thereof. Target cells or tissues of the subject may be characterized by examining host cell nucleic acid sequences (e.g., RNA sequences, e.g., mRNA sequences), e.g., by Southern blotting or PCR analysis, to detect or quantify the presence (e.g., persistence) of the delivered therapeutic sequence. Alternatively, expression of the therapeutic sequence in the subject may be characterized (e.g., quantitatively or qualitatively) by monitoring the progression of a disease (e.g., associated with a defect or mutation targeted by the therapeutic sequence) being treated by delivery of the therapeutic sequence. In some embodiments, transcription or expression (e.g., sustained transcription or sustained expression) of the therapeutic sequence is confirmed by observing a diminution of one or more symptoms associated with the disease.

[0301] Thus, the present invention provides a method for treating a disease in a subject by administering to the subject any of the therapeutic circular DNA vectors or pharmaceutical compositions thereof described herein. Any of the therapeutic circular DNA vectors or pharmaceutical compositions thereof described herein may be administered in a concentration of 1 μg to 10 mg of DNA (e.g., 5 μg to 5.0 mg, 10 μg to 2.0 mg, or 100 μg to 1.0 mg of DNA, e.g., 10 μg to 20 μg, 20 μg to 30 μg, 30 μg to 40 μg, 40 μg to 50 μg, 50 μg to 75 μg, 75 μg to 100 μg, 100 μg to 200 μg, 200 μg to 300 μg, 300 μg to 400 μg, 400 μg to 500 μg, 500 μg to 1.0 mg, 1.0 mg to 5 The subject may be administered a dosage of about 1.0 mg, or 5.0 mg to 10 mg of DNA, for example, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 750 μg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5 mg, or about 10 mg of DNA.

[0302] In some embodiments, administration of a therapeutic circular DNA vector or pharmaceutical composition thereof of the present invention is less likely to elicit an immune response in a subject compared to administration of other gene therapy vectors (e.g., plasmid DNA vectors and viral vectors).

[0303] In some examples, the therapeutic circular DNA vectors and pharmaceutical compositions thereof provided herein are suitable for repeated administration due to their ability to transfect target cells without causing an immune response or with a reduced immune response compared to reference vectors such as plasmid DNA vectors or AAV vectors, as described above. Thus, the present invention provides a method for repeatedly administering the therapeutic circular DNA vectors and pharmaceutical compositions described herein. Any of the above-mentioned dosages can be repeated at a suitable frequency and duration. In some embodiments, the subject is administered about twice a day, about once a day, about five times a week, about four times a week, about three times a week, about twice a week, about once a week, about twice a month, about once a month, about once every six weeks, about once every two months, about once every three months, about once every four months, twice a year, once a year, or less frequently. In some embodiments, the number and frequency of administrations are consistent with the turnover rate of the target cells. It will be understood that in the long-lived postmitotic target cells transfected with the vectors described herein, a single administration of the vector may be sufficient to maintain the expression of heterologous genes in the target cells for a long period of time.Therefore, in other embodiments, the therapeutic circular DNA vector provided herein can be administered to a subject in a single administration.The number of occasions that the therapeutic circular DNA vector is delivered to a subject can be the number of times required to maintain clinical effect (e.g., therapeutic effect).

[0304] The method of the present invention includes administration of the therapeutic circular DNA vector or pharmaceutical composition thereof by any suitable route. The therapeutic circular DNA vector or pharmaceutical composition thereof can be administered systemically or locally, for example, intravenously, ophthalmically (e.g., intravitreally, subretinal, by eye drop, intraocularly, intraorbitally), intramuscularly, intravitreally (e.g., by intravitreal injection), intradermally, intrahepatically, intracerebrally, intramuscularly, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intraarachnoidally, intranasally, intravaginally, intrarectally, intratumorally, subcutaneously, subconjunctivally, intravesically, mucosally, intrapericardially, intraumbilically, orally, topically, transdermally, by inhalation, by aerosol administration, by injection (e.g., by jet injection), by electroporation, by implantation, by injection (e.g., by continuous infusion), by local perfusion directly bathing the target cells, by catheter, by lavage, by cream, or by lipid composition.

[0305] The therapeutic circular DNA vectors described herein can be delivered to cells by in vivo electrical transfer (e.g., in vivo electroporation). In vivo electroporation has been demonstrated in certain tissues, such as the eye, skin, skeletal muscle, certain tumor types, and lung epithelium. Naked DNA delivery to cells by in vivo electroporation involves administering DNA to the target tissue, followed by applying an electric field to temporarily increase cell membrane permeability in the tissue by creating pores, thereby allowing DNA molecules to pass through the cell membrane. As an example, delivery to skin using in vivo electroporation is described in Cha & Daud Hum.Vaccin.Immunother.2012,8(11):1734-1738, which is incorporated by reference in its entirety. In vivo electroporation of skeletal muscle is described in Sokolowska & Blachnio-Zabielska, Int. J. Molecular Sci. 2019, 20:2776, which is incorporated by reference in its entirety. Intratumoral delivery using in vivo electroporation is described in Aung et al. Gene Therapy 2009, 16:830-839, which is incorporated by reference in its entirety. DNA electroporation into lung cells in vivo is described in Pringle et al. J. Gene Med. 2007, 9:369-380, which is incorporated by reference in its entirety. In vivo electrical introduction of circular DNA vectors into cells of the eye (e.g., retinal cells and / or photoreceptor cells) is described in International Patent Publication No. WO2022 / 198138, which is incorporated by reference in its entirety. In some instances, following administration of the circular DNA vector to the eye, an electrode can be placed within the eye (e.g., within about 1 mm of the retina) and an electric field can be delivered through the electrode to the target ocular tissue under conditions suitable for electrical transfer of the circular DNA vector to the target cells (e.g., by applying 6-10 pulses of 10-100 V each).Devices and systems with electrodes suitable for delivering an electric field to mammalian tissue are commercially available and may be useful in the methods disclosed herein. In some examples, the electric field is delivered through an electrode with a needle (e.g., a needle placed in the vitreous humor or subretinal space). Suitable needle electrodes include the CLINIPORATOR™ electrode sold by IGEA™ and the needle electrode sold by AMBU™. The methods of the present invention include administration of any of the therapeutic circular DNA vectors described herein or pharmaceutical compositions thereof to skin, skeletal muscle, tumors (including, for example, melanoma), eyes, and lungs by electrical transfer in vivo.

[0306] Additionally or alternatively, the therapeutic circular DNA vector or pharmaceutical composition thereof may be administered to host cells ex vivo, for example, by ex vivo culturing of cells from an individual patient, followed by reimplantation of the host cells into the patient, for example, after selection of cells that have taken up the vector. Thus, in some aspects, the present disclosure provides transfected host cells and methods of administration thereof for treating disease.

[0307] Additionally or alternatively, the invention includes a method of treating a subject having a disease or disorder by administering to the subject an isolated DNA vector of the invention (or a composition thereof).

[0308] Evaluation of transfection efficiency of any of the therapeutic circular DNA vectors described herein can be performed using any method known in the art or described herein. Isolation of transfected cells can also be performed by standard techniques. For example, cells containing a therapeutic gene can express a visible marker, such as a fluorescent protein (e.g., GFP) or other reporter protein encoded by the sequence of the heterologous gene, which aids in the identification and isolation of a cell or cells containing a heterologous gene. Cells carrying a therapeutic gene can also be characterized by examining the nucleic acid sequence (e.g., RNA sequence, e.g., mRNA sequence) of the host cell, for example, by Southern blotting or PCR analysis, to analyze the presence of the heterologous gene contained in the vector.

[0309] Thus, the methods of the invention include administering to a subject any of the therapeutic circular DNA vectors described herein, followed by detecting expression of the heterologous gene in the subject. Expression may be detected 1 week to 4 weeks after administration, 1 month to 4 months after administration, 4 months to 1 year after administration, 1 year to 5 years after administration, or 5 years to 20 years after administration (e.g., at least 1 week, at least 2 weeks, at least 1 month, at least 4 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years after administration). At any of these detection time points, persistence of the DNA vector (e.g., episomal persistence) may be observed. In some embodiments, the persistence of the circular DNA vector is 5% to 50% higher, 50% to 100% higher, 1 to 5 times higher, or 5 to 10 times higher (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 1 times higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 6 times higher, 7 times higher, 8 times higher, 9 times higher, 10 times higher, or more) compared to a reference vector (e.g., a circular vector produced in bacteria or having one or more bacterial signatures not present in the vectors of the invention).

[0310] Additionally or alternatively, any of the therapeutic circular DNA vectors of the present invention can be administered to host cells ex vivo, for example, by ex vivo culturing of cells from an individual patient, followed by reimplanting the host cells into the patient, for example, after selecting cells that have incorporated the vector. Thus, in some aspects, the present disclosure provides transfected host cells (e.g., electrically transfected host cells), methods of transfecting host cells, and methods of administering host cells to a subject, for example, to treat a disease in the subject. In some examples, any of the therapeutic circular DNA vectors described herein can be transfected into host cells by electroporation using known methods and devices (e.g., by NEON transfection (Thermo Fisher) or a flow electroporation chamber (e.g., as described in U.S. Patent No. 9,546,350 or U.S. Patent Publication No. 2020 / 0131500, each of which is incorporated by reference).

[0311] VII. KITS AND ARTICLES OF MANUFACTURE In another aspect of the present invention, an article of manufacture or kit comprising any of the therapeutic circular DNA vectors or pharmaceutical compositions thereof described herein. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing, and / or diagnosing a condition by itself or in combination with another composition that is effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is the therapeutic circular DNA vector of the present invention or a pharmaceutical composition comprising the therapeutic circular DNA vector. The label or package insert indicates that the composition is used to treat a condition treatable by its contents. Additionally, the article of manufacture may include (a) a first container having a composition contained therein, the composition comprising a therapeutic circular DNA vector or a pharmaceutical composition thereof; and (b) a second container having a composition contained therein, the composition comprising an additional therapeutic agent. The article of manufacture may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising a pharma-ceutically acceptable carrier, such as sterile water for injection (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, or any of the pharma-ceutically acceptable carriers disclosed above. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, or other delivery devices.

[0312] In some examples, the kits provided herein include any of the therapeutic circular DNA vectors described herein (or generated by the methods described herein) or compositions (e.g., pharmaceutical compositions) thereof, and instructions for expressing the therapeutic circular DNA vector in a cell or culture of cells using electroporation (e.g., in vitro or ex vivo electroporation) or electrotransfer (e.g., in vivo electrotransfer). EXAMPLES

[0313] Example 1. Creation and purification of closed circular DNA using two restriction enzymes Therapeutic circular DNA vectors can be generated by a cell-free process using a plasmid DNA vector as a template, which can be amplified into concatemers by rolling circle amplification. This example compares a process that includes gel extraction of digested DNA with a streamlined process in which gel extraction is not required. In the streamlined process, ligation is immediately followed by restriction enzyme digestion (i.e., without gel extraction), and undesired products are purified using a second restriction enzyme instead of gel extraction.

[0314] The process described in this example requires two different restriction enzymes: a first restriction enzyme that cuts at two sites between the plasmid backbone and the desired DNA sequence, and a second restriction enzyme that cuts at least once within the plasmid backbone to digest the plasmid backbone.

[0315] 1A-1E show such a dual-enzyme process. First, a plasmid DNA with two EcoRI restriction sites flanking the sequence of the closed circular DNA and two PvuII restriction sites within the plasmid DNA sequence is amplified in a rolling circle amplification reaction with Phi29. The sample is heat inactivated at 65°C. EcoRI is then added to the sample to excise the closed circular DNA insert of interest and separate it from the plasmid DNA backbone. The reaction is stopped by another heat inactivation step at 65°C. T4 ligase is then used to intramolecularly self-ligate both linear products, and another heat inactivation step is performed at 65°C. The sample is then contacted with PvuII to digest the plasmid backbone and leave the closed circular DNA intact. Gyrase may be used to supercoil the closed circular DNA, and an exonuclease, e.g., T5 exonuclease or Plasmid-Safe (Lucigen), may be used to purify the closed circular DNA from the plasmid backbone fragment.

[0316] Example 2. Process for generating and purifying closed circular DNA using a single Type IIS restriction enzyme that cuts two sites on the template DNA Therapeutic circular DNA vectors are generated from plasmid DNA using a single type IIS restriction enzyme that cuts two sites flanking the therapeutic sequence. Figures 2A-2F show such a single-enzyme process.

[0317] Plasmid DNA is used as a template. The plasmid contains the therapeutic sequence (shown in Figures 2A and 2B as the C3 region / payload) and the backbone sequence (shown in Figures 2A and 2B as the sequence with the origin of replication, resistance gene, and BsaI recognition site). Prior to restriction digestion, the template may be amplified by rolling circle amplification (e.g., using Phi29 polymerase).

[0318] Next, BsaI and ligase are added. The BsaI enzyme recognizes two BsaI recognition sites in the backbone and cleaves the template (which can be a circular template or an amplified concatemer obtained by rolling circle amplification) at a cleavage site between the therapeutic sequence and the recognition site, generating a linear therapeutic fragment and a linear backbone fragment. The linear therapeutic fragment has the therapeutic sequence and the linear backbone fragment has the backbone sequence and two BsaI recognition sites. Upon ligation, the linear therapeutic fragment circularizes into a therapeutic circular DNA vector as shown in Figures 2C and 2D, and the linear backbone fragment circularizes as shown in Figures 2E and 2F. Because the circular backbone fragment has a BsaI site and ligation occurs in the presence of the BsaI enzyme, BsaI can cleave the backbone but not the therapeutic circular DNA vector, driving the reaction to generate purer therapeutic circular DNA. Exonuclease is added to digest the remaining linear backbone, and gyrase is added to supercoil the therapeutic circular DNA vector.

[0319] Example 3. Process for generating and purifying closed circular DNA using a single Type IIS restriction enzyme that cuts four sites on the template DNA Therapeutic circular DNA vectors were generated from plasmid DNA using a single type IIS restriction enzyme to digest the plasmid backbone, (1) cutting two sites flanking the desired DNA sequence and (2) cutting twice within the plasmid backbone.

[0320] Figures 3A-3D show such a single-enzyme process. First, a plasmid DNA with two BsaI restriction sites flanking the sequence of the closed circular DNA and two PvuII restriction sites within the plasmid DNA sequence was amplified in a rolling circle amplification reaction with Phi29. The sample was heat inactivated at 65°C. BsaI was then added to the sample to cleave four sites within each amplicon to excise the closed circular DNA insert of interest, separate it from the plasmid DNA backbone, and digest the plasmid backbone. The reaction was stopped by heat inactivation at 80°C. T4 ligase was added to self-ligate the closed circular DNA. During the self-ligation of the closed circular DNA, the fragments of the plasmid DNA self-ligate. Therefore, after heat inactivation of the ligation reaction at 65°C, a final BsaI digestion step was performed to digest the plasmid backbone. Gyrase was then used to supercoil the closed circular DNA, and Plasmid-Safe was used to purify the closed circular DNA from the plasmid backbone fragments.

[0321] Figures 4A-4C show three closed circular DNA constructs generated as described above. Construct 1103 is a 1431 bp single transcription unit (TU) vector containing a single CMV promoter (pCMV), transgene, and polyA tail. Construct 1147 is a 6293 bp compound TU vector containing four transgenes each flanked by a promoter and polyA tail. Construct 1258 is a 5065 bp polycistronic vector containing three transgenes flanked by a single promoter and a single polyA tail.

[0322] Various reaction conditions were tested with the three constructs. Animal-free BsaI (New England Biolabs) was compared to standard BsaI. In addition, random hexamer primers were tested in parallel with the specific primers, but primer amounts were not equal (more random primers than specific primers were used). A digestion control was included in which a Pvull cleavage site was included within the closed circular DNA transgene (lanes 7 and 10). Table 1 below describes the reaction conditions for each well. [Table 1]

[0323] A simulated gel showing the theoretical bands after the digestion step is shown in Figure 5B and its corresponding real gel is shown in Figure 5A. Simulation lane 1 corresponds to the expected band pattern in lanes 1-4 of the real gel, simulation gel lane 2 corresponds to the expected band pattern in lanes 5, 6, 8, and 9 of the real gel, simulation gel lane 3 corresponds to the expected band pattern in lanes 7 and 10 of the real gel, and simulation gel lane 4 corresponds to the expected band pattern in lanes 11 and 12 of the real gel. All band patterns appeared to be as expected.

[0324] A gel showing the banding pattern after in vitro ligation is shown in Figure 5C. Ligation intermediates were observed in each well.

[0325] FIG. 5D shows the banding patterns after exonuclease digestion. Lanes 1-4 each had a single prominent band corresponding to a DNA size of 1431 bp, the expected size of the closed circular DNA of sample 1103. Lanes 5 and 6 each had a single prominent band corresponding to a DNA size of 6293 bp, the expected size of the closed circular DNA of sample 1147. Lanes 11 and 12 each had a single prominent band corresponding to a DNA size of 5065 bp, the expected size of the closed circular DNA of sample 1258. The digestion controls in lanes 7 and 10 did not have a single prominent band, as expected due to the presence of a PvuII cut site within the closed circular DNA transgene.

[0326] Example 4. Streamlined process for the generation and purification of c3DNA using a single Type IIS restriction enzyme As described in Example 3, closed circular DNA was generated from plasmid DNA using a single Type IIS restriction enzyme (referred to in this example as "Condition 1") and compared to a streamlined variant process in which BsaI restriction digestion was combined with a ligation step ("Condition 2"). Conditions 1 and 2 are shown in FIG. 6.

[0327] A streamlined process was achieved using a type IIS restriction enzyme (BsaI) by utilizing its ability to cleave outside of its recognition sequence. Applicants utilized this property to ensure that upon self-ligation of the closed circular DNA, the recognition site was not present in the closed circular DNA. An exemplary template plasmid DNA vector for such a process is shown in Figures 7A and 7B. Upon self-ligation of the plasmid backbone, the BsaI site is confined to the backbone by-product, which results in enzymatic cleavage of the plasmid backbone. As a result, the combined digestion / ligation reaction proceeds to digest the plasmid backbone while the self-ligated closed circular DNA accumulates without further digestion.

[0328] This streamlined process eliminates the need to heat inactivate the restriction digest prior to ligation. Instead, heat inactivation was performed after ligation. By increasing the reaction temperature to 65°C, which is sufficient to inactivate T4 ligase but not enough to inactivate BsaI, T4 ligase was inactivated while leaving BsaI activity intact.

[0329] In this experiment, random primers and animal-free BsaI were used. Heat inactivation was performed for 10 min, followed by the addition of DTT to a final concentration of 1 mM and T5 exonuclease. Figure 8A shows a simulation of the band pattern, and Figure 8B shows the actual gel. Lanes 1-3 contain products from condition 1, while lanes 4-6 contain products from condition 2. Lanes 1 and 4 contain construct 1103 (1431 bp), lanes 2 and 5 contain construct 1147 (6293 bp), and lanes 3 and 6 contain construct 1258 (5065 bp). 1Kb Plus DNA ladder (left marker, Invitrogen, Waltham, MA) and supercoiled DNA ladder (right marker, New England Biolabs, Ipswich, MA) were used. Both conditions resulted in the generation of c3DNA of the correct size for all three constructs tested. Thus, by utilizing the unique properties of type IIS restriction enzymes such as BsaI, it was confirmed that pre-supercoiled c3DNA generation was performed in a single reaction vessel without buffer exchange, making c3DNA generation simpler and enabling higher throughput.

[0330] Example 5. Cell-free generation of c3DNA using a single restriction digest A. Method The following reagents were mixed in 1x Phi29 buffer (New England Biolabs) to prepare a rolling circle amplification (RCA) solution: plasmid DNA (5 μg / mL final concentration); random primers (50 μM final concentration); NaOH (10 mM final concentration); dNTPs (2 mM final concentration); bovine serum albumin (BSA) (0.2 mg / mL final concentration); Phi29 DNA polymerase (200 U / mL final concentration); and pyrophosphatase stock solution (New England Biolabs; 0.4 mU / mL final concentration). The RCA solution was kept mixing for 18 hours at 30°C.

[0331] After incubation, the RCA solution was heat inactivated for 45 minutes by increasing the temperature to 65° C. The temperature of the inactivated RCA solution was then reduced to 25° C.

[0332] To make the BsaI solution, the inactivated RCA solution (0.2 mg DNA / mL final concentration) was added to rCutSmart buffer (New England Biolabs; 1x final concentration) containing BsaI (2.5 U / μg DNA final concentration). The BsaI solution was kept mixing for 2 hours at 37°C. No heat inactivation was performed on the BsaI solution. The temperature of the digested BsaI solution was reduced to 25°C.

[0333] To make the ligation solution, the digested BsaI solution (final concentration of 40 μg DNA / mL) was added to rCutSmart buffer containing T4 ligase (10 U T4 ligase per μg DNA) and riboATP (final concentration of 1 mM). The ligation solution was incubated at 25° C. for 2 hours.

[0334] After incubation, the ligation solution was heat inactivated for 45 minutes by increasing the temperature to 65° C. The temperature of the inactivated ligation solution was then reduced to 37° C.

[0335] To make the supercoiling solution, the ligation solution was added to a gyrase buffer containing DNA gyrase (1.5 U of gyrase per μg of DNA). The gyrase buffer contains 35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl, 1 mM ATP, 2 mM DTT, 1.8 mM spermidine, 6.5% glycerol (w / v), and 100 μg / mL BSA. The supercoiling solution was kept mixing for at least 2 hours at 37° C. No heat inactivation was performed on the supercoiling solution.

[0336] The supercoiling solution was then added to potassium acetate buffer (50 mM final concentration of potassium acetate) containing T5 exonuclease (2.5 U T5 exonuclease per μg DNA) to create the cleanup solution. The cleanup solution was kept mixing for at least 2 hours at 37° C. No heat inactivation was performed on the cleanup solution.

[0337] The cleanup solution was then sterile filtered through a 0.22 μm filter and diluted 1:1 in a buffer containing 1.5 M NaCl, 100 mM MOPS, 30% isopropyl alcohol (IPA), and 0.3% Triton X-100 (v / v) to give a final concentration of 750 mM NaCl, 50 mM MOPS, 15% isopropyl alcohol (IPA), and 0.15% Triton X-100 (v / v). The diluted cleanup solution was applied to a Qiagen plasmid prep column, the DNA was washed with QC buffer, and the DNA was eluted with QN buffer. The eluate was diluted with IPA (40 v / v%) and centrifuged at 15,000 g for 30 min at 4° C. The pellet was washed with 70% EtOH and centrifuged again at 15,000 g for 30 min at 4° C. After the second centrifugation, the pellet was resuspended in PBS at a concentration of 1.0 mg / mL to 2.0 mg / mL.

[0338] For the scaled-up c3DNA production described below, supercoiled monomers were calculated by densitometric analysis of electrophoretic gel samples using Image Lab software (BIO-RAD®). 200 ng of c3DNA samples were loaded onto a Tris-Acetate-EDTA gel and electrophoresis was performed at 110V for 40 min, followed by staining with 1% EtBr for 20 min. For each c3DNA sample, target bands were identified by their size compared to the supercoiled ladder and the "Band Detection Sensitivity" in "Detection Settings" was set to a value of 50 as "Custom Sensitivity".

[0339] B. Results Generation of 12.75kb c3DNA The method described above was adapted for bench-scale production of a 12.75 kb c3 DNA construct starting from 0.15 mg template, where two lots of gyrase were tested. Figure 9 shows the bands corresponding to the 12.75 kb c3 DNA recovered after T5 exonuclease digestion for both gyrase lots (lanes 1 and 2).

[0340] Large-scale generation of c3DNA One advantage of the streamlined cell-free processes of the embodiments disclosed herein, e.g., those that do not include intermediate gel extraction, is that they are suitable for scalability to large-scale production of therapeutic circular DNA vectors (i.e., producing at least 2 mg of circular DNA vector per batch). The reaction volume in the large-scale production method can be at least 100 mL, at least 1 L, at least 10 L, at least 100 L, at least 500 L, or more, and the yield of therapeutic circular DNA vector can be at least 2 mg per liter of initial reaction volume (e.g., rolling circle amplification solution).

[0341] The method described in Example 5A above was scaled up to generate larger quantities of c3DNA with various therapeutic sequences and differing in size, number of transcription units, and number of regulatory elements. An exemplary large-scale production involves a volume of 150 mL of RCA solution, a volume of 500 mL of BsaI solution, a volume of 2.5 L of ligation solution, a volume of about 3.2 L of supercoiling solution, a volume of about 3.2 L of cleanup solution, and a volume of 2-20 mL of c3DNA product.

[0342] The amount of c3DNA in each lot after a single purification step exceeded 2 mg, representing at least a three-fold increase in the amount of c3DNA product compared to the plasmid DNA vector from which it was generated. Each lot of c3DNA was confirmed to have in vitro protein expression (data not shown), endotoxin levels below 0.5 EU / mL, and a supercoiled monomer percentage above 70%. [Table 2]

[0343] Example 6. Effect of different ligation conditions Ligation reactions were performed on linear DNA after digestion with restriction enzymes. The total DNA concentrations in the reactions ranged from 40 ng / μL to 100 ng / μL. As shown in FIG. 10, 20 ng / μL of plasmid DNA (pDNA) was treated with ligase as a reference (lanes 2 and 3), and 20 ng / μL of closed circular DNA was treated with ligase as a reference (lanes 4 and 5). Lanes 6 and 7 show 40 ng / μL of linear DNA, lanes 8 and 9 show 100 ng / μL of linear DNA, and lanes 10 and 11 show 40 ng / μL of linear DNA without buffer. As shown in lanes 8 and 9, a large undesired band appears, indicating undesired intermolecular ligation. In contrast, lanes 6 and 7 show less undesired products, indicating that reducing the linear DNA concentration reduced (undesired) intermolecular ligation and increased (desired) intramolecular self-ligation.

[0344] Figure 11 shows another ligation experiment in which the amount of ligase enzyme was varied. On the left side of the gel are control reactions of markers and linear DNA previously treated with restriction enzyme. Lanes 1-3 show 20 μg / mL DNA treated with 100 U / μg ligase, 20 U / μg ligase, and 5 U / μg ligase, respectively. Lanes 4-6 show 40 μg / mL DNA treated with 100 U / μg ligase, 20 U / μg ligase, and 5 U / μg ligase, respectively. Lanes 7-9 show 100 μg / mL DNA treated with 100 U / μg ligase, 20 U / μg ligase, and 5 U / μg ligase, respectively. Samples 4-9 were diluted to 20 μg / mL before loading the samples so that lanes 1-9 all contained the same amount of total DNA. As shown in lanes 3, 6, and 9, reducing the ligase concentration to 5 U / μg ligase resulted in the greatest amount of the desired intramolecular ligation product, shown as the band of interest.

[0345] Example 7. Comparison of ligase compositions In this study, synthetic C 3 Three different ligase enzymes (T3, T4, and T7) were purchased from New England Biolabs for comparison as reagents for DNA purification. BsaI digestion was carried out for 3 hours 42 minutes at a BsaI concentration of 2.5 U BsaI per μg DNA (500 U / mL). Ligation reactions were carried out on BsaI-digested DNA samples (construct size of 9,542 bp) in rCutSmart® buffer containing ATP (1 mM) and no polyethylene glycol (PEG). The conditions for each sample are summarized in Table 3 below. [Table 3]

[0346] For each sample, the ligase reaction was carried out over various time courses and after ligation was completed the samples were subjected to enzyme heat inactivation. Samples were collected at various time points and for analysis after gyrase treatment and subsequent T5 exonuclease digestion.

[0347] Figure 13 shows the gel profile of each sample after ligation. The band of the therapeutic vector of interest (C3DNA) is shown in the black box. Samples 1-5 (T3 ligase and T4 ligase) showed similar gel profiles, while samples 6 and 7 (T7 ligase) showed fewer bands and the band of interest was nearly invisible.

[0348] Figures 14 and 15 show gel profiles of time course experiments with T4 ligase (Figure 14) and T3 and T7 ligases (Figure 15). The results are presented as curves in Figure 16, showing the ligation kinetics for each sample as a decrease in linear DNA over time. This study suggests that T4 ligase showed the fastest ligation kinetics, while T7 ligase showed the least ligation activity. T3 showed effective ligase activity, albeit with slower kinetics than T4 ligase. Taken together, these results indicate that both T3 and T4 were suitable ligases for self-ligation of synthetic circular DNA.

[0349] Example 8. Maintaining high efficiency ligation through streamlined modifications In an attempt to improve manufacturing efficiency by reducing the reaction volume and decreasing the process time, applicants systematically studied the impact on product yield of two process modifications: (1) increasing the DNA concentration in the ligation reaction, and (2) removing the post-ligation heat inactivation. Specifically, applicants attempted to (1) reduce the ligation reaction volume by increasing the concentration of DNA in a smaller ligation reaction volume, and (2) speed up the process by removing the post-ligation heat inactivation step (a time-consuming step that can take up to 2 hours). Each of these two process modifications was expected to have a negative impact on the yield of ligated DNA. Surprisingly, the application of both of these process modifications did not have a significant negative impact on the yield. This indicates that a process incorporating both modifications can greatly improve manufacturability without sacrificing efficiency. Details of the study are provided below.

[0350] In the first experiment of this study, the effect of post-ligation heat killing was evaluated on constructs of different sizes: 5,065 kb and 8,656 kb (SEQ ID NO: 1). In summary, the generation was performed as follows: DNA was amplified using Phi29 rolling circle amplification for 18 hours 14 minutes starting with plasmid DNA at a concentration of 5 μg / mL, Phi29 at 200 U / mL, and dNTPs at 2 mM. BsaI digestion was performed for 2 hours 10 minutes using BsaI at a concentration of 2.5 U / ug (500 U / mL), and ligation was performed on 40 μg / mL DNA using T4 ligase at 10 U / ug. Post-ligation heat inactivation was performed only for the samples indicated. All samples were then supercoiled using gyrase, followed by T5 exonuclease digestion and purification according to the method described above. Each construct was tested both with and without heat killing immediately after ligation, and samples were tested in duplicate. Sample identification information is summarized in Table 4 below. [Table 4]

[0351] Figure 17 is a gel showing the banding patterns after ligation and before heat killing. Bands of interest are indicated by boxes. As expected, no differences were observed between the types of constructs.

[0352] The post-gyrase gels are shown in Figure 18A for samples 1-4 and Figure 18B for samples 5-8, and the post-exonuclease gels are shown in Figure 19A for samples 1-4 and Figure 19B for samples 5-8. The post-purification gel results were quantified and are shown in Table 5 below and Figure 20. The yields were C 3 The mass of the total DNA product was calculated by dividing it by the mass of the total DNA after BsaI digestion. [Table 5]

[0353] The elimination of heat killing had no adverse effect on yield. In fact, yields were unexpectedly improved in all samples in which heat killing was eliminated. Specifically, the yield of the 5,065 bp construct improved by 59% and the yield of the 8,656 bp construct improved by 91% (averaged across duplicates). This result supports the conclusion that synthetic C 3 We show that the elimination of heat killing from the DNA production process can improve manufacturing efficiency by meaningfully reducing process time (previously heat killing took 1.5-2 hours).

[0354] Next, the effect of increasing DNA concentration (enhanced ligation) in the ligation reaction was evaluated using the 8,656 kb construct as a model construct. The relative order of supercoiling by gyrase and exonuclease digestion by T5 exonuclease (i.e., gyrase before T5 exonuclease vs. T5 exonuclease before gyrase) was also compared for each DNA concentration. Other conditions (amplification, BsaI digestion, and ligation) were the same as for the heat death exclusion experiment, except for the DNA concentration at ligation. The conditions for each sample are shown in Table 6 below. DNA was diluted to each given concentration from 133 μg / mL, which was the DNA concentration immediately after BsaI digestion as measured by Qubit. [Table 6]

[0355] Purification was carried out on the samples up to gyrase / exonuclease treatment and the samples were run on a gel before purification. The percentage supercoiled monomer for each sample measured after exonuclease / before purification is shown below in Table 7. [Table 7]

[0356] As shown in the gel profiles (Figure 21) and relative quantification of yields (Figure 22), doubling the DNA concentration from 40 to 80 μg / mL during the ligation step had only a minor impact on yield and purity compared to sample 1, whereas a second doubling of the DNA concentration to 160 μg / mL had a larger (negative) impact on yield (see samples 6 and 7 in Figure 22). Furthermore, placing T5 exonuclease digestion before gyrase had a minor impact on yield (Figure 22) while increasing purity (Table 7).

[0357] Downstream purification was then performed on selected samples to produce drug substance. Elimination of heat inactivation resulted in a large increase in drug substance yield, as shown in the gel profile (Figure 23) and relative quantification of yield (Figure 24). By performing a T5 exonuclease step prior to gyrase-mediated supercoiling, the previous purity was maintained while completely removing low molecular weight species.

[0358] Taken together, these results surprisingly show that purity and yield can be maintained despite (a) increasing DNA concentration in the ligation reaction to reduce the required reaction volume, and (b) eliminating the time-consuming heat inactivation step after ligation. Thus, a process involving these modifications (and optionally performing exonuclease digestion prior to supercoiling) offers manufacturability advantages (e.g., smaller reactors and shorter processing times required, as well as the ability to use disposable vessels that are not compatible with heat killing) without sacrificing product quality, representing a major improvement in synthetic DNA manufacturing.

[0359] Example 9. Improving gyrase efficiency In the process described above, which included supercoiling prior to exonuclease digestion, the lowest concentration of gyrase used was 1.5 U / ug. This example describes a titration experiment aimed at determining whether lower concentrations of gyrase would be possible in light of new process modifications, such as implementing exonuclease digestion prior to supercoiling.

[0360] The production was carried out as follows: Phi29 rolling circle amplification was used to generate amplified DNA using a plasmid DNA starting concentration of 5 μg / mL, a Phi29 concentration of 200 U / mL, a dNTP concentration of 2 mM, and a time of 18 hours 49 minutes. BsaI digestion was carried out for 4 hours 5 minutes using a BsaI concentration of 2.5 U / μg DNA (500 U / mL) and a DNA concentration of 200 μg / mL. Ligation was carried out on 80 μg / mL DNA using 10 U / ug T4 ligase. No heat inactivation was performed after ligation. Instead, T5 exonuclease was added immediately after ligation at a concentration of 2.5 U / ug DNA. Three concentrations of gyrase were then tested: 1.5 U / ug, 1.0 U / μg, and 0.5 U / μg. Results were observed by gel electrophoresis (relative quantification and average corrected concentrations by Qubit).

[0361] Notably, no major changes in the intensity or purity of the desired product were observed with decreasing gyrase concentrations (Figure 25 and Table 8). In addition, minor changes in the intensity of the undesired bands were observed across the three samples (Figure 25), suggesting minor impact of lower gyrase concentrations on product quality. [Table 8]

[0362] Importantly, these results suggest that when exonuclease digestion is performed prior to supercoiling, process efficiency can be significantly improved by reducing the minimum effective amount of gyrase used.

[0363] Example 10. Effect of Type IIS overhang sequence and number of cleavage sites In the previous examples, overhang sequence AAAA was used as the BsaI overhang sequence adjacent to the therapeutic sequence. Overhang sequence AAAA was selected due to its low efficiency, which, without being bound by theory, was hypothesized to be advantageous in favor of favoring kinetics toward intramolecular ligation (self-ligation (desired)) rather than intermolecular ligation (ligation with another therapeutic sequence (undesired)). However, the Type IIS restriction process described herein allows for the selection of desirable overhang sequences. Applicants therefore tested a second overhang sequence, AACC.

[0364] This study also included an assessment of the impact of the number of BsaI cleavage sites in the template plasmid (i.e., whether the template plasmid had only two BsaI cleavage sites flanking the therapeutic sequence or, alternatively, more than two BsaI cleavage sites, where additional cleavage sites were present in the backbone).

[0365] Constructs with two different sizes were tested, and the experimental design is shown in Table 9 below. [Table 9]

[0366] In this experiment, the purification was generally carried out as follows: Phi29 amplification (starting DNA concentration of 5 μg / mL, Phi29 concentration of 200 U / mL, dNTP concentration of 2 mM, and primer concentration of 50 μM, taking 18 hours 55 minutes) → BsaI digestion (2.5 U / ug (500 U / mL), starting DNA concentration of 200 μg / mL, taking 4 hours 18 minutes) → ligation (DNA concentration of 40 μg / mL, T4 ligase concentration of 10 U / ug DNA) → heat killing → supercoiling → T5 exonuclease digestion → column purification. Samples were stopped at various points during the BsaI digestion step, ligation step, and T5 exonuclease step to compare reaction rates between samples. After purification was completed, the percentage of supercoiled monomer in the final product was quantified using the gel analysis method described above.

[0367] The results of the BsaI digestion time course experiment show similar banding profiles and intensities between the 30, 60, and 120 minute time points (Figure 26). No differences in digestion kinetics were observed from this gel. These results indicate that the reaction with BsaI is highly efficient and occurs primarily within the first 30 minutes of the reaction.

[0368] The results of the ligation time course experiment (Figures 27A and 27B) show that the construct with two BsaI cleavage sites and AACC overhangs showed a stronger desired monomer band (indicated by white arrow) at 18 hours (Figure 27A). Sample 5 showed high ligation efficiency with similar banding profiles between 1 hour, 3 hours, and 18 hours (Figure 27A). Sample 4 showed similar banding profile and ligation efficiency as Sample 3 (Figure 27A). Samples 1, 3, and 4 showed high ligation efficiency from 3 hours to 24 hours. 3Enhancement of DNA monomer band intensity was observed (FIG. 27B). Samples with AACC overhangs resulted in faster self-ligation kinetics compared to AAAA samples 1, 3, and 4 for both construct sizes. No differences were visually observed between the 3 and 24 hour time points for any samples with AACC overhangs. These results indicate that the AACC overhangs resulted in substantially faster self-ligation kinetics, which may explain the reduction in overall process time and C 3 This may allow for increased DNA monomer yields.

[0369] The effect of the number of cleavage sites was also observed, although to a lesser extent than the overhang sequence. The lower band in the white box of sample 4 in Figure 27B represents the 8.4 kb linear C 3 The lower band corresponds to DNA. In sample 4 (AAAA with two cleavage sites), the intensity of the lower band decreases as the reaction progresses from 3 hours to 18 hours to 24 hours. In contrast, the corresponding band in sample 3 (AAAA with five cleavage sites) remained unchanged throughout the 24 hour time course. These results indicate that the reaction kinetics of sample 4 was faster than that of sample 3. In sample 5 (AACC with two cleavage sites), the corresponding band was not visible at any time point, indicating faster self-ligation kinetics than either sample 3 or 4.

[0370] Table 10 below shows the C 3 Relative quantification of DNA monomer bands is shown. Relative quantification was performed using the five cleavage site reference sequences as a reference. 3 Runs were performed on the DNA monomer band (sample was a reference for sample 2, sample 3 was a reference for samples 4 and 5). [Table 10]

[0371] These results indicate that during the first 18 hours of the ligation reaction, (i) the reduction to two BsaI cleavage sites was 3(ii) confirming the visual observation that changing the overhang from AAAA to AACC greatly promoted self-ligation formation.

[0372] FIG. 28A shows the gel profiles of samples 1-5 after T5 exonuclease treatment, where sample 2 (AACC with two cleavage sites) shows a significantly stronger C cleavage than sample 1 (AAAA with five cleavage sites). 3 The DNA monomer bands (the darkest bands in each lane) were not significantly different visually between samples 3-5. Relative quantification was performed on the post-exonuclease samples, as shown in Table 11 below. References were made as above. [Table 11]

[0373] The AACC overhang sequence resulted in higher yields compared to the AAAA overhangs in both constructs.

[0374] Results were further quantified by Qubit at post-exonuclease time points, with different operators yielding results for each replicate (Figure 28B). Results were comparable between operators, demonstrating reproducibility. Similar to gel quantification, Qubit results also showed that changing the overhang from AAAA to AACC resulted in substantially higher counts after 18 hours of exonuclease digestion. Additionally, reducing the number of BsaI cleavage sites from 5 to 2 (sample 4 vs. sample 3) without changing the overhang resulted in comparable counts as measured by Qubit.

[0375] FIG. 29 shows Qubit results after 18 hours of exonuclease treatment, reproduced by two operators, A and B. Without being bound by theory, the decrease in counts over time reflects the consumption of non-supercoiled DNA (linear and nicked DNA) into nucleotides by the exonuclease. The trend line suggests that the rate of exonuclease digestion slowed (or plateaued) between 3 and 18 hours (36.7%-48.7% (with one outlier) decrease in counts within the first 3 hours vs. over 88% decrease by 18 hours). The total decrease in counts over 18 hours of exonuclease digestion across all operators is shown below in Table 12. [Table 12]

[0376] Final product yields were determined for two operators and the average and standard deviation for each sample are shown in Table 13 below. [Table 13]

[0377] For both construct sizes, the C was significantly higher in samples with AACC overhangs with two cleavage sites compared to AAAA overhangs with five cleavage sites. 3 The total yield of DNA product was increased, an effect that was more pronounced in the 10,927 bp construct compared to the 8,425 bp construct (30% increase versus 4.4% increase).

[0378] Taken together, these results indicate that the AACC overhang sequence 3 The DNA contains a C 3 These results show that, compared to DNA, it can be produced with unexpectedly faster kinetics and improved product yields. Reducing the number of cleavage sites may also improve manufacturability, although the effect of this modification did not appear to be as great as the AACC overhang in this experiment.

[0379] Example 11. Head-to-head comparison of AAAA+ 5 cleavage sites vs. AACC+ 2 cleavage sites under various conditions In this experiment, two constructs of different sizes were generated by two different restriction procedures, and each restriction procedure was tested under four different conditions. The two constructs were CpG1 and CpG2 of 8,656 bp ("8.7 kb construct") and 10,300 bp ("10.3 kb construct"). 3 The sizes of the DNA vectors correspond to those shown. Both constructs have a CAG promoter and ABCA4 coding sequence, with the 10.3 kb construct containing additional regulatory elements downstream of the ABCA coding sequence (Figure 30). Each construct was made with two restriction treatments: (1) a BsaI overhang of AAAA combined with four backbone fragments (AAAA) and (2) a BsaI overhang of AACC combined with one backbone fragment (AACC). The constructs and restriction treatments are shown in Figure 30. Plasmid maps of the 8.7 kb construct with AAAA restriction treatment (Figure 31; SEQ ID NO:2) and the 8.7 kb construct with AACC restriction treatment (Figure 32; SEQ ID NO:4) are shown. The final therapeutic circular C 3 The nucleic acid sequence of the DNA vector is shown.

[0380] The four conditions were: (1) Phi29 amplification → BsaI digestion → ligation (40 μg / mL T4 ligase) → heat killing → supercoiling → T5 exonuclease digestion → column purification; (2) Phi29 amplification → BsaI digestion → ligation (80 μg / mL T4 ligase) (no heat killing) → supercoiling → T5 exonuclease digestion → column purification; (3) Phi29 amplification → BsaI digestion → ligation (40 μg / mL T4 ligase) (no heat killing) → T5 exonuclease digestion → supercoiling → column purification; and (4) Phi29 amplification → BsaI digestion → ligation (40 μg / mL T4 ligase) (no heat killing) → supercoiling → T5 exonuclease digestion → column purification.

[0381] For each condition, Phi29 amplification was performed with starting plasmid DNA at a concentration of 5 μg / mL (90 μg starting plasmid DNA in each sample), 50 μM random hexamer primers, 2 mM dNTPs, 200 U / mL Phi29 polymerase for approximately 19 hours, BsaI digestion was performed with a BsaI concentration of 2.5 U / ug DNA (500 U / mL) for approximately 3 hours, supercoiling was performed with 1.5 U of gyrase per μg DNA, DNA concentrations ranging from 3 to 10 μg / mL for 4 hours, and T5 exonuclease digestion was performed with 2.5 U of T5 exonuclease per μg DNA for approximately 18 hours.

[0382] In addition to the differences above, condition 4 included a smaller amplification step (⅓ the amount of DNA template at the start of amplification) compared to conditions 1-3, and included alternative buffer conditions (conditions 1-3 included buffers as described in Example 5). Samples were taken after the ligation, gyrase, and T5 exonuclease steps and run on a gel to quantify yields.

[0383] 8.7kb construct Gel profiles of the 8,656 bp construct at end of ligation (EOR) are shown in Figure 33, gyrase EOR under conditions 1, 2, and 4 in Figure 34, T5 exonuclease EOR under condition 3 in Figure 35, and T5 exonuclease EOR under conditions 1, 2, and 4 as well as gyrase EOR under condition 3 in Figure 36. At each EOR, the band of interest at AACC appears to be more intense compared to AAAA under all conditions (Figures 33-36). Band intensities are quantified in Figure 36 and yields are shown in Table 14 below. [Table 14]

[0384] Yield results were also quantified for each sample with the Qubit assay. The mass values ​​quantified by Qubit were multiplied by the band % in Table 14 to calculate the improvement (of the desired product) as shown in Table 15 below. [Table 15]

[0385] As shown in Table 15, the yield improvement was also captured by the Qubit assay, with improvements to a similar extent between the AAAA and AACC restriction treatments. The AACC restriction treatment produced significantly less yield of the desired product (C) in all four conditions. 3 Overall, AACC showed a 20-40% yield improvement compared to AAAA in all conditions.

[0386] 10.3kb construct Gel profiles of the 10.3 kb construct of ligase EOR are shown in Figure 37, gyrase EOR under conditions 1, 2, and 4 in Figure 38, T5 exonuclease EOR under conditions 1, 2, and 4 as well as gyrase EOR under condition 3 in Figure 39. In general, the band of interest for AACC appears to be stronger than AAAA. Band intensities are quantified in Figure 39 and yields are shown in Table 16 below. [Table 16]

[0387] A Qubit assay was run on each of the 10.3 kb samples. The mass values ​​quantified by Qubit were multiplied by the % bands in Table 16 to calculate the improvement (of the desired product) as shown in Table 17 below. [Table 17]

[0388] As shown in Table 17, and similar to the 8.7 kb construct, improvements in 10.3 kb construct yield were also captured by the Qubit assay, with improvements in a similar range between AAAA and AACC restriction treatments. AACC restriction showed increased yield of the desired product (C3 DNA) in conditions 1-3.

[0389] The amount of DNA for each construct and condition was quantified at various time points along the production process. Each sample had an initial DNA content of 90 μg (plasmid DNA). 3 Measure DNA mass, C 3 To show the ratio of DNA product to initial amount, C 3 The DNA mass was divided by the initial plasmid DNA mass. The results are shown in Table 18 below. [Table 18]

[0390] Taken together, these results across both the 8.7 kb and 10.3 kb constructs and the various conditions described throughout this specification are consistent with the observations in Example 10, and demonstrate that restriction of AACC (combination of the AACC overhang with one backbone fragment) enhances C 3 It further suggests that DNA yields can be increased, providing an unexpected and useful improvement in the ease of manufacture of synthetic circular DNA.

[0391] Numbered Items 1. A method for generating a therapeutic circular DNA vector, comprising: (a) providing a sample comprising a template DNA vector comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) digesting the linear concatemer with a type IIS restriction enzyme that cleaves at a first site and a second site for each unit of the linear concatemer, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs, the digestion producing a linear therapeutic fragment and a linear backbone fragment, the linear therapeutic fragment comprising the therapeutic sequence, and the linear backbone fragment comprising at least a portion of the backbone sequence and a type IIS restriction site; (d) contacting the linear backbone fragment and the linear therapeutic fragment with a ligase to generate a circular backbone comprising the type IIS restriction site and a therapeutic circular DNA vector lacking a type IIS restriction site.

[0392] 2. The method of claim 1, wherein the type IIS restriction enzyme cleaves the circular backbone and does not cleave the therapeutic circular DNA vector.

[0393] 3. A method for generating a therapeutic circular DNA vector, comprising: (a) providing a sample comprising a template DNA vector comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) digesting the linear concatemer with one or more restriction enzymes that cleave at least a first site, a second site, and a third site for each unit of the linear concatemer, wherein (i) the first and second sites flank the therapeutic sequence and form self-complementary overhangs, and (ii) the third site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second sites, wherein the digestion produces a linear therapeutic fragment that comprises the therapeutic sequence and at least two linear scaffold fragments, each comprising a portion of the scaffold sequence; (d) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0394] 4. The method of claim 3, wherein the linear concatemer is digested with a single restriction enzyme that cuts the first site, the second site, and the third site.

[0395] 5. The method of claim 3, wherein the one or more restriction enzymes cleave the linear concatemer unit-by-unit at a fourth site, the fourth site being within the scaffold sequence and forming a non-complementary overhang at the first or second site, and the digestion produces at least three linear scaffold fragments, each of which contains a portion of the scaffold sequence.

[0396] 6. The method of claim 5, wherein the single restriction enzyme cleaves the linear concatemer unit-by-unit at a fourth site, the fourth site being within the scaffold sequence and forming a non-complementary overhang at the first or second site, and the digestion produces at least three linear scaffold fragments, each of which contains a portion of the scaffold sequence.

[0397] 7. The method according to any one of items 1, 2, 4, and 6, wherein the restriction enzyme is a type IIS restriction enzyme.

[0398] 8. The method according to item 7, wherein the type IIS restriction enzyme is BsaI.

[0399] 9. The method according to any one of items 1 to 8, wherein step (d) is not preceded by a restriction enzyme inactivation step.

[0400] 10. The method according to any one of items 1 to 9, wherein no temperature increase is performed between steps (c) and (d).

[0401] 11. The method according to any one of items 1 to 10, wherein steps (c) and (d) are carried out simultaneously.

[0402] 12. The method according to any one of items 1 to 11, further comprising increasing the temperature of the solution containing the therapeutic circular DNA vector to about 65°C.

[0403] 13. The method of any one of items 1 to 12, further comprising (e) contacting the therapeutic circular DNA vector with a topoisomerase or helicase.

[0404] 14. The method according to item 13, wherein step (e) is carried out at about 37°C.

[0405] 15. The method of any one of items 1 to 14, further comprising (f) contacting the linear backbone fragment with an exonuclease.

[0406] 16. The method according to item 15, wherein step (f) is carried out at about 37°C.

[0407] 17. (e) contacting the therapeutic circular DNA vector with a topoisomerase or helicase; 13. The method of any one of items 1 to 12, further comprising (f) contacting the linear backbone fragment with an exonuclease, wherein no enzyme inactivation step is performed between steps (e) and (f).

[0408] 18. The method according to item 17, wherein step (e) is carried out before step (f).

[0409] 19. The method according to any one of items 1 to 18, wherein the restriction enzyme is provided at a concentration of about 0.5 U / μg to about 20 U / μg.

[0410] 20. The method of claim 19, wherein the restriction enzyme is provided at a concentration of about 2.5 U / μg.

[0411] 21. The method according to any one of items 1 to 20, wherein step (c) comprises incubation for 1 to 12 hours.

[0412] 22. The method according to item 21, wherein step (c) comprises incubation for about 1 hour.

[0413] 23. The method according to any one of items 1 to 22, wherein the ligase is provided at a concentration of 20 U ligase per μg of DNA (U / μg) or less.

[0414] 24. The method according to any one of items 1 to 23, wherein the ligase is T4 ligase.

[0415] 25. The method according to any one of items 13 to 24, wherein the topoisomerase is provided at a concentration of 10 U of topoisomerase per μg of DNA (U / μg) or less.

[0416] 26. The method according to any one of items 13 to 25, wherein the topoisomerase is a type II topoisomerase.

[0417] 27. The method according to any one of items 13 to 26, wherein the topoisomerase is gyrase or topoisomerase IV.

[0418] 28. The method according to any one of items 15 to 27, wherein the exonuclease is provided at a concentration of about 0.5 U / μg to about 20 U / μg.

[0419] 29. The method according to any one of items 15 to 28, wherein step (f) is carried out two or more times.

[0420] 30. The method according to any one of items 15 to 29, wherein step (f) comprises incubation for 1 hour to 12 hours.

[0421] 31. The method according to any one of items 15 to 30, wherein the exonuclease is T5 exonuclease.

[0422] 32. (g) passing the therapeutic circular DNA vector through a column; and / or (h) The method according to any one of items 1 to 31, further comprising precipitating the therapeutic circular DNA vector with isopropyl alcohol.

[0423] 33. The method according to any one of items 1 to 32, wherein step (b) is carried out using a site-specific primer.

[0424] 34. The method according to any one of items 1 to 33, wherein step (b) is carried out using random primers.

[0425] 35. The method according to any one of items 1 to 34, wherein the amount of therapeutic circular DNA vector produced is at least 5-fold the amount of plasmid DNA vector in the sample of step (a).

[0426] 36. The method according to any one of items 1 to 35, wherein no DNA purification or gel extraction step is performed prior to step (d).

[0427] 37. The method according to any one of items 1 to 36, wherein the amount of the therapeutic circular DNA in the solution in step (d) is at least 2.0% by weight of the amount of the linear concatemers in step (b).

[0428] 38. The method according to any one of items 1 to 37, wherein the amount of the therapeutic circular DNA produced in step (d) is at least 1.0 mg.

[0429] 39. The method according to any one of items 1 to 38, wherein the concentration of the therapeutic circular DNA in the solution after step (d) is at least 5 μg / mL without any purification or concentration being performed.

[0430] 40. The method according to any one of items 1 to 39, wherein the volume of the solution in step (d) is at least 5 liters.

[0431] 41. The method according to any one of items 1 to 40, wherein steps (b) to (d) are carried out in a reaction vessel having a volume of at least 1 liter.

[0432] 42. The method according to any one of items 1 to 41, wherein the amount of the therapeutic circular DNA produced in step (d) is at least 5-fold the amount of the template DNA vector provided in step (a).

[0433] 43. A method for removing a backbone sequence from a DNA molecule to generate a therapeutic circular DNA vector, the DNA molecule comprising a backbone sequence and a therapeutic sequence, the method comprising: (a) digesting the DNA molecule with a type IIS restriction enzyme that cleaves at a first site and a second site per linear concatemer unit, the first and second sites being adjacent to the therapeutic sequence and forming self-complementary overhangs, the digestion producing a linear therapeutic fragment and a linear backbone fragment, the linear therapeutic fragment comprising the therapeutic sequence, and the linear backbone fragment comprising at least a portion of the backbone sequence and a type IIS restriction site; (b) contacting the linear backbone fragment and the linear therapeutic fragment with a ligase to generate a circular backbone comprising the type IIS restriction site and a therapeutic circular DNA vector lacking a type IIS restriction site.

[0434] 44. A method for removing a backbone sequence from a DNA molecule to generate a therapeutic circular DNA vector, the DNA molecule comprising a backbone sequence and a therapeutic sequence, the method comprising: (a) digesting the DNA molecule with one or more restriction enzymes that cleave at least a first site, a second site, and a third site per unit of the DNA molecule, wherein (i) the first and second sites flank the therapeutic sequence and form self-complementary overhangs, and (ii) the third site is within the scaffold sequence and forms an overhang that is non-complementary to the first or second sites, wherein the digestion produces a linear therapeutic fragment that comprises the therapeutic sequence and at least two linear scaffold fragments, each comprising a portion of the scaffold sequence; (b) contacting the linear therapeutic fragment with a ligase to generate a therapeutic circular DNA vector in solution.

[0435] 45. The method according to item 44, wherein the linear concatemer is digested with a single restriction enzyme that cuts the first site, the second site, and the third site.

[0436] 46. ​​The method of item 44, wherein the one or more restriction enzymes cleave the DNA molecule at a fourth site, the fourth site being within the scaffold sequence and forming a non-complementary overhang to the first or second site, and the digestion generates at least three linear scaffold fragments, each of which contains a portion of the scaffold sequence.

[0437] 47. The method of item 45, wherein the single restriction enzyme cleaves the DNA molecule at a fourth site, the fourth si...

Claims

1. 1. A method for generating a therapeutic circular DNA vector, comprising: (a) providing a sample comprising a template DNA vector comprising a therapeutic sequence and a backbone sequence; (b) amplifying the template DNA vector using polymerase-mediated rolling circle amplification to generate linear concatemers; (c) digesting the linear concatemers with a Type IIS restriction enzyme that cleaves at a first site and a second site for each unit of the linear concatemer, wherein the first and second sites flank the therapeutic sequence and form self-complementary overhangs, and wherein the digestion produces linear therapeutic fragments and linear backbone fragments, wherein the linear therapeutic fragments comprise the therapeutic sequence and the linear backbone fragments comprise the backbone sequence or a portion thereof; (d) contacting the linear backbone fragment and the linear therapeutic fragment with a ligase to generate a circular backbone and a therapeutic circular DNA vector lacking type IIS restriction sites; The method comprising:

2. 2. The method of claim 1, wherein the linear backbone fragment of (c) comprises a Type IIS restriction site and the circular backbone of (d) comprises the Type IIS restriction site, and the Type IIS restriction enzyme cleaves the circular backbone but does not cleave the therapeutic circular DNA vector.

3. The method described in claim 1, wherein the type IIS restriction enzyme further cleaves a third site for each unit of the linear concatemer, the third site being present within the backbone sequence and forming an overhang that is non-complementary to the first or second site.

4. 10. The method of claim 1, wherein the method further comprises diluting the DNA between steps (c) and (d).

5. 2. The method of claim 1, wherein the DNA concentration at the start of step (d) is greater than or equal to 20 μg / mL but less than 160 μg / mL.

6. The method of claim 1, wherein the ligase is T4 ligase.

7. 4. The method of claim 3, wherein the linear concatemers are digested with a single restriction enzyme that cuts the first site, the second site, and the third site.

8. 8. The method of claim 7, wherein the one or more restriction enzymes cleave the linear concatemer unit-by-unit at a fourth site, the fourth site being within the scaffold sequence and forming an overhang that is non-complementary to the first or second site, and the digestion produces at least three linear scaffold fragments, each comprising a portion of the scaffold sequence.

9. 9. The method of claim 8, wherein the single restriction enzyme cleaves the linear concatemer unit-by-unit at a fourth site, the fourth site being within the scaffold sequence and forming an overhang that is non-complementary to the first or second site, and the digestion produces at least three linear scaffold fragments, each comprising a portion of the scaffold sequence.

10. The method of claim 1, wherein the type IIS restriction enzyme is BsaI.

11. step (d) is not preceded by a restriction enzyme inactivation step, and / or No temperature increase is performed between steps (c) and (d); and / or step (d) is not immediately followed by a temperature increase; and / or Steps (c) and (d) are performed simultaneously; The method of claim 1.

12. 10. The method of claim 1, further comprising: (e) contacting the therapeutic circular DNA vector with a topoisomerase or helicase.

13. 10. The method of claim 1, further comprising: (f) contacting said linear backbone fragment with an exonuclease.

14. the therapeutic sequence comprises two or more transcription units, and / or the therapeutic sequence encodes one or more therapeutic proteins; The method of claim 1.

15. The method of claim 1 , wherein the therapeutic sequence encodes a therapeutic nucleic acid.

16. the therapeutic nucleic acid is an RNA molecule; Optionally, the RNA molecule is a self-replicating RNA molecule, a short hairpin RNA, or a microRNA.

16. The method of claim 15.

17. further comprising formulating the therapeutic circular DNA vector in a pharmaceutically acceptable carrier to produce a pharmaceutical composition; Optionally, the pharmaceutical composition comprises at least 1.0 mg of the therapeutic circular DNA vector in a pharmaceutically acceptable carrier; Optionally, the therapeutic circular DNA vector in the pharmaceutical composition is at least 70% supercoiled monomeric; and / or Optionally, the pharmaceutical composition contains no more than 1.0% residual protein or backbone sequence. The method of claim 1.

18. 18. The method of claim 17, wherein the pharmaceutical composition comprises a protein content of less than 1.0% by weight, an RNA content of less than 1.0% by weight, and an endotoxin content of less than 5 EU / mg.

19. 18. A pharmaceutical composition produced by the method of claim 17.

20. 20. The pharmaceutical composition of claim 19 for expressing a therapeutic sequence in an individual.

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