CLOSED-ENDED DNA VECTORS OBTAINABLE FROM CELL-FREE SYNTHESIS AND PROCESS FOR OBTAINING ceDNA VECTORS

Abstract

To provide closed-ended DNA vectors obtainable from cell-free synthesis, and to provide a process for obtaining ceDNA vectors.SOLUTION: The application describes methods for synthetic synthesis and cell-free synthesis of DNA vectors, particularly closed-ended DNA vectors (e.g., ceDNA vectors) having linear and continuous structure for delivery and expression of a transgene. The present invention relates to an in vitro process for production of closed-ended DNA vectors, corresponding DNA vector products produced by the methods and uses thereof, and oligonucleotides and kits useful in the process of the invention. DNA vectors produced using the methods described herein are free from unwanted side effects due to contaminants introduced during production in cell lines, for example, bacterial or insect cell lines.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 619,392, filed on January 19, 2018, under 35 U.S.C. § 119(e), the content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is being filed electronically in ASCII format and includes a sequence listing which is incorporated herein by reference in its entirety. The aforementioned ASCII copy, created on January 17, 2019, is named 080170 - 091310 - WOPT_SL.txt and is 102,804 bytes in size.

[0003] The present invention relates to the field of gene therapy, including the production of non - viral vectors for the purpose of expressing transgenes or isolated polynucleotides in a subject or cell. For example, the present disclosure provides a cell - free method for synthesizing non - viral DNA vectors. The present disclosure also relates to the nucleic acid constructs produced thereby and methods of using them.

Background Art

[0004] Gene therapy aims to improve the clinical outcome of patients suffering from either genetic mutations or acquired diseases caused by abnormalities in gene expression profiles. Gene therapy includes the treatment or prevention of defective genes, or medical conditions resulting from abnormal regulation or expression, such as underexpression or overexpression, that can lead to disorders, diseases, malignancies, etc. For example, diseases or disorders caused by defective genes can be treated, prevented, or ameliorated by delivering corrective genetic material to the patient, or by modifying or silencing the defective genes, such as by using corrective genetic material in the patient to effect a therapeutic expression of the genetic material in the patient's body.

[0005] The basis of gene therapy is to supply an active gene product (which may be referred to as a transgene) to a transcription cassette, which can, for example, result in a positive gain-of-function effect, a negative loss-of-function effect, or another outcome. Gene therapy can also be used to treat diseases or malignancies caused by other factors. Human single-gene disorders can be treated by the delivery and expression of a normal gene to target cells. The delivery and expression of a corrective gene in a patient's target cells can be carried out via many methods, including the use of engineered viruses and viral gene delivery vectors. Among the many virus-derived vectors that can be used (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated virus (rAAV) has gained popularity as a versatile vector in gene therapy.

[0006] Adeno-associated virus (AAV) belongs to the parvovirus family and more specifically constitutes the genus Dependoparvovirus. AAV-derived vectors (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for delivering genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons, (ii) they lack viral structural genes, thereby reducing host cell responses to viral infection, such as interferon-mediated responses, (iii) wild-type virus is considered non-pathogenic in humans, (iv) in contrast to wild-type AAV, which can integrate into the host cell genome, replication-deficient AAV vectors lack the rep gene and generally persist as episomes, thus limiting the risk of insertional mutagenesis or genotoxicity, and (v) AAV vectors are generally considered relatively poor immunogens and thus do not induce a significant immune response (see ii), thereby obtaining vector DNA for the therapeutic transgene and potentially long-term expression persistence.

[0007] However, the use of AAV particles as gene delivery vectors has several significant drawbacks. One major drawback associated with rAAV is its limited viral packaging capacity for heterologous DNA of approximately 4.5 kb (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), and as a result, the use of AAV vectors is restricted to protein coding capacities of less than 150,000 Da. A second drawback is that as a result of the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy need to be screened for the presence of neutralizing antibodies that eliminate the vector from patients. A third drawback relates to capsid immunogenicity that prevents readministration to patients who were not excluded from the initial treatment. The immune system in patients can be stimulated to generate high-titer anti-AAV antibodies that interfere with future treatment by reacting to vectors that function effectively as "booster" shots. Some recent reports have shown a relationship with immunogenicity in high-dose situations. Considering that single-stranded AAV DNA must be converted to double-stranded DNA before heterologous gene expression, another significant drawback is that the onset of AAV-mediated gene expression is relatively slow.

[0008] Additionally, conventional AAV virions with capsids are produced by introducing one or more plasmids containing the AAV genome, the rep gene, and the cap gene (Grimm et al., 1998). However, such encapsidated AAV viral vectors have been found to inefficiently transduce certain cell and tissue types, and the capsids also induce immune responses.

[0009] Therefore, the use of adeno-associated virus (AAV) vectors for gene therapy is restricted by single administration to patients (due to the patient immune response), the limited range of transgene genetic material suitable for delivery in AAV vectors due to the minimal viral packaging capacity (approximately 4.5 kb), and the slow AAV-mediated gene expression.

[0010] Closed - ended DNA vectors have been developed that can deliver one or more desired transgenes in vivo for therapeutic or other purposes and avoid the above - mentioned drawbacks of AAV and other viral vector systems. However, methods for producing such ceDNA vectors have relied on traditional bacterial or insect cell production methods. Such methods can introduce contaminants (e.g., nucleic acid contaminants) from the cells used to produce the vector, which are inconvenient or costly to remove and, if present in a ceDNA therapeutic formulation, can have undesirable side effects. Thus, there is a need for a field of technology that enables the generation of recombinant vectors for use in methods of controlling gene expression with minimal off - target effects, such as those introduced by such contaminants or other artifacts of purification methods. The methods provided herein mitigate or avoid such problems. Summary of the Invention Means for Solving the Problems

[0011] Conventional methods for producing viruses and virus-derived DNA typically use eukaryotic cells, such as mammalian or insect cells. One commonly used insect cell line is Sf9. However, these cells contain not only enzymes and other proteins that can have a harmful effect on the DNA being replicated, but also cellular nucleic acids introduced by the process of purifying the desired DNA from the cell lysate, the presence of which can make the purification of the desired DNA product more difficult. Furthermore, such impurities or contaminants can have a series of harmful and / or undesirable effects in the subject to whom the desired DNA is administered. Additionally, such traditional cell-based production methods can pose problems with respect to the amount of DNA vector product produced, and it is not uncommon for significant engineering of the cell line itself or the production technology to be required to produce a desirable yield. The techniques described herein relate to synthetic production methods that can readily produce DNA vectors containing closed-loop hairpin loops, such as, but not limited to, closed-ended DNA vectors (ceDNA vectors), with higher purity and in greater amounts than conventional means, and avoid the concerns detailed above.

[0012] The invention described herein provides a synthetic production method for producing closed-ended DNA vectors using a synthetic production system that can be a cell-free system. In some embodiments, the closed-ended DNA vector is a ceDNA vector that can be used in methods of controlling gene expression in a cell, tissue, or system, or for introducing new genetic material into a desired cell, tissue, or system. In one particular embodiment, the techniques described herein relate to a novel cell-free method of creating a DNA vector containing modified AAV inverted terminal repeats (ITRs) and, for example, one or more expressible transgenes. Using the methods disclosed herein, DNA vectors containing any closed-ended hairpin loop can be produced in a cell-free system, and herein refers to ceDNA vectors formed from single-stranded DNA (linear, continuous, and non-capsidated structures) having covalently closed ends.

[0013] One exemplary synthetic production method for generating a closed - ended DNA vector, illustrated using the production of the ceDNA vectors disclosed herein, relates to excising the entire molecule that forms the closed - ended DNA vector from a double - stranded DNA construct. In such embodiments, the double - stranded DNA construct is provided, in order from 5´ to 3´, with a first restriction endonuclease site, an upstream ITR, an expression cassette, a downstream ITR, and a second restriction endonuclease site. The double - stranded DNA construct is then contacted with one or more restriction endonucleases to generate a double - strand break at both of the restriction endonuclease cleavage sites. It is possible for one endonuclease to target both sites, or for each site to be targeted by a different endonuclease, so long as the restriction sites do not lie within the closed - ended vector template region. This results in the sequence between the restriction endonuclease sites being excised from the remaining double - stranded DNA construct. This excised molecule has free 5´ and 3´ ends, which are then ligated to form the ceDNA vector. In some embodiments, the excised molecule is first annealed to promote hairpin formation prior to ligation of the free 5´ and 3´ ends. In some embodiments, the unwanted double - stranded DNA construct backbone is cleaved by one or more restriction endonucleases specific for unique cleavage sites in the backbone, so that it is degraded during purification and more easily removed.

[0014] Another exemplary method of producing a DNA vector, e.g., a ceDNA vector, using the synthetic production methods disclosed herein involves the assembly of various oligonucleotides to form a complete vector. In such embodiments, in some embodiments, 5´ oligonucleotides and 3´ ITR oligonucleotides in a hairpin or other three-dimensional configuration (e.g., a Holliday junction configuration) are synthesized and produced by ligating the 5´ and 3´ ITR oligonucleotides to a double-stranded polynucleotide comprising an expression cassette or a heterologous nucleic acid sequence. Optionally, a step of subjecting the oligo(s) to conditions that facilitate folding the oligo into a three-dimensional configuration is added prior to the ligation step. FIG. 11B shows an exemplary method of generating a ceDNA vector that includes ligating 5´ ITR oligonucleotides and 3´ ITR oligonucleotides to a double-stranded polynucleotide comprising an expression cassette. In some embodiments, the 5´ and 3´ ITR oligonucleotides are 5´ and 3´ hairpin oligonucleotides or have a hairpin structure or a different three-dimensional configuration (e.g., a T or Y-shaped Holliday junction) and can optionally be provided by in vitro DNA synthesis. In some embodiments, the 5´ and 3´ ITR oligonucleotides are cleaved with a restriction endonuclease to have sticky ends complementary to a double-stranded polynucleotide having corresponding restriction endonuclease sticky ends. In some embodiments, the ends of the hairpin of the 5´ ITR oligonucleotide have sticky ends complementary to the 5´ sense strand and the 3´ antisense strand of the double-stranded polynucleotide. In some embodiments, the ends of the hairpin of the 3´ ITR oligonucleotide have sticky ends complementary to the 3´ sense strand and the 5´ antisense strand of the double-stranded polynucleotide. In some embodiments, the ends of the hairpins of the 5´ ITR oligonucleotide and the 3´ ITR oligonucleotide have different restriction endonuclease sticky ends such that directional ligation to each end of the double-stranded polynucleotide can be achieved.In some embodiments, one or both ends of the ITR oligonucleotide do not have overhangs, and such ITR oligo(s) are ligated to the double-stranded polynucleotide by blunt-end ligation. In some aspects, the unwanted double-stranded DNA polynucleotide backbone is cleaved by one or more restriction endonucleases specific for unique cleavage sites in the backbone, so that it is degraded during purification and more easily eliminated.

[0015] Another exemplary method of producing a DNA vector (e.g., a ceDNA vector) involves the formation of single-stranded linear DNA containing an expression cassette and subsequent ligation to close the DNA molecule. In this embodiment, the DNA vector comprises, in the 5' to 3' direction, a first sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense second ITR, an antisense expression cassette sequence, and an antisense first ITR, and then the single-stranded linear DNA whose free ends are ligated to form a closed-end ceDNA vector is prepared by synthesizing it by any means known in the art. In one embodiment, using the production of a ceDNA vector as an exemplary DNA vector produced, the single-stranded DNA molecule obtained for the production of the ceDNA vector comprises, from 5' to 3', a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense second ITR, an antisense expression cassette sequence, and an antisense first ITR.

[0016] In this exemplary method, in one embodiment, an oligonucleotide can be synthesized that includes one or more of a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense second ITR, an antisense expression cassette sequence, and an antisense first ITR. One or more of such oligonucleotides can be ligated to form a single-stranded DNA molecule as shown above. Once the single-stranded DNA molecule is formed, the free 3' and 5' ends of the molecule can be joined by ligation to form a ceDNA vector.

[0017] Another exemplary method of producing a closed-end DNA vector is by synthesis of a single-stranded sequence that includes at least one ITR adjacent to an expression cassette sequence and also includes an antisense expression cassette sequence. In a non-limiting example, a ceDNA vector is produced by the following method.

[0018] In order from 5' to 3', a sense first ITR, a sense expression cassette sequence, a sense second ITR, and a single-stranded sequence including an antisense expression cassette sequence is provided. In one embodiment, the single-stranded sequence can be directly synthesized by any method known in the art. In another embodiment, the single-stranded sequence can be constructed by ligating two or more oligos including one or more of a sense first ITR, a sense expression cassette sequence, a sense second ITR, and an antisense expression cassette sequence.

[0019] In yet another embodiment, a single-stranded sequence can be obtained by excision of a sequence from a double-stranded DNA construct followed by separation of the strands from the excised double-stranded fragment. More specifically, a double-stranded DNA construct is provided that sequentially includes, from 5' to 3', a first restriction site, a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense expression cassette sequence, and a second restriction site. The region between the two restriction endonuclease cleavage sites is excised by cleavage with at least one restriction endonuclease that recognizes such cleavage site(s). The resulting excised double-stranded DNA fragment is processed such that the sense and antisense strands are separated into the desired single-stranded sequence fragments.

[0020] The single-stranded sequence is subjected to an annealing step to promote formation of one or more hairpin loops by the sense first ITR and / or the sense second ITR, and complementary binding of the sense expression cassette sequence to the antisense expression cassette sequence. The result is a closed-end structure that does not require ligation to form. Annealing parameters and techniques are well known in the art.

[0021] In all aspects of the synthetic production method for generating a DNA vector as disclosed herein, the ligation step can be a chemical ligation step or an enzymatic ligation step. In some embodiments, ligation is performed using a ligation-competent enzyme, such as a DNA ligase, and can ligate, for example, 5' and 3' sticky overhangs, or blunt ends. In some embodiments, the ligation enzyme is a ligase enzyme other than the Rep protein. In some embodiments, the ligation enzyme is an AAV Rep protein.

[0022] In all aspects of the synthetic method for generating a DNA vector as disclosed herein, the method is an in vitro method. In a preferred embodiment, the method is a cell-free method, i.e., it is not performed in or in the presence of cells, such as insect cells.

[0023] One or more of the enzyme or oligonucleotide components for the synthetic production method can be produced from cells and used in the method of the present invention in purified form, which will be understood by those skilled in the art. Thus, in some embodiments, the synthetic production method is a cell-free method, but restriction enzymes and / or ligase enzymes can be produced from cells. In one embodiment, cells such as bacterial cells may be present that contain an expression vector expressing one or more of the restriction endonucleases or ligase enzymes. Thus, the methods disclosed herein are primarily directed to cell-free synthesis methods for generating the DNA vectors disclosed herein, but in one embodiment also include synthetic production methods where cells, e.g., bacterial cells rather than insect cells, are present and can be used to express one or more of the enzymes required for the method.

[0024] One aspect of the technology described herein is to generate a ceDNA vector using a synthetic production method. The ceDNA vectors described herein are capsid-free linear double-stranded DNA molecules formed from continuous strands of complementary DNA having covalently closed ends (linear, continuous, and non-capsidated structures), and include 5' inverted terminal repeat (ITR) sequences and 3' ITR sequences, where the 5' ITR and 3' ITR can have the same symmetric three-dimensional configuration (i.e., symmetric or substantially symmetric) relative to each other, or alternatively the 5' ITR and 3' ITR can have different three-dimensional configurations (i.e., asymmetric ITRs) relative to each other. Further, the ITRs can be derived from the same or different serotypes. In some embodiments, the ceDNA vector can include ITR sequences having a symmetric three-dimensional spatial configuration such that their structures are the same shape in geometric space or have the same A, C-C' and B-B' loops in three-dimensional space (i.e., they are the same or mirror images of each other). In some embodiments, one ITR can be derived from one AAV serotype and the other ITR can be derived from a different AAV serotype.

[0025] Accordingly, some aspects of the technology described herein relate to the synthetic production of a ceDNA vector comprising an ITR sequence selected from any of (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITRs) having different three-dimensional spatial configurations relative to each other, (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration.

[0026] Aspects of the invention relate to a synthetic production method for producing a ceDNA vector useful for the expression of a desired transgene in a cell, tissue, organ, system, or subject as described herein. In particular, provided herein are methods for producing a closed-ended DNA vector, including but not limited to, a ceDNA vector, in a cell-free environment, thereby limiting the amount of impurities and preventing the introduction of contaminants during the production process that can affect the efficacy and / or safety of a given vector product. Using such methods, a DNA vector that expresses any desired transgene, e.g., a ceDNA vector, can be synthesized. The transgene can be selected for the treatment of a given disease, the promotion of optimal health, the prevention of the onset of a disease, for diagnostic purposes, or as desired by one of ordinary skill in the art for a given application.

[0027] In another embodiment of this aspect and all other aspects provided herein, the transgene encodes a protein of interest, e.g., the protein of interest is a receptor, toxin, hormone, enzyme, or cell surface protein. In another embodiment of this aspect and all other aspects provided herein, the protein of interest is a receptor. In another embodiment of this aspect and all other aspects provided herein, the protein of interest is an enzyme. Exemplary genes and proteins of interest that are targeted are described in detail in the Methods of Use and Methods of Treatment sections of this specification.

[0028] In some embodiments, the present application may be defined in any of the following paragraphs. 1. A method of preparing a closed - end DNA vector, comprising: (i) providing a first single - stranded ITR molecule comprising a first ITR; (ii) providing a second single - stranded ITR molecule comprising a second ITR; and (iii) providing a double - stranded polynucleotide comprising an expression cassette sequence, ligating the 5' and 3' ends of the first ITR molecule to the first end of the double - stranded molecule, and ligating the 5' and 3' ends of the second ITR molecule to the second end of the double - stranded molecule to form a DNA vector. 2. A method of preparing a closed - end DNA vector, comprising: (i) an expression cassette, (ii) a first ITR upstream (5' end) of the expression cassette, (iii) a second ITR downstream (3' end) of the expression cassette, and (iii) contacting a double - stranded DNA construct comprising at least two restriction endonuclease cleavage sites adjacent to the ITRs such that the restriction endonuclease is distal to the expression cassette, cleaving the double - stranded DNA construct at the restriction endonuclease cleavage sites and contacting it with one or more restriction endonucleases capable of excising the sequence between the restriction endonuclease cleavage sites from the double - stranded DNA construct, and ligating the 5' and 3' ends of the excised sequence to form a closed - end DNA vector. 3. A method of preparing a DNA vector, comprising: in order from 5' to 3': a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense second ITR, an antisense expression cassette sequence, and synthesizing a single - stranded DNA molecule comprising an antisense first ITR. A method comprising forming a polynucleotide containing a hairpin from a single-stranded molecule and ligating the 5' and 3' ends to form a closed-end DNA vector. 4. A method for preparing a closed-end DNA vector, sequentially in the 5' to 3' direction, a sense first ITR, a sense expression cassette sequence, a sense second ITR, and synthesizing a single-stranded DNA molecule containing an antisense expression cassette sequence, and annealing the molecule. 5. A method for preparing a closed-end DNA vector, sequentially in the 5' to 3' direction, a first restriction endonuclease cleavage site, a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense expression cassette sequence, and providing a double-stranded DNA construct containing a second restriction endonuclease cleavage site, contacting the double-stranded DNA construct with one or more restriction endonucleases capable of cleaving the double-stranded DNA construct at the first restriction endonuclease cleavage site and the second restriction endonuclease cleavage site to excise the double-stranded sequence between the restriction endonuclease cleavage sites from the double-stranded polynucleotide, separating the excised double-stranded sequence into a sense strand and an antisense strand, and performing an annealing step in which each of the sense strand and the antisense strand forms a closed-end DNA vector. 6. The method according to any one of paragraphs 1 to 5, wherein the double-stranded DNA construct is a bacmid, plasmid, minicircle, or linear double-stranded DNA molecule. 7. The method according to any one of paragraphs 1 to 6, wherein a single restriction endonuclease is used for excision. 8. The method according to any one of paragraphs 1-7, wherein two different restriction endonucleases are used for excision. 9. The method according to any one of paragraphs 1-8, wherein at least one of the sense first ITR, sense expression cassette sequence, sense second ITR, antisense second ITR, antisense expression cassette sequence, and antisense first ITR is synthesized. 10. The method according to any one of paragraphs 1-9, wherein a single-stranded DNA molecule is constructed by synthesizing one or more of the sense first ITR, sense expression cassette sequence, sense second ITR, antisense second ITR, antisense expression cassette sequence, and antisense first ITR as oligonucleotides and ligating such oligonucleotides to form a single-stranded DNA molecule. 11. The method according to any one of paragraphs 1-10, wherein a single-stranded DNA molecule is provided by excision of a molecule from a double-stranded DNA polynucleotide followed by denaturation of the excised double-stranded fragment to produce a single-stranded DNA molecule. 12. The method according to any one of paragraphs 1-11, wherein the step of forming a polynucleotide containing a hairpin from a single-stranded molecule is performed by annealing the single-stranded molecule under conditions in which one or more of the ITRs form a hairpin loop. 13. The method according to any one of paragraphs 1-12, wherein at least one of the first ITR and the second ITR is synthesized. 14. The method according to any one of paragraphs 1-13, wherein the double-stranded expression cassette sequence is obtained by excision from a double-stranded DNA construct containing the expression cassette sequence. 15. The method according to any one of paragraphs 1-14, wherein within the double-stranded DNA construct, the expression cassette sequence is adjacent to a first restriction endonuclease cleavage site at the 5' end and adjacent to a second restriction endonuclease cleavage site at the 3' end. 16. The method according to any one of paragraphs 1-15, wherein the double-stranded DNA construct is a bacmid, plasmid, minicircle, or linear double-stranded DNA molecule. 17. The method according to any one of paragraphs 1 to 16, wherein the first restriction endonuclease and the second restriction endonuclease are the same restriction endonuclease. 18. The method according to any one of paragraphs 1 to 17, wherein the first restriction endonuclease and the second restriction endonuclease are different restriction endonucleases. 19. The method according to any one of paragraphs 1 to 18, wherein at least one of the first ITR and the second ITR is annealed before ligation to the expression cassette sequence. 20. The method according to any one of paragraphs 1 to 19, wherein at least one of the first ITR and the second ITR each comprises an overhang region complementary to the first end of the expression cassette sequence or the second end of the expression cassette sequence. 21. The method according to any one of paragraphs 1 to 20, wherein the ligation is selected from chemical ligation and protein-assisted ligation. 22. The method according to any one of paragraphs 1 to 21, wherein the ligation is performed by T4 ligase or AAV Rep protein. 23. The method according to any one of paragraphs 1 to 22, wherein the first ITR is selected from a wild-type ITR and a modified ITR. 24. The method according to any one of paragraphs 1 to 23, wherein the second ITR is selected from a wild-type ITR and a modified ITR. 25. The method according to any one of paragraphs 1 to 24, wherein at least one of the first ITR and the second ITR comprises at least one RBE site. 26. The method according to any one of paragraphs 1 to 25, wherein at least one of the first ITR and the second ITR is an AAV ITR or an ITR derived from AAV. 27. The method according to any one of paragraphs 1 to 26, wherein the sequence of the first ITR is selected from any of the left ITR sequences shown in Table 3, Table 4B, or Table 5 or SEQ ID NOs: 2, 5 to 9, 32 to 48. 28. The method according to any one of paragraphs 1 to 27, wherein the second ITR sequence is selected from any of the right ITR sequences shown in Table 3, Table 4A, or Table 5 or SEQ ID NOs: 1, 3, 10-14, 15-31. 29. The method according to any one of paragraphs 1 to 28, wherein the expression cassette sequence comprises at least one cis-regulatory element. 30. The method according to any one of paragraphs 1 to 29, wherein the cis-regulatory element is selected from the group consisting of a promoter, an enhancer, a post-transcriptional regulatory element, and a polyadenylation signal. 31. The method according to any one of paragraphs 1 to 30, wherein the post-transcriptional regulatory element comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). 32. The method according to any one of paragraphs 1 to 31, wherein the promoter is selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter. 33. The method according to any one of paragraphs 1 to 32, wherein the expression cassette sequence comprises a transgene sequence. 34. The method according to any one of paragraphs 1 to 33, wherein the transgene sequence is at least 2000 nucleotides in length. 35. The method according to any one of paragraphs 1 to 34, wherein the transgene sequence encodes a protein. 36. The method according to any one of paragraphs 1 to 35, wherein the transgene sequence encodes a reporter protein, a therapeutic protein, an antigen, a gene editing protein, or a cytotoxic protein. 37. The method according to any one of paragraphs 1 to 36, wherein the transgene sequence is a functional nucleotide sequence. 38. The method according to any one of paragraphs 1 to 37, wherein the closed-ended DNA vector is a ceDNA vector. 39. The method according to any one of paragraphs 1 to 38, wherein the ceDNA vector is purified. 40. A closed-ended DNA vector produced by the method according to any one of paragraphs 1 to 39. 41. A pharmaceutical composition comprising the closed - end DNA vector according to any one of paragraphs 1 to 40 and optionally an excipient. 42. An isolated closed - end DNA vector obtainable or obtained by the process according to any one of paragraphs 1 to 6 or 6 to 39. 43. Genetic medicine comprising an isolated closed - end DNA vector obtained by the process according to any one of paragraphs 1 to 42. 44. A cell comprising the closed - end DNA vector according to paragraph 40. 45. A transgenic animal comprising the closed - end DNA vector according to paragraph 40. 46. A method of treating a subject by administering a closed - end DNA vector obtainable or obtained by the process according to any one of paragraphs 1 to 5 or 6 to 39. 47. A method for delivering a therapeutic protein to a subject, comprising administering to the subject a composition comprising the closed - end DNA vector according to claim 40, or a closed - end DNA vector obtainable or obtained by the process according to any one of paragraphs 1 to 5 or 6 to 39, wherein at least one heterologous nucleotide sequence encodes a transgene or a therapeutic protein. 48. The method according to paragraph 47, wherein the therapeutic protein is a therapeutic antibody, a reporter protein, a therapeutic protein, an antigen, a gene - editing protein, or a cytotoxic protein. 49. A kit comprising the closed - end DNA vector according to claim 40, or a closed - end DNA vector obtainable or obtained by the process according to any one of paragraphs 1 to 5 or 6 to 39, packaged in a container together with a packet insert and a nanocarrier. 50. A kit for producing a closed - end DNA vector obtainable or obtained by the process according to any one of paragraphs 1 to 5 or 6 to 39. A kit for producing a closed - end DNA vector obtained by, or obtainable by, the process according to any one of paragraphs 1 - 39, comprising a first single - stranded ITR molecule comprising a first ITR, a second single - stranded ITR molecule comprising a second ITR, and at least one reagent for ligating the first single - stranded ITR molecule and the second single - stranded ITR molecule into a double - stranded polynucleotide molecule. 52. (i) A double - stranded DNA construct comprising an expression cassette, a first ITR upstream (5´ end) of the expression cassette, a second ITR downstream (3´ end) of the expression cassette, and at least two restriction endonuclease cleavage sites adjacent to the ITRs such that the restriction endonucleases are distal to the expression cassette, wherein the expression cassette has a restriction endonuclease for insertion of a transgene; (ii) at least one ligation reagent for ligation. A kit for producing a closed - end DNA vector obtained by, or obtainable by, the process according to any one of paragraphs 2 - 39. 53. (i) A single - stranded DNA molecule comprising, in order from 5´ to 3´, a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense second ITR, an antisense expression cassette sequence, and an antisense first ITR, wherein the sense expression cassette sequence and the antisense expression cassette sequence have restriction endonuclease sites for insertion of a transgene; (ii) at least one ligation reagent for ligation. A kit for producing a closed - end DNA vector obtained by, or obtainable by, the process according to any one of paragraphs 3 - 39. 54. (i) A single-stranded DNA molecule comprising, in order from 5' to 3', a sense first ITR, a sense expression cassette sequence, a sense second ITR, and an antisense expression cassette sequence, wherein the sense expression cassette sequence and the antisense expression cassette sequence have restriction endonuclease sites for the insertion of a transgene, and (ii) at least one ligation reagent for ligation, which is obtained by or can be obtained by the process described in any one of paragraphs 4 to 39, a kit for producing a closed-end DNA vector. 55. (i) A double-stranded DNA construct comprising, in order from 5' to 3', a first restriction endonuclease cleavage site, a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense expression cassette sequence, and a second restriction endonuclease cleavage site, wherein the sense expression cassette sequence and the antisense expression cassette sequence have restriction endonuclease sites for the insertion of a transgene, and (ii) at least one ligation reagent for ligation, which is obtained by or can be obtained by the process described in any one of paragraphs 5 to 39, a kit for producing a closed-end DNA vector. 56. The kit according to any one of paragraphs 49 to 55, wherein at least one reagent for ligation is a reagent for chemical ligation. 57. The kit according to any one of paragraphs 49 to 56, wherein at least one reagent for ligation is a reagent for protein-assisted ligation. 58. The kit according to any one of paragraphs 49 to 57, wherein ligation is performed by T4 ligation or an AAV Rep protein. 59. The kit according to any one of paragraphs 49 to 58, wherein the first single-stranded ITR molecule and the second single-stranded ITR molecule comprise restriction endonuclease cleavage sites at their ends. 60. The kit according to any one of paragraphs 49 to 59, wherein the kit further comprises at least one restriction endonuclease enzyme.

[0029] In some embodiments, one aspect of the technology described herein relates to a synthetically produced non-viral capsid-free DNA vector (ceDNA vector) having a covalently closed end, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between wild-type inverted terminal repeats, optionally the heterologous nucleic acid sequence encodes a transgene, and the vector is not present in a viral capsid.

[0030] In some embodiments, one aspect of the technology described herein relates to a synthetically produced non-viral capsid-free DNA vector (ceDNA vector) having a covalently closed end, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between asymmetric inverted terminal repeats (asymmetric ITRs), at least one of the asymmetric ITRs comprising a functional terminal resolution site and a Rep binding site, optionally the heterologous nucleic acid sequence encodes a transgene, and the vector is not present in a viral capsid.

[0031] In some embodiments, one aspect of the technology described herein relates to a synthetically produced non-viral capsid-free DNA vector (ceDNA vector) having a covalently closed end, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between symmetric mutant inverted terminal repeats, at least one of the ITRs comprising a functional terminal resolution site and a Rep binding site, optionally the heterologous nucleic acid sequence encodes a transgene, and the vector is not present in a viral capsid.

[0032] These and other aspects of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to the exemplary embodiments of the present disclosure depicted in the accompanying drawings. However, the accompanying drawings show only typical embodiments of the present disclosure and, therefore, should not be regarded as limiting the scope, as the present disclosure can recognize other equally valid embodiments.

[0034]

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Mode for Carrying Out the Invention

[0035] The methods and compositions provided herein are, in part, based on the discovery of synthetic production methods useful for generating closed - end DNA vectors that, among other things, but not limited to, have fewer impurities and / or higher yields compared to DNA vectors produced in insect cell lines such as the Sf9 cell line, and / or the production process is simplified or made more efficient or cost - effective compared to traditional cell - based production methods. In one embodiment, cells are not used to replicate the DNA vector, and thus, the production is cell - free. Accordingly, provided herein are methods for synthesizing closed - end DNA vectors without using cells. In some embodiments, methods for synthesizing closed - end DNA vectors without using insect cells are provided herein. Also provided herein are closed - end DNA vector compositions produced using the synthetic production methods herein, including ceDNA vector compositions, as well as the use of such closed - end DNA vectors and ceDNA vectors.

[0036] The present invention relates to in vitro processes for the production of closed - end DNA vectors, the corresponding DNA vector products produced by the methods and their uses herein, and oligonucleotides and kits useful in the processes of the present invention.

[0037] Closed - end DNA vectors produced by the methods described herein are advantageous over other vectors in that they can be used more safely to express transgenes in cells, tissues, or subjects. That is, the resulting vectors do not contain bacterial or insect cell contaminants, and thus, by generating linear vectors by such cell - free methods, undesirable side effects can potentially be minimized. The synthetic production methods can also result in more highly purified desired vectors. The synthetic production methods can also be more efficient and / or cost - effective than traditional cell - based production methods of such vectors.

[0038] The vectors synthesized as described in this specification can express any desired transgene, for example, a transgene for treating or curing a given disease. One of ordinary skill in the art will readily recognize that any transgene used in conventional gene therapy methods using conventional recombinant vectors is suitable for expression by, for example, a ceDNA vector produced by the synthetic methods described herein.

[0039] I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc., described herein and may vary as such. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-1 9-3), Robert S. Porter et al. (eds.), Fields Virology, 6 th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA(2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999 - 2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1 - 56081 - 569 - 8), Immunology by Werner Luttmann, published by Elsevier, 2006, Janeway‘s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor&Francis Limited, 2014 (IS BN 0815345305, 9780815345305), Lewin’s Genes XI, published by Jones& Bartlett Publi shers, 2014 (ISBN - 1449659055), Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414), Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X), Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), and the contents of all of these are hereby incorporated by reference in their entirety.

[0040] As used herein, the terms “cell-free production,” “synthetic closed-ended DNA vector production,” and “synthetic production,” and their grammatically related counterparts, are used interchangeably and refer to the production of one or more molecules in a manner that does not involve replication or other propagation of the molecules by, within, or using cell extracts. Synthetic production avoids contamination of the produced molecules by cell contaminants (e.g., cell proteins or cell nucleic acids) and further avoids unwanted cell-specific modifications (e.g., methylation or glycosylation or other post-translational modifications) of the molecules during the production process.

[0041] As used herein, the terms “heterologous nucleotide sequence” and “transgene” are used interchangeably and refer to nucleic acids of interest (other than nucleic acids encoding capsid polypeptides) that can be incorporated into and thereby delivered and expressed by the ceDNA vectors disclosed herein.

[0042] As used herein, the terms "expression cassette", "transcription cassette", and "gene expression unit" are used interchangeably and refer to a linear stretch of nucleic acid that contains a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct the transcription of the transgene, but does not contain a sequence encoding a capsid, other vector sequences, or inverted terminal repeats. An expression cassette may additionally contain one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0043] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. "Oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides as applicable to the described embodiments.

[0044] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain segments of nucleic acids in a manner that does not occur naturally, or is synthetic. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences necessary for the expression of the coding sequences of the present disclosure. An "expression cassette" contains a DNA coding sequence operably linked to a promoter.

[0045] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) is capable of non-covalently binding, i.e., forming Watson-Crick base pairs and / or G / U base pairs, to another nucleic acid in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength such that the nucleic acid "anneals" or "hybridizes" to the other nucleic acid (i.e., the nucleic acid specifically binds to the complementary nucleic acid). As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and cytosine (C) pairing with guanine (G). Further, it is known in the art that for hybridization between two RNA molecules (e.g., dsRNA), a guanine (G) base pairs with uracil (U). For example, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pairing with codons in mRNA. In the context of this disclosure, a guanine (G) in the protein-binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of interest is considered complementary to uracil (U), and vice versa. Thus, if a G / U base pair can be made at a given nucleotide position in the protein-binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of interest, that position is not considered non-complementary but rather complementary.

[0046] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length that can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0047] A DNA sequence that "encodes" a particular RNA or protein gene product is a DNA nucleic acid sequence that is transcribed into the particular RNA and / or protein. A DNA polynucleotide can encode an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA-targeted RNA, also referred to as "non-coding" RNA or "ncRNA").

[0048] As used herein, the term "genomic safe harbor gene" or "safe harbor gene" refers to a gene or locus into which a nucleic acid sequence can be inserted such that it can integrate and function in a predictable manner (e.g., express a protein of interest) without significantly adversely affecting endogenous gene activity or promoting cancer. In some embodiments, a safe harbor gene is also a locus or gene at which the inserted nucleic acid sequence can be expressed more efficiently and at a higher level than at non-safe harbor sites.

[0049] As used herein, the term "gene delivery" means the process by which foreign DNA is introduced into a host cell for the application of gene therapy.

[0050] As used herein, the term "terminal repeat" or "TR" includes any viral terminal repeat sequence or synthetic sequence that includes at least one minimal origin of replication and a region that includes a palindromic hairpin structure. A Rep binding sequence ("RBS") (also referred to as an RBE (Rep binding element)) and a terminal resolution site ("TRS") together constitute the "minimal origin of replication", and thus a TR includes at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically each referred to as an "inverted terminal repeat" or "ITR". In the context of a virus, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found in the present invention herein, a TR that is not a reverse complement over its full length can still perform the conventional functions of an ITR, and thus the term ITR is used herein to refer to a TR in a ceDNA genome or ceDNA vector that can mediate replication of the ceDNA vector. It will be understood by those skilled in the art that more than two ITRs or asymmetric ITR pairs can be present in a complex ceDNA vector construct. An ITR can be an AAV ITR or a non-AAV ITR, or can be derived from an AAV ITR or a non-AAV ITR. For example, an ITR can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, murine parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as an origin of SV40 replication, can be further modified by cleavage, substitution, deletion, insertion, and / or addition and used as an ITR. Parvoviridae family viruses are composed of two subfamilies, Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the viral family of adeno-associated viruses (AAV), which are capable of replicating in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.For convenience in this specification, the ITR located 5' (upstream thereof) relative to the expression cassette in the ceDNA vector is referred to as the "5' ITR" or "left ITR", and the ITR located 3' (downstream thereof) relative to the expression cassette in the ceDNA vector is referred to as the "3' ITR" or "right ITR".

[0051] "Wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in AAV or other dependoviruses that retains, for example, Rep binding activity and Rep nicking ability. The nucleotide sequence of the WT-ITR from any AAV serotype may vary slightly from the naturally occurring canonical sequence due to degeneracy of the genetic code or drift, and thus the WT-ITR sequences included for use in this specification include WT-ITR sequences as a result of naturally occurring changes (e.g., replication errors) that occur during the production process.

[0052] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of WT-ITRs within a single ceDNA genome or ceDNA vector that are wild-type ITRs having reverse complementary sequences over their entire lengths. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, provided the changes do not affect the properties of the sequence and the overall three-dimensional structure. In some embodiments, the deviating nucleotides represent conservative sequence changes. As a non-limiting example, the sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., measured using BLAST with default settings) to the canonical sequence and has a three-dimensional spatial configuration that is symmetric to other WT-ITRs such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric WT-ITRs have the same A, C-C', and B-B' loops in three-dimensional space. Substantially symmetric WT-ITRs can be functionally confirmed as WT by determining that they have operable Rep binding sites (RBE or RBE') and terminal resolution sites (trs) that pair with the appropriate Rep protein. Optionally, other functions, including transgene expression under permissive conditions, can be tested.

[0053] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably herein and refer to an ITR having a mutation in at least one or more nucleotides as compared to a WT-ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, B' regions of the ITR and can result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) as compared to the three-dimensional spatial configuration of the WT-ITR of the same serotype.

[0054] As used herein, the term "asymmetric ITR pair", also referred to as "asymmetric ITR", refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are not reverse complementary strands over their entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetric three-dimensional spatial configuration relative to its cognate ITRs such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair has a different overall geometric structure, i.e., different configurations of their A, C-C', and B-B' loops in three-dimensional space (e.g., one ITR may have a shorter C-C' arm and / or a shorter B-B' loop compared to its cognate ITR). The sequence difference between the two ITRs can be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair can be a wild-type AAV ITR sequence and the other ITR can be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence and the two ITRs are modified ITRs having different shapes (i.e., different overall geometric structures) in geometric space. In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm and the other ITR can have different modifications (e.g., a single arm, or a short B-B' arm, etc.) such that they have a different three-dimensional spatial configuration compared to their cognate asymmetric mod-ITR.

[0055] As used herein, the term "symmetric ITR" refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are mutated or modified relative to the wild-type adeno-associated virus ITR sequences and are reverse complements over their entire length. Neither of the ITRs is the wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs) and may have a sequence different from the wild-type ITR due to nucleotide addition, deletion, substitution, cleavage, or point mutation. For convenience herein, the ITR located 5' (upstream) relative to the expression cassette in the ceDNA vector is referred to as the "5' ITR" or "left ITR", and the ITR located 3' (downstream) relative to the expression cassette in the ceDNA vector is referred to as the "3' ITR" or "right ITR".

[0056] As used herein, the terms "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refer to a pair of modified ITRs within a single ceDNA genome or ceDNA vector, both of which have inverted complementary sequences over their entire length. For example, a modified ITR can be considered substantially symmetric even if there are some nucleotide sequences that deviate from the inverted complementary sequence, as long as the variations do not affect the characteristics and overall shape. As a non-limiting example, the sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) to a reference sequence and have a symmetric three-dimensional spatial configuration relative to their cognate modified ITRs such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C’, and B-B’ loops configured in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair can have different inverted complementary strand nucleotide sequences but can still have the same symmetric three-dimensional spatial configuration. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR of a mod-ITR pair (e.g., the 5’ ITR) can be derived from one serotype, and the other ITR (e.g., the 3’ ITR) can be derived from a different serotype, but both can have the same corresponding mutations (e.g., if the 5’ ITR has a deletion in the C region, the cognate modified 3’ ITR of a different serotype has a deletion at the corresponding position in the C’ region), such that the modified ITR pair has the same symmetric three-dimensional spatial configuration. In such embodiments, each ITR of the modified ITR pair can be derived from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected at the corresponding position of the cognate ITR of a different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) as long as the differences in the nucleotide sequences between the ITRs do not affect the characteristics or overall shape and they have substantially the same shape in three-dimensional space.As a non-limiting example, the mod-ITR has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the canonical mod-ITR, determined by standard means well known in the art such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and has a symmetric three-dimensional spatial configuration such that their three-dimensional structures are of the same shape in geometric space. Substantially symmetric mod-ITR pairs have the same A, C-C’, and B-B’ loops in three-dimensional space. For example, if a modified ITR of a substantially symmetric mod-ITR pair has a deletion in the C-C’ arm, the cognate mod-ITR has a corresponding deletion in the C-C’ loop and has a similar three-dimensional structure of the remaining A and B-B’ loops of the same shape in the geometric space of the cognate mod-ITR.

[0057] The term "adjacent" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, A and C are adjacent to B on both sides. The same applies to the arrangement AxBxC. Thus, an adjacent sequence follows before or after the sequence to which it is adjacent, but does not have to be contiguous with, or immediately adjacent to, the sequence to which it is adjacent. In one embodiment, the term "adjacent" refers to the terminal repeats at each end of a linear double-stranded ceDNA vector.

[0058] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further include one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded DNA polynucleotide.

[0059] As used herein, the term "ceDNA spacer region" refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. In some embodiments, the ceDNA spacer region holds two functional elements for a desired process for optimal functionality. In some embodiments, the ceDNA spacer region provides, or increases, the genetic stability of the ceDNA genome, for example, within a plasmid or baculovirus. In some embodiments, the ceDNA spacer region facilitates easy genetic manipulation of the ceDNA genome by providing a convenient location, such as a cloning site. For example, in certain aspects, an oligonucleotide "polylinker" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to have no known protein (e.g., transcription factor) binding sites, is positioned in the ceDNA genome to separate cis-acting factors, for example, inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc. between a terminal resolution site and an upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between a polyadenylation signal sequence and a 3' terminal resolution site.

[0060] As used herein, the terms "Rep binding site", "Rep binding element", "RBE", and "RBS" are used interchangeably and refer to the binding site of a Rep protein (e.g., AAV Rep78 or AAV Rep68), such that upon binding by the Rep protein, the Rep protein can exercise its site-specific endonuclease activity on the sequence incorporating the RBS. The RBS sequence and its reverse complement together form a single RBS. RBS sequences are known in the art and include, for example, the RBS sequence identified in AAV2, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60). Any known RBS sequence can be used in embodiments of the invention, including other known AAV RBS sequences and other naturally known or synthetic RBS sequences. Without being bound by theory, the nuclease domain of the Rep protein binds to the double-stranded nucleotide sequence GCTC, and thus two known AAV Rep proteins are thought to bind directly to and stably assemble with the double-stranded oligonucleotide, 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO: 60). In addition, soluble aggregated conformational isomers (i.e., a variable number of interrelated Rep proteins) dissociate and bind to an oligonucleotide containing the Rep binding site. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interaction with the nitrogenous bases provides sequence specificity, while the interaction with the phosphodiester backbone is non-sequence specific or low-sequence specific and stabilizes the protein-DNA complex.

[0061] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein, and Rep refers to the region that forms a tyrosine-phosphodiester bond with 5'-thymidine that generates a 3'OH that serves as a substrate for DNA elongation via cellular DNA polymerases, such as DNA pol δ or DNA pol ε. Alternatively, the Rep-thymidine complex may be involved in a coordination ligation reaction. In some embodiments, the TRS includes a minimal non-base paired thymidine. In some embodiments, the nicking efficiency of the TRS can be at least partially controlled by its distance within the same molecule from the RBS. When the acceptor substrate is a complementary ITR, the resulting product is an intermolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' (SEQ ID NO: 61) identified in AAV2. Any known TRS sequence, including other known AAV TRS sequences, such as AGTT (SEQ ID NO: 62), GGTTGG (SEQ ID NO: 63), AGTTGG (SEQ ID NO: 64), AGTTGA (SEQ ID NO: 65), and other naturally known or synthetic TRS sequences, and other motifs such as RRTTRR (SEQ ID NO: 66), can be used in embodiments of the present invention.

[0062] As used herein, the term "ceDNA-plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0063] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can propagate as a plasmid in E. coli and can thereby be manipulated as a shuttle vector for baculoviruses.

[0064] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0065] As used herein, the terms "ceDNA-baculovirus infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells infected with ceDNA-baculovirus, including but not limited to insect cells (e.g., Sf9 cells).

[0066] As used herein, the term "closed-end DNA vector" refers to a capsid-free DNA vector that has at least one covalent closed end and at least a portion of the vector has an intramolecular double-stranded structure.

[0067] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector that contains at least one terminal palindrome. In some embodiments, the ceDNA contains two covalent closed ends.

[0068] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism can be readily observed. For example, a fluorescent protein fluoresces a cell when excited with light of a specific wavelength, luciferase catalyzes a reaction that produces light in a cell, and an enzyme such as β-galactosidase converts a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP), and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0069] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide or, more preferably, as a polypeptide that kills cells, such as a toxin, or as an agent that renders cells more susceptible to killing by a selected agent or deletion thereof. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins include restriction endonucleases that target host cell DNA sequences (whether genomic or episomal), proteases that degrade polypeptide targets required for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins, but are not limited thereto. In some embodiments, the expression of an effector protein controlled by a synthetic biological circuit described herein can participate as a factor in another synthetic biological circuit, thereby expanding the range and complexity of the responsiveness of the biological circuitry.

[0070] Transcription regulators refer to transcription activators and repressors that activate or repress the transcription of a gene of interest. A promoter is a region of a nucleic acid that initiates the transcription of a particular gene. Transcription activators typically bind near a transcription promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to the transcription promoter and sterically impede the initiation of transcription by RNA polymerase. Other transcription regulators can serve as either activators or repressors depending on where they bind and on cellular and environmental conditions. Non-limiting examples of the transcription regulator class include homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine-zipper proteins, but are not limited thereto.

[0071] As used herein, a "repressor protein" or an "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates the transcription of a sequence operably linked to the regulatory sequence element, respectively. Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins described herein are, for example, modules in the form of separable DNA-binding and input-agent binding, or containing reactive elements or domains.

[0072] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce a toxic, allergic, or similar adverse reaction when administered to a host.

[0073] As used herein, an "input-agent reactive domain" is a domain of a transcription factor that binds to a condition or an input agent, or otherwise renders a linked DNA-binding fusion domain reactive to the presence of that condition or input, such that the presence of the condition or input results in a conformational change in the input-agent reactive domain or the protein to which it is fused, modifying the transcriptional regulatory activity of the transcription factor.

[0074] The term "in vivo" refers to an assay or process that occurs in or within an organism such as a multicellular animal. In some of the aspects described herein, a method or use may be said to occur "in vivo" when a unicellular organism such as a bacterium is used. The term "ex vivo" refers to methods and uses that are performed using living cells having an intact membrane outside the body of a multicellular animal or plant, such as, inter alia, explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells). The term "in vitro" refers to assays and methods that do not require the presence of cells having an intact membrane, such as cell extracts, and may refer to introducing a programmable synthetic biological circuit into a cell-free medium, such as a medium that does not contain a cell or cell line, such as a cell extract.

[0075] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of a nucleic acid sequence, which can be a heterologous target gene encoding a protein or RNA. A promoter can be constitutive, inducible, repressive, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence are controlled. A promoter can also contain gene elements to which regulatory proteins and molecules such as RNA polymerase and other transcription factors can bind. In some embodiments of the aspects described herein, a promoter can drive the expression of a transcription factor that regulates the expression of the promoter itself. Within a promoter sequence, a transcription start site, as well as protein-binding domains involved in the binding of RNA polymerase, will be found. Eukaryotic promoters often, but not always, contain a "TATA" box and a "CAT" box. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the ceDNA vectors disclosed herein. A promoter sequence is joined at its 3' end by a transcription start site and extends upstream (5' orientation) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background.

[0076] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase the transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream or downstream of the gene start site they regulate. Enhancers can be located within the intron region or exon region of an unrelated gene.

[0077] A promoter can be said to drive or initiate transcription of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably joined," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence and regulates to control the initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which the nucleic acid sequence is in an inverted orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences are used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in combination with an enhancer.

[0078] A promoter can be obtained by isolating the 5' non-coding sequence that is located upstream of the coding segment and / or exon of a given gene or sequence and can be naturally associated with the gene or sequence. Such a promoter can be referred to as "endogenous." Similarly, in some embodiments, an enhancer can be naturally associated with the nucleic acid sequence and can be located either downstream or upstream of that sequence.

[0079] In some embodiments, the coding nucleic acid segment is positioned under the control of a “recombinant promoter” or “heterologous promoter,” both of which refer to a promoter that is not normally associated with the encoded nucleic acid sequence operably linked in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such a promoter or enhancer can include a promoter or enhancer of another gene, a promoter or enhancer isolated from any other prokaryotic, viral, or eukaryotic cell, and a synthetic promoter or enhancer that is not “naturally occurring,” i.e., can include different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic manipulation known in the art. In addition to synthetically producing the nucleic acid sequences of promoters and enhancers, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques including PCR for the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906, each of which is incorporated herein by reference). Further, it is contemplated that control sequences that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc. can be used as well.

[0080] As used herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity in the presence of, when affected by, or when contacted by an inducer or inducing agent. As defined herein, an "inducer" or "inducing agent" can be endogenous or a usually exogenous compound or protein administered in such a way as to be active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducing agent, i.e., a chemical, compound, or protein, can itself be the result of the transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of an inducible promoter. In some embodiments, the inducible promoter is induced in the absence of a particular agent such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionein, ecdysone, mammalian viruses (e.g., adenovirus late promoter, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), and other steroid-responsive promoters, rapamycin-responsive promoters, etc.

[0081] The terms "DNA regulatory sequence", "control element", and "regulatory element" as used interchangeably herein refer to transcriptional and translational control sequences such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., which provide and / or regulate the transcription of non-coding sequences (e.g., DNA-targeted RNA) or coding sequences (e.g., site-specific modified polypeptides or Cas9 / Csn1 polypeptides) and / or regulate the translation of the encoded polypeptide.

[0082] "Operably linked" refers to a tandem in which the components so described are in a relationship that permits them to function in the intended manner. For example, if a promoter affects its transcription or expression, the promoter is operably linked to the coding sequence. An "expression cassette" contains a heterologous DNA sequence that is operably linked to a promoter or other regulatory sequence sufficient to direct transcription of the transgene in the ceDNA vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0083] As used herein, the term "subject" refers to a human or animal to whom treatment, including prophylactic treatment with a ceDNA vector according to the present invention, is provided. Typically, the animal is a vertebrate such as, but not limited to, a primate, rodent, domestic animal, or game animal. Examples of primates include, but are not limited to, chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques, such as the rhesus macaque. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon, but are not limited thereto. In certain embodiments of aspects described herein, the subject is a mammal such as, for example, a primate or a human. The subject can be male or female. Additionally, the subject can be an infant or a child. In some embodiments, the subject can be a neonatal or fetal subject, for example, the subject is present in utero. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Non-human mammals can be advantageously used as subjects representing animal models of diseases and disorders. Additionally, the methods and compositions described herein can be used in domestic animals and / or pets. Human subjects can be of any age, sex, race, or ethnic group, such as, for example, Caucasian, Asian, African, Black, African American, Afro-European, Spanish, Middle Eastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject has already received treatment. In some embodiments, the subject is an embryo, fetus, neonate, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonate, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.

[0084] As used herein, the term "host cell" includes any cell type that is susceptible to transformation, transfection, transduction, etc. by a nucleic acid construct or ceDNA expression vector of the present disclosure. By way of non-limiting example, a host cell can be any of an isolated primary cell, a pluripotent stem cell, a CD34 + cell), an induced pluripotent stem cell, or some immortalized cell lines (e.g., HepG2 cells). Alternatively, a host cell can be a cell in situ or in vivo in a tissue, organ, or organism.

[0085] The term "exogenous" refers to a substance that is present in a cell other than its natural source. As used herein, the term "exogenous" refers to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that is not normally found and has been introduced into a biological system such as a cell or organism by a process involving human intervention, where it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that is present in relatively small amounts and has been introduced into a biological system such as a cell or organism by a process involving human intervention, where it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to effect ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0086] The term "sequence identity" refers to the relatedness between two nucleotide sequences. For the purposes of the present disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably as implemented in version 3.0.0 or later, and the Needleman-Wunsch algorithm (Needleman and determined using the Needleman and Wunsch, 1970 (supra). Any parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (number of identical deoxyribonucleotides × 100) / (length of the alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0087] As used herein, the terms "homology" or "homologous" are defined as the percentage of nucleotide residues of a homologous arm that are identical to the nucleotide residues of the corresponding sequence on the target chromosome after aligning the sequences as necessary and introducing gaps to achieve the maximum percent sequence identity. Alignments for the purpose of determining percent nucleotide sequence homology can be achieved in a variety of ways within the skill in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. In some embodiments, for example, the nucleic acid sequence (e.g., DNA sequence) of the homology arm of the repair template is considered "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.

[0088] As used herein, the term "heterologous" means a nucleotide or polypeptide sequence that is not found in natural nucleic acids or proteins, respectively. A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleotide sequence encoding a fusion variant polypeptide.

[0089] A "vector" or "expression vector" is a replicon, such as a plasmid, bacteriomid, phage, virus, virion, or cosmid, into which another DNA segment, i.e., an "insert", can be ligated to effect replication of the ligated segment in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin and / or final form, but for the purposes of the present disclosure, the term "vector" generally refers to a ceDNA vector when the term is used herein. The term "vector" encompasses any genetic element that can replicate when associated with appropriate control elements and can transfer a gene sequence into a cell. In some embodiments, the vector can be an expression vector or a recombinant vector.

[0090] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or a polypeptide from a sequence ligated to transcriptional regulatory sequences on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. Expression vectors can contain additional elements; for example, an expression vector can have two replication systems so that it can be maintained in two organisms, e.g., human cells in the case of expression, and a prokaryotic host in the case of cloning and amplification. The term "expression" refers to cell processes involved in the production of RNA and proteins and, if necessary, secreted proteins, including, but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions before and after the coding region, e.g., 5'untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, and intervening sequences (introns) between individual coding segments (exons).

[0091] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of suitable episomal vectors provides a means to maintain the nucleotide of interest in the subject as extrachromosomal DNA at high copy number, thereby eliminating the potential impact of chromosomal integration.

[0092] As used herein, the term "hereditary disease" refers to a disease that is caused, in whole or in part, directly or indirectly, by one or more abnormalities in the genome, particularly a condition that is present from birth. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence of a gene or its regulatory sequence. Hereditary diseases can include, but are not limited to, DMD, hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, hereditary diseases of liver metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom syndrome, retinoblastoma, and Tay-Sachs disease.

[0093] As used herein, the terms "comprising" or "comprises" are used with respect to a method or composition and its respective components, if any, and are open-ended with respect to the inclusion of unspecified elements, whether essential or not, in that composition or method.

[0094] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics, if any, of that embodiment. The use of "comprising" indicates inclusion rather than limitation.

[0095] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any element not recited in the description of an embodiment.

[0096] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics, if any, of the embodiments of the present invention.

[0097] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a method" includes one or more methods of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading the present disclosure and / or steps. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with "for example".

[0098] Except where operating examples or otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about". The term "about" when used in connection with percentages may mean ±1%. The invention is further described in the following examples, which are not to be construed as limiting the scope of the invention.

[0099] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as a limitation. Members of each group can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in or deleted from the group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to include the group as modified, and thus to satisfy all written group descriptions used in the appended claims.

[0100] In some embodiments of any aspect, the disclosure described herein is not related to the process of human cloning, the process of modifying the genetic identity of the human germ line, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without any substantial medical benefit to humans or animals, and processes for modifying the genetic identity of animals resulting from such processes.

[0101] In this specification, other terms are defined within the description of the various aspects of the invention.

[0102] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and pending patent applications, are hereby expressly incorporated by reference herein for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technologies described herein. These publications are provided only for their disclosure prior to the filing date of this application. Nothing in this regard shall be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the content of these documents are based on the information available to the applicant, and do not constitute any admission as to the accuracy of the date or content of these documents.

[0103] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the exact forms disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as will be recognized by those of ordinary skill in the art, various equivalent modifications are possible within the scope of the present disclosure. For example, the steps or functions of a method are presented in a given order, but alternative embodiments may perform the functions in a different order or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as needed. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as needed, to provide further embodiments using the configurations, functions, and concepts of the above references and applications. Additionally, considering the equivalence of biological functions, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action. In light of the detailed description, these and other changes can be made to the present disclosure. All such modifications are intended to be included within the scope of the appended claims.

[0104] Any particular element of any of the foregoing embodiments can be combined with or replaced by an element of other embodiments. Further, while the advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages since all embodiments are within the scope of the present disclosure.

[0105] The techniques described herein are further illustrated by the following examples and should in no way be construed as further limiting. It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and can vary as such. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0106] II. Detailed synthetic production method of circular DNA vectors containing ceDNA vectors. The techniques described herein generally target methods for generating closed-end DNA vectors in the absence of cells or cell lines. As such, the resulting vectors have fewer impurities than equivalent vectors produced using conventional cell production methodologies.

[0107] A. General synthetic production method In some embodiments, a process for the synthesis of closed-end DNA vectors containing ceDNA vectors that does not require the use of any microbiological steps is disclosed herein. In some embodiments, the process enables the synthesis of closed-end DNA vectors in a system using an enzymatic cleavage step using a restriction endonuclease and a ligation step to generate a closed-end DNA vector. In almost all embodiments, the synthetic system for DNA vector production is a cell-free system. In some embodiments, the cell-free system is an insect cell-free system.

[0108] One of ordinary skill in the art will understand that one or more of the enzyme or oligonucleotide components for the synthetic production method can be produced from cells and used in the methods of the present invention in purified form. Thus, in some embodiments, the synthetic production method is a cell-free method, but restriction enzymes and / or ligase enzymes can be produced from cells.

[0109] In one embodiment, the restriction endonuclease and / or ligation competent protein can be expressed or provided from an expression vector in a cell, such as a bacterial cell. In one embodiment, there can be a cell, such as a bacterial cell, that contains an expression vector that expresses one or more of the restriction endonuclease or ligase enzymes. Thus, the methods disclosed herein are primarily directed to cell-free synthesis methods for generating the DNA vectors disclosed herein, but in one embodiment, also include synthesis production methods where there are cells, such as bacterial cells rather than insect cells, that can be used to express one or more of the enzymes required for the method. In such embodiments, the cells that express the restriction endonuclease and / or ligation competent protein are not insect cells. In all embodiments where there are cells that express one or more restriction endonucleases or ligation competent proteins, the cells do not replicate the closed-ended DNA vector. In other words, the intracellular machinery of the cell is not replicated or involved in the replication of the DNA vector.

[0110] In some embodiments, the synthesis of the closed - ended DNA vectors (e.g., ceDNA vectors) described herein is performed by an in vitro cell - free process starting from either a double - stranded DNA construct or one or more oligonucleotides. The double - stranded DNA construct or one or more oligonucleotides are cleaved with a restriction endonuclease and ligated to form a DNA molecule. In some embodiments, since they are oligonucleotides that can be chemically synthesized, the use of a large starting template encoding the entire desired sequence that typically needs to replicate within bacteria is avoided. Once the desired DNA sequence is synthesized, it can be cleaved and ligated to other oligonucleotides as disclosed herein. The use of multiple oligonucleotides in the generation of closed - ended DNA vectors using the methods disclosed herein allows for a modular approach to DNA vector generation, enabling the tuning and / or specific selection of terminal repeats, such as ITRs, and the spacing of terminal repeats, as well as the selection of heterologous nucleic acid sequences within the synthetically produced closed - ended DNA vectors.

[0111] B. Methods of synthetic production of DNA vectors Certain methods for the production of ceDNA vectors, including those having various ITR configurations, using cell - based methods are described in Example 1 of International Application No. PCT / US18 / 49996, filed on September 7, 2018, and International Application No. PCT / US2018 / 064242, filed on December 6, 2018, each of which is hereby incorporated by reference in its entirety.

[0112] In contrast, the methods provided herein relate to synthetic production methods, e.g., in some embodiments, cell - free production methods, also referred to herein as "synthetic closed - ended DNA vector production" or "synthetic production".

[0113] In this specification, a method for the synthetic production of a closed - end DNA vector is exemplified and described using the synthetic production of a ceDNA vector. In some embodiments, the synthetic production method is a cell - free method, such as an insect - cell - free method. In some embodiments, the synthetic production method is performed in the absence of a bacmid, or a baculovirus, or both. In alternative embodiments, the synthetic production method may involve the use of cells, such as bacterial cells, e.g., cells expressing a restriction endonuclease, and / or the use of ligation - competent Rep proteins. In such embodiments, the cells can be a cell line in which the polynucleotide vector template is stably integrated, and the restriction endonuclease protein and / or ligation - competent protein, e.g., but not limited to, a Rep protein, etc., can be used to introduce into a reaction mixture containing the oligonucleotides used in the synthetic production methods described herein.

[0114] Examples of processes for generating and isolating ceDNA vectors produced using the synthetic production methods disclosed herein are described in FIGS. 4A - 4E and in certain examples of the following Examples section.

[0115] In all aspects of the synthetic production method for generating a closed - end DNA vector as disclosed herein, the ligation step can be a chemical ligation step or an enzymatic ligation step. In some embodiments, the ligation is performed using a ligation - competent enzyme, such as DNA ligase, and can ligate, for example, 5´ and 3´ sticky overhangs, or blunt ends. In some embodiments, the ligation enzyme is a ligase enzyme other than the Rep protein. In some embodiments, the ligation enzyme is an AAV Rep protein.

[0116] In all aspects of the synthetic method for generating a closed-end DNA vector as disclosed herein, the method is an in vitro method. In a preferred embodiment, the method is a cell-free method, i.e., it is not carried out in or in the presence of cells, such as insect cells. In an alternative embodiment, one or more of the enzymes for the synthetic production method can be produced or expressed from cells, such as non-insect cells. For example, in some embodiments, cells, such as bacterial cells, may be present that contain an expression vector that expresses one or more of the restriction endonuclease or ligase enzymes. Thus, the methods disclosed herein are primarily directed to cell-free synthetic methods for generating the closed-end DNA vectors disclosed herein, but also encompass synthetic production methods that can use cells, such as bacterial cells, to express one or more of the enzymes required for the method.

[0117] (i) Synthetic production method from a double-stranded DNA construct In one aspect, a closed - end DNA vector is generated by excising the entire molecule that forms the closed - end DNA vector from a double - stranded DNA construct and then ligating the ends to close the molecule. In such embodiments, the double - stranded DNA construct is provided, in order from 5´ to 3´, with a first restriction endonuclease site, an upstream ITR, an expression cassette, a downstream ITR, and a second restriction endonuclease site. The double - stranded DNA construct is then contacted with one or more restriction endonucleases to generate a double - strand break at both of the restriction endonuclease cleavage sites. It is possible for one endonuclease to target both sites, or for each site to be targeted by a different endonuclease, provided that the restriction sites do not lie within the closed - end vector template region. This results in the excision of the sequence between the restriction endonuclease sites from the remaining double - stranded DNA construct. This excised molecule has free 5´ and 3´ ends, which are then ligated to form the closed - end DNA vector. Ligation can be performed, for example, by using a protein with ligation functionality such as Rep or a phage protein, or by chemical ligation. In some aspects, the vector length in the 5’ to 3’ direction exceeds the maximum length known to be encapsidated in an AAV virion. In some aspects, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb. In some aspects, the excised molecule is first annealed to promote hairpin formation prior to ligation of the free 5´ and 3´ ends. In some aspects, the unwanted double - stranded DNA construct backbone is cleaved by one or more restriction endonucleases specific for unique cleavage sites in the backbone, and thus degraded and more easily removed during purification. In some aspects, the method described above can further include heating or melting the excised dsDNA molecule to form single - stranded polynucleotides prior to the ligation step. In some aspects, the two restriction endonuclease sites have the same sequence. In some aspects, the two restriction endonuclease sites can be cleaved to provide blunt ends.

[0118] (ii) Method of synthetic production from single-stranded molecules (Variant 1) Another exemplary method of producing a closed-ended DNA vector, such as a ceDNA vector, using the synthetic production method disclosed herein is to use a single-stranded linear DNA having a closed end and include two ITRs adjacent to the expression cassette, first in the sense direction and then in the antisense direction. Thus, in some embodiments, the method comprises: a) synthesizing a single-stranded molecule comprising, from 5' to 3', a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense second ITR, an antisense expression cassette sequence, and an antisense first ITR; b) facilitating the formation of at least one hairpin loop within the single-stranded molecule; and c) ligating the 5' and 3' ends to form a ceDNA vector. Various methods of synthesizing oligonucleotides and polynucleotides, such as in vitro or in silico synthesis of oligonucleotides, are known in the art, and any method known in the art can be used in step a). The terms "sense" and "antisense" in the foregoing method refer to the orientation of structural elements on the polynucleotide. The sense and antisense versions of the element are complementary strands that are reverse of each other. The hairpin loop sequence can be any nucleotide sequence that preferably does not hybridize to form dsDNA along its entire length. Methods of ligating DNA to form a linear double-stranded structure are known in the art, and in non-limiting examples, viral proteins such as Rep, or phage, or pox, proteins, or chemical ligation are used.

[0119] In this embodiment, a closed-ended DNA vector, such as a ceDNA vector, is produced by providing a single-stranded linear DNA sequence encoding an expression cassette with adjacent sense and antisense ITRs, and then made closed-ended by ligation. Using the production of a ceDNA vector as an exemplary closed-ended DNA vector produced, the single-stranded DNA molecule for the production of the ceDNA vector comprises, from 5' to 3', Sense first ITR, Sense expression cassette array, Sense second ITR, Antisense second ITR, Antisense expression cassette array, and Antisense first ITR.

[0120] In this exemplary method, the oligonucleotides are ligated in order as shown above, where the antisense first ITR is complementary to the sense first ITR, and similarly the antisense second ITR and antisense expression cassette array are complementary to the sense second ITR and sense expression cassette array, respectively. The ligation step joins the free 5' and 3' ends, resulting in the formation of a closed-ended DNA vector, ceDNA.

[0121] In all aspects of the synthetic production method for generating a closed-ended DNA vector as disclosed herein, the ligation step can be a chemical ligation step or an enzymatic ligation step. In some embodiments, the ligation is performed using a ligation-competent enzyme, such as a DNA ligase, and can ligate, for example, 5' and 3' sticky overhangs, or blunt ends. In some embodiments, the ligation enzyme is a ligase enzyme other than the Rep protein. In some embodiments, the ligation enzyme is the AAV Rep protein.

[0122] (iii) Synthetic production using 5' and 3' ITR oligonucleotides Another aspect includes: a) synthesizing (and / or providing) a first single-stranded ITR molecule comprising a first ITR; b) synthesizing (and / or providing) a second single-stranded ITR molecule comprising a second ITR; c) providing a double-stranded polynucleotide comprising an expression cassette sequence; d) ligating the 5' and 3' ends of the first ITR molecule to a first end of the double-stranded molecule and ligating the 5' and 3' ends of the second ITR molecule to a second end of the double-stranded molecule to form a DNA vector. Prior to the ligation step, the ITR molecule and / or the double-stranded polynucleotide can be contacted with a restriction enzyme to generate compatible ends, such as overhangs, to ensure proper ligation at the desired positions. In some embodiments, the three elements have blunt ends. The ligation of each ITR to the double-stranded polynucleotide can be sequential or simultaneous. In one embodiment, the ligation step involves the ligation of a single-stranded 5' to 3' oligo that forms a hairpin.

[0123] In such embodiments, a closed-ended DNA vector, such as a ceDNA vector, is produced in some embodiments by synthesizing 5´ and 3´ ITR oligonucleotides in a hairpin or other three-dimensional configuration (e.g., a T- or Y-Holliday junction configuration) and ligating the 5´ and 3´ ITR oligonucleotides to a double-stranded polynucleotide comprising an expression cassette or a heterologous nucleic acid sequence. Optionally, a step of subjecting the oligo(s) to conditions that facilitate folding the oligo into a three-dimensional configuration is added prior to the ligation step. FIG. 11B shows an exemplary method of generating a ceDNA vector that includes ligating 5´ ITR oligonucleotides and 3´ ITR oligonucleotides to a double-stranded polynucleotide comprising an expression cassette. In some embodiments, the 5´ and 3´ ITR oligonucleotides are 5´ and 3´ hairpin oligonucleotides or have different three-dimensional configurations (e.g., Holliday junctions) and can optionally be provided by in vitro DNA synthesis. In some embodiments, the 5´ and 3´ ITR oligonucleotides are cleaved with a restriction endonuclease to have sticky ends complementary to a double-stranded polynucleotide having corresponding restriction endonuclease sticky ends. In some embodiments, the ends of the hairpin of the 5´ ITR oligonucleotide have sticky ends that are complementary to the 5´ sense strand and the 3´ antisense strand of the double-stranded polynucleotide. In some embodiments, the ends of the hairpin of the 3´ ITR oligonucleotide have sticky ends that are complementary to the 3´ sense strand and the 5´ antisense strand of the double-stranded polynucleotide. In some embodiments, the ends of the hairpins of the 5´ ITR oligonucleotide and the 3´ ITR oligonucleotide have different restriction endonuclease sticky ends such that directional ligation to the double-stranded polynucleotide can be achieved. In some embodiments, one or both ends of the ITR oligonucleotide(s) do not have overhangs and such ITR oligo(s) are ligated to the double-stranded polynucleotide by blunt-end ligation.The ITR molecules in the foregoing method can be synthesized and / or ligated by any method known in the art. Various methods for synthesizing oligonucleotides and polynucleotides, such as solid-phase DNA synthesis, phosphoramidite DNA synthesis, and PCR, are known in the art. The ITR molecules can also be excised from a DNA construct containing the ITR. Various methods for ligating nucleic acids, such as chemical ligation or ligation with ligation-competent proteins, such as ligase, AAV Rep, or topoisomerase, are known in the art.

[0124] (iv) Synthetic production methods that do not require ligation In some embodiments, the synthetic production of a closed-end DNA vector is by synthesis of a single-stranded sequence that includes at least one ITR adjacent to the expression cassette sequence and also includes an antisense expression cassette sequence. In one non-limiting example, the ceDNA vector is produced by the following method.

[0125] In order from 5´ to 3´, the sense first ITR, the sense expression cassette sequence, the sense second ITR, and a single-stranded sequence containing the antisense expression cassette sequence is provided. In one embodiment, the single-stranded sequence can be directly synthesized by any method known in the art. In another embodiment, the single-stranded sequence can be constructed by ligating together two or more oligos that include one or more of the sense first ITR, the sense expression cassette sequence, the sense second ITR, and the antisense expression cassette sequence.

[0126] In yet another embodiment, a single-stranded sequence can be obtained by excision of a sequence from a double-stranded DNA construct followed by separation of the strands from the excised double-stranded fragment. More specifically, a double-stranded DNA construct is provided that contains, in order from 5´ to 3´, a first restriction site, a sense first ITR, a sense expression cassette sequence, a sense second ITR, an antisense expression cassette sequence, and a second restriction site. The region between the two restriction endonuclease cleavage sites is excised by cleavage with at least one restriction endonuclease that recognizes such cleavage site(s). The resulting excised double-stranded DNA fragment is processed such that the sense and antisense strands are separated into the desired single-stranded sequence fragment.

[0127] The single-stranded sequence is subjected to an annealing step to promote formation of one or more hairpin loops by the sense first ITR and / or the sense second ITR and complementary binding of the sense expression cassette sequence to the antisense expression cassette sequence. The result is a closed-end structure that does not require ligation to form. Annealing parameters and techniques are well known in the art.

[0128] In some embodiments, the modified ITR contains the polynucleotide of SEQ ID NO: 4 and the wild-type ITR contains the polynucleotide of SEQ ID NO: 1.

[0129] The DNA vectors produced by the methods provided herein preferably have a linear and continuous structure rather than a discontinuous structure as determined by restriction enzyme digestion assays (Figure 4C). The linear and continuous structure is considered to be more stable against attack by cellular endonucleases and at the same time less likely to be recombined to cause mutagenesis. Thus, in some embodiments, vectors with a linear and continuous structure are preferred. The double-stranded DNA vectors within a continuous linear single-stranded molecule may be covalently bound at the termini without a sequence encoding an AAV capsid protein. These DNA vectors are structurally different from plasmids, which are circular double-stranded nucleic acid molecules of bacterial origin. The complementary strands of a plasmid may separate after denaturation, but these DNA vectors have complementary strands and are single DNA molecules. Preferably, the vectors can be produced without prokaryotic-type DNA base methylation, unlike plasmids.

[0130] Figure 5 is a gel confirming the production of ceDNA from multiple ceDNA plasmid constructs using the method described in the examples. ceDNA is confirmed by the characteristic band pattern in the gel, as discussed with respect to Figure 4C of the examples above.

[0131] C. Isolation and purification of ceDNA vectors: Methods for generating and isolating ceDNA vectors, which are exemplary closed-end DNA vectors, are described herein. For example, closed-end DNA vectors produced by the synthetic methods described herein, such as ceDNA vectors, can be harvested or collected at an appropriate time point after the final ligation reaction and optimized to achieve high-yield production of ceDNA vectors. Closed-end DNA vectors, such as ceDNA vectors, can be purified by any means known to those skilled in the art for DNA purification. In one embodiment, the ceDNA vector is purified as a DNA molecule. Generally, any nucleic acid purification method known in the art, as well as commercially available DNA extraction kits, can be employed.

[0132] Alternatively, purification can be carried out by subjecting the reaction mixture to chromatographic separation. As a non-limiting example, this process involves loading the reaction mixture onto an ion exchange column that retains nucleic acids (e.g., SARTOBIND Q®), then eluting (e.g., with a 1.2 M NaCl solution), and performing further chromatographic purification on a gel filtration column (e.g., 6 High Load GE). The DNA vector, e.g., the ceDNA vector, is then recovered, e.g., by precipitation.

[0133] The presence of the ceDNA vector can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector and using gel electrophoresis to analyze both the digested DNA material and the undigested DNA material to confirm the presence of linear and continuous DNA with characteristic bands as compared to linear and discontinuous DNA. Figures 4B and 4C show one embodiment for identifying the presence of the closed-end ceDNA vector produced by the process herein.

[0134] Figure 5 of International Application No. PCT / US18 / 49996 shows a gel confirming the production of ceDNA from multiple ceDNA plasmid constructs using the method described in the Examples. The ceDNA is confirmed by the characteristic band pattern in the gel as discussed with respect to Figure 4C of the Examples.

[0135] In some embodiments, the closed - ended DNA vectors produced by the synthetic production methods disclosed herein can be delivered to target cells in vitro or in vivo by various suitable methods as discussed herein. Only the vector can be applied or injected. The vector can be delivered to cells without the aid of transfection reagents or other physical means. Alternatively, the vector can be delivered using transfection reagents or other physical means that facilitate the entry of DNA into cells, such as liposomes, alcohol, poly - lysine compounds, poly - arginine compounds, calcium phosphate, microvesicles, microinjection, etc.

[0136] D. Circular DNA vectors produced using synthetic production methods As provided herein are various methods for the in vitro production of DNA molecules and closed - ended DNA vectors. In some embodiments, the closed - ended DNA vector is a ceDNA vector as described herein. In alternative embodiments, the closed - ended DNA vector is, for example, a dumbbell DNA vector or a dog - bone DNA vector (see, e.g., WO2010 / 0086626, the entire contents of which are incorporated herein by reference). (2017):65.

[0137] III. ceDNA vectors in general In some embodiments, the closed - ended DNA vectors produced using the synthetic processes described herein are ceDNA vectors that can express a transgene and include ceDNA vectors. The ceDNA vectors described herein are not limited by size, thereby enabling, for example, the expression of all the components necessary for the expression of a transgene from a single vector. The ceDNA vector is preferably double - stranded, for example, self - complementary, over at least a portion of a molecule such as an expression cassette (e.g., ceDNA is not a double - stranded circular molecule). The ceDNA vector has a covalently closed end and is thus, for example, resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37 °C for 1 hour or more.

[0138] Generally, a ceDNA vector produced using the synthetic processes described herein includes, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR. The ITR sequences are selected from any of (i) at least one wild-type (WT) ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITRs) having different three-dimensional spatial configurations relative to each other, (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric mod-ITR pair in which each mod-ITR has the same three-dimensional spatial configuration.

[0139] Methods and compositions comprising a ceDNA vector produced using the synthetic processes described herein are included herein and may further include, but are not limited to, a delivery system such as a liposomal nanoparticle delivery system. Non-limiting exemplary liposomal nanoparticle systems for use are disclosed herein. In some aspects, the disclosure provides lipid nanoparticles comprising ceDNA and ionizable lipids. For example, a lipid nanoparticle formulation made and loaded using ceDNA obtained by the process is disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, and incorporated herein by reference.

[0140] There are no packaging constraints imposed by the limited space within the viral capsid on a ceDNA vector produced using the synthetic processes described herein. This permits the insertion of regulatory elements such as regulatory switches, large transgenes, multiple transgenes, etc., disclosed herein.

[0141] Figures 1A - 1E show schematic diagrams of non - limiting exemplary ceDNA vectors or the sequences of the corresponding ceDNA plasmids. The ceDNA vector does not contain a capsid and can be obtained from a plasmid encoding a first ITR, an expression cassette containing a transgene, and a second ITR in this order. The expression cassette may contain one or more regulatory sequences that enable and / or control the expression of the transgene. For example, the expression cassette may contain, in this order, an enhancer / promoter, an ORF reporter (transgene), a post - transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA).

[0142] The expression cassette may also contain an internal ribosome entry site (IRES) and / or a 2A element. Cis - regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir - regulatory elements, post - transcriptional regulatory elements, tissue - and cell - type - specific promoters, and enhancers. In some embodiments, the ITR may act as a promoter for the transgene. In some embodiments, the ceDNA vector contains additional components for regulating the expression of the transgene, such as a regulatory switch for controlling and regulating the expression of the transgene, described in the section herein titled "Regulatory Switches", and may contain, if desired, a regulatory switch that is a kill switch enabling controlled cell death of cells containing the ceDNA vector.

[0143] The expression cassette can include more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range between about 4000 - 10,000 nucleotides, or 10,000 - 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can include a transgene in the range of 500 - 50,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene in the range of 500 - 75,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene that is in the range of 500 - 10,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene that is in the range of 1000 - 10,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene that is in the range of 500 - 5,000 nucleotides in length. The ceDNA vector does not have the size limitation of the capsidated AAV vector, and thus can enable the delivery of large-size expression cassettes to result in efficient transgenes. In some embodiments, the ceDNA vector lacks prokaryotic cell-specific methylation.

[0144] The ceDNA expression cassette can include an expressible exogenous sequence (e.g., open reading frame) or transgene that encodes a protein that is inactive, or has insufficient activity, for example, a protein that does not exist in the recipient subject, or a gene that encodes a protein having a desired biological or therapeutic effect. The transgene can encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that results in a therapeutic effect when overexpression is considered within the scope of the present disclosure.

[0145] The expression cassette can contain any transgene useful for treating a subject disease or disorder. Using the ceDNA vector produced using the synthetic process described herein, nucleic acids encoding polypeptides, or non-coding nucleic acids (e.g., RNAi, miR, etc.), as well as exogenous genes and nucleotide sequences including viral sequences in the subject's genome, such as HIV viral sequences, etc., can be delivered to and expressed in the subject for any gene of interest, including but not limited to these. Preferably, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or for immunogenic polypeptides. In certain embodiments, the ceDNA vector is useful for expressing in a subject any gene of interest, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNAs (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)), antibodies, antigen-binding fragments, or any combination thereof.

[0146] The expression cassette can also encode a polypeptide, sense or antisense oligonucleotide, or RNA (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). The expression cassette can contain exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well-known in the art.

[0147] The sequences provided by the expression cassette, the expression construct of the ceDNA vector described herein, may be codons optimized for the target host cell. As used herein, the term "optimized codon" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing the codons of at least one, two or more, or a significant number of native sequences (e.g., prokaryotic cell sequences) with codons that are more frequently or most frequently used in the genes of that vertebrate, for enhanced expression in cells of a vertebrate of interest, such as a mouse or human cell. Various species exhibit a particular bias for certain codons of a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database.

[0148] In some embodiments, the transgene expressed by the ceDNA vector is a therapeutic gene. In some embodiments, the therapeutic gene is an antibody, or an antibody fragment, or an antigen-binding fragment thereof, such as, for example, a neutralizing antibody or antibody fragment.

[0149] In particular, the therapeutic gene is, for example, one or more therapeutic agents (plural) including, but not limited to, proteins (plural), polypeptides (plural), peptides (plural), enzymes (plural), antibodies, antigen-binding fragments, and variants and / or active fragments thereof, for use in the treatment, prevention, and / or amelioration of one or more symptoms of a disease, dysfunction, injury, and / or disorder. Exemplary therapeutic genes are described in the section entitled "Methods of Treatment" herein.

[0150] There are many structural features of the ceDNA vector that are different from plasmid-based expression vectors. The ceDNA vectors produced by the synthetic methods herein have the following features: deletion of the original (i.e., uninserted) bacterial DNA, deletion of the prokaryotic origin of replication, self-closing (i.e., they do not require any sequences other than the two ITRs containing the Rep binding and terminal resolution sites (RBS and TRS), nor exogenous sequences between the ITRs), the presence of ITR sequences that form hairpins of eukaryotic origin (i.e., they are produced in eukaryotic cells), and may have one or more of the absence of bacterial-type DNA methylation, or any other methylation that is actually associated with production in a given cell type and is considered abnormal by mammalian hosts. In general, it is preferred that the vector does not contain any prokaryotic cell DNA, although it is contemplated that some prokaryotic cell DNA may be inserted as an exogenous sequence, as a non-limiting example, into a promoter or enhancer region. Another important feature that distinguishes the ceDNA vector from plasmid expression vectors is that the ceDNA vector is single-stranded linear DNA with a closed end, while plasmids are always double-stranded DNA.

[0151] The ceDNA vectors produced by the synthetic methods provided herein preferably have a linear and continuous structure rather than a discontinuous structure as determined by restriction enzyme digestion assays (Figure 4C). The linear and continuous structure is thought to be more stable against attack by cellular endonucleases and at the same time less likely to be recombined to cause mutagenesis. Thus, ceDNA vectors with a linear and continuous structure are a preferred embodiment. The continuous linear single-stranded intramolecular double-stranded ceDNA vectors may be covalently bound at the termini without a sequence encoding an AAV capsid protein. These ceDNA vectors are structurally different from plasmids, which are circular double-stranded nucleic acid molecules of bacterial origin (including the ceDNA plasmids described herein). The complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, whereas in contrast, a ceDNA vector has complementary strands but is a single DNA molecule and thus remains a single molecule even when denatured. In some embodiments, the ceDNA vectors described herein can be produced without prokaryotic-type DNA base methylation, unlike plasmids. Thus, ceDNA vectors and ceDNA-plasmids differ both with respect to their structure (in particular, linear versus circular) and the methods used to produce and purify these different objects (see below), and also with respect to their DNA methylation, which is of the prokaryotic cell type for ceDNA-plasmids and of the eukaryotic cell type for ceDNA vectors.

[0152] There are several advantages to using the ceDNA vectors described herein over plasmid-based expression vectors, such advantages including, but not limited to, the following: 1) Plasmids contain bacterial DNA sequences and are subject to prokaryotic cell-specific methylation, such as 6-methyladenosine and 5-methylcytosine methylation, whereas the capsid-free AAV vector sequences are of eukaryotic cell origin and are not subject to prokaryotic cell-specific methylation. As a result, capsid-free AAV vectors are less likely to induce inflammation and immune responses compared to plasmids. 2) Plasmids require the presence of resistance genes during the production process, whereas ceDNA vectors do not. 3) Circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements, namely ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. The minimum defined elements essential for ITR function are hypothesized to be the Rep binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAV2) and the terminal resolution site (TRS, 5'-AGTTGG-3' (SEQ ID NO: 64) for AAV2), in addition to variable palindromic sequences that allow hairpin formation. 4) ceDNA vectors do not have the overrepresentation of CpG dinucleotides often found in prokaryotic cell-derived plasmids that bind to members of the Toll-like family of receptors and induce T cell-mediated immune responses, according to reports. In contrast, transduction with the capsid-free AAV vectors disclosed herein can efficiently target cell and tissue types that are difficult to transduce with conventional AAV virions using various delivery reagents.

[0153] IV.ITR As disclosed herein, a ceDNA vector includes a transgene or heterologous nucleic acid sequence positioned between two inverted terminal repeat (ITR) sequences, and as these terms are defined herein, the ITR sequences can be asymmetric ITR pairs or symmetric or substantially symmetric ITR pairs. The ceDNA vectors disclosed herein can include ITR sequences selected from any of (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITRs) where the mod-ITR pairs have different three-dimensional spatial configurations relative to each other, (iii) symmetric or substantially symmetric WT-WT ITR pairs where each WT-ITR has the same three-dimensional spatial configuration, or (iv) symmetric or substantially symmetric modified ITR pairs where each mod-ITR has the same three-dimensional spatial configuration, and the methods of the disclosure can further include, but are not limited to, delivery systems such as liposomal nanoparticle delivery systems.

[0154] In some embodiments, the ITR sequences can be derived from viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. The Parvovirinae subfamily (referred to as parvoviruses) includes the Dependovirus genus, members of which require co-infection with a helper virus such as an adenovirus or herpesvirus for productive infection under most conditions. The Dependovirus genus includes adeno-associated viruses (AAVs) that typically infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 FIELDS VIROLOGY (3d Ed. 1996).

[0155] The ITRs are illustrated in the specification, and the examples herein are AAV2 WT-ITRs. However, those skilled in the art will recognize that, as described above, any known parvovirus, such as Dependovirus, such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and ITRs from the AAV-DJ8 genome) can be used. For example, NCBI: NC002077; NC001401; NC001729; NC001829; NC006152; NC006260; NC006261), chimeric ITRs, or ITRs from any synthetic AAV. In some embodiments, the AAV can infect warm-blooded animals, such as birds (AAAV), cows (BAAV), dogs, horses, and sheep adeno-associated viruses. In some embodiments, the ITRs are derived from parvovirus B19 (GenBank accession number NC000883), minute virus of mice (MVM) from mice (GenBank accession number NC001510), goose parvovirus (GenBank accession number NC001701), and snake parvovirus 1 (GenBank accession number NC006148). In some embodiments, as discussed herein, the 5’ WT-ITR can be derived from one serotype and the 3’ WT-ITR can be derived from a different serotype.

[0156] Those skilled in the art will know that the ITR sequence has the general structure of a double-stranded Holliday junction and typically has a T-shaped or Y-shaped hairpin structure (see, for example, FIGS. 2A and 3A), and each WT-ITR is formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), as well as a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). See, for example, structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6). Grimm et al., J. Virology, 2006;80(1);426-439, Yan et al., J. Virology, 2005;364-379, Duan et al., Virology 1999;261;8-14. Those skilled in the art can readily determine the WT-ITR sequence from any AAV serotype for use in a ceDNA vector or ceDNA-plasmid based on the exemplary AAV2 ITR sequence provided herein. See, for example, sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006;80(1);426-439. This shows the percent identity of the AAV2 left ITR to the left ITRs from other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%).

[0157] A. Symmetric ITR pair In some embodiments, the ceDNA vectors described herein include, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5' ITR) and the second ITR (3' ITR) are symmetric or substantially symmetric with respect to each other, i.e., the ceDNA vector may include ITR sequences having a symmetric three-dimensional spatial configuration, whereby their structures are of the same shape in geometric space or have the same A, C-C', B-B' loops in three-dimensional space. In such embodiments, the symmetric ITR pair, or substantially symmetric ITR pair, may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have one or more modifications from the wild-type ITR and may have the same sequences that are reverse complementary (inverted) to each other. In alternative embodiments, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetric three-dimensional shapes.

[0158] (i) wild-type ITR In some embodiments, the symmetric ITR, or substantially symmetric ITR, is wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but they do not necessarily have to be WT-ITRs of the same AAV serotype. That is, in some embodiments, one WT-ITR may be derived from one AAV serotype and the other WT-ITR may be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they can have one or more conservative nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0159] Thus, as disclosed herein, a ceDNA vector comprises a transgene or heterologous nucleic acid sequence positioned between two adjacent wild-type inverted terminal repeat (WT-ITR) sequences that are either inversely complementary (inverted) to each other or alternatively substantially symmetric to each other. That is, the WT-ITR pair has a symmetric three-dimensional configuration. In some embodiments, the wild-type ITR sequence (e.g., AAV WT-ITR) comprises a functional Rep binding site (RBS, e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 60) and a functional terminal resolution site (TRS, e.g., 5'-AGTT-3', SEQ ID NO: 62).

[0160] In one aspect, a ceDNA vector is obtainable from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two WT inverted terminal repeat sequences (WT-ITRs) (e.g., AAV WT-ITRs). That is, both ITRs have wild-type sequences, but do not necessarily have to be WT-ITRs of the same AAV serotype. That is, in some embodiments, one WT-ITR can be derived from one AAV serotype and the other WT-ITR can be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they can have one or more conservative nucleotide modifications while maintaining a symmetric three-dimensional configuration. In some embodiments, the 5' WT-ITR is derived from one AAV serotype and the 3' WT-ITR is derived from the same or a different AAV serotype. In some embodiments, the 5' WT-ITR and the 3' WT-ITR are mirror images of each other, i.e., they are symmetric. In some embodiments, the 5' WT-ITR and the 3' WT-ITR are derived from the same AAV serotype.

[0161] The WT ITR is well-known. In one embodiment, the two ITRs are derived from the same AAV2 serotype. In certain embodiments, the WT from other serotypes can be used. There are several serotypes that are homologous, such as AAV2, AAV4, AAV6, AAV8. In one embodiment, closely homologous ITRs (e.g., ITRs having similar loop structures) can be used. In another embodiment, more diverse AAV WT ITRs, such as AAV2 and AAV5, can be used, and in yet another embodiment, ITRs that are substantially WT can be used, i.e., it has the basic loop structure of the WT but has some conservative nucleotide changes that do not alter or affect the properties. When using WT-ITRs derived from the same viral serotype, one or more regulatory sequences can further be used. In certain embodiments, the regulatory sequence is a regulatory switch that enables the regulation of the activity of the ceDNA.

[0162] In some embodiments, one aspect of the techniques described herein relates to synthetically produced ceDNA vectors, where the ceDNA vector comprises at least one heterologous nucleotide sequence operably positioned between two wild-type inverted terminal repeats (WT-ITRs), and the WT-ITRs can be derived from the same serotype, different serotypes, or can be substantially symmetric with respect to each other (i.e., having a symmetric three-dimensional spatial configuration such that their structures are the same shape in geometric space, or having the same A, C-C’, and B-B’ loops in three-dimensional space). In some embodiments, the symmetric WT-ITRs comprise a functional terminal resolution site and a Rep binding site. In some embodiments, the heterologous nucleic acid sequence encodes a transgene and the vector is not a viral capsid.

[0163] In some embodiments, the WT-ITRs are the same but are reverse complementary strands of each other. For example, the sequence AACG of the 5’ ITR can be CGTT (i.e., the reverse complementary strand) of the 3’ ITR of the corresponding site. In one example, the 5’ WT-ITR sense strand comprises the sequence ATCGATCG, and the corresponding 3’ WT-ITR sense strand is

Chem.

[0164] Exemplary WT-ITR sequences for use in a ceDNA vector comprising a WT-ITR are shown in Table 2 herein, which shows the pair of WT-ITRs (5’ WT-ITR and 3’ WT-ITR).

[0165] As an exemplary embodiment, the present disclosure provides a synthetically produced ceDNA vector comprising a promoter operably linked to a transgene (e.g., a gene editing sequence), with or without a regulatory switch, wherein the ceDNA lacks a capsid protein and (a) is produced from a ceDNA-plasmid encoding a WT-ITR (see, e.g., FIGS. 1F-1G), each WT-ITR having the same number of intramolecular duplex base pairs in its hairpin secondary structure (preferably excluding any deletion of the AAA or TTT terminal loop of this configuration as compared to these reference sequences), and (b) is identified as ceDNA using an assay for the identification of ceDNA by agarose gel electrophoresis under non-denaturing and denaturing conditions of the native gel of Example 1.

[0166] In some embodiments, adjacent WT-ITRs are substantially symmetric to each other. In this embodiment, the 5’ WT-ITR may be derived from one serotype of AAV and the 3’ WT-ITR may be derived from a different serotype of AAV so that the WT-ITRs are not the same inverted complementary strand. For example, the 5’ WT-ITR may be derived from AAV2 and the 3’ WT-ITR may be derived from a different serotype (e.g., AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12). In some embodiments, the WT-ITRs may be selected from two different parvoviruses selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, a hepadnavirus (e.g., Royal Palm Parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or an insect AAV. In some embodiments, such a combination of WT ITRs is a combination of WT-ITRs from AAV2 and AAV6. In one embodiment, the substantially symmetric WT-ITRs are at least 90% identical, at least 95% identical, at least 96%... 97%... 98%... 99%... 99.5%, and all points in between, and have the same symmetric three-dimensional spatial configuration when one is inverted with respect to the other. In some embodiments, the WT-ITR pair is substantially symmetric because they have the same three-dimensional configuration of, for example, the A, C-C’, B-B’, and D arms, such that they have a symmetric three-dimensional spatial configuration. In one embodiment, the substantially symmetric WT-ITR pair is inverted with respect to the other and is at least 95% identical, at least 96%... 97%... 98%... 99%... 99.5%, and all points in between, and one of the WT-ITRs retains the Rep binding site (RBS) and the terminal resolution site (trs) of 5´-GCGCGCTCGCTCGCTC-3´ (SEQ ID NO: 60).In some embodiments, substantially symmetric WT-ITR pairs are inverted relative to each other and are at least 95% identical, at least 96%... 97%... 98%... 99%... 99.5%, and all points in between, and one of the WT-ITRs retains a Rep binding site (RBS) and a terminal resolution site (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. Homology can be determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool), BLASTN with default settings.

[0167] In some embodiments, the structural elements of the ITR can be any structural elements involved in the functional interaction of the ITR with a large Rep protein (e.g., Rep78 or Rep68). In certain embodiments, the structural elements provide selectivity for the interaction of the ITR with a large Rep protein. That is, at least in part, they determine which Rep protein interacts functionally with the ITR. In other embodiments, the structural elements physically interact with the large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleotide sequence of the ITR, the space between two or more elements, or any combination of the above. In one embodiment, the structural elements are selected from the group consisting of the A and A' arms, the B and B' arms, the C and C' arms, the D arm, the Rep binding sites (RBE) and RBE' (i.e., complementary RBE sequences), and the terminal resolution site (trs).

[0168] As a mere example, Table 1 shows exemplary combinations of WT-ITRs.

[0169] Table 1: Exemplary combinations of WT-ITRs from the same serotype or different serotypes, or different parvoviruses. The order shown does not indicate the ITR position; for example, "AAV1, AAV2" indicates that the ceDNA can contain the WT-AAV1 ITR at the 5' position and the WT-AAV2 ITR at the 3' position, or vice versa, the WT-AAV2 ITR at the 5' position and the WT-AAV1 ITR at the 3' position. Abbreviations: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12); AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes (e.g., NCBI: NC002077; NC001401; NC001729; NC001829; NC006152; NC006260; NC006261), ITRs from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV), canine, equine, and ovine AAV), ITRs from parvovirus B19 (GenBank accession number: NC000883), minute virus of mice (MVM) from mice (GenBank accession number NC001510); goose: goose parvovirus (GenBank accession number: NC001701); snake: snake parvovirus 1 (GenBank accession number NC006148).

Table 1-1

Table 1-2

Table 1-3

[0170] As a mere example, Table 2 shows exemplary sequences of WT-ITRs from several different AAV serotypes.

Table 2

[0171] In some embodiments, the nucleotide sequence of the WT-ITR can be modified (e.g., by modifying 1, 2, 3, 4, or 5 or more nucleotides or any range therein), whereby the modification is a substitution of complementary nucleotides, e.g., G for C and vice versa, T for A and vice versa.

[0172] In certain embodiments of the invention, the synthetically produced ceDNA vector does not have a WT-ITR consisting of a nucleotide sequence selected from any of SEQ ID NOs: 1, 2, 5-14. In alternative embodiments of the invention, when the ceDNA vector has a WT-ITR comprising a nucleotide sequence selected from any of SEQ ID NOs: 1, 2, 5-14, the adjacent ITRs are also WT, and the ceDNA contains, for example, regulatory switches as disclosed herein and in International Application No. PCT / US18 / 49996 (see, e.g., Table 11 of PCT / US18 / 49996). In some embodiments, the ceDNA vector contains a selected WT-ITR having a regulatory switch as disclosed herein and a nucleotide sequence selected from any of the group consisting of SEQ ID NOs: 1, 2, 5-14.

[0173] The ceDNA vectors described herein may include a WT-ITR structure that retains operable RBE, trs, and RBE' moieties. Figures 2A and 2B, which use the wild-type ITR for illustrative purposes, show one possible mechanism for the manipulation of the trs site within the wild-type ITR structure portion of the ceDNA vector. In some embodiments, the ceDNA vector includes one or more functional WT-ITR polynucleotide sequences that include a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAV2) and a terminal resolution site (TRS, 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one WT-ITR is functional. In an alternative embodiment where the ceDNA vector includes two WT-ITRs that are substantially symmetric to each other, at least one WT-ITR is functional and at least one WT-ITR is non-functional.

[0174] B. General modified ITRs (mod-ITRs) of ceDNA vectors containing asymmetric ITR pairs or symmetric ITR pairs As discussed herein, synthetically produced ceDNA vectors may include symmetric ITR pairs or asymmetric ITR pairs. In either case, one or both of the ITRs may be modified ITRs, and the difference is that in the first case (i.e., symmetric mod-ITR), the mod-ITRs have the same three-dimensional spatial configuration (i.e., have the same A-A', C-C', and B-B' arm configurations), but in the second case (i.e., asymmetric mod-ITR), the mod-ITRs have different three-dimensional spatial configurations (i.e., have different configurations of the A-A', C-C', and B-B' arms).

[0175] In some embodiments, a modified ITR is an ITR modified by deletions, insertions, and / or substitutions as compared to a wild-type ITR sequence (e.g., AAV ITR). In some embodiments, at least one of the ITRs of the ceDNA vector comprises a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 60) and a functional terminal resolution site (TRS; e.g., 5'-AGTT-3', SEQ ID NO: 62). In one embodiment, at least one of the ITRs is a non-functional ITR. In one embodiment, the different or modified ITRs are each not wild-type ITRs from different serotypes.

[0176] Certain alterations and mutations of ITRs are described in detail herein, but in the context of ITRs, "altered", "mutated", or "modified" indicates that a nucleotide has been inserted, deleted, and / or substituted relative to a wild-type, reference, or original ITR sequence. An altered or mutated ITR can be an engineered ITR. As used herein, "engineered" refers to a manner that has been manipulated by human hand. For example, a polypeptide is considered "engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by human hand and is different from the manner in which it exists in nature.

[0177] In some embodiments, the mod-ITR can be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITR does not contain AAV-based sequences. In yet another embodiment, the synthetic ITR preserves the above ITR structure but has few or no AAV source sequences. In some aspects, the synthetic ITR can preferably interact with wild-type Rep or Rep of a particular serotype, or in some cases, is not recognized by wild-type Rep and is only recognized by a mutated Rep.

[0178] One skilled in the art can determine the corresponding sequences in other serotypes by known means. For example, it is determined whether there are changes in the A, A’, B, B’, C, C’, or D regions, and the corresponding regions in another serotype are determined. The corresponding sequences can be determined using BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs in their default states. The present invention further provides a population containing mod-ITRs and a plurality of ceDNA vectors from combinations of different AAV serotypes. That is, one mod-ITR can be derived from one AAV serotype, and another mod-ITR can be derived from a different serotype. Without being bound by theory, in one embodiment, one ITR can be derived from or based on the AAV2 ITR sequence, and the other ITRs of the ceDNA vector can be derived from or based on any one or more of the ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[0179] Any parvovirus ITR can be used for modification as an ITR or as a base ITR. Preferably, the parvovirus is a dependovirus. More preferably, it is AAV. The serotype selected can be based on the tissue tropism of that serotype. AAV2 has a broad tissue tropism, AAV1 preferably targets neurons and skeletal muscle, and AAV5 targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 preferably targets skeletal muscle and lung. AAV8 preferably targets liver, skeletal muscle, heart, and pancreatic tissues. AAV9 preferably targets liver, bone, and lung tissues. In one embodiment, the modified ITR is based on the AAV2 ITR.

[0180] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleotide sequence of a structural element can be modified as compared to the wild-type sequence of the ITR. In one embodiment, the structural elements of the ITR (e.g., A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from different parvoviruses. For example, the replacement structure can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, a hepadnavirus (e.g., Royal Pyson parvovirus), a bovine parvovirus, a caprine parvovirus, an avian parvovirus, a canine parvovirus, an equine parvovirus, a shrimp parvovirus, a porcine parvovirus, or an insect AAV. For example, the ITR can be an AAV2 ITR, and the A or A' arm or RBE can be replaced with a structural element from AAV5. In another example, the ITR can be an AAV5 ITR, and the C or C' arm, RBE, and trs can be replaced with structural elements from AAV2. In another example, the AAV ITR can be an AAV5 ITR, and the B and B' arms can be replaced with the AAV2 ITR B and B' arms.

[0181] As a mere example, Table 3 shows exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in the region of the modified ITR, where X represents a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any of the regions of C and / or C’ and / or B and / or B’ retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. For example, if the modification results in either a single-arm ITR (e.g., a single C-C’ arm, or a single B-B’ arm), or a modified C-B’ arm or C’-B arm, or a two-arm ITR with at least one cleaved arm (e.g., a cleaved C-C’ arm and / or a cleaved B-B’ arm), then at least the single arm, or at least one of the arms of the two-arm ITR (one arm can be cleaved) retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In some embodiments, the cleaved C-C’ arm and / or the cleaved B-B’ arm has three consecutive T nucleotides (i.e., TTT) in the terminal loop.

Table 3

[0182] In some embodiments, the mod-ITR for use in a synthetically produced ceDNA vector comprising an asymmetric ITR pair or a symmetric mod-ITR pair disclosed herein can be any one of the combinations of modifications shown in Table 3, or can include modifications of at least one nucleotide in any one or more of the regions selected from between A’ and C, between C and C’, between C’ and B, between B and B’. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the C or C’ or B or B’ region still preserves the terminal loop of the stem-loop. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C’ and / or between B and B’ retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In an alternative embodiment, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C’ and / or between B and B’ retains three consecutive A nucleotides (i.e., AAA) in at least one terminal loop. In some embodiments, the modified ITR for use herein can be any one of the combinations of modifications shown in Table 3, or can include modifications (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any one or more of the regions selected from A’, A, and / or D. For example, in some embodiments, the modified ITR for use herein can be any one of the combinations of modifications shown in Table 3 and can also include at least one modification (e.g., deletion, insertion, and / or substitution) in the A region. In some embodiments, the modified ITR for use herein can be any one of the combinations of modifications shown in Table 3 and can include modifications (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A’ region.In some embodiments, a modified ITR for use herein may comprise any one of the combinations of modifications shown in Table 3, and / or a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A and / or A' region. In some embodiments, a modified ITR for use herein may comprise any one of the combinations of modifications shown in Table 3, and / or a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the D region.

[0183] In one embodiment, the nucleotide sequence of the structural element can be modified (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein) to produce a modified structural element. In one embodiment, specific modifications to the ITR are exemplified herein (e.g., SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187), or are shown in FIGS. 7A-7B of PCT / US2018 / 064242, filed Dec. 6, 2018 (e.g., SEQ ID NOs: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 of PCT / US2018 / 064242). In some embodiments, the ITR can be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein). In other embodiments, the ITR is one of the modified ITRs of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with the RBE-containing sections of the A-A' arm and the C-C' and B-B' arms shown in Tables 2-9 of International Application No. PCT / US18 / 49996, which is hereby incorporated by reference in its entirety (i.e., SEQ ID NOs: 110-112, 115-190, 200-468).

[0184] In some embodiments, the modified ITR can include, for example, removal or deletion of all or a portion of a particular arm, such as all or a portion of the A-A' arm, or all or a portion of the B-B' arm, or all or a portion of the C-C' arm, or removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs forming the stem of the loop (see, e.g., ITR-21 of FIG. 7A of PCT / US2018 / 064242 filed Dec. 6, 2018) as long as the final loop that caps the stem (e.g., a single arm) still exists. In some embodiments, the modified ITR can include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, the modified ITR can include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm (see, e.g., ITR-1 of FIG. 3B or ITR-45 of FIG. 7A of PCT / US2018 / 064242). In some embodiments, the modified ITR can include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. Any combination of base pair removals is contemplated, for example, 6 base pairs in the C-C' arm and 2 base pairs in the B-B' arm can be removed. As an exemplary embodiment, FIG. 3B shows an exemplary modified ITR having at least 7 base pairs deleted from each of the C and C' portions such that the modified ITR includes two arms where at least one arm (e.g., C-C') is cleaved, nucleotide substitutions in the loop between the C and C' regions, and at least 1 base pair deletion from each of the B and B' regions. In some embodiments, the modified ITR also includes at least 1 base pair deletion from each of the B and B' regions, thereby also cleaving the B-B' arm relative to the WT ITR.

[0185] In some embodiments, the modified ITR may have a 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR may have a 1-30 nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR has a 2-20 nucleotide deletion relative to the full-length wild-type ITR sequence.

[0186] In some embodiments, the modified ITR does not contain any nucleotide deletions in the RBE-containing portion of the A or A' region so as not to interfere with DNA replication (e.g., binding of the Rep protein to the RBE or nicking at the terminal resolution site). In some embodiments, the modified ITRs included for use herein have one or more deletions in the B, B', C, and / or C' regions described herein.

[0187] In some embodiments, a synthetically produced ceDNA vector comprising a symmetric ITR pair or an asymmetric ITR pair comprises at least one selected modified ITR having a nucleotide sequence selected from the group consisting of a regulatory switch as disclosed herein and SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187.

[0188] In another embodiment, the structure of the structural element can be modified. For example, the structural element can vary in the height of the stem and / or the number of nucleotides in the loop. For example, the height of the stem can be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. In one embodiment, the height of the stem can be from about 5 nucleotides to about 9 nucleotides and can interact functionally with Rep. In another embodiment, the height of the stem can be about 7 nucleotides and can interact functionally with Rep. In another example, the loop can have 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0189] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites in the RBE or extended RBE can be increased or decreased. In one example, the RBE or extended RBE can include 1, 2, 3, 4, 5, or 6 or more GAGY binding sites, or any range therein. Each GAGY binding site can independently be an exact GAGY sequence or a sequence similar to GAGY as long as the sequence is sufficient to bind to the Rep protein.

[0190] In another embodiment, the space between two elements (such as, but not limited to, the RBE and the hairpin) can be altered (e.g., increased or decreased) to change the functional interaction with the large Rep protein. For example, the space can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any range therein.

[0191] The synthetically produced ceDNA vectors described herein may include ITR structures modified with respect to the wild-type AAV2 ITR structures disclosed herein, but still retain operable RBE, trs, and RBE' moieties. Figures 2A and 2B show one possible mechanism for the manipulation of the trs site within the wild-type ITR construct of the ceDNA vector. In some embodiments, the ceDNA vector includes one or more functional ITR polynucleotide sequences comprising a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAV2) and a terminal resolution site (TRS, 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one ITR (wild-type or modified ITR) is functional. In alternative embodiments where the ceDNA vector includes two modified ITRs that are different from each other or asymmetric, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0192] In some embodiments, the synthetically produced ceDNA vector does not have a modified ITR selected from any sequence consisting of or consisting essentially of SEQ ID NOs: 500-529, as provided herein. In some embodiments, the ceDNA vector does not have an ITR selected from any sequence selected from SEQ ID NOs: 500-529.

[0193] In some embodiments, the modified ITRs (e.g., left or right ITRs) of the synthetically produced ceDNA vectors described herein have modifications within the loop arm, cleavage arm, or spacer. Exemplary sequences of ITRs having modifications within the loop arm, cleavage arm, or spacer are set forth in Table 2 (i.e., SEQ ID NOs: 135-190, 200-233); Table 3 (e.g., SEQ ID NOs: 234-263); Table 4 (e.g., SEQ ID NOs: 264-293); Table 5 (e.g., SEQ ID NOs: 294-318 herein); Table 6 (e.g., SEQ ID NOs: 319-468); and Tables 7-9 (e.g., SEQ ID NOs: 101-110, 111-112, 115-134) or Tables 10A or 10B (e.g., SEQ ID NOs: 9, 100, 469-483, 484-499) of International Application No. PCT / US18 / 49996, which is hereby incorporated by reference in its entirety.

[0194] In some embodiments, the modified ITRs for use in synthetically produced ceDNA vectors that include asymmetric ITR pairs or symmetric mod-ITR pairs are selected from any of those shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, and 10A-10B of International Application No. PCT / US18 / 49996, which is hereby incorporated by reference in its entirety, or combinations thereof.

[0195] Additional exemplary modified ITRs for use in synthetically produced ceDNA vectors that include asymmetric ITR pairs or symmetric mod-ITR pairs for each of the above classes are shown in Tables 4A and 4B. The predicted secondary structure of the right modified ITR of Table 4A is shown in FIG. 7A of International Application No. PCT / US2018 / 064242, filed Dec. 6, 2018, and the predicted secondary structure of the left modified ITR of Table 4B is shown in FIG. 7B of International Application No. PCT / US2018 / 064242, which is hereby incorporated by reference in its entirety, filed Dec. 6, 2018.

[0196] Tables 4A and 4B show exemplary right and left modified ITRs.

[0197] Table 4A: Exemplary Modified Right ITR These exemplary modified right ITRs may include the RBE GCGCGCTCGCTCGCTC-3’ (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70), and the RBE’ (i.e., the complement to the RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71). [Table 4A]

[0198] Table 4B: Exemplary Modified Left ITR These exemplary modified left ITRs may include the RBE GCGCGCTCGCTCGCTC-3’ (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70), and the RBE complement (RBE’) GAGCGAGCGAGCGCGC (SEQ ID NO: 71). [Table 4B]

[0199] In one embodiment, a synthetically produced ceDNA vector comprises, in the 5´ to 3´ direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5´ ITR) and the second ITR (3´ ITR) are asymmetric with respect to each other. That is, they have different three-dimensional spatial configurations. As an exemplary embodiment, the first ITR can be a wild-type ITR, the second ITR can be a mutant or modified ITR, or vice versa, the first ITR can be a mutant or modified ITR, and the second ITR can be a wild-type ITR. In some embodiments, both the first ITR and the second ITR are mod-ITRs, but have different sequences or different modifications, and thus are not the same modified-type ITR and have different three-dimensional spatial configurations. In other words, a ceDNA vector using asymmetric ITRs contains ITRs where any change in one ITR with respect to the WT-ITR is not reflected in the other ITR, or, in the case of having an asymmetric ITR pair where the ITRs are modified, can have different sequences and different three-dimensional shapes with respect to each other. Exemplary asymmetric ITRs for use in generating ceDNA-plasmids in the ceDNA vector are shown in Tables 4A and 4B.

[0200] In an alternative embodiment, a synthetically produced ceDNA vector comprises two symmetric mod-ITRs. That is, both ITRs have the same sequence but are reverse complementary strands (inverted) of each other. In some embodiments, the symmetric mod-ITR pair comprises at least one or any combination of deletions, insertions, or substitutions compared to the wild-type ITR sequence from the same AAV serotype. The addition, deletion, or substitution of the symmetric ITRs is the same but is the reverse complementary strand of each other. For example, the insertion of three nucleotides into the C region of the 5’ ITR is reflected by the insertion of three reverse complementary nucleotides into the corresponding section of the C’ region of the 3’ ITR. For illustrative purposes only, if the addition is AACG in the 5’ ITR, the addition is CGTT in the 3’ ITR at the corresponding site. For example, if the 5’ ITR sense strand is [Chemical formula] and there is [Chemical formula] with the addition of, resulting in the sequence [Chemical formula] in the case of bringing about. The corresponding 3' ITR sense strand is [Chemical formula] and there is [Chemical formula] (i.e., the reverse complementary strand of AACG) with the addition of, resulting in the sequence [Chemical formula] bringing about.

[0201] In an alternative embodiment, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetric three-dimensional shapes. For example, one modified ITR may be derived from one serotype and another modified ITR may be derived from a different serotype, but they have the same mutation (e.g., nucleotide insertion, deletion, or substitution) in the same region. In other words, for illustrative purposes only, the 5' mod-ITR may be derived from AAV2 and have a deletion in the C region, and the 3' mod-ITR may be derived from AAV5 and have a corresponding deletion in the C' region. They are included for use as a modified ITR pair herein provided that the 5' mod-ITR and the 3' mod-ITR have the same or symmetric three-dimensional spatial configurations.

[0202] In some embodiments, substantially symmetric mod-ITR pairs have the same A, C-C’, and B-B’ loops in three-dimensional space. For example, if a modified ITR of a substantially symmetric mod-ITR pair has a deletion in the C-C’ arm, the cognate mod-ITR has a corresponding deletion in the C-C’ loop and has a similar three-dimensional structure of the remaining A and B-B’ loops of the same shape in the geometric space of the cognate mod-ITR. As a mere example, substantially symmetric ITRs can have a symmetric spatial configuration such that their structures are of the same shape in geometric space. This can occur, for example, when a G-C pair is modified to, for example, a C-G pair, or vice versa, or when an A-T pair is modified to a T-A pair, or vice versa. Thus,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0203] Table 5 shows exemplary symmetrically modified ITR pairs (i.e., left-modified ITR and symmetric right-modified ITR). The bold (red) portions of the sequences identify the partial ITR sequences (i.e., the sequences of the A-A’, C-C’, and B-B’ loops) also shown in FIGS. 31A - 46B. These exemplary modified ITRs can include the RBE GCGCGCTCGCTCGCTC-3’ (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70), and the RBE’ (i.e., the complement to the RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 5-1

Table 5-2

Table 5-3

[0204] In some embodiments, a ceDNA vector comprising an asymmetric ITR pair can have an ITR sequence or ITR subsequence shown in any one or more of Tables 4A - 4B herein, or as shown in FIG. 7A or 7B of International Application No. PCT / US2018 / 064242, filed on December 6, 2018 (which is incorporated herein by reference in its entirety), or can have a modification corresponding to any of the modifications in the sequences disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9, or 10A - 10B of International Application No. PCT / US18 / 49996, filed on September 7, 2018 (which is incorporated herein by reference in its entirety).

[0205] V. Exemplary ceDNA Vectors As described above, the present disclosure relates to a synthetically produced recombinant ceDNA expression vector and a ceDNA vector encoding a transgene comprising any one of the above asymmetric ITR pairs, symmetric ITR pairs, or substantially symmetric ITR pairs. In certain embodiments, the present disclosure relates to a synthetically produced recombinant ceDNA vector having adjacent ITR sequences and a transgene, wherein the ITR sequences are asymmetric, symmetric, or substantially symmetric relative to each other as defined herein, and the ceDNA further comprises a nucleotide sequence of interest (e.g., an expression cassette comprising a nucleic acid of the transgene) located between the adjacent ITRs, and the nucleic acid molecule lacks a viral capsid protein coding sequence.

[0206] A synthetically produced ceDNA expression vector can be any ceDNA vector that is amenable to recombinant DNA procedures comprising the nucleotide sequence(s) described herein, if at least one ITR is modified. The synthetically produced ceDNA vector of the present disclosure is compatible with the host cell into which the ceDNA vector is introduced. In certain embodiments, the synthetically produced ceDNA vector may be linear. In certain embodiments, the synthetically produced ceDNA vector may exist as an episomal entity. In certain embodiments, the synthetically produced ceDNA vector of the present disclosure may comprise element(s) that enable integration of the donor sequence into the genome of the host cell. As used herein, "transgene" and "heterologous nucleotide sequence" are synonymous.

[0207] Referring now to FIGS. 1A-1G, there are shown schematic diagrams of the functional components of two non-limiting plasmids useful for synthetically producing the ceDNA vectors of the present disclosure. FIGS. 1A, 1B, 1D, 1F show the constructs of the ceDNA vectors or the corresponding sequences of the ceDNA plasmids, where the first and second ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. In some embodiments, an expressible transgene cassette optionally includes an enhancer / promoter, one or more homology arms, a donor sequence, a post-transcriptional regulatory element (e.g., WPRE, e.g., SEQ ID NO: 67), as well as a polyadenylation and termination signal (e.g., BGH polyA, e.g., SEQ ID NO: 68).

[0208] FIG. 5 is a gel confirming the production of ceDNA vectors produced using the synthetic process as described herein and in the examples. The generation of the ceDNA vectors is confirmed by the characteristic band pattern in the gel, as discussed with respect to FIG. 4B above and in the examples.

[0209] A. Regulatory Element The ceDNA vectors described herein and produced using the synthetic processes described herein may include asymmetric ITR pairs or symmetric ITR pairs as defined herein, and may further include specific combinations of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments, the ITR may act as a promoter for the transgene. In some embodiments, the ceDNA vector includes additional components for regulating the expression of the transgene, such as regulatory switches described herein, and regulates the expression of a kill switch that can kill the transgene or the cell containing the ceDNA vector. Regulatory elements including regulatory switches that may be used in the present invention are more fully discussed in International Application No. PCT / US18 / 49996, which is incorporated herein by reference in its entirety.

[0210] In an embodiment, the second nucleotide sequence comprises a regulatory sequence and a nucleotide sequence encoding a nuclease. In certain embodiments, the gene regulatory sequence is operably linked to the nucleotide sequence encoding the nuclease. In certain embodiments, the regulatory sequence is suitable for controlling the expression of the nuclease in a host cell. In certain embodiments, the regulatory sequence comprises a suitable promoter sequence capable of directing the transcription of a gene operably linked to a nucleotide sequence encoding a nuclease (s) of the present disclosure, such as a promoter sequence. In certain embodiments, the second nucleotide sequence comprises an intron sequence linked to the 5' end of the nucleotide sequence encoding the nuclease. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the efficacy of the promoter. In certain embodiments, the regulatory sequence comprises an enhancer and a promoter, the second nucleotide sequence comprises an intron sequence upstream of the nucleotide sequence encoding the nuclease, the intron comprises one or more nuclease cleavage sites (s), and the promoter is operably linked to the nucleotide sequence encoding the nuclease.

[0211] ceDNA vectors produced using the synthetic processes described herein may further comprise specific combinations of cis-regulatory elements such as the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67) and the BGH polyA (SEQ ID NO: 68). Suitable expression cassettes for use in expression constructs are not limited by the packaging constraints imposed by the viral capsid.

[0212] (i). Promoter: Those skilled in the art will understand that the promoters used in the synthetically produced ceDNA vectors of the present invention should be appropriately adjusted for the specific sequences they promote. For example, guide RNAs may not require a promoter at all because their function is to form a duplex with a specific target sequence on native DNA to generate recombination events. In contrast, nucleases encoded by ceDNA vectors can be expressed efficiently and optionally regulably from the vector because they benefit from a promoter.

[0213] The expression cassette of the present invention includes a promoter that can affect cell specificity along with the overall expression level. In the case of transgene expression, they may include very active early promoters derived from viruses. The expression cassette may contain a tissue-specific eukaryotic cell promoter to restrict transgene expression to specific cell types and reduce toxic effects and immune responses resulting from unregulated ectopic expression. In a preferred embodiment, the expression cassette may contain a synthetic regulatory element such as the CAG promoter (SEQ ID NO: 72). The CAG promoter includes (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and the first intron of the chicken β-actin gene, and (iii) a splice acceptor of the rabbit β-globin gene. Alternatively, the expression cassette may contain the α-1-antitrypsin (AAT) promoter (SEQ ID NO: 73 or SEQ ID NO: 74), the liver-specific (LP1) promoter (SEQ ID NO: 75 or SEQ ID NO: 76), or the human elongation factor-1α (EF1a) promoter (e.g., SEQ ID NO: 77 or SEQ ID NO: 78). In some embodiments, the expression cassette includes one or more constitutive promoters, such as the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), or the cytomegalovirus (CMV) early promoter (optionally having a CMV enhancer, e.g., SEQ ID NO: 79). Alternatively, inducible promoters, native promoters of transgenes, tissue-specific promoters, or various promoters known in the art can be used.

[0214] Suitable promoters, including those described above, may be derived from viruses and thus may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Using suitable promoters, any RNA polymerase (e.g., pol It can be driven by expression by I, pol II, and pol III. Exemplary promoters include the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6, e.g., SEQ ID NO: 80) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), the human H1 promoter (H1) (e.g., SEQ ID NO: 81 or SEQ ID NO: 155), the CAG promoter, the human α1-antitrypsin (HAAT) promoter (e.g., SEQ ID NO: 82), etc., but are not limited thereto. In certain embodiments, these promoters are modified at their downstream intron-containing ends and contain one or more nuclease cleavage sites. In certain embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.

[0215] In one embodiment, the promoter used is the native promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of each gene encoding the therapeutic protein are known and characterized. The promoter region used may further contain one or more additional regulatory sequences (e.g., native), such as enhancers (e.g., SEQ ID NO: 79 and SEQ ID NO: 83).

[0216] Non-limiting examples of suitable promoters for use according to the present invention include, for example, the CAG promoter (SEQ ID NO: 72), the HAAT promoter (SEQ ID NO: 82), the human EF1-α promoter (SEQ ID NO: 77), or the EF1a promoter (SEQ ID NO: 78), the IE2 promoter (e.g., SEQ ID NO: 84), and the rat EF1-α promoter (SEQ ID NO: 85), or the 1E1 promoter fragment (SEQ ID NO: 125).

[0217] (ii). Polyadenylation sequence: Sequences encoding polyadenylation sequences can be included in the ceDNA vector to stabilize the mRNA expressed from synthetically produced ceDNA vectors and to assist in nuclear transport and translation. In one embodiment, the synthetically produced ceDNA vector does not contain a polyadenylation sequence. In other embodiments, the vector contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine nucleotides. In some embodiments, the polyadenylation sequence includes about 43 nucleotides, about 40 - 50 nucleotides, about 40 - 55 nucleotides, about 45 - 50 nucleotides, about 35 - 50 nucleotides, or any range therebetween.

[0218] Expression cassettes can include naturally occurring sequences isolated from polyadenylation sequences known in the art or variants thereof, such as bovine BGHpA (e.g., SEQ ID NO: 68) or viral SV40pA (e.g., SEQ ID NO: 86), or synthetic sequences (e.g., SEQ ID NO: 87). Some expression cassettes can also include the SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the USE can be used in combination with SV40pA or a heterologous polyA signal.

[0219] The expression cassette may also include post-transcriptional elements to increase the expression of the transgene. In some embodiments, the Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67) is used to increase the expression of the transgene. Other post-transcriptional processing elements, such as post-transcriptional elements from the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV), can be used. The secretion sequence can be linked to the transgene, e.g., the VH-02 and VK-A26 sequences, e.g., SEQ ID NO: 88 and SEQ ID NO: 89.

[0220] (iii). Nuclear localization sequence In some embodiments, vectors encoding RNA-guided endonucleases include one or more nuclear localization sequences (NLSs), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, one or more NLSs are located at or near the amino terminus, at or near the carboxy terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and / or one or more NLSs at the carboxy terminus). When multiple NLSs are present, each can be selected independently of the other NLSs, such that a single NLS is present in multiple copies and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs are shown in Table 6.

Table 6

[0221] E. Additional components of the ceDNA vector ceDNA vectors produced using the synthetic processes described herein may contain nucleotides encoding other components for gene expression. For example, to select for a particular gene targeting event, a protective shRNA can be embedded within a microRNA and inserted into a recombinant ceDNA vector designed to site-specifically integrate into a highly active locus such as the albumin locus. Such embodiments may provide a system for in vivo selection and expansion of genetically modified hepatocytes in any genetic background, as described in Nygaard et al., A universal system to select gene-modified hepatocytes in vivo, Gene Therapy, June 8, 2016. The ceDNA vectors of the present disclosure may contain one or more selectable markers that enable the selection of cells that have been transformed, transfected, transduced, etc. Selectable markers are genes whose products provide biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophy, NeoR, etc. In certain embodiments, a positive selectable marker is incorporated into a donor sequence such as NeoR. A negative selectable marker can be incorporated downstream of the donor sequence. For example, the nucleic acid sequence HSV-tk encoding a negative selectable marker can be incorporated into a nucleic acid construct downstream of the donor sequence.

[0222] In embodiments, ceDNA vectors produced using the synthetic processes described herein can be used for gene editing, as disclosed, for example, in International Application No. PCT / US2018 / 064242, filed December 6, 2018, which is hereby incorporated by reference in its entirety, and may contain one or more of a 5' homology arm, a 3' homology arm, upstream of the polyadenylation site, and proximal to the 5' homology arm. Exemplary homology arms are the 5' and 3' albumin homology arms (SEQ ID NOs: 151 and 152) or the CCR5 5' and 3' homology arms (e.g., SEQ ID NOs: 153, 154).

[0223] F. Regulatory Switch A molecular regulatory switch is one that generates a measurable change in state in response to a signal. Such a regulatory switch can be usefully combined with a ceDNA vector produced using the synthetic processes described herein to control the output of transgene expression from the ceDNA vector. In some embodiments, the ceDNA vector includes a regulatory switch that serves to finely tune the expression of the transgene. For example, it can serve as a biological containment function of the ceDNA vector. In some embodiments, the switch is an “on / off” switch designed to initiate or stop (i.e., shut down) the controllable and regulatable expression of a gene of interest in the ceDNA vector. In some embodiments, the switch can include a “kill switch” that, once activated, can instruct a cell containing the ceDNA vector to undergo programmed cell death. Exemplary regulatory switches included for use with ceDNA vectors can be used to regulate the expression of transgenes and are more fully discussed in International Application No. PCT / US18 / 49996, which is hereby incorporated by reference in its entirety.

[0224] (i) Binary regulatory switch In some embodiments, the ceDNA vectors produced using the synthetic processes described herein include regulatory switches that can help controllably regulate the expression of transgenes. For example, an expression cassette located between the ITRs of a ceDNA vector can additionally include a regulatory region operably linked to a gene of interest, such as a promoter, cis-element, repressor, enhancer, etc., and this regulatory region is regulated by one or more cofactors or exogenous agents. By way of mere example, the regulatory region can be regulated by a small molecule switch or an inducible or repressible promoter. Non-limiting examples of inducible promoters are hormone-inducible or metal-inducible promoters. Other exemplary inducible promoter / enhancer elements include, but are not limited to, the RU486-inducible promoter, the ecdysone-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

[0225] (ii) Small molecule regulatory switches Various small molecule-based regulatory switches known in the art can be combined with the synthetically produced ceDNA vectors disclosed herein to form ceDNA vectors controlled by the regulatory switches. In some embodiments, the regulatory switch is an orthogonal ligand / nuclear receptor pair, such as an artificial promoter that controls the expression of an operably linked transgene, such as those disclosed in Taylor, et al., BMC Biotechnology 10(2010):15; an engineered steroid receptor, such as a modified progesterone receptor having a C-terminal truncation that cannot bind progesterone but can bind RU486 (mifepristone) (U.S. Patent No. 5,364,791); the ecdysone receptor from Drosophila and their ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517), or Sando R 3 rd;It may be selected from any one or a combination of the switches controlled by the antibiotic trimethoprim (TMP) disclosed in Nat Methods. 2013, 10(11): 1085-8. In some embodiments, the regulatory switch for controlling the transgene expressed by the ceDNA vector is a prodrug activation switch such as those disclosed in US Patent Nos. 8,771,679 and 6,339,070.

[0226] (iii) "Passcode" regulatory switch In some embodiments, the regulatory switch can be a "passcode switch" or a "passcode circuit". The passcode switch enables fine-tuning of the control of transgene expression from the synthetically produced ceDNA vector when certain conditions occur. That is, for transgene expression and / or suppression to occur, a combination of conditions must exist. For example, for transgene expression to occur, at least conditions A and B must occur. The passcode regulatory switch can be any number of conditions. For example, for transgene expression to occur, at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or more conditions must exist. In some embodiments, at least two conditions (e.g., conditions A, B) must occur, and in some embodiments, at least three conditions must occur (e.g., A, B, and C or A, B, and D). As a mere example, for gene expression from ceDNA having a passcode "ABC" regulatory switch to occur, conditions A, B, and C must be present. Conditions A, B, and C can be as follows. Condition A is the presence of a pathological condition or disease, condition B is a hormonal response, and condition C is a response to transgene expression. For example, when the transgene edits a defective EPO gene, condition A is the presence of chronic kidney disease (CKD), condition B occurs when the subject has a hypoxic state in the kidney, and condition C is either insufficient mobilization of erythropoietin-producing cells (EPCs) in the kidney or alternatively insufficient activation of HIF-2. Once the oxygen level increases or the desired EPO level is reached, the transgene turns off again until the three conditions occur and then turns back on.

[0227] In some embodiments, the passcode regulatory switch or “passcode circuit” incorporated for use in synthetically produced ceDNA vectors includes hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biological containment conditions. In contrast to a dead man switch that triggers cell death in the presence of a default condition, the “passcode circuit” enables cell survival or transgene expression in the presence of a specific “passcode” and can be readily reprogrammed to allow transgene expression and / or cell survival only when a given environmental condition or passcode is present.

[0228] Any and all combinations of the regulatory switches disclosed herein, such as small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulatory radiation control switches, hypoxia-mediated switches, and other regulatory switches known to those of skill in the art disclosed herein, can be used in the passcode regulatory switches disclosed herein. The regulatory switches incorporated for use are also discussed in the review article Kis et al., J R Soc Interface. 12:20141000 (2015) and summarized in Table 1 of Kis. In some embodiments, the regulatory switch for use in a passcode system can be selected from any of the switches in Table 11, or combinations thereof.

[0229] (iv). Nucleic acid-based regulatory switches for controlling transgene expression In some embodiments, the regulatory switch for controlling the transgene expressed by the synthetically produced ceDNA vector is based on nucleic acid-based regulatory mechanisms. Exemplary nucleic acid regulatory mechanisms are known in the art and are contemplated for use. For example, such mechanisms include riboswitches, such as those disclosed in US2009 / 0305253, US2008 / 0269258, US2017 / 0204477, WO2018 / 026762A1, U.S. Patent No. 9,222,093, and European Patent Application No. EP288071, as well as those disclosed in the review by Villa JK et al., Microbiol Spectr. 2018 May;6(3). Also included are metabolite-responsive transcriptional biosensors, such as those disclosed in WO2018 / 075486 and WO2017 / 147585. Other mechanisms known in the art that are contemplated for use include silencing of the transgene by siRNA or RNAi molecules (e.g., miR, shRNA). For example, the ceDNA vector may include a regulatory switch that encodes an RNAi molecule that is complementary to the transgene expressed by the ceDNA vector. The transgene will be silenced by the complementary RNAi molecule if such RNAi is expressed even when the transgene is expressed by the ceDNA vector, and the transgene will not be silenced by RNAi if RNAi is not expressed when the transgene is expressed by the ceDNA vector.

[0230] In some embodiments, the regulatory switch is, for example, the tissue-specific self-inactivating regulatory switch disclosed in US2002 / 0022018, whereby the regulatory switch intentionally switches off transgene expression at sites where transgene expression could otherwise be disadvantageous. In some embodiments, the regulatory switch is, for example, the recombinase-reversible gene expression system disclosed in US2014 / 0127162 and U.S. Patent No. 8,324,436.

[0231] (v). Post-transcriptional and post-translational regulatory switches. In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the synthetically produced ceDNA vector is a post-transcriptional modification system. For example, such a regulatory switch can be an aptazyme riboswitch sensitive to tetracycline or theophylline, as disclosed in US2018 / 0119156, GB2011 / 07768, WO2001 / 064956A3, European Patent No. 2707487, and Beilstein et al., ACS Synth. Biol., 2015, 4(5), pp 526 - 534, Zhong et al., Elife. 2016 Nov 2;5.pii:e18858. In some embodiments, one of ordinary skill in the art would assume that it is possible to encode both the transgene and inhibitory siRNAs containing a ligand-sensitive (off-switch) aptamer, the end result of which is a ligand-sensitive on-switch.

[0232] (vi). Other exemplary regulatory switches Any known regulatory switch can be used in the synthetically produced ceDNA vector to control the gene expression of the transgene expressed by the ceDNA vector, including those triggered by environmental changes. Additional examples include, but are not limited to, the BOC method of Suzuki et al., Scientific Reports 8;10051(2018), genetic code expansion and non-canonical amino acids, radiation-controlled or ultrasound-controlled on / off switches (e.g., Scott S et (see, e.g., al., Gene Ther. 2000 Jul;7(13):1121-5, U.S. Patent Nos. 5,612,318, 5,571,797, 5,770,581, 5,817,636, and WO1999 / 025385A1). In some embodiments, the regulatory switch is controlled by an implantable system, such as that disclosed in U.S. Patent No. 7,840,263, US2007 / 0190028A1, and gene expression is controlled by one or more forms of energy, including electromagnetic energy that activates a promoter operably linked to the transgene in the ceDNA vector.

[0233] In some embodiments, regulatory switches contemplated for use in synthetically produced ceDNA vectors are hypoxia-mediated or stress-activated switches, such as those disclosed in WO1999 / 060142A2, U.S. Patent Nos. 5,834,306, 6,218,179, 6,709,858, US2015 / 0322410, Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD, and NRSE elements, and conditionally inducible silencing elements (including, e.g., hypoxia response elements (HREs), inflammatory response elements (IREs), and shear stress activation elements (SSAEs) disclosed in U.S. Patent No. 9,394,526). Such embodiments are useful for turning on the expression of transgenes from ceDNA vectors after ischemia or in ischemic tissue and / or tumors.

[0234] (iv). Kill switch Other embodiments of the invention relate to synthetically produced ceDNA vectors that include a kill switch. The kill switches disclosed herein can be used to cause cells containing the ceDNA vector to be killed or undergo programmed cell death as a means of permanently removing the introduced ceDNA vector from the subject system. It will be understood by those skilled in the art that the use of kill switches in the synthetically produced ceDNA vectors of the invention is typically associated with targeting the ceDNA vector to a limited number of cells that the subject can tolerably lose, or cell types in which apoptosis is desirable (e.g., cancer cells). In all aspects, the "kill switch" disclosed herein is designed to effect rapid and robust cell killing of cells containing the ceDNA vector in the absence of an input survival signal or other specified condition. In other words, the kill switch encoded by the ceDNA vector herein can restrict the cell survival of cells containing the ceDNA vector to an environment defined by a specific input signal. Such kill switches serve as a biological containment function when it is desirable to ensure removal of the synthetically produced ceDNA vector from the subject or non-expression of the encoded transgene.

[0235] VI. Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition includes a closed-ended DNA vector, such as a ceDNA vector, produced using the synthetic processes described herein, and a pharmaceutically acceptable carrier or diluent.

[0236] A closed - end DNA vector containing a ceDNA vector produced using the synthetic process described herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to the cells, tissues, or organs of the subject. Typically, the pharmaceutical composition contains a ceDNA vector disclosed herein and a pharmaceutically acceptable carrier. For example, a closed - end DNA vector containing a ceDNA vector produced using the synthetic process described herein can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transfection via high - pressure intravenous or intra - arterial injection, as well as intracellular injection, such as nuclear microinjection or cytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high - concentration synthetically produced closed - end DNA vectors, such as ceDNA vectors. Sterile injectable solutions can be prepared by incorporating the required amount of a synthetically produced closed - end DNA vector, such as a ceDNA vector compound, into a suitable buffer, along with one or a combination of the ingredients listed above, followed by filter sterilization to incorporate the ceDNA vector, and formulated to deliver the transgene in the nucleic acid to the recipient cells, which can result in the therapeutic expression of the transgene or donor sequence therein. This composition can also contain a pharmaceutically acceptable carrier.

[0237] A pharmaceutically active composition containing a closed - end DNA vector containing a ceDNA vector produced using the synthetic process described herein can be formulated to deliver transgenes for various purposes to cells, such as the cells of a subject.

[0238] Pharmaceutical compositions for therapeutic purposes are typically sterile and must be stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high-concentration synthetically produced closed-ended DNA vectors, such as ceDNA vectors. Sterile injectable solutions can be prepared by incorporating the required amount of the synthetically produced closed-ended DNA vector, such as a ceDNA vector compound, in a suitable buffer into one or a combination of the ingredients listed above and then filter sterilizing.

[0239] As disclosed herein, closed-ended DNA vectors containing ceDNA vectors produced using the synthetic processes described herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retrobulbar, intraretinal, subretinal, choroidal, subchoroidal, interstitial, anterior chamber, and vitreous), intravestibular, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transfection via high-pressure intravenous or intra-arterial injection, as well as intracellular injection, such as nuclear microinjection or cytoplasmic injection, are also contemplated.

[0240] In some embodiments, the methods provided herein include delivering to a host cell one or more closed - end DNA vectors comprising a ceDNA vector produced using the synthetic processes described herein. Also provided herein are cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. Methods of nucleic acid delivery can include lipofection, nucleofection, microinjection, biolistic, liposomes, immunoliposomes, polycation or lipid:nucleic acid complexes, naked DNA, and drug - enhanced uptake with DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Delivery can be to cells (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration).

[0241] A variety of techniques and methods for delivering nucleic acids to cells are known in the art. For example, closed - end DNA vectors comprising a ceDNA vector produced using the synthetic processes described herein can be formulated into lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core - shell nanoparticles. Typically, LNPs are composed of a nucleic acid (e.g., ceDNA) molecule, one or more ionizable or cationic lipids (or salts thereof), one or more non - ionizable or neutral lipids (e.g., phospholipids), a molecule to prevent aggregation (e.g., PEG or PEG - lipid conjugate), and optionally a sterol (e.g., cholesterol).

[0242] Another method for delivering a closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein to cells is by conjugating a ligand that is internalized by the cells to the nucleic acid. For example, the ligand can bind to a receptor on the cell surface and be internalized via endocytosis. The ligand can be covalently attached to a nucleotide in the nucleic acid. Exemplary conjugates for delivering nucleic acids into cells are described, for example, in WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515, and WO2017 / 177326.

[0243] Nucleic acids, and closed - end DNA vectors containing ceDNA vectors produced using the synthetic processes described herein, can also be delivered to cells by transfection. Useful transfection methods include, but are not limited to, lipid - mediated transfection, cationic - polymer - mediated transfection, or calcium phosphate precipitation. Transfection reagents are well - known in the art, such as the TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS (trademark) P protein transfection reagent (New England Biolabs), CHARIOT (trademark) protein delivery reagent (Active Motif), PROTEOJUICE (trademark) protein transfection reagent (EMD Millipore), 293fectin, LIPOFECTAMINE (trademark) 2000, LIPOFECTAMINE (trademark) 3000 (Thermo Fisher Scientific), LIPOFECTAMINE (trademark) (Thermo Fisher Scientific), LIPOFECTIN (trademark) (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN (trademark) (Thermo Fisher Scientific), OLIGOFECTAMINE (trademark) (Thermo Fisher Scientific), LIPOFECTACE (trademark), FUGENE (trademark) (Roche, Basel, Switzerland), FUGENE (trademark) HD (Roche), TRANSFECTAM (trademark) (Transfectam, Promega, Madison, Wis.), TFX-10 (trademark) (Promega), TFX-20 (trademark) (Promega), TFX-50 (trademark) (Promega), TRANSFECTIN (trademark) (BioRad, Hercules, Calif.), SILENTFECT (trademark) (Bio-Rad), Effectene (trademark) (Qiagen, Valencia, Calif.), DC-chol (Avanti Polar Lipids), GENEPORTER (trademark) (Gene Therapy Systems, San Diego, Calif.), DHARMAFECT 1 (trademark) (Dharmacon, Lafayette, Colo.), DHARMAFECT 2 (trademark) (Dharmacon), DHARMAFECT 3 (trademark) (Dharmacon), DHARMAFECT 4 (trademark) (Dharmacon), ESCORT (trademark) III (Sigma, St. Louis, Mo.) and ESCORT™ IV (Sigma Chemical Co.), among others, but not limited thereto. Nucleic acids such as ceDNA can also be delivered to cells via microfluidics methods known to those skilled in the art.

[0244] A closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein can also be directly administered to an organism for in vivo cell transduction. Administration can be by any of the routes commonly used to ultimately bring the molecule into contact with blood or tissue cells, including, but not limited to, infusion, injection, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well - known to those skilled in the art, and two or more routes can be used to administer a particular composition, although a particular route is often more immediate and can result in a more effective response than another route.

[0245] Methods for the introduction of a closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein can be delivered to hematopoietic stem cells, for example, by the method described in U.S. Patent No. 5,928,638.

[0246] Closed - end DNA vectors containing ceDNA vectors produced using the synthetic processes described herein can be added to liposomes for delivery to target cells or target organs. Liposomes are vesicles having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of pharmaceutical development. They act by fusing with the cell membrane and rearranging their lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposome compositions for such delivery are composed of compounds having phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids. Exemplary liposomes and liposome formulations are disclosed in International Application No. PCT / US2018 / 050042 filed on September 7, 2018 and International Application No. PCT / US2018 / 064242 filed on December 6, 2018. See, for example, the section entitled "Pharmaceutical Formulations".

[0247] Using various delivery methods known in the art or modifications thereof, closed - end DNA vectors containing ceDNA vectors produced in vitro or in vivo using the synthetic processes described herein can be delivered. For example, in some embodiments, the ceDNA vector is delivered by creating a transient pore in the cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser - based energy, thereby facilitating DNA entry into the targeted cells. For example, the ceDNA vector can be delivered by squeezing cells through size - restricted channels or by transiently disrupting the cell membrane by other means known in the art. In some cases, the ceDNA vector is injected directly into the skin, thyroid, myocardium, skeletal muscle, or liver cells as naked DNA alone. In some cases, the ceDNA vector is delivered by a gene gun. Gold or tungsten spherical particles (1 - 3 μm in diameter) coated with a capsid - free AAV vector can be accelerated at high speed by a pressurized gas and penetrate into the target tissue cells.

[0248] Compositions comprising a closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein and a pharmaceutically acceptable carrier are specifically contemplated herein. In some embodiments, the ceDNA vector is formulated with a lipid delivery system, such as the liposomes described herein. In some embodiments, such compositions are administered by any route desired by a skilled practitioner. The compositions can be administered to a subject by different routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, intrabuccal administration, intrapleural, intravenous, intra - arterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intra - articular or combinations thereof. For veterinary use, the compositions can be administered as appropriately acceptable formulations according to normal veterinary practice. A veterinarian can readily determine the most appropriate dosing schedule and route of administration for a particular animal. The compositions can be administered by conventional syringes, needle - free injection devices, "microprojectile bombardment gene gun", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

[0249] In some cases, a closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein is delivered by hydrodynamic injection, which is a simple and very efficient method for direct intracellular delivery of any water - soluble compound and particles to skeletal muscle throughout the viscera and limbs.

[0250] In some cases, a closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein is delivered by ultrasound by creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles to cells of the viscera or tumors. The size and concentration of the closed - end DNA play a major role in the efficiency of this system. In some cases, a closed - end DNA vector containing a ceDNA vector produced using the synthetic processes described herein is delivered by magnetofection using a magnetic field to concentrate the particles containing the nucleic acid to the target cells.

[0251] In some cases, the chemical delivery system can be used by using, for example, a nanocomposite comprising the compression of a negatively charged nucleic acid by cationic liposomes / micelles or polycationic nanoparticle particles such as polycationic polymers. Examples of cationic lipids used in the delivery method include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol derivative compounds, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.

[0252] A. Exosomes: In some embodiments, a closed-end DNA vector containing a ceDNA vector produced using the synthetic process described herein is delivered by being encapsulated in exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following the fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the cell membrane of the donor cell, they contain the cytosol from the s cell that produced the exosome, and present membrane proteins from the parental cell on the surface. Exosomes are produced by various cell types including epithelial cells, B and T lymphocytes, mast cells (MC), and dendritic cells (DC). In some embodiments, exosomes having diameters of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, 50 nm to 100 nm are contemplated for use. Exosomes can be isolated for delivery to target cells either by using their donor cells or by introducing specific nucleic acids into them. Exosomes containing the capsid-free AAV vector of the present invention can be produced using various approaches known in the art.

[0253] B. Microparticles / Nanoparticles: In some embodiments, a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein is delivered by lipid nanoparticles. Generally, lipid nanoparticles include, for example, ionizable amino lipids (e.g., heptatriaconta - 6,9,28,31 - tetraene - 19 - yl 4 - (dimethylamino) butanoate, DLin - MC3 - DMA), phosphatidylcholine (1,2 - distearoyl - sn - glycero - 3 - phosphocholine, DSPC), cholesterol, and coating lipids (polyethylene glycol - dimyristoyl glycerol, PEG - DMG) as disclosed by, for example, Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498 - 507.

[0254] In some embodiments, the lipid nanoparticles have an average diameter between about 10 and about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter between about 10 and about 300 nm. In some embodiments, the lipid nanoparticles have a diameter less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter between about 25 and about 200 nm. In some embodiments, a lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution and an average size (e.g., diameter) is between about 70 nm and about 200 nm, and more typically, the average size is about 100 nm or less.

[0255] A variety of lipid nanoparticles known in the art can be used to deliver a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein. For example, various delivery methods using lipid nanoparticles are described in U.S. Patent Nos. 9,404,127, 9,006,417, and 9,518,272.

[0256] In some embodiments, the closed - end DNA vectors comprising the ceDNA vectors produced using the synthetic processes described herein are delivered by gold nanoparticles. Generally, nucleic acids can be covalently or non - covalently attached to gold nanoparticles (e.g., attached by charge - charge interactions), as described, for example, by Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 22(6);1075 - 1083. In some embodiments, the gold nanoparticle - nucleic acid complexes are produced using, for example, the methods described in U.S. Patent No. 6,812,334.

[0257] C. Complexes In some embodiments, as disclosed herein, the closed - end DNA vectors comprising the ceDNA vectors produced using the synthetic processes described herein are complexed (e.g., covalently attached to an agent that increases cell uptake). An "agent that increases cell uptake" is a molecule that facilitates the transport of nucleic acids across the lipid membrane. For example, nucleic acids can be complexed with lipophilic compounds (e.g., cholesterol, tocopherol, etc.), cell - penetrating peptides (CPPs) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine). Further examples of agents that increase cell uptake are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7);791 - 809.

[0258] In some embodiments, as disclosed herein, a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein is complexed with a polymer (e.g., a polymer molecule) or a folate molecule (e.g., a folic acid molecule). Generally, the delivery of nucleic acids complexed with polymers is known in the art, as described, for example, in WO2000 / 34343 and WO2008 / 022309. In some embodiments, the ceDNA vectors disclosed herein are complexed with poly(amide) polymers, as described by, for example, U.S. Patent No. 8,987,377. In some embodiments, the nucleic acids described by the present disclosure are complexed with folic acid molecules, as described in U.S. Patent No. 8,507,455.

[0259] In some embodiments, as disclosed herein, a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein is complexed with a carbohydrate, as described, for example, in U.S. Patent No. 8,450,467.

[0260] D. Nanocapsules Alternatively, as disclosed herein, a nanocapsule formulation of a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein can be used. Nanocapsules can generally capture substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such microparticles (approximately 0.1 μm in size) should be designed to be degraded in vivo using a polymer. Biodegradable polyalkyl - cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0261] E. Liposomes A closed - end DNA vector containing a ceDNA vector produced using the synthetic process described herein can be added to liposomes for delivery to a target cell or target organ. Liposomes are vesicles having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of pharmaceutical development. They act by fusing with the cell membrane and rearranging their lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposome compositions for such delivery are composed of compounds having phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0262] The formation and use of liposomes are generally known to those skilled in the art. Liposomes having improved serum stability and circulating half - life have been developed (U.S. Patent No. 5,741,516). Further, various methods of liposomes and liposome - like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0263] F. Exemplary Liposome and Lipid Nanoparticle (LNP) Compositions A closed - end DNA vector containing a ceDNA vector produced using the synthetic process described herein can be added to liposomes for delivery to cells, such as cells that require the expression of a transgene. Liposomes are vesicles having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of pharmaceutical development. They act by fusing with the cell membrane and rearranging their lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposome compositions for such delivery are composed of compounds having phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0264] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, and International Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is hereby incorporated by reference in its entirety and is contemplated for use in the methods and compositions disclosed herein.

[0265] In some embodiments, the disclosure provides liposomal formulations comprising one or more compounds having polyethylene glycol (PEG) functional groups (so-called "PEGylated compounds") that reduce immunogenicity / antigenicity, provide hydrophilicity and hydrophobicity to the compound(s), and can reduce the frequency of administration. Alternatively, the liposomal formulation simply comprises a polyethylene glycol (PEG) polymer as an additional component. In such embodiments, the molecular weight of the PEG or PEG functional group can be from 62 Da to about 5,000 Da.

[0266] In some embodiments, the disclosure provides liposomal formulations that deliver an API having an extended release or controlled release profile over a period of hours to weeks. In some related embodiments, the liposomal formulation can comprise an aqueous chamber bound by a lipid bilayer. In other related embodiments, the liposomal formulation encapsulates an API having a component that undergoes a physical transition at elevated temperature to release the API over a period of hours to weeks.

[0267] In some embodiments, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein. In some embodiments, the liposomal formulation comprises Optisome.

[0268] In some embodiments, the present disclosure provides a liposomal formulation comprising one or more lipids selected from N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol) composite lipid, HSPC (hydrogenated soybean phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoyl phosphatidylcholine); DOPC (dioleoyl phosphatidylcholine); DPPG (dipalmitoyl phosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleoyl phosphatidylserine); POPC (palmitoyl oleoyl phosphatidylcholine); SM (sphingomyelin); MPEG (methoxypolyethylene glycol); DMPC (dimyristoyl phosphatidylcholine); DMPG (dimyristoyl phosphatidylglycerol); DSPG (distearoyl phosphatidylglycerol); DEPC (dielcoyl phosphatidylcholine); DOPE (dioleoyl-sn-glycero-phosphoethanolamine), cholesteryl sulfate (CS), dipalmitoyl phosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphatidylcholine), or any combination thereof.

[0269] In some embodiments, the present disclosure provides a liposomal formulation comprising phospholipids, cholesterol, and PEGylated lipids in a molar ratio of 56:38:5. In some embodiments, the total lipid content of the liposomal formulation is 2 - 16 mg / mL. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. In some embodiments, the present disclosure provides a liposomal formulation comprising a phosphatidylcholine functional group, an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol. In some embodiments, the PEGylated lipid is PEG-2000-DSPE. In some embodiments, the present disclosure provides a liposomal formulation comprising DPPG, soybean PC, MPEG-DSPE lipid complex, and cholesterol.

[0270] In some embodiments, the present disclosure provides a liposomal formulation comprising one or more lipids containing a phosphatidylcholine functional group and one or more lipids containing an ethanolamine functional group. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing one or more phosphatidylcholine functional groups, a lipid containing an ethanolamine functional group, and a sterol, such as cholesterol. In some embodiments, the liposomal formulation comprises DOPC / DEPC and DOPE.

[0271] In some embodiments, the present disclosure provides a liposomal formulation further comprising one or more pharmaceutical excipients, such as sucrose and / or glycine.

[0272] In some embodiments, the present disclosure provides a liposome formulation that is either a single lamellar structure or a multi-lamellar structure. In some embodiments, the present disclosure provides a liposome formulation comprising polyparticle and / or foamed particles. In some embodiments, the present disclosure provides a liposome formulation that is larger in relative size to common nanoparticles and has a size of about 150 - 250 nm. In some embodiments, the liposome formulation is a lyophilized powder.

[0273] In some embodiments, the present disclosure provides a loaded liposome formulation made with the ceDNA vectors disclosed or described herein by adding a weak base to a mixture having ceDNA isolated outside the liposome. This addition increases the pH outside the liposome to approximately 7.3 and drives the API into the liposome. In some embodiments, the present disclosure provides a liposome formulation having a pH that is acidic inside the liposome. In such cases, the inside of the liposome can be pH 4 - 6.9, more preferably pH 6.5. In other embodiments, the present disclosure provides a liposome formulation made by using a liposome drug stabilization technique inside the liposome. In such cases, a polymer or non-polymeric highly charged anion and a liposome entrapping agent, such as polyphosphate or sucrose octasulfate, are utilized.

[0274] In some embodiments, the present disclosure provides lipid nanoparticles comprising a DNA vector comprising a ceDNA vector produced using the synthetic process described herein, and an ionizable lipid. For example, a lipid nanoparticle formulation made and loaded with ceDNA obtained by the process is disclosed in International Application No. PCT / US2018 / 050042, filed on September 7, 2018, and incorporated herein. This can be achieved by high-energy mixing of an ethanol lipid and aqueous ceDNA at low pH, which protonates the ionizable lipid and provides a favorable energetics for particle ceDNA / lipid association and nucleation. The particles can be further stabilized by aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.

[0275] Generally, lipid particles are prepared at a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 30:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and ceDNA can be adjusted to provide a desired N / P ratio, such as an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid particle formulation can be in the range of about 5 mg / mL to about 30 mg / mL.

[0276] Ionizable lipids are typically used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and membrane fusion. Generally, an ionizable lipid is a lipid that is positively charged or contains at least one amino group that is protonated under acidic conditions, such as at a pH of 6.5 or less. Ionizable lipids are also referred to herein as cationic lipids.

[0277] Exemplary ionizable lipids are described in International PCT Patent Publications WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO2012 / 099755, WO2013 / 049328, WO2013 / 086322, WO2013 / 086373, WO2011 / 071860, WO2009 / 132131, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO2010 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346,and WO2013 / 086354, and US Patent Publication Nos. US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, US2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673, US2012 / 0128760, US2010 / 0324120, US2014 / 0200257, US2015 / 0203446, US2018 / 0005363, US2014 / 0308304, US2013 / 0338210, US2012 / 0101148, US2012 / 0027796, US2012 / 0058144, US2013 / 0323269, US2011 / 0117125, US2011 / 0256175, US2012 / 0202871, US2011 / 0076335, US2006 / 0083780, US2013 / 0123338, US2015 / 0064242, US2006 / 0051405, US2013 / 0065939, US2006 / 0008910, US2003 / 0022649, US2010 / 0130588, US2013 / 0116307, US2010 / 0062967, US2013 / 0202684, US2014 / 0141070, US2014 / 0255472, US2014 / 0039032, US2018 / 0028664, US2016 / 0317458, US2013 / 0195920, the entire contents of all of which are hereby incorporated by reference in their entirety).

[0278] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3) having the following structure:

Chemical formula

[0279] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the content of which is incorporated herein by reference in its entirety.

[0280] In some embodiments, the ionizable lipid is lipid ATX-002 as described in WO2015 / 074085, the content of which is incorporated herein by reference in its entirety.

[0281] In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32) as described in WO2012 / 040184, the content of which is incorporated herein by reference in its entirety.

[0282] In some embodiments, the ionizable lipid is Compound 6 or Compound 22 as described in WO2015 / 199952, the content of which is incorporated herein by reference in its entirety.

[0283] Without limitation, the ionizable lipid can constitute 20-90% (mol) of the total lipids present in the lipid nanoparticles. For example, the molar content of the ionizable lipid can be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the ionizable lipid comprises about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticles.

[0284] In some embodiments, the lipid nanoparticles may further comprise a non-cationic lipid. Non-ionic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid can be a neutral non-charged, zwitterionic, or anionic lipid. Non-cationic lipids are typically used to enhance membrane fusion.

[0285] Exemplary non-cationic lipids contemplated for use in methods and compositions comprising a DNA vector comprising a ceDNA vector produced using the synthetic processes described herein are described in International Application No. PCT / US2018 / 050042, filed September 7, 2018, and International Application No. PCT / US2018 / 064242, filed December 6, 2018.

[0286] Exemplary non-cationic lipids are described in International Application Publication No. WO2017 / 099823 and U.S. Patent Publication No. US2018 / 0028664, the contents of both of which are incorporated herein by reference in their entirety.

[0287] The non-cationic lipid can constitute 0 to 30% (mol) of the total lipids present in the lipid nanoparticles. For example, the non-cationic lipid content is 5 to 20% (mol) or 10 to 15% (mol) of the total lipids present in the lipid nanoparticles. In various embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1.

[0288] In some embodiments, the lipid nanoparticles do not contain any phospholipids. In some embodiments, the lipid nanoparticles may further comprise components such as sterols to provide membrane integration.

[0289] One exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Exemplary cholesterol derivatives are described in International Application No. WO2009 / 127060 and U.S. Patent Publication No. US2010 / 0130588, the contents of both of which are hereby incorporated by reference in their entirety.

[0290] Components that provide membrane integration, such as sterols, can constitute from 0 to 50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, such components are 20 to 50% (mol), 30 to 40% (mol) of the total lipid content of the lipid nanoparticles.

[0291] In some embodiments, the lipid nanoparticles may further comprise polyethylene glycol (PEG) or a composite lipid molecule. Generally, these are used to inhibit aggregation of the lipid nanoparticles and / or provide steric stabilization. Exemplary composite lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the composite lipid molecule is a PEG-lipid conjugate, for example, a (methoxypolyethylene glycol)-composite lipid. Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic acid diacylglycerol (PEGS-DAG) (4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropyl carbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5,885,613, US6,287,591, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904, the entire contents of all of which are incorporated herein by reference.

[0292] In some embodiments, the PEG-lipid is a compound disclosed in US2018 / 0028664, the entire contents of which are incorporated herein by reference.

[0293] In some embodiments, the PEG-lipid is disclosed in US2015 / 0376115 or US2016 / 0376224, the entire contents of both of which are incorporated herein by reference in their entirety.

[0294] The PEG-DAA conjugate can be, for example, PEG-dilauroxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauryl glycamide, PEG-dimyristyl glycamide, PEG-dipalmitoyl glycamide, PEG-distearyl glycamide, PEG-cholesterol (1-[8’-(cholest-5-en-3[β]-oxy) carboxamido-3’,6’-dioxaoctanyl] carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and one or more of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0295] Lipids complexed with molecules other than PEG can also be used instead of PEG-lipids. For example, polyoxazoline (POZ)-lipid complexes, polyamide-lipid complexes (such as ATTA-lipid complexes), and cationic-polymer lipid (CPL) complexes can be used instead of or in addition to PEG-lipids. Exemplary complex lipids, namely, PEG-lipids, (POZ)-lipid complexes, ATTA-lipid complexes, and cationic polymer-lipids are described in International Patent Application Publication Nos. WO1996 / 010392, WO1998 / 051278, WO2002 / 087541, WO2005 / 026372, WO2008 / 147438, WO2009 / 086558, WO2012 / 000104, WO2017 / 117528, WO2017 / 099823, WO2015 / 199952, WO2017 / 004143, WO2015 / 095346, WO2012 / 000104, WO2012 / 000104, and WO2010 / 006282, U.S. Patent Application Publication Nos. US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2013 / 0303587, US2018 / 0028664, US2015 / 0376115, US2016 / 0376224, US2016 / 0317458, US2013 / 0303587, US2013 / 0303587, and US20110123453, and U.S. Patents Nos. US5,885,613, US6,287,591, US6,320,017, and US6,586,559, the entire contents of all of which are hereby incorporated by reference in their entirety.

[0296] In some embodiments, one or more additional compounds can be therapeutic agents. The therapeutic agent can be selected from any class suitable for therapeutic purposes. In other words, the therapeutic agent can be selected from any class suitable for therapeutic purposes. In other words, the therapeutic agent can be selected according to the desired therapeutic purpose and biological effect. For example, if the ceDNA within the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, small molecules, antibodies, or antibody-drug conjugates)). In another example, if the LNP containing ceDNA is useful for treating an infectious disease, the additional compound can be an antibacterial agent (e.g., an antibiotic or an antiviral compound). In yet another example, if the LNP containing ceDNA is useful for treating an immune disease or disorder, the additional compound can be a compound that modulates the immune response (e.g., an immunosuppressive agent, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as ceDNA encoding different proteins or different compounds (such as therapeutic agents), can be used in the compositions and methods of the present invention.

[0297] In some embodiments, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressive agent. In some embodiments, the additional compound is an immunostimulatory agent.

[0298] Also provided herein are pharmaceutical compositions comprising a synthetically produced ceDNA vector encapsulated in a lipid nanoparticle, and a pharmaceutically acceptable carrier or excipient.

[0299] In some aspects, the disclosure provides lipid nanoparticle formulations further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, Tris, trehalose, and / or glycine.

[0300] Closed - end DNA vectors containing ceDNA vectors produced using the synthetic processes described herein can complex with the lipid portion of the particles or be encapsulated at the lipid position of lipid nanoparticles. In some embodiments, DNA vectors containing ceDNA produced using the synthetic processes described herein can be fully encapsulated at the lipid position of lipid nanoparticles, thereby protecting it, for example, from degradation by nucleases in an aqueous solution. In some embodiments, DNA vectors containing ceDNA vectors produced using the synthetic processes described herein in lipid nanoparticles are not substantially degraded after exposure of the lipid nanoparticles to nucleases at 37 °C for at least about 20, 30, 45, or 60 minutes. In some embodiments, the ceDNA in lipid nanoparticles is not substantially degraded after incubation of the particles in serum at 37 °C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0301] In certain embodiments, the lipid nanoparticles are substantially non - toxic to a subject, such as a mammal, e.g., a human. In some aspects, the lipid nanoparticle formulation is a lyophilized powder.

[0302] In some embodiments, the lipid nanoparticles are solid - core particles having at least one lipid bilayer. In other embodiments, the lipid nanoparticles have a non - bilayer structure, i.e., a non - lamellar (i.e., non - bilayer) form. Without limitation, non - bilayer forms can include, for example, three - dimensional tubes, rods, cubic symmetry, etc. For example, the morphology (lamellar vs. non - lamellar) of the lipid nanoparticles can be readily evaluated and characterized using Cryo - TEM analysis as described in US2010 / 0130588, the content of which is incorporated herein by reference in its entirety.

[0303] In some further embodiments, the lipid nanoparticles having a non-lamellar morphology are of high electron density. In some aspects, the present disclosure provides lipid nanoparticles that are either of a single lamellar structure or a multi-lamellar structure. In some aspects, the present disclosure provides lipid nanoparticles that include polyhedral particles and / or vesicular particles.

[0304] By controlling the composition and concentration of the lipid components, the rate at which lipid complexes exchange outside the lipid particles and, in turn, the rate at which the lipid nanoparticles become membrane-fusogenic can be controlled. Further, other variables, such as, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid nanoparticles become membrane-fusogenic. Other methods that can be used to control the rate at which lipid nanoparticles become membrane-fusogenic will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that the lipid particle size can be controlled by controlling the composition and concentration of the lipid complexes.

[0305] The pKa of the formulated cationic lipid can correlate with the efficacy of the LNP for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529 - 8533, Semple et al, Nature Biotechnology 28, 172 - 176 (2010); both are incorporated herein by reference in their entirety). The preferred range of pKa is from about 5 to about 7. The pKa of the cationic lipid can be determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS).

[0306] VII. Method for Delivering Closed-End DNA Vectors In some embodiments, closed - end DNA vectors comprising a ceDNA vector produced using the synthetic processes described herein can be delivered to target cells in vitro or in vivo by a variety of suitable methods. Only the closed - end DNA vectors comprising a ceDNA vector produced using the synthetic processes described herein can be applied or injected. Closed - end DNA vectors comprising a ceDNA vector produced using the synthetic processes described herein can be delivered to cells without the aid of transfection reagents or other physical means. Alternatively, the closed - end DNA comprising a ceDNA vector produced using the synthetic processes described herein can be delivered using any transfection reagent known in the art that facilitates entry of the DNA into the cell or other physical means known in the art, such as liposomes, alcohol, poly - lysine compounds, poly - arginine compounds, calcium phosphate, microvesicles, microinjection, electroporation, and the like.

[0307] In another embodiment, a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein is administered to the CNS (e.g., the brain or the eye). For example, the ceDNA vector may be introduced into the spinal cord, the brainstem (medulla oblongata, pons), the midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), the cerebellum, the telencephalon (including the cerebrum, which includes the striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe, cortex, basal ganglia, hippocampus, and amygdala), the limbic system, the neocortex, the striatum, the cerebrum, and the inferior colliculus. The ceDNA vector may also be administered to different regions of the eye, such as the retina, the cornea, and / or the optic nerve. The ceDNA vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture). The ceDNA vector may further be administered intravascularly to the CNS in situations where the blood - brain barrier is disrupted (e.g., in a brain tumor or a cerebral infarction).

[0308] In some embodiments, a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein can be administered to a desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra - auricular, intraocular (e.g., intravitreal, subretinal, anterior chamber), and peri - ocular (e.g., sub - Tenon's space) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0309] In some embodiments, a closed - end DNA vector comprising a ceDNA vector produced using the synthetic processes described herein is administered as a liquid formulation by direct injection (e.g., stereotactic injection) into a desired region or compartment of the CNS. In other embodiments, for example, a synthetically produced ceDNA vector can be provided by topical application to a desired region or by intranasal administration of an aerosol formulation. Administration to the eye may be by topical application of droplets. As a further alternative, for example, a ceDNA vector can be administered as a solid sustained - release formulation (see, e.g., U.S. Patent No. 7,201,898). In yet additional embodiments, for example, a synthetically produced ceDNA vector is used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). By way of example, for instance, a synthetically produced ceDNA vector can be delivered to muscle tissue and move therefrom into neurons.

[0310] VIII. Additional Uses of ceDNA Vectors Compositions produced using the synthetic processes described herein, and closed - end DNA vectors containing a ceDNA vector, can be used to express a target gene or transgene for various purposes. In some embodiments, the resulting transgene encodes a protein or functional RNA that is intended to be used for research purposes, for example, to create a somatic transgenic animal model containing the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of a disease. In some embodiments, the resulting transgene encodes one or more peptides, polypeptides, or proteins that are useful for the treatment, prevention, or amelioration of a disease state or disorder in a mammalian subject. The resulting transgene can be introduced (e.g., expressed) into a subject in an amount sufficient to treat a disease associated with reduced gene expression, loss of expression, or dysfunction. In some embodiments, the resulting transgene can be expressed in a subject in an amount sufficient to treat a disease associated with increased expression, activity of the gene product, or inappropriate up - regulation of a gene (where the resulting transgene suppresses or otherwise reduces its expression). In yet other embodiments, the resulting transgene replaces or supplements a defective copy of an endogenous gene. It will be understood by those skilled in the art that the transgene need not be the open reading frame of a gene that is itself transcribed. Instead, it can be a promoter region or repressor region of a target gene, and the ceDNA vector can modify such a region as a result of thus modulating the expression of the gene of interest.

[0311] In some embodiments, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for the treatment or prevention of a disease state in a mammalian subject. The transgene can be introduced (e.g., expressed) into a patient in an amount sufficient to treat a disease associated with reduced gene expression, loss of expression, or dysfunction.

[0312] IX. Methods of Use Synthetically produced closed-ended DNA vectors, such as the ceDNA vectors as disclosed herein, can also be used in methods for delivering a nucleotide sequence of interest (e.g., a transgene) to a target cell (e.g., a host cell). This method can be a method for delivering a transgene, in particular, to cells of a subject in need thereof and for treating a disease of interest. The present invention enables in vivo expression of a transgene encoded by a ceDNA vector in a cell of a subject, such as a nucleic acid such as a protein, an antibody, an miRNA, etc., such that a therapeutic effect of the expression of the transgene occurs. These results are seen in both in vivo and in vitro forms of closed-ended DNA vector (e.g., ceDNA vector) delivery.

[0313] In addition, the present invention provides a method for delivering a gene editing molecule into cells of a subject in need thereof, comprising multiple administrations of the synthetically produced closed-ended DNA vector (e.g., ceDNA vector) of the present invention comprising the nucleic acid or transgene of interest. Since the ceDNA vectors of the present invention do not induce an immune response as typically observed for capsid-formed viral vectors, such multiple administration strategies will be more successful in ceDNA-based systems.

[0314] Synthetically produced closed - ended DNA vectors (e.g., ceDNA vectors) nucleic acid(s) are administered in an amount sufficient to transfect cells of a desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intravenous (e.g., liposomal formulations), direct delivery to a selected organ (e.g., intra - portal delivery to the liver), intramuscular, and other routes of administration. If desired, routes of administration can be combined.

[0315] Delivery of closed - ended DNA vectors (e.g., ceDNA vectors) is not limited to delivery gene replacement. For example, the synthetically produced closed - ended DNA vectors (e.g., ceDNA vectors) described herein may be used in conjunction with other delivery systems provided to provide a part of gene therapy. One non - limiting example of a system that can be combined with a ceDNA vector synthetically produced according to the present disclosure includes a system that separately delivers one or more co - factors or immunosuppressive factors for effective gene expression of a transgene.

[0316] The present invention also provides a method of treating a disease in a subject, comprising introducing a therapeutically effective amount of a synthetically produced closed - ended DNA vector (e.g., ceDNA vector), optionally together with a pharmaceutically acceptable carrier, into target cells (particularly muscle cells or tissues) of a subject in need thereof. The synthetically produced ceDNA vector can be introduced in the presence of a carrier, but such a carrier is not required. For example, the synthetically produced ceDNA vector selected contains a nucleotide sequence of interest useful for treating a disease. In particular, for example, the synthetically produced ceDNA vector can contain a desired exogenous DNA sequence operably linked to a control element capable of directing the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject. For example, the synthetically produced ceDNA vector can be administered via any suitable route provided above and elsewhere in this specification.

[0317] Using the synthetically produced compositions and vectors provided herein, transgenes can be delivered for a variety of purposes. In some embodiments, the transgene is intended to be used for research purposes, for example, to create a somatic transgenic animal model containing the transgene, for example, to study the function of the transgene product, by encoding a protein or functional RNA. In another example, the transgene encodes a protein or functional RNA intended to be used to create an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for the treatment or prevention of a disease state in a mammalian subject. The transgene can be introduced (e.g., expressed) into a patient in an amount sufficient to treat a disease associated with reduced gene expression, loss of expression, or dysfunction.

[0318] In principle, the expression cassette can contain a nucleic acid or any transgene that encodes a protein or polypeptide that is reduced or absent by mutation, or that provides a therapeutic effect when overexpression is considered to be within the scope of the present invention.

[0319] The synthetically produced ceDNA vectors are not limited to one type of ceDNA vector. As such, in another aspect, multiple ceDNA vectors that are operably linked to different transgenes, or different promoters or cis-regulatory elements but contain the same transgene, can be delivered to target cells, tissues, organs, or subjects simultaneously or sequentially. Thus, with this strategy, gene therapy or gene delivery of multiple genes can be performed simultaneously. It is also possible to separate different parts of the transgene into separate ceDNA vectors (e.g., different domains and / or cofactors required for the function of the transgene), which can be administered simultaneously or at different times and can be separately regulatable, thereby adding an additional level of control over the expression of the transgene. Delivery can also be performed multiple times and, importantly, in a clinical setting for gene therapy at doses that increase or decrease later, assuming the absence of an anti-capsid host immune response due to the absence of the viral capsid. Since there is no capsid, no anti-capsid reaction is expected to occur.

[0320] The present invention also provides a method of treating a disease in a subject, comprising introducing a therapeutically effective amount of the synthetically produced ceDNA vector disclosed herein, optionally together with a pharmaceutically acceptable carrier, into target cells (particularly muscle cells or tissues) of the subject in need of treatment. The ceDNA vector can be introduced in the presence of a carrier, but such a carrier is not required. The implemented ceDNA vector contains a nucleotide sequence of interest useful for treating the disease. In particular, the ceDNA vector can contain a desired exogenous DNA sequence operably linked to a control element capable of directing the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject. The synthetically produced ceDNA vector can be administered via any suitable route provided above and elsewhere in this specification.

[0321] X. Method of Treatment The technology described herein also demonstrates methods for making the disclosed synthetically produced ceDNA vectors, as well as methods of using them in various ways (e.g., ex situ, in vitro and in vivo applications, methodologies, diagnostic procedures, and / or gene therapy regimens).

[0322] Provided herein is a method of treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of a synthetically produced ceDNA vector, optionally together with a pharmaceutically acceptable carrier, into target cells (e.g., muscle cells or tissues, or other diseased cell types) of a subject in need of treatment. The ceDNA vector can be introduced in the presence of a carrier, although such a carrier is not required. The synthetically produced ceDNA vector being implemented comprises a nucleotide sequence of interest useful for treating the disease. In particular, the synthetically produced ceDNA vector can comprise a desired exogenous DNA sequence operably linked to control elements capable of directing the transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into the subject. The synthetically produced ceDNA vector can be administered via any suitable route provided above and elsewhere herein.

[0323] Disclosed herein are ceDNA vector compositions and formulations comprising one or more of the synthetically produced ceDNA vectors of the invention, together with one or more pharmaceutically acceptable buffers, diluents, or excipients. Such compositions can be included in one or more diagnostic or therapeutic kits for diagnosing, preventing, treating, or ameliorating one or more symptoms of a disease, injury, disorder, trauma, or dysfunction. In one aspect, the disease, injury, disorder, trauma, or dysfunction is a human disease, injury, disorder, trauma, or dysfunction.

[0324] Another aspect of the technology described herein provides a method for providing a diagnostically or therapeutically effective amount of a synthetically produced ceDNA vector to a subject in need thereof, the method comprising providing a disclosed amount of the synthetically produced ceDNA vector to a cell, tissue, or organ of the subject in need thereof for a time effective to permit expression of the transgene from the ceDNA vector, thereby providing to the subject a protein, peptide, or nucleic acid expressed by a diagnostically or therapeutically effective amount of the ceDNA vector. In a further aspect, the subject is human.

[0325] Another aspect of the technology described herein provides a method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject. Generally and in general terms, the method comprises administering to the subject in need thereof at least one or more of the disclosed synthetically produced ceDNA vectors in an amount and for a time effective to diagnose, prevent, treat, or ameliorate one or more symptoms of the subject's disease, disorder, dysfunction, injury, abnormal condition, or trauma. In a further aspect, the subject is human.

[0326] Another aspect is the use of synthetically produced ceDNA vectors as tools for treating or reducing one or more symptoms of a disease or disease state. There are several genetic diseases in which the defective gene is known, typically falling into two classes: a deficiency state of an enzyme that is usually inherited in a generally recessive manner, and a dysregulated state that can involve regulatory or structural proteins, but is not typically always inherited in a dominant manner. In the case of deficiency state diseases, synthetically produced ceDNA vectors are used to deliver a transgene to carry a normal gene into diseased tissue for replacement therapy, and in some embodiments, antisense mutations can be used to create animal models of the disease. In the case of dysregulated disease states, synthetically produced ceDNA vectors can be used to create a pathology in a model system, which can then be used in attempts to address that pathology. Thus, the synthetically produced ceDNA vectors and methods disclosed herein enable the treatment of genetic diseases. As used herein, a pathology is treated by partially or wholly rectifying a deficiency or dysregulation that causes or exacerbates the disease.

[0327] A. Host cell: In some embodiments, the synthetically produced ceDNA vector delivers a transgene to a target host cell. In some embodiments, the target host cell is a human host cell, such as, for example, a blood cell, a stem cell, a hematopoietic cell, a CD34 + cell, a hepatocyte, a cancer cell, a vascular cell, a muscle cell, a pancreatic cell, a nerve cell, an eye or retinal cell, an epithelial or endothelial cell, a dendritic cell, a fibroblast, or any other cell of mammalian origin (including, without limitation, hepatocytes (i.e., liver cells), lung cells, heart cells, pancreatic cells, intestinal cells, diaphragm cells, kidney (i.e., renal) cells, neuron cells, blood cells, bone marrow cells, or any one or more selected tissues of interest for which gene therapy is contemplated). In one aspect, the target host cell is a human host cell.

[0328] The present disclosure also relates to recombinant host cells as described above that contain synthetically produced ceDNA vectors described herein. Thus, as will be apparent to those skilled in the art, multiple host cells can be used depending on the purpose. A construct containing a donor sequence or a synthetically produced ceDNA vector is introduced into a host cell such that the donor sequence is maintained as a chromosomal integrant as described above. The term host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell depends largely on the donor sequence and its source. The host cell may also be a eukaryote such as a mammalian, insect, plant, or fungal cell. In one embodiment, the host cell is a human cell (e.g., a primary cell, a stem cell, or an immortalized cell line). In some embodiments, the host cell can be administered with a synthetically produced ceDNA vector ex vivo and then delivered to a subject after a gene therapy event. The host cell can be any cell type, such as a somatic cell or a stem cell, an induced pluripotent stem cell, or a blood cell, such as a T cell or a B cell, or a bone marrow cell. In certain embodiments, the host cell is an autologous cell. For example, T cell genome engineering is useful for disease modulation such as cancer immunotherapy, HIV therapy (e.g., receptor knockout such as CXCR4 and CCR5), and immunodeficiency therapy. MHC receptors on B cells can be targets for immunotherapy. In some embodiments, genetically modified host cells, such as bone marrow stem cells, such as CD34 + cells, or induced pluripotent stem cells can be transplanted back into a patient for the expression of a therapeutic protein.

[0329] B. Exemplary Transgenes and Diseases Treated with ceDNA Vectors Closed-ended DNA vectors containing ceDNA vectors produced using the synthetic processes described herein are also useful for correcting defective genes. As a non-limiting example, the DMD gene for Duchenne muscular dystrophy can be delivered using a synthetically produced ceDNA vector as disclosed herein.

[0330] The synthetically produced ceDNA vector or its composition can be used for the treatment of any genetic disease. As a non-limiting example, the synthetically produced ceDNA vector or its composition can be used for the treatment of transthyretin amyloidosis (ATTR), a rare disease in which mutant proteins misfold and aggregate in the nerves, heart, gastrointestinal system, etc. It is contemplated herein that the synthetically produced ceDNA vector described herein can be used to treat a disease by deletion of the mutant disease gene (mutTTR). Such treatment of genetic diseases can halt the progression of the disease and may allow for regression of an established disease or reduction of at least 10% of at least one symptom of the disease.

[0331] In another embodiment, the synthetically produced ceDNA vector or its composition can be used for the treatment of ornithine transcarbamylase deficiency (OTC deficiency), hyperammonemia, or other urea cycle disorders that impair the ability of newborns or infants to detoxify ammonia. Similar to all diseases of inborn metabolism, even partial restoration of enzyme activity compared to wild-type controls (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) may be sufficient to reduce at least one symptom of OTC and / or improve the quality of life in subjects with OTC deficiency. In one embodiment, the nucleic acid encoding OTC can be inserted behind the albumin endogenous promoter for in vivo protein replacement.

[0332] In another embodiment, a synthetically produced ceDNA vector or a composition thereof is used in the treatment of phenylketonuria (PKU) by delivering a nucleic acid sequence encoding the phenylalanine hydroxylase enzyme to reduce the accumulation of dietary phenylalanine, which can be toxic to PKU patients. As with all diseases of inborn metabolism, even a partial restoration of enzyme activity compared to wild-type controls (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) may be sufficient to reduce at least one symptom of PKU and / or improve the quality of life in subjects with PKU. In one embodiment, a nucleic acid encoding phenylalanine hydroxylase can be inserted behind the albumin endogenous promoter for in vivo protein replacement.

[0333] In another embodiment, the synthetically produced ceDNA vector or composition thereof can be used for the treatment of glycogen storage disease (GSD) by delivering a nucleic acid sequence encoding an enzyme to correct abnormal glycogen synthesis or breakdown in a subject having GSD. Non-limiting examples of enzymes that can be delivered and expressed using the synthetically produced ceDNA vectors and methods described herein include glycogen synthase, glucose-6-phosphatase, acid-α-glucosidase, glycogen branching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter-2 (GLUT-2), aldolase A, β-enolase, phosphoglucomutase-1 (PGM-1), and glycogenin-1. As with all diseases of inborn metabolism, even a partial restoration of enzyme activity compared to wild-type controls (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) may be sufficient to reduce at least one symptom of GSD and / or improve the quality of life of a subject having GSD. In one embodiment, the nucleic acid encoding the enzyme can be inserted behind the albumin endogenous promoter for in vivo protein replacement to correct abnormal glycogen storage.

[0334] The synthetically produced ceDNA vectors described herein are also contemplated for use in the treatment of any of Leber congenital amaurosis (LCA), polyglutamine diseases containing polyQ repeats, and alpha-1 antitrypsin deficiency (A1AT). LCA is a rare congenital eye disease that causes blindness and can be caused by mutations in any one of the following genes: GUCY2D, RPE65, SPATA7, AIPL1, LCA5, RPGRIP1, CRX, CRB1, NMNAT1, CEP290, IMPDH1, RD3, RDH12, LRAT, TULP1, KCNJ13, GDF6, and / or PRPH2. It is contemplated herein that the ceDNA vectors and compositions and methods described herein can be adapted for delivery of one or more genes associated with LCA to correct errors in the gene(s) that cause the symptoms of LCA. Polyglutamine diseases include, but are not limited to, dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3 (also known as Machado-Joseph disease), 6, 7, and 17. A1AT deficiency is a genetic disorder that causes poor production of alpha-1 antitrypsin, leading to reduced enzyme activity in the blood and lungs, which can in turn lead to emphysema or chronic obstructive pulmonary disease in affected subjects. Treatment of subjects with A1AT deficiency using a ceDNA vector or composition thereof as outlined herein is specifically contemplated herein. It is contemplated herein that a ceDNA vector containing a nucleic acid encoding a desired protein for the treatment of LCA, polyglutamine disease, or A1AT deficiency can be administered to a subject in need of treatment.

[0335] In further embodiments, compositions comprising the synthetically produced ceDNA vectors described herein can be used to deliver, among other things, viral sequences, pathogen sequences, chromosomal sequences, translocation junctions (e.g., translocations associated with cancer), non-coding RNA genes or RNA sequences, disease-associated genes.

[0336] Any nucleic acid or target gene of interest can be delivered or expressed by the synthetically produced ceDNA vectors disclosed herein. Target nucleic acids and target genes include, but are not limited to, nucleic acids encoding polypeptides, or non-coding nucleic acids (e.g., RNAi, miR, etc.), preferably therapeutic agents (e.g., for medical, diagnostic, or veterinary use), or immunogenic polypeptides (e.g., for vaccines). In certain embodiments, the target nucleic acid or target gene targeted by the synthetically produced ceDNA vectors described herein encodes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0337] In particular, gene targets or transgenes for expression by the synthetically produced ceDNA vectors disclosed herein can encode, for example, but not limited to, proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, and variants and / or active fragments thereof for use in the treatment, prevention, and / or amelioration of one or more symptoms of a disease, dysfunction, injury, and / or disorder. In one aspect, the disease, dysfunction, trauma, injury, and / or disorder is a human disease, dysfunction, trauma, injury, and / or disorder.

[0338] The expression cassette can also encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). The expression cassette can include exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0339] The expression cassette, the sequence provided by the expression construct of the ceDNA vector described herein, can be codons optimized for the host cell. As used herein, the term "optimized codon" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing the codons of at least one, two or more, or a significant number of unmodified sequences (e.g., prokaryotic cell sequences) with codons that are more frequently or most frequently used in the genes of the vertebrate of interest, e.g., a mouse or human cell, for enhanced expression in the vertebrate cell of interest. Different species exhibit a particular bias for certain codons for a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database.

[0340] Many organisms exhibit a bias for using certain codons to encode the insertion of a particular amino acid in the growing peptide chain. Codon preference or codon bias, which is the difference in codon usage frequency between organisms, is brought about by the degeneracy of the genetic code and is well documented among many organisms. Codon bias is often correlated with the efficiency of messenger RNA (mRNA) translation and is thought to depend particularly on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be adjusted for optimal gene expression in a given organism.

[0341] Considering the numerous gene sequences available in a wide variety of animal, plant, and microorganism species, it is possible to calculate the relative frequencies of codon usage (Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000)).

[0342] As described herein, the synthetically produced ceDNA vectors disclosed herein can encode a protein or peptide, or a therapeutic nucleic acid sequence, and as therapeutic agents, one or more agonists, antagonists, anti-apoptotic factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietin agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neurotrophic peptides, neurotrophic peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, enzymes, receptor-binding proteins, transport proteins or one or more inhibitors thereof, serotonin receptors, or one or more uptake inhibitors thereof, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, cytotoxins, or any combination thereof, but are not limited thereto.

[0343] Synthetically produced ceDNA gene vectors are also useful for gene expression knockdown. For example, in one embodiment, ceDNA vectors can be used to express antisense nucleic acids or functional RNAs to induce knockdown of target genes. As a non-limiting example, the expression of CXCR4 and CCR5, which are HIV receptors, has been successfully excised in primary human T cells. See Schumann et al. (2015), PNAS 112(33):10437-10442, which is incorporated herein by reference in its entirety. Another gene for targeted inhibition is PD-1, and synthetically produced ceDNA vectors can express inhibitory nucleic acids or RNAi or functional RNAs to inhibit the expression of PD-1. PD-1 expresses an immune checkpoint cell surface receptor on chronically active T cells that occur in malignant tumors. See Schumann et al. supra.

[0344] In some embodiments, synthetically produced ceDNA gene vectors are useful for correcting defective genes by expressing a transgene that targets the affected gene. Non-limiting examples of diseases or disorders susceptible to treatment with the synthetically produced ceDNA vectors disclosed herein are shown in Tables A-C along with those genes and their related genes of US Patent Publication No. 2...

Claims

【Claim 1】 The invention described in the specification.

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