RU2800914C9 - Non-viral dna vectors and their use for the production of antibodies and fusion proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to scDNA vectors having a linear and continuous structure for transgene delivery and expression; scDNA vectors contain an expression cassette flanked by two ITR sequences, the expression cassette encodes a transgene. Some scDNA vectors additionally contain cis regulatory elements, including regulatory switches. This document further provides methods and cell lines for robust gene expression in vitro, ex vivo and in vivo using scDNA vectors. Methods and compositions containing scDNA vectors useful for expressing an antibody or fusion protein in a cell, tissue, or subject are provided in this document.

EFFECT: such antibodies or fusion proteins may be expressed to treat a disease or, alternatively, to produce antibodies or fusion proteins commercially.

43 cl, 12 dwg, 13 tbl, 13 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 USC §119(e) based on US Provisional Application No. 62/630670, filed February 14, 2018, US Provisional Application No. 62/680087, filed June 4, 2018, US Provisional Application No. 62/630676, filed February 14, 2018, and US Provisional Application No. 62/680092, filed June 4, 2018, the contents of each of which are incorporated herein by reference in their entirety.

LIST OF SEQUENCES

[0002] This application includes a sequence listing that was submitted electronically in ASCII format and is hereby incorporated herein by reference in its entirety. The ASCII copy in question, created on February 13, 2019, is named 080170-091100-WOPT_SL.txt and is 128788 bytes in size.

TECHNICAL FIELD

[0003] This invention relates to the field of gene therapy, including non-viral vectors for expressing a transgene or isolated polynucleotides in a subject or cell. This invention also relates to nucleic acid constructs, promoters, vectors and host cells that contain polynucleotides, as well as methods for delivering exogenous DNA sequences to a target cell, tissue, organ or organism. For example, the present invention provides methods for using non-viral DNA vectors to express antibodies, such as therapeutic antibodies, from a cell. The present invention also provides methods for using non-viral DNA vectors to express fusion proteins, such as therapeutic fusion proteins, from a cell. The methods and compositions can be used, for example, for the commercial production of antibodies or fusion proteins, or for the purpose of treating a disease by expressing a therapeutic antibody or fusion protein in a cell or tissue of a subject in need thereof.

BACKGROUND OF THE ART

[0004] The goal of gene therapy is to improve clinical outcomes for patients suffering from genetic mutations or acquired diseases caused by aberrations in the gene expression profile. Gene therapy involves the treatment or prevention of medical conditions caused by defective genes or abnormal regulation or expression, such as underexpression or overexpression, which can lead to a disorder, disease, cancer, etc. For example, a disease or disorder caused by a defective gene. may be treated, prevented, or ameliorated by delivering corrective genetic material to a patient, or may be treated, prevented, or alleviated by modifying or suppressing a defective gene in a patient, for example, by providing corrective genetic material resulting in therapeutic expression of the genetic material in the patient.

[0005] The basis of gene therapy is the introduction of a transcription cassette with an active gene product (sometimes called a transgene), which, for example, can lead to a positive gain-of-function effect, a negative loss-of-function effect, or other outcome. Such results can be explained by the expression of an activating antibody or fusion protein or an inhibitory (neutralizing) antibody or fusion protein. Gene therapy can also be used to treat a disease or cancer caused by other factors. Human monogenic disorders can be treated by delivering and expressing a normal gene into target cells. Delivery and expression of the corrective gene in the patient's target cells can be accomplished by numerous methods, including the use of genetically engineered viruses and viral gene delivery vectors. Among the many viral vectors available (eg, recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, etc.), recombinant adeno-associated virus (rAAV) is gaining popularity as a versatile vector for gene therapy.

[0006] Adeno-associated viruses (AAVs) belong to the family Parvoviridae and, more specifically, constitute the genus Dependoparvoviruses. AAV-derived vectors (i.e., recombinant AAV (rAAV) or AAV vectors) are an attractive method of delivering genetic material because (i) they are capable of infecting (transducing) a wide range of nondividing and dividing cell types, including myocytes and neurons; (ii) they lack viral structural genes, thereby reducing host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered nonpathological to humans; (iv) unlike wild-type AAV, which is able to integrate into the host cell genome, replication-defective AAV vectors lack the rep gene and usually persist as episomes, which limits the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively weak immunogens and therefore do not elicit a significant immune response (see ii), thus ensuring vector DNA persistence and potentially long-term expression of therapeutic transgenes.

[0007] However, the use of AAV particles as a vector for gene delivery has a number of serious disadvantages. One of the major disadvantages associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb. heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), as a result of which the use of AAV vectors is limited by the ability to encode proteins of less than 150,000 Da. A second disadvantage is that, as a result of the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy must be screened for the presence of neutralizing antibodies that eliminate the vector from the patient. A third disadvantage relates to the immunogenicity of the capsid, which prevents re-administration in patients not excluded from primary treatment. The patient's immune system may respond to the vector, which effectively acts as a "booster" agent, stimulating the immune system to generate high titers of anti-AAV antibodies, precluding future treatment. Some recent reports indicate problems with immunogenicity in high-dose situations. Another notable disadvantage is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA before expression of the heterologous gene.

[0008] In addition, conventional AAV capsid virions are produced by introducing a plasmid or plasmids containing the AAV genome, rep genes and cap genes (Grimm et al., 1998). However, such capsid-enclosed viral AAV vectors have been found to ineffectively transduce certain types of cells and tissues, and these capsids also induce an immune response.

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

[0010] There is a need in the art for technology that allows expression of a therapeutic antibody (eg, secreted antibody or intrabody) or fusion protein (eg, receptor extracellular domain-Fc fusion) in a cell, tissue or subject, or alternatively, with for the purpose of producing antibodies or fusion proteins in vitro or in vivo for purification and/or commercial production. In addition, there remains an important unmet need for controlled recombinant DNA vectors with improved production and/or expression characteristics to improve the production of antibodies (eg, therapeutic antibodies) and fusion proteins (eg, therapeutic fusion proteins) compared to existing or conventional methods, or vectors.

BRIEF DESCRIPTION OF THE INVENTION

[0011] The technology described herein relates to methods and compositions for expressing antibodies and fusion proteins (such as therapeutic antibodies and fusion proteins) using a capsidless (e.g., non-viral) DNA vector with covalently closed ends (referred to herein as "DNA -closed-end vector" or "ccDNA vector"). Such cccDNA vectors can be used for the production of antibodies and fusion proteins for the treatment of diseases, cancer treatment, monitoring and diagnosis, as well as for the commercial production of antibodies or fusion proteins. One exemplary example of an antibody is an anti-tumor necrosis factor antibody or antigen-binding fragment thereof, including, without limitation, the monoclonal antibody adalimumab (Humira™), which can be expressed in a cell or tissue of a subject using the cccDNA vectors described herein . Such a therapeutic antibody can be used to treat rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and Crohn's disease.

[0012] Accordingly, the invention described herein relates to a capsid-free (e.g., non-viral) covalently closed-end DNA vector (referred to herein as a “closed-end DNA vector” or “ccDNA vector”) containing a heterogeneous a gene encoding an antibody (eg, light chains, heavy chains, scaffolds, Fabs', single chain antibodies), or an antigen binding fragment thereof, to enable expression of the antibody in a cell, eg, a secreted antibody or intrabody. The invention also relates to a cccDNA vector containing a heterogeneous gene encoding a fusion protein to enable expression of the fusion protein in a cell. Such antibodies or fusion proteins to be expressed may be therapeutic antibodies or fusion proteins, and/or the methods employed may be used in the production of antibodies or fusion proteins for commercial purposes. In particular, the technology described herein relates to the improved production of antibodies and fusion proteins using cccDNA vectors.

[0013] cccDNA vectors for the production of antibodies and fusion proteins, as described herein, are capsidless linear duplex DNA molecules formed by a continuous strand of complementary DNA with covalently closed ends (linear, continuous and unencapsidated structure) that contain a 5' sequence inverted terminal repeat (ITR) and a 3'-ITR sequence, wherein the 5'-ITR and 3'-ITR may have the same symmetrical three-dimensional organization relative to each other (i.e., symmetrical or substantially symmetrical) or, alternatively, a 5'-ITR The ITR and 3'-ITR may have different three-dimensional organizations relative to each other (ie, asymmetric ITRs). In addition, ITRs may belong to the same or different serotypes. In some embodiments, the cccDNA vector may contain ITR sequences that have a symmetrical three-dimensional spatial organization such that their structure has 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 are mirror images of each other). In some embodiments, one ITR may belong to one AAV serotype, and the other ITR may belong to a different AAV serotype.

[0014] Accordingly, certain aspects of the technology described herein relate to a cccDNA vector for improved expression and/or production of an antibody or fusion protein that contains ITR sequences selected from any of: (i) at least one wild-type ITR type (WT) and at least one modified inverted terminal repeat (ITR) of AAV (eg, asymmetric modified ITR); (ii) two modified ITRs, wherein the mod-ITR pair has a different three-dimensional spatial organization relative to each other (e.g., asymmetric modified ITRs), or (iii) a symmetric or substantially symmetric WT-WT ITR pair, wherein each WT-ITR has the same three-dimensional spatial organization, or (iv) a symmetric or substantially symmetric pair of modified ITRs, each mod-ITR having the same three-dimensional spatial organization. The cccDNA vectors disclosed herein can be produced in eukaryotic cells and thus are free of prokaryotic DNA modifications and bacterial endotoxin contamination in insect cells.

[0015] The methods and compositions described herein relate in particular to the discovery of non-viral capsid-free, covalently closed-end DNA vectors (ccDNA vectors) that can be used to express at least one antibody and/or fusion protein, or more than one antibody and/or fusion protein, from a cell. The methods and compositions may be used, for example, for the commercial production of an antibody or fusion protein, or for the purpose of treating a disease with a therapeutic antibody or fusion protein.

[0016] Accordingly, provided herein in one aspect are DNA vectors (e.g., cccDNA vectors) comprising at least one heterologous nucleic acid sequence encoding at least one transgene encoding an antibody or antigen binding fragment thereof, or fusions thereof proteins operably linked to a promoter located between two different AAV inverted terminal repeat (ITR) sequences, one of the ITRs containing a functional AAV terminal resolution site and a Rep binding site, and one of the ITRs containing a deletion, insertion or substitution relative to the other ITR; wherein the transgene is an antibody or a fragment thereof (eg, an antigen binding fragment thereof) or a fusion protein; and wherein DNA, when digested by a restriction enzyme having a single recognition site on the DNA vector, is distinguished by the presence of characteristic bands of linear and continuous DNA compared to controls of linear and discontinuous DNA when analyzed on a non-denaturing gel. Other aspects include delivering a therapeutic antibody or fusion protein by expressing it in vivo from a cccDNA vector as described herein and, further, treating various diseases using such antibodies or fusion proteins. This document also provides for cells containing the cDNA vector described herein.

[0017] Aspects of the present invention relate to methods for producing cccDNA vectors useful for producing an antibody or fusion protein, or expressing an antibody or fusion protein in a cell, as described herein. Other embodiments relate to a cDNA vector produced by the method presented herein. In one embodiment, a capsid-free (e.g., non-viral) DNA vector (ccDNA vector) for producing an antibody or fusion protein is derived from a plasmid (referred to herein as a “ccDNA plasmid”) containing a polynucleotide expression construct template containing, in this order : first 5' inverted terminal repeat (e.g. ITR AAB); heterologous nucleic acid sequence; and 3'-ITR (eg, AAB ITR), wherein the 5'-ITR and 3'-ITR can be asymmetric to each other or symmetric (eg, WT-ITR or modified symmetric ITR) as defined herein.

[0018] The cDNA vector for producing antibodies or fusion proteins as disclosed herein can be produced by several methods that will be known to one of ordinary skill in the art after reading this disclosure. For example, the polynucleotide expression construct template used to generate the ccDNA vectors of the present invention may be a ccDNA plasmid, a ccDNA bacmid, and/or a ccDNA baculovirus. In one embodiment, the cccDNA plasmid contains a restriction cloning site (e.g., SEQ ID NO: 123 and/or 124) operably located between the ITR, into which an expression cassette can be inserted containing, for example, a promoter operably linked to the transgene, e.g. , a nucleic acid encoding an antibody or an antigen-binding fragment thereof or a fusion protein and/or a reporter gene). In some embodiments, cccDNA vectors for producing antibodies or fusion proteins are derived from a polynucleotide template (e.g., ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus) containing symmetric or asymmetric ITRs (modified or WT-ITR).

[0019] In a permissive host cell, in the presence of, for example, Rep, a polynucleotide template having at least two ITRs is replicated to produce ccDNA vectors for producing antibodies or fusion proteins. Production of a cccDNA vector occurs in two stages: first, excision (“rescue”) of the template from the template backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) using Rep proteins and, second, Rep-mediated replication of the excised cccDNA vector. The Rep proteins and Rep binding sites of various AAV serotypes are well known to those skilled in the art. One skilled in the art will appreciate that the Rep protein should be selected from a serotype that binds to and replicates a nucleic acid sequence based on at least one functional ITR. For example, if a replication competent ITR is an AAV serotype 2, the corresponding Rep will be from an AAV serotype that interacts with that serotype, for example, the AAB2 ITR with Rep AAB2 or AAB4, but not with Rep AAB5, which does not have this ability. After replication, the cccDNA vector with covalently closed ends continues to accumulate in permissive cells, and the cccDNA vector is preferably sufficiently stable over time in the presence of the Rep protein under standard replication conditions, for example, to accumulate in an amount of at least 1 pg/cell, preferably at least 2 pg/cell, preferably at least 3 pg/cell, more preferably at least 4 pg/cell, even more preferably at least 5 pg/cell.

[0020] Accordingly, one aspect of the present invention relates to a method of producing a cccDNA vector for producing an antibody or fusion protein, comprising the steps of: a) incubating a population of host cells (eg, insect cells) bearing a polynucleotide expression construct template (eg, cccDNA- plasmid, cccDNA bacmid and/or ccDNA baculovirus) that lacks sequences encoding the viral capsid, in the presence of the Rep protein under conditions ensuring effectiveness and for a time sufficient to induce production of the cccDNA vector in host cells, wherein the cells -hosts do not contain viral capsid coding sequences; and b) collecting and isolating the cDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide with a modified ITR to produce a cDNA vector for producing antibodies or a fusion protein in a host cell. However, viral particles (eg, AAV virions) are not expressed. Thus, there are no size restrictions imposed by the virion.

[0021] The presence of a cccDNA vector useful for producing antibodies or a fusion protein isolated from host cells can be confirmed by digesting DNA isolated from the host cell with a restriction enzyme having a single recognition site on the ccDNA vector and analyzing the digested material DNA on denaturing and non-denaturing gels to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[0022] For purposes of this invention, the transgene expressed by the cDNA encodes an antibody or antibody binding fragment or fusion protein. Antibodies and fusion proteins are well known in the art and can bind to any protein of interest, including, without limitation, a ligand, receptor, toxin, hormone, enzyme, or cell surface protein, or pathogen, or viral protein, or antigen, as well as pre- and post-translationally modified proteins such as glycoproteins or sumoylated proteins (eg anti-SUMO2/3 antibody), etc. Antibodies and antigen-binding fragments also include antibodies that bind to any antigen, including, but not limited to, nucleic acids, e.g., DNA (eg, anti-dsDNA antibodies), RNA (eg, anti-RNA binding antibodies). In some embodiments, the antibodies produced by the cDNA vectors disclosed herein are neutralizing antibodies or antigen binding fragments thereof. Representative examples of target genes and proteins of interest are described in detail in the Applications and Treatments sections of this document.

[0023] This document also provides methods for expressing an antibody or fusion protein that has therapeutic use using a cccDNA vector in a cell or subject. Such antibodies or fusion proteins can be used to treat a disease. Accordingly, provided herein are methods of treating a disease comprising administering a cccDNA vector encoding a therapeutic antibody or fusion protein to a subject in need thereof. In other embodiments, the therapeutic antibody or fusion protein can be used to target malignant cells, monitor specific proteins, or for diagnostic purposes.

[0024] In some embodiments, this claim may be defined by any of the following paragraphs:

1. A non-capsid closed-end DNA vector (ccDNA) containing:

at least one heterologous nucleotide sequence between the flanking inverted terminal repeats (ITRs), wherein the at least one heterologous nucleotide sequence encodes at least one antibody and/or fusion protein.

1. cccDNA vector according to claim 1, in which at least one heterologous nucleotide sequence encodes an antibody.

2. The cccDNA vector of claim 2, wherein the antibody is a full-length antibody, a Fab, a Fab', a single domain antibody, or a single chain antibody (scFv).

3. cccDNA vector according to claim 3, in which at least one heterologous nucleotide sequence encodes a single-domain antibody or a single-chain antibody.

4. The cccDNA vector according to claim 4, wherein the at least one heterologous nucleotide sequence further encodes a secretory leader sequence preceding the single domain antibody or single chain antibody.

5. cccDNA vector according to any one of paragraphs. 1-3, wherein the first heterologous nucleotide sequence encodes a heavy chain variable region and the second heterologous nucleotide sequence encodes a light chain variable region.

6. cccDNA vector according to claim 4, in which the first heterologous nucleotide sequence encodes a heavy chain variable region and a heavy chain constant region or a part thereof, and the second heterologous nucleotide sequence encodes a light chain variable region and a light chain constant region or a part thereof.

7. The cccDNA vector according to claim 6 or claim 7, wherein the first heterologous nucleotide sequence and/or the second heterologous nucleotide sequence further encodes a secretory leader sequence preceding the heavy chain variable region and/or the light chain variable region.

8. cccDNA vector according to any one of paragraphs. 1-8, wherein the antibody is a human or humanized antibody.

9. cccDNA vector according to any one of paragraphs. 1-9, wherein the antibody is an IgG, IgA, IgD, IgM or IgE antibody.

10. The cDNA vector according to claim 10, wherein the antibody is an IgG antibody.

11. The cccDNA vector according to claim 11, wherein the IgG antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

12. cccDNA vector according to any one of paragraphs. 1-12, in which the antibody binds to at least one target selected from the targets listed in Tables 1, 2, 3A, 3B, 4 and 5.

13. cccDNA vector according to claim 1, in which at least one heterologous nucleotide sequence encodes a fusion protein.

14. The cccDNA vector of claim 14, wherein the at least one heterologous nucleotide sequence further encodes a secretory leader sequence preceding the fusion protein.

15. The cccDNA vector of claim 14 or claim 15, wherein the fusion protein contains at least one extracellular domain of the receptor fused to an Fc region.

16. The cccDNA vector of claim 16, wherein the extracellular domain of the receptor is an extracellular domain of a receptor selected from CTLA-4, VEGFR1, VEGFR2, LFA-3, TNFR, IL-1R1, IL-1R1, IL-1RAcP and ACVR2A .

17. cccDNA vector according to any one of paragraphs. 1-17, wherein the antibody or fusion protein is selected from the antibodies and fusion proteins from Tables 1, 2, 3A, 3B, 4 or 5.

18. cccDNA vector according to any one of paragraphs. 1-18, in which the cDNA vector contains one or more poly(A) sites.

19. cccDNA vector to any of paragraphs. 1-19, in which the cDNA vector contains at least one promoter operably linked to at least one heterologous nucleotide sequence.

20. cccDNA vector according to any one of paragraphs. 1-20, wherein the at least one heterologous nucleotide sequence is a cDNA.

21. cccDNA vector according to any one of paragraphs. 1-21, wherein at least one ITR contains a functional end resolution site and a Rep binding site.

22. cccDNA vector according to any one of paragraphs. 1-22, in which one or both ITRs are taken from a virus selected from parvovirus, dependovirus and adeno-associated virus (AAV).

23. cccDNA vector according to any one of paragraphs. 1-23, in which the flanking ITRs are symmetric or asymmetric.

24. The cccDNA vector of claim 24, wherein the flanking ITRs are symmetrical or substantially symmetrical.

25. cccDNA vector according to claim 24, in which the flanking ITRs are asymmetric.

26. cccDNA vector according to any one of paragraphs. 1-26, in which one or both ITRs are wild type, or in which both ITRs are wild type.

27. cccDNA vector according to any one of paragraphs. 1-27, in which the flanking ITRs belong to different viral serotypes.

28. cccDNA vector according to any one of paragraphs. 1-28, in which the flanking ITRs belong to the pair of viral serotypes listed in Table 6.

29. cccDNA vector according to any one of paragraphs. 1-29, wherein one or both of the ITRs contain a sequence selected from the sequences in Table 7.

30. cccDNA vector according to any one of paragraphs. 1-30, wherein at least one of the ITRs is altered relative to the wild-type AAB ITR sequence by deletion, addition, or substitution that affects the overall three-dimensional conformation of the ITR.

31. cccDNA vector according to any one of paragraphs. 1-31, wherein one or both ITRs are derived from an AAV serotype selected from AAB1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAB12.

32. cccDNA vector according to any one of paragraphs. 1-32, in which one or both ITRs are synthetic.

33. cccDNA vector according to any one of paragraphs. 1-33, in which one or both ITRs are not wild-type ITRs, or in which both ITRs are not wild-type.

34. cccDNA vector according to any one of paragraphs. 1-34, wherein one or both ITRs are modified by deletion, insertion and/or substitution in at least one of the ITR regions selected from A, A', B, B', C, C', D and D'.

35. The cccDNA vector of claim 35, wherein the deletion, insertion and/or substitution removes all or part of the stem-loop structure typically formed by regions A, A', B, B', C or C'.

36. cccDNA vector according to any one of paragraphs. 1-36, wherein one or both ITRs are modified by deletion, insertion and/or substitution that remove all or part of the stem-loop structure typically formed by regions B and B'.

37. cccDNA vector according to any one of paragraphs. 1-37, wherein one or both ITRs are modified by deletion, insertion and/or substitution that remove all or part of the stem-loop structure typically formed by the C and C' regions.

38. cccDNA vector according to any one of paragraphs. 1-38, wherein one or both ITRs are modified by deletion, insertion and/or substitution that removes part of the stem-loop structure typically formed by regions B and B' and/or part of the stem-loop structure typically formed by regions C and C'.

39. cccDNA vector according to any one of paragraphs. 1-39, in which one or both ITRs contain a single stem-loop structure in a region typically containing a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'.

40. cccDNA vector according to any one of paragraphs. 1-40, in which one or both ITRs contain one stem and two loops in a region typically containing a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'.

41. cccDNA vector according to any one of paragraphs. 1-41, in which one or both ITRs contain one stem and one loop in a region typically containing a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'.

42. cccDNA vector according to any one of paragraphs. 1-42, in which both ITRs are changed in such a way that it results in an overall three-dimensional symmetry when the ITRs are inverted with respect to each other.

43. cccDNA vector according to any one of paragraphs. 1-43, in which one or both ITRs contain a sequence selected from the sequences in Tables 7, 9A, 9B and 10.

44. cccDNA vector according to any one of paragraphs. 1-44, wherein the at least one heterologous nucleotide sequence is under the control of at least one regulatory switch.

45. The cccDNA vector of claim 45, wherein the at least one regulatory switch is selected from a binary regulatory switch, a small molecule regulatory switch, a password-controlled regulatory switch, a nucleic acid-based regulatory switch, a post-transcriptional regulatory switch, radiation-controlled or controllable ultrasound regulatory switch, hypoxia-mediated regulatory switch, inflammatory response regulatory switch, shift-activated regulatory switch and "safety switch".

46. A method for expressing an antibody or fusion protein in a cell, comprising bringing the cell into contact with a cDNA vector according to any one of claims. 1-46.

47. The method of claim 47, wherein the contacting cell is a eukaryotic cell.

48. The method according to claim 47 or 48, in which the cell is in vitro or in vivo .

49. Method according to any one of paragraphs. 47-49, wherein the at least one heterologous nucleotide sequence is codon-optimized for expression in a eukaryotic cell.

50. Method according to any one of paragraphs. 47-50, in which the antibody or fusion protein is secreted from the cell.

51. Method according to any one of paragraphs. 47-50, in which the antibody or fusion protein is retained in a cell.

52. A method of treating a subject with a therapeutic antibody or therapeutic fusion protein, comprising administering to the subject a cccDNA vector according to any one of claims. 1-46, wherein at least one heterologous nucleotide sequence encodes a therapeutic antibody or therapeutic fusion protein.

53. The method of claim 53, wherein the subject has a disease or disorder selected from cancer, an autoimmune disease, a neurodegenerative disorder, hypercholesterolemia, acute organ rejection, multiple sclerosis, postmenopausal osteoporosis, skin diseases, asthma, or hemophilia.

54. The method of claim 53, wherein the cancer is selected from solid tumor, soft tissue sarcoma, lymphoma and leukemia.

55. The method of claim 53, wherein the autoimmune disease is selected from rheumatoid arthritis and Crohn's disease.

56. The method of claim 53, wherein the skin disease is selected from psoriasis and atopic dermatitis.

57. The method of claim 53, wherein the neurodegenerative disorder is Alzheimer's disease.

58. Pharmaceutical composition containing the cDNA vector according to any one of paragraphs. 1-46.

59. A cell containing a cccDNA vector according to any one of paragraphs. 1-46.

60. A composition containing a cccDNA vector according to any one of paragraphs. 1-46 and lipid.

61. The composition according to claim 61, in which the lipid is a lipid nanoparticle (LNP).

62. A kit containing a cccDNA vector according to any one of paragraphs. 1-46 or the composition according to paragraph 61 or 62, or the cell according to paragraph 65.

63. A method for producing an antibody or fusion protein, comprising culturing the cell of claim 60 under conditions suitable for producing the antibody or fusion protein.

64. The method of claim 64, further comprising isolating the antibody or fusion protein.

[0025] In some embodiments, one aspect of the technology described herein relates to a non-viral, capsid-free, covalently closed-end DNA vector (ccDNA vector), wherein the cccDNA vector contains at least one heterologous nucleotide sequence operably located between two inverted terminal repeat sequences, wherein the ITR sequences may be asymmetric, or symmetric, or substantially symmetric, as those terms are defined herein, wherein at least one of the ITRs contains a functional end resolution site and a Rep binding site, and optionally , a heterologous nucleic acid sequence encodes a transgene (eg, an antibody or fusion protein), and the vector is not contained within a viral capsid.

[0026] These and other aspects of the present invention are described in more detail below.

DESCRIPTION OF GRAPHIC MATERIALS

[0027] The embodiments of the present invention summarized above and described in more detail below will be understood with reference to the illustrative embodiments of the invention shown in the accompanying drawings. However, the accompanying drawings illustrate only exemplary embodiments of the invention and, therefore, should not be construed as limiting the scope thereof, since the disclosure may permit other equally effective embodiments.

[0028] The embodiments of the present invention summarized above and described in more detail below will be understood with reference to the illustrative embodiments of the invention shown in the accompanying drawings. However, the accompanying drawings illustrate only exemplary embodiments of the invention and, therefore, should not be construed as limiting the scope thereof, since the disclosure may permit other equally effective embodiments.

[0029] FIG. 1A shows a typical example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing asymmetric ITRs. In this embodiment, a typical cccDNA vector comprises an expression cassette containing a CAG promoter, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a BGHpA (bovine growth hormone polyadenylation signal). An open reading frame (ORF) encoding a transgene, such as a nucleic acid encoding an antibody or fusion protein, can be inserted into a cloning site (R3/R4) between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - the wild-type AAB2 ITR to the left of (at the 5' end) and the modified ITR to the right of (at the 3' end) the expression cassette, thus the two ITRs flanking the expression cassette are asymmetrical relative to each other.

[0030] FIG. 1B shows a typical example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing asymmetric ITRs with an expression cassette containing a CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a transgene, such as a nucleic acid encoding an antibody or fusion protein, can be inserted into a cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs), a modified ITR to the left of (at the 5' end) and a wild-type ITR to the right of (the 3' end of) the expression cassette.

[0031] FIG. 1C shows a typical example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing asymmetric ITRs, with an expression cassette containing an enhancer/promoter, a transgene, a post-transcriptional element (WPRE), and a poly(A) signal. An open reading frame (ORF) allows the insertion of a transgene, such as a nucleic acid encoding an antibody or fusion protein, into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs), which are asymmetrical relative to each other - a modified ITR to the left of (at the 5' end) and a modified ITR to the right of (at the 3' end) the expression cassette, with the 5' ITR and The 3'-ITRs are both modified ITRs, but have different modifications (ie they do not have the same modifications).

[0032] FIG. 1D shows a typical example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing symmetrical modified ITRs or substantially symmetrical modified ITRs as defined herein, with an expression cassette containing a CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a transgene, such as a nucleic acid encoding an antibody or fusion protein, is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs), the modified 5'-ITR and the modified 3'-ITR being symmetrical or substantially symmetrical.

[0033] FIG. 1E shows a representative example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing symmetrical modified ITRs or substantially symmetrical modified ITRs as defined herein, with an expression cassette containing an enhancer/promoter, transgene, post-transcriptional element (WPRE) and poly(A) signal. An open reading frame (ORF) allows the insertion of a transgene, such as a nucleic acid encoding an antibody or fusion protein, into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs), the modified 5'-ITR and the modified 3'-ITR being symmetrical or substantially symmetrical.

[0034] FIG. 1F shows a representative example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing symmetrical WT-ITRs or substantially symmetrical WT-ITRs as defined herein, with an expression cassette containing a CAG promoter, WPRE, and BGHpA. An open reading frame (ORF) encoding a transgene, such as a nucleic acid encoding an antibody or fusion protein, is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two wild-type inverted terminal repeats (WT-ITRs), with the 5' WT-ITR and 3' WT-ITR being symmetrical or substantially symmetrical.

[0035] FIG. 1G shows a representative example of the structure of a cccDNA vector for producing an antibody or fusion protein, as described herein, containing symmetrical modified ITRs or substantially symmetrical modified ITRs as defined herein, with an expression cassette containing an enhancer/promoter, transgene, post-transcriptional element (WPRE) and poly(A) signal. An open reading frame (ORF) allows the insertion of a transgene, such as a nucleic acid encoding an antibody or fusion protein, into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two wild-type inverted terminal repeats (WT-ITRs), with the 5' WT-ITR and 3' WT-ITR being symmetrical or substantially symmetrical.

[0036] In FIG. 2A shows the T-shaped stem-loop structure of the left ITR of wild-type AAB2 (SEQ ID NO: 52) with the A-A' arm, B-B' arm, C-C' arm, and two Rep-binding sites indicated ( RBE and RBE'), as well as with the specified terminal resolution site ( trs ). RBE contains a series of 4 duplex tetramers that are believed to interact with either Rep 78 or Rep 68. In addition, RBE' is also thought to interact with the Rep complex assembled on a wild-type ITR or a mutated ITR construct. Regions D and D' contain transcription factor binding sites and other conserved structures. Fig. 2B shows the putative Rep-catalyzed nicking and ligation activities of the wild-type left ITR (SEQ ID NO: 53), including the T-shaped stem-loop structure of the wild-type left ITR of AAB2 with arm A-A', arm B-B', arm C designated -C', two Rep-binding sites (RBE and RBE'), as well as the indicated terminal resolution site ( trs ) and a region D and D' containing several transcription factor binding sites and other conserved structures.

[0037] In FIG. 3A shows the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the AA' arm and the CC' and BB' arms of the left ITR of wild-type AAB2 (SEQ ID NO: 54). Fig. 3B shows a typical example of a mutated ITR (also called modified ITR) sequence for the left ITR. Shown are the primary structure (left) and predicted secondary structure (right) of the RBE-containing portion of arm A-A', arm C, and arm B-B' of a representative example of a mutated left ITR (ITR-1, left) (SEQ ID NO: 113). Fig. 3C shows the primary structure (left) and secondary structure (right) of the RBE-containing portion of the AA' loop and arms BB' and CC' of the right ITR of wild-type AAB2 (SEQ ID NO: 55). Fig. 3D shows a typical example of a right modified ITR. Shown are the primary structure (left) and predicted secondary structure (right) of the RBE-containing portion of the A-A' arm, BB' and C arms of a representative example of a mutant right ITR (ITR-1, right) (SEQ ID NO: 114). Any combination of left and right ITRs (eg, AAB2 or other viral serotype ITRs, or synthetic ITRs) as described herein can be used. Each of the polynucleotide sequences in FIG. 3A-3D refer to sequences used in the genome of a plasmid or bacmid/baculovirus used to produce cccDNA as described herein. Also each of FIGS. 3A-3D include the corresponding cccDNA secondary structures derived from the configurations of the ccDNA vector in the plasmid or bacmid/baculovirus genome and predicted Gibbs free energy values.

[0038] FIG. 4A is a schematic representation of an upstream process for producing baculovirus-infected insect cells (BIICs) that are useful for producing a cccDNA vector for producing an antibody or fusion protein in the manner schematically represented in FIG. 4B. In FIG. 4B schematically illustrates an example of a method for obtaining cccDNA, and FIG. 4C shows a biochemical method and process for confirming the production of a cccDNA vector. In FIG. 4D and Fig. 4E is a schematic diagram describing the process of identifying the presence of cccDNA in DNA collected from cell pellets obtained during the ccDNA production process of FIG. 4B. In FIG. 4D is a schematic representation of the expected bands for a typical example of cccDNA undigested or digested with a restriction endonuclease and then subjected to electrophoresis on a native or denaturing gel. The leftmost diagram shows a native gel with several bands, indicating that in duplex and uncut form, cccDNA exists in at least monomeric and dimeric states, which appear as a faster migrating monomer of smaller size and a slower migrating dimer of larger size 2 times more than the monomer. The second schematic from the left demonstrates that when cccDNA is cut by a restriction endonuclease, the original bands disappear and faster migrating bands (eg, smaller) appear that correspond to the expected fragment sizes remaining after digestion. Under denaturing conditions, the original duplex DNA is single-stranded and migrates as a compound twice the size of that observed on a native gel because the complementary strands are covalently linked. Accordingly, in the second diagram from the right, digested ccDNA exhibits a band distribution similar to that observed on the native gel, but the bands migrate as fragments twice the size of their native gel counterparts. In the rightmost diagram, uncut cccDNA under denaturing conditions migrates as a single-stranded open ring and therefore the bands observed are twice as large as under native conditions when the ring is not open. In this figure, “t.p.o.” used to indicate the relative size of nucleotide molecules, based, depending on the context, on either the length of the nucleotide chain (for example, for single-stranded molecules observed under denaturing conditions) or the number of base pairs (for example, for double-stranded molecules observed under native conditions) . Fig. 4E shows DNA having a discontinuous structure. cDNA can be cut by a restriction endonuclease that has a single recognition site on the cDNA vector to produce two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. Fig. 4E also shows cccDNA having a linear and continuous structure. The cccDNA vector can be cut with a restriction endonuclease and generate two DNA fragments that migrate as 1 kb. and 2 kb. under neutral conditions, but under denaturing conditions the chains remain linked and form single chains that migrate as 2 kb. and 4 kb.

[0039] FIG. 5 shows an illustrative denaturing gel run of representative examples of cccDNA vectors with (+) or without (-) endonuclease digestion (EcoRI for ccDNA constructs 1 and 2; BamH1 for ccDNA constructs 3 and 4; SpeI for ccDNA constructs 5 and 6 and XhoI for cccDNA constructs 7 and 8). Constructs 1-8 are described in Example 1 of international application PCT/US18/49996, which is incorporated herein by reference in its entirety. The dimensions for the stripes highlighted with an asterisk have been determined and are given at the bottom of the drawing.

[0040] FIG. 6A-6C show representative examples of constructs and plasmids for creating a cccDNA vector for producing an antibody or fusion protein, and for illustrative purposes, a ccDNA vector encoding aducanumab is shown. One of ordinary skill in the art can easily replace the nucleic acid encoding aducanumab with any other nucleic acid encoding another antibody or fusion protein. Fig. 6A shows a typical example of a ccDNA plasmid (pFBdual-ccDNA-adecanumab; SEQ ID NO: 56) for producing a ccDNA vector expressing aducanumab (full IgG1). This cccDNA plasmid contains a nucleic acid sequence for the expression of aducanumab, which has been codon-optimized, flanked between a pair of asymmetric ITRs (ie, WT 5'-ITR (wild type ITR) and 3' mod-ITR (right asymmetric ITR). This ITR pair can be easily replaced by another asymmetric ITR pair or a symmetric ITR pair as described herein.In addition, this plasmid contains, flanked between the ITR pair, in the 5' to 3' direction: the SV40 enhancer (SEQ ID NO: 126 ), the human EF1 alpha promoter (SEQ ID NO: 77) or fragment thereof (SEQ ID NO: 78) and the VH1-02 secretory leader sequence (SEQ ID NO: 88), the optimized heavy chain (HC) nucleic acid sequence of aducanumab ( SEQ ID NO: 57), SV40 poly(A) sequence (SEQ ID NO: 86) and, located upstream of the aducanumab light chain (LC) sequence, are the following: CMV enhancer (SEQ ID NO: 83), rEF1 promoter (SEQ ID NO : 85 or SEQ ID NO: 150), VK A26 leader sequence (SEQ ID NO: 89), optimized aducanumab light chain (LC) nucleic acid sequence (SEQ ID NO: 58) and BGH polyadenylation sequence (SEQ ID NO: 68 or SEQ ID NO: 148). The optimized aducanumab heavy chain (HC) sequence and the optimized aducanumab light chain (LC) nucleic acid sequence can be easily replaced by any other antibody heavy chain or light chain sequences described herein, for example, see Tables 1-5 herein. Fig. 6B shows a typical example of an insert that can be used as a modular component for insertion into a desired ccDNA vector to produce the plasmid shown in FIG. 6A. Fig. 6C shows a linearized view of the region of the plasmid ceDNA-Adu-full-IgG1 (ccDNA-Adu-full-IgG1) containing the sequences for producing aducanumab.

[0041] FIG. 7A-7G show exemplary cccDNA vectors that can express a variety of different antibodies or antigen binding fragments or fusion proteins as disclosed herein. The exemplary cccDNA vectors also illustrate a variety of configurations with respect to the use of IRES sequences, promoter sequences, enhancer sequences, linker sequences, and polyadenylation sequences. Fig. 7A shows an embodiment of a cccDNA vector construct for producing antibodies with a poly(A) sequence located after the heavy chain sequence and, optionally, an enhancer before the light chain nucleic acid sequence. Fig. 7B shows an embodiment of a cccDNA vector construct for producing antibodies with a poly(A) sequence after the heavy chain sequence, and an IRES before the light chain nucleic acid sequence. Fig. 7C shows a variant of a cccDNA vector construct for producing antibody fragments (eg, antigen binding fragments) similar to FIG. 7A, including those containing a poly(A) sequence after a heavy chain Fab fragment sequence and, optionally, an enhancer before a light chain fragment nucleic acid sequence. Fig. 7D shows an embodiment of a cccDNA vector construct for producing an antibody as disclosed herein, including a poly(A) sequence located after the light chain sequence. Fig. 7E shows an embodiment of a cccDNA vector construct for antibody production as described herein, including a poly(A) sequence located after the heavy chain sequence. Fig. 7F shows an embodiment of a cccDNA vector construct for producing a single domain antibody (dAb) as described herein, including a poly(A) sequence located after the dAb sequence. Fig. 7G shows an embodiment of a cccDNA vector construct for producing an antibody fragment, such as a single chain variable fragment (scFv) fusion protein or single chain antibody as disclosed herein, including a poly(A) sequence located after the scFv sequence of the single chain antibody sequence. . One skilled in the art will appreciate that cccDNA vectors for producing antibodies, as described herein, can be used in a modular manner such that desired regulatory sequences or heterologous nucleic acids encoding an antibody or fragment thereof can be replaced by other desired sequences. Thus, cccDNA vectors can be tailored for desired applications. Fig. 7A-7G also show embodiments in which the variable chain (V _H and V _L ) and constant chain ( _CH and C _L ) nucleic acid sequences and Fc sequences are proximal to each other, or alternatively, the Fc may be attached to the V _H and V _L coding sequence via a linker sequence as disclosed herein.

[0042] FIG. 8A-8B show a typical example of SDS-PAGE ( Figure 8A ) and Western blotting ( Figure 8B ) analysis of the expression of aducanumab antibody (full IgG1) expressed from a cDNA-IgG1- construct. Adu as described in Example 9, after one-step purification of the expressed protein. Fig. 8A shows an SDS-PAGE gel image of the expressed antibody. In the lanes: protein marker M1 (Takara, cat. no. 3452), and purified aducanumab is shown under reducing conditions (lane 1) and non-reducing conditions (lane 2). The presence of two bands under reducing conditions, but only one band under non-reducing conditions, is consistent with the proposal that the protein is an antibody with a heavy and light chain that migrates as a single band under non-reducing conditions, and as constituent heavy and light chains in restorative conditions. Fig. 8B shows a Western blot image immunostained with anti-human IgG antibody. In lanes: protein marker M2 (GenScript, cat. no. M00521), and P indicates positive control human IgG1 antibody (Sigma).

[0043] FIG. 9A-9B show the expression of cccDNA expressing GFP antibody or aducanumab (full IgG1) expressed by ccDNA vector-IgG1-Adu. Fig. 9A shows fluorescence microscopic images of HEK293T cells transfected with ccDNA-GFP plasmid (top panel) and ccDNA vector-GFP (bottom panel) as described in Example 8. The presence of abundant fluorescence in both images indicates that significant transfection and expression of transgenic GFP has occurred in cells treated with any cDNA. Fig. 9B shows two different images of the same membrane transport of cell samples separated by SDS-PAGE as described in Example 8. Bottom panel is Ponceau-stained membrane showing total protein content; the top panel is a Western blot in which visible bands reflect the presence of human antibody. In lanes 7-10, the antibody heavy chain migrates at approximately 50 kDa and the antibody light chain migrates at approximately 25 kDa; both circuits are visible on all four tracks.

[0044] FIG. 10A-10B show characterization of a cDNA-generated antibody to aducanumab. Fig. 10A shows the results of the HPLC analysis described in Example 9 showing a single peak corresponding to aducanumab cDNA production. Fig. 10B shows the results of an ELISA assay assessing the ability of a purified anti-aducanumab antibody to recognize immobilized beta-amyloid (1-42) ligand as described in Example 9.

[0045] FIG. 11 graphically depicts the results of the experiments described in Example 10. Negative control samples from mice treated with cccDNA constructs lacking aducanumab transgenes (labeled negative control ccDNA) were at or below the lower limit of quantitation of the assay. In contrast, sera from mice treated with the cccDNA-IgG construct had high levels of human immunoglobulin present on both days 3 and 7.

[0046] FIG. 12 shows results for two different exposure times of the same membrane transfer of cell samples separated by SDS-PAGE as described in Example 12. The top panel corresponds to a 6 second exposure and the bottom panel to a 20 second exposure. Bands corresponding to intact antibody are visible at the top of the gel (and a limited amount of reduced heavy and light chains migrating at ca. 50 kDa and ca. 25 kDa, respectively) are visible in lanes 5, 7 (both aducanumab) and 11 (bevacizumab ) (indicated by arrows). In lane 9, the presence of the Fc fusion protein is observed at the top of the lane and, as expected, no lower molecular weight products are observed.

DETAILED DESCRIPTION OF THE INVENTION

[0047] Provided herein are cccDNA vectors for producing antibodies, as described herein, containing one or more heterologous nucleic acids that encode an antibody (e.g., heavy chains, light chains, scaffold, Fab', scAb (single chain antibody) ). Also provided herein are cccDNA vectors for producing a fusion protein, as described herein, containing one or more heterologous nucleic acids encoding the fusion protein. Such vectors can be used in the production of commercial antibodies or fusion proteins, or in the delivery of a therapeutic antibody or fusion protein, as described herein, by intracellular expression from a cDNA vector. In some embodiments, expression of an antibody or fusion protein may involve secreting the antibody or fusion protein from the cell in which it is expressed, or alternatively, in some embodiments, the expressed antibody or fusion protein may be targeted to a protein within the cell in which it is expressed (e.g. , the antibody is an intrabody). In some embodiments, the cccDNA vector expresses the antibody or antigen binding fragment or fusion protein thereof in muscle (e.g., skeletal muscle) of the subject, which can act as a depot for the production and secretion of the antibody or fusion protein into multiple systemic compartments.

I. Definitions

[0048] Unless otherwise defined herein, scientific and technical terms used in connection with this application will have the meanings commonly understood by those skilled in the art to which this invention relates. It should be understood that the present invention is not limited to the specific methodology, protocols, reagents, etc. described herein, which may in fact vary. The terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. Definitions of common immunology and molecular biology terms can be found in the following sources: The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th 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, Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, ^4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al ., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe (eds.), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737 ), the contents of which are incorporated herein by reference in their entirety.

[0049] As used herein, the terms “heterologous nucleotide sequence” and “transgene” are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that is included in and can be delivered and expressed by cccDNA -vector as described in this document.

[0050] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear fragment of nucleic acids that includes a transgene operably linked to one or more promoters or other regulatory sequences sufficient to allow direct transcription of the transgene, but does not contain capsid coding sequences, other vector sequences or inverted terminal repeat regions. The expression cassette may further comprise one or more cis- acting sequences (eg, promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0051] The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to the polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the term includes single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. "Oligonucleotide" generally refers to polynucleotides of about 5 to about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this invention, no upper limit to the length of the oligonucleotide has been established. 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, for the purposes of the described embodiments, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0052] As used herein, the term “nucleic acid construct” refers to a nucleic acid molecule, single-stranded or double-stranded, that is isolated from a naturally occurring gene or is modified to contain nucleic acid segments in a manner not found in nature or is synthetic. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains control sequences necessary for expression of the coding sequence of the present invention. An "expression cassette" includes a DNA coding sequence operably linked to a promoter.

[0053] By “hybridizable” or “complementary” or “substantially complementary” it is meant that the nucleic acid (eg, RNA) includes a nucleotide sequence that allows it to bind non-covalently, i.e. form Watson-Crick base pairs and/or G/U base pairs, undergo "annealing" or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) in appropriate in vitro and/or in vivo conditions of temperature and ionic strength of the solution. As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, it is also known in the art that when two RNA molecules (eg, dsRNA) are hybridized, a guanine (G) base is paired with a 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 the base pairing of tRNA anti-codons with codons in mRNA. In the context of the present invention, the guanine (G) of the protein-binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of the present invention is considered complementary to uracil (U), and vice versa. Thus, when a G/U base pair can be produced at a given nucleotide position with the protein binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of the present invention, this position is not considered non-complementary, but is considered complementary.

[0054] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to the polymeric form of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids. and polypeptides having modified peptide backbones.

[0055] As used herein, the term “antibody” includes any antibody or antibody fragment (ie, a functional antibody fragment) or antigen binding fragment that retains antigen binding activity for a desired antigen or epitope. In one embodiment, the antibody or antigen-binding fragment thereof comprises an immunoglobulin chain or fragment thereof and at least one immunoglobulin variable domain sequence. Examples of antibodies include, but are not limited to, scFv, Fab fragment, Fab', F(ab') ₂ , single domain antibody (dAb), heavy chain, light chain, heavy and light chain, complete antibody (eg, including each of Fc, Fab , heavy chains, light chains, variable regions, etc.), bispecific antibody, diabody, linear antibody, single chain antibody, intrabody, monoclonal antibody, chimeric antibody or multimeric antibody. In addition, the antibody can be obtained from any mammal, such as primates, humans, rats, mice, horses, goats, etc. In one embodiment, the antibody is human or humanized. In some embodiments, the antibody is a modified antibody. In some embodiments, the antibody components may be expressed separately such that the antibody self-assembles upon expression of the protein components. In some embodiments, the antibody has a desired function, such as interacting with and inhibiting a desired protein, to treat a disease or symptom of a disease. In one embodiment, the antibody or antigen-binding fragment thereof comprises a framework region or F _c region. An antibody fragment may retain 10-99% of the activity of the full antibody (e.g., 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10 -20%, 50-99%, 50-90%, 50-80%, 50-70%, 50-60%, 20-99%, 30-99%, 40-99%, 60-99%, 70 -99%, 80-99%, 90-99% or any activity value between the specified values). It is also contemplated herein that the functional antibody fragment contains activity that is greater than that of the intact antibody (eg, at least 2-fold or greater). In another embodiment, the antibody fragment contains an affinity for its target that is substantially similar to the affinity of an intact antibody for the same target (eg, epitope). An antibody may be "activating" so that it increases the activity of the target protein, or "inhibitory" (eg, a neutralizing or blocking antibody) so that it reduces the activity of the target protein.

[0056] As used herein, the term “antigen binding domain” of an antibody molecule refers to the portion of an antibody molecule, such as an immunoglobulin (Ig) molecule, that is involved in binding an antigen. In embodiments, the antigen binding site is formed by amino acid residues of the heavy (H) and light (L) chain variable (V) regions. Three highly divergent regions in the variable regions of the heavy and light chains, called hypervariable regions, are located between more conserved flanking regions called "framework regions" (FR). FRs are amino acid sequences that occur naturally between and adjacent to hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are positioned relative to each other in three-dimensional space such that they form an antigen-binding surface complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions of each of the heavy and light chains are called “complementarity determining regions” or “CDRs”. The framework region and CDR have been defined and described, for example, in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (eg, a variable heavy chain and a variable light chain) typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus, in the amino acid sequence order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. As used herein, the terms “complementarity determining region” and “CDR” refer to the amino acid sequences in the variable regions of an antibody that confer antigen specificity and binding affinity. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The precise boundaries of the amino acid sequence of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (Chothia numbering scheme). As used herein, CDRs defined in accordance with the Chotia numbering scheme are also sometimes referred to as “hypervariable loops.” For example, according to the Kabat scheme, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). In the Chotia scheme, the CDR amino acids in VH are numbered 26–32 (HCDR1), 52–56 (HCDR2), and 95–102 (HCDR3); and amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). Each VH and VL typically includes three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0057] As used herein, the term “full-length antibody” refers, for example, to an immunoglobulin (Ig) molecule (eg, IgG, IgE, IgM antibody) that occurs naturally and is formed by recombination processes of a fragment of a normal immunoglobulin gene.

[0058] As used herein, the terms “functional antibody fragment” or “antigen binding fragment” are used interchangeably and refer to an antibody fragment that binds to the same antigen or epitope that is recognized by an intact (eg, full-length) antibody. The terms "antibody fragment" or "functional fragment" also include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, or recombinant single chain polypeptide molecules in which the light and heavy variable regions are linked peptide linker (“scFv proteins”). In some embodiments, the antibody fragment does not include portions of antibodies without antigen-binding activity, such as Fc fragments or individual amino acid residues. In some embodiments, the functional antibody fragment retains at least 20% of the activity of the intact or full-length antibody, for example, when assessed by measuring the degree of activation or inhibition of a target protein. In other embodiments, the functional antibody fragment retains at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% (ie substantially the same amount) of activity compared to an intact antibody. It is also contemplated herein that the functional antibody fragment will have increased activity compared to the intact antibody (e.g., at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 100 times or more).

[0059] As used herein, the term “immunoglobulin variable domain sequence” refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N- or C-terminal amino acids, or may include other changes that are compatible with the formation of the protein structure.

[0060] As used herein, the term "framework" or "framework sequence" refers to the remaining variable region sequences excluding the CDRs. Since the precise definition of the CDR sequence may be subject to different systems, the meaning of the framework sequence will be determined according to different interpretations. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 light chain and CDR-H1, CDR-H2 and CDR-H3 heavy chain) also divide the light chain and heavy chain framework regions into four subregions (FR1, FR2, FR3 and FR4) on each strand, with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying specific subregions as FR1, FR2, FR3 or FR4, the framework region, in accordance with other views, denotes combined FRs within a variable region of a single naturally occurring immunoglobulin chain. As used herein, a single FR (a FR) represents one of four subregions, and multiple FRs (FRs) represent two or more of the four subregions constituting a framework region.

[0061] The DNA sequence that "encodes" a particular antibody or antigen binding fragment is a DNA nucleic acid sequence that is transcribed into a particular RNA and/or protein. A DNA polynucleotide may encode RNA (mRNA) that is translated into a protein, or a DNA polynucleotide may encode RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA-targeting RNA; also called "non-coding RNA" or "ncRNA") .

[0001] As used herein, the term “fusion protein”, as used herein, refers to a polypeptide containing protein domains from at least two different proteins. For example, a fusion protein may comprise (i) an antibody or fragment thereof (eg, an antigen binding portion or antigen binding fragment of an antibody) or a ligand binding domain, and (ii) at least one non-antibody protein. Fusion proteins covered by this document include, without limitation, an antibody or Fc or antigen binding fragment of an antibody fused to a protein of interest, for example, a receptor extracellular domain, ligand, enzyme or peptide. The antibody or antigen binding fragment thereof that is part of the fusion protein may be a monospecific antibody or a bispecific or multispecific antibody.

[0062] As used herein, the term “genomic safe harbor gene” or “safe harbor gene” refers to a gene or loci into which a nucleic acid sequence can be inserted such that the sequence can integrate and function in a predictable manner (e.g., to express a protein, of interest) without significant detrimental effects on endogenous gene activity or promotion of cancer development. In some embodiments, a safe harbor gene is also a locus or gene at which the inserted nucleic acid sequence can be expressed efficiently and at higher levels than at a non-safe harbor site.

[0063] As used herein, the term “gene delivery” refers to the process by which foreign DNA is transferred into host cells for gene therapy use.

[0064] As used herein, the term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that contains at least one minimum essential origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence (“RBS”) (also called the RBE (Rep binding element)) and the terminal resolution site (“TRS”) together constitute the “minimum required origin of replication” and, accordingly, the TR contains at least one RBS and at least one TRS. TRs that are inversely complementary to each other along a given stretch of polynucleotide sequence are commonly referred to as “inverted terminal repeats” or “ITRs.” In the context of a virus, ITRs mediate viral replication, viral packaging, integration, and proviral rescue. It has been surprisingly discovered by the inventors that TRs that are not reverse complementary throughout their entire length can still perform traditional ITR functions and, accordingly, the term ITR is used herein to refer to a TR in a cccDNA genome or ccDNA vector , which is capable of mediating the replication of the cDNA vector. One skilled in the art will appreciate that cccDNA vectors of complex configuration may contain more than two ITRs or pairs of asymmetric ITRs. The ITR may be an AAB ITR or a non-AAB ITR, or may be derived from an AAB ITR or a non-AAB ITR. For example, the ITR may be from a virus in 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 the SV40 origin of replication. may be used as an ITR, which may be further modified by truncation, substitution, deletion, insertion and/or addition. Viruses of the Parvoviridae family consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that are capable of replication in vertebrate hosts, including, but not limited to, human, primate, bovine, canine, equine, and sheep species. For convenience, in this document the ITR located 5' (upstream) of the expression cassette in a cccDNA vector is referred to as the "5'-ITR" or "left ITR", and the ITR located 3' (downstream) of the expression cassette in the cccDNA vector, referred to as “3'-ITR” or “right ITR”.

[0065] “Wild-type ITR” or “WT-ITR” refers to a naturally occurring ITR sequence in an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. The WT-ITR nucleotide sequence from any AAV serotype may differ slightly from the canonical naturally occurring sequence due to degeneracy or drift of the genetic code and, thus, WT-ITR sequences suitable for use in this invention include WT-ITR sequences generated as a result of natural changes that occur during production (for example, replication errors).

[0066] As used herein, the term “substantially symmetrical WT-ITRs” or “substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRs in a single ccDNA genome or ccDNA vector that are both wild-type ITRs having reverse complementary sequences along their entire length. For example, an ITR may be considered a wild-type sequence even if it has one or more nucleotides that deviate from the canonical sequence found in nature, as long as these changes do not affect the properties and overall three-dimensional structure of the sequence. In some aspects, deviant nucleotides are conserved sequence changes. As one non-limiting example, a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with a canonical sequence (measured, for example, using BLAST at default settings) also has a symmetrical three-dimensional spatial organization on the other WT-ITR, so that their three-dimensional structures have the same shape in geometric space. Essentially symmetrical WT-ITRs have identical A, C-C' and B-B' loops in three-dimensional space. An essentially symmetrical WT-ITR can be functionally confirmed to be wild type (WT) by determining that it has a functional Rep binding site (RBE or RBE') and a terminal resolution site (trs) that binds to the corresponding Rep protein. Additionally, other functions can be tested, including transgene expression under permissive conditions.

[0067] As used herein, the phrases “modified ITR” or “mod-ITR” or “mutant ITR” are used interchangeably and refer to an ITR that has a mutation in at least one or more nucleotides compared to a WT-ITR from the same serotype. The mutation may result in a change in one or more of the A, C, C', B, B' regions of the ITR, and may result in a change in the three-dimensional spatial organization (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial organization WT-ITR of the same serotype.

[0068] As used herein, the term “asymmetric ITRs,” also referred to as “asymmetric ITR pairs,” refers to a pair of ITRs in the same ccDNA genome or ccDNA vector that are not reverse complements in their full length. As one non-limiting example, an ITR in an asymmetric pair does not share a symmetrical three-dimensional spatial organization with its partner ITR, such that their three-dimensional structures have different shapes in geometric space. In other words, the ITRs in an asymmetric pair have different overall geometric structures, i.e. they have different organization of their A, C-C'- and B-B'-loops in three-dimensional space (for example, one ITR may have a short C-C' arm and/or a short B-B' arm compared to its partner ITR). The sequence difference between two ITRs may be due to addition, deletion, truncation, or point mutation of one or more nucleotides. In one embodiment, one ITR of an asymmetric ITR pair may be a wild-type AAB ITR sequence and the other ITR a modified ITR as defined herein (eg, a non-wild-type or synthetic ITR sequence). In another embodiment, neither of the ITRs of an asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs that have different shapes in geometric space (ie, different overall geometric structure). In some embodiments, one mod-ITR of an asymmetric ITR pair may have a short C-C' arm, and the other ITR may have a different modification (e.g., single arm or short B-B' arm, etc.), such that they have a different three-dimensional spatial organization compared to its paired asymmetric mod-ITR.

[0069] As used herein, the term “symmetrical ITRs” refers to a pair of ITRs in a single cccDNA genome or cccDNA vector that are mutated or modified relative to wild-type dependoviral ITR sequences and are reverse complementary throughout their entire length. Neither ITR is the wild-type AAB2 ITR sequence (ie, they are modified ITRs, also called mutant ITRs), and may differ from the wild-type ITR sequence due to addition, deletion, substitution, truncation, or point mutation of nucleotides. For convenience, in this document the ITR located 5' (upstream) of the expression cassette in a cccDNA vector is referred to as the "5'-ITR" or "left ITR", and the ITR located 3' (downstream) of the expression cassette in the cccDNA vector, referred to as “3'-ITR” or “right ITR”.

[0070] As used herein, the term “substantially symmetric modified ITRs” or “substantially symmetric mod-ITR pair” refers to a pair of modified ITRs in a single ccDNA genome or ccDNA vector that both have reverse complementary sequences throughout their entire length. For example, a modified ITR may be considered substantially symmetrical even though it has some nucleotide sequences that deviate from the reverse complementary sequence, as long as the changes do not affect the properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the canonical sequence (measured using BLAST at default settings) also has a symmetrical three-dimensional spatial organization with a paired modified ITR such that their three-dimensional structures have the same shape in geometric space. In other words, an essentially symmetric pair of modified ITRs has the same organization of A, C-C' and B-B' loops in three-dimensional space. In some embodiments, ITRs from a mod-ITR pair may have different reverse complementary nucleotide sequences, but still have the same symmetrical three-dimensional spatial organization, i.e. both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., 5'-ITR) in a mod-ITR pair may belong to one serotype, and the other ITR (e.g., 3'-ITR) may belong to a different serotype, but both may have the same corresponding mutations (e.g., if a 5'ITR has a deletion in region C, then a related modified 3'ITR of a different serotype has a deletion at the corresponding position in region C'), so that the pair of modified ITRs has the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in a modified ITR pair may belong to a different serotype (e.g., AAB1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAB2 and AAB6, with a modification in one ITR appearing at a corresponding position in a partner ITR from another serotype. In one embodiment, a pair of substantially symmetrical modified ITRs refers to a pair of modified ITRs (mod-ITRs), provided that the difference in nucleotide sequences between the ITRs does not affect properties or overall shape and they have substantially the same shape in three-dimensional space. As a non-limiting example, a mod-ITR has at least 95%, 96%, 97%, 98%, or 99% sequence identity with the canonical mod-ITR, as determined by standard tools well known to those skilled in the art, such as BLAST (Basic Local Alignment Search Tool or BLASTN with default settings, and also has a symmetrical 3D spatial organization so that their 3D structures have the same shape in geometric space. An essentially symmetric mod-ITR pair has identical A, C-C' and B-B' loops in three-dimensional space, for example, if a modified ITR in an essentially symmetric mod-ITR pair has a deletion of the C-C' arm, then its paired mod-ITR has a corresponding deletion of loop C-C', and also has a similar three-dimensional structure of the remaining loops A and B-B' of the same shape in geometric space with its paired mod-ITR.

[0071] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. In general, in the ABC sequence, A and C flank B. The same applies to the AxBxC arrangement. Thus, the flanking sequence precedes or follows the flanking sequence, but does not have to be adjacent to or immediately adjacent to the flanking sequence. In one embodiment, the term flanking refers to the terminal repeats at each end of a linear duplex ccDNA vector.

[0072] As used herein, the term “ccDNA genome” refers to an expression cassette that further includes at least one inverted terminal repeat region. The cccDNA genome may further comprise one or more spacer regions. In some embodiments, the cccDNA genome is included as an intermolecular duplex DNA polynucleotide in a plasmid or viral genome.

[0073] As used herein, the term “ccDNA spacer region” refers to an intervening sequence that separates functional elements in a cccDNA vector or ccDNA genome. In some embodiments, cccDNA spacer regions hold two functional elements at a desired distance for optimal functionality. In some embodiments, the cDNA spacer regions provide or increase the genetic stability of the cDNA genome within, for example, a plasmid or baculovirus. In some embodiments, cccDNA spacer regions facilitate genetic manipulation of the cccDNA genome by providing convenient locations for cloning sites and the like. For example, in certain aspects, an oligonucleotide "polylinker" containing multiple restriction endonuclease sites, or a non-open reading frame sequence designed to not contain known protein (e.g., transcription factor) binding sites, can be placed in the cccDNA genome to separate cis -acting factors, for example, by insertions of 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc., between the terminal resolution site and the upstream one transcription regulatory element. Likewise, a spacer may be included between the polyadenylation signal sequence and the 3' end resolution site.

[0074] 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., Rep 78 AAB or Rep 68 AAB) that upon binding by the Rep protein, allows the Rep protein to exert its site-specific endonuclease activity on the sequence including the RBS. The RBS sequence and its reverse complement together form one RBS. RBS sequences are known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), which is the RBS sequence identified in AAB2. Any known RBS sequence can be used in embodiments of the invention, including other known AAB RBS sequences and other known natural or synthetic RBS sequences. Without wishing to be bound by any theory, it is believed that the nuclease domain of the Rep protein binds to the duplex nucleotide sequence GCTC and, accordingly, direct binding and stable assembly of two known Rep proteins of AAB on the duplex oligonucleotide 5'-(GCGC)(GCTC)(GCTC) occurs )(GCTC)-3' (SEQ ID NO: 60). In addition, soluble aggregated conformers (i.e., an indefinite number of interconnected Rep proteins) dissociate and bind to oligonucleotides that contain Rep binding sites. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone of each chain. Interactions with nitrogenous bases provide sequence specificity, while interactions with the phosphodiester backbone are nonspecific or less sequence specific and stabilize the protein-DNA complex.

[0075] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region in which Rep forms a tyrosine phosphodiester bond with a 5'-thymidine to form a 3'-OH that serves a substrate for DNA extension by a cellular DNA polymerase, such as DNA polymerase delta or DNA polymerase epsilon. Alternatively, the Rep-thymidine complex may participate in a coordinated ligation reaction. In some embodiments, the TRS encompasses at least an unbase-paired thymidine. In some embodiments, the nicking efficiency of a TRS can be controlled, at least in part, by its distance from the RBS in the same molecule. When the acceptor substrate is complementary to the ITR, the resulting product is an intramolecular duplex. TRS sequences are known in the art and include, for example, 5'-GGTTGA-3' (SEQ ID NO: 61), which is a hexanucleotide sequence identified in AAB2. Any known TRS sequence can be used in embodiments of the present invention, including other known AAB TRS sequences and other known natural or synthetic 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 motifs such as RRTTRR (SEQ ID NO: 66).

[0076] As used herein, the term “ccDNA plasmid” refers to a plasmid that contains the cccDNA genome as an intermolecular duplex.

[0077] As used herein, the term “ccDNA bacmid” refers to an infectious baculovirus genome containing the cccDNA genome as an intermolecular duplex that is capable of propagation in E. coli as a plasmid and thus can act as a shuttle vector for the baculovirus .

[0078] As used herein, the term “cDNA-baculovirus” refers to a baculovirus that contains the cDNA genome as an intermolecular duplex within the baculovirus genome.

[0079] As used herein, the terms “ccDNA-baculovirus-infected insect cell” and “ccDNA-BIIC” are used interchangeably and refer to an invertebrate animal host cell (including, without limitation, an insect cell (e.g., an Sf9 cell)), infected with cDNA baculovirus.

[0080] As used herein, the term “closed-end DNA vector” refers to a non-capsid DNA vector with at least one covalently closed end, wherein at least a portion of the vector has an intramolecular duplex structure.

[0081] As used herein, the terms “ccDNA vector” and “ccDNA” are used interchangeably and refer to a closed-ended DNA vector containing at least one terminal palindrome. In some embodiments, the cccDNA contains two covalently closed ends.

[0082] As defined herein, “reporters” refer to proteins that can be used to provide detectable readouts. Reporters typically generate a measurable signal, such as fluorescence, color, or luminescence. The coding sequences of reporter proteins encode proteins whose presence in a cell or organism can be easily observed. For example, fluorescent proteins cause a cell to fluoresce when excited by light of a specific wavelength, luciferases cause the cell to catalyze a reaction that generates light, and enzymes such as β-galactosidase convert a substrate into a colored product. Typical examples of 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.

[0083] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, either, for example, as a reporter polypeptide, or, more preferably, as a cell-killing polypeptide, such as a toxin, or a cell-sensing agent. to killing by a specific agent or in the absence of a specific agent. Effector proteins include any protein or peptide that directly targets or damages the DNA and/or RNA of a host cell. For example, effector proteins may include, but are not limited to, a restriction endonuclease that targets a host cell DNA sequence (whether genomic or extrachromosomal element), a protease that causes degradation of a target polypeptide essential for cell survival, a DNA gyrase inhibitor, and a ribonuclease toxin. type. In some embodiments, expression of an effector protein controlled by a synthetic biological circuit as described herein can act as a factor in another synthetic biological circuit, thereby expanding the range and complexity of the response of the biological circuit system.

[0084] Transcription regulators refer to transcriptional activators and repressors that either activate or repress transcription of a gene of interest. Promoters are regions of nucleic acid that initiate transcription of a particular gene. Transcription activators typically bind in the vicinity of transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically hinder the initiation of transcription by RNA polymerase. Other transcriptional regulators can serve as either an activator or a repressor, depending on where they bind and the conditions in the cell and environment. Non-limiting examples of classes of transcription regulators include, but are not limited to, homeodomain proteins, zinc finger proteins, forkhead proteins, and leucine zipper proteins.

[0085] As used herein, the term “repressor protein” or “inducer protein” is a protein that binds to a regulatory sequence element and inhibits or activates, respectively, the transcription of sequences operably associated with said regulatory sequence element. Preferred repressor and inducer proteins as described herein are sensitive to the presence or absence of at least one administered agent or environmental input. Preferred proteins, as described herein, are modular in form and contain, for example, detachable DNA-binding and input-binding or responsive elements or domains.

[0086] As used herein, the term "carrier" includes any solvents, dispersion media, carrier media, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retarding agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Additional active ingredients may also be included in these compositions. The expression "pharmaceutically acceptable" refers to molecular substances and compositions that do not cause a toxic, allergic or similar adverse reaction when administered to a host.

[0087] As used herein, the term “injector-responsive domain” is a transcription factor domain that binds to, or otherwise responds to, a condition or injector by causing the DNA-binding domain fused thereto to respond to the presence of said condition or input influence. In one embodiment, the presence of a condition or input leads to a conformational change in the input-responsive domain or fusion protein that modifies the transcription-modulating activity of the transcription factor.

[0088] The term " in vivo " refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some aspects described herein, the method or use may be said to occur " in vivo " when a single cell organism, such as a bacterium, is used. The term " ex vivo " refers to methods and applications that are carried out using a living cell with an intact membrane located outside the body of a multicellular animal or plant, for example, explants, cultured cells, including primary cells and cell lines, transformed cell lines and extracted tissues or cells, including blood cells, among others. The term " in vitro " refers to assays and methods that do not require the presence of a cell with an intact membrane, such as those performed on cell extracts, and may refer to the introduction of a programmable synthetic biological circuit into a non-cellular system, such as a cell-free environment or cellular systems, such as cell extracts.

[0089] As used herein, the term “promoter” refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by directing the transcription of said nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressible, tissue specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence that controls the initiation and level of transcription of the rest of the nucleic acid sequence. A promoter may also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments of the aspects described herein, the promoter may drive the expression of a transcription factor that regulates the expression of the promoter itself. The promoter sequence will contain a transcription initiation site, as well as protein binding domains responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain TATA boxes and CAT boxes. Various promoters, including inducible promoters, can be used to drive the expression of transgenes in the cDNA vectors described herein. The promoter sequence may be limited at its 3' end by the transcription initiation site and extends upstream (towards the 5' end), including the minimum number of bases or elements necessary to initiate transcription at levels detected above background.

[0090] As used herein, the term “enhancer” refers to a cis-acting regulatory sequence (e.g., 50-1500 base pairs) that binds one or more proteins (e.g., activator proteins or a transcription factor) to enhance transcriptional activation of a nucleic acid sequence acids. Enhancers can be located up to 1,000,000 base pairs upstream of the start site of the gene or downstream of the start site of the gene they regulate. An enhancer may be located in an intronic region or in an exonic region of an unrelated gene.

[0091] A promoter can be said to drive the expression or drive the transcription of the nucleic acid sequence that it regulates. The expressions "operably linked", "functionally located", "operably linked", "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 that it regulates. to control the initiation of transcription and/or expression of this sequence. "Inverted promoter" as used herein refers to a promoter whose nucleic acid sequence is in reverse orientation such that the coding strand becomes non-coding and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of the switch. Additionally, in various embodiments, a promoter may be used in conjunction with an enhancer.

[0092] A promoter can be naturally associated with a gene or sequence, which can be achieved by isolating 5' non-coding sequences located upstream of the coding segment and/or exon of the gene or sequence. Such a promoter can be called “endogenous”. Likewise, in some embodiments, an enhancer may be naturally associated with a nucleic acid sequence located either after or before the specified sequence.

[0093] In some embodiments, a coding segment of a nucleic acid is placed under the control of a “recombinant promoter” or a “heterologous promoter,” both terms referring to a promoter associated with a coding nucleic acid sequence other than the one to which it is operably linked under its conditions. natural environment. A recombinant or heterologous enhancer refers to an enhancer not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring”, i.e. contain various elements of different transcriptional regulatory regions and/or expression-altering mutations introduced using genetic engineering methods known in the art. In addition to obtaining promoter and enhancer nucleic acid sequences synthetically, promoter sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR, in relation to the synthetic biological circuits and modules disclosed herein (see, for example, US Patent No. 4,683,202, US Pat. No. 5,928,906, each of which is incorporated herein by reference). In addition, it is also possible to use control sequences that direct transcription and/or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, and the like.

[0094] As described herein, an “inducible promoter” is a promoter that is characterized by initiation or enhancement of transcriptional activity in the presence of, under the influence of, or upon contact with, an inducer or inducing agent. An "inducer" or "inducing agent" as defined herein can be an endogenous or normally exogenous compound or protein that is administered so as to have the activity of inducing the transcriptional activity of an inducible promoter. In some embodiments, the inducer or inducing agent, i.e. chemical, compound or protein may itself be produced by transcription or expression of a nucleic acid sequence (ie, the inducer may be an inducer protein expressed by another component or module), which may itself be under the control of an inducible promoter. In some embodiments, the inducible promoter is induced in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothioneine, ecdysone, mammalian viruses (eg, adenovirus late promoter and murine mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, etc. .P.

[0095] The terms "DNA regulatory sequences", "control elements" and "regulatory elements", used interchangeably herein, refer to transcriptional and translational control sequences such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, etc. .p., which provide and/or regulate the transcription of a non-coding sequence (eg, DNA-targeting RNA) or coding sequence (eg, a site-directed modifying polypeptide or Cas9/Csn1 polypeptide) and/or regulate the translation of the encoded polypeptide.

[0096] "Operably coupled" refers to placement in close proximity such that the described components are in a relationship that allows them to function as intended. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. An "expression cassette" includes a heterologous DNA sequence that is operably linked to a promoter or other regulatory sequence sufficient to direct transcription of the transgene in the cDNA vector. Suitable promoters include, for example, tissue-specific promoters. Promoters can also be derived from AAV.

[0097] As used herein, the term “subject” refers to a human or animal being treated, including prophylactic treatment, with a cccDNA vector in accordance with this invention. Typically the animal is a vertebrate such as, but not limited to, a primate, rodent, pet or game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, koats, and macaques such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, felines such as the domestic cat, canid species such as dogs, foxes, wolves, bird species such as chickens, emus, ostriches , and fish such as trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject may be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a newborn or unborn subject, for example, an in utero subject. Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, without limitation to these examples. Mammals other than humans can be advantageously used as animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and/or pets. A person can be of any age, gender, belong to any race or ethnic group, for example, he can be Caucasian (white), Asian, African, black, African American, Afro-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment. In some embodiments, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a human newborn, a human infant, a human child, an adolescent, or an adult. In some embodiments, the subject is an animal embryo or a non-human embryo or a non-human primate embryo. In some embodiments, the subject is a human embryo.

[0098] As used herein, the term “host cell” includes any type of cell that is susceptible to transformation, transfection, transduction, or the like. using a nucleic acid construct or cDNA expression vector of the present invention. By way of non-limiting examples, the host cell may be an isolated primary cell, pluripotent stem cells, CD34 ⁺ cells), induced pluripotent stem cells, or any of a number of immortalized cell lines (eg, HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ, or organism.

[0099] The term "exogenous" refers to a substance present in a cell other than its natural source. The term “exogenous” as used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced through a human process into a biological system, such as a cell or organism, in which it are not typically present and introduction of the nucleic acid or polypeptide into such a cell or organism is desired. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that has been introduced through a human-related process into a biological system, such as a cell or organism, in which it is present in relatively small quantities when increasing amounts of the nucleic acid or polypeptide in a specified cell or organism, for example, to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell.

[00100] The term "sequence identity" refers to the affinity between two nucleotide sequences. For the purposes of this invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000 above), preferably version 3.0.0 or later. The optional parameters used are a gap opening penalty of 10, a gap expansion penalty of 0.5, and the substitution matrix EDNAFULL (EMBOSS NCBI NUC4.4 version). The Needle output parameter, designated as "longest identity" (obtained using the -nobrief parameter), is used as the percent identity and is calculated as follows: (Identical deoxyribonucleotides x 100) / (Alignment length - Total number of gaps in alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[00101] The term "homology" or "homologous" as used herein is defined as the percentage of nucleotide residues in a homologous arm that are identical to nucleotide residues in the corresponding sequence on the target chromosome after sequence alignment and introduction of gaps, if necessary, to achieve maximum percent sequence identity. Alignment to determine percent homology of nucleotide sequences can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as the BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) applications. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, a nucleic acid sequence (e.g., a DNA sequence), such as a homologous arm, is considered “homologous” when the sequence is at least 70% identical, at least 75%, at least 80%, at least 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, corresponding to the native or unedited nucleic acid sequence (eg, genomic sequence) of the host cell.

[00102] The term “heterologous” as used herein means a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. A heterologous nucleic acid sequence can be linked to a naturally occurring nucleic acid sequence (or variant thereof) (eg, by genetic engineering) to produce a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence can be linked to a variant polypeptide (eg, by genetic engineering) to produce a nucleotide sequence encoding the variant fusion polypeptide.

[00103] A "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion or cosmid, to which another DNA segment can be attached, i.e. "insertion" to cause the attached segment to replicate in the cell. A vector may be a nucleic acid construct designed for delivery into a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral in origin and/or final form, however, for purposes of this invention, “vector” generally refers to a cccDNA vector as that term is used herein. The term "vector" covers any genetic element that is capable of replication when associated with appropriate control elements and that can transfer gene sequences into cells. In some embodiments, the vector may be an expression vector or a recombinant vector.

[00104] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptide from sequences linked to transcriptional regulatory sequences in the vector. The expressed sequences will often, but not necessarily, be heterologous to the cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, allowing it to be maintained in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins and, if necessary, the secretion of proteins, including, where applicable, without limitation, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene and polypeptides produced by translation of mRNA transcribed from the gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5'-untranslated (5'UTR) or "leader" sequences and 3'-UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[00105] By "recombinant vector" is meant a vector that includes a heterologous nucleic acid sequence, or "transgene", capable of expression in vivo . It should be understood that the vectors described herein, in some embodiments, can be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the subject's nucleotide of interest in multicopy extrachromosomal DNA, thereby eliminating the potential effects of chromosome integration.

[00106] As used herein, the expression “genetic disease” refers to a disease, partially or wholly, directly or indirectly, caused by one or more abnormalities in the genome, especially a condition present from birth. The abnormality may be a mutation, insertion, or deletion. The abnormality may affect the coding sequence of a gene or its regulatory sequence. Genetic disease may include, but is not limited to, DMD (Duchenne muscular dystrophy), hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma and Tay-Sachs disease.

[00107] As used herein, the term “comprising” or “comprises” is used to refer to compositions, methods and their respective components that are essential to the method or composition but allow for the inclusion of unspecified elements, whether essential or not.

[00108] As used herein, the term “consisting essentially of” refers to those elements that are necessary for a given embodiment. This term allows for the presence of elements that do not significantly affect the essential and novel or functional characteristics of the embodiment. The use of the word “comprising” indicates inclusion rather than limitation.

[00109] The term “consisting of” refers to compositions, methods and their respective components as described herein, which exclude any element not specified in this embodiment description.

[00110] As used in this description and the accompanying claims, the singular forms, including those accompanied by the definition “specified” (in the English text - with the articles “a”, “an” and “the”) include references to the plural forms, if the context does not clearly indicate otherwise.Thus, for example, references to “method” include one or more methods and/or steps of the type described herein and/or those that will become apparent to those skilled in the art upon reading the present invention , etc. Likewise, the term “or” is intended to include “and” unless the context clearly indicates otherwise, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention , suitable methods and materials are described below: Abbreviation “eg.” is used herein to describe a non-limiting example. Thus, the abbreviation “eg.” is synonymous with the term "for example".

[00111] Except in the working examples or as otherwise indicated, all numbers indicating amounts of ingredients or reaction conditions according to this invention are in all cases to be understood as modified by the term “about.” The term "about" when referring to a percentage may mean ±1%. The present invention is further illustrated in more detail in the following examples, but the scope of the present invention is not limited to them.

[00112] The groups of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and stated individually or in any combination with other members of the specified group or other elements mentioned herein. One or more members of the group may be included or excluded from the group for reasons of convenience and/or patentability. Any such inclusion or deletion herein shall be deemed to contain the modified group, thereby satisfying the requirement of a written description of all Markush groups used in the appended claims.

[00113] In some embodiments of any of the aspects, the invention described herein does not relate to the process of cloning humans, processes for modifying the genetic identity of the germline of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are likely to may cause them suffering without any significant medical benefit to humans or animals, as well as animals resulting from such processes.

[00114] Definitions of other terms are provided herein in the description of various aspects of the invention.

[00115] All patents and other publications; including references, issued patents, published patent applications and pending patent applications; mentioned in this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methodologies described in such publications that may be used in connection with the technology described herein. These publications are presented solely by reason of their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors have no rights in an earlier invention with respect to such disclosure by reason of prior invention or for any other reason. All statements as to the date or representations as to the contents of these documents are based on information available to the applicants and do not imply any admission as to the correctness of the dates or contents of these documents.

[00116] The description of embodiments of the invention is not to be construed as exhaustive or limiting of the invention to the precise form disclosed. Although specific embodiments and examples of the disclosure have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be understood by those skilled in the relevant art. For example, although the steps or functions of a method are presented in the order shown, in alternative embodiments the functions may be performed in a different order, or the functions may be performed substantially simultaneously. The principles of the invention presented herein may be applied to other procedures or methods as appropriate. Various implementations described herein may be combined to provide additional implementations. Aspects of the disclosure may be modified, as necessary, to utilize the compositions, functions and concepts of the above references and uses to provide additional embodiments of the invention. In addition, for reasons of biological functional equivalence, some changes in protein structure may be made that do not affect the biological or chemical effect in natural or quantitative terms. These and other changes may be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00117] Specific elements of any of the above embodiments may be combined or replaced with elements of other embodiments. In addition, although the advantages associated with some embodiments of the invention have been described in the context of these embodiments, other embodiments may also demonstrate such advantages, and not all embodiments must demonstrate such advantages to fall within the scope of the disclosure.

[00118] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting. It should be understood that the present invention is not limited to the specific methodology, protocols, reagents, etc. described herein, which may in fact vary. The terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

II. Expression of an antibody or fusion protein from a cccDNA vector

[00119] The technology described herein is generally directed to the expression and/or production of an antibody or fusion protein in a cell from a non-viral DNA vector, such as a cccDNA vector, as described herein. cccDNA vectors for the production of antibodies or fusion proteins are described herein in the section entitled “ccDNA vectors in general.” In particular, cccDNA vectors for producing antibodies or fusion proteins contain a pair of ITRs (e.g., symmetric or asymmetric, as described herein) and between the pair of ITRs, a nucleic acid encoding the antibody or fusion protein, as described herein, operably linked with a promoter or regulatory sequence. An obvious advantage of cccDNA vectors for the production of antibodies or fusion proteins compared to traditional AAV vectors and even lentiviral vectors is that there are no size restrictions on the heterologous nucleic acid sequences encoding the desired protein. Thus, even a full-length antibody containing, for example, two heavy chain Fc regions, a linker and a Fab fragment, can be expressed from a single cDNA vector. In particular, for the expression and/or production of antibodies, given their size, a cccDNA vector may be preferable to traditional vectors due to ease of use and lack of size restrictions, which allows antibodies (including multimeric antibodies) with different domains to be easily expressed. structures in a controlled manner. In addition, multiple administrations can be performed, allowing the administration of cocktails of different cDNA vectors expressing different antibodies or fusion proteins. Thus, the cccDNA vectors described herein can be used to express a therapeutic antibody or fusion protein in a subject in need thereof. Alternatively, cDNA vectors can be used in the production of antibodies or fusion proteins in a commercial setting, for example, using a bioreactor, or for production in a desired host.

[00120] It should be understood that the cDNA vector technologies described herein can be adapted to any level of complexity or can be used in a modular manner where the expression of different antibody or fusion protein components can be controlled independently. For example, it is specifically contemplated that the cccDNA vector technologies developed herein may be as simple as using a single cccDNA vector to express a single heterologous gene sequence (e.g., the heavy chain or light chain of a desired antibody) or may be as complex as the use of multiple cccDNA vectors, where each vector expresses multiple antibodies or antibody components, each independently controlled by different promoters. The embodiments described below are specifically contemplated herein and may be adapted by one skilled in the art as desired.

[00121] In one embodiment, a single cDNA vector can be used to express one antibody component or fusion protein, such as a heavy chain or a light chain. Alternatively, a single cccDNA vector can be used to express multiple components (e.g., at least 2) of an antibody or fusion protein under the control of a single promoter (e.g., a strong promoter), optionally using IRES sequence(s) to ensure proper expression of each from components.

[00122] This document also provides, in another embodiment, a single cDNA vector containing at least two inserts (eg, expressing a heavy chain or a light chain), the expression of each insert being under the control of its own promoter. Promoters may include multiple copies of the same promoter, multiple different promoters, or any combination thereof. One skilled in the art will appreciate that it is often desirable to express antibody components at different expression levels, thereby controlling the stoichiometry of the individual expressed components to ensure efficient folding and combination of antibodies in the cell.

[00123] One skilled in the art may envision additional variations on cDNA vector technologies or adapt them to antibody production methods using traditional vectors.

A. Heterogeneous sequences for expression of an antibody or fusion protein

[00124] Essentially any antibody or antigen binding fragment (eg, functional fragment) or fusion protein thereof can be expressed from a cDNA vector as described herein. One skilled in the art will appreciate that an antibody fragment contains, at a minimum, the amino acids necessary for antigen or epitope binding (eg, scAb, Fab, F(ab') ₂ , dAb and Fv). For example, an antibody molecule may include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In one embodiment, the antibody molecule contains, or consists of, a heavy chain and a light chain (referred to herein as a half-antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen binding sites, such as Fab, Fab', F(ab') ₂ , Fc, Fd , Fd', Fv, single chain antibodies (eg, scFv), single variable domain antibodies, diabodies (Dab) (divalent and bispecific), and chimeric (eg, humanized) antibodies, which can be obtained by modifying whole antibodies, or synthesized de novo using recombinant DNA technologies. Such functional antibody fragments retain the ability to selectively bind to their corresponding antigen or epitope and activate or inhibit the target protein.

[00125] This document also covers cccDNA vectors expressing fusion proteins. In some embodiments, the fusion protein is a biofunctional fusion protein. In some embodiments, the fusion protein is suitable for decoy technology, such as antibody-ligand decoys, in which the fusion protein comprises an antibody (e.g., a monospecific or bispecific antibody) or antibody fragment (e.g., an antigen-binding fragment) fused to a peptide or ligand-binding domain or receptor domain that captures a ligand, thereby inhibiting the activity of the ligand. Accordingly, in some embodiments, the cDNA vector may encode and express a fusion protein, which is referred to in the art as a trap or Y-trap. In some embodiments, a cccDNA vector, as described herein, is used to express a fusion protein, e.g., a VEGF-Trap fusion protein, an IGF-trap fusion protein (see Vaniotis et al., Sci Rep, 2018, 8(1) , 17361) or TGFβ-Trap fusion protein. A typical example of a TGFβ trap is a Y-trap, such as a bifunctional antibody-ligand trap (Y-trap) containing an antibody targeting CTLA-4 and/or PD-L1 fused to a TGFβ II receptor ectodomain sequence that simultaneously blocks autocrine /paracrine TGFβ in the target cell microenvironment (a-CTLA4-TGFβRIIecd and a-PDL1-TGFβRIIecd) (see, for example, Ravi et al., Nat Comm 2018, 9(1); 741). In some embodiments, a fusion protein included for use herein and expressed by a cccDNA vector is an antibody-ligand trap, wherein the fusion protein comprises an antibody or antibody fragment (e.g., an antigen-binding fragment) fused to a ligand-binding domain or receptor domain (e.g., an extracellular receptor domain) that captures the ligand, wherein the ligand is selected from any generally known growth factors or ligands, including, without limitation, IGF, VEGF, TGFβ, TNFα, EGF, NGF, PDGF, LFA-3, CTLA-4 , IL-1, TPO.

[00126] Representative examples of fusion proteins include, but are not limited to, etanercept (ENBREL®), which contains a 75 kDa soluble extracellular domain (ECD) of the tumor necrosis factor (TNF) receptor II fused to human IgG1 Fc; alefacept (AMEVIVE®), which contains the first ECD of lymphocyte function-associated antigen 3 (LFA-3) fused to human IgG1 Fc; abatacept (ORENCIA®), which contains the human cytotoxic T lymphocyte-4 (CTLA-4) ECD molecule fused to human IgG1 Fc; rilonacept (ARCALYST®), which contains two chains, each of which contains the C-terminus of the ligand binding region of the accessory protein-IL-1R fused to the N-terminus of the IL-1RI ECD fused to the Fc of human IgG1; romiplostim (NPLATE®), which contains a peptide thrombopoietin (TPO) mimetic fused to the C-terminus of an aglycosylated human IgG1 Fc; belatacept (NULOJIX®), which contains the CTLA-4 ECD fused to the human IgG1 Fc and differs from abatacept by two amino acid substitutions (L104E, A29Y) in the CTLA-4 region.

[00127] Antibodies and antibody fragments can belong to any class of antibodies, including, without limitation, IgG, IgA, IgM, IgD and IgE, and any subclass of antibodies (for example, IgG1, IgG2, IgG3 and IgG4). The antibody may be monoclonal. The antibody produced using the methods described herein may be a human, humanized, CDR-grafted or in vitro generated antibody. The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3 or IgG4. The antibody may also have a light chain selected from, for example, kappa or lambda chains. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein. By inserting the coding sequences of such antibodies into a cDNA vector, almost any antibody can be obtained. In one embodiment, the light chain and heavy chain genes are under the control of a regulatory switch. In the same or alternative embodiments, the light and heavy chain genes are linked to an IRES sequence (eg, SEQ ID NO: 190).

[00128] Typically, the antibody or fusion protein gene also encodes a secretory sequence so that the antibody is directed to the Golgi apparatus and endoplasmic reticulum (ER), where the antibody is folded into the correct conformation by chaperone molecules as it passes through the ER and exits the cell. Representative examples of secretory sequences include, but are not limited to, VH-02 (SEQ ID NO: 88) and (abd) VK-A26 (SEQ ID NO: 89) and the Igĸ signal sequence (SEQ ID NO: 126), as well as the Gluc secretory signal , allowing the tagged protein to be secreted from the cytosol (SEQ ID NO: 188), the secretory sequence TMD-ST, directing the tagged protein to the Golgi apparatus (SEQ ID NO: 189).

[00129] When it is desired to produce an intrabody, the nucleic acid or gene encoding the antibody typically does not encode a secretory sequence. In some cases, they may encode a secretory sequence, but also have a putative targeting sequence, such as, but not limited to, the KDEL sequence, for its retention in the cell. In other embodiments, the intrabody genes encode a different intracellular targeting sequence, such as a nuclear localization sequence.

[00130] Regulatory switches can be used to fine-tune the expression of antibodies (including intrabodies) or fusion proteins so that the antibody is expressed in a desired manner, including, without limitation, expressing the antibody or fusion protein at a desired level of expression or amount, or alternatively, if the presence or absence of a specific signal, including a cell signaling event. For example, as described herein, expression of an antibody or fusion protein from a cDNA vector can be turned on or off when a certain condition occurs, as described in the section herein entitled “Regulatory Switches”.

[00131] For example, and for purposes of illustration only, antibodies or fusion proteins can be used to turn off an adverse reaction, as is the case with anti-TNFα antibodies such as adalimumab. In other cases, the antibody or fusion protein may help enhance the immune response. For example, in relation to a malignant cell, for example, a tumor. The antibody gene may contain a tumor-associated marker for transferring the antibody to the desired cell. However, in any situation it may be desirable to regulate the expression of the antibody or fusion protein. cccDNA vectors allow regulatory switches to be easily tailored for use with antibodies. cccDNA vectors also allow control of the stoichiometry of the heavy and light chains. Examples of fusion proteins include, but are not limited to, VEGF-trap and TGFβ-trap technologies.

[00132] Antibody molecules include intact molecules as well as antigen-binding fragments thereof and functional fragments thereof. The constant regions of antibody molecules can be changed, eg, mutated, to modify the properties of the antibody (eg, to increase or decrease one or more of: Fc receptor binding, number of cysteine residues, etc.). Examples of antigen binding fragments of an antibody molecule include, but are not limited to: (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of the antibody, (v) a diabody fragment (dAb) which consists of a VH domain; (vi) camelized or camelized antibody variable domain; (vii) single chain Fv (scFv), see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) single domain antibody. These antibody fragments are produced using cDNA vectors and can be screened for eligibility in the same manner as intact antibodies.

[00133] An undoubted advantage of cDNA vectors compared to traditional AAV vectors and even lentiviral vectors is the absence of restrictions on the size of heterologous nucleic acid sequences encoding the desired protein. Thus, even a full-length antibody containing two heavy chain Fc regions, a linker and a Fab fragment can be expressed from a single cccDNA vector. In addition, depending on the desired stoichiometry, multiple segments of the same antibody or fusion protein can be expressed, such as a light chain and a heavy chain, and the same or different promoters can be used, and regulatory switches can be used to fine-tune the expression of each region . For example, as shown in the Examples, a cccDNA vector containing a dual promoter system can be used such that a separate promoter is used for each of the heavy chain and light chain of the aducanumab antibody. Use of a cccDNA plasmid to produce an antibody or fusion protein may include a unique combination of heavy and light chain expression promoters that results in the proper ratios of heavy and light chains to produce a functional antibody or fusion protein. Accordingly, in some embodiments, the cDNA vector can be used to express different regions of the antibody or fusion protein separately (eg, under the control of a different promoter). In some embodiments, a nucleic acid encoding a heavy chain may be operably linked to a first promoter or a first regulatory switch, and a nucleic acid encoding a light chain may be operably linked to a second promoter or a second regulatory switch, thereby providing controlled or regulated expression heavy chain and light chain independently of each other, allowing control of the ratios of heavy chain and light chain to produce a functional antibody or fusion protein.

[00134] Expression of an antibody or fusion protein from a cccDNA vector can be achieved both spatially and temporally using one or more inducible or repressible promoters.

[00135] Antibody molecules can also be single domain antibodies. Single-domain antibodies may include antibodies whose complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional 4 chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibodies may be any known in the art or any single domain antibodies that will be developed in the future. Single domain antibodies can be obtained from any species, including, without limitation, mouse, human, camel, llama, fish, shark, goat, rabbit and ruminants.

[00136] In some embodiments, the antibody is a multispecific antibody that contains two or more variable regions for binding to at least two different epitopes, for example, on the same target protein, or for simultaneously targeting at least two different proteins. Thus, the epitopes recognized by a multispecific antibody may be on the same or different targets.

[00137] In other embodiments, the antibody is a bispecific antibody that can recognize and bind to at least two different epitopes or targets (see, e.g. , Riethmuller, G Cancer Immunity (2012) 12: 12-18; Schaefer W et al PNAS (2011) 108(27): 11187-92 for typical structures of bispecific antibodies). Second generation bispecific antibodies, eg, "trifunctional bispecific" antibodies, are also provided with the methods and compositions described herein.

[00138] In certain embodiments, the antibody provided is a multispecific antibody, including a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. A typical example of a bispecific antibody is an antibody in which one of the binding specificities is for beta-amyloid peptide and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different beta-amyloid epitopes. Bispecific antibodies can also be used to localize cytotoxic agents to cells. Bispecific antibodies can be produced as full-length antibodies or antibody fragments.

[00139] In some embodiments, the cccDNA vector for producing a multispecific antibody includes coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and the tongue-to-groove technology (see, for example, US Pat. No. 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic targeting effects to produce Fc heterodimeric antibody molecules (WO 2009/089004A1); linking two or more antibodies or fragments (see, for example, US Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); the use of diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single chain Fv dimers (sFv) (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[00140] In some embodiments, the cccDNA vector encodes an engineered antibody with three or more functional antigen binding sites, including octopus antibodies, which are also included herein (see, for example, US 2006/0025576A1). In some embodiments, the cccDNA vector encodes an antibody or fusion protein that is a "dual-action FAb" or "DAF" containing an antigen-binding site that binds to beta-amyloid peptide as well as to another non-antigen (see eg US 2008/0069820).

[00141] In one embodiment, an antibody is a “variant antibody,” which refers to an antibody having an altered amino acid sequence, composition, or structure compared to its corresponding native antibody. For example, a variant antibody may contain a non-native secretion signal to allow the antibody to be secreted from a host cell.

[00142] In some embodiments, the cccDNA vector encodes a cysteine modified antibody variant, such as a "thioMAb", in which one or more antibody residues are replaced with cysteine residues. In certain embodiments, the replaced residues are at accessible sites on the antibody. By replacing these residues with cysteine, the reactive thiol groups are thus located at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or drug-linker moieties, to create an immunoconjugate, as described herein below . In some embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) light chain; A118 (EU numbering) heavy chain; and S400 (EU numbering) heavy chain Fc region. Cysteine-modified antibodies can be prepared as described, for example, in US Pat. No. 7,521,541.

[00143] In another embodiment, the antibody may be a miniaturized antibody that is a monovalent or divalent antibody comprising a variable light chain, a variable heavy chain antigen binding domain, and optionally one or more effector domains (e.g., tissue-specific targeting). Although the use of miniaturized antibodies is specifically contemplated herein, cccDNA vectors are not limited in size with respect to heterologous nucleic acid sequences and therefore have the advantage of being able to express even a full-length antibody.

[00144] In another embodiment, the antibody or fusion protein expressed from the cDNA vectors further comprises additional functionality, such as fluorescence, enzymatic activity, a secretion signal, or an immune cell activator.

[00145] In some embodiments, the antibody encoded by the cccDNA vector comprises a diabody (bispecific single chain antibodies) or a unitbody (an IgG4 molecule lacking a hinge region to reduce the risk of immune activation).

[00146] cccDNA vectors for antibody production, as described herein, are also useful for the expression of fusion proteins or intrabodies (i.e., intracellular antibodies) that can target intracellular proteins that affect cellular function (e.g., metabolism, division cells, transcription, translation, etc.). The intrabody may be a scFv. Intrabodies can be targeted to a specific cellular compartment by incorporating signaling motifs such as a C-terminal ER retention signal (eg, KDEL), a mitochondrial targeting sequence, a nuclear localization sequence, etc.

[00147] Intrabodies are particularly useful for treating diseases associated with misfolded proteins, such as Alzheimer's disease, Parkinson's disease, prion disease, Huntington's disease, etc.

[00148] In some embodiments, the antibody or fusion protein may further comprise, for example, a linker domain. As used herein, the term “linker domain” refers to an oligo- or polypeptide region of about 2 to 100 amino acids in length that binds any domains/regions of an antibody as described herein. In some embodiments, linkers may include or be composed of flexible residues, such as glycine and serine, such that adjacent protein domains can move freely relative to each other. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not sterically interfere with each other. Linkers can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (eg, T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. The linker may be a T2A linker region derived from Thosea asigna virus.

[00149] One skilled in the art can take known and/or publicly available protein sequences, e.g., a fusion protein, heavy chain, light chain, variable region, etc., and reverse engineer a cDNA sequence to encode such a protein (e.g., a fusion protein) or antibodies. The cDNA can then be codon-optimized to match the intended host cell and inserted into a cDNA vector as described herein.

[00150] In one embodiment, the antibody coding sequence can be obtained from an existing hybridoma cell line, for example, by reverse transcribing mRNA obtained from the hybridoma cell line and amplifying the sequence using PCR.

B. cccDNA vectors expressing antibody or fusion protein

[00151] A cccDNA vector for producing an antibody or fusion protein having one or more sequences encoding the desired antibody may contain regulatory sequences such as promoters, secretion signals, poly(A) regions and enhancers. At a minimum, the cccDNA vector contains one or more heterologous sequences encoding an antibody or fusion protein. Typical cccDNA vectors for producing antibodies or fusion proteins are shown in FIG. 7A-7G .

[00152] To achieve highly efficient and accurate assembly of an antibody or fusion protein, some embodiments specifically provide that the antibody, fusion protein, or individual domains of the antibody comprise an endoplasmic reticulum (ER) ER leader sequence to direct it to the ER where protein folding occurs . For example, a sequence that directs the expressed protein(s) to the ER for folding.

[00153] In some embodiments, a cellular or extracellular localization signal (e.g., a secretory signal, a nuclear localization signal, a mitochondrial localization signal, etc.) is contained in a cccDNA vector (see, e.g. , FIG. 10G ) to direct secretion or the desired subcellular localization of the antibody or fusion protein so that the antibody or fusion protein can bind to intracellular target(s) (eg, in the case of an intrabody) or extracellular target(s).

[00154] In some embodiments, the cDNA antibody production vector may encode an intrabody, and in some embodiments, the intrabody may be a full-length antibody as well as a single chain. Intrabodies can be used in a wide range of applications, including the treatment of viral disorders and cellular disorders such as cancer, see, for example, US Pat. No. 6,004,940.

[00155] In some embodiments, a cDNA vector for producing an antibody or fusion protein, as described herein (e.g., see the exemplary example of a cDNA vector shown in FIG. 6A ), allows the assembly and expression of any desired antibody or fusion protein in a modular way. As used herein, the term “modular” refers to elements in the cDNA expression plasmid that can be readily removed from the construct. For example, the modular elements in a cccDNA generating plasmid contain unique pairs of restriction sites flanking each element of the construct, allowing for exceptional ability to manipulate individual elements (see, e.g. , Figures 7A-7G ). Thus, the cccDNA vector platform can allow the expression and assembly of any desired antibody or fusion protein configuration. Disclosed herein in various embodiments are plasmid cccDNA vectors that can reduce and/or minimize the amount of manipulation required to assemble a desired ccDNA vector encoding an antibody or fusion protein.

C. Representative Examples of Antibodies and Fusion Proteins Expressed by cccDNA Vectors

[00156] In particular, a cccDNA vector for producing antibodies or fusion proteins as described herein may encode, for example, without limitation, antibodies, antigen binding fragments, fusion proteins, as well as variants and/or active fragments thereof, for use in the treatment, prevention and/or alleviation of one or more symptoms of disease, dysfunction, injury and/or disorder. In one aspect, the disease, dysfunction, injury, impairment, and/or disorder is a human disease, dysfunction, injury, impairment, and/or disorder.

[00157] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins as described herein may also encode cofactors or other polypeptides, sense or antisense oligonucleotides, or RNA (coding or noncoding; e.g., siRNA, small hairpin RNAs (shRNAs), microRNAs and their antisense analogues (eg, antagomir (antagoMiR)), which can be used in combination with an antibody or fusion protein expressed from cccDNA. In addition, expression cassettes containing the sequence encoding the antibody or fusion protein may also include an exogenous sequence that encodes a reporter protein intended for use in 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.

[00158] In certain embodiments, a variety of different antibodies and/or fusion proteins can be introduced using one or more cccDNA vectors. Thus, it is specifically contemplated that a desired amount of antibodies and/or fusion proteins may be expressed in a “cocktail” in a cell, tissue, or subject.

[00159] The cccDNA vectors described herein can be used to deliver antibodies and fusion proteins for the treatment of, for example, cancer, autoimmune disease (e.g., rheumatoid arthritis, Crohn's disease), Alzheimer's disease, hypercholesterolemia, acute organ rejection, multiple sclerosis, postmenopausal osteoporosis, skin diseases (eg psoriasis, atopic dermatitis), asthma or hemophilia.

[00160] cccDNA vectors, as described herein, can be used to express any desired therapeutic antibody or fusion protein. Typical examples of therapeutic antibodies and fusion proteins include, but are not limited to, abciximab, abaloparatide, adalimumab, adalimumab-atto, ado-trastuzumab, emtansine, aducanumab, alemtuzumab, alirocumab, atezolizumab, avelumab, bapineuzumab, basiliximab, belimumab, bevacizumab, bezlotoxumab, blinatumomab, blosozumab, bococizumab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cetuximab, concizumab, daclizumab, daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, ecallantide, eculizumab, elotuzumab, emtansine, um icizumab, evolocumab, evinacumab, factor IX - Fc antibody, factor VIII-Fc antibody, golimumab, ibritumomab tiuxetan, idarucizumab, inclacumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, lanadelumab, lodelcizumab, mepolizumab, natalizumab, necitumumab, nivolum b, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, orticumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rituximab, roledumab, romosozumab, secukinumab, siltuximab, solanezumab, sotatercept, tadocizumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, sarilumab, rituximab, guselkumab, inotuzumab ozogamicin, adalimumab-adbm, gemtuzumab ozogamicin, bevacizumab-awwb, benralizumab, emicizumab-kxwh, trastuzumab-dkst.

[00161] In one embodiment, the cccDNA vector contains a nucleic acid sequence for expression of a therapeutic antibody or fusion protein that is functional for treating a disease. In a preferred embodiment, the therapeutic antibody or fusion protein does not cause an immune system response unless desired.

[00162] In one embodiment, the antibody is a therapeutic antibody or fusion protein expressed by a cccDNA vector that targets an immune checkpoint inhibitor (e.g., PDL1) and can be used to treat, for example, cancer (e.g., solid tumors, cancer breast, lymphomas, liver cancer, ovarian cancer, lung cancer, colorectal cancer, leukemia, hematological cancers, skin cancer, multiple myeloma, etc.). In one embodiment, the therapeutic antibody or fusion protein targets a checkpoint inhibitor such as PDL1, CD47, mesothelin, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2/neu, telomerase, SAP-1, survivin, NY-ESO-1/LAGE-1, PRAME, SSX-2, Melan-A/MART-1, Gp100/pmel17, tyrosinase, TRP-1/-2 , MC1R, β-catenin, BRCA1/2, CDK4, CML66, fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, β-catenin, CDK4, CDC27, CD47, α-actinin-4, TRP1/gp75, TRP2, gp100, Melan-A/MART1, gangliosides, WT1, EphA3, epidermal growth factor receptor (EGFR), CD20, MART-2, MART-1, MUC1, MUC2, MUC16, MUM1, MUM2, MUMS, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor alpha, L1-CAM, CAIX , EGFRvIII, gpA33, GD3, GM2, VEGFR, integrins (integrin alphaVbeta3, integrin alpha5beta1), carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, fibroblast-activating protease (FAP), TGF-beta, hyaluronic acid, collagen (eg, collagen IV, tenascin C or tenascin W), CD19, CD33, CD47, CD123, CD20, CD99, CD30, BCMA, CD38, CD22, SLAMF7 or NY-ESO1.

[00163] In one embodiment, the cccDNA vector expresses a monoclonal antibody to evolocumab and is used to treat hyperlipidemia. Evolocumab inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is a protein that targets LDL receptors for degradation, thereby reducing the liver's ability to clear the LDL-cholesterol complex (LDL-C), or "bad" cholesterol, from the blood. Evolocumab is further described in US 8,999,341, which is incorporated herein by reference in its entirety.

[00164] Typical examples of antibodies and fusion proteins expressed from a cccDNA vector for use in the methods and compositions described herein may be any of the antibodies or fusion proteins listed in Table 1, Table 2, Table 3A, Table 3B, Table 4 or Table 5 in this document.

[00165]

Table 1: FDA-approved antibodies and fusion proteins as representative examples of antibodies and fusion proteins. BLASTN CHEMICAL (GENERIC) NAME PATENTED NAME REFERENCE(S), SEQ ID NO IN REFERENCE 125118 abatacept ORENCIA® US20130330338A1, SEQ ID NO: 6
WO2017125402A1, SEQ ID NO: 2 103575 abciximab REOPRO® US20170002060A1, SEQ ID NO: 1,2 125057 adalimumab HUMIRA® US 8921526
US20180126000A1, SEQ ID NO: 66.73
WO2017112536A1, SEQ ID NO: 23.24 125387 aflibercept EYLEA® WO2014006113A1, SEQ ID NO: 1
WO2016073894A1, SEQ ID NO: 54 103948 alemtuzumab Campath (CAMPATH), Lemtrada (LEMTRADA®) US8592149B2, SEQ ID NO: 14
US20150112045A1, SEQ ID NO: 15.16 125559 alirocumab Praluent® US 8062640
US20180363000A1, SEQ ID NO: 11
US20170002060A1, SEQ ID NO: 160,161 761034 atezolizumab TECENTRIQ® US20170239351A1, SEQ ID NO: 66.67
US8217149B2, SEQ ID NO: 20.21 761049 avelumab Bavencio® US20170239351A1, SEQ ID NO: 76.77
US20140341917A1, SEQ ID NO: 24.25 103764 basiliximab SIMULECT® US20150314007A1, SEQ ID NO: 20.21
US20170002060A1, SEQ ID NO: 15.16 125370 belimumab benlysta® US20160281106A1, SEQ ID NO: 25.26
US20170002060A1, SEQ ID NO: 17 761070 benralizumab FASENRA® US20170002060A1, SEQ ID NO: 183,184,185,186
WO2008143878A1, SEQ ID NO: 1,2,3,4 125085 bevacizumab Avastin® WO2018129533A1, SEQ ID NO: 65.66
US20190016817A1, SEQ ID NO: 1,2
WO2016073894A1, SEQ ID NO: 55.56 761046 bezlotoxumab zinplava (ZINPLAVA®) WO2018111662A1, SEQ ID NO: 1.5
US9181632B1, SEQ ID NO: 11,12 125557 blinatumomab BLINCYTO® US20190038730A1, SEQ ID NO: 19
US20170362325A1, SEQ ID NO: 5
WO2016036678A1, SEQ ID NO: 25 125388 brentuximab vedotin adcetris (ADCETRIS®) US20180086734A1, SEQ ID NO: 10.11
US20180036425A1, SEQ ID NO: 3.4 761032 brodalumab silica (SILIQ®) US20170002060A1, SEQ ID NO: 238,239
WO2006013107A1, SEQ ID NO: 427,429 761068 burosumab-twza CRYSVITA® WO2015191312A1, SEQ ID NO: 7.8, as KRN23 125319 canakinumab ILARIS® WO2018235056A1, SEQ ID NO: 17.18
US20180186882A1, SEQ ID NO: 349,350
US20130122008A1, SEQ ID NO: 1,2 125160 certolizumab pegol CIMZIA® WO2017112536A1, SEQ ID NO: 25.26
US20170002060A1, SEQ ID NO: 39.40 125084 cetuximab Erbitux® WO2018204717A1, SEQ ID NO: 124,125
WO2017205465A2, SEQ ID NO: 1,2
WO2017120536A1, SEQ ID NO: 7.8 103749 daclizumab Zenapax® US20170226223A1, SEQ ID NO: 45.46
US20170002060A1, SEQ ID NO: 41.42
US20180256732A1, SEQ ID NO: 29.30 761036 daratumumab darzalex (DARZALEX®) US20170044265A1, SEQ ID NO: 4.5
WO2018002181A1, SEQ ID NO: 4.5
WO2017162890A1, SEQ ID NO: 22.29 125320 denosumab prolia (PROLIA®), exjiva (XGEVA®) US 8962807, US 7528236, US 7364736, US 8058418, US 8409578
US20170002060A1, SEQ ID NO: 49.50 125516 dinutuximab unituxin (UNITUXIN®) US20140170155A1, SEQ ID NO: 3.4 761055 dupilumab DUPIXENT® WO2018190990A1, SEQ ID NO: 345,346
US20190040147A1, SEQ ID NO: 9.10
US20170002060A1, SEQ ID NO: 359,364 761069 durvalumab imfinzi (IMFINZI®) US20190031766A1, SEQ ID NO: 643,644
WO2018172286A1, SEQ ID NO: 13.14
WO2018129533A1, SEQ ID NO: 25.26 125166 eculizumab SOLIRIS® WO2018195034A1, SEQ ID NO: 10.11
WO2018075758A1, SEQ ID NO: 5.6
US20180311345A1, SEQ ID NO: 7.8 761035 elotuzumab empliciti® US20180222979A1, SEQ ID NO: 36.37
US20170002060A1, SEQ ID NO: 289,290
US20180185348A1, SEQ ID NO: 1,2 761083 emicizumab-kxwh hemlibra® US20180011114A1, SEQ ID NO: 9,10,11,12 as ACE910 761077 erenumab-aooe AIMOVIG® US9102731B2 103795 etanercept Enbrel® US20180291084A1, SEQ ID NO: 10
WO2016009049A1, SEQ ID NO: 4
US20160160279A1, SEQ ID NO: 1 125522 evolocumab repata (REPATHA®) US 8030457
US20170002060A1, SEQ ID NO: 158,159 761060 gemtuzumab ozogamicin Mylotarg® WO2018129533A1, SEQ ID NO: 67.68
US20190002560A1, SEQ ID NO: 248,249
US20180148514A1, SEQ ID NO: 243,244 125289 golimumab SIMPONI® WO2018147915A1, SEQ ID NO: 36.37
US7250165B2, cited in US20170002060A1 125433 golimumab SIMPONI ARIA® US20170002060A1, SEQ ID NO: 63.64
US7250165B2, SEQ ID NO: 7.8
WO2013087912A1, SEQ ID NO: 6.7
All - VH/VL 761061 guselkumab tremfya (TREMFYA®) WO2018093841A1, SEQ ID NO: 106,116
US9803010B2, SEQ ID NO: 44.45 761065 ibalizumab-uiyk trogarzo (TROGARZO®) US8637024B2, SEQ ID NO: 15.19, VH/VL 125019 ibritumomab tiuxetan Zevalin® WO2018129533A1, SEQ ID NO: 59.60
US20170002060A1, SEQ ID NO: 71.72
US9683985B2, SEQ ID NO: 41.42 761025 idarucizumab PRAXBIND® US 8486398 103772 infliximab REMICADE® US20160153020A1, SEQ ID NO: 41.42
US20180256732A1, SEQ ID NO: 22.23 761040 inotuzumab ozogamicin BESPONSA® WO2018193231A1, SEQ ID NO: 25.26
US20170002060A1, SEQ ID NO: 299,304
US8153768B2, SEQ ID NO: 28.30 125377 ipilimumab YERVOY® US20150283234A1
WO2018204343A1, SEQ ID NO: 84.85
WO2018049042A1, SEQ ID NO: 1,2 125521 ixekizumab TALTZ® WO2019027828A1, SEQ ID NO: 4.5
US 7838638
US 8110191 125526 mepolizumab NUCALA® WO2009068649A2, SEQ ID NO: 65.66
WO2009120927A2, SEQ ID NO: 19.21
link in US20170002060A1 125104 natalizumab TYSABRI® US5840299A, SEQ ID NO: 2.4 VL/VH, reference in US20170002060A1
WO2010121141A1, SEQ ID NO: 1,2 125547 necitumumab Portrazza® US20170002060A1, SEQ ID NO: 320,324 125554 nivolumab OPDIVO® US20190031766A1, SEQ ID NO: 535,536
WO2018183408A1, SEQ ID NO: 23.24
WO2018150326A1, SEQ ID NO: 98.99 125509 obiltoxaximab ANTHIM® WO2017186928A1, SEQ ID NO: 616,617 125486 obinutuzumab GAZYVA® US20190008869A1, SEQ ID NO: 3.4
US20190022092A1, SEQ ID NO: 367,368 761053 ocrelizumab OCREVUS® US8679767B2, SEQ ID NO: 11.19
US20180327505A1, SEQ ID NO: 13.14 125326 ofatumumab ARZERRA® US20180169189A1, SEQ ID NO: 23.24 VH/VL
WO2009086072A2, SEQ ID NO: 1.2 VH/VL 761038 olaratumab lartruvo (LARTRUVO®) WO2018022407A1, SEQ ID NO: 26.28
US20170137523A1, SEQ ID NO: 11,12 103976 omalizumab XOLAIR® US20170002060A1, SEQ ID NO: 110,111 VH/VL
US20150314007A1, SEQ ID NO: 22.23
US20150140608A1, SEQ ID NO: 1,2 103770 palivizumab Synagis® WO2018013483A1, SEQ ID NO: 1.2 VH/VL
US7786273B2, SEQ ID NO: NO: 1.2 VH/VL 125147 panitumumab VECTIBIX® WO2018156802A1, SEQ ID NO: 1.4
WO2017060322A2, SEQ ID NO: 237,238 125514 pembrolizumab KEYTRUDA® WO2018183408A1, SEQ ID NO: 21.22
WO2018150326A1, SEQ ID NO: 50.51
WO2018106588A1, SEQ ID NO: 32.33 125409 pertuzumab Perjeta (PERJETA®) WO2018160654A2, SEQ ID NO: 11,12
WO2018140831A2, SEQ ID NO: 11.16 US20120107391A1, SEQ ID NO: 16.17 125477 ramucirumab Cyramza® WO2018093668A1, SEQ ID NO: 1.2 VH/VL
WO2018081512A1, SEQ ID NO: 1.2 VH/VL 125156 ranibizumab Lucentis® WO2018211529A1, SEQ ID NO: 2.4
WO2017117464A1, SEQ ID NO: 5.6 761033 reslizumab synkea (CINQAIR®) US20170002060A1, SEQ ID NO: 181,182
WO1995035375A1, referenced in US20170002060A1 103705 rituximab Rituxan® WO2018129533A1, SEQ ID NO: 55.56
WO2018033482A1, SEQ ID NO: 4.5
US20190008869A1, SEQ ID NO: 1,2 761037 sarilumab kevzara (KEVZARA®) US20170166646A1, SEQ ID NO: 2.3
US20170252434A1, SEQ ID NO: 1,2
US20170002060A1, SEQ ID NO: 207,208 125504 secukinumab cosentix (COSENTYX®) WO2006013107A1
US20170002060A1, SEQ ID NO: 225,226
US20170304439A1, SEQ ID NO: 8.10, VH/VL 125496 siltuximab SYLVANT® US20170002060A1, SEQ ID NO: 197,198
US20180236032A1, SEQ ID NO: 4.5 761067 tildrakizumab-asmn Ilumya® US20170002060A1, SEQ ID NO: 245,246
US8263748B2, SEQ ID NO: 7.17 125276 tocilizumab Actemra® WO2017202387A1, SEQ ID NO: 1,2
US20170074890A1, SEQ ID NO: 1,2
US20170152319A1, SEQ ID NO: 1,2 103792 trastuzumab Herceptin® WO2018160654A2, SEQ ID NO: 13.14
WO2018129533A1, SEQ ID NO: 57.58 761044 ustekinumab Stelara® WO2018024770A1, SEQ ID NO: 1,2
US20190025325A1, SEQ ID NO: 1,2
WO2017112536A1, SEQ ID NO: 19.20 125476 vedolizumab entyvio® US20170002060A1, SEQ ID NO: 154,155
US20120282249A1, SEQ ID NO: 2.4
WO2008115504A2, SEQ ID NO: 11,12

[00166]

Table 2: Representative examples of antibodies and fusion proteins for expression by cccDNA vectors suitable for use in the methods and compositions described herein. Target Examples of antibodies Information about links Transferrin receptor 2015/0110791 A1 Integrin alpha-4 Natalizumab, Tysabri (TYSABRI®) 5840299; 6033665; 6602503; 5168062; 5385839; 5730978 EGFR Cetuximab, erbitux (ERBITUX®), panitumumab, necitumumab, portrazza (PORTRAZZA®) US6217866B1 CD25 (IL-2 receptor alpha chain) Basiliximab, SIMULECT®, daclizumab, ZINBRYTA® US6521230B1 TNF-alpha Remicade (REMICADE®), Simponi (SIMPONI®), Adalimumab, Humira (HUMIRA®), Certolizumab pegol, CIMZIA®, Golimumab, Infliximab US20080033149A1, US20120258114A1, US20030157061 CTLA4 Orencia, ipilimumab, YERVOY® WO1993000431A1, US8784815B2 IgE Xolair (XOLAIR®), omalizumab, ligelizumab WO1993004173A1 C5 Eculizumab, SOLIRIS®, REGN3918, pexelizumab WO1995029697A1 IL-6R Sarilumab, satralizumab, Actemra (ACTEMRA®), tocilizumab, atlizumab US8017121B2 BAFF (BLyS) Belimumab, benlysta (BENLYSTA®) WO1998018921A1, WO2003055979A2 VEGF-A Ranibizumab, Lucentis®, Avastin (AVASTIN®), Brolucizumab, Bevacizumab, Ramucirumab, Cyramza (CYRAMZA®) WO1998045331A2, WO1994010202A1, US20140086829, US2013067098, Lien and Lowman, in: Chemajovsky, 2008, Therapeutic Antibodies. Handbook of Experimental Pharmacology 181, Springer-Verlag, Berlin Heidelberg 131-150 P-selectin Inclacumab, LC1004-002, R04905417 US 7563441; US 8039596; US 7432359 HER2 Trastuzumab, Herceptin (HERCEPTIN®), Pertuzumab, Perjeta (PERJETA®), Kadcyla (KADCYLA®) US 7879325 PCSK9 Alirocumab, bococizumab, evolocumab, repata (REPATHA®), lodelcizumab, ralpancizumab US 8030457, US 8710192 PD-L1 Atezolizumab, Tecentriq (TENCENTRIQ®), Avelumab, Bavencio (BAVENCIO®), Durvalumab, Imfinzi (IMFINZI®) US 8217149 oxLDL Orticumab, BI-204, MLDL-1278A, R7418 US 8318161 Dabigatran Idarucizumab, praxbind (PRAXBIND®) US 8486398 Parathyroid hormone-related protein (PTHrP) Abaloparatide, thymlos (TYMLOS®) US 8747382, US 7803770; US 8148333 RANKL Denosumab, Prolia (PROLIA®), ExGiva (XGEVA®) US 8962807; US 7528236; US 7364736; US 8058418; US 8409578 NGF Ranezumab, fasinumab US20040237124 PD1 receptor Pembrolizumab, lambrolizumab, Keytruda (KEYTRUDA®) US2012135408 Alpha4beta7 integrin Vedolizumab, Entyvio (ENTYVIO®) US2012151248 Integrin alpha4beta7 Vedolizumab, Entyvio (ENTYVIO®) US2012151248 IL17a Taltz (TALTZ®), ixekizumab, secukinumab, Cosentix (COSENTYX®) US20130202610 CSa BNJ378, BNJ383 US20130224187 PD1 Cemiplimab, nivolumab, opdivo (OPDIVO®) US2013173223 PDGFR-beta 3299N, olaratumab, lartruvo (LARTRUVO®) US20140193402 TiKA BXhVH5VLl US20150183885 TrkA GBR, VH5(P60A, T62S), VL1 US20150183885 Beta amyloid crenezumab, MEDI1814, gantenerumab, aducanumab, BAN2401, solenezumab, LY3002813, bapinezumab US20150246963 Activin REGN 2477, bimagrumab (BIMAGRUMAB®), garetosmab US20170260275, WO2015162590 CD33 Gemtuzumab, mylotarg (MYLOTARG®) US5585089 CD19 Blinatumomab, BLINCYTO®, inbilizumab US7235641; US7575923; US7635472; US8247194 IL-6 Siltuxumab, silvant (SYLVANT®) US7612182 NGF Fulraintin, 4D4, AMG-403, lN.142160443 US7601818 Fulranumab; 4D4, AMG-403, JNJ- US7988967 US8552157 US8048421 CD20 Ocrelizumab, Ocrevus (OCREVUS®), Ofatumumab, Arzerra (ARZERRA®), Rituximab, Rituxan (RITUXAN®), Obinutuzumab, Ibritumomab, US8337847, WO1994011026A3 GFRa3 H4H2364S US8968736 CORP 01, cluster headache US9115194 CGRP galcanezumab, erenumab US9115194, US20120294802, US9102731 TrkA BXhVHl® W02009098238 TrkA HUVHWOV® W02009098238 Annexin IV or phospholipid; and (b) complement inhibitor W02014116880 Nav 1.7 H1Hl015B W02014159595 Factor D Fab238, lampalizumab WO2009134711, US8273352 Rh antigen Roledumab, dmatria (DMATRIA®) WO2010100383 TGF-beta DOM23h-271-12 WO2011012609 Alpha-synuclein BIIB054 WO2018178950A1, SEQ ID NO: 8.9 VH/VL RG7935 US20160324852A1 Protective antigen of Bacillus anthracis Raxibacumab US20130028920A1 Obiltoxaximab, antim (ANTHIM®) WO2017186928A1, SEQ ID NO: 616,617 ANGPTL-3 Evinacumab US20170312359A1, SEQ ID NO: 1.5 VH/VL WO2017151783A1, SEQ ID NO: 10.11 B7RP1 Prezalumab WO2017186928A1 BAFF VAY736 US8106163B2, SEQ ID NO: 71.75 BDCA2 BIIB059 WO2017189827A1, SEQ ID NO: 9.10 US20180362652A1, SEQ ID NO: 3.4 C1 BIVV009 WO2018170145A1, SEQ ID NO: 22.23 CD22 Inotuzumab WO2018193231A1, SEQ ID NO: 25.26; WO2018193231A1, SEQ ID NO: 25.26; US8153768B2, SEQ ID NO: 28.30 CD30 Brentuximab vedotin, adcetris (ADCETRIS®) US20180086734A1, SEQ ID NO: 10,11; US20180036425A1, SEQ ID NO: 3.4 CD38 Daratumumab, Darzalex (DARZALEX®) US20170044265A1, SEQ ID NO: 4.5; WO2018002181A1, SEQ ID NO: 4.5; WO2017162890A1, SEQ ID NO: 22.29 CD52 Alemtuzumab, Lemtrada® US8592149B2, SEQ ID NO: 14; US20150112045A1, SEQ ID NO: 15.16 CD319 (SLAMF7) Elotuzumab, EMPLICITI® US20180222979A1, SEQ ID NO: 36.37; US20170002060A1, SEQ ID NO: 289,290; US20180185348A1, SEQ ID NO: 1,2 Clostridia toxins A and B Bezlotoxumab, zinplava (ZINPLAVA®) WO2018111662A1, SEQ ID NO: 1.5; US9181632B1, SEQ ID NO: 11,12 Fel d1 REGN1908 WO2018118713A1, SEQ ID NO: 18.26 VH/VL REGN1909 WO2018118713A1, SEQ ID NO: 306,314 VH/VL GDF8 Trevogrumab US8840894B2 WO2016168613A1, SEQ ID NO: 360.368 VH/VL Glycolipid GD2 Dinutuximab, unituxin (UNITUXIN®), dinutuximab beta, isquet (ISQUETTE®) US20140170155A1, SEQ ID NO: 3.4 Glycoprotein IIb/IIa receptor Abciximab, Reopro (REOPRO®) US20170002060A1, SEQ ID NO: 1,2 GM-CSF Namilumab WO2017076804A1, SEQ ID NO: 11,12 Mavrilimumab US8263075B2 US8506960B2 WO2017202879A1, SEQ ID NO: 2.3 IFN-alpha receptor Anifrolumab US20110059078A1 US20160251441A1, SEQ ID NO: 1.2 VH/VL as MEDI-546 IG2-kappa SHP647 IL-1beta Canakinumab, ILARIS® WO2018235056A1, SEQ ID NO: 17,18; US20180186882A1, SEQ ID NO: 349,350; US20130122008A1, SEQ ID NO: 1,2 IL-3R Dupilumab WO2018190990A1, SEQ ID NO: 345,346; US20190040147A1, SEQ ID NO: 9,10; US20170002060A1, SEQ ID NO: 359,364 IL-5 Fensenra®, mepolizumab, GS3511294, reslizumab, sinqir® WO2009068649A2, SEQ ID NO: 65.66; WO2009120927A2, SEQ ID NO: 19.21; link in US20170002060A1 IL-5R (CD125) Benralizumab, fasrenra (FASRENRA®) US20170002060A1, SEQ ID NO: 183,184,185,186 WO2008143878A1, SEQ ID NO: 1,2,3,4 IL-17 receptor Brodalumab, silica (SILIQ®) US20170002060A1, SEQ ID NO: 238,239 WO2006013107A1, SEQ ID NO: 427,429 IL-23 Tremfya®, guselkumab WO2018093841A1, SEQ ID NO: 106.116 US9803010B2, SEQ ID NO: 44.45 Mirikizumab WO2019012531A1 IL-31 Nemolizumab US20180346543A1 IL-33 REGN3500 WO2018102597A1, SEQ ID NO: 274,282 VH/VL MEDI3506 GSK3772847 (formally CNTO 716) Influenza A MEDI8852 Integrin alphaIIbbeta3 Tadocizumab WO2016011264A1 Kallikrein SHP643 EP3390439A1 Ecallantide, kalbitor (KALBITOR®) US20160252527A1, SEQ ID NO: 3 US20070213275A1, SEQ ID NO: 1 Lanadelumab US20180306807A1, SEQ ID NO: 81.82 VH/VL DX-2930 US20180118851A1, SEQ ID NO: 9.10 US20150362492A1, SEQ ID NO: 9.10 VH/VL LAG-3 REGN3767 WO2018222722A2 LEPR LINGO-1 Opininumab Middle East respiratory coronavirus REGN3048, REGN3049, REGN3050, REGN3051 US20190030187A1 Myostatin Domagrozumab, RG6206 NGF 42160443 PSMA Capromab penetide, prostascint (PROSTASCINT®) WO2006076525A2 RSV F protein Palivizumab, synagis (SYNAGIS®) WO2018013483A1, SEQ ID NO: 1.2 VH/VL US7786273B2, SEQ ID NO: NO: 1.2 VH/VL MEDI8897 WO2018160722A1, SEQ ID NO: 1,2 SP0232 Sklerostin Romosozumab WO2017153541A1, SEQ ID NO: 11,12 SOST Blosozumab WO2018115880A1, SEQ ID NO: 70.81, possible VH/VL Staphylococcus aureus toxin (Staph. aureus) Suvratoxumab WO2017075188A2, SEQ ID NO: 9,10, as MEDI4893 Tau protein BIIB092 WO2018231254A1, SEQ ID NO: 14.15 RG6100 WO2019025595A1 TFPI Concizumab WO2017186928A1, SEQ ID NO: 176,177 NN7415 Thymic stromal lymphoprotein Tezepelumab WO2018191479A1, SEQ ID NO: 105,106 Targets of bispecific antibodies Alpha3beta7/alphaEbeta7 Etrolizumab US20180086833A1 WO2017019673A2, SEQ ID NO: 31.32 VH/VL CD20/CD3 REGN1979 Factor IX/Factor X Emicizumab, hemlibra (HEMLIBRA®) US20180011114A1, SEQ ID NO: 9,10,11,12 as ACE910 IL-4/IL-13 SAR156597 US20170145089A1, SEQ ID NO: 1,2,3,4 VH/VL IL-12/IL-23 Ustekinumab, Stelara (STELARA®) WO2018024770A1, SEQ ID NO: 1.2 US20190025325A1, SEQ ID NO: 1.2 WO2017112536A1, SEQ ID NO: 19.20 IL-33/STR2 AMG 282, RG6149 Psl/PcrV MEDI3902 VEGF-A/Ang2 RG7716 WO2017218977A2, SEQ ID NO: 13,14,15,16 Antibody-like proteins Activin receptor type 2A-IgG-Fc fusion protein (functions as a TGF-β ligand decoy) ACE-011, SOTATERCEPT® US20180161426A1, SEQ ID NO: 7 US20170304397A1, SEQ ID NO: 7

[00167]

Table 3A . Representative examples of antibodies for expression by cccDNA vectors, including, but not limited to, antibody therapeutics approved in the European Union or the United States. International nonproprietary name BRAND NAME Target; Format First approved indication Brodalumab silica (SILIQ®), lumisef (LUMICEF®), kinteum (KYNTHEUM®) IL-17RA; human IgG2 Plaque psoriasis Avelumab Bavencio® PD-L1; human IgG1 Merkel cell carcinoma Dupilumab DUPIXENT® IL-4Rα; human IgG4 Atopic dermatitis Ocrelizumab OCREVUS® CD20; humanized IgG1 Multiple sclerosis Durvalumab imfinzi (IMFINZI®) PD-L1; human IgG1 Bladder cancer Sarilumab kevzara (KEVZARA®) IL-6R; human IgG1 Rheumatoid arthritis Guselkumab tremfya (TREMFYA®) IL-23 p19; human IgG1 Plaque psoriasis Inotuzumab ozogamicin BESPONSA® CD22; humanized IgG4; ADC (antibody-drug conjugate) Acute lymphoblastic leukemia Benralizumab FASENRA® IL-5Rα; humanized IgG1 Asthma Emicizumab hemlibra® Factor IXa, X; humanized IgG4, bispecific Hemophilia A

[00168]

Table 3B. Representative examples of antibodies for expression by cccDNA vectors, including, but not limited to, antibody-based drugs undergoing regulatory testing in the European Union or the United States. International nonproprietary name Suggested brand name Target; Format Indication in question Ibalizumab (not completed) CD4; humanized IgG4 HIV infection Burosumab (not completed) FGF23; human IgG1 X-linked hypophosphatemia Tildrakizumab (not completed) IL-23 p19; humanized IgG1 Plaque psoriasis Caplacizumab (not completed) von Willebrand factor; humanized nanobody Acquired thrombotic thrombocytopenic purpura Erenumab AIMOVIG® CGRP receptor; human IgG2 Migraine prevention Fremanezumab (not completed) CGRP; humanized IgG2 Migraine prevention Galcanezumab (not completed) CGRP; humanized IgG4 Migraine prevention Romosozumab EVENITY® Sclerostin; humanized IgG2 Osteoporosis in postmenopausal women with increased risk of fractures Mogamulizumab potiligio® (POTELIGEO®) CCR4; humanized IgG1 Cutaneous T-cell lymphoma

[00169] Table 4: Representative examples of antibodies for expression by cccDNA vectors, including, but not limited to, antibody therapeutics for non-cancer indications, in late-stage clinical trials. Companies conducting commercial development or clinical testing of antibodies: 1. Novartis, 2. LFB Group, 3. Shire, 4. Prothena Therapeutics Ltd., 5. Omeros Corporation, 6. Alexion Pharmaceuticals Inc., 7. AstraZeneca/MedImmune LLC, 8 Boehringer Ingelheim Pharmaceuticals, AbbVie, 9. Genentech, 10. Shire, 11. R-Pharm, 12. Chugai Pharmaceuticals/Roche, 13. NovImmune SA, 14. CytoDyn, 15. Biogen, 16. Hoffmann-La Roche, 17. Alder Biopharmaceuticals, 18. Regeneron Pharmaceuticals, 19. Pfizer; Eli Lilly & Company, 20. Horizon Pharma USA

Table 4 International Nonproprietary Name (INN) or Code Name Molecular format Target Late stage indication(s) Crizanlizumab ¹ Humanized IgG2 CD62 (aka P-selectin) Sickle cell anemia Roledumab ² Human IgG1 Rh D Hemolytic disease Lanadelumab ³ Human IgG1 Plasma kallikrein Attacks of hereditary angioedema NEOD001 ⁴ Humanized IgG1 Amyloid Primary systemic amyloidosis OMS721 ⁵ Human monoclonal antibody MASP-2 Atypical hemolytic uremic syndrome Ravulizumab (ALXN1210) ⁶ Humanized monoclonal antibody C5 Paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome Tezepelumab ⁷ Human IgG2 Thymic stromal lymphopoietin Severe uncontrolled asthma Tralokinumab ⁷ Human IgG4 IL-13 Atopic dermatitis Risankizumab ⁸ Humanized IgG1 Il-23 p19 Psoriasis; Crohn's disease Anifrolumab ⁷ Human IgG1 Interferon receptor (IFN) α, β, ω type 1 Systemic lupus erythematosus Etrolizumab ⁹ Humanized IgG1 Integrin receptor α4-β7/αE-β7 Nonspecific ulcerative colitis (UC); Crohn's disease SHP-647 ¹⁰ Human IgG2 Mucosal cell adhesion molecule addressin-1 Nonspecific ulcerative colitis (UC); Crohn's disease Olokizumab ¹¹ Humanized IgG4 IL-6 Rheumatoid arthritis Inebilizumab ⁷ Humanized IgG1 CD19 Neuromyelitis optica and neuromyelitis optica spectrum disorders Satralizumab ¹² Humanized IgG2 IL-6R Neuromyelitis optica and neuromyelitis optica spectrum disorders Emapalumab ¹³ Human IgG1 IFNγ Primary hemophagocytic lymphohistiocytosis PRO-140, PA14 ¹⁴ Humanized IgG4 CCR5 HIV infection Aducanumab ¹⁵ Human IgG1 Amyloid β Alzheimer's disease Crenezumab ⁹ Humanized IgG4 Amyloid β Alzheimer's disease Gantenerumab ¹⁶ Human IgG1 Amyloid β Alzheimer's disease Eptinezumab ¹⁷ Humanized IgG1 CGRP Migraine prevention, chronic migraine Fasinumab ¹⁸ Human IgG4 NGF (nerve growth factor) Knee or hip pain due to osteoarthritis, chronic low back pain Tanezumab ¹⁹ Humanized IgG2 NGF (nerve growth factor) Knee or hip pain due to osteoarthritis, chronic low back pain, cancer pain due to bone metastases Teprotumumab ²⁰ Human IgG1 IGF-1R Endocrine ophthalmopathy Lampalizumab ⁹ Humanized Fab IgG1 Complement factor D Geographic atrophy associated with dry age-related macular degeneration Brolucizumab ¹ Humanized scFv VEGF-A Neovascular age-related macular degeneration

[00170] Table 5 : Representative examples of antibodies for expression by cccDNA vectors, including, without limitation, antibody therapeutics for cancer-related indications in late-stage clinical trials. Companies conducting commercial development or clinical testing of antibodies in Table 4: 1. Actinium Pharmaceuticals, 2. Sanofi, 3. TG Therapeutics, 5. MorphoSys, 6. Pfizer, 8. Viventia Bio, 10. Jiangsu HengRui Medicine Co., Ltd, 11. MacroGenics, 16. Gilead Sciences, 18. AstraZeneca/MedImmune LLC, 19. Recombio SL, 20. Regeneron Pharmaceuticals, 21. Innovent Biologics (Suzhou) Co. Ltd., 22. BeiGene, 24. Biocad, 25. Novartis, 26. Philogen SpA, 27. Tracon.

TABLE 5 International Nonproprietary Name (INN) or Code Name Molecular format Target Late stage indication(s) I-131-BC8, Iomab-B (Iomab-B) ¹ Radiolabeled mouse IgG1 CD45 Bone marrow ablation before hematopoietic cell transplantation in patients with acute myelogenous leukemia (AML) Isatuximab ² Humanized* IgG1 CD38 Multiple myeloma Ublituximab ³ Chimeric IgG1 CD20 Chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple sclerosis XMAB-5574, MOR208 ⁵ Humanized IgG1 CD19 Diffuse large B-cell lymphoma Utomilumab ⁶ Human IgG2 4–1BB (CD137) Diffuse large B-cell lymphoma Oportuzumab Monatox ⁸ Humanized scFv immunotoxin EpCAM Bladder cancer Camrelizumab ¹⁰ Humanized IgG4 PD-1 Hepatocellular carcinoma, esophageal cancer Margetuximab ¹¹ Chimeric IgG1 HER2 Mammary cancer Andecaliximab ¹⁶ Humanized* IgG4 MMP-9 Gastric cancer or adenocarcinoma of the esophagogastric junction Tremelimumab ¹⁸ Human IgG2 CTLA-4 Non-small cell lung cancer, head and neck cancer, urothelial cancer, hepatocellular carcinoma Racotumomab ¹⁹ Mouse IgG1 NGcGM3 Non-small cell lung cancer Cemiplimab ²⁰ Human monoclonal antibody PD-1 Cutaneous squamous cell carcinoma; non-small cell lung cancer, cervical cancer IBI308 ²¹ Human monoclonal antibody PD-1 Squamous cell non-small cell lung cancer BGB-A317 ²² Humanized monoclonal antibody PD-1 Non-small cell lung cancer BCD-100 ²⁴ Human monoclonal antibody PD-1 Melanoma PDR001 ²⁵ Humanized IgG4 PD-1 Melanoma L19IL2/L19TNF ²⁶ scFv immunoconjugates Extradomain B of fibronectin Melanoma Carotuximab ²⁷ Chimeric IgG1 Endoglin Soft tissue sarcoma, angiosarcoma, renal cell carcinoma, wet age-related macular degeneration

[00171] Additional exemplary examples of antibodies and fusion proteins for expression by cccDNA vectors include, but are not limited to, those described below:

[00172] Brodalumab (SILIQ®, LUMICEF®, KYNTHEUM®, AMG-827) is a human IgG2 antibody that targets IL-17 receptor A (IL-17RA) and prevents inflammatory transmission signaling of proinflammatory cytokines IL-17A, IL-17F and IL-17C through IL-17RA. Brodalumab is approved in the US (SILIQ®), EU (KYNTHEUM®) and Japan (LUMICEF®). Brodalumab is indicated for the treatment of adult patients with moderate to severe plaque psoriasis who are candidates for systemic therapy or phototherapy and who have not responded or become unresponsive to other systemic treatments.

[00173] Dupilumab (DUPIXENT®, REGN668/SAR231893) is a human IgG4 monoclonal antibody (mAb) targeting the IL-4 receptor (IL4R), thereby blocking inflammatory responses mediated by IL-4 and IL-13. This MAb has been approved in the US and EU for patients with atopic dermatitis.

[00174] Ocrelizumab (ocrevum (OCREVUS®)) is a humanized IgG1 antibody that targets CD20-positive B cells. Such B cells play a role in myelin damage and the pathogenesis of multiple sclerosis.

[00175] Ocrelizumab has received approval for the treatment of relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS) in the United States.

[00176] Sarilumab (kevzara (KEVZARA®), SAR153191, REGN88) is a human IgG1 antibody targeting the IL-6 receptor (IL-6R) and has been approved in Canada, the US and the EU for patients with rheumatoid arthritis (RA) moderate to severe, demonstrating inadequate response to or intolerance to one or more disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX).

[00177] Benralizumab (Fazenra®, MEDI-563) is an afucosylated IgG1 mAb targeting the α-subunit of IL-5R present on eosinophils that has received FDA approval for the adjunctive maintenance treatment of patients with severe asthma aged 12 years and older.

[00178] Emicizumab (hemlibra (HEMLIBRA®), emicizumab-kxwh, ACE910, RO5534262) is a bispecific IgG4 mAb targeting factors IXa and X that has received FDA approval. This drug has been approved to prevent or reduce the frequency of bleeding episodes in adult and pediatric patients with hemophilia A who develop factor VIII inhibitors. As of December 1, 2017, a total of 9 antibody therapeutics were being evaluated in the US or EU. Of these, 8 (ibalizumab, burosumab, tildrakizumab, caplacizumab, erenumab, fremanezumab, galcanezumab, romosozumab) have not yet received marketing authorization. Mogamulizumab received its first global approval in Japan on March 20, 2012.

[00179] Ibalizumab is an IgG4 mAb targeting CD4 that is currently being evaluated by the FDA as a treatment for multidrug-resistant human immunodeficiency virus (HIV).

[00180] Burosumab (KRN23) is a human IgG1 mAb that targets fibroblast growth factor 23 (FGF23), which is a hormone that regulates phosphate excretion and active vitamin D production in the kidneys.

[00181] Tildrakizumab (SCH 900222/MK-3222) is a humanized IgG1 mAb targeting IL-23p19. Applications for marketing authorization for tildrakizumab for the treatment of moderate to severe plaque psoriasis have been submitted to the EU and the US.

[00182] Caplacizumab (ALX-0081) is a bivalent single domain antibody (Nanobody®) targeting von Willebrand factor and is currently in regulatory testing as a treatment for acquired thrombotic thrombocytopenic purpura (aTTP), a rare life-threatening disease. bleeding disorders associated with the formation of microclots leading to low platelet counts, tissue ischemia and organ dysfunction in patients with pTTP.

[00183] Erenumab (AIMOVIG™, AMG 334) is an IgG2 mAb targeting the calcitonin gene-related peptide (CGRP) receptor, which is involved in the development of sensitized nociceptive neurons. Applications for marketing authorization of erenumab for the prevention of migraine in patients experiencing four or more migraine days per month have been submitted in the EU and the US.

[00184] Fremanezumab (TEV-48125) is an IgG2 mAb targeting CGRP that is currently in regulatory testing for the prophylactic treatment of migraine.

[00185] Galcanezumab (LY2951742) is an IgG4 mAb targeting CGRP that is currently in regulatory testing for the prevention of episodic and chronic migraine in adults.

[00186] Romosozumab (EVENITY™, AMG785) is a humanized IgG2 mAb targeting sclerostin that is currently being evaluated as a treatment for osteoporosis in women and men.

[00187] Mogamulizumab (KW-0761, potiligeo (POTELIGEO®)) is an afucosylated humanized IgG1 mAb targeting CC chemokine receptor 4 (CCR4) expressed on tumor cells from patients with cutaneous T-cell leukemia/lymphoma (CTCL), including fungal mycoses and Sezary syndrome.

[00188] Lanadelumab (SHP643, DX-2930) is a human IgG1 mAb that targets plasma kallikrein and thereby prevents the production of bradykinin.

[00189] Crizanlizumab (SEG101) is a humanized mAb that targets P-selectin, also known as CD62, and is currently being evaluated as a treatment for sickle cell-associated pain crisis (SCPC) caused by vaso-occlusion in patients with sickle cell disease. anemia.

[00190] Ravulizumab (ALXN1210) is a complement component 5 (C5) targeting humanized mAb that is being evaluated in two phase 3 studies in patients with paroxysmal nocturnal hemoglobinuria (PNH).

[00191] Eptinezumab (ALD403) is an IgG1 mAb targeting calcitonin gene-related peptide (CGRP) and is currently being evaluated for use in the prevention of migraine.

[00192] Risankizumab (ABBV066, BI655066) is an IgG1 mAb targeting the p19 subunit of IL-23, which is involved in the pathogenesis of psoriasis.

[00193] Satralizumab (SA237) is a humanized IgG2 targeting the IL-6R that is being evaluated in two phase 3 studies in patients with neuromyelitis optica (NMO) or NMO spectrum disorder.

[00194] Brolucizumab (RTH258) is a single chain variable fragment (scFv) targeting vascular endothelial growth factor (VEGF)-A.

[00195] PRO140 is a humanized IgG4 mAb that blocks human immunodeficiency virus (HIV) coreceptor CCR5 on T cells, thereby preventing viral entry.

[00196] Lampalizumab (RG7417, FCFD4514S) is a humanized antigen-binding fragment (Fab) that inhibits the activation and amplification of the alternative complement pathway by binding complement factor D.

[00197] Roledumab (LFB-R593) is an anti-human IgG1 Rh (Rh)D mAb produced using LFB S.A.'s EMABLING® platform technology, which modifies fucosylation, resulting in more efficient antibody binding to effector cells. The antibody is intended to prevent certain pregnancy-associated alloimmunization conditions, namely, in RhD-negative pregnant women with an RhD-positive fetus.

[00198] Fasinumab (REGN475) is a human IgG4 mAb targeting nerve growth factor that is currently being evaluated in multiple late-stage studies as a treatment for moderate to severe osteoarthritic pain of the hip or knee and chronic low back pain .

[00199] Etrolizumab (RH7413) is a humanized mAb that binds the β7 subunit of integrin heterodimers α4β7 and αEβ7, thereby inhibiting interactions with their ligands MAdCAM-1 and E-cadherin, respectively.

[00200] NEOD001 is a humanized IgG1 mAb that targets soluble and insoluble light chain aggregates that cause amyloid light chain (AL) amyloidosis, which is a disorder characterized by excessive accumulation of protein aggregates in tissues and organs, including the heart, kidneys, and liver.

[00201] Gantenerumab (RO4909832) is a human mAb targeting fibrillar amyloid-β that is being investigated as a treatment for Alzheimer's disease.

[00202] Anifrolumab (MEDI-546) is a human IgG1 mAb targeting interferon (IFN) receptor type I subunit 1 that is being evaluated as a treatment for systemic lupus erythematosus (SLE).

[00203] Moxetumomab pasudotox (HA22, CAT-8015) is a recombinant immunotoxin containing the 38-kDa cytotoxic moiety of Pseudomonas exotoxin A fused to an antibody variable fragment targeting CD22.

[00204] Cemiplimab (REGN2810, SAR439684), a human antibody that targets programmed death protein 1 (PD1), is being evaluated as a treatment for metastatic or unresectable cutaneous squamous cell carcinoma (CSCC).

[00205] Ublituximab (LFB-R603, TGT-1101, TGTX-1101) is a glycoengineered chimeric mAb targeting CD20.

[00206] Tremelimumab (CP-675,206) is a human IgG2 antibody targeting cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Under physiological conditions, CD28 interacts with B7 ligands (CD80, CD86), leading to T cell activation, proliferation and up-regulation of CTLA-4. CTLA-4 binds B7 ligands with higher affinity than CD28 and terminates T cell responses.

[00207] Isatuximab (SAR650984) is a chimeric anti-CD38 IgG1 antibody that is being evaluated for the treatment of patients with relapsed and refractory multiple myeloma (MM).

[00208] BCD-100 is a human antibody targeting programmed cell death protein-1 (PD-1)

[00209] Carotuximab (TRC105) is a chimeric IgG1 antibody targeting endoglin (CD105), which is a protein highly expressed in angiogenic and proliferative endothelial cells. This MAb binds human CD105 on proliferating endothelium with a dissociation constant (KD) of 1-2 ng/ml and induces antibody-mediated cellular cytotoxicity (ADCC) of human umbilical vein endothelial cells.

[00210] Camrelizumab (INCSHR-1210, SHR-1210) is a humanized IgG4κ antibody targeting PD-1.

[00211] Glembatumumab vedotin (CDX-011, CR011-vcMMAE) is a human IgG2 antibody targeting the non-metastatic transmembrane glycoprotein B (gpNMB) gene conjugated to monomethyl auristatin E, a cytotoxic drug that can cause death when released in cancer cells tumor cells.

[00212] Mirvetuximab soravtansine (IMGN853) is an antibody targeting folate receptor alpha (FRα) that is conjugated to 3-4 molecules of the maytansinoid drug DM4, an antimitotic agent.

[00213] Oportuzumab Monatox (VICINIUM™, VB4-845) is a recombinant humanized anti-epithelial cell adhesion molecule (EpCAM) antibody scFv fragment conjugated to Pseudomonas aeruginosa exotoxin A.

[00214] L19IL2/L19TNF (DAROMUN) is a fusion protein consisting of scFv antibody L19 targeting the extradomain B of fibronectin fused to either human IL2 or human TNF.

[00215] In addition, additional exemplary antibodies and fusion proteins may be selected from any of the following: benilizumab, MEDI-8968, anifrolumab, MEDI7183, sifalimumab, MEDI-575, tralokinumab from AstraZeneca and MedImmune; BAN2401 manufactured by Biogen Idec/Eisai Co. LTD (“Eisai”)/BioArctic Neuroscience AB; CDP7657 Fab fragment of monovalent pegylated anti-CD40L, STX-100, anti-avB6 mAb, BIIB059, anti-TWEAK (BIIB023) and BIIB022 from Biogen; fulranumab manufactured by Janssen and Amgen; BI-204/RG7418 manufactured by BioInvent International/Genentech; BT-062 (indatuximab ravtansine) from Biotest Pharmaceuticals Corporation; XmAb from Boehringer Ingelheim/Xencor; anti-IP10 from Bristol-Myers Squibb; J 591 Lu-177 manufactured by BZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX-0401 from Celldex Therapeutics; forovirumab manufactured by Crucell; tigatuzumab manufactured by Daiichi Sankyo Company Limited; MORAb-004, MORAb-009 (amatuximab) manufactured by Eisai; LY2382770 manufactured by Eli Lilly; DI17E6 manufactured by EMD Serono Inc; zanolimumab from Emergent BioSolutions, Inc.; FG-3019 manufactured by FibroGen, Inc.; catumaxomab manufactured by Fresenius SE & Co. KGaA; pateklizumab, rontalizumab manufactured by Genentech; frezolimumab manufactured by Genzyme &Sanofi; GS-6624 (simtuzumab) from Gilead; CNTO-328, bapineuzumab (AAB-001), carlumab, CNTO-136 from Janssen; KB003 manufactured by KaloBios Pharmaceuticals, Inc.; ASKP1240 manufactured by Kyowa; RN-307 manufactured by Labrys Biologics Inc.; Ecromeximab manufactured by Life Science Pharmaceuticals; LY2495655, LY2928057, LY3015014, LY2951742 manufactured by Eli Lilly; MBL-HCV1 from MassBiologics; AME-133v manufactured by MENTRIK Biotech, LLC; abituzumab manufactured by Merck KGaA; MM-121 from Merrimack Pharmaceuticals, Inc.; MCS 110, QAX576, QBX258, QGE031 manufactured by Novartis AG; HCD122 manufactured by Novartis AG and XOMA Corporation (“XOMA”); NN8555 manufactured by Novo Nordisk; bavituximab, Cotara from Peregrine Pharmaceuticals, Inc.; PSMA-ADC from Progenies Pharmaceuticals, Inc.; oregovomab manufactured by Quest Pharmatech, Inc.; fasinumab (REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331, RG7745 manufactured by Roche; ibalizumab (TMB-355) from TaiMed Biologics Inc.; TCN-032 manufactured by Theraclone Sciences; TRC105 manufactured by TRACON Pharmaceuticals, Inc.; UB-421 manufactured by United Biomedical Inc.; VB4-845 from Viventia Bio, Inc.; ABT-110 manufactured by AbbVie; caplacizumab, ozoralizumab manufactured by Ablynx; PRO 140 manufactured by CytoDyn, Inc.; GS-CDA1, MDX-1388 manufactured by Medarex, Inc.; AMG 827, AMG 888 manufactured by Amgen; ublituximab manufactured by TG Therapeutics Inc.; TOL101 manufactured by Tolera Therapeutics, Inc.; huN901-DM1 (lorvotuzumab mertansine) manufactured by ImmunoGen Inc.; the combination epratuzumab Y-90/veltuzumab (IMMU-102) manufactured by Immunomedics, Inc.; anti-fibrin mAb/3B6/22 Tc-99m manufactured by Agenix, Limited; ALD403 from Alder Biopharmaceuticals, Inc.; RN6G/PF-04382923 manufactured by Pfizer; CG201 manufactured by CG Therapeutics, Inc.; KB001-A manufactured by KaloBios Pharmaceuticals/Sanofi; KRN-23 manufactured by Kyowa; Y-90 hPAM 4 manufactured by Immunomedics, Inc.; tarextumab manufactured by Morphosys AG & OncoMed Pharmaceuticals, Inc.; LFG316 manufactured by Morphosys AG & Novartis AG; CNTO3157, CNTO6785 manufactured by Morphosys AG &Jannsen; RG6013 manufactured by Roche &Chugai; MM-111 manufactured by Merrimack Pharmaceuticals, Inc. ("Merrimack"); GSK2862277 manufactured by GlaxoSmithKline; AMG 282, AMG 172, AMG 595, AMG 745, AMG 761 manufactured by Amgen; BVX-20 manufactured by Biocon; CT-P19, CT-P24, CT-P25, CT-P26, CT-P27, CT-P4 manufactured by Celltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776A manufactured by GlaxoSmithKline; anetumab ravtansine manufactured by Morphosys AG & Bayer AG; BI-836845 manufactured by Morphosys AG & Boehringer Ingelheim; NOV-7, NOV-8 manufactured by Morphosys AG and Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151 from Merrimack, RG7882 from Roche & Seattle Genetics; RG7841 manufactured by Roche/Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412, PF-05280586 manufactured by Pfizer; RG7716, RG7936, gentenerumab, RG7444 manufactured by Roche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212, MEDI-5117, MEDI-7814 manufactured by Astrazeneca; ulocuplumab, Adceptin PCSK9 from Bristol-Myers Squibb; FPA009, FPA145 manufactured by FivePrime Therapeutics, Inc.; GS-5745 manufactured by Gilead; BIW-8962, KHK4083, KHK6640 manufactured by Kyowa Hakko Kirin; MM-141 manufactured by Merck KGaA; REGN1154, REGN1193, REGN1400, REGN1500, REGN1908-1909, REGN2009, REGN2176-3, REGN728 manufactured by Regeneron; SAR307746 manufactured by Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141, ALX-0171 manufactured by Ablynx; milatuzumab-DOX, milatuzumab, TF2, manufactured by Immunomedics, Inc.; MLNO264 manufactured by Millennium; ABT-981 manufactured by AbbVie; AbGn-168H from AbGenomics International Inc.; ficlatuzumab manufactured by AVEO; BI-505 manufactured by BioInvent International; CDX-1127, CDX-301 manufactured by Celldex Therapeutics; CLT-008 manufactured by Cellerant Therapeutics Inc.; VGX-100 manufactured by Circadian; U3-1565 manufactured by Daiichi Sankyo Company Limited; DKN-01 manufactured by Dekkun Corp.; flanvotumab (TYRP1 protein), IL-1 β antibody, IMC-CS4 from Eli Lilly; mAb VEGFR3, IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; Anthim manufactured by Elusys Therapeutics Inc.; HuL2G7 manufactured by Galaxy Biotech LLC; IMGB853, IMGN529 manufactured by ImmunoGen Inc.; CNTO-5, CNTO-5825 manufactured by Janssen; KD-247 manufactured by Kaketsuken; KB004 manufactured by KaloBios Pharmaceuticals; MGA271, MGAH22 manufactured by MacroGenics, Inc.; XmAb5574 from MorphoSys AG/Xencor; ensituximab (NPC-1C) from Neogenix Oncology, Inc.; LFA102 manufactured by Novartis AG and XOMA; ATI355 manufactured by Novartis AG; SAN-300 manufactured by Santarus Inc.; SelG1 manufactured by Selexys; HuM195/rGel from Targa Therapeutics, Corp.; VX15 manufactured by Teva Pharmaceuticals, Industries Ltd. (“Teva”) and Vaccinex Inc.; TCN-202 from Theraclone Sciences; XmAb2513, XmAb5872 manufactured by Xencor; XOMA 3AB manufactured by XOMA and the National Institute for Allergy and Infectious Diseases; neuroblastoma antibody vaccine from MabVax Therapeutics; cytolin manufactured by CytoDyn, Inc.; Travixa from Emergent BioSolutions Inc.; and FB 301 from Cytovance Biologics; anti-rabies mAb manufactured by Janssen and Sanofi; influenza mAb from Janssen, with partial funding from the National Institutes of Health; MB-003 and ZMapp from Mapp Biopharmaceutical, Inc.; and ZMAb manufactured by Defyrus IncG.

III. cccDNA vector in general for use in the production of antibodies and fusion proteins

[00216] Embodiments of the invention are based on methods and compositions containing linear closed-end duplex vectors (ccDNA) that can express a transgene (eg, an antibody or fusion protein). In some embodiments, the transgene is a sequence encoding an antibody or fusion protein. cccDNA vectors for the production of antibodies or fusion proteins, as described herein, are not limited in size, allowing, for example, the expression of all components necessary for transgene expression from a single vector. The cccDNA vector for producing antibodies or fusion proteins is preferably duplex, eg, self-complementary on at least part of the molecule, such as an expression cassette. (eg, cctDNA is not a double-stranded circular molecule). The cccDNA vector has covalently closed ends and is thus resistant to digestion by an exonuclease (eg exonuclease I or exonuclease III), for example, within an hour at 37°C.

[00217] Typically, a cccDNA vector for the production of antibodies or fusion proteins, as described herein, contains, in the 5' to 3' direction: the first inverted terminal repeat (ITR) of an adeno-associated virus (AAV), a nucleotide sequence representing interest (eg, an expression cassette as described herein) and a second AAV ITR. The ITR sequences are selected from any of: (i) at least one WT ITR and at least one modified AAB inverted terminal repeat (mod-ITR) (eg, asymmetric modified ITRs); (ii) two modified ITRs, wherein the mod-ITR pair has a different three-dimensional spatial organization relative to each other (e.g., asymmetric modified ITRs), or (iii) a symmetric or substantially symmetrical WT-WT ITR pair, wherein each WT-ITR has the same three-dimensional spatial organization, or (iv) pairs of symmetric or substantially symmetric modified ITRs, each mod-ITR having the same three-dimensional spatial organization.

[00218] This document covers methods and compositions containing a cccDNA vector for the production of antibodies or fusion proteins, which may further include a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system. Non-limiting examples of liposome nanoparticle systems contemplated for use are disclosed herein. In some aspects, the present invention relates to a lipid nanoparticle comprising cccDNA and an ionizable lipid. For example, the composition of lipid nanoparticles that are manufactured and loaded with a cccDNA vector obtained by this method is disclosed in international application PCT/US2018/050042, filed September 7, 2018, which is incorporated herein.

[00219] cDNA vectors for the production of antibodies or fusion proteins, as described herein, do not have the packaging restrictions imposed by limited space within the viral capsid. cccDNA vectors provide a viable eukaryotic-produced alternative to prokaryotic-produced plasmid DNA vectors as opposed to encapsulated AAV genomes. This allows the insertion of control elements, such as regulatory switches as described herein, large transgenes, multiple transgenes, etc.

[00220] FIG. 1A-1E show diagrams of non-limiting examples of cccDNA vectors for producing antibodies or fusion proteins, or corresponding ccDNA plasmid sequence. cccDNA vectors for the production of antibodies or fusion proteins are non-capsid and can be derived from a plasmid encoding, in this order: a first ITR, an expression cassette containing the transgene, and a second ITR. An expression cassette may include one or more regulatory sequences that enable and/or control expression of a transgene, for example, where the expression cassette may contain one or more of the following, in that order: enhancer/promoter, ORF reporter (transgene), post-transcriptional regulatory element (eg, WPRE) and polyadenylation and termination signal (eg, poly(A) BGH).

[00221] 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, a promoter, a riboswitch, an insulator, a mir-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, the ITR may act as a promoter for a transgene, such as an antibody or fusion protein. In some embodiments, the cccDNA vector contains additional components for regulating expression of the transgene, such as a regulatory switch, which is described herein in the section entitled “Regulatory Switches” for controlling and regulating expression of the antibody or fusion protein, and may include, as appropriate, , a regulatory switch that is a “safety switch” that ensures controlled death of the cell containing the cccDNA vector.

[00222] The expression cassette may contain more than 4000 nucleotides, 5000 nucleotides, 10000 nucleotides or 20000 nucleotides, or 30000 nucleotides, or 40000 nucleotides, or 50000 nucleotides, or any range from about 4000 to 10000 nucleotides or 100,000,000 nucleotides up to more than 50,000 nucleotides . In some embodiments, the expression cassette may contain a transgene having a length in the range of 500 to 50,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length ranging from 500 to 75,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 500 to 10,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 1000 to 10,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 500 to 5000 nucleotides. cccDNA vectors have no restrictions on the size of encapsidated AAV vectors, which allows delivery of a large expression cassette to ensure efficient transgene expression. In some embodiments, the cDNA vector lacks prokaryote-specific methylation.

[00223] The cccDNA expression cassette may include, for example, an exogenous expressed sequence (e.g., an open reading frame) or a transgene encoding a protein that is either absent, inactive, or deficient in activity in the recipient subject, or a gene that encodes a protein having a desired biological or therapeutic effect. The transgene may encode a gene product that may function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene encoding a protein, polypeptide or RNA that is either reduced or absent due to mutation, or that provides a therapeutic benefit when overexpression is considered within the scope of the invention.

[00224] The expression cassette may contain any transgene (eg, encoding an antibody or fusion protein), for example, an antibody or fusion protein useful for treating a disease or disorder in a subject, i.e. therapeutic antibody or fusion protein. The cccDNA vector can be used to deliver and express any antibody or fusion protein of interest in a subject, alone or in combination with polypeptide-encoding nucleic acids or non-coding nucleic acids (e.g., RNAi, mature microRNAs (miR), etc. .d.), as well as exogenous genes and nucleotide sequences, including viral sequences in the subject's genome, for example, sequences of the HIV virus, etc. Preferably, the cDNA vector disclosed herein is used for therapeutic purposes (eg, medical, diagnostic or veterinary applications), or immunogenic polypeptides. In some embodiments, the cccDNA vector is suitable for expressing in a subject any gene of interest, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAi, antisense oligonucleotides, antisense polynucleotides, or RNAs (encoding or non-coding eg, siRNAs, short hairpin RNAs (shRNAs), microRNAs and their antisense analogues (eg, antagomir (antagoMiR)), antibodies, antigen-binding fragments, or any combination thereof.

[00225] The expression cassette may also encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., siRNAs, short hairpin RNAs, microRNAs, and their antisense counterparts (e.g., antagomir)). Expression cassettes may include an exogenous sequence that encodes a reporter protein for use in 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.

[00226] The sequences present in the expression cassette, cDNA vector expression construct for producing an antibody, or fusion protein described herein can be codon optimized for the target host cell. As used herein, the term “codon-optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in cells of a vertebrate animal of interest, e.g., mouse or human, by replacing at least one, more than one, or a significant number of codons from a native sequence (eg, a prokaryotic sequence) to codons that are more frequently or most frequently used in the genes of that vertebrate animal. Different species exhibit a particular affinity 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 using, for example, the Gene Forge® codon optimization and custom gene synthesis platform from Aptagen (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or other publicly available database. In some embodiments, the nucleic acid encoding the antibody or fusion protein is optimized for expression in humans and/or is a human antibody or humanized antibody or antigen binding fragment thereof, as known in the art.

[00227] In some embodiments, the antibody or fusion protein expressed by the cccDNA vector is a therapeutic antibody or fusion protein, including therapeutic activating antibodies or fusion proteins or therapeutic neutralizing (e.g., blocking or inhibitory) antibodies or fusion proteins.

[00228] A transgene expressed by a cccDNA vector to produce an antibody or fusion protein as disclosed herein encodes the antibody or fusion protein. Antibodies and fusion proteins are well known in the art and can bind to any protein of interest, including, without limitation, a ligand, receptor, toxin, hormone, enzyme, or cell surface protein, or pathogen, or viral protein, or antigen, as well as pre- and post-translationally modified proteins such as glycoproteins or sumoylated proteins (eg anti-SUMO2/3 antibody), etc. Antibodies also include antibodies that bind to any antigen, including, but not limited to, nucleic acids, eg, DNA (eg, anti-dsDNA antibodies), RNA (eg, RNA-binding antibodies). In some embodiments, the antibodies or fusion proteins produced by the cDNA vectors disclosed herein are neutralizing antibodies or fusion proteins. Representative examples of target genes and proteins of interest are described in detail in the Applications and Treatments sections of this document. Antibodies, fusion proteins, and variants and/or active fragments thereof for use in the treatment, prevention and/or alleviation of one or more symptoms of a disease, dysfunction, lesion and/or disorder are provided for use in cccDNA vectors for the purpose of producing antibodies or fusion proteins disclosed herein. Representative examples of therapeutic genes are described herein in the section entitled “Method of Treatment.”

[00229] There are many structural features of cDNA vectors for producing antibodies or fusion proteins that differ from plasmid-based expression vectors. cccDNA vectors may have one or more of the following features: lack of parent (i.e., uninserted) bacterial DNA, lack of a prokaryotic origin of replication, self-sufficiency, i.e. they do not require any sequences other than the two ITRs, including Rep and end resolution binding sites (RBS and TRS), and exogenous sequence between the ITRs, the presence of hairpin-forming ITR sequences, and the absence of bacterial-type DNA methylation or any other methylation , considered abnormal in the mammalian host. In general, it is preferred that the vectors of the present invention do not contain any prokaryotic DNA, but it is contemplated that some prokaryotic DNA may be inserted as an exogenous sequence, by way of non-limiting example, in a promoter or enhancer region. Another important feature that distinguishes cccDNA vectors from plasmid expression vectors is that cccDNA vectors are single-stranded linear DNA with closed ends, whereas plasmids are always double-stranded DNA.

[00230] cccDNA vectors for the production of antibodies or fusion proteins produced by the methods presented herein preferably have a linear and continuous structure, rather than a discontinuous structure, as determined by a restriction enzyme digestion assay ( Figure 4D ). It is believed that a linear and continuous structure is more resistant to the effects of cellular endonucleases, and is also less likely to undergo recombination and cause mutagenesis. Thus, a cccDNA vector with a linear and continuous structure is the preferred implementation option. A continuous linear single-stranded cccDNA vector with an intramolecular duplex may have covalently linked terminal ends, without sequences encoding AAV capsid proteins. These cDNA vectors are structurally different from plasmids (including the cDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. The complementary strands of plasmids can be separated after denaturation to form two nucleic acid molecules, while cccDNA vectors, on the contrary, although they have complementary strands, represent a single DNA molecule and therefore remain a single molecule even in a denatured state. In some embodiments, cccDNA vectors, as described herein, can be produced without prokaryotic DNA base methylation, unlike plasmids. Thus, cccDNA vectors and ccDNA plasmids differ both in structure (in particular, linear or circular), and in the methods used to obtain and purify these various objects (see below), as well as in their DNA methylation patterns, which occurs in a prokaryotic type in cDNA plasmids and in a eukaryotic type in a cDNA vector.

[00231] There are several advantages to using a cccDNA vector to produce antibodies or fusion proteins as described herein over plasmid-based expression vectors, such advantages include, but are not limited to: 1) plasmids contain bacterial DNA sequences and are subjected to prokaryotic-specific methylation, for example, methylation of 6-methyladenosine and 5-methylcytosine, whereas capsidless AAV vector sequences are of eukaryotic origin and do not undergo prokaryotic-specific methylation; as a result, capsidless AAV vectors are less likely to induce inflammatory and immune responses compared to plasmids; 2) if plasmids require the presence of a resistance gene during production, then cccDNA vectors do not require it; 3) if the circular plasmid is not delivered to the nucleus when introduced into the cell and requires overload in order to avoid degradation by cellular nucleases, then ccDNA vectors contain viral cis-elements, i.e. ITRs, which confer nuclease resistance and can be engineered to enable nuclear targeting and delivery. It has been proposed that the minimum defining elements required for ITR function are the Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAB2) and the terminal resolution site (TRS; 5'-AGTTGG -3' (SEQ ID NO: 64) for AAB2) plus a variable palindromic sequence enabling hairpin formation; and 4) cccDNA vectors do not have the overrepresentation of CpG (cytosine phosphate guanine) dinucleotides often observed in plasmids of prokaryotic origin, which are known to bind to a member of the Toll-like receptor family, inducing a T cell-mediated immune response. In contrast, transduction with capsidless AAV vectors disclosed herein can provide effective targeting of cell and tissue types that are difficult to transduce with standard AAV virions using a variety of delivery reagents.

IV. ITR

[00232] As disclosed herein, cccDNA vectors for producing antibodies or fusion proteins contain a transgene or heterologous nucleic acid sequence located between two inverted terminal repeat (ITR) sequences, wherein the ITR sequences can be an asymmetric ITR pair or a symmetric or substantially a symmetrical ITR pair as those terms are defined herein. The cccDNA vector, as described herein, may contain ITR sequences that are selected from any of: (i) at least one wild-type (WT) ITR and at least one modified inverted terminal repeat (mod-ITR) AAV ( for example, asymmetric modified ITR); (ii) two modified ITRs, wherein the mod-ITR pair has a different three-dimensional spatial organization relative to each other (e.g., asymmetric modified ITRs), or (iii) a symmetric or substantially symmetric WT-WT ITR pair, in which each WT-ITR has the same three-dimensional spatial organization, or (iv) pairs of symmetrical or substantially symmetrical modified ITRs, wherein each mod-ITR has the same three-dimensional spatial organization, wherein the methods of this invention may further include a delivery system, such as, but not limited to, a delivery system based on liposomal nanoparticles.

[00233] In some embodiments, the ITR sequence may belong to viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. The subfamily Parvovirinae (called parvoviruses) includes the genus Dependovirus, which in most cases requires coinfection with a helper virus, such as an adenovirus or herpes virus, for productive infection. The genus Dependovirus includes adeno-associated virus (AAV), which typically infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g. adeno-associated viruses of cattle, canines, horses and sheep). Parvoviruses and other members of the family Parvoviridae are described generally in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[00234] Although the ITRs provided for illustration herein in the Description and Examples are the WT-ITR AAB2, one skilled in the art will appreciate that, as stated above, ITRs from any known parvovirus, such as dependovirus, can be used. such as AAV (for example, from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8. For example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITRs or ITRs from any synthetic AAV. In some embodiments, AAV can infect warm-blooded animals, such as avian adeno-associated virus (AAAV), bovine adeno-associated virus (BAAV), dog, horse, and sheep. In some embodiments, said ITR is derived from parvovirus B19 (GenBank Accession No. NC 000883), mouse minute virus (MVM) (GenBank Accession No. NC 001510); goose parvovirus (GenBank accession no. NC 001701); snake parvovirus 1 (GenBank accession no. NC 006148). In some embodiments, the 5' WT-ITR may be from one serotype and the 3' WT-ITR from a different serotype, as described herein.

[00235] One of ordinary skill in the art will recognize that ITR sequences contain, as a common structural element, a double-stranded Holliday structure, which is typically a T- or Y-hairpin (see, for example , FIG. 2A and Fig. 3A ), with each WT-ITR formed by two palindromic arms or loops (BB' and C-C') embedded within a larger palindromic arm (A-A'), and a single-stranded sequence D (where the order of said palindromic arms sequences determines the direction of ITR orientation in one direction or the other (flip or flop)). See, for example, for a description of the structural analysis and comparison of ITR sequences from different AAV serotypes (AAV1-AAV6) in Grimm et al., J. Virology, 2006; 80(1); 426-439; Yan et al., J. Virology, 2005; 364-379; Duan et al., Virology 1999; 261; 8-14. One skilled in the art can readily determine WT-ITR sequences from any AAV serotype for use in a cccDNA vector or ccDNA plasmid based on the examples of AAB2 ITR sequences provided herein. See, for example, comparison of ITR sequences from different AAV serotypes (AAV1-AAV6 and avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006; 80(1); 426-439; where is the % identity of the left ITR of AAV2 with the left ITR 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%).

A. Symmetrical ITR pairs

[00236] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins as described herein comprises, in the 5' to 3' direction: an adeno-associated virus (AAV) first inverted terminal repeat (ITR), a nucleotide of interest sequence (eg, an expression cassette as described herein), and a second AAB ITR, wherein the first ITR (5'-ITR) and the second ITR (3'-ITR) are symmetrical or substantially symmetrical with respect to each other, i.e. . The cccDNA vector may contain ITR sequences that have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space, or has the same A, C-C' and B-B' loops in three-dimensional space. In such an embodiment, the symmetrical ITR pair or substantially symmetrical ITR pair may be modified ITRs (eg, mod-ITRs) that are not wild-type ITRs. A mod-ITR pair may have identical sequences that contain one or more modifications compared to the wild-type ITR and are reverse complementary (inverted) to each other. In alternative embodiments, the pair of modified ITRs is substantially symmetrical as defined herein, i.e. a pair of modified ITRs may have different sequences but a similar or identical symmetrical three-dimensional shape.

[00237] (i) Wild type ITR

[00238] In some embodiments, symmetric ITRs or substantially symmetric ITRs are wild type (WT-ITR), as described herein. That is, both ITRs have wild-type sequence, but do not have to be WT-ITRs belonging to the same AAV serotype. Thus, in some embodiments, one WT-ITR may belong to one AAV serotype, and another WT-ITR may belong to a different AAV serotype. In such an implementation, the WT-ITR pair is substantially symmetric as defined herein, i.e. they can have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial organization.

[00239] Accordingly, as described herein, cccDNA vectors contain a transgene or heterologous nucleic acid sequence located between two wild-type inverted terminal repeat (WT-ITR) flanking sequences that are either reverse complementary (inverted) to each other, or, alternatively, are substantially symmetrical with respect to each other - i.e. the WT-ITR pair has a symmetrical three-dimensional spatial organization. In some embodiments, the wild-type ITR sequence (e.g., AAB WT-ITR) contains a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAB2, SEQ ID NO: 60) and a functional terminal resolution site (TRS) ; e.g. 5'-AGTT-3', SEQ ID NO: 62).

[00240] In one aspect, cccDNA vectors for producing antibodies or fusion proteins can be derived from a vector polynucleotide that encodes a heterologous nucleic acid operably located between two wild-type inverted terminal repeat (WT-ITR) sequences (e.g., AAB WT-ITR) . That is, both ITRs have wild-type sequence, but do not have to be WT-ITRs belonging to the same AAV serotype. Thus, in some embodiments, one WT-ITR may belong to one AAV serotype, and another WT-ITR may belong to a different AAV serotype. In such an implementation, the WT-ITR pair is substantially symmetric as defined herein, i.e. they can have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial organization. In some embodiments, the 5' WT-ITR is from one AAV serotype and the 3' WT-ITR is from the same or a different AAV serotype. In some embodiments, the 5'-WT-ITR and 3'WT-ITR are mirror images of each other, i.e. they are symmetrical. In some embodiments, the 5' WT-ITR and 3' WT-ITR are of the same AAV serotype.

[00241] WT ITRs are well known. In one embodiment, the two ITRs are of the same AAB2 serotype. In some embodiments, wild-type (WT) sequences from other serotypes may be used. There are a number of serotypes that are homologous, for example, AAV2, AAV4, AAV6, AAV8. In one embodiment, closely homologous ITRs (eg, ITRs with a similar loop structure) may be used. In another embodiment, a WT AAB ITR with a greater degree of variation may be used, such as AAB2 and AAB5, and in yet another embodiment, an ITR that is substantially WT may be used, i.e. has the basic loop structure of WT, but contains some conservative nucleotide changes that do not change or affect properties. When using WT-ITR from the same viral serotype, one or more regulatory sequences may be additionally used. In certain embodiments, the regulatory sequence is a regulatory switch that allows modulation of the activity of the cccDNA, such as the expression of an encoded antibody or fusion protein.

[00242] In some embodiments, one aspect of the technology described herein relates to a cccDNA vector for producing antibodies or fusion proteins, wherein the ccDNA vector contains at least one heterologous nucleotide sequence operably located between two inverted terminal sequences wild-type repeats (WT-ITR), where WT-ITR may be the same serotype, different serotypes, or be substantially symmetrical with respect to each other (i.e. have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space, or have the same A, C-C' and B-B' loops in three-dimensional space). In some embodiments, symmetrical WT-ITRs contain a functional end resolution site and a Rep binding site. In some embodiments, the heterologous nucleic acid sequence encodes a transgene and wherein the vector is not enclosed within a viral capsid.

[00243] In some embodiments, the WT-ITRs are the same but inversely complementary to each other. For example, the sequence AACG in the 5'-ITR may correspond to CGTT (ie, reverse complement) at the corresponding site in the 3'-ITR. The 5'-WT-ITR contains the sequence ATCGATCG, and the corresponding sense strand 3'-WT-ITR contains the CG A TCGAT (ie, the reverse complement of ATCGATCG). In some embodiments of the cccDNA, the WT-ITR further comprises a termination site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), such as a Rep binding site.

[00244] Typical WT-ITR sequences for use in cccDNA vectors for the production of antibodies or fusion proteins containing WT-ITR are shown in Table 7 , which shows WT-ITR pairs (5' WT-ITR and 3' WT-ITR) .

[00245] As an exemplary example, the present invention provides a cccDNA vector for producing an antibody or fusion protein comprising a promoter operably linked to a transgene (e.g., a heterologous nucleic acid sequence), with or without a regulatory switch, wherein the cccDNA is devoid of capsid proteins and: (a) produced from a cccDNA plasmid (e.g., see Figures 1F-1G ) that encodes a WT-ITR, each WT-ITR having the same number of base pairs of intramolecular duplexes in its hairpin secondary configuration (preferably excluding deletion any AAA or TTT terminal loop in this configuration compared to such reference sequences), and (b) identified as cccDNA using the cccDNA identification assay using native agarose gel electrophoresis and denaturing conditions in Example 1.

[00246] In some embodiments, the flanking WT-ITRs are substantially symmetrical to each other. In this embodiment, the 5' WT-ITR may be from one AAV serotype and the 3' WT-ITR ITR from a different AAV serotype such that the WT-ITRs are not identical reverse complements. For example, the 5' WT-ITR may belong to AAB2, and the 3' WT-ITR may belong to another serotype (eg, AAB1, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12). In some embodiments, the WT-ITR may be selected from two different parvoviruses selected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g. , royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus or insect AAV. In some embodiments, such a WT ITR combination is a combination of the WT ITRs of AAB2 and AAB6. In one embodiment, WT-ITRs are substantially symmetrical when one of them is inverted with respect to another ITR that is at least 90% identical, at least 95% identical, at least 96% ... 97% identical. .. 98%... 99%... 99.5%, including all intermediate values, and has the same symmetrical three-dimensional spatial organization. In some embodiments, a WT-ITR pair is substantially symmetrical in that they have a symmetrical three-dimensional spatial organization, such as having the same three-dimensional organization of the A, C-C', B-B', and D arms. In one embodiment, a substantially symmetrical WT-ITR pair is inverted with respect to each other and is at least 95% identical, at least 96% ... 97% ... 98% ... 99% .... 99.5%, including all intermediate values, and one WT-ITR retains the Rep binding site (RBS) 5´-GCGCGCTCGCTCGCTC-3´ (SEQ ID NO: 60) and the terminal resolution site (trs). In some embodiments, a substantially symmetrical WT-ITR pair is inverted with respect to each other, and is at least 95% identical, at least 96%...97%...98%...99%... .. 99.5%, including all intermediate values, and one WT-ITR retains the Rep binding site (RBS) 5´-GCGCGCTCGCTCGCTC-3´ (SEQ ID NO: 60) and the terminal resolution site (trs), and in addition to a variable palindromic sequence that allows the formation of a secondary hairpin structure. Homology can be determined by standard tools well known in the art, such as BLAST (Basic Local Alignment Search ToolTool), BLASTN, with default settings.

[00247] In some embodiments, the ITR structural element can be any structural element that is involved in the functional interaction of the ITR with the large Rep protein (eg, Rep 78 or Rep 68). In some embodiments, the structural element provides selectivity for the interaction of the ITR with the large Rep protein, i.e. determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, the structural element physically interacts with a large Rep protein upon binding of said Rep protein to the ITR. Each structural element may be, for example, an ITR secondary structure, an ITR nucleotide sequence, an intermediate sequence between two or more elements, or a combination of any of the above. In one embodiment, the constructs are selected from the group consisting of arm A and A', arm B and B', arm C and C', arm D, a Rep-binding site (RBE), and an RBE' (i.e., the sequence complementary RBE), and a terminal resolution site (trs).

[00248] By way of example only, Table 6 provides examples of WT-ITR combinations.

[00249] Table 6: Illustrative examples of WT-ITR combinations of the same serotype, or different serotypes, or different parvoviruses. The given order does not indicate the position of the ITR, for example, “AAB1, AAB2” demonstrates that ccDNA can contain the WT-ITR AAB1 at the 5' position and the WT-ITR AAB2 at the 3' position or, conversely, the WT-ITR AAB2 at the 5' position and WT-ITR AAB1 at position 3'. 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); genome AABrh8, AABrh10, AAV-DJ and AAV-DJ8 (for example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC001829; NC006152; NC 006260; NC 006261), ITR of warm-blooded animals (avian y AAV (AAAV), bull AAV (bovine AAV) (BAAV), canine, equine and sheep AAV), parvovirus B19 ITR (GenBank, accession no. NC 000883), mouse minute virus (MVM) (GenBank, accession no. NC 001510); geese: goose parvovirus (GenBank, accession no.: NC 001701); snakes: snake parvovirus 1 (GenBank Accession No.: NC 006148).

[00250]

Table 6 AAV1, AAV1 AAV2, AAV2 AAV3, AAV3 AAV4, AAV4 AAV5, AAV5 AAV1, AAV2 AAV2, AAV3 AAV3, AAV4 AAV4, AAV5 AAV5, AAV6 AAV1, AAV3 AAV2, AAV4 AAV3, AAV5 AAV4, AAV6 AAV5, AAV7 AAV1, AAV4 AAV2, AAV5 AAV3, AAV6 AAV4, AAV7 AAV5, AAV8 AAV1, AAV5 AAV2, AAV6 AAV3, AAV7 AAV4, AAV8 AAV5, AAV9 AAV1, AAV6 AAV2, AAV7 AAV3, AAV8 AAV4, AAV9 AAV5, AAV10 AAV1, AAV7 AAV2, AAV8 AAV3, AAV9 AAV4, AAV10 AAV5, AAV11 AAV1, AAV8 AAV2, AAV9 AAV3, AAV10 AAV4, AAV11 AAV5, AAV12 AAV1, AAV9 AAV2, AAV10 AAV3, AAV11 AAV4, AAV12 AAB5, AABRH8 AAV1, AAV10 AAV2, AAV11 AAV3, AAV12 AAB4, AABRH8 AAB5, AABRH10 AAV1, AAV11 AAV2, AAV12 AAB3, AABRH8 AAV4, AAVRH10 AAV5, AAV13 AAV1, AAV12 AAB2, AABRH8 AAV3, AAVRH10 AAV4, AAV13 AAB5, AABDJ AAB1, AABRH8 AAV2, AAVRH10 AAV3, AAV13 AAB4, AABDJ AAB5, AAVDJ8 AAB1, AAVRH10 AAV2, AAV13 AAB3, AABDJ AAB4, AAVDJ8 AAV5, BIRD AAV1, AAV13 AAB2, AABDJ AAB3, AAVDJ8 AAV4, BIRD AAB5, BULLISH AAB1, AABDJ AAB2, AAVDJ8 AAV3, BIRD AAB4, BULLISH AAV5, CANINE AAB1, AAVDJ8 AAV2, BIRD AAB3, BULLISH AAV4, CANINE AAV5, HORSES AAV1, BIRD AAB2, BULLISH AAV3, CANINE AAV4, HORSES AAV5, GOAT AAB1, BULLISH AAV2, CANINE AAV3, HORSES AAV4, GOAT AAV5, SHRIMP AAV1, CANINE AAV2, HORSES AAV3, GOAT AAB4, SHRIMP AAV5, PIGS AAV1, HORSES AAV2, GOAT AAB3, SHRIMP AAV4, PIGS AAV5, INSECTS AAV1, GOAT AAB2, SHRIMP AAV3, PIGS AAV4, INSECTS AAB5, SHEEP AAB1, SHRIMP AAV2, PIGS AAV3, INSECTS AAB4, SHEEP AAV5, B19 AAV1, PIGS AAV2, INSECTS AAB3, SHEEP AAV4, B19 AAB5, MVM AAV1, INSECTS AAV2, SHEEP AAV3, B19 AAB4, MVM AAV5, GEESE AAB1, SHEEP AAV2, B19 AAB3, MVM AAV4, GEESE AAV5, SERPENT AAV1, B19 AAB2, MVM AAV3, GEESE AAV4, SERPENT AAB1, MVM AAV2, GEESE AAV3, SERPENT AAV1, GEESE AAV2, SERPENT AAV1, SERPENT AAV6, AAV6 AAV7, AAV7 AAV8, AAV8 AAV9, AAV9 AAV10, AAV10 AAV6, AAV7 AAV7, AAV8 AAV8, AAV9 AAV9, AAV10 AAV10, AAV11 AAV6, AAV8 AAV7, AAV9 AAV8, AAV10 AAV9, AAV11 AAV10, AAV12 AAV6, AAV9 AAV7, AAV10 AAV8, AAV11 AAV9, AAV12 AAB10, AABRH8 AAV6, AAV10 AAV7, AAV11 AAV8, AAV12 AAB9, AABRH8 AAB10, AAVRH10 AAV6, AAV11 AAV7, AAV12 AAB8, AABRH8 AAB9, AABRH10 AAV10, AAV13 AAV6, AAV12 AAB7, AABRH8 AAB8, AABRH10 AAV9, AAV13 AAB10, AABDJ AAB6, AABRH8 AAB7, AABRH10 AAV8, AAV13 AAB9, AABDJ AAB10, AAVDJ8 AAV6, AAVRH10 AAV7, AAV13 AAB8, AABDJ AAB9, AAVDJ8 AAV10, BIRD AAV6, AAV13 AAB7, AABDJ AAB8, AAVDJ8 AAV9, BIRD AAB10, BULLISH AAB6, AABDJ AAB7, AAVDJ8 AAV8, BIRD AAB9, BULLISH AAV10, CANINE AAB6, AAVDJ8 AAV7, BIRD AAB8, BULLISH AAV9, CANINE AAV10, HORSES AAV6, BIRD AAB7, BULLISH AAV8, CANINE AAV9, HORSES AAV10, GOAT AAB6, BULLISH AAV7, CANINE AAV8, HORSES AAV9, GOAT AAB10, SHRIMP AAV6, CANINE AAV7, HORSES AAV8, GOAT AAV9, SHRIMP AAV10, PIGS AAV6, HORSES AAV7, GOAT AAB8, SHRIMP AAV9, PIGS AAV10, INSECTS AAV6, GOAT AAB7, SHRIMP AAV8, PIGS AAV9, INSECTS AAB10, SHEEP AAB6, SHRIMP AAV7, PIGS AAV8, INSECTS AAB9, SHEEP AAV10, B19 AAV6, PIGS AAV7, INSECTS AAB8, SHEEP AAV9, B19 AAB10, MVM AAV6, INSECTS AAB7, SHEEP AAV8, B19 AAB9, MVM AAV10, GEESE AAB6, SHEEP AAV7, B19 AAB8, MVM AAV9, GEESE AAV10, SERPENT AAV6, B19 AAB7, MVM AAV8, GEESE AAV9, SERPENT AAB6, MVM AAV7, GEESE AAV8, SERPENT AAV6, GEESE AAV7, SERPENT AAV6, SERPENT AAV11, AAV11 AAV12, AAV12 AAVRH8, AAVRH8 AAVRH10, AAVRH10 AAV13, AAV13 AAV11, AAV12 AAB12, AABRH8 AAVRH8, AAVRH10 ААВRH10, ААВ13 AAB13, AABDJ AAB11, AABRH8 AAB12, AAVRH10 ААВRH8, ААВ13 ААВRH10, ААВDJ AAB13, AAVDJ8 AAB11, AAVRH10 AAV12, AAV13 ААВRH8, ААВDJ ААВRH10, ААВDJ8 AAV13, BIRD AAV11, AAV13 AAB12, AAVDJ ААВRH8, ААВDJ8 AABRH10, BIRD AAB13, BULLISH AAB11, AAVDJ AAB12, AAVDJ8 AABRH8, BIRD AABRH10, BULLISH AAV13, CANINE AAB11, AAVDJ8 AAV12, BIRD AABRH8, BULLISH AABRH10, CANINE AAV13, HORSES AAV11, BIRD AAB12, BULLISH AABRH8, CANINE AABRH10, HORSES AAV13, GOAT AAB11, BULLISH AAV12, CANINE AABRH8, HORSES AABRH10, GOAT AAV13, SHRIMP AAV11, CANINE AAV12, HORSES AABRH8, GOAT AABRH10, SHRIMP AAV13, PIGS AAV11, HORSES AAV12, GOAT AABRH8, SHRIMP AABRH10, PIGS AAV13, INSECTS AAV11, GOAT AAV12, SHRIMP AABRH8, PIGS AABRH10, INSECTS AAV13, SHEEP AAB11, SHRIMP AAV12, PIGS AABRH8, INSECTS AABRH10, SHEEP AAV13, B19 AAV11, PIGS AAV12, INSECTS AABRH8, SHEEP AABRH10, B19 AAV13, MVM AAV11, INSECTS AAB12, SHEEP AABRH8, B19 AABRH10, MVM AAV13, GEESE AAB11, SHEEP AAV12, B19 AABRH8, MVM AABRH10, GEESE AAV13, SERPENT AAV11, B19 AAV12, MVM AABRH8, GEESE AABRH10, SERPENT AAV11, MVM AAV12, GEESE AABRH8, SERPENT AAV11, GEESE AAV12, SERPENT AAV11, SERPENT ААВDJ, ААВDJ ААВDJ8, AVVDJ8 BIRD, BIRD BULLY, BULLY DOGGY, DOGGY ААВDJ, ААВDJ8 ААВDJ8, BIRD BIRD, BULL BULL, DOG DOGS, HORSES AABDJ, BIRD ААВDJ8, BULLISH BIRD, CANINE BULL, HORSES DOG, GOAT AABDJ, BULLISH ААВDJ8, CANINE BIRD, HORSES BULL, GOAT DOG, PRAWN AABDJ, CANINE AABDJ8, HORSES BIRD, GOAT BULL, PRAWN DOGS, PIGS AABDJ, HORSES AABDJ8, GOAT POULTRY, PRAWN BULL, PIGS CANINE, INSECTS AABDJ, GOAT AABDJ8, PRAWN POULTRY, PIGS BULL, INSECTS CANINE, SHEEP AABDJ, PRAWN AABDJ8, PIGS BIRD, INSECTS BULL, SHEEP DOG, B19 AABDJ, PIGS AABDJ8, INSECTS BIRD, SHEEP BULLISH, B19 CANINE, MVM AABDJ, INSECTS AABDJ8, SHEEP BIRD, B19 BULLISH, MVM DOG, GEESE AABDJ, SHEEP ААВDJ8, B19 BIRD, MVM BULL, GOOSE DOG, SNAKE ААВDJ, B19 ААВDJ8, MVM BIRD, GEESE BULL, SNAKE ААВDJ, MVM ААВDJ8, GEESE BIRD, SNAKE ААВDJ, GUSEY ААВDJ8, SERPENT AABDJ, SERPENT HORSES, HORSES GOAT, GOAT SHRIMP, PRAWN PIGS, PIGS INSECTS, INSECTS HORSES, GOAT GOAT, PRAWN PRAWNS, PIGS PIGS, INSECTS INSECTS, SHEEP HORSES, PRAWNS GOAT, PIGS PRAWNS, INSECTS PIGS, SHEEP INSECTS, B19 HORSES, PIGS GOAT, INSECTS SHRIMP, SHEEP PIGS, B19 INSECTS, MVM HORSES, INSECTS GOAT, SHEEP SHRIMP, B19 PIGS, MVM INSECTS, GEESE HORSES, SHEEP GOAT, B19 SHRIMP, MVM PIGS, GEESE INSECTS, SNAKES HORSES, B19 GOAT, MVM SHRIMP, GEESE PIGS, SNAKES HORSES, MVM GOAT, GEESE SHRIMP, SNAKE HORSES, GEESE GOAT, SERPENT HORSES, SNAKES SHEEP, SHEEP B19, B19 MVM, MVM GEESE, GEESE SNAKE, SNAKE SHEEP, B19 B19, MVM MVM, GEESE GEESE, SNAKE SHEEP, MVM B19, GEESE MVM, SERPENT SHEEP, GEESE B19, SERPENT SHEEP, SNAKE

[00251] By way of example only, Table 7 shows sequences of representative WT-ITRs from several different AAV serotypes.

[00252]

[00253] In some embodiments, the WT-ITR nucleotide sequence may be modified (e.g., by modifying 1, 2, 3, 4, or 5 or more nucleotides, or any range thereof), wherein the modification is a substitution with a complementary nucleotide , for example, G to C and vice versa, and T to A and vice versa.

[00254] In certain embodiments of the present invention, the cccDNA vector for producing an antibody or fusion protein does not have a WT-ITR consisting of a nucleotide sequence selected from any of: SEQ ID NO: 1, 2, 5-14. In alternative embodiments of the present invention, if the cccDNA vector has a WT-ITR comprising a nucleotide sequence selected from any of: SEQ ID NO: 1, 2, 5-14, then the flanking ITR is also wild type (WT), and The cccDNA vector contains a regulatory switch, for example, as described herein and in international application PCT/US18/49996 (for example, see Table 11 of PCT/US18/49996). In some embodiments, the cccDNA vector for producing an antibody or fusion protein comprises a regulatory switch as described herein, and the selected WT-ITR has a nucleotide sequence selected from any member of the group consisting of: SEQ ID NO: 1, 2, 5 -14.

[00255] An cccDNA vector for producing antibodies or fusion proteins as described herein may include WT-ITR structures that retain the functional portions of RBE, trs, and RBE´. Fig. 2A and Fig. 2B demonstrates, using the wild-type ITR for illustration, one possible mechanism of function of the trs site in the wild-type ITR portion of the ccDNA vector. In some embodiments, the cccDNA vector for producing an antibody or fusion protein comprises one or more functional WT-ITR polynucleotide sequences that contain a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAB2) and a terminal resolution site (TRS; 5'-AGTT (SEQ ID NO: 62)). In some implementations, at least one WT-ITR is functional. In alternative embodiments, when the cccDNA vector for producing an antibody or fusion protein contains two WT-ITRs that are substantially symmetrical to each other, at least one WT-ITR is functional and at least one WT-ITR is non-functional.

B. Modified ITRs (mod-ITRs) in general for cccDNA vectors containing asymmetric ITR pairs or symmetric ITR pairs

[00256] As discussed herein, a cDNA vector for producing an antibody or fusion protein may contain a symmetric ITR pair or an asymmetric ITR pair. In both cases, one or both of the ITRs can be modified ITRs - the difference is that in the first case (i.e. symmetric mod-ITRs) said mod-ITRs have the same three-dimensional spatial organization (i.e. have the same configurations A -A', C-C' and B-B' arms), while in the second case (i.e. asymmetric mod-ITRs) these mod-ITRs have different three-dimensional spatial organization (i.e. have different A-A configurations ', C-C' and B-B'-shoulders).

[00257] In some embodiments, a modified ITR is an ITR that is modified by deletion, insertion, and/or substitution from the wild-type ITR sequence (eg, the AAB ITR). In some embodiments, at least one of the ITRs in the cccDNA vector contains a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAB2, 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, each of the different or modified ITRs is not a wild-type ITR from different serotypes.

[00258] Specific changes and mutations in the ITR are described in detail herein, but, in the context of the ITR, “altered” or “mutated” or “modified” indicates that nucleotides have been inserted, deleted and/or substituted relative to the wild-type sequence , reference or original ITR sequence. The altered or mutated ITR may be a recombinant ITR. As used herein, the term “recombinant” refers to the human manipulation aspect. For example, a polypeptide is considered "recombinant" when at least one aspect of the polypeptide, such as its sequence, has been manipulated by humans to be different from the aspect in which it exists in nature.

[00259] In some embodiments, mod-ITR may be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from more than one AAV serotype. In another embodiment, the synthetic ITR does not include an AAV-based sequence. In yet another embodiment, the synthetic ITR retains the ITR structure described above, although it contains only some or no sequences from AAB. In some aspects, the synthetic ITR may interact preferentially with wild-type Rep or Rep of a particular serotype, or, in some cases, it will not be recognized by wild-type Rep and will only be recognized by mutated Rep.

[00260] One skilled in the art can determine the corresponding sequence in other serotypes by known methods. For example, by determining whether the change is in region A, A', B, B', C, C', or D, and identifying the corresponding region in a different serotype. BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs can be used with default settings to determine the corresponding sequence. The invention further provides populations and pluralities of cccDNA vectors containing mod-ITR from a combination of different AAV serotypes, i.e. one mod-ITR may belong to one AAV serotype, and another mod-ITR may belong to a different serotype. Without wishing to be bound by any theory, in one embodiment, one ITR may be taken from or based on the AAB2 ITR sequence, and the other cDNA vector ITR may be taken from or be based on any one or more sequences ITR 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).

[00261] Any parvovirus ITR can be used as an ITR or as a base ITR for modification. Preferably, the parvovirus is a dependovirus. More preferably, it is AAB. The serotype selected may be based on the tissue tropism of the serotype. AAB2 has broad tissue tropism, AAB1 predominantly targets neuronal tissues and skeletal muscle, and AAV5 predominantly targets neuronal tissues, retinal pigmented epithelium, and photoreceptors. AAB6 primarily targets skeletal muscle and lungs. AAB8 targets predominantly tissues of the liver, skeletal muscle, heart, and pancreas. AAV9 primarily targets liver, skeletal muscle, and lung tissues. In one embodiment, the modified ITR is based on the AAB2 ITR.

[00262] More specifically, the ability of a structural element to operably interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleotide sequence of the structural element may be modified compared to the wild-type ITR sequence. In one embodiment, a structural element (e.g., arm A, arm A, arm B, arm B, arm C, arm C, arm D, RBE, RBE', and TRS) of the ITR can be removed and replaced with a wild-type structural element from another parvovirus . For example, the replacement structure may be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (eg, royal python parvovirus), bovine parvovirus, parvovirus goat, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus or insect AAV. For example, the ITR may be an AAB2 ITR, and the A or A' arm or RBE may be replaced by a structural element from AAB5. In another example, the ITR may be an AAB5 ITR, and arms C or C', RBE, and TRS may be replaced by a building block from AAB2. In another example, the AAB ITR may be an AAB5 ITR in which arms B and B' are replaced by arms B and B' of the AAB2 ITR.

[00263] By way of example only, Table 8 provides representative examples of modifications of at least one nucleotide (e.g., deletions, insertions, and/or substitutions) in modified ITR regions, where X indicates modification of at least one nucleic acid (e.g., deletion, insertion and/or substitution) in the indicated region relative to the corresponding wild-type ITR. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any of the C and/or C' and/or B and/or B' regions preserves three consecutive T nucleotides (i.e. .TTT) in at least one terminal loop. For example, if the modification results in any of: a single arm ITR (e.g. single arm C-C' or single arm B-B'), or a modified arm C-B' or arm C'-B, or an ITR with two arms , containing at least one truncated arm (for example, a truncated arm C-C' and/or a truncated arm B-B'), then in at least the specified single arm, or in at least one of the ITR arms with two arms (wherein one arm may be truncated) three consecutive T nucleotides (i.e., TTT) are retained in at least one terminal loop. In some embodiments, the CC' truncated arm and/or the BB' truncated arm contains three consecutive T nucleotides (ie, TTT) in the terminal loop.

[00264]

Table 8: Examples of combinations of modifications to at least one nucleotide (e.g., deletion, insertion, and/or substitution) in different B-B' and C-C' regions or ITR arms (X indicates a nucleotide modification, e.g., addition, deletion, or substitution of at least one nucleotide per region). Area B Region B' Area C Region C' X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

[00265] In some embodiments, a mod-ITR for use in a cDNA vector for producing an antibody or fusion protein containing an asymmetric ITR pair or a symmetric mod-ITR pair as described herein may contain any one of the combinations of modifications specified in Table 8 , as well as modification of at least one nucleotide in any of one or more regions selected from: between A' and C, between C and C', between C' and B, between B and B', and between B' and A. In some embodiments, any modification of at least one nucleotide (eg, deletion, insertion, and/or substitution) in regions C or C' or B or B' still retains the terminal loop of the stem-loop structure. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' preserves three consecutive T nucleotides (i.e., TTT) at least in one end loop. In alternative embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' preserves three consecutive nucleotides of A (i.e., AAA) in at least one end loop. In some embodiments, a modified ITR for use with the present invention may comprise any one of the combinations of modifications set forth in Table 8, as well as modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any one or more regions , selected from: A', A, and/or D. For example, in some embodiments, a modified ITR for use in this invention may contain any of the combinations of modifications listed in Table 8 , as well as a modification of at least one nucleotide (for example, a deletion , insertion, and/or substitution) in region A. In some embodiments, a modified ITR for use in this invention may contain any one of the combinations of modifications listed in Table 8 , as well as modification of at least one nucleotide (e.g., deletion, insertion, and /or replacement) in area A'. In some embodiments, a modified ITR for use with the present invention may comprise any one of the combinations of modifications set forth in Table 8 , as well as modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in the A and/or A region '. In some embodiments, a modified ITR for use in this invention may contain any one of the combinations of modifications listed in Table 8 , as well as a modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in region D.

[00266] In one embodiment, the nucleotide sequence of the building block may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19 or 20 or more nucleotides, or any range of values) to obtain a modified structural element. Examples of specific modifications to the ITR according to one embodiment are provided herein (e.g., SEQ ID NO: 3, 4, 15-47, 101-116, or 165-187, or shown in Fig. 7A-7B PCT/US2018/064242, filed December 6, 2018 (e.g., SEQ ID NO: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 in PCT/US2018/064242) In some implementations, the ITR may be modified (for example, 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 of values).In other embodiments, the ITR may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least at least 98%, at least 99% or greater sequence identity to one of the modified ITRs of SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187, or the RBE-containing arm A-A' region and arms C-C' and B-B' according to SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187, or those shown in Tables 2-9 (i.e. SEQ ID NO: 110-112, 115-190, 200-468) of international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00267] In some embodiments, the modified ITR may, for example, comprise the removal or deletion of all or part of an arm A-A', or all or part of an arm B-B', or all or part of an arm CC', or , alternatively, removing 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs forming the stem of the loop, while maintaining the presence of a capping loop at the end of the stem (e.g., one arm) (e.g., see ITR-21 in Fig. 7A of application PCT/US2018/064242 filed December 6, 2018). In some embodiments, the modified ITR may comprise removing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, the modified ITR may comprise deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the CC' arm (see, e.g. , ITR-1 in Fig. 3B or ITR- 45 in Fig. 7A of application PCT/US2018/064242, filed December 6, 2018). In some embodiments, the modified ITR may comprise deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the CC' arm and deleting 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from arm B-B'. Any combination of base pair deletions is contemplated, for example, 6 base pairs in the CC' arm and 2 base pairs in the B-B' arm may be deleted. As an illustrative example, FIG. 3B shows an example of a modified ITR with a deletion of at least 7 base pairs from each of region C and region C', a loop nucleotide substitution in the region between C and C', and a deletion of at least one base pair from each of regions B and B ', such that the modified ITR contains two arms, at least one of which (eg, C-C') is truncated. In some embodiments, the modified ITR also contains a deletion of at least one base pair from each of the B and B' regions such that the BB' arm is also truncated compared to the wild type ITR.

[00268] In some embodiments, the modified ITR may have from 1 to 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 deletions relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR may have 1 to 30 nucleotide deletions relative to the full-length WT ITR sequence. In some embodiments, the modified ITR has 2 to 20 nucleotide deletions relative to the full-length wild-type ITR sequence.

[00269] In some embodiments, the modified ITR does not contain any nucleotide deletions in the RBE-containing portion of the A or A' regions so as not to interfere with DNA replication (eg, Rep protein binding to the RBE or nicking at the end resolution site). In some embodiments, a modified ITR useful for use in the present invention has one or more deletions in the B, B', C, and/or C region, as described herein.

[00270] In some embodiments, a cccDNA vector for producing an antibody or fusion protein comprising a symmetric ITR pair or an asymmetric ITR pair comprises a regulatory switch as described herein, and at least one modified ITR selected from having a nucleotide sequence selected from any of the group consisting of: SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187.

[00271] In another embodiment, the structure of the structural element may be modified. For example, a structural element (has) a change in stem height and/or number of nucleotides in a loop. For example, the stem height may be about 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides or more, or any range of values. In one embodiment, the stem may be from about 5 nucleotides to about 9 nucleotides in height and operably interacts with Rep. In another embodiment, the stem may be about 7 nucleotides in height and operably interacts with Rep. In another example, the loop may contain 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or more, or any range thereof.

[00272] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites within an RBE or extended RBE may be increased or decreased. In one example, an RBE or extended RBE may contain 1, 2, 3, 4, 5, or 6 or more GAGY binding sites, or any range thereof. Each GAGY binding site may independently be the exact GAGY sequence or a sequence similar to GAGY, provided that the sequence is sufficient for binding of the Rep protein.

[00273] In another embodiment, the spacing between two elements (such as, but not limited to, an RBE and a hairpin) can be changed (eg, increased or decreased) to change the functional interaction with the large Rep protein. For example, the spacing may 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 of specified values.

[00274] The cccDNA vector for producing antibodies or fusion proteins described herein may include an ITR structure that is modified from the wild-type AAB2 ITR structure disclosed herein but still retains the functional portion of RBE, trs, and RBE´. In FIG. 2A and Fig. Figure 2B demonstrates one possible mechanism for the trs site to operate as part of the wild-type ITR structure of a cDNA vector for the production of antibodies or fusion proteins. In some embodiments, the cDNA vector for producing an antibody or fusion protein comprises one or more functional ITR polynucleotide sequences that comprise a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAB2) and a site end resolution (TRS; 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one ITR (wild type or modified ITR) is functional. In alternative embodiments, when the cccDNA vector contains two modified ITRs that are different from or asymmetrical with respect to each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[00275] In some embodiments, the modified ITR (e.g., left or right ITR) of a cccDNA vector for producing an antibody or fusion protein, as described herein, has modifications in a loop arm, truncated arm, or spacer. Examples of ITR sequences having modifications in the loop arm, truncated arm or spacer are listed in Table 2 (ie SEQ ID NO: 135-190, 200-233); Table 3 (eg SEQ ID NO: 234-263); Table 4 (eg SEQ ID NO: 264-293); Table 5 (eg, SEQ ID NO: 294-318 herein); Table 6 (e.g., SEQ ID NO: 319-468; and Tables 7-9 (e.g., SEQ ID NO: 101-110, 111-112, 115-134) or Table 10A or 10B (e.g., SEQ ID NO: 9 , 100, 469-483, 484-499) of international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00276] In some embodiments, the modified ITR for use in a cccDNA vector for producing antibodies or fusion proteins containing an asymmetric ITR pair or a symmetric mod-ITR pair is selected from any or a combination of ITRs listed in Tables 2, 3, 4. 5, 6, 7, 8, 9 and 10A-10B of international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00277] Additional examples of modified ITRs for use in a cccDNA vector for producing antibodies or fusion proteins containing an asymmetric ITR pair or a symmetric mod-ITR pair in each of the above classes are presented in Tables 9A and 9B . The predicted secondary structures of the right modified ITRs listed in Table 9A are presented in Fig. 7A of international application PCT/US2018/064242, filed December 6, 2018, and the predicted secondary structures of the left-modified ITRs listed in Table 9B are presented in FIG. 7B of international application PCT/US2018/064242, filed December 6, 2018, which is incorporated herein in its entirety.

[00278] Table 9A and Table 9B provide examples of right and left modified ITRs.

[00279] Table 9A: Examples of right-hand modified ITRs. These examples of right-handed modified ITRs may contain the RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), the ACTGAGGC spacer (SEQ ID NO: 69), the complement of the GCCTCAGT spacer (SEQ ID NO: 70) and the RBE' (i.e., the complement of the RBE ) GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 9A : Examples of right-hand modified ITRs ITR construct Subsequence SEQ ID NO: ITR-18 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCGCACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 15 ITR-19 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 16 ITR-20 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 17 ITR-21 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCTTTGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 18 ITR-22 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACAAAGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 19 ITR-23 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAAATCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 20 ITR-24 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAACGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 21 ITR-25 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCAAAGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 22 ITR-26 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 23 ITR-27 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGTTTCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 24 ITR-28 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGTTTCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 25 ITR-29 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCTTTGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 26 ITR-30 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCTTTGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 27 ITR-31 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCTTTGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 28 ITR-32 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGTTTCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 29 ITR-49 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGGCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG thirty ITR-50 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 31

[00280] TABLE 9B : Examples of left modified ITRs. These examples of left-handed modified ITRs may contain the RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), spacer ACTGAGGC (SEQ ID NO: 69), spacer complement GCCTCAGT (SEQ ID NO: 70) and complement RBE (RBE') GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 9B : Examples of left modified ITRs ITR-33 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTGCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 32 ITR-34 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 33 ITR-35 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 34 ITR-36 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 35 ITR-37 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCAAAGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 36 ITR-38 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACTTTGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 37 ITR-39 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGATTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 38 ITR-40 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 39 ITR-41 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCTTTGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 40 ITR-42 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 41 ITR-43 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGAAACCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 42 ITR-44 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGAAACGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 43 ITR-45 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCAAAGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 44 ITR-46 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCAAAGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 45 ITR-47 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCAAAGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 46 ITR-48 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGAAACGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 47

[00281] In one embodiment, the cccDNA vector for producing an antibody or fusion protein contains, in the 5' to 3' direction: an adeno-associated virus (AAV) first inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette , as described herein), and a second ITR of AAB, wherein the first ITR (5'-ITR) and the second ITR (3'-ITR) are asymmetric relative to each other, i.e. they have different three-dimensional spatial configurations compared to each other. As an example embodiment, the first ITR may be a wild-type ITR and the second ITR may be a mutated or modified ITR, or conversely, the first ITR may be a mutated or modified ITR and the second ITR may be a wild-type ITR. In some embodiments, the first ITR and the second ITR are both mod-ITRs, but have different sequences, or have different modifications, and thus are not the same modified ITRs and have different 3D spatial configurations. In other words, a cccDNA vector with asymmetric ITRs contains ITRs in which any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or, alternatively, in which the asymmetric ITRs are a modified asymmetric pair of ITRs that may have different sequences and different three-dimensional shapes relative to each other. Typical examples of asymmetric ITRs in a cDNA vector for the production of antibodies or fusion proteins and for use in the preparation of a cDNA plasmid are presented in Tables 9A and 9B.

[00282] In an alternative embodiment, the cDNA vector for producing an antibody or fusion protein contains two symmetric mod-ITRs, i.e. both ITRs have the same sequence but are reverse complements (inverted forms) of each other. In some embodiments, the symmetric mod-ITR pair contains at least one of, or any combination of, a deletion, insertion, or substitution relative to a wild-type ITR sequence from the same AAV serotype. Additions, deletions or substitutions in symmetrical ITRs are the same but inversely complementary to each other. For example, an insertion of 3 nucleotides in the C region of the 5'-ITR will be mapped to an insertion of 3 reverse complementary nucleotides in the corresponding region of the C' region of the 3'-ITR. For purposes of illustration only, if in the 5'-ITR the addition is AACG, then in the 3'-ITR the addition will be CGTT at the corresponding site. For example, if the sense strand of the 5'-ITR is ATC GA TCG with the addition of AACG between G and A, the resulting sequence would be ATC G AACG A TCG (SEQ ID NO: 51). The corresponding sense strand of the 3'-ITR is CGA TC GAT (reverse complement ATC GA TCG) with the addition of CGTT (i.e. reverse complement AACG) between T and C, resulting in the sequence CGA T CGTT C GAT (SEQ ID NO: 49) (reverse complement ATC G AACG A TCG) (SEQ ID NO: 51).

[00283] In alternative embodiments, a pair of modified ITRs is substantially symmetrical as defined herein, i.e. a pair of modified ITRs may have different sequences but have a corresponding or identical symmetrical three-dimensional shape. For example, one modified ITR may belong to one serotype and another modified ITR may belong to a different serotype, but they have the same mutation (eg, insertion, deletion, or nucleotide substitution) in the same region. In other words, for illustrative purposes only, if the 5' mod-ITR belongs to AAB2 and has a deletion in the C region, and the 3' mod-ITR belongs to AAB5 and has a corresponding deletion in the C' region, then provided that the 5' mod-ITR and 3' mod-ITRs have the same or symmetrical three-dimensional spatial organization, they will be included for use in this invention as a pair of modified ITRs.

[00284] In some embodiments, a substantially symmetric mod-ITR pair has identical A, CC', and BB' loops in three-dimensional space, e.g., if a modified ITR in a substantially symmetric mod-ITR pair has a C-C' arm deletion, then the cognate mod-ITR has a corresponding deletion of the C-C' loop and also has a similar three-dimensional structure of the remaining A and BB' loops of the same shape in geometric space as its cognate mod-ITR. By way of example only, substantially symmetrical ITRs may have a symmetrical spatial organization such that their structure has the same shape in geometric space. This may occur, for example, when a GC pair is modified, for example, into a CG pair, or vice versa, or an AT pair is modified into a TA pair, or vice versa. Therefore, using the above illustrative example of a modified 5'-ITR as ATC G AACG A TCG (SEQ ID NO: 51), and a modified 3'-ITR as CGA T CGTT C GAT (SEQ ID NO: 49) ( i.e. reverse complement ATC G AACG A TCG (SEQ ID NO: 51)), these modified ITRs will remain symmetrical if, for example, the 5'-ITR has the sequence ATC G AAC C A TCG (SEQ ID NO: 50) wherein the G in addition is modified to C, and the substantially symmetrical 3'-ITR has the sequence CGA T CGTT C GAT (SEQ ID NO: 49), without a corresponding modification of the T in addition to A. In some embodiments, such a pair of modified ITRs is essentially symmetrical, since the modified ITR pair has symmetrical stereochemistry.

[00285] Table 10 provides examples of symmetric modified ITR pairs (ie, modified left ITR and symmetric right modified ITR) for use in a ccDNA vector for producing antibodies or fusion proteins. The bold (red) portion of the sequences identifies partial ITR sequences (ie, loop sequences A-A', CC', and B-B'), also depicted in FIG. 31A-46B. These examples of modified ITRs may contain 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 RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 10: Typical examples of symmetrically modified ITR pairs in a cDNA vector for the production of antibodies or fusion proteins LEFT modified ITR
(modified 5'-ITR) Symmetrical RIGHT modified ITR
(modified 3'-ITR) SEQ ID NO: 32 (ITR-33, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTGCGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 15 (ITR-18, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCGCACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 33 (ITR-34, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 48 (ITR-51, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 34 (ITR-35, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 16 (ITR-19, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 35 (ITR-36, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 17 (ITR-20, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 36 (ITR-37, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCAAAGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 18 (ITR-21, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCTTTGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 37 (ITR-38, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACTTTGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 19 (ITR-22 on the right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACAAAGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 38 (ITR-39, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGATTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 20 (ITR-23, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAAATCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 39 (ITR-40, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 21 (ITR-24, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAACGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 40 (ITR-41, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCTTTGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 22 (ITR-25 on the right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCAAAGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 41 (ITR-42, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 23 (ITR-26 on the right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 42 (ITR-43, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGAAACCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 24 (ITR-27 on the right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGTTTCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 43 (ITR-44, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCGAAACGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 25 (ITR-28 on the right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGTTTCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 44 (ITR-45, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCCAAAGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 26 (ITR-29, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCTTTGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 45 (ITR-46, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCCAAAGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 27 (ITR-30, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCTTTGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 46 (ITR-47, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGCAAAGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 28 (ITR-31, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCTTTGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG SEQ ID NO: 47 (ITR-48, left) CCTGCAGGCAGCT GCGCGCTCGCTCGCTCACTGAGGCCGAAACGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 29 (ITR-32 on the right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGTTTCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG

[00286] In some embodiments, a cccDNA vector for producing an antibody or fusion protein containing a pair of asymmetric ITRs may contain an ITR with a modification corresponding to any of the modifications of the ITR sequences or partial ITR sequences shown in any one or more of Tables 9A-9B herein, or the sequences presented in FIG. 7A-7B of international application PCT/US2018/064242, filed December 6, 2018, which is incorporated herein in its entirety, or disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9 or 10A-10B international application PCT/US18/49996, filed September 7, 2018, which is incorporated herein by reference in its entirety.

V. Typical examples of cDNA vectors

[00287] As described above, this invention relates to recombinant cccDNA expression vectors and cccDNA vectors that encode an antibody or fusion protein, for producing an antibody or fusion protein containing any of: asymmetric ITR pairs, symmetric ITR pairs, or substantially symmetrical ITRs as described above. In some embodiments, the invention provides recombinant cccDNA vectors for producing antibodies or fusion proteins having ITR and transgene flanking sequences, wherein the ITR sequences are asymmetric, symmetric, or substantially symmetrical with respect to each other as defined herein, and the ccDNA further comprises a nucleotide sequence of interest (eg, an expression cassette containing a transgene nucleic acid) located between flanking ITRs, wherein said nucleic acid molecule lacks viral capsid protein coding sequences.

[00288] The cDNA expression vector for producing an antibody or fusion protein may be any cDNA vector that can be conveniently subjected to recombinant DNA procedures, including the nucleotide sequence(s) as described herein, provided that at least one ITR is modified. The cccDNA vectors for producing antibodies or fusion proteins of the present invention are compatible with the host cell into which the cccDNA vector is to be introduced. In certain embodiments, the cDNA vectors may be linear. In some embodiments, the cDNA vectors may exist as an extrachromosomal entity. In certain embodiments, the cDNA vectors of this invention may contain element(s) that allow integration of a donor sequence into the genome of a host cell. As used herein, the terms “transgene” and “heterologous nucleotide sequence” are synonymous and encode an antibody or fusion protein as described herein.

[00289] In FIG. 1A-1G show diagrams of the functional components of two non-limiting examples of plasmids useful for producing a cccDNA vector for the production of antibodies or fusion proteins. Fig. 1A, 1B, 1D, 1F depict cDNA vector constructs or corresponding cDNA plasmid sequences for producing antibodies or fusion proteins. cccDNA vectors are capsidless and can be derived from a plasmid encoding, in this order: a first ITR, a transgene expression cassette, and a second ITR, wherein the first and second ITR sequences are asymmetrical, symmetrical, or substantially symmetrical with respect to each other, as defined herein document. cccDNA vectors for the production of antibodies or fusion proteins are capsidless and can be derived from a plasmid encoding, in this order: a first ITR, an expressed transgene (protein or nucleic acid), and a second ITR, wherein the first and second ITR sequences are asymmetric, symmetric, or substantially symmetrical with respect to each other, as defined herein. In some embodiments, the transgene expression cassette includes, as appropriate, an enhancer/promoter, one or more homologous arms, a donor sequence, a post-transcriptional regulatory element (e.g., WPRE, e.g., SEQ ID NO: 67), and a polyadenylation and termination signal (e.g., poly (A) BGH, e.g. SEQ ID NO: 68).

[00290] FIG. 5 depicts a gel confirming the production of cccDNA from multiple plasmid constructs using the method described in the examples. Preparation of the cccDNA vector is confirmed by a representative gel electropherogram as described for FIG. 4A above and in the examples.

A. Regulatory elements

[00291] cccDNA vectors for producing antibodies or fusion proteins, as described herein, containing an asymmetric ITR pair or a symmetric ITR pair, as defined herein, may further contain a particular combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a mir-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, the ITR may act as a promoter for a transgene, such as an antibody or fusion protein. In some embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, contains additional components for regulating transgene expression, such as regulatory switches, as described herein, for regulating transgene expression, or a "safety switch" which can kill a cell containing a cDNA vector encoding an antibody or an antigen-binding fragment thereof. Control elements, including control switches, that can be used in this invention are described in more detail in international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00292] In embodiments, the second nucleotide sequence includes a regulatory sequence and a nucleotide sequence encoding a nuclease. In some embodiments, the gene regulatory sequence is operably linked to a nucleotide sequence encoding a nuclease. In certain embodiments, the regulatory sequence is useful for controlling the expression of a nuclease in a host cell. In some embodiments, the regulatory sequence includes a suitable promoter sequence capable of directing transcription of a gene operably linked to the promoter sequence, such as a nucleotide sequence encoding the nuclease(s) of the present invention. In some embodiments, the second nucleotide sequence includes an intronic sequence associated with the 5' end of the nucleotide sequence encoding the nuclease. In some embodiments, an enhancer sequence is provided upstream of the promoter to improve promoter efficiency. In some embodiments, the regulatory sequence includes an enhancer and a promoter, wherein the second nucleotide sequence includes an intronic sequence preceding a nucleotide sequence encoding a nuclease, wherein the intron includes one or more nuclease cleavage sites, and the promoter is operably linked to the nucleotide sequence encoding the nuclease.

[00293] cccDNA vectors for the production of antibodies or fusion proteins, produced synthetically or using a cell-based production method, as described herein in the Examples, may further contain a specific combination of cis-regulatory elements, such as a post-transcriptional hepatitis virus regulatory element North American woodchuck (WPRE) (eg, SEQ ID NO: 67) and poly(A) BGH (SEQ ID NO: 68). Suitable expression cassettes for use in expression constructs do not have the packaging restrictions imposed by the viral capsid.

(i). Promoters:

[00294] One skilled in the art will appreciate that promoters used in cDNA vectors for producing antibodies or fusion proteins, as described herein, must be tailored to match the specific sequences that they stimulate.

[00295] The cDNA vector expression cassettes for producing antibodies or fusion proteins may include a promoter that can influence overall expression levels as well as cell specificity. For transgene expression, eg antibody or antigen binding fragment expression, they may include a highly active viral-derived immediate early promoter. Expression cassettes may contain tissue-specific eukaryotic promoters to restrict transgene expression to certain cell types and reduce toxic effects and immune responses resulting from unregulated ectopic expression. In some embodiments, the expression cassette may comprise a synthetic regulatory element, such as a CAG promoter (SEQ ID NO: 72). The CAG promoter includes (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of the chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette may comprise an alpha-1 antitrypsin (AAT) promoter (SEQ ID NO: 73 or SEQ ID NO: 74), a liver-specific (LP1) promoter (SEQ ID NO: 75 or SEQ ID NO: 76), or human elongation factor-1 alpha (EF1a) promoter (eg, SEQ ID NO: 77 or SEQ ID NO: 78). In some embodiments, the expression cassette includes one or more constitutive promoters, such as the Rous sarcoma virus (RSV) long terminal repeat (LTR) retroviral promoter (optionally with an RSV enhancer), or the cytomegalovirus (CMV) immediate early promoter (optionally with an enhancer CMV, e.g. SEQ ID NO: 79). Alternatively, an inducible promoter, a native transgene promoter, a tissue-specific promoter, or various promoters known in the art may be used.

[00296] Suitable promoters, including those described above, may be derived from viruses and may therefore be referred to as viral promoters, or they may be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive the expression of any RNA polymerase (eg, pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early (CMVIE) promoter region, Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6, e.g. SEQ ID NO: 80) ( Miyagishi et al. , Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g. Xia et al. , Nucleic Acids Res. 2003 Sep. 1;31 (17)), human H1 promoter (H1) ( e.g., SEQ ID NO: 81 or SEQ ID NO: 155), CAG promoter, human alpha 1 antitypsin (HAAT) promoter (e.g., SEQ ID NO: 82), and the like. In some embodiments, these promoters are modified at their downstream intron-containing end to include one or more nuclease cleavage sites. In some embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.

[00297] In one embodiment, the promoter used is the native promoter of a gene encoding a therapeutic protein. Promoters and other regulatory sequences for the corresponding genes encoding therapeutic proteins are known and have been characterized. The promoter region used may further include one or more additional regulatory sequences (eg, native), such as enhancers (eg, SEQ ID NO: 79 and SEQ ID NO: 83), including the SV40 enhancer (SEQ ID NO: 126).

[00298] In some embodiments, the promoter may also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a liver-specific promoter such as human alpha-1 antitypsin (HAAT), natural or synthetic. In one embodiment, delivery to the liver can be achieved using endogenous ApoE-specific targeting of a cccDNA vector-containing composition to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[00299] Non-limiting examples of suitable promoters for use in accordance with this invention include the CAG promoter, for example, (SEQ ID NO: 72), the HAAT promoter (SEQ ID NO: 82), the human EF1-α promoter (SEQ ID NO: 77) or a fragment of the EF1a promoter (SEQ ID NO: 78), an IE2 promoter (e.g., SEQ ID NO: 84) and a rat EF1-α promoter (SEQ ID NO: 85), a mEF1 promoter (SEQ ID NO: 59) or a fragment of the 1E1 promoter (SEQ ID NO: 125).

(ii). Polyadenylation sequences:

[00300] A sequence encoding a polyadenylation sequence may be included in a cccDNA vector to produce antibodies or a fusion protein to stabilize the mRNA expressed from the ccDNA vector and to assist in nuclear export and translation. In one embodiment, the cDNA vector does not include a polyadenylation sequence. In other embodiments, the cccDNA vector for producing an antibody or fusion protein includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine dinucleotides. In some embodiments, the polyadenylation sequence comprises about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween. As shown in FIG. 10A-10G , the polyadenylation sequence may be located 3' of the transgene encoding the antibody or antibody fragment. In some embodiments, a cccDNA vector for producing an antibody or fusion protein that encodes a complete IgG or a complete antibody may comprise an IRES (internal ribosome entry site) sequence (SEQ ID NO: 190), wherein the IRES sequence is located, for example, on the side 3' of the polyadenylation sequence such that a second transgene (e.g., antibody or antigen binding fragment) located 3' of the first transgene is translated and expressed by the same ccDNA vector so that said ccDNA vector can express the complete antibody (see, for example, Fig. 10B ).

[00301] Expression cassettes may include a polyadenylation sequence known in the art, or a variant thereof, such as a naturally occurring sequence isolated from bovine BGHpA (e.g., SEQ ID NO: 68) or viral SV40pA (e.g., SEQ ID NO: 86 ), or a synthetic sequence (eg, SEQ ID NO: 87). Some expression cassettes may also include an upstream enhancer (USE) sequence of the SV40 late poly(A) signal. In some embodiments, USE can be used in combination with SV40pA or a heterologous poly-A signal.

[00302] Expression cassettes may also include a post-transcriptional element to increase expression of the transgene. In some embodiments, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is used to increase expression of the transgene (eg, SEQ ID NO: 67). Other post-transcriptional processing elements may be used, such as a post-transcriptional element from the herpes simplex virus or hepatitis B virus (HBV) thymidine kinase gene. Secretory sequences, such as VH-02 and VK-A26 sequences, such as SEQ ID NO: 88 and SEQ ID NO: 89, may be associated with the transgenes.

(iii). Nuclear localization sequences

[00303] In some embodiments, the cDNA vector for producing antibodies or fusion proteins contains one or more nuclear localization sequences (NLS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. 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-terminal). If more than one NLS is present, each may be selected independently of the others, such that one NLS will be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLS are given in Table 11.

[00304]

Table 11: Nuclear localization signals SOURCE SUBSEQUENCE SEQ ID NO. SV40 large T antigen PKKKRKV (encoded by the sequence CCCAAGAAGAAGAGGAAGGTG; SEQ ID NO: 91) 90 nucleoplasmin KRPAATKKAGQAKKKK 92 c-myc PAAKRVKLD 93 RQRRNELKRSP 94 hRNPA1 M9 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY 95 IBB domain of importin-alpha RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV 96 fibroid protein T VSRKRPRP 97 PPKKARED 98 human p53 PQPKKKPL 99 mouse c-abl IV SALIKKKKKMAP 100 NS1 influenza virus DRLRR 117 PKQKKRK 118 hepatitis delta virus antigen RKLKKKIKKL 119 mouse Mx1 protein REKKKFLKRR 120 human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK 121 glucocorticoid (human) steroid hormone receptor RKCLQAGMNLEARKTKK 122

B. Additional components of cccDNA vectors

[00305] cccDNA vectors for producing antibodies or fusion proteins of the present invention may contain nucleotides that encode other components for gene expression. For example, to select specific gene targeting events, a protective shRNA can be engineered into a microRNA and inserted into a recombinant cDNA vector designed for integration in a site-specific manner at 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, such as that described in Nygaard et al. , A universal system to select gene-modified hepatocytes in vivo, Gene Therapy , June 8, 2016. cccDNA vectors of this invention may contain one or more selectable markers that allow the selection of transformed, transfected, transduced or the like cells. A selectable marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophy to auxotrophs, NeoR, etc. In some embodiments, positive selection markers, such as NeoR, are included in the donor sequences. Negative selection markers may be included after the donor sequence, for example, an HSV-tk nucleic acid sequence encoding a negative selection marker may be included in the nucleic acid construct after the donor sequence.

C. Regulatory switches

[00306] A molecular regulatory switch is a switch that generates a measurable change of state in response to a signal. Such regulatory switches can be effectively combined with cccDNA vectors to produce antibodies or fusion proteins, as described herein, to control the release of expression of an antibody or antigen binding fragment from the ccDNA vector. In some embodiments, the cDNA vector for producing an antibody or fusion protein comprises a regulatory switch that serves to fine-tune the expression of the antibody or antigen-binding fragment. For example, it may serve to provide a biocontainment function to the cDNA vector. In some embodiments, the switch is a two-position (“ON/OFF”) switch that is configured to start or stop (ie, terminate) expression of an antibody or antigen binding fragment in a cccDNA vector in a controlled and regulated manner. In some embodiments, the switch may include a "kill switch" that may instruct the cell containing the cccDNA vector to enter programmed cell death upon activation of the switch. Exemplary examples of regulatory switches provided for use in a cccDNA vector for producing antibodies or fusion proteins can be used to regulate transgene expression and are described in more detail in international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

(i) Binary control switches

[00307] In some embodiments, the cDNA vector for producing an antibody or fusion protein comprises a regulatory switch that can serve to controllably modulate the expression of the antibody or antigen binding fragment. For example, an expression cassette located between the ITRs of a cccDNA vector may further comprise a regulatory region, e.g., a promoter, a cis element, a repressor, an enhancer, etc., that is operably linked to an antibody or antigen binding fragment, wherein said regulatory region is regulated by one or by several cofactors or exogenous agents. By way of example only, regulatory regions can be modulated by small molecule switches or inducible or repressible promoters. Non-limiting examples of inducible promoters are hormone inducible or metal inducible promoters. Other exemplary inducible promoters/enhancer elements include, but are not limited to, the RU486 inducible promoter, the ecdysone inducible promoter, the rapamycin inducible promoter, and the metallothionein promoter.

(ii) Small molecule-based regulatory switches

[00308] Various small molecule regulatory switches are known in the art and can be combined with cccDNA vectors to produce antibodies or fusion proteins as described herein to produce a cccDNA vector controlled by a regulatory switch. In some embodiments, the regulatory switch may be selected from any one or combination of the following: an orthogonal ligand/nuclear receptor pair, for example, a retinoid receptor variant/LG335 and GRQCIMFI, together with an artificial promoter controlling the expression of an operably linked transgene, as disclosed in Taylor et al. BMC Biotechnology 10 (2010): 15; recombinant steroid receptors, for example, a modified C-terminal truncated progesterone receptor that cannot bind progesterone but binds RU486 (mifepristone) (US Pat. No. 5,364,791); the Drosophila ecdysone receptor and its ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517); or a switch controlled by the antibiotic trimethoprim (TMP) as described in Sando R 3 ^rd ; Nat Methods, 2013, 10(11):1085-8. In some embodiments, the regulatory switch for controlling the transgene or expressed by the cccDNA vector is a prodrug activation switch, such as disclosed in US Pat. Nos. 8,771,679 and 6,339,070.

(iii) Regulatory switches with "password"

[00309] In some embodiments, said control switch may be a "passcode switch" or a "password circuit." Password switches allow for fine-tuning control of transgene expression from a cccDNA vector when specific conditions arise, i.e. combinations of conditions whose presence is necessary for expression and/or repression of the transgene. For example, at least conditions A and B must be present for transgene expression to occur. A password control switch may control the presence of any number of conditions, such as 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 necessary for transgene expression to occur. In some embodiments, at least 2 conditions must be met (e.g., conditions A, B), and in some embodiments, at least 3 conditions must be met (e.g., A, B, and C, or A, B, and D) . By way of example only, to effect expression of a gene from cccDNA containing a regulatory switch with the password "ABC", conditions A, B and C must be present. Conditions A, B and C may be as follows; condition A represents the presence of a condition or disease, condition B represents a hormonal response, and condition C represents a response to transgene expression. For example, if a transgene edits a defective EPO gene, then condition A is the presence of chronic kidney disease (CKD), condition B is satisfied if the subject has hypoxic conditions in the kidneys, condition C is impaired recruitment of erythropoietin-producing cells (EPC) to the kidneys; or, alternatively, impaired HIF-2 activation. When oxygen levels rise or the required EPO level is reached, the transgene turns off again until 3 conditions are met that turn it back on.

[00310] In some embodiments, a regulatory password switch or “password circuit” provided for use in a cccDNA vector contains hybrid transcription factors (TFs) to expand the range and level of complexity of environmental signals used to determine biocontainment conditions. Unlike a blocking switch, which initiates cell death when a predetermined condition is met, a “password circuit” allows cell survival or transgene expression to occur if a specific “password” is present, and can be easily reprogrammed to allow transgene expression and/or survival cells only in the presence of a predefined environmental condition or password.

[00311] Any and all combinations of regulatory switches disclosed herein, e.g., small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulation, radiation-controlled switches, hypoxia-mediated switches switches and other control switches known to those skilled in the art disclosed herein may be used in a password control switch as disclosed herein. The control switches envisaged for the application are also described in the review article by Kis et al., JR Soc Interface, 12: 20141000 (2015) and are summarized in Table 1 of the Kis article. In some embodiments, the control switch for use in a password system may be selected from any of the switches, or combinations thereof, set forth in Table 11 of International Patent Application PCT/US18/49996, which is incorporated herein by reference in its entirety.

(iv). Nucleic acid-based regulatory switches to control transgene expression

[00312] In some embodiments, the regulatory switch for controlling an antibody or antigen binding fragment expressed by cccDNA is based on a nucleic acid-based control mechanism. Typical examples of nucleic acid-based control mechanisms are known in the art and are contemplated for use. For example, such mechanisms include riboswitches such as those disclosed, for example, in US2009/0305253, US2008/0269258, US2017/0204477, WO2018026762A1, US patent 9222093 and European patent application EP288071, as well as the review by Villa JK et al. Microbiol Spectr., 2018 May;6(3). Also included are metabolite-sensitive transcriptional biosensors such as those disclosed in WO2018/075486 and WO2017/147585. Other mechanisms contemplated in the art include silencing of said transgene by siRNA or RNA interference (RNAi) molecule (eg, mature microRNA (miR), shRNA). For example, the cccDNA vector may contain a regulatory switch encoding an RNAi molecule that is complementary to a portion of the transgene expressed by the cccDNA vector. When such RNAi is expressed, even when the transgene is expressed by a ccDNA vector, its silencing occurs due to the action of a complementary RNA interference molecule, and in the absence of RNAi expression, if the specified transgene is expressed by a ccDNA vector, silencing of the specified transgene due to RNA interference does not occur.

[00313] In some embodiments, said regulatory switch is a tissue-specific self-inactivating regulatory switch, for example, as disclosed in US2002/0022018, wherein the regulatory switch specifically turns off transgene expression at a site where transgene expression may otherwise be disadvantageous. In some embodiments, the regulatory switch is a recombinase reversible gene expression system, for example, as disclosed in US2014/0127162 and US Pat. No. 8,324,436.

(v). Post-transcriptional and post-translational regulatory switches.

[00314] In some embodiments, the regulatory switch for controlling an antibody or antigen binding fragment expressed by a cccDNA vector is a post-transcriptional modification system. For example, such a regulatory switch may be an aptazyme riboswitch that is sensitive to tetracycline or theophylline, as disclosed in US2018/0119156, GB201107768, WO2001/064956A3, EP 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 can encode both a transgene and an inhibitory siRNA that contains a ligand-sensitive (OFF-switch) aptamer, resulting in a ligand-sensitive ON-switch ).

(vi). Other examples of control switches

[00315] Any known regulatory switch can be used in the cccDNA vector to control the expression of an antibody gene or antigen binding fragment expressed by the ccDNA vector, including those triggered by environmental changes. Additional examples include, without limitation; BOC method according to Suzuki et al., Scientific Reports 8; 10051 (2018); genetic code expansion and non-physiological amino acid; radiation-controlled or ultrasonic-controlled switches (see, for example, Scott S. et al. , Gene Ther. 2000 Jul; 7 (13): 1121-5; US patents 5612318; 5571797; 5770581; 5817636; WO1999/025385A1. B In some embodiments, the regulatory switch is controlled by an implantable system, for example, as disclosed in US Pat. No. 7,840,263; US2007/0190028A1, in which gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activates promoters operably linked to the transgene in the cccDNA vector.

[00316] In some embodiments, the regulatory switch for use in the cDNA vector is a hypoxia-mediated or stress-activated switch, such as those described in WO1999060142A2, US Pat. No. 5,834,306; 6218179; 6709858; 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 hypoxia response elements (HRE), inflammatory response elements (IRE) and shear stress activated elements (SSAE), for example, as disclosed in US Pat. No. 9,394,526. This embodiment is suitable for enabling expression of said transgene from a cDNA vector after ischemia or in ischemic tissues and/or tumors.

(iv). "Emergency switches"

[00317] Other embodiments described herein relate to a cccDNA vector for producing an antibody or fusion protein, as described herein, comprising a "kill switch". A "kill switch" as described herein allows the cell containing the cccDNA vector to be killed or to enter programmed death as a means of permanently removing the introduced cccDNA vector from the subject's system. One skilled in the art will appreciate that the use of "kill switches" in cccDNA vectors to produce antibodies or fusion proteins will typically involve targeting the cccDNA vector to a limited number of cells that are acceptable to be lost by the subject, or to a cell type that is undergoing apoptosis. which is desired (for example, on cancer cells). In all aspects, the kill switch disclosed herein is intended to provide rapid and reliable killing of a cell containing the cccDNA vector in the absence of a survival input or other specified condition. In other words, the “safety switch” encoded by the cccDNA vector for producing antibodies or fusion proteins described herein may limit the survival of the cell containing the ccDNA vector to an environment determined by specific input signals. Such “kill switches” serve to provide a biological biocontainment function when it is necessary to remove a cccDNA vector expressing an antibody or antigen binding fragment from a subject, or to ensure that the encoded antibody or antigen binding fragment is not expressed.

[00318] The cccDNA vector for producing antibodies or fusion proteins as described herein contemplates the use of other "kill switches" known to one skilled in the art, for example, as disclosed in US 2010/0175141; US2013/0009799; US2011/0172826; US2013/0109568, as well as the “kill switches” disclosed in Jusiak et al., Reviews in Cell Biology and molecular Medicine; 2014; 1-56; Kobayashi et al., PNAS, 2004; 101; 8419-9; Marchisio et al., Int. Journal of Biochem and Cell Biol., 2011; 43; 310-319; and in Reinshagen et al., Science Translational Medicine, 2018, 11.

[00319] Accordingly, in some embodiments, a cccDNA vector for producing an antibody or fusion protein may comprise a "kill switch" nucleic acid construct that contains a nucleic acid encoding an effector toxin or reporter protein, wherein expression of the effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, a predetermined condition may be the presence of an environmental agent, such as, for example, an exogenous agent, without which the cell will by default express an effector toxin (eg, a death protein) and die. In alternative embodiments, the predetermined condition is the presence of two or more environmental agents, for example, the cell will survive only when supplied with two or more required exogenous agents, and without any of them, the cell containing the cccDNA vector will die.

[00320] In some embodiments, the cccDNA vector for producing an antibody or fusion protein is modified to include a "safety switch" to destroy cells containing the cccDNA vector to effectively terminate in vivo expression of the transgene expressed by the cccDNA vector (e.g., a full-length antibody , Fab, single chain antibody (scAb)). Specifically, the cccDNA vector is further genetically engineered to express a switch protein that does not function in mammalian cells under normal physiological conditions. Cells expressing a switch protein will only be destroyed upon administration of a drug or exposure to an environmental condition that specifically targets the switch protein, thereby stopping expression of the therapeutic protein or peptide. For example, it has been reported that cells expressing HSV thymidine kinase can be killed by the administration of drugs such as ganciclovir and cytosine deaminase. See, for example, Dey and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in: Targets in Gene Therapy, ed. You (2011); and Beltinger et al., Proc. Natl. Acad. Sci. USA 96(15):8699–8704 (1999). In some embodiments, the cccDNA vector may contain a miRNA “kill switch” called DISE (death caused by survival gene elimination) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo ).

VI. Detailed description of the method for obtaining the cDNA vector

A. Receipt in general

[00321] Certain methods for producing a cccDNA vector for producing an antibody or fusion protein containing an asymmetric ITR pair or a symmetric ITR pair as defined herein are described in Section IV of International Application PCT/US18/49996, filed September 7, 2018. which is incorporated herein by reference in its entirety. In some embodiments, a cDNA vector for producing antibodies or fusion proteins as described herein can be produced using insect cells as described herein. In alternative embodiments, the cDNA vector for producing antibodies or fusion proteins as described herein can be produced synthetically and, in some embodiments, cell-free, as described in international application PCT/US19/14122, filed January 18, 2019 g., which is incorporated herein by reference in its entirety.

[00322] As described herein, in one embodiment, a cccDNA vector for producing antibodies or fusion proteins can be produced, for example, by a process comprising the steps of: a) incubating a population of host cells (e.g., insect cells) bearing a polynucleotide template of an expression construct (e.g., cccDNA plasmid, ccDNA bacmid, and/or ccDNA baculovirus) that lacks viral capsid coding sequences, in the presence of the Rep protein under conditions ensuring effectiveness and for a time sufficient to induce the production of cccDNA- vectors in host cells, wherein the host cells do not contain viral capsid coding sequences; and b) collecting and isolating the cDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide with a modified ITR to produce a cccDNA vector in the host cell. However, viral particles (eg, AAV virions) are not expressed. Thus, there are no size restrictions such as those imposed naturally in AAV or other viral vectors.

[00323] The presence of a cccDNA vector isolated from host cells can be confirmed by digesting DNA isolated from a host cell with a restriction enzyme having a single recognition site on the ccDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[00324] In another aspect, the present invention provides for the use of host cell lines having a polynucleotide DNA expression vector template (ccDNA template) stably integrated into its genome in the production of a non-viral DNA vector, for example, as described in Lee, L. et. al. (2013) Plos One 8(8): e69879. Preferably, Rep is added to host cells at a multiplicity of infection (MOI) of about 3. When the host cell line is a mammalian cell line, such as HEK293 cells, the cell lines may have a polynucleotide vector array stably integrated, and a second vector, such as a herpes virus, can be used to introduce Rep protein into cells, allowing cctDNA to be excised and amplified in the presence of Rep and helper virus.

[00325] In one embodiment, the host cells used to produce cccDNA vectors for producing antibodies or fusion proteins as described herein are insect cells, and a baculovirus is used to deliver both the polynucleotide encoding the Rep protein and the template a non-viral polynucleotide DNA vector expression construct for cccDNA, for example, as described in FIG. 4A-4C and in Example 1. In some embodiments, the host cell is modified to express the Rep protein.

[00326] The cccDNA vector is then collected and isolated from the host cells. The timing of collection and isolation from cells of the cccDNA vectors described herein can be selected and optimized to achieve high-throughput production of cccDNA vectors. For example, the collection time may be selected based on cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after a period of time sufficient to produce cccDNA vectors after infection with the baculovirus, but before the majority of the cells begin to die due to baculovirus toxicity. DNA vectors can be isolated using plasmid purification kits such as Qiagen Endo-Free Plasmid kits. Other methods developed for plasmid isolation can also be adapted for DNA vectors. In general, any method for purifying nucleic acids can be used.

[00327] DNA vectors can be purified by any methods known to those skilled in the art for purifying DNA. In one embodiment, the cDNA vectors are purified as DNA molecules. In another embodiment, cccDNA vectors are purified into exosomes or microparticles.

[00328] The presence of a cccDNA vector for producing antibodies or fusion proteins can be confirmed by digesting the DNA vector isolated from cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing the digested and uncleaved DNA material using gel electrophoresis to confirming the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA. Fig. 4C and FIG. 4D shows one option for identifying the presence of closed-end cccDNA vectors produced by the methods of this invention.

B. cDNA plasmid

[00329] A cDNA plasmid is a plasmid used to subsequently produce a cDNA vector for the production of antibodies or fusion proteins. In some embodiments, the cDNA plasmid can be constructed using known techniques to provide at least the following operably linked components in the direction of transcription: (1) a modified 5'-ITR sequence; (2) an expression cassette containing a cis-regulatory element, for example, a promoter, an inducible promoter, a regulatory switch, enhancers, and the like; and (3) a modified 3'-ITR sequence, wherein the 3'-ITR sequence is symmetrical with respect to the 5'-ITR sequence. In some embodiments, the expression cassette flanked by the ITR contains a cloning site for introducing an exogenous sequence. The expression cassette replaces the rep and cap coding regions of the AAV genomes.

[00330] In one aspect, a ccDNA vector for producing an antibody or fusion protein is derived from a plasmid, referred to herein as a "ccDNA plasmid", encoding, in this order: a first inverted terminal repeat (ITR) adeno-associated virus (AAV) expression cassette , containing a transgene and a mutated or modified AAV ITR, wherein said cDNA plasmid is devoid of coding sequences for the AAV capsid protein. In alternative embodiments, the ccDNA plasmid encodes, in that order: a first (or 5') modified or mutated AAV ITR, an expression cassette containing the transgene, and a second (or 3') modified AAV ITR, wherein the ccDNA plasmid is devoid of coding sequences capsid protein AAV, and the 5'- and 3'-ITRs are symmetrical relative to each other. In alternative embodiments, the ccDNA plasmid encodes, in that order: a first (or 5') modified or mutated AAV ITR, an expression cassette containing the transgene, and a second (or 3') mutated or modified AAV ITR, wherein said ccDNA plasmid lacks coding sequences of the AAV capsid protein, and the modified 5'- and 3'-ITRs have the same modifications (ie, they are reverse complementary or symmetrical with respect to each other).

[00331] In a further embodiment, the cDNA plasmid system lacks viral capsid protein coding sequences (i.e., it lacks AAV capsid genes as well as capsid genes of other viruses). Additionally, in a particular embodiment, the cDNA plasmid also lacks Rep AAB protein coding sequences. Accordingly, in a preferred embodiment, the ccDNA plasmid lacks functional cap AAV and rep AAV genes (GG-3' for AAV2) plus a variable palindromic sequence allowing hairpin formation.

[00332] The cDNA plasmid of this invention can be obtained using natural nucleotide sequences from the genomes of any AAV serotypes well known in the art. In one embodiment, the backbone of the cccDNA plasmid is derived from the genome of AAB1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 . For example, NCBI: NC 002077; NC001401; NC001729; NC001829; NC006152; NC 006260; NC 006261; Kotin and Smith, The Springer Index of Viruses, available from a URL maintained by Springer (at www: oesys.springer.de/viruses/database/mkchapter.asp?virID=42.04.) ( Note - URL or database references refer to the contents of the URL or database as of the effective filing date of this application). In a specific embodiment, the cDNA plasmid backbone is derived from the AAB2 genome. In another specific embodiment, the cDNA plasmid backbone is a synthetic backbone genetically engineered to include 5' and 3' ITRs derived from one of the AAV genomes.

[00333] The cccDNA plasmid may optionally include a selectable or selectable marker for use in creating a cell line that produces the cccDNA vector. In one embodiment, the selection marker may be inserted after (ie, on the 3' side) the 3' ITR sequence. In another embodiment, a selection marker may be inserted upstream (ie, 5' side) of the 5' ITR sequence. Suitable selection markers include, for example, markers that confer drug resistance. Selection markers can be, for example, a gene for resistance to blasticidin S, kanamycin, geneticin, etc. In a preferred embodiment, the drug selection marker is a blasticidin S resistance gene.

[00334] A typical cDNA vector (eg, rAAV0) for producing antibodies or fusion proteins is derived from the rAAV plasmid. A method for producing an rAAV vector may include: (a) providing a host cell with an rAAV plasmid as described above, wherein both the host cell and the plasmid are devoid of genes encoding the capsid protein, (b) culturing the host cell under conditions to obtain the genome cccDNA, and (c) collecting cells and isolating the AAV genome obtained from said cells.

C. An example of a method for obtaining cDNA vectors from cDNA plasmids

[00335] This document also provides methods for producing capsidless cccDNA vectors for the production of antibodies or fusion proteins, in particular, a method with sufficiently high yield to obtain sufficient quantities of the vector for in vivo experiments.

[00336] In some embodiments, a method of producing a cccDNA vector for producing an antibody or fusion protein includes the steps of: (1) introducing a nucleic acid construct comprising an expression cassette and two symmetrical ITR sequences into a host cell (e.g., Sf9 cells), ( 2) optionally, establishing a clonal cell line, for example, using a selection marker present on a plasmid, (3) introducing a gene encoding Rep (by transfection or infection with a baculovirus carrying said gene) into said insect cells, and (4) collecting cells and cDNA vector purification. The nucleic acid construct containing the expression cassette and two ITR sequences described above to produce a cccDNA vector may be in the form of a ccDNA plasmid or a bacmid or baculovirus produced using a ccDNA plasmid as described below. The nucleic acid construct can be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

D. Cell lines :

[00337] The host cell lines used to obtain the cccDNA vector for production of antibodies or fusion proteins may include insect cell lines derived from the corn fall armyworm (Spodoptera frugiperda), such as Sf9, Sf21, or cabbage moth (Trichoplusia ni) cells ), or other invertebrate, vertebrate, or other eukaryotic cell lines, including mammalian cells. Other cell lines known to one skilled in the art may also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes, and mature and immature dendritic cells. Host cell lines can be transfected to stably express the cccDNA plasmid to produce a ccDNA vector in high yield.

[00338] cccDNA plasmids can be introduced into Sf9 cells by transient transfection using reagents (eg, liposomal, calcium phosphate) or physical means (eg, electroporation) known in the art. Alternatively, stable Sf9 cell lines can be generated with the cccDNA plasmid stably integrated into the genomes. Such stable cell lines can be created by incorporating a selection marker into a cccDNA plasmid as described above. If the cccDNA plasmid used to transfect a cell line includes a selection marker, such as an antibiotic, then cells transfected with such ccDNA plasmid and having the ccDNA plasmid DNA integrated into their genome can be selected by adding the antibiotic to the cell growth medium . Resistant cell clones can then be isolated by clonal propagation or colony transfer methods and expanded.

E. Isolation and purification of cDNA vectors :

[00339] Examples of the method for preparing and isolating cccDNA vectors are described in FIG. 4A-4E and in the specific examples below. The cccDNA vectors for producing antibodies or fusion proteins described herein can be obtained from a producer cell expressing the AAV Rep protein(s), further transformed with a ccDNA plasmid, a ccDNA bacmid, or a ccDNA baculovirus. Plasmids useful for producing cccDNA vectors include plasmids encoding an antibody heavy chain and/or an antibody light chain, or plasmids encoding one or more (moe) REP proteins. A typical example of a cDNA plasmid is shown in FIG. 6A, wherein the transgene encoding the heavy chain (HC) of aducanumab and the transgene encoding the light chain (LC) of aducanumab can be replaced by nucleic acid sequences with the heavy chain and/or light chain of the antibody or fusion protein of interest, for example, see. Tables 1-5.

[00340] In one aspect, the polynucleotide encodes an AAV Rep protein (Rep 78 or 68) delivered to the producing cell on a plasmid (Rep-plasmid), bacmid (Rep-bacmid), or baculovirus (Rep-baculovirus). Rep plasmid, Rep bacmid and Rep baculovirus can be produced by the methods described above.

[00341] Methods for producing a cccDNA vector for the production of antibodies or fusion proteins are described herein. The expression constructs used to obtain a cccDNA vector for producing an antibody or fusion protein as described herein may be a plasmid (eg, cccDNA plasmids), a bacmid (eg, ccDNA-bacmid), and/or a baculovirus (eg, cccDNA -baculovirus). By way of example only, a cDNA vector can be prepared from cells coinfected with a cDNA baculovirus and a Rep baculovirus. Rep proteins produced from Rep baculovirus can replicate cccDNA baculovirus to produce cccDNA vectors. Alternatively, cccDNA vectors for the production of antibodies or fusion proteins can be obtained from cells stably transfected with a construct containing the sequence encoding the AAV Rep protein (Rep78/52) delivered in Rep plasmids, Rep bacmids or Rep baculovirus. The cccDNA baculovirus can be transiently transfected into cells, replicated by the Rep protein, and produce cccDNA vectors.

[00342] A bacmid (e.g., cccDNA bacmid) can be transfected into permissive insect cells such as Sf9, Sf21 cells, Tni (Trichoplusia ni) cells, High Five cells, and generate a cccDNA baculovirus, which is a recombinant baculovirus comprising sequences containing symmetrical ITRs and an expression cassette. Insect cells can be reinfected with cDNA baculovirus to produce the next generation of recombinant baculovirus. Optionally, this step can be repeated one or more times to obtain a larger quantity of recombinant baculovirus.

[00343] The timing of collection and isolation from cells of cccDNA vectors for the production of antibodies or fusion proteins, as described herein, can be selected and optimized to provide cccDNA vectors with high yield. For example, the collection time may be selected based on cell viability, cell morphology, cell growth, etc. Typically, cells can be harvested a period of time after baculovirus infection sufficient to allow production of cDNA vectors (e.g., cDNA vectors) but before most cells begin to die due to viral toxicity. cccDNA vectors can be isolated from Sf9 cells using plasmid purification kits such as the Qiagen ENDO-FREE PLASMID® kits. Other methods developed for plasmid isolation can also be adapted for cDNA vectors. Typically, any nucleic acid purification methods known in the art, as well as commercially available DNA extraction kits, can be used.

[00344] Alternatively, purification can be accomplished by performing an alkaline cell pellet lysis process, centrifuging the resulting lysate, and performing chromatographic separation. As one non-limiting example, the process can be carried out by loading the supernatant onto an ion exchange column (eg, SARTOBIND Q®) that retains nucleic acids, and then eluting (eg, 1.2 M NaCl) and further chromatographic purification on a column to gel filtration (for example, 6 Fast Flow GE). The capsidless AAV vector is then recovered, for example by sedimentation.

[00345] In some embodiments, cccDNA vectors for producing antibodies or fusion proteins can also be purified as exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins, but also complex protein/nucleic acid cargoes through shedding of membrane microvesicles (Cocucci et al., 2009; EP 10306226.1). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles) containing proteins and RNA as cargo. Microvesicles are formed by direct budding of the plasma membrane, and exosomes are released into the extracellular environment by fusion of multivesicular endosomes with the plasma membrane. Thus, microvesicles and/or exosomes containing the cccDNA vector can be isolated from cells transduced with the ccDNA plasmid or a bacmid or baculovirus generated with the ccDNA plasmid.

[00346] Microvesicles can be isolated by filtering or ultracentrifuging the culture medium at 20,000 x g, and exosomes at 100,000 x g. The optimal duration of ultracentrifugation can be determined experimentally and will depend on the specific cell type secreting the vesicles. Preferably, the culture medium is first clarified by low-speed centrifugation (eg at 2000 x g for 5-20 minutes) and subjected to centrifugal concentration using, for example, an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified by FACS (Fluorescence Activated Cell Sorting) or MACS (Magnetic Cell Sorting System) methods using specific antibodies that recognize specific surface antigens present on microvesicles and exosomes. Other methods for purifying microvesicles and exosomes include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. After purification, the vesicles are washed, for example, with phosphate buffered saline. One advantage of using microvesicles or exosomes to deliver cccDNA-containing vesicles is that such vesicles can be targeted to different cell types by incorporating proteins into their membranes that are recognized by specific receptors on the relevant cell types. (See also EP 10306226).

[00347] Another aspect of the invention disclosed herein relates to methods for purifying cccDNA vectors from host cell lines that have the cccDNA construct stably integrated into their genome. In one embodiment, the cDNA vectors are purified as DNA molecules. In another embodiment, cccDNA vectors are purified into exosomes or microparticles.

[00348] FIG. 5 of international application PCT/US18/49996 shows a gel confirming the production of cccDNA from a variety of ccDNA plasmid constructs using the method described in the examples. cccDNA is confirmed by a characteristic banding pattern in the gel, as described with reference to FIG. 4D in examples.

VII. Pharmaceutical compositions

[00349] In another aspect, pharmaceutical compositions are provided. The pharmaceutical composition contains a cccDNA vector for the production of antibodies or fusion proteins, as described herein, and a pharmaceutically acceptable carrier or diluent.

[00350] cccDNA vectors for the production of antibodies or fusion proteins, as described herein, can be included in pharmaceutical compositions suitable for administration to a subject for in vivo delivery to cells, tissues or organs of the subject. Typically, the pharmaceutical composition contains a cccDNA vector as described herein and a pharmaceutically acceptable carrier. For example, cDNA vectors for the production of antibodies or fusion proteins, as described herein, can be included in a pharmaceutical composition suitable for the desired route of therapeutic administration (eg, parenteral administration). Passive tissue transduction through high-pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes may be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable to provide a high concentration of cccDNA vector. Sterile injection solutions containing the cccDNA vector can be prepared by incorporating the cccDNA vector formulation in the required amount into an appropriate buffer with one or a combination of the ingredients listed above, if necessary, followed by filter sterilization, to deliver the transgene as nucleic acid to recipient cells, leading to therapeutic expression of the transgene or donor sequence in it. The composition may also include a pharmaceutically acceptable carrier.

[00351] Pharmaceutically active compositions containing a cccDNA vector for the production of antibodies or fusion proteins can be formulated to deliver a transgene for various purposes into a cell, for example, into the cells of a subject.

[00352] Pharmaceutical compositions for therapeutic purposes generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure suitable for providing a high concentration of cccDNA vector. Sterile injectable solutions can be prepared by incorporating the cccDNA vector formulation in the required amount into an appropriate buffer with one or a combination of the ingredients listed above, if necessary, followed by filter sterilization.

[00353] An cccDNA vector for the production of antibodies or fusion proteins as described herein may be included in a pharmaceutical composition suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, interstitial (eg, intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (eg, extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, intracameral, and intravitreal), intracochlear, and mucosal (eg , oral, rectal, nasal). Passive tissue transduction through high-pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated.

[00354] In some aspects, the methods provided herein include delivering one or more cccDNA vectors for producing an antibody or fusion protein as described herein into a host cell. Also provided herein are cells produced by such methods and organisms (such as animals, plants or fungi) containing or derived from such cells. Methods for delivering nucleic acids may include lipofection, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, and agent-enhanced DNA uptake. Lipofection is described, for example, in US patent No. 5049386, 4946787; and 4897355), and lipofection reagents are sold commercially (eg, Transfectam™ and Lipofectin™). Delivery can be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration).

[00355] Various methods and methods for delivering nucleic acids into cells are known in the art. For example, nucleic acids, such as cccDNA for producing antibodies or fusion proteins, can be formulated as lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core-shell nanoparticles. Typically, LNPs consist of a nucleic acid molecule (e.g., cccDNA), one or more ionizable or cationic lipids (or salts thereof), one or more nonionic or neutral lipids (e.g., a phospholipid), a molecule that prevents aggregation (e.g., PEG or a PEG-lipid conjugate) and optionally a sterol (eg cholesterol).

[00356] Another method of delivering nucleic acids, such as cccDNA, into a cell to produce antibodies or a fusion protein is by conjugating the nucleic acid to a ligand that is internalized by the cell. For example, a ligand may bind to a cell surface receptor and be internalized through endocytosis. A ligand may be covalently linked to a nucleotide in a nucleic acid. Typical examples of conjugates for delivering nucleic acids into cells are described, for example, in WO2015/006740, WO2014/025805, WO2012/037254, WO2009/082606, WO2009/073809, WO2009/018332, WO2006/11287 2, WO2004/090108, WO2004/091515 and WO2017/177326.

[00357] Nucleic acids, such as cDNA vectors for the production of antibodies or fusion proteins, can also be delivered into cells by transfection. Suitable transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include, but are not limited to, TurboFect Transfection Reagent (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASS™ P Protein Transfection Reagent (New England Biolabs), Protein Reagent CHARIOT™ (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMD Millipore), 293-Fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific), LIPOFECTAMINE ™, Thermo Fisher Scientific), lipofectin (LIPOFECTIN™, Thermo Fisher Scientific), DMRIE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific), LIPOFECTACE™, FUGENE™ (Roche, Basel, Switzerland), FUGENE™ HD ( Roche), TRANSFECTAM™ (Transfectam, Promega, Madison, WI), TFX-10™ (Promega), TFX-20™ (Promega), TFX-50™ (Promega), Transfectin (TRANSFECTIN™, BioRad, Hercules, CA) , SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen, Valencia, CA), DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems, San Diego, CA), DHARMAFECT 1™ (Dharmacon, Lafayette, Colorado), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, MO), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids, such as cctDNA, can also be delivered into cells using microfluidics techniques known to those skilled in the art.

[00358] cccDNA vectors for producing antibodies or fusion proteins, as described herein, can also be administered directly into the body to transduce cells in vivo . Administration is by any route commonly used to bring a molecule into direct contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art, and although more than one route may be used to administer a particular composition, a particular route can often provide a faster and more efficient reaction than another route.

[00359] Methods of administering a cccDNA vector-type nucleic acid vector to produce an antibody or fusion protein as described herein may include delivery to hematopoietic stem cells, for example, by methods described, for example, in US Pat. No. 5,928,638.

[00360] cccDNA vectors for producing antibodies or fusion proteins in accordance with this invention can be added to liposomes for delivery to a cell or target organ in a subject. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are typically used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They act by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds having a phosphatidylcholine group, however, these compositions may also include other lipids. Exemplary liposomes and liposome formulations, including, but not limited to, compounds containing polyethylene glycol (PEG) functional groups, are disclosed in International Application PCT/US2018/050042, filed September 7, 2018, and International Application PCT/US2018/064242, filed 6 December 2018, for example, see the section entitled “Pharmaceutical Compositions.”

[00361] Various delivery methods known in the art, or modifications thereof, can be used to deliver cccDNA vectors in vitro or in vivo. For example, in some embodiments, cccDNA vectors for producing antibodies or fusion proteins are delivered by transiently penetrating the cell membrane using mechanical, electrical, ultrasonic, hydrodynamic, or laser energy so as to facilitate entry of DNA into target cells. For example, the cccDNA vector can be delivered by transiently disrupting the cell membrane by squeezing the cell through a size-limited channel, or other methods known in the art. In some cases, a single cccDNA vector is administered by direct injection of naked DNA into the skin, thymus, cardiac muscle, skeletal muscle or liver cells. In some cases, the cccDNA vector is delivered using a gene gun. Spherical gold or tungsten particles (1–3 μm in diameter) coated with capsidless AAV vectors can be accelerated to high speed using compressed gas to penetrate target tissue cells.

[00362] Compositions containing a cDNA vector for producing antibodies or fusion proteins and a pharmaceutically acceptable carrier are specifically provided for herein. In some embodiments, the cDNA vector consists of a lipid delivery system, such as liposomes, as described herein. In some embodiments, such compositions are administered by any route desired by a skilled practitioner. The compositions may be administered to a subject by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or their combinations. For veterinary use, the composition may be administered in a reasonably acceptable composition in accordance with normal veterinary practice. The veterinarian can easily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The compositions can be administered using traditional syringes, needleless injection devices, microparticle bombardment gene guns, or other physical methods such as electroporation (“EP”), hydrodynamic methods, or the use of ultrasound.

[00363] In some cases, the cccDNA vector for producing antibodies or fusion proteins is delivered by hydrodynamic injection, which is a simple and highly effective method for direct intracellular delivery of any water-soluble compounds and particles to internal organs and skeletal muscle throughout the limb.

[00364] In some cases, cccDNA vectors for the production of antibodies or fusion proteins are delivered using ultrasound by creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles into internal organ or tumor cells, so the size and concentration of plasmid DNA is of great importance for the effectiveness of the system . In some cases, cccDNA vectors are delivered by magnetofection, using magnetic fields to concentrate particles containing the nucleic acid into target cells.

[00365] In some cases, chemical delivery systems can be used, for example, using nanometer complexes, which involve compaction of negatively charged nucleic acid with polycationic nanometer particles belonging to cationic liposomes/micelles, or cationic polymers. Cationic lipids used for the delivery method include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol-derived compounds, cationic polymers (e.g., poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers ) and a lipid-polymer hybrid.

A. Exosomes:

[00366] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, is delivered packaged in an exosome. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after 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 cytosol from the exosome-producing cell, and they display membrane proteins from the parent cell on the surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). Some embodiments include the use of exosomes with a diameter of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, 50 nm to 100 nm. Exosomes can be isolated for delivery to target cells either using their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing the capsidless AAV vectors of this invention.

B. Microparticles/nanoparticles:

[00367] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, is delivered via a lipid nanoparticle. Typically, lipid nanoparticles contain an ionizable aminolipid (e.g., heptatriakonta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3 β-phosphocholine, DSPC), cholesterol and envelope lipid (polyethylene glycol-dimyristol glycerol, PEG-DMG), for example, as described by Tam et al. (2013), Advances in Lipid Nanoparticles for siRNA delivery . Pharmaceuticals 5(3): 498-507.

[00368] In some embodiments, the lipid nanoparticle has an average diameter of from about 10 nm to about 1000 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 300 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 10 nm to about 300 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 200 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 25 nm to about 200 nm. In some embodiments, the lipid nanoparticle formulation (e.g., a composition containing a plurality of lipid nanoparticles) has a size distribution such that the average size (e.g., diameter) is from about 70 nm to about 200 nm, and more typically the average size is about 100 nm or less.

[00369] Various lipid nanoparticles known in the art can be used to deliver a cccDNA vector for the production of an antibody or fusion protein, as described herein. For example, various delivery methods using lipid nanoparticles are described in US Patent Nos. 9404127, 9006417 and 9518272.

[00370] In some embodiments, the cDNA vector for producing antibodies or fusion proteins, as described herein, is delivered using gold nanoparticles. Typically, the nucleic acid may be covalently associated with the gold nanoparticle or non-covalently associated with the gold nanoparticle (eg, associated through charge-charge interaction), for example, as described by Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery . Mol. Ther. 22(6); 1075-1083. In some embodiments, gold nanoparticle nucleic acid conjugates are prepared using methods described, for example, in US Pat. No. 6,812,334.

C. Conjugates

[00371] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins as described herein is conjugated (eg, covalently linked) to an agent that enhances cellular uptake. A "cellular uptake enhancing agent" is a molecule that facilitates the transport of nucleic acid across a lipid membrane. For example, the nucleic acid may be conjugated to a lipophilic compound (e.g. cholesterol, tocopherol, etc.), cell penetrating peptide (CPP) (e.g. penetratin, TAT, Syn1B, etc.) and polyamines (e.g. spermine ). Additional examples of agents that increase cellular uptake are disclosed, for example, in Winkler (2013), Oligonucleotide conjugates for therapeutic applications . Ther. Deliv. 4(7); 791-809.

[00372] In some embodiments, a cccDNA vector for producing an antibody or fusion protein as described herein is conjugated to a polymer (eg, a polymer molecule) or a folate molecule (eg, a folic acid molecule). In general, delivery of nucleic acids conjugated to polymers is known in the art, for example, as described in WO2000/34343 and WO2008/022309. In some embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, is conjugated to a poly(amide) polymer, for example, as described in US Pat. No. 8,987,377. In some embodiments, the nucleic acid described herein is conjugated to a folic acid molecule as described in US Pat. No. 8,507,455.

[00373] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins as described herein is conjugated to a carbohydrate, for example, as described in US Pat. No. 8,450,467.

D. Nanocapsule

[00374] Alternatively, cDNA vector nanocapsule compositions can be used to produce antibodies or fusion proteins as described herein. Nanocapsules can typically entrap substances in a stable and reproducible manner. To avoid side effects caused by excessive intracellular loading of polymers, such ultrafine particles (about 0.1 μm in size) must be developed using polymers that can be degraded in vivo . The use of biodegradable polyalkyl cyanoacrylate nanoparticles that meet such requirements is envisaged.

E. Liposomes

[00375] cccDNA vectors for producing antibodies or fusion proteins in accordance with this invention can be added to liposomes for delivery to a cell or target organ in a subject. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are typically used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They act by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds having a phosphatidylcholine group, however, these compositions may also include other lipids.

[00376] The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulation half-life have been developed (US Patent No. 5,741,516). In addition, various methods for preparing liposomes and liposome-like preparations as potential drug carriers have been described (US Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

F. Typical compositions of liposomes and lipid nanoparticles (LNPs)

[00377] cccDNA vectors for producing antibodies or fusion proteins in accordance with this invention can be added to liposomes for delivery into a cell, for example, into a cell in need of transgene expression. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are typically used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They act by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds having a phosphatidylcholine group, however, these compositions may also include other lipids.

[00378] Lipid nanoparticles (LNPs) containing cccDNA vectors are disclosed in International Application PCT/US2018/050042, filed September 7, 2018, and International Application PCT/US2018/064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety and is intended for use in the methods and compositions disclosed herein.

[00379] In some aspects, the present invention provides a liposome composition that includes one or more polyethylene glycol (PEG) functional compounds (so-called “PEGylated compounds”) that can reduce immunogenicity/antigenicity, provide hydrophilicity and hydrophobicity to the compound(s), and reduce dosing frequency. In other cases, the liposomal composition simply contains a polyethylene glycol (PEG) polymer as an additional component. In such aspects, the molecular weight of the PEG or PEG functionality may range from 62 Da to about 5000 Da.

[00380] In some aspects, the present invention provides a liposome composition that delivers an API with a sustained or controlled release profile over a period of time ranging from hours to weeks. In some related aspects, the liposome composition may comprise aqueous chambers defined by lipid bilayers. In other related aspects, the liposomal composition encapsulates the API with components that undergo a physical transition at elevated temperature to release the API over a period of hours to weeks.

[00381] In some aspects, the liposome composition contains sphingomyelin and one or more lipids disclosed herein. In some aspects, the liposome composition contains optisomes.

[00382] In some aspects, the present invention provides a liposomal composition that includes one or more lipids selected from: N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn- glycerophosphoethanolamine), MPEG (methoxypolyethylene glycol)-conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycerophosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleoylphosphatidylserine); POPC (palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxy polyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); DOPE (dioleoyl-sn-glycerophosphoethanolamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycerophosphatidylcholine) or any combination thereof.

[00383] In some aspects, the present invention provides a liposomal composition comprising a phospholipid, cholesterol, and PEGylated lipid in a molar ratio of 56:38:5. In some aspects, the total lipid content of the liposome composition is from 2 to 16 mg/ml. In some aspects, the present invention provides a liposomal composition comprising a phosphatidylcholine-functional lipid, an ethanolamine-functional lipid, and a PEGylated lipid. In some aspects, the present invention provides a liposomal composition comprising a phosphatidylcholine-functional lipid, an ethanolamine-functional lipid, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. In some aspects, the present invention provides a liposomal composition comprising a lipid containing a phosphatidylcholine functional group, cholesterol, and a pegylated lipid. In some aspects, the present invention provides a liposomal composition comprising a phosphatidylcholine functional group-containing lipid and cholesterol. In some aspects, the PEGylated lipid is PEG-2000-DSPE. In some aspects, the present invention provides a liposomal composition containing DPPG, soy phosphatidylcholine (PC), MPEG-DSPE lipid conjugate and cholesterol.

[00384] In some aspects, the present invention provides a liposomal composition comprising one or more phosphatidylcholine-functional lipids and one or more ethanolamine-functional lipids. In some aspects, the present invention provides a liposomal composition comprising one or more of: phosphatidylcholine functional lipids, ethanolamine functional lipids, and sterols, such as cholesterol. In some aspects, the liposomal composition contains DOPC/DEPC; and DOPE.

[00385] In some aspects, the present invention provides a liposomal composition further comprising one or more pharmaceutical excipients, such as sucrose and/or glycine.

[00386] In some aspects, the present invention provides a liposomal composition that is either single-layer or multi-layer in structure. In some aspects, the present invention provides a liposomal composition that includes multivesicular particles and/or foam particles. In some aspects, the present invention provides a composition of liposomes that are larger in size than conventional nanoparticles, ranging from about 150 to 250 nm. In some aspects, the liposomal composition is a lyophilized powder.

[00387] In some aspects, the present invention provides a liposomal composition that is prepared and loaded with cccDNA vectors disclosed or described herein by adding a weak base to a mixture containing isolated cccDNA outside a liposome. This addition raises the pH outside the liposomes to approximately 7.3 and directs the API into the liposome. In some aspects, the present invention provides a liposomal composition having an acidic pH within the liposomes. In such cases, the pH value within the liposome may be 4-6.9, more preferably 6.5. In other aspects, the present invention provides a liposomal composition prepared using intraliposomal drug stabilization technology. In such cases, polymeric or non-polymeric highly charged anions and intraliposomal capture agents, such as polyphosphate or sucrose octasulfate, are used.

[00388] In some aspects, the present invention provides a lipid nanoparticle comprising cccDNA and an ionizable lipid. For example, a lipid nanoparticle composition that is prepared and loaded with cccDNA obtained by the method disclosed in international application PCT/US2018/050042, filed September 7, 2018, which is incorporated herein. This can be achieved by high-energy mixing of ethanol solutions of lipids with aqueous cccDNA at low pH values that protonate the ionizable lipid and provide favorable energetics for ccDNA/lipid association and particle nucleation. The particles can be further stabilized by diluting with water and removing the organic solvent. Particles can be concentrated to the desired level.

[00389] Typically, lipid particles are prepared at a ratio of total lipid to cccDNA (w/w) of about 10:1 to 30:1. In some embodiments, the lipid to cccDNA ratio (mass ratio; w/w ratio) may range from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3: 1 to 15:1, 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 9:1. The amount of lipids and cccDNA can be adjusted to provide a desired N/P ratio, for example, an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Typically, the total lipid content of the lipid particles may range from about 5 to about 30 mg/ml.

[00390] An ionizable lipid is typically used to condense a nucleic acid cargo, such as cccDNA, at low pH and to promote membrane association and fusogenicity. Typically, ionizable lipids are lipids containing at least one amino group that is positively charged or protonated under acidic conditions, such as pH 6.5 or lower. Ionizable lipids are also referred to herein as cationic lipids.

[00391] Typical examples of ionizable lipids are described in PCT international patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/0001 04, WO2015/074085, WO2016/081029, WO2017 /004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO 2011/090965, WO2013/016058, WO2012/162210, WO2008/042973 , WO2010/129709, WO2010/144740, WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/0 48536, WO2010/088537, WO2010/054401, WO2010/054406, WO2010 /054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO 2013/126803, WO2006/007712, WO2011/038160, WO2005/121348 , WO2011/066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/0 99823, WO2015/095346 and WO2013/086354, and US Patent Publications US2016 /0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US 2016/0376224, US2017/0119904, US2012/0149894, US2015/0057373 , US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/02 03446, US2018/0005363, US2014/0308304, US2013/0338210, US2012 /0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US 2013/0123338, US2015/0064242, US2006/0051405, US2013/0065939 , US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/00 39032, US2018/0028664, US2016/0317458 and US2013/0195920, contents which are incorporated herein by reference in their entirety.

[00392] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) having the following structure:

.

[00393] The DLin-MC3-DMA lipid is described in Jayaraman et al. Angew. Chem. Int. Ed Engl. (2012), 51(34): 8529-8533, the contents of which are incorporated herein by reference in their entirety.

[00394] In some embodiments, the ionizable lipid is lipid ATX-002, as described in WO 2015/074085, the contents of which are incorporated herein by reference in their entirety.

[00395] 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 incorporated herein by reference in its entirety.

[00396] In some embodiments, the ionizable lipid is compound 6 or compound 22, as described in WO 2015/1992, the contents of which are incorporated herein by reference in their entirety.

[00397] Without limitation, the ionizable lipid may comprise 20-90 mol% of the total lipid present in the lipid nanoparticle. For example, the molar content of the ionizable lipid may be 20-70 mol%, 30-60 mol%, or 40-50 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the ionizable lipid is from about 50 mol%. up to approximately 90 mol.% of the total amount of lipid present in the lipid nanoparticle.

[00398] In some aspects, the lipid nanoparticle may further comprise a non-cationic lipid. Non-cationic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non-cationic lipid may be a neutral, uncharged, zwitterionic or anionic lipid. Non-cationic lipids are commonly used to enhance fusogenicity.

[00399] Representative examples of non-cationic lipids for use in the methods and compositions disclosed herein are described in international application PCT/US2018/050042, filed September 7, 2018, and PCT/US2018/064242, filed December 6, 2018 ., which are fully included in this document. Exemplary non-cationic lipids are described in International Application Publication WO2017/099823 and US Patent Publication US2018/0028664, the contents of which are incorporated herein by reference in their entirety.

[00400] The non-cationic lipid may comprise 0-30 mol% of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 5-20 mol% or 10-15 mol% of the total lipid present in the lipid nanoparticle. In various embodiments, the molar ratio of ionizable lipid to neutral lipid is from about 2:1 to about 8:1.

[00401] In some embodiments, the lipid nanoparticles do not contain any phospholipids. In some aspects, the lipid nanoparticle may further contain a component, such as a sterol, to provide membrane integrity.

[00402] One example of a sterol that can be used in a lipid nanoparticle is cholesterol and its derivatives. Exemplary cholesterol derivatives are described in International Application WO2009/127060 and US Patent Publication US2010/0130588, the contents of which are incorporated herein by reference in their entirety.

[00403] The membrane integrity component, such as a sterol, may comprise 0-50 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, such component constitutes 20-50 mol% and 30-40 mol% of the total lipid content of the lipid nanoparticle.

[00404] In some aspects, the lipid nanoparticle may further comprise polyethylene glycol (PEG) or a conjugated lipid molecule. They are typically used to inhibit lipid nanoparticle aggregation and/or provide steric stabilization. Typical conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer-lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as (methoxypolyethylene glycol)-conjugated lipids. Typical PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O)-(w-methoxy (polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG-dialkoxypropylcarbam, N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional examples of PEG-lipid conjugates are described, for example, in US5885613, US6287591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2 016/0376224 and US2017/0119904, the contents of which are included incorporated into this description by reference in its entirety.

[00405] In some embodiments, the PEG lipid is the compound disclosed in US2018/0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG lipid is disclosed in US20150376115 or US2016/0376224, the contents of which are incorporated herein by reference in their entirety.

[00406] The PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmythyloxypropyl, or PEG-distearyloxypropyl. The PEG lipid may be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1 -[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methylpoly(ethylene glycol), PEG-DMB (3,4-ether ditetradecoxybenzyl-[omega]-methylpoly(ethylene glycol)) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG lipid may be selected from the group consisting of from PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[00407] Instead of a PEG lipid, lipids conjugated to a molecule other than PEG can also be used. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer-lipid (CPL) conjugates can be used instead of or in addition to PEG-lipid. Typical examples of conjugated lipids, i.e. PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and (cationic polymer)-lipids are described in international patent application publications WO1996/010392, WO1998/051278, WO2002/087541, WO2005/026372, WO2008/147438, WO2 009 /086558, WO2012/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO2017/004143, WO2015/095346, WO2012/000104, WO2012/000104 and WO 2010/006282, US patent application publications US2003/0077829, US2005/0175682 , US2008/0020058, US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115, US2016/0376224, US2016/0317458, US2013/0303587, US2013/03 03587 and US20110123453, and US patents US5885613, US6287591, US6320017 and US6586559, the contents of which are incorporated herein in their entirety by reference.

[00408] In some embodiments, one or more additional compounds may be therapeutic agents. The therapeutic agent may be selected from any class suitable to achieve the therapeutic goal. In other words, the therapeutic agent may be selected from any class suitable to achieve the therapeutic goal. In other words, the therapeutic agent can be selected according to the purpose of treatment and the desired biological effect. For example, if the cccDNA in the LNP is for the treatment of cancer, the additional compound may be an anticancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, antibody, or antibody-drug conjugate). In another example, if The cccDNA-containing LNPs are intended to treat an infection, the additional compound may be an antimicrobial agent (eg, an antibiotic or an antiviral compound).In yet another example, if the cccDNA-containing LNPs are intended to treat an immune disease or disorder, the additional compound may be a compound that modulates an immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways).In some embodiments, the compositions and methods of the invention may use various cocktails of different lipid nanoparticles containing different compounds, such as cccDNA encoding another protein, or another compound, such as a therapeutic agent.

[00409] In some embodiments, the additional compound is an immunomodulator. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is an immunostimulating agent. Also provided herein is a pharmaceutical composition comprising, encapsulated in a lipid nanoparticle, an insect cell-produced or synthetically produced cccDNA vector or for producing an antibody or fusion protein as described herein, and a pharmaceutically acceptable carrier or excipient.

[00410] In some aspects, the present invention provides a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle composition further comprises sucrose, tris, trehalose, and/or glycine.

[00411] The cccDNA vector can form complexes with a lipid portion (portion) of a particle or be encapsulated in a lipid portion (position) of a lipid nanoparticle. In some embodiments, the cccDNA may be completely encapsulated in the lipid portion of the lipid nanoparticle, thereby protecting it from degradation by a nuclease, for example, in an aqueous solution. In some embodiments, the cccDNA in the lipid nanoparticle is not significantly degraded after the lipid nanoparticle is exposed to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments, the cccDNA in the lipid nanoparticle is not significantly degraded after the particle is incubated 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.

[00412] In certain embodiments, the lipid nanoparticles are substantially non-toxic to a subject, such as a mammal, such as a human. In some aspects, the lipid nanoparticle composition is a lyophilized powder.

[00413] 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. non-lamellar (i.e. non-bilayer) morphology. Without limitation, non-bilayer morphologies may include, for example, three-dimensional tubes, rods, cubic particles, etc. For example, the morphology of lipid nanoparticles (lamellar and non-lamellar) can be easily assessed and characterized using, for example, cryo-TEM (cryotransmission electron microscopy) analysis as described in US2010/0130588, the contents of which are incorporated herein by reference in full.

[00414] In some other embodiments, lipid nanoparticles having a non-lamellar morphology are electron dense. In some aspects, the present invention provides a lipid nanoparticle that is either monolamellar or multilamellar in structure. In some aspects, this invention relates to a lipid nanoparticle composition that includes multivesicular particles and/or foam-based particles.

[00415] By controlling the composition and concentration of lipid components, it is possible to control the rate of release of the lipid conjugate from the lipid particle by the metabolic mechanism and, in turn, the rate of conversion of the lipid nanoparticle into a fusogenic one. In addition, other variables, including, for example, pH, temperature or ionic strength, can be used to change and/or control the rate of conversion of a lipid nanoparticle to a fusogenic one. Other methods that can be used to control the rate of conversion of a lipid nanoparticle to a fusogenic one will be apparent to those skilled in the art based on the present invention. It will also be apparent that by controlling the composition and concentration of the lipid conjugate, the size of the lipid particles can be controlled.

[00416] The pKa value of formulated cationic lipid compositions may correlate with the efficiency of LNPs for delivering nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entirety). The preferred range of pKa values is from about 5 to about 7. The pKa value of a cationic lipid in lipid nanoparticles can be determined using an assay based on 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) fluorescence.

VIII. Methods of application

[00417] A cccDNA vector for producing antibodies or fusion proteins, as described herein, can also be used in a method of delivering a nucleotide sequence of interest (e.g., encoding an antibody or fusion protein) to a target cell (e.g., a host cell) . The method, in particular, may be a method of delivering an antibody or antigen-binding fragment to a cell of a subject in need thereof and treating a disease of interest. The invention allows for in vivo expression of an antibody or fusion protein encoded in a cDNA vector in a cell in a subject such that the therapeutic effect of expression of the antibody or fusion protein is manifested. Such results are observed both in vivo and in vitro methods of cDNA vector delivery.

[00418] The invention further provides a method of delivering an antibody or fusion protein into a cell of a subject in need thereof, comprising repeated administration of a cccDNA vector of the invention encoding said antibody or fusion protein. Since the cccDNA vector of the invention does not induce an immune response similar to that typically observed against encapsidated viral vectors, such a multiple administration strategy is likely to be more successful in a cccDNA-based system.

[00419] The cccDNA vector is administered in sufficient quantities to transfect cells of the desired tissue and to provide sufficient levels of gene transfer and antibody or fusion protein expression without undesirable side effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous (eg, liposomes), direct organ delivery (eg, intraportal liver), intramuscular, and other parental routes. If necessary, routes of administration can be combined.

[00420] Delivery of a cccDNA vector for the production of antibodies or fusion proteins, as described herein, is not limited to delivery of the expressed antibody or antigen binding fragment. For example, those produced by conventional methods (e.g., using a cell-based production method (e.g., insect cell production methods)) or synthetically produced cccDNA vectors, as described herein, can be used in conjunction with other delivery systems designed to provide part gene therapy. One non-limiting example of a system that can be combined with cccDNA vectors in accordance with the present invention includes systems that separately deliver one or more cofactors or immunosuppressants for efficient gene expression of a cccDNA vector expressing an antibody or fusion protein.

[00421] The invention also provides a method of treating a disease in a subject, comprising administering to a target cell in need thereof (particularly a muscle cell or tissue) of the subject a therapeutically effective amount of a cccDNA vector, optionally with a pharmaceutically acceptable carrier. Although the cDNA vector can be introduced in the presence of a carrier, such a carrier is not necessary. The selected cccDNA vector contains a nucleotide sequence encoding an antibody or fusion protein useful for treating a disease. In particular, the cccDNA vector may contain a desired antibody or fusion protein sequence operably linked to control elements capable, when administered to a subject, of directing transcription of the desired antibody or fusion protein encoded by the exogenous DNA sequence. The cccDNA vector can be introduced by any suitable route, as described above and elsewhere in this document.

[00422] The compositions and vectors provided herein can be used to deliver an antibody or fusion protein for a variety of purposes. In some embodiments, the transgene encodes an antibody or fusion protein that is intended to be used for research purposes, for example, to create a somatic model of a transgenic animal carrying the transgene, for example, to study the function of the antibody or fusion protein product. In another example, the transgene encodes an antibody or fusion protein that is intended to be used to create an animal model of a disease. In some embodiments, the encoded antibody or fusion protein is useful for treating or preventing a disease state in a mammalian subject. The antibody or fusion protein can be transferred to a patient (eg, expressed) in an amount sufficient to treat a disease associated with reduced expression, lack of expression, or dysfunction of a gene.

[00423] In principle, an expression cassette may include a nucleic acid or any transgene encoding an antibody or fusion protein that is either reduced or absent due to mutation, or that provides a therapeutic benefit when overexpressed, and is considered to be within the scope of the invention. Preferably, no non-inserted bacterial DNA is present, and preferably, the cccDNA compositions provided herein do not contain bacterial DNA.

[00424] The cccDNA vector is not limited to one kind of ccDNA vector. As such, in another aspect, multiple cccDNA vectors expressing different antibodies or fusion proteins, or the same antibody or fusion protein but operably linked to different promoters or cis-regulatory elements, can be delivered simultaneously or sequentially to the target cell, tissue , organ or subject. Thus, this strategy may enable gene therapy or gene delivery of multiple antibodies and/or fusion proteins simultaneously. It is also possible to house different parts of an antibody in separate cccDNA vectors (e.g., different domains and/or cofactors required for the functionality of the antibody or antigen-binding fragment), which can be administered simultaneously or at different times, and which can be regulated separately, thereby adding an additional layer controlling the expression of the antibody or fusion protein. Delivery can also be done repeatedly and, importantly for gene therapy in a clinical setting, with sequentially increasing or decreasing doses, given the lack of host immune response against the capsid due to the absence of the viral capsid. It is expected that no reaction against the capsid will occur due to the absence of the capsid.

[00425] The invention also provides a method of treating a disease in a subject, comprising administering to a target cell in need thereof (particularly a muscle cell or tissue) of the subject a therapeutically effective amount of a cccDNA vector as described herein, optionally with a pharmaceutically acceptable carrier. Although the cDNA vector can be introduced in the presence of a carrier, such a carrier is not necessary. The cDNA vector provided in this invention contains a nucleotide sequence of interest useful for treating a disease. In particular, the cccDNA vector may contain a desired exogenous DNA sequence operably linked to control elements capable, when administered to a subject, of directing transcription of the desired polypeptide, protein or oligonucleotide encoded by the exogenous DNA sequence. The cccDNA vector can be introduced by any suitable route, as described above and elsewhere in this document.

IX. Methods for delivering cDNA vectors for the production of antibodies or fusion proteins

[00426] In some embodiments, a cccDNA vector for producing an antibody or fusion protein can be delivered to a target cell in vitro or in vivo by various suitable methods. Separate cccDNA vectors may be used or administered by injection. cccDNA vectors can be delivered into cells without the aid of a transfection reagent or other physical means. Alternatively, cccDNA vectors for the production of antibodies or fusion proteins can be delivered using any transfection reagent known in the art or other physical means known in the art that facilitate entry of DNA into the cell, such as liposomes, alcohols, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, microinjections, electroporation, etc.

[00427] cccDNA vectors for the production of antibodies or fusion proteins, as described herein, can effectively target cell and tissue types that are typically difficult to transduce with conventional AAV virions using various delivery reagents.

[00428] One aspect of the technology described herein relates to a method of delivering an antibody or antigen binding fragment into a cell. In general, for in vivo and in vitro methods, a cccDNA vector for producing antibodies or fusion proteins as described herein can be introduced into a cell using the methods disclosed herein as well as other methods known in the art. . The cDNA vector for producing antibodies or fusion proteins as described herein is preferably introduced into the cell in a biologically effective amount. If a cccDNA vector is introduced into a cell in vivo (eg, a subject), then a biologically effective amount of cccDNA vector is sufficient to result in transduction and expression of an antibody or antigen binding fragment in the target cell.

[00429] Typical examples of methods of administering a cccDNA vector to produce an antibody or fusion protein as described herein include oral, rectal, transmucosal, intranasal, inhalation (e.g., aerosol), buccal (e.g., sublingual), vaginal, intrathecal , intraocular, percutaneous, intraendothelial, intrauterine (in utero) (or in ovo (“into the egg”)), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including skeletal, diaphragmatic and/or cardiac muscle], intrapleural, intracerebral and intra-articular), local (for example, on the surface of the skin and mucous membranes, including the surface of the respiratory tract, and percutaneous administration), intralymphatic administration, etc., as well as direct injection into a tissue or organ (eg, but not limited to, liver, eye, muscle, including skeletal muscle, cardiac muscle, diaphragmatic muscle, or brain).

[00430] Administration of the cccDNA vector can be performed at any site in a subject, including, without limitation, a site selected from the group consisting of brain, skeletal muscle, smooth muscle, heart, diaphragm, respiratory epithelium, liver, kidney, spleen , pancreas, skin and eyes. Introduction of the cccDNA vector can also be done into a tumor (eg, into or near a tumor or lymph node).

[00431] The most appropriate route in any particular case will depend on the nature and severity of the condition being treated, ameliorated and/or prevented, and the nature of the particular cccDNA vector used. In addition, cccDNA allows the introduction of more than one antibody in one vector or multiple cccDNA vectors (eg, cccDNA cocktail).

A. Intramuscular injection of cDNA vector

[00432] In some embodiments, a method of treating a disease in a subject includes administering to a target cell in need thereof (such as a muscle cell or tissue) of the subject a therapeutically effective amount of a cccDNA vector encoding an antibody or antigen-binding fragment thereof, optionally with a pharmaceutically acceptable carrier. In some embodiments, the cDNA vector for producing antibodies or fusion proteins is introduced into muscle tissue of a subject.

[00433] In some embodiments, administration of the cccDNA vector may be administered to any site in a subject, including, but not limited to, a site selected from the group consisting of skeletal muscle, smooth muscle, heart, diaphragm, or ocular muscle. In some embodiments, the cDNA vector of the present invention is administered to skeletal muscle, diaphragm muscle, and/or cardiac muscle (e.g., to treat, alleviate, and/or prevent muscular dystrophy or heart disease (e.g., peripheral arterial disease (PAD) or congestive heart failure).

[00434] Administration of a cccDNA vector for producing antibodies or a fusion protein as described herein into skeletal muscle in accordance with the present invention includes, but is not limited to, administration into skeletal muscle of the extremities (e.g., shoulder, forearm, thigh, and/or lower leg), back, neck, head (eg tongue), chest, abdomen, pelvis/perineum and/or fingers. The cccDNA vector, as described herein, can be delivered to skeletal muscle by intravenous injection, intra-arterial injection, intraperitoneal injection, limb perfusion (optionally, isolated limb perfusion - leg and/or arm; see, for example, Arruda et al. , (2005) Blood 105: 3458-3464) and/or direct intramuscular injection. In specific embodiments, the cccDNA vector described herein is introduced into a limb (arm and/or leg) of a subject (e.g., a subject with a muscular dystrophy, such as Duchenne muscular dystrophy (DMD)) by perfusing the limb, optionally perfusing an isolated limb (for example, by intravenous or intra-articular administration). In embodiments, the cccDNA vector, as described herein, can be introduced without the use of “hydrodynamic” methods. For example, delivery to tissue (eg, muscle) of conventional viral vectors is often enhanced by hydrodynamic techniques (eg, high-volume intravenous/intravenous administration), which increase pressure in the vasculature and enhance the ability of the viral vector to cross the endothelial cell barrier. In specific embodiments, the cccDNA vectors described herein can be administered in the absence of hydrodynamic techniques, such as large volume infusions and/or increased intravascular pressure (e.g., above normal systolic pressure, e.g., increased intravascular pressure relative to normal systolic pressure less than 5%, 10%, 15%, 20%, 25%, or an amount equal to the specified value). Such techniques may reduce or avoid side effects associated with hydrodynamic techniques, such as edema, nerve damage, and/or compartment syndrome.

[00435] Additionally, a composition containing a cccDNA vector for producing antibodies or fusion proteins as described herein that is administered to skeletal muscle may be administered to skeletal muscle of the extremities (e.g., shoulder, forearm, thigh, and/or lower leg), back, neck, head (eg tongue), chest, abdomen, pelvis/perineum and/or fingers. Suitable skeletal muscles include, but are not limited to, abductor digiti minimi (hand), abductor pollicis (foot), abductor pollicis, abductor pollicis 5, abductor pollicis brevis, abductor pollicis longus , adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, articular knee, biceps brachii, biceps femoris, brachialis, brachioradialis muscle, buccal muscle, coracobrachialis muscle, corrugator brow muscle, deltoid muscle, depressor anguli oris muscle, depressor labii inferioris muscle, digastric muscle, dorsal interosseous muscles (hands), dorsal interosseous muscles (feet), extensor radialis brevis wrist, extensor carpi radialis longus, extensor carpi ulnaris, extensor of the little finger, extensor digitorum, extensor digitorum brevis, extensor digitorum longus, extensor hallucis brevis, extensor hallucis longus, extensor index finger, extensor pollicis brevis, long extensor pollicis, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum brevis, flexor digitorum brevis, flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis foot, flexor hallucis longus, flexor hallucis brevis, flexor hallucis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis neck, iliocostalis lumborum, iliocostalis pectoralis, iliacus, inferior gemellus, inferior oblique, inferior rectus, infraspinatus, interspinous muscles, intertransverse muscles, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superioris, levator labii superioris and alae nasi, levator superior pallidum, levator scapulae, long rotator cuff, longissimus capitis, longissimus colli, longissimus thoracis, longus capitis muscle, longus colli muscle, lumbrical muscles (hands), lumbrical muscles (feet), masseter muscle, medial pterygoid muscle, medial rectus muscle, middle scalenus muscle, multifidus muscles, mylohyoid muscle, inferior oblique muscle capitis, superior oblique muscle capitis, obturator externus, obturator internus, occipital muscle, omohyoid muscle, opponus little finger muscle, opponus pollicis muscle, orbicularis oculi muscle, orbicularis oris muscle, palmar interosseous muscle, palmaris brevis, palmaris longus muscle, pectineus muscle, pectoralis major muscle, pectoralis minor muscle, peroneus brevis, peroneus longus muscle, peroneus 3 muscle, piriformis muscle, plantar interosseous muscles, plantaris muscle, subcutaneous neck muscle, popliteus muscle, posterior scalenus muscle, pronator quadratus muscle, pronator teres muscle muscle, psoas major, quadratus femoris, quadratus plantaris, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, laughter muscles, sartorius muscle, scalenus minor, semimembranosus, semispinalis capitis, semispinalis cervicis, semispinalis pectoralis, semitendinosus, serratus anterior, rotator brevis, soleus, spinalis capitis, spinalis cervicis, spinalis pectoralis, splenius head, splenius neck muscle, sternocleidomastoid muscle, sternohyoid muscle, sternothyroid muscle, stylohyoid muscle, subclavian muscle, subscapularis muscle, superior gemellus muscle, superior oblique muscle, superior rectus muscle, supinator muscle, supraspinatus muscle , temporalis, tensor fasciae lata, teres major, teres minor, chest muscles, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis , vastus medialis, zygomaticus major and zygomaticus minor, and any other suitable skeletal muscles known in the art.

[00436] Administration of a cccDNA vector for producing an antibody or fusion protein as described herein into the diaphragmatic muscle can be done by any suitable route, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration. In some embodiments, delivery of a transgene expressed from a cccDNA vector to a target tissue may also be accomplished by delivery of a synthetic depot containing the cccDNA vector, wherein the depot containing the cccDNA vector is implanted into skeletal, smooth, cardiac, and/or diaphragmatic muscle. the tissue or muscle tissue may be contacted with a film or other matrix containing the cDNA vector, as described herein. Such implantable matrices or carriers are described in US Pat. No. 7,201,898.

[00437] Administration of a cccDNA vector for producing antibodies or a fusion protein as described herein into cardiac muscle includes administration into the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The cccDNA vector as described herein can be delivered to the cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct injection into the heart (eg, left atrium, right atrium, left ventricle, right ventricle), and/ or coronary artery perfusion.

[00438] Administration of a cccDNA vector for producing antibodies or a fusion protein as described herein into smooth muscle can be accomplished by any suitable route, including intravenous administration, intraarterial administration, and/or intraperitoneal administration. In one embodiment, administration may be to endothelial cells present in, adjacent to, and/or on smooth muscle. Non-limiting examples of smooth muscle include the iris, bronchioles of the lungs, muscles of the larynx (vocal cords), muscular layers of the stomach, esophagus, small and large intestines of the gastrointestinal tract, ureter, detrusor muscle of the bladder, uterine myometrium, penis, or prostate gland.

[00439] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, is introduced into skeletal muscle, diaphragmatic muscle, and/or cardiac muscle (e.g., to treat, alleviate, and/or prevent muscular dystrophy or disease heart (eg, peripheral arterial disease (PAD) or congestive heart failure)). In exemplary embodiments, the cccDNA vector of the present invention is used to treat and/or prevent skeletal, cardiac, and/or diaphragmatic muscle disorders.

[00440] In particular, it is contemplated that a composition containing a cccDNA vector for the production of antibodies or fusion proteins as described herein can be delivered to one or more muscles of the eye (e.g., lateral rectus, medial rectus, superior rectus, inferior rectus rectus, superior oblique, inferior oblique), facial muscles (for example, occipitofrontal muscle, temporoparietal muscle, proud muscle, nasal muscle, depressor septum nasal muscle, orbicularis oculi muscle, corrugator muscle, depressor brow muscle, anterior auricularis, orbicularis oris, depressor anguli oris, laughter muscle, zygomaticus major, zygomaticus minor, levator labii superioris, levator labii superioris and alae nasalis, depressor labii inferioris, levator anguli mouth, buccal muscle, mentalis muscle) or muscles of the tongue (eg, genioglossus, hyoglossus, cartilaginous, styloglossus, palatoglossus, superior longitudinal muscle, inferior longitudinal muscle, vertical muscle and transverse muscle).

[00441] (i) Intramuscular injection: In some embodiments, a composition comprising a cccDNA vector for the production of antibodies or fusion proteins, as described herein, can be administered to a subject by injection into one or more sites of a given muscle, such as skeletal muscle ( eg deltoid, vastus lateralis, gluteus medius, gluteus minimus, or (of) gluteus maximus, or anterolateral thigh for infants) using a needle. A composition containing cccDNA can be introduced into other muscle cell subtypes. Non-limiting examples of muscle cell subtypes include skeletal muscle cells, cardiac muscle cells, smooth muscle cells and/or diaphragmatic muscle cells.

[00442] Intramuscular injection methods are known to those skilled in the art and, as such, are not described in detail herein. However, when performing an intramuscular injection, the proper needle size must be determined based on the age and size of the patient, the viscosity of the composition, and the injection site. Table 12 provides recommendations for approximate injection sites and corresponding needle sizes:

Table 12: Recommendations for intramuscular administration in humans Injection site Needle gauge Needle size Maximum composition volume Ventroglutheal region (gluteus medius and gluteus minimus) Aqueous solutions: caliber 20-25
Viscous or oily solution : 18-21 gauge Thin adults: 15 to 25 mm
Average size adults: 25 mm
Large adults (over 68 kg (150 lb)): 25 to 38 mm.
Children and Infants: Requires a smaller needle 3 ml Vastus lateralis muscle Aqueous solutions: caliber 20-25
Viscous or oily solution : 18-21 gauge
Children/infants : 22 to 25 gauge Adults: 25 to 38 mm 3 ml Deltoid Caliber from 22 to 25 Men:
59-118 kg (130-260 lb): 25 mm
Women:
<59 kg (<130 lb): 16 mm
59-91 kg (130-200 lb): 25 mm
>91 kg (>200 lbs): 38 mm 1 ml

[00443] In certain embodiments, the cDNA vector composition for producing antibodies or fusion proteins, as described herein, is formulated in a small volume, such as the representative volume example listed in Table 12 for a given subject. In some embodiments, if necessary, a general or local anesthetic may be administered to the subject prior to injection. This is especially desirable if multiple injections are required, or if the injection is performed into a deeper muscle rather than the usual injection sites noted above.

[00444] In some embodiments, intramuscular injection can be combined with electroporation, pressure delivery, or the use of transfection reagents to enhance cellular uptake of the cccDNA vector.

[00445] (ii) Transfection reagents: In some embodiments, cccDNA vector compositions for the production of antibodies or fusion proteins, as described herein, are formulated containing one or more transfection reagents to enhance the uptake of the vectors into myotubes or muscle tissue. Thus, in one embodiment, the nucleic acids described herein are introduced into a muscle cell, myotube, or muscle tissue by transfection using methods described elsewhere in this document.

[00446] (iii) Electroporation: In some embodiments, the cccDNA vector for producing antibodies or fusion proteins, as described herein, is administered in the absence of a vehicle to facilitate entry of the ccDNA into cells or in a physiologically inert pharmaceutically acceptable carrier (i.e. any vehicle that does not improve or increase the uptake of capsid-free non-viral vectors into myotubes). In such embodiments, uptake of the capsidless nonviral vector may be facilitated by electroporation of the cell or tissue.

[00447] Cell membranes naturally prevent the passage of extracellular substances into the cytoplasm of the cell. One way to temporarily reduce this resistance is "electroporation", in which electrical fields are used to create pores in cells without causing permanent cell damage. These pores are large enough to allow DNA vectors, pharmaceuticals, DNA and other polar compounds to gain access to the interior of the cell. Over time, the pores in the cell membrane close and the cell becomes impenetrable again.

[00448] Electroporation can be used for both in vitro and in vivo applications by introducing, for example, exogenous DNA into living cells. In vitro applications typically mix a sample of living cells with a composition containing, for example, DNA. The cells are then placed between electrodes, such as parallel plates, and an electric field is applied to the cell/composition mixture.

[00449] There are a number of in vivo electroporation methods; the electrodes can be made in various configurations, such as, for example, in the form of a caliper, which is used to clamp the epidermis covering the area of cells to be treated. Alternatively, needle-shaped electrodes can be inserted into tissue to access deeper cells. In any case, after the composition containing, for example, nucleic acids is injected into the treatment area, the electrodes create an electric field in the specified area. In some electroporation applications, such an electric field contains a single rectangular pulse of the order of 100-500 V/cm. lasting approximately 10 to 60 ms. Such a pulse can be generated, for example, in known applications, using an Electro Square Porator T820 manufactured by the BTX Division of Genetronics, Inc.

[00450] Typically, successful uptake of, for example, nucleic acids occurs only if the muscle is electrically stimulated immediately or shortly after administration of the composition, for example, by injection into the muscle.

[00451] In some embodiments, electroporation is provided using pulsed electric fields or using low voltage/long pulse processing modes (eg, using a square pulse electroporation system). Examples of pulse generators capable of generating a pulsed electric field include, for example, the ECM600, which can generate an exponential waveform, and the ElectroSquarePorator (T820), which can generate square wave pulses, both of which are available from the BTX division of Genetronics, Inc. (San Diego, California). Square wave electroporation systems produce controlled electrical pulses that rise rapidly to a set voltage value, remain at that level for a set amount of time (pulse duration), and then quickly fall to zero.

[00452] In some embodiments, a local anesthetic is administered, for example by injection at the treatment site, to reduce pain that may be associated with electroporation of tissue in the presence of a composition comprising a capsidless non-viral vector, as described herein. In addition, one skilled in the art will appreciate that the dosage of the composition should be selected to minimize and/or prevent excessive tissue damage leading to fibrosis, necrosis, or inflammation of the muscle.

[00453] (iv) Delivery under d pressure : In some embodiments, delivery of a cccDNA vector for producing an antibody or fusion protein, as described herein, to muscle tissue is facilitated by pressure delivery, which uses a combination of high volumes and rapid administration into an artery supplying a limb (eg, iliac artery). This route of administration can be accomplished by a variety of methods that involve infusion into the limb vessels of a composition containing the cccDNA vector, typically by isolating the muscle from the systemic circulation using a tourniquet of vasofixators. In one method, the composition circulates in the vasculature of the limb to allow extravasation into cells. In another method, intravascular hydrodynamic pressure is increased to dilate the vasculature and increase uptake of the cDNA vector into muscle cells or tissues. In one embodiment, the cccDNA composition is administered into an artery.

[00454] (v) Lipid nanoparticle compositions: In some embodiments, a cccDNA vector for producing antibodies or fusion proteins as described herein for intramuscular delivery is formulated as a composition containing a liposome as described elsewhere herein.

[00455] (vi) Systemic administration of a cccDNA vector targeting muscle tissue. In some embodiments, a cccDNA vector for antibody production or fusion protein, as described herein, is formulated to target muscle through indirect delivery, in which the cccDNA is transported to the muscle rather than the liver. Accordingly, the technology described herein covers indirect administration of compositions containing a cccDNA vector to produce an antibody or fusion protein as described herein into muscle tissue, for example, by systemic administration. Such compositions may be administered topically, intravenously (by bolus or continuous infusion), intracellular injection, interstitial injection, orally, by inhalation, intraperitoneally, subcutaneously, intracavitarily, and, if desired, may be delivered by peristaltic devices or other means known to those skilled in the art. this area. The agent can be administered systemically, for example, by intravenous infusion, if desired.

[00456] In some embodiments, the uptake of a cccDNA vector for producing an antibody or fusion protein as described herein into muscle cells/tissues is increased by the use of a targeting agent or moiety that preferentially targets the vector to muscle tissue. Thus, in some embodiments, the capsidless cccDNA vector may be concentrated in muscle tissue relative to the amount of capsidless cccDNA vectors present in other cells or tissues of the body.

[00457] In some embodiments, a composition comprising a cDNA vector for producing an antibody or fusion protein, as described herein, further comprises a muscle cell targeting moiety. In other embodiments, the expressed gene product contains a targeting moiety that is specific to the tissue in which its action is desired. A targeting moiety may include any molecule or complex of molecules that is capable of targeting, interacting with, combining with and/or binding to an intracellular, cell surface or extracellular biomarker of a cell or tissue. The biomarker may include, for example, a cellular protease, kinase, protein, cell surface receptor, lipid and/or fatty acid. Other examples of biomarkers that targeting moieties can target, interact, couple and/or bind include molecules associated with a particular disease. For example, biomarkers may include cell surface receptors involved in cancer development, such as epidermal growth factor receptor and transferrin receptor. Targeting moieties may include, but are not limited to, synthetic compounds, natural compounds or products, macromolecular fragments, bioengineered molecules (e.g., polypeptides, lipids, polynucleotides, antibodies, antibody fragments), and small entities (e.g., small molecules, neurotransmitters, substrates, ligands, hormones and elemental compounds) that bind to molecules expressed in target muscle tissue.

[00458] In some embodiments, the targeting moiety may further comprise a receptor molecule, including, for example, receptors that naturally recognize a particular desired target cell molecule. Such receptor molecules include receptors that have been modified to increase the specificity of their interaction with a target molecule, receptors that have been modified to interact with a desired target molecule not naturally recognized by the receptor, and fragments of such receptors (see, e.g. , Skerra, 2000, J. Molecular Recognition, 13:167-187). The preferred receptor is the chemokine receptor. Representative chemokine receptors have been described, for example, in Lapidot et al., 2002, Exp Hematol, 30:973-81 and Onuffer et al., 2002, Trends Pharmacol Sci, 23:459-67.

[00459] In other embodiments, the additional targeting moiety may comprise a ligand molecule, including, for example, ligands that naturally recognize a specific desired target cell receptor, such as a transferrin (Tf) ligand. Such ligand molecules include ligands that have been modified to increase the specificity of their interaction with a target receptor, ligands that have been modified to interact with a desired receptor not naturally recognized by a ligand, and fragments of such ligands.

[00460] In other embodiments, the targeting moiety may comprise an aptamer. Aptamers are oligonucleotides that are selected to specifically bind to the desired molecular structure of the target cell. Aptamers are typically the products of an affinity selection process similar to phage display affinity selection (also known as in vitro molecular evolution). The process involves performing multiple tandem iterations of affinity separation, for example using a solid support to which a disease-associated immunogen is attached, followed by polymerase chain reaction (PCR) to amplify nucleic acids that bind to the immunogens. Thus, each round of affinity separation enriches the nucleic acid population with molecules that successfully bind to the desired immunogen. In this way, a random pool of nucleic acids can be “trained” to produce aptamers that specifically bind target molecules. Aptamers are typically RNA, but may be DNA, or analogs or derivatives thereof, such as, but not limited to, peptide nucleic acids (PNAs) and phosphorothioate nucleic acids.

[00461] In some embodiments, the targeting moiety may comprise a photodegradable ligand (i.e., a caged ligand) that is released, for example, upon exposure to a focused beam of light to target capsidless nonviral vectors or gene product to a specific tissue.

[00462] It is also contemplated herein that the compositions will be delivered to multiple sites in one or more muscles of a subject. Thus, injections can be performed in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 , at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 places. Such sites may be distributed over the area of one muscle or may be distributed among several muscles.

B. Introduction of cccDNA vector to produce antibodies or fusion protein into non-muscle areas

[00463] In another embodiment, the cDNA vector for producing antibodies or fusion proteins is introduced into the CNS (eg, the brain or eye). The cccDNA vector can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, cerebrum, including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and portal amygdala (portaamygdala), limbic system, neocortex, striatum, cerebrum and inferior colliculus. The cccDNA vector can also be introduced into various areas of the eye, such as the retina, cornea and/or optic nerve. The cccDNA vector can be delivered into the cerebrospinal fluid (eg, via lumbar puncture). The cccDNA vector for production of antibodies or fusion proteins can be additionally introduced into the CNS intravascularly in situations where the blood-brain barrier is disrupted (eg, brain tumor or cerebral infarction).

[00464] In some embodiments, the cccDNA vector for producing antibodies or fusion proteins can be administered to the desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g. , in the presence of a sugar such as mannitol), intranasal, intraauricular, intraocular (eg, intravitreous, subretinal, anterior chamber) and periocular (eg, sub-Tenon's region) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[00465] In some embodiments, the cccDNA vector for producing antibodies or fusion proteins is administered in a liquid composition by direct injection (eg, stereotactic injection) into the desired region or compartment of the CNS. In other embodiments, the cccDNA vector can be administered by topical application to the desired area or by intranasal administration of an aerosol composition. Administration to the eye can be accomplished by topical application of liquid drops. As a further alternative, the cDNA vector can be administered as a sustained release solid composition (see, for example , US Pat. No. 7,201,898). In additional embodiments, the cccDNA vector can be used for retrograde transport to treat, alleviate, and/or prevent motor neuron-related diseases and disorders (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc. ). For example, the cccDNA vector can be delivered to muscle tissue, from which it can migrate into neurons.

C. Treatment ex vivo

[00466] In some embodiments, the cells are removed from the subject, a cDNA vector is introduced into them to produce antibodies or a fusion protein as described herein, and the cells are then returned to the subject. Methods for extracting cells from a subject for treatment ex vivo and then reintroducing them back to the subject are known in the art (see, for example , US Pat. No. 5,399,346, the disclosure of which is incorporated herein in its entirety). Alternatively, the cccDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.

[00467] Cells transduced with a cccDNA vector to produce antibodies or fusion proteins as described herein are preferably administered to a subject in a "therapeutically effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that therapeutic effects do not have to be complete or provide a cure, as long as the subject receives some benefit.

[00468] In some embodiments, a cccDNA vector for producing an antibody or fusion protein as described herein may encode an antibody or fusion protein as described herein (sometimes referred to as a transgene or heterologous nucleotide sequence) to be produced in a cell in vitro, ex vivo or in vivo . For example, in contrast to the use of cccDNA vectors described herein in a method of treatment discussed herein, in some embodiments, a cccDNA vector for producing an antibody or fusion protein can be introduced into cultured cells, and the antibody or fusion protein expressed can be isolated from cells, for example, to produce antibodies and fusion proteins. In some embodiments, cultured cells containing a cccDNA vector for producing antibodies or fusion proteins, as described herein, can be used for commercial production of antibodies or fusion proteins, for example, as a source of cells for small or large scale biomanufacturing of antibodies or fusion proteins. In alternative embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, is introduced into cells of a non-human host subject for the production of antibodies or fusion proteins in vivo , including small scale production, as well as for commercialization. large-scale production of an antibody or fusion protein.

[00469] cccDNA vectors for the production of antibodies or fusion proteins, as described herein, can be used in both veterinary and human medicine. Suitable subjects for ex vivo gene delivery methods as described above include both birds (eg, chickens, ducks, geese, quail, turkeys and pheasants) and mammals (eg, humans, cattle, sheep, goats, equines, felids, canids and lagomorphs), with mammals being preferred. People are the most preferred subjects. People include newborns, children, adolescents and adults.

D. Dose Ranges

[00470] Provided herein are methods of treatment comprising administering to a subject an effective amount of a composition comprising a cccDNA vector encoding an antibody or fusion protein as described herein. As will be understood by one skilled in the art, the term “effective amount” refers to that amount of the cccDNA composition administered that results in expression of the antibody or fusion protein in a “therapeutically effective amount” to treat a disease.

[00471] In vivo and/or in vitro assays can optionally be used to determine optimal dosage ranges for use. The exact dosage to be used in the composition will also depend on the route of administration and the severity of the condition and should be determined by the judgment of one skilled in the art and taking into account the circumstances of each subject. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

[00472] cccDNA vectors for the production of antibodies or fusion proteins, as described herein, are administered in quantities sufficient to transfect cells of the desired tissue and to ensure sufficient levels of gene transfer and expression without undesirable side effects. Common and pharmaceutically acceptable routes of administration include, but are not limited to, those described above in the Introduction section, such as direct delivery to the target organ (eg, intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral and other routes of parenteral administration. The routes of administration can be combined if desired.

[00473] The dosage amount of cccDNA vectors to produce an antibody or fusion protein, as described herein, required to achieve a particular “therapeutic effect” will vary depending on several factors, including, but not limited to: route of administration of the nucleic acid, level of expression gene or RNA required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene(s), RNA product(s), or resulting protein(s) expressed. One skilled in the art can readily determine a dosage range of a cccDNA vector for treating a patient with a particular disease or disorder based on the above factors as well as other factors that are well known in the art.

[00474] The dosage regimen may be adjusted to provide an optimal therapeutic response. For example, the oligonucleotide can be administered repeatedly, for example, multiple doses can be administered daily, or the dose can be proportionally reduced based on the dictated therapeutic situation. One skilled in the art will readily be able to determine appropriate dosages and administration schedules for the oligonucleotides of this invention, both for administration to cells and for administration to subjects.

[00475] A "therapeutically effective dose" will be within a relatively wide range, which can be determined through clinical trials, and will depend on the specific application (nerve cells will require very small amounts, while systemic injections will require large amounts). For example, for direct in vivo injection into human skeletal or cardiac muscle, the therapeutically effective dose would range from about 1 μg to 100 g of cccDNA vector. If exosomes or microparticles are used to deliver the cccDNA vector, the therapeutically effective dose can be determined experimentally but is expected to deliver between 1 μg and approximately 100 g of vector. In addition, a therapeutically effective dose is an amount of cccDNA vector that expresses a sufficient amount of the transgene to cause an effect in a subject that results in a reduction in one or more symptoms of the disease, but does not significantly affect off-target areas or cause significant undesirable side effects. In one embodiment, a “therapeutically effective amount” is an amount of expressed antibody or fusion protein sufficient to produce a statistically significant measurable change in the expression of a disease biomarker or a reduction in a given disease symptom. Such effective amounts for a given cDNA vector composition can be determined in clinical trials as well as in animal studies.

[00476] Formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosage and treatment regimens when using the specific compositions described herein in various treatment regimens.

[00477] For in vitro transfection, the effective amount of cccDNA vectors for the production of antibodies or fusion proteins as described herein to be delivered into cells (1×10 ⁶ cells) will be on the order of 0.1-100 μg of ccDNA vector , preferably 1 to 20 μg, and more preferably 1 to 15 μg or 8 to 10 μg. Larger cDNA vectors will require higher doses. If exosomes or microparticles are used, the effective in vitro dose can be determined experimentally, but must deliver generally the same amount of cccDNA vector.

[00478] The appropriate dosage for treating a disease of a cccDNA vector that expresses an antibody or fusion protein as described herein will depend on the specific type of disease being treated, the type of antibody, the severity and course of the disease, prior therapy, the patient's medical history, and reaction to the antibody, and is determined at the discretion of the attending physician. The antibody may be administered to the patient at one time or over a series of treatments. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple dosing at different time points, bolus dosing, and pulse infusion.

[00479] Depending on the type and severity of the disease, the cccDNA vector is administered in such an amount that the encoded antibody or fusion protein is expressed in an amount from about 0.3 mg/kg to 100 mg/kg (e.g., 15-100 mg/kg, or at any dosage within the specified range), through one or more separate administration sessions, or by continuous infusion. One typical daily dose of a cccDNA vector is sufficient to induce expression of the encoded antibody or fusion protein ranging from about 15 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. One exemplary dose of cccDNA vector is an amount sufficient to result in expression of the encoded antibody or fusion protein as described herein, ranging from about 10 mg/kg to about 50 mg/kg. Thus, the patient may be administered one or more doses of the cccDNA vector in an amount sufficient to result in expression of the encoded antibody or fusion protein of approximately 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg. kg, 2.0 mg/kg, 3 mg/kg, 4.0 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg , 35 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg (or any combination thereof). In some embodiments, the cccDNA vector is administered in an amount sufficient to express the encoded antibody or fusion protein at a total dose ranging from 50 mg to 2500 mg. An exemplary dose of the cDNA vector is an amount sufficient to result in total expression of the encoded antibody or fusion protein of about 50 mg, about 100 mg, 200 mg, 300 mg, 400 mg, about 500 mg, about 600 mg. about 700 mg, about 720 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2050 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, or about 2500 mg (or any combination thereof). Since expression of an antibody or fusion protein from a cccDNA vector can be precisely controlled using regulatory switches as described herein or, alternatively, by repeatedly administering doses of a cccDNA vector to a subject, expression of an antibody or fusion protein from a cccDNA vector can be controlled in such a manner that so that doses of the antibody or fusion protein expressed from the cccDNA vector can be administered periodically, for example, once a week, every two weeks, every three weeks, every four weeks, once a month, every two months, every three months or once every six months. The effectiveness of such therapy can be monitored using traditional methods and tests.

[00480] In some embodiments, the cccDNA vector is administered in an amount sufficient to result in expression of the encoded antibody or fusion protein at a dose of 15 mg/kg, 30 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg or a fixed dose such as 300 mg, 500 mg, 700 mg, 800 mg or higher. In some embodiments, expression of the antibody or fusion protein from the cccDNA vector is controlled such that the antibody or fusion protein is expressed every day, every other day, every week, every 2 weeks, or every 4 weeks, for a period of time. In some embodiments, expression of an antibody or fusion protein from a cccDNA vector is controlled such that the antibody or fusion protein is expressed every 2 weeks or every 4 weeks over a period of time. In some embodiments, the time period is 6 months, one year, eighteen months, two years, five years, ten years, 15 years, 20 years, or the lifespan of the patient.

[00481] Treatment may include administration of a single dose or multiple doses. In some embodiments, more than one dose may be administered to a subject; in fact, multiple doses can be administered if necessary because the cccDNA vector does not induce a host immune response against the capsid due to the absence of a viral capsid. Thus, a person skilled in the art can easily determine the appropriate number of doses. The number of doses administered may be, for example, on the order of 1-100, preferably 2-20 doses.

[00482] Without wishing to be bound by any particular theory, the absence of the typical antiviral immune response caused by administration of a cccDNA vector as described in this invention (i.e., the absence of capsid components) allows repeated administration of the cccDNA vector to the host to produce antibodies or fusion proteins. In some embodiments, the number of times the heterologous nucleic acid is administered to a subject ranges from 2 to 10 times (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the cccDNA vector is delivered to a subject more than 10 times.

[00483] In some embodiments, a dose of a cccDNA vector to produce an antibody or fusion protein, as described herein, is administered to a subject no more than once per calendar day (eg, per 24-hour period). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of a cccDNA vector to produce an antibody or fusion protein, as described herein, is administered to a subject no more than once per calendar week (eg, 7 calendar days). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once every two weeks (eg, once every two calendar weeks). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once per calendar month (eg, once every 30 calendar days). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once every six calendar months. In some embodiments, a dose of the cDNA vector is administered to a subject no more than once during a calendar year (eg, 365 days, or 366 days in a leap year).

[00484] In certain embodiments, more than one administration (e.g., two, three, four, or more administrations) of a cccDNA vector to produce an antibody or fusion protein, as described herein, can be used to achieve the desired level of gene expression over varying periods time, such as daily, weekly, monthly, annually, etc.

[00485] In some embodiments, a therapeutic antibody encoded by a cccDNA vector as described herein can be regulated by a regulatory switch, an inducible or a repressible promoter such that it is expressed in a subject for at least 1 hour, according to at least 2 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 12 months/one year, at least 2 years, at least at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years or more. In one embodiment, expression can be achieved by reintroducing the cDNA vectors described herein at predetermined or desired intervals. Alternatively, the cDNA vector for producing antibodies or fusion proteins as described herein may further contain gene editing system components (e.g., CRISPR/Cas, TALENs (transcription activator-like effector nucleases), zinc finger endonucleases, etc. ) to enable the insertion of one or more nucleic acid sequences encoding an antibody to substantially permanently treat or "cure" a disease. Such cccDNA vectors containing gene editing components are disclosed in international application PCT/US18/64242 and may include 5' and 3' homologous arms (for example, SEQ ID NO: 151-154, or sequences with at least 40% , 50%, 60%, 70%, or 80% homology thereto) to insert an antibody-encoding nucleic acid into a safe harbor region such as, but not including, the albumin gene or the CCR5 gene.

[00486] The duration of treatment depends on the subject's clinical progress and responsiveness to therapy. Provides continuous administration of relatively low maintenance doses after an initial higher therapeutic dose.

E. Unit dosage forms

[00487] In some embodiments, pharmaceutical compositions containing a cccDNA vector for the production of antibodies or fusion proteins, as described herein, can be conveniently presented in a unit dosage form. The unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration via a vaporizer. In some embodiments, the unit dosage form is adapted for administration via a nebulizer. In some embodiments, the unit dosage form is adapted for administration using an aerosol generator. In some embodiments, the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical use. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form is typically that amount of said compound that produces a therapeutic effect.

X. Treatment methods

[00488] The technology described herein also demonstrates methods for producing, as well as methods for using, the disclosed cccDNA vectors to produce antibodies or fusion proteins in various ways, including, for example, ex vivo , ex situ , in vitro and in vivo applications, methodologies, diagnostic gene therapy procedures and/or regimens.

[00489] In one embodiment, a therapeutic antibody expressed from a cccDNA vector as described herein is functional for treating a disease. In a preferred embodiment, the therapeutic antibody does not cause an immune system response unless desired.

[00490] Provided herein is a method of treating a disease or disorder in a subject, comprising administering to a target cell in need thereof (e.g., a muscle cell or tissue, or other type of diseased cell) of the subject a therapeutically effective amount of a cccDNA vector for producing antibodies or fusions. proteins as described herein, optionally with a pharmaceutically acceptable carrier. Although the cDNA vector can be introduced in the presence of a carrier, such a carrier is not necessary. The provided cccDNA vector contains a nucleotide sequence encoding an antibody or antigen binding fragment, as described herein, useful for treating a disease. In particular, a cccDNA vector for producing an antibody or fusion protein, as described herein, may contain a desired antibody or antigen binding fragment DNA sequence operably linked to control elements capable of directing transcription of the desired antibody or antigen binding fragment encoded by the exogenous DNA sequence, at introduction to the subject. The cDNA vector for producing antibodies or fusion proteins as described herein can be administered by any suitable route as described above and elsewhere in this document.

[00491] Disclosed herein are compositions and compositions of cccDNA vectors for the production of antibodies or fusion proteins as described herein, which include one or more ccDNA vectors of the present invention along with one or more pharmaceutically acceptable buffers, diluents or excipients. Such compositions may be included in one or more diagnostic or therapeutic kits for diagnosing, preventing, treating, or alleviating one or more symptoms of a disease, lesion, disorder, injury, or dysfunction. In one aspect, the disease, lesion, disorder, injury, or dysfunction is a disease, lesion, disorder, injury, or dysfunction in a human.

[00492] Another aspect of the technology described herein provides a method of administering to a subject in need thereof a diagnostically or therapeutically effective amount of a cDNA vector to produce an antibody or fusion protein as described herein, the method comprising providing to a cell, tissue or organ the subject in need thereof, a quantity of cDNA vector, as described herein; and for a period of time allowing efficient expression of the transgene from the cccDNA vector, thereby providing the subject with a diagnostically or therapeutically effective amount of the antibody or antigen binding fragment expressed by the cccDNA vector. In an additional aspect, the subject is a person.

[00493] Another aspect of the technology described herein provides a method for diagnosing, preventing, treating, or alleviating at least one or more symptoms of a disease, disorder, dysfunction, lesion, abnormal condition, or injury in a subject. In general, the method includes at least the step of administering to a subject in need thereof one or more disclosed cccDNA vectors for producing antibodies or fusion proteins, in an amount and for a time sufficient to diagnose, prevent, treat, or alleviate one or more several symptoms of a disease, disorder, dysfunction, lesion, abnormal condition, or injury in a subject. In such an embodiment, a subject may be assessed for the effectiveness of the antibody/antigen-binding fragment or, alternatively, for detection of an antigen or antigen-binding fragment at a specific protein (protien) or tissue site (including cellular and subcellular location) in the subject. As such, a cccDNA vector for producing antibodies or fusion proteins as described herein can be used as an in vivo diagnostic tool, for example, for the detection of cancer or other indications. In an additional aspect, the subject is a person.

[00494] Another aspect is the use of a cccDNA vector to produce an antibody or fusion protein, as described herein, as a tool for treating or ameliorating one or more symptoms of a disease or disease state. There are a number of inherited diseases with known defective genes, which generally fall into two classes: deficiency states, usually of enzymes, which are usually inherited in a recessive manner, and imbalanced conditions, which may involve regulatory or structural proteins and which are usually but not always, inherited dominantly. In deficient diseases, a cccDNA vector for producing antibodies or fusion proteins, as described herein, can be used to deliver antibodies or fusion proteins that neutralize the pathway protein, resulting in increased expression of the normal gene for replacement therapy, and also, in in some embodiments, to create animal models of diseases using cccDNA vectors expressing neutralizing antibodies or fusion proteins. For unbalanced disease states, a cDNA vector for producing antibodies or fusion proteins, as described herein, can be used to create the disease state in a model system, which can then be used to counteract the disease state. Thus, a cccDNA vector for the production of antibodies or fusion proteins, as described herein, allows the treatment of genetic diseases. As used herein, a disease state is treated by partially or completely correcting the deficiency or imbalance that causes the disease or makes it more severe.

[00495] In one embodiment, the present invention provides that intramuscular delivery of a transgene to express an antibody in muscle can be used to treat muscle-specific diseases or, alternatively, as a protein production depot for exposure of a therapeutic transgene product at a distant site. The cccDNA vectors described herein can be used to express an antibody or fusion protein in muscle. In some embodiments, the gene product increases the expression and/or activity of a target gene. In other embodiments, the gene product reduces the expression and/or activity of a target gene.

A. Host cells:

[00496] In some embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, delivers an antibody or antigen binding fragment transgene to a host cell of the invention. In some embodiments, the host cell of the present invention is a human host cell, including, for example, blood cells, stem cells, hematopoietic cells, CD34 ⁺ cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, nerve cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts or any other cells derived from mammals, including, without limitation, hepatocytes (i.e. liver cells), lung cells, heart cells, pancreatic cells glands, intestinal cells, diaphragmatic cells, renal cells (ie, kidney cells), nerve cells, blood cells, bone marrow cells, or any one or more selected tissues of the subject for whom gene therapy is to be performed. In one aspect, the host cell of this invention is a human host cell.

[00497] This invention also relates to recombinant host cells, as defined above, including a cDNA vector for the production of antibodies or fusion proteins, as described herein. Thus, multiple host cells can be used depending on the purpose, as will be appreciated by one skilled in the art. A construct or cDNA vector for producing antibodies or fusion proteins as described herein, including a donor sequence, is introduced into a host cell such that the donor sequence is maintained as a chromosomal integrant as previously described. The term "host cell" covers any progeny of a parent cell that is not identical to the parent cell due to mutations occurring during replication. The choice of host cell will largely depend on the donor sequence and its source.

[00498] The host cell may also be eukaryotic, such as a mammalian, insect, plant, or fungal cell. In one embodiment, the host cell is a human cell (eg, a primary cell, a stem cell, or an immortalized cell line). In some embodiments, a cccDNA vector may be introduced into a host cell ex vivo to produce antibodies or fusion proteins, as described herein, followed by delivery to the subject after the gene therapy event. The host cell can be any type of cell, for example a somatic cell or a stem cell, an induced pluripotent stem cell or a blood cell, for example a T cell or a B cell, or a bone marrow cell. In certain embodiments, the host cell is an allogeneic cell. For example, T cell genomic engineering is useful for cancer immunotherapies, disease modulation such as HIV therapy (eg, receptor knockout such as CXCR4 and CCR5), and immunodeficiency therapies. The targets of immunotherapy may be MHC receptors (major histocompatibility complex) on B cells. 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 the patient to express the therapeutic protein.

C. Additional diseases for gene therapy:

[00499] In general, a cccDNA vector for producing an antibody or fusion protein as described herein can be used to deliver any antibody or antigen binding fragment as described above to treat, prevent, or alleviate symptoms associated with any disorder associated with aberrant protein expression or gene expression in a subject. Illustrative examples of disease conditions include, but are not limited to: cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes, muscular dystrophies (eg, Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic defects, degenerative retinal diseases (and other eye diseases), mitochondria (eg, Leber hereditary optic neuropathy (LHON), Leigh syndrome and subacute sclerosing encephalopathy), myopathies (eg, facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (eg, brain, liver, kidney, heart); and so on. In some embodiments, the cccDNA vectors described herein may be advantageously used in the treatment of individuals with metabolic disorders (eg, ornithine transcarbamylase deficiency).

[00500] In some embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, can be used to treat, ameliorate, and/or prevent a disease or disorder caused by a mutation in a gene or gene product. Typical examples of diseases or disorders that can be treated with cccDNA vectors include, but are not limited to, metabolic diseases or disorders (eg, Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); diseases or disorders of the urea cycle (eg, ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (eg, metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Hunter syndrome)); liver diseases or disorders (eg, progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (eg, hemophilia (A and B), thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (eg, cystic fibrosis) .

[00501] In some embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, can be used to deliver the antibody or fusion protein to skeletal, cardiac, or diaphragmatic muscle, to produce the antibody or fusion protein for secretion and circulation in blood or for systemic delivery to other tissues for the treatment, amelioration and/or prevention of a disorder (eg, metabolic disorder such as diabetes (eg, insulin), hemophilia (eg, factor VIII), mucopolysaccharidosis disorder (eg, Sly syndrome, Hurler syndrome , Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteaux-Lamy syndrome, etc.) or lysosomal storage disorder (such as Gaucher disease [glucocerebrosidase], Pompe disease [lysosomal acid alpha-glucosidase] or Fabry disease [alpha-galactosidase A]) or glycogen storage disorders (such as Pompe disease [lysosomal acid alpha-glucosidase]). Other suitable proteins for treating, alleviating and/or preventing metabolic disorders are described above.

[00502] In other embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, can be used to deliver an antibody or antigen binding fragment in a method of treating, alleviating, and/or preventing a metabolic disorder in a subject in need thereof. Illustrative examples of metabolic disorders and antibodies or antigen binding fragments are described herein. Optionally, the polypeptide is secreted (eg, a polypeptide that is a secreted polypeptide in its native state, or that has been engineered to be secreted, for example, through functional association with a secretory signal sequence, as is known in the art).

[00503] An cccDNA vector for producing antibodies or fusion proteins, as described herein, can be administered to the lungs of a subject by any suitable method, optionally by administering an aerosol suspension of inhalable particles containing the cccDNA vectors, which the subject inhales. Inhaled particles can be liquid or solid. Aerosols of liquid particles containing cccDNA vectors can be produced by any suitable means, for example, using a pneumatic aerosol nebulizer or an ultrasonic nebulizer, as known to those skilled in the art. See, for example, US Pat. No. 4,501,729. Particulate aerosols containing cccDNA vectors can also be produced using any particulate drug aerosol generator by methods known in the pharmaceutical art.

[00504] In some embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, can be introduced into CNS tissues (eg, brain, eye). In specific embodiments, cccDNA vectors, as described herein, can be administered to treat, ameliorate, or prevent CNS diseases, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Illustrative examples of CNS diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis , pseudoparalytic myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarctions, mental disorders, including mood disorders (for example, depression, bipolar affective disorder, persistent affective disorder disorder, secondary mood disorder), schizophrenia, drug dependence (for example, alcoholism and addiction to other substances), neuroses (for example, anxiety, obsessive disorder, somatoform disorder, dissociative disorder, grief, postpartum depression), psychosis (for example, hallucinations and delirium), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleep disorders, pain-related disorders, eating or weight-related disorders (eg, obesity, cachexia, anorexia nervosa, and bulimia), and cancers and tumors of the central nervous system (CNS). for example, pituitary tumors).

[00505] Ophthalmic disorders that can be treated, ameliorated, or prevented using a cccDNA vector for producing antibodies or fusion proteins as described herein include ophthalmic disorders affecting the retina, posterior tract, and optic nerve (eg, retinitis pigmentosa, diabetic retinopathy and other degenerative diseases of the retina, uveitis, age-related macular degeneration, glaucoma). Many ophthalmic diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. In some embodiments, a cccDNA vector, as described herein, can be used to deliver antiangiogenic factors; anti-inflammatory factors; factors that slow down cell degeneration, promote cell preservation or promote cell growth, and combinations of the above. For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more antiangiogenic antibodies or fusion proteins either intraocularly (eg, into the vitreous) or periocularly (eg, into the sub-Tenon's space). Additional ocular diseases that may be treated, ameliorated, or prevented by the cccDNA vectors of the invention include geographic atrophy, vascular or wet macular degeneration, Stargardt disease, Leber congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), X-linked retinitis pigmentosa (XLRP), X-linked retinoschisis (XLRS), choroideremia, Leber congenital optic neuropathy (LHON), achromatopsia, rod-cone dystrophy, Fuchs' corneal endothelial dystrophy, diabetic macular edema, and eye cancers and tumors .

[00506] In some embodiments, inflammatory ocular diseases or disorders (eg, uveitis) can be treated, alleviated, or prevented using a cccDNA vector for producing antibodies or fusion proteins, as described herein. One or more anti-inflammatory antibodies or fusion proteins can be expressed by intraocular (eg, intravitreal or anterior chamber) administration of a cccDNA vector as described herein.

[00507] In some embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, may encode an antibody or antigen binding fragment that is linked to a transgene encoding a reporter polypeptide (e.g., an enzyme such as green fluorescent protein or alkaline phosphatase). In some embodiments, the transgene that encodes a reporter protein useful for experimental or diagnostic purposes is selected from any of: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT) , luciferase and others well known in the art. In some aspects, cccDNA vectors expressing an antibody or antigen binding fragment linked to a reporter polypeptide can be used for diagnostic purposes or as markers of cccDNA vector activity in a subject to which they are administered.

[00508] In some embodiments, a cccDNA vector for producing an antibody or fusion protein, as described herein, can express an antibody or antigen binding fragment that specifically binds to an immunogenic polypeptide or immunogen in a subject. The antibody or antigen binding fragment may specifically bind to any immunogen of interest known in the art, including, without limitation, immunogens from human immunodeficiency virus, influenza virus, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. .

D. Testing for successful gene expression using cccDNA vector

[00509] Assays well known in the art can be used to test the efficiency of gene delivery of an antibody or antigen binding fragment by a cDNA vector and can be performed in both in vitro and in vivo models. One of ordinary skill in the art can assess the levels of expression of an antibody or antigen binding fragment by cccDNA by measuring the levels of mRNA and protein of the antibody or antigen binding fragment (eg, reverse transcription PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA)). In one embodiment, the cccDNA contains a reporter protein that can be used to assess the expression of an antibody or antigen binding fragment, for example, by examining the expression of the reporter protein by fluorescence microscopy or using a fluorescent plate reader. For in vivo applications, protein function assays can be used to test the functionality of a given antibody or antigen binding fragment to determine whether gene expression has been successful. One skilled in the art will be able to determine the best test to measure the functionality of the antibody or antigen binding fragment expressed by the cccDNA vector in vitro or in vivo.

[00510] It is provided herein that the effects of gene expression of an antibody or antigen binding fragment from a cccDNA vector in a cell or subject may last for at least 1 month, at least 2 months, at least 3 months, at least four months, at least 5 months, at least six months, at least 10 months, at least 12 months, at least 18 months, at least 2 years, at least 5 years, at least 10 years, at least 20 years, or may be permanent.

[00511] In some embodiments, an antibody or antigen binding fragment in an expression cassette, expression construct, or cDNA vector described herein may be codon optimized for a host cell. As used herein, the term “codon-optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in vertebrate cells of interest, e.g., mouse or human (e.g., humanized), by replacing at least one more than one or a significant number of codons from a native sequence (eg, a prokaryotic sequence) to the codons that are most or most frequently used in the genes of a given vertebrate. Different species exhibit a particular affinity 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 using, for example, the Gene Forge® codon optimization and custom gene synthesis platform from Aptagen (Aptagen, Inc.) or other publicly available database.

E. Determination of efficacy by assessing antibody expression from cccDNA vector

[00512] Essentially any method for determining protein expression known in the art can be used to analyze the expression of a desired antibody from a cccDNA vector. Non-limiting examples of such methods/assays include enzyme-linked immunosorbent assay (ELISA), affinity ELISA, enzyme-linked immunospot test (ELISPOT), serial dilution, flow cytometry, surface plasmon resonance assay, kinetic exclusion assay, mass spectrometry, Western blotting, immunoprecipitation and PCR.

[00513] To assess antibody expression in vivo , a biological sample may be obtained from the subject for analysis. Typical biological samples include a body fluid sample, a body fluid sample, blood (including whole blood), serum, plasma, urine, saliva, biopsy and/or tissue sample, etc. A biological or tissue sample may also refer to a sample of tissue or fluid obtained from an individual, including, but not limited to, tumor biopsy, stool, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, external skin, respiratory tract, intestinal tract, and genitourinary tract, tears, saliva, breast milk, cells (including, but not limited to, blood cells), tumors, organs, and samples of in vitro cell culture components. This term also includes a mixture of the above samples. The term "sample" also includes unprocessed or preprocessed (or preprocessed) biological samples. In some embodiments, the sample used for the assays and methods described herein comprises a serum sample collected from the subject to be tested.

F. Determination of the effectiveness of the expressed antibody by clinical parameters.

[00514] The effectiveness of a given antibody or antigen binding fragment expressed by a cccDNA vector for a given disease (i.e., functional expression), such as rheumatoid arthritis or cancer (including, but not limited to, breast cancer, melanoma, etc.) , can be determined by an experienced physician. However, treatment is considered “effective treatment” as that term is used herein if any one or all of the signs or symptoms of the cancer change for the better, or if other clinically accepted symptoms or markers of the disease are improved or relieved, e.g. , by at least 10% after treatment with a cccDNA vector encoding a therapeutic antibody as described herein. Efficacy can also be measured by the individual's lack of deterioration, which is assessed by stabilization of the disease or need for medical intervention (ie, progression of the disease stops or at least slows down). Methods for measuring these indicators are known to those skilled in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include a human or mammal) and includes: (1) inhibition of the disease, eg, stopping or slowing the progression of the disease (eg, arthritis, cancer); or (2) relief of disease, such as caused by regression of symptoms; and (3) preventing or reducing the likelihood of developing a disease, or preventing secondary diseases/disorders associated with the disease (eg, hand deformities due to rheumatoid arthritis or cancer metastasis).

[00515] An effective amount for treating a disease means an amount that, when administered to a mammal in need thereof, is sufficient to provide effective treatment, as that term is defined herein, of that disease. The effectiveness of an agent can be determined by assessing physical parameters that are specific to a given disease. For example, physical indicators for cancer include, but are not limited to, pain, tumor size, tumor growth rate, blood cell count, etc.

XI. Various applications of cccDNA vectors expressing antibodies or fusion proteins

[00516] As disclosed herein, compositions and cDNA vectors for producing an antibody or fusion protein as described herein can be used to express the antibody or fusion protein for a variety of purposes. In one embodiment, a cccDNA vector expressing an antibody or fusion protein can be used to create a somatic model of a transgenic animal carrying a transgene, for example, to study the function of a target of the antibody or fusion protein. In some embodiments, a cccDNA vector expressing an antibody or fusion protein is useful for treating, preventing, or alleviating a disease state or disorder in a mammalian subject.

[00517] In some embodiments, the antibody or fusion protein can be expressed from a cccDNA vector in a subject in an amount sufficient to treat a disease associated with overexpression, overactivity of the gene product, or inappropriate activation of the gene. In such an embodiment, the expressed antibody or fusion protein may be a blocking or neutralizing antibody or fusion protein that causes inhibition or suppression or other reduction in the activity of a protein or gene product that is targeted or to which the antibody or fusion protein specifically binds.

[00518] In some embodiments, an antibody or fusion protein can be expressed from a cccDNA vector in a subject in an amount sufficient to treat a disease associated with decreased expression, lack of expression, or impaired function of the protein. For example, the expressed antibody or fusion protein is an activating antibody or fusion protein and can increase the activity or function of a protein with decreased expression and/or activity in a subject, for example, by agonizing the protein or inhibiting a repressor of the protein.

[00519] One skilled in the art will appreciate that the transgene may not be the open reading frame of a gene that itself must be transcribed; instead, it may be a promoter region or a repressor region of a target gene, and the cDNA vector may modify such a region, resulting in modulation of expression of the gene of interest.

[00520] Compositions and cccDNA vectors for producing antibodies or fusion proteins, as described herein, can be used to deliver an antibody or antigen binding fragment for various purposes, as described above.

[00521] In some embodiments, the transgene encodes one or more antibodies or fusion proteins that are useful for treating, ameliorating, or preventing disease states in a mammalian subject. An antibody or fusion protein expressed by a cccDNA vector is administered to a patient in an amount sufficient to treat a disease associated with an abnormal gene sequence that may result in any one or more of the following: increased protein expression, excessive protein activity, decreased expression, no expression or dysfunction of a target gene or protein.

[00522] In some embodiments, cccDNA vectors for producing antibodies or fusion proteins, as described herein, are for use in diagnostic and screening methods, wherein the antibody or antigen binding fragment is transiently or stably expressed in a cell culture or, alternatively, transgenic system animal model.

[00523] Another aspect of the technology described herein provides a method for transducing a population of mammalian cells with a cccDNA vector to produce antibodies or fusion proteins as described herein. In general, the method includes at least the step of introducing, into one or more cells of the population, a composition containing an effective amount of one or more cccDNA vectors for producing antibodies or fusion proteins, as described herein.

[00524] In addition, this invention relates to compositions, as well as therapeutic and/or diagnostic kits, that include one or more disclosed cccDNA vectors for producing antibodies or fusion proteins as described herein, or cccDNA compositions formulated with one or more additional ingredients, or prepared in accordance with one or more instructions for use thereof.

[00525] The cell for introducing the cccDNA vector to produce an antibody or fusion protein as described herein can be of any type, including, but not limited to, neural cells (including cells of the peripheral and central nervous system, particularly brain cells) , lung cells, retinal cells, epithelial cells (eg, intestinal and respiratory epithelial cells), muscle cells, dendritic cells, pancreatic cells (including islet cells), liver cells, myocardial cells, bone cells (eg, bone marrow stem cells) , hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. Alternatively, the cell may be any progenitor cell. As a further alternative, the cell may be a stem cell (eg, neural stem cell, liver stem cell). As yet another alternative, the cell may be a cancer or tumor cell. Additionally, the cells may be of any species origin, as noted above.

A. cccDNA vectors for commercial production of antibodies or fusion proteins

[00526] In some embodiments, the cccDNA vectors described herein can be used to produce antibodies or fusion proteins in a commercial setting, such as using a bioreactor or for production in a desired host.

[00527] For example, cells containing a cccDNA vector for the production of antibodies or fusion proteins, as described herein, can be used for commercial production of antibodies or fusion proteins, for example, as a source of cells for small-scale or large-scale bioproduction of antibodies or fusion proteins. In alternative embodiments, a cccDNA vector for producing antibodies or fusion proteins, as described herein, is introduced into cells of a non-human host subject for the production of antibodies or fusion proteins in vivo , including small scale production, as well as for commercialization. large-scale production of an antibody or fusion protein. For example, in some embodiments, the cccDNA vectors described herein can be used to produce antibodies or fusion proteins in vivo , such as in rats, mice, horses, goats, etc., using ascites tumors.

[00528] In some embodiments, cccDNA vectors encoding an antibody or fusion protein can be used, for example, to generate a chimeric antigen receptor (CAR), which can then be used to generate CAR T cells. CARs are fusion proteins of a selected single chain variable fragment from a specific monoclonal antibody and one or more intracellular T cell receptor signaling domains. Such genetic modification of T cells can be carried out using cccDNA vectors as described herein. Thus, this document specifically provides that a cccDNA vector expressing a chimeric antigen receptor can be introduced, for example, into a T cell ex vivo to generate a CAR T cell for the treatment of cancers such as, but not limited to, leukemia, mammary cancer. lung cancer, ovarian cancer, etc.

B. Antibody or Fusion Protein Production and Purification

[00529] The cccDNA vectors described herein are intended for use in the production of antibodies or fusion proteins in vitro or in vivo . Antibodies or fusion proteins produced in this manner can be isolated, tested for the desired function, and purified for further use in research or as therapeutic treatments. Each antibody or fusion protein production system has its own advantages/disadvantages. Although antibodies or fusion proteins produced in vitro can be easily purified and can produce antibodies or fusion proteins in a short time, antibodies or fusion proteins produced in vivo may have post-translational modifications such as glycosylation.

[00530] Conventional methods for producing antibodies, such as immunization and display library (eg, phage display), can be adapted to encode an antibody or antibody component by using cccDNA instead of a traditional vector (eg, plasmid, viral, etc.). In addition, cccDNA vectors, as described herein, can replace traditional vectors in bioreactors, bioreactor production methods, or in the production of antibodies in a desired host, cell, tissue or organ. Such techniques are known to those skilled in the art and are not described in detail herein.

[00531] The cccDNA vectors described herein can be used to express the desired antibody in a hybridoma cell line. Methods for generating hybridoma cell lines are known in the art. For large-scale antibody production, hybridoma cells can be grown in either a static or agitated cell suspension culture, a roller bottle culture, or a bioreactor (eg, a hollow fiber bioreactor). For in vitro production of antibodies, membrane culture systems can also be used, in which the cell culture is separated from nutrients by a special gas membrane that enhances the transfer of oxygen and gas. Alternatively, a matrix culture system is provided in which the hybridoma cells are immobilized on a matrix and are continuously supplied with fresh culture medium.

[00532] Antibodies produced using cDNA vectors can be purified by any method known to those skilled in the art, such as ion exchange chromatography, affinity chromatography, precipitation, or electrophoresis.

[00533] Antibody produced by the methods described herein and compositions thereof can be tested for binding to the desired target protein.

XIII. Various other implementation options

[00534] In some embodiments, this application may be defined by any of the following:

[00535] 1. A DNA vector containing at least one heterologous nucleic acid sequence encoding at least one transgene thereof operably linked to a promoter located between two AAV inverted terminal repeat (ITR) sequences, wherein the ITRs may not necessarily be the same or different ITRs, and wherein they are different ITRs, one of the ITRs contains a functional AAV end resolution site and a Rep binding site, and one of the ITRs contains a deletion, insertion or substitution relative to the other ITR; wherein the transgene is an antibody or a fragment or fusion protein thereof; and where DNA, when digested with a restriction enzyme having a single recognition site on the DNA vector, has characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

[00536] 2. The vector of claim 1, wherein the antibody is a full-length antibody or a fragment thereof.

[00537] 3. The vector of claim 2, wherein the antibody is a monoclonal antibody, a single chain antibody, a Fab' fragment, or a single domain antibody (dAb).

[00538] 4. The vector according to any one of claims. 1-3, in which the DNA vector contains a promoter operably linked to a first transgene encoding a secretory sequence and a heavy chain protein, and a second promoter operably linked to a second transgene encoding a light chain protein.

[00539] 5. The vector according to any one of claims. 1-4, in which the transgene encodes a single chain variable fragment (scFv) fusion protein.

[00540] 6. The vector according to any one of claims. 1-5, wherein the antibody is an antibody selected from Tables 1-5.

[00541]. Vector according to any one of paragraphs. 1-6, wherein the ITR contains a functional end-resolution site and the Rep-binding site is a wild-type AAB ITR, or wherein the two ITRs are symmetrical or substantially symmetrical, or wherein the two ITRs are asymmetrical, or wherein the two ITRs selected from any listed in Tables 7, 9A, 9B and 10.

[00542] 8. The vector of claim 1, wherein the antibody is aducanumab.

[00543] 9. The vector of any one of the preceding claims, wherein the antibody is a human or humanized antibody.

[00544] 10. The vector of any one of the preceding claims, wherein the antibody is an IgG, IgA, IgD, IgM, or IgE antibody.

[00545] 11. A method of expressing an antibody in a cell or its population, including introducing into the cell or its population an effective amount of a DNA vector according to claims. 1-10 and culturing the cell or population thereof under conditions necessary for expression of the antibody in the cell.

[00546] 12. The method of claim 11, wherein the DNA vector or cccDNA vector is administered in combination with a pharmaceutically acceptable carrier.

[00547] 13. The method of claim 11 or claim 12, wherein the antibody or fragment thereof is secreted from the cell.

[00548] 14. The method according to any one of paragraphs. 11-13, in which the antibody or its fragment is retained intracellularly in the form of an intrabody.

[00549] 15. The method according to any one of paragraphs. 11-14, in which the cell is a mammalian cell.

[00550] 16. The method of claim 15, wherein the cell is a human cell.

[00551] 17. The method of claim 15, wherein the antibody is isolated from the cell and purified.

[00552] 18. A method of delivering a therapeutic antibody to a subject, comprising: administering to the subject a composition containing a DNA vector according to claims. 1-10.

[00553] 19. A method of treating a disease in a subject, comprising: administering to the subject a composition containing a cccDNA vector according to claims. 1-10, thereby resulting in expression of the therapeutic antibody in the subject and treatment of the disease.

[00554] 20. The method of claim 18 or claim 19, wherein the cccDNA vector is administered in combination with a pharmaceutically acceptable carrier.

[00555] 21. The method according to any one of paragraphs. 18-20, in which the therapeutic antibody is secreted from the cell in which it was expressed.

[00556] 22. The method according to any one of paragraphs. 18-22, in which the therapeutic antibody is retained in the cell in which it was expressed.

[00557] 23. The method according to any one of paragraphs. 11-17, in which the cell or population thereof is cultured in a bioreactor.

[00558] 24. A composition comprising a vector according to any one of claims. 1-10 for use in treating a disease in a subject.

[00559] 25. Use of a composition containing a vector according to any one of claims. 1-10, for treating a disease in a subject.

[00560] 26. Use of a composition containing a vector according to any one of claims. 1-10, for preparing a medicament for treating a disease in a subject.

EXAMPLES

[00561] The following examples are provided by way of illustration and not limitation. One skilled in the art will appreciate that cccDNA vectors can be constructed from any of the wild-type ITRs or modified ITRs described herein, and that the following example methods can be used to construct and evaluate the activity of such ccDNA vectors. Although the methods are illustrated with specific cDNA vectors, they are applicable to any cDNA vector as described.

Example 1: Construction of cDNA vectors using an insect cell-based method.

[00562] The production of cccDNA vectors using a polynucleotide construct template is described in Example 1 of PCT/US18/49996, which is incorporated herein by reference in its entirety. For example, the polynucleotide construct template used to produce the cDNA vectors of the present invention may be a cDNA plasmid, a cDNA bacmid, and/or a cDNA baculovirus. Without being limited by theory, in a permissive host cell, in the presence of, for example, Rep, a template polynucleotide construct having two symmetrical ITRs and an expression construct, at least one of the ITRs modified relative to the wild-type ITR sequence, is replicated to produce cccDNA vectors. Production of a cccDNA vector occurs in two stages: firstly, excision (“rescue”) of the template from the matrix backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) through Rep proteins and, secondly, -second, Rep-mediated replication of the excised cDNA vector.

[00563] A typical example of a method for producing cccDNA vectors is the use of a ccDNA plasmid, as described herein. In FIG. 1A and 1B, the polynucleotide construct template of each ccDNA plasmid includes both a left modified ITR and a right modified ITR, with the following sequences between the ITRs: (i) enhancer/promoter; (ii) transgene cloning site; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (eg, from the bovine growth hormone gene (BGHpA)). Unique restriction endonuclease recognition sites (R1-R6) (shown in Fig. 1A and Fig. 1B ) were also introduced between all components to facilitate the introduction of new genetic components into specific sites of the construct. Enzyme sites R3 (PmeI) GTTTAAAC (SEQ ID NO: 123) and R4 (PacI) TTAATTAA (SEQ ID NO: 124) are inserted into the cloning site to introduce the open reading frame of the transgene. These sequences were cloned into plasmid pFastBac HT B obtained from ThermoFisher Scientific.

[00564] Production of cccDNA-bactmid:

[00565] DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells, Thermo Fisher) were transformed with test or control plasmids according to the protocol according to the manufacturer's instructions. To obtain recombinant cccDNA bacmids, recombination between the plasmid and the baculovirus shuttle vector was induced in DH10Bac cells. Recombinant bacteria were selected by positive selection screening based on blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics for selection of transformants and maintenance of bacmids and transposase plasmids. White colonies resulting from transposition that disrupts the β-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00566] Recombinant cccDNA bacteria were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 ml of medium in T25 flasks at 25 °C. After four days, the culture medium (containing P0 virus) was separated from the cells and filtered through a 0.45 μm filter, separating infectious baculovirus particles from cells or cellular debris.

[00567] Optionally, first generation Rep baculovirus (P0) was amplified by infecting naïve insect cells with Sf9 or Sf21, and cultured in 50-500 ml of medium. Cells were maintained in suspension cultures in an orbital shaker incubator at 130 rpm at 25°C, monitoring cell diameter and viability until cells reached a diameter of 18–19 nm (from a naïve diameter of 14–15 nm) and a density of ca. 4.0E+6 cells/ml. Between 3 and 8 days postinfection, baculovirus P1 particles in the medium were collected after centrifugation to remove cells and debris and then filtered through a 0.45-μm filter.

[00568] The cDNA baculovirus containing the test constructs was collected and the infectivity or titer of the baculovirus was determined. More specifically, four 20 ml Sf9 cell cultures at 2.5E+6 cells/ml were treated with baculovirus P1 at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated at 25-27 °C. Infectivity was determined by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability, every day for 4-5 days.

[00569] "Rep plasmid" as disclosed in FIG. 8A of application PCT/US18/49996, which is incorporated herein by reference in its entirety, was prepared in the expression vector pFASTBAC ^TM -Dual (ThermoFisher) containing either Rep78 (SEQ ID NO: 131 or 133) and Rep52 (SEQ ID NO: : 132), or Rep68 (SEQ ID NO: 130) and Rep40 (SEQ ID NO: 129). The Rep plasmid was transformed into DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells (Thermo Fisher)) according to the protocol provided by the manufacturer. Recombination between the Rep plasmid and the baculovirus shuttle vector was induced in DH10Bac cells to generate recombinant bactmids (“Rep-bactmid”). Recombinant bacmids were selected by positive selection, which included blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. Isolated white colonies were collected and inoculated into 10 ml of selection medium (kanamycin, gentamicin, tetracycline in Luria-Bertani (LB) broth). Recombinant bacmids (Rep bacmids) were isolated from E. coli, and Rep bacmids were transfected into Sf9 or Sf21 insect cells to produce infectious baculovirus.

[00570] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days and an infectious recombinant baculovirus (“Rep-baculovirus”) was isolated from the culture. Optionally, first generation Rep baculovirus (P0) was amplified by infecting naïve insect Sf9 or Sf21 cells and cultured in 50-500 ml of medium. Between 3 and 8 days postinfection, baculovirus P1 particles in the medium were collected by isolating cells either by centrifugation, filtration, or other fractionation process. Rep-baculovirus was collected and the infectivity of the baculovirus was determined. In particular, four 20 ml Sf9 cell cultures at 2.5×10 ⁶ cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability, every day for 4-5 days.

[00571] Preparation and characterization of cccDNA vector

[00572] As indicated in FIG. 4B , Sf9 insect cell culture medium containing either (1) a sample containing cccDNA bacmid or cccDNA baculovirus or (2) Rep baculovirus described above was then added to a fresh Sf9 cell culture (2.5E+6 cells /ml, 20 ml) in a ratio of 1:1000 and 1:10000, respectively. Cells were then cultured at 130 rpm at 25°C. Cell diameter and viability were determined 4–5 days after coinfection. When the cell diameter reached 18-20 nm with a viability of approx. 70-80%, cell cultures were centrifuged, the medium was removed and cell pellets were collected. Cell pellets were first resuspended in a sufficient volume of aqueous medium, either water or buffer. The cccDNA vector was isolated and purified from cells using the Qiagen MIDI PLUS™ purification protocol (Qiagen, treatment 0.2 mg cell pellet per column).

[00573] Yields of cccDNA vectors derived from Sf9 insect cells and purified were initially determined based on UV absorbance at 260 nm.

[00574] Evaluation of cccDNA vectors can be accomplished by identification by agarose gel electrophoresis under native or denaturing conditions, as shown in FIG. 4D , with (a) the presence of characteristic bands on denaturing gels migrating at twice the size compared to native gels after restriction endonuclease digestion and gel electrophoretic analysis, and (b) the presence of monomeric and dimeric (2x) bands on denaturing gels for undigested material is a characteristic feature of the presence of the cDNA vector.

[00575] The structures of the isolated cccDNA vectors were further analyzed by digesting DNA obtained from coinfected Sf9 cells (as described herein) with restriction endonucleases selected for a) the presence of only one cleavage site in the cccDNA vectors, and b ) the formation of fragments large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel (>800 bp). As shown in FIG. 4D and 4E , linear discontinuous DNA vectors and linear and continuous cccDNA vectors may vary in the size of their reaction products—for example, a discontinuous DNA vector is expected to produce 1 kb fragments. and 2 kb, while for a cDNA vector with a continuous structure the formation of fragments of 2 kb is expected. and 4 kb.

[00576] Thus, in order to qualitatively demonstrate that the isolated cccDNA vectors have covalently closed ends, as required by definition, samples were digested with a restriction endonuclease identified, in the context of the sequence of the particular DNA vector, as having a single restriction site, preferably resulting in two cleavage products of unequal size (eg, 1000 bp and 2000 bp). After digestion and electrophoresis in a denaturing gel (which separates two complementary DNA strands), linear DNA that does not have covalently closed ends will be separated into 1000 bp fragments. and 2000 bp, while covalently closed DNA (i.e. cDNA vector) will be divided into fragments 2 times larger (2000 bp and 4000 bp), since two chains The DNA is linked and, when unrolled, will be twice as long (even though it is single-stranded). In addition, cleavage of the monomeric, dimeric, and n- mer forms of DNA vectors will in all cases produce fragments of the same size due to the end-to-end binding of multimeric DNA vectors (see Fig. 4D ).

[00577] As used herein, the phrase “assay for identification of DNA vectors by agarose gel electrophoresis under native gel and denaturing conditions” refers to an assay for assessing the degree of closure of ccDNA by performing restriction endonuclease digestion followed by electrophoretic evaluation of the digestion products. The following is one such example of analysis, although one of ordinary skill in the art will appreciate that many variations of this example known in the art are possible. The restriction endonuclease is selected to be an enzyme that makes a single cut of the cDNA vector of interest, which will generate products having a length of approximately 1/3 and 2/3 of the length of the DNA vector. This ensures separation of bands on both the native and denaturing gels. It is important to remove the buffer from the sample before denaturation. A Qiagen PCR product purification kit or desalting “spun columns,” such as the GE Healthcare ILUSTRA™ MicroSpin™ G-25 columns, are some of the options known in the art for endonuclease digestion. The analysis involves, for example, i) digestion of DNA with suitable restriction endonuclease(s), 2) application, for example, to a Qiagen PCR product purification kit, eluting with distilled water, iii) addition of a 10× denaturing solution (10× = 0.5 M NaOH, 10 mM EDTA), adding 10X unbuffered dye solution and running the assay together with DNA molecular weight markers obtained by adding 10X denaturing solution in a 4 replicate experiment, at 0.8-1.0 % gel, pre-incubated with 1 mM EDTA and 200 mM NaOH to ensure homogeneity of NaOH concentration in the gel and gel chamber, and running the gel in the presence of 1× denaturing solution (50 mM NaOH, 1 mM EDTA). One skilled in the art will understand what voltage to use to perform electrophoresis based on the size and desired time to obtain results. After electrophoresis, the gels are dried and neutralized in 1× TBE or TAE and transferred to distilled water or 1× TBE/TAE with 1× SYBR Gold. The bands can then be visualized, for example, using Thermo Fisher's SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm).

[00578] The purity of the resulting cDNA vector can be assessed using any method known in the art. As one non-limiting example of a method, the contribution of the cDNA plasmid to the total UV absorbance of the sample can be assessed by comparing the fluorescence intensity of the cDNA vector with a standard. For example, if, based on UV absorbance determination, 4 μg of ccDNA vector was loaded onto the gel, and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 μg, then this corresponds to the presence of 1 μg of cDNA vector, and the cDNA vector constitutes 25% of the total amount of UV absorbing material. The intensity of the band on the gel is then plotted against the calculated input amount to which the band corresponds - for example, if the full ccDNA vector is 8 kb and the excised reference band is 2 kb, then the intensity of the band is on the graph will be 25% of the total amount administered, which in this case will correspond to 0.25 μg when 1.0 μg is administered. Using titration of the cccDNA vector plasmid to construct a calibration curve, the amount of ccDNA vector band is then calculated from a linear regression equation, which can then be used to determine the percentage of the ccDNA vector in the total input, or percentage purity.

[00579] For purposes of comparison, Example 1 describes the preparation of cccDNA vectors using an insect cell-based method and a polynucleotide construct template, which is also described in Example 1 of PCT/US18/49996, incorporated herein by reference in its entirety. For example, the template polynucleotide construct used to produce the ccDNA vectors of the present invention in accordance with Example 1 may be a ccDNA plasmid, a ccDNA bacmid, and/or a ccDNA baculovirus. Without being limited by theory, in a permissive host cell, in the presence of, for example, Rep, a template polynucleotide construct having two symmetrical ITRs and an expression construct, at least one of the ITRs modified relative to the wild-type ITR sequence, is replicated to produce cccDNA vectors. Production of a cccDNA vector occurs in two stages: firstly, excision (“rescue”) of the template from the matrix backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) through Rep proteins and, secondly, -second, Rep-mediated replication of the excised cDNA vector.

[00580] An example of a method for producing cccDNA vectors using an insect cell is its production from a ccDNA plasmid, as described herein. In FIG. 1A and 1B , the polynucleotide construct template of each ccDNA plasmid includes both a left modified ITR and a right modified ITR, with the following sequences between the ITRs: (i) enhancer/promoter; (ii) transgene cloning site; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (eg, from the bovine growth hormone gene (BGHpA)). Also, unique restriction endonuclease recognition sites (R1-R6) (depicted in Fig. 1A and Fig. 1B ) were introduced between all components to facilitate the introduction of new genetic components into specific sites of the construct. Enzyme sites R3 (PmeI) GTTTAAAC (SEQ ID NO: 123) and R4 (PacI) TTAATTAA (SEQ ID NO: 124) were inserted into the cloning site to introduce the open reading frame of the transgene. These sequences were cloned into plasmid pFastBac HT B obtained from ThermoFisher Scientific.

[00581] Production of cccDNA-bactmid:

[00582] DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells, Thermo Fisher) were transformed with test or control plasmids according to the protocol in accordance with the manufacturer's instructions. To obtain recombinant cccDNA bacmids, recombination between the plasmid and the baculovirus shuttle vector was induced in DH10Bac cells. Recombinant bacteria were selected by positive selection screening based on blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics for selection of transformants and maintenance of bacmids and transposase plasmids. White colonies resulting from transposition that disrupts the β-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00583] Recombinant cccDNA bacteria were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 ml of medium in T25 flasks at 25 °C. After four days, the culture medium (containing P0 virus) was separated from the cells and filtered through a 0.45 μm filter, separating infectious baculovirus particles from cells or cellular debris.

[00584] Optionally, first generation Rep baculovirus (P0) was amplified by infecting naïve insect cells with Sf9 or Sf21, and cultured in 50-500 ml of medium. Cells were maintained in suspension cultures in an orbital shaker incubator at 130 rpm at 25°C, monitoring cell diameter and viability until cells reached a diameter of 18–19 nm (from a naïve diameter of 14–15 nm) and a density of ca. 4.0E+6 cells/ml. Between 3 and 8 days postinfection, baculovirus P1 particles in the medium were collected after centrifugation to remove cells and debris and then filtered through a 0.45-μm filter.

[00585] The cDNA baculovirus containing the test constructs was collected and the infectivity or titer of the baculovirus was determined. More specifically, four 20 ml Sf9 cell cultures at 2.5E+6 cells/ml were treated with baculovirus P1 at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated at 25-27 °C. Infectivity was determined by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability, every day for 4-5 days.

[00586] The "Rep plasmid" was generated in the expression vector pFASTBAC ^TM -Dual (ThermoFisher) containing either Rep78 (SEQ ID NO: 131 or 133) and Rep52 (SEQ ID NO: 132) or Rep68 (SEQ ID NO: 130) and Rep40 (SEQ ID NO: 129). The Rep plasmid was transformed into DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells (Thermo Fisher)) according to the protocol provided by the manufacturer. Recombination between the Rep plasmid and the baculovirus shuttle vector was induced in DH10Bac cells to generate recombinant bactmids (“Rep-bactmid”). Recombinant bacmids were selected by positive selection, which involved blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. Isolated white colonies were collected and inoculated into 10 ml of selection medium (kanamycin, gentamicin, tetracycline in Luria-Bertani (LB) broth). Recombinant bacmids (Rep bacmids) were isolated from E. coli, and Rep bacmids were transfected into Sf9 or Sf21 insect cells to produce infectious baculovirus.

[00587] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days and an infectious recombinant baculovirus (“Rep-baculovirus”) was isolated from the culture. Optionally, first generation Rep baculovirus (P0) was amplified by infecting naïve insect Sf9 or Sf21 cells and cultured in 50-500 ml of medium. Between 3 and 8 days postinfection, baculovirus P1 particles in the medium were collected by isolating cells either by centrifugation, filtration, or other fractionation process. Rep-baculovirus was collected and the infectivity of the baculovirus was determined. In particular, four 20 ml Sf9 cell cultures at 2.5×10 ⁶ cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability, every day for 4-5 days.

[00588] Preparation and characterization of cDNA vector

[00589] Sf9 insect cell culture medium containing either (1) a sample containing cccDNA bacmid or cccDNA baculovirus and (2) Rep baculovirus described above was then added to a fresh Sf9 cell culture (2.5E+6 cells/ml, 20 ml) in a ratio of 1:1000 and 1:10000, respectively. Cells were then cultured at 130 rpm at 25°C. Cell diameter and viability were determined 4–5 days after coinfection. When the cell diameter reached 18-20 nm with a viability of approx. 70-80%, cell cultures were centrifuged, the medium was removed and cell pellets were collected. Cell pellets were first resuspended in a sufficient volume of aqueous medium, either water or buffer. The cccDNA vector was isolated and purified from cells using the Qiagen MIDI PLUS™ purification protocol (Qiagen, treatment 0.2 mg cell pellet per column).

[00590] Yields of cccDNA vectors derived from Sf9 insect cells and purified were initially determined based on UV absorbance at 260 nm. The correct configuration of purified closed-end cccDNA vectors can be assessed using the electrophoretic methodology described in Example 5.

EXAMPLE 2: Production of synthetic cccDNA by excision of double-stranded DNA from a molecule

[00591] A synthetic method for producing cccDNA vectors is described in Examples 2-6 of International Application PCT/US19/14122, filed January 18, 2019, which is incorporated herein by reference in its entirety. One typical example of a method for producing a cDNA vector using a synthetic method involves cutting out a double-stranded DNA molecule. Briefly, a cDNA vector can be produced using a double-stranded DNA construct, for example, see FIG. 7A-8E of application PCT/US19/14122. In some embodiments, the double-stranded DNA construct is a ccDNA plasmid, see, for example , FIG. 6 of international patent application PCT/US2018/064242, filed December 6, 2018).

[00592] In some embodiments, the cDNA vector construct comprises a regulatory switch, as described herein.

[00593] For illustrative purposes, Example 2 describes the preparation of cccDNA vectors as examples of closed-end DNA vectors prepared using this method. However, although cccDNA vectors are described in this Example as an example illustrating in vitro synthetic production methods for producing a closed-end DNA vector by excision of a double-stranded polynucleotide containing an ITR and an expression cassette (e.g., a heterologous nucleic acid sequence), followed by ligation free 3' and 5' ends as described herein, one skilled in the art will know that, as stated above, a double-stranded DNA polynucleotide molecule can be modified to produce any desired closed-end DNA vector. including, without limitation, doggybone DNA, dumbbell DNA, and the like. Representative examples of cccDNA vectors for the production of antibodies or fusion proteins that can be produced by the synthesis method described in Example 2 are discussed in the section entitled “III. cccDNA vectors in general.” Representative examples of antibodies and fusion proteins expressed by cccDNA vectors are described in the section entitled “II.C. Typical examples of antibodies and fusion proteins expressed by cccDNA vectors.”

[00594] The method includes (i) excision of a sequence encoding an expression cassette from a double-stranded DNA construct, and (ii) formation of hairpin structures in one or more ITRs, and (iii) joining the free 5' and 3' ends by ligation, for example, using T4 DNA ligase.

[00595] The double-stranded DNA construct contains, in order from 5' to 3': a first restriction endonuclease site; left (5' direction) ITR; expression cassette right (in the 3' direction) ITR; and a second restriction endonuclease site. The double-stranded DNA construct is then contacted with one or more restriction endonucleases to generate double-strand breaks at both restriction endonuclease sites. Both sites may be targeted by a single endonuclease, or each site may be targeted by a separate endonuclease, provided that the restriction sites are not part of the cccDNA vector template. This results in the excision of the sequence between the restriction endonuclease sites from the rest of the double-stranded DNA construct (see FIG. 9 of PCT/US19/14122). After ligation, a DNA vector with closed ends is formed.

[00596] One or both of the ITRs used in the method may be wild-type ITRs. Modified ITRs may also be used, and the modification may include deletion, insertion or substitution of one or more nucleotides from the wild-type ITR in the sequences forming arms B and B' and/or arms C and C' (see, for example, Fig. 6- 8 and 10, Fig. 11B of application PCT/US19/14122), and may have two or more hairpin loops (see, for example , Fig. 6-8, Fig. 11B of application PCT/US19/14122) or one hairpin loop ( see, for example, Fig. 10A-10B, Fig. 11B of application PCT/US19/14122). A modified hairpin loop ITR can be produced by genetic modification of an existing oligonucleotide (oligo) or by biological and/or chemical de novo synthesis.

[00597] In a non-limiting example, the left and right ITR-6 (SEQ ID NOs: 111 and 112) include deletions of 40 nucleotides in arms B-B' and C-C' compared to the wild-type AAB2 ITR. The nucleotides remaining in the modified ITR are predicted to form a single hairpin structure. The Gibbs free energy of structure expansion is about -54.4 kcal/mol. Other ITR modifications can also be made, including optional deletion of a functional Rep binding site or Trs site.

EXAMPLE 3 : Production of ccDNA by Design of Oligonucleotides

[00598] Another example of a method for producing a cccDNA vector using a synthetic method involving the assembly of various oligonucleotides is presented in Example 3 of PCT/US19/14122, in which a ccDNA vector is prepared by synthesizing 5' and 3' ITR oligonucleotides and ligation ITR oligonucleotides with a double-stranded polynucleotide containing an expression cassette. Fig. 11B of application PCT/US19/14122 shows an example of a method for ligating a 5'-ITR oligonucleotide and a 3'-ITR oligonucleotide to a double-stranded polynucleotide containing an expression cassette.

[00599] As disclosed herein, ITR oligonucleotides may comprise WT-ITR (eg, see Figure 3A , Figure 3C ) or modified ITRs (eg, see Figure 3B and Figure 3D ). (See also, for example, Figs. 6A, 6B, 7A and 7B of PCT/US19/14122, which is incorporated herein in its entirety.) Exemplary ITR oligonucleotides include, but are not limited to, SEQ ID NO: 134-145 (eg, see Table 7 in PCT/US19/14122). Modified ITRs may include the deletion, insertion or substitution of one or more nucleotides relative to the wild-type ITR in the sequences forming arms B and B' and/or arms C and C'. ITR oligonucleotides containing WT-ITR or mod-ITR, as described herein, intended for use in cell-free synthesis, can be obtained by genetic modification or biological and/or chemical synthesis. As described herein, the ITR oligonucleotides in Examples 2 and 3 may contain WT-ITR or modified ITR (mod-ITR) in symmetric or asymmetric configurations, as discussed herein.

EXAMPLE 4: Production of ccDNA using a single-stranded DNA molecule

[00600] Another example of a method for producing a cccDNA vector using a synthetic method, presented in Example 4 of PCT/US19/14122, uses single-stranded linear DNA containing two sense ITRs that flank a sense expression cassette sequence and are covalently attached to two antisense ITRs , which flank the antisense expression cassette, and the ends of the single-stranded linear DNA are then ligated to form a single-stranded molecule with closed ends. One non-limiting example involves synthesizing and/or producing a single-stranded DNA molecule, annealing the portions of the molecule to form a single linear DNA molecule that has one or more base-paired secondary structure regions, and then ligating the free 5' and 3' ends to each other to form a closed single-chain molecule.

[00601] A typical single-stranded DNA molecule for producing a cDNA vector contains, in the 5' to 3' direction:

first semantic ITR;

semantic sequence of the expression cassette;

second semantic ITR;

second antisense ITR;

antisense sequence of the expression cassette; And

first antisense ITR.

[00602] The single-stranded DNA molecule for use in an exemplary method of Example 4 can be prepared using any DNA synthesis methodology described herein, for example, in vitro DNA synthesis, or by digestion of a DNA construct (eg, a plasmid) with nucleases and melting the resulting dsDNA fragments to form single-stranded DNA (ssDNA) fragments.

[00603] Annealing can be accomplished by lowering the temperature to below the calculated melting temperatures of sense and antisense sequence pairs. The melting point depends on the content of the specific nucleotide bases and the characteristics of the solution used, such as salt concentration. One skilled in the art can readily calculate melting temperatures for any given combination of sequence and solution.

[00604] The free 5' and 3' ends of the annealed molecule can be ligated to each other or ligated to a hairpin molecule to form a cccDNA vector. Typical examples of suitable ligation techniques and hairpin molecules are described in Examples 2 and 3.

EXAMPLE 5: Purification and/or confirmation of cccDNA production

[00605] Any of the DNA vector products produced by the methods described herein, for example, including the insect cell-based production methods described in Example 1, or the synthetic production methods described in Examples 2-4, can be purified, for example , to remove impurities, unused components, or by-products using methods generally known to those skilled in the art; and/or can be analyzed to confirm that the produced DNA vector (in this case, the ccDNA vector) is the desired molecule. An example of a method for purifying a DNA vector, such as cccDNA, is using the Qiagen Midi Plus purification protocol (Qiagen) and/or gel purification.

[00606] The following is a typical example of a method for confirming the identity of cccDNA vectors.

[00607] Evaluation of cccDNA vectors can be accomplished by identification by agarose gel electrophoresis under native or denaturing conditions, as shown in FIG. 4D , with (a) the presence of characteristic bands on denaturing gels migrating at twice the size compared to native gels after restriction endonuclease digestion and gel electrophoretic analysis, and (b) the presence of monomeric and dimeric (2x) bands on denaturing gels for undigested material is a characteristic feature of the presence of the cDNA vector.

[00608] The structures of the isolated cccDNA vectors were further analyzed by digesting the purified DNA with restriction endonucleases selected to ensure a) the presence of only one cut site in the ccDNA vectors, and b) the resulting fragments are large enough to be clearly visible when fractionated into 0.8% denaturing agarose gel (>800 bp). As shown in FIG. 4C and 4D , linear discontinuous DNA vectors and linear and continuous cccDNA vectors can be distinguished by the size of their reaction products—for example, a discontinuous DNA vector is expected to produce 1 kb fragments. and 2 kb, while for a cDNA vector with a continuous structure one would expect the formation of fragments of 2 kb in size. and 4 kb.

[00609] Thus, in order to qualitatively demonstrate that the isolated cccDNA vectors have covalently closed ends, as required by definition, samples were digested with a restriction endonuclease identified, in the context of the sequence of the particular DNA vector, as having a single restriction site, preferably resulting in two cleavage products of unequal size (eg, 1000 bp and 2000 bp). After digestion and electrophoresis in a denaturing gel (which separates two complementary DNA strands), linear DNA that does not have covalently closed ends will be separated into 1000 bp fragments. and 2000 bp, while covalently closed DNA (i.e. cDNA vector) will be divided into fragments 2 times larger (2000 bp and 4000 bp), since two chains The DNA is linked and when unrolled will be twice as long (but single stranded). Additionally, digestion of the monomeric, dimeric, and n -mer forms of DNA vectors will in all cases produce fragments of the same size due to the end-to-end binding of multimeric DNA vectors (see Fig. 4E ).

[00610] As used herein, the expression “assay for identification of DNA vectors by agarose gel electrophoresis under native gel and denaturing conditions” refers to an assay for assessing the degree of closure of ccDNA by performing restriction endonuclease digestion followed by electrophoretic assessment of the digestion products. The following is one such example of analysis, although one of ordinary skill in the art will appreciate that many variations of this example known in the art are possible. The restriction endonuclease is selected to be an enzyme that makes a single cut of the cDNA vector of interest, which will generate products having a length of approximately 1/3 and 2/3 of the length of the DNA vector. This ensures separation of bands on both the native and denaturing gels. It is important to remove the buffer from the sample before denaturation. A Qiagen PCR product purification kit or desalting “spun columns,” such as the GE Healthcare ILUSTRA™ MicroSpin™ G-25 columns, are some of the options known in the art for endonuclease digestion. The analysis involves, for example, i) digestion of DNA with suitable restriction endonuclease(s), 2) application, for example, to a Qiagen PCR product purification kit, eluting with distilled water, iii) addition of a 10× denaturing solution (10× = 0.5 M NaOH, 10 mM EDTA), adding 10X unbuffered dye solution and running the assay together with DNA molecular weight markers obtained by adding 10X denaturing solution in a 4 replicate experiment, at 0.8-1.0 % gel, pre-incubated with 1 mM EDTA and 200 mM NaOH to ensure homogeneity of NaOH concentration in the gel and gel chamber, and running the gel in the presence of 1× denaturing solution (50 mM NaOH, 1 mM EDTA). One skilled in the art will understand what voltage to use to perform electrophoresis based on the size and desired time to obtain results. After electrophoresis, the gels are dried and neutralized in 1× TBE or TAE and transferred to distilled water or 1× TBE/TAE with 1× SYBR Gold. The bands can then be visualized, for example, using Thermo Fisher's SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm). The above gel-based method can be adapted for purification purposes by isolating the cDNA vector from the gel band and allowing it to be renatured.

[00611] The purity of the resulting cccDNA vector can be assessed using any method known in the art. As one non-limiting example of a method, the contribution of the cDNA plasmid to the total UV absorbance of the sample can be assessed by comparing the fluorescence intensity of the cDNA vector with a standard. For example, if, based on UV absorbance determination, 4 μg of ccDNA vector was loaded onto the gel, and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 μg, then this corresponds to the presence of 1 μg of cDNA vector, and the cDNA vector constitutes 25% of the total amount of UV absorbing material. The intensity of the band on the gel is then plotted against the calculated input amount to which the band corresponds - for example, if the full ccDNA vector is 8 kb and the excised reference band is 2 kb, then the intensity of the band is on the graph will be 25% of the total amount administered, which in this case will correspond to 0.25 μg when 1.0 μg is administered. Using titration of the cccDNA vector plasmid to construct a calibration curve, the amount of ccDNA vector band is then calculated from a linear regression equation, which can then be used to determine the percentage of the ccDNA vector in the total input, or percentage purity.

EXAMPLE 6: Preparation of antibodies or fusion proteins from cDNA plasmid

[00612] To demonstrate the ability of the cDNA vector to express an antibody, cDNA plasmids encoding the monoclonal antibody aducanumab were generated.

[00613] Aducanumab is a human monoclonal antibody that is being studied for the treatment of Alzheimer's disease by targeting aggregated forms of amyloid beta protein. In FIG. 6A shows a representative example of a plasmid that was generated and determined to express full-length aducanumab. Fig. 6B shows a design of a ccDNA vector that can be produced using the ccDNA plasmid of FIG. 6A . The expression cassette in FIG. 6B shows a dual promoter system, with a different promoter used for each of the heavy chain and light chain of the anti-aducanumab antibody. The cccDNA plasmid used to generate the aducanumab antibody includes a unique combination of heavy and light chain promoters that ensures the proper ratio of heavy and light chains to produce a functional antibody. One skilled in the art will appreciate how to select promoters for each of the light and heavy chains of a desired antibody to produce the desired ratios of heavy and/or light chains expressed to facilitate efficient production of an intact antibody.

[00614] a ccDNA plasmid, referred to as “pFBdual-ccDNA-aducanumab plasmid” or “ccDNA-IgG plasmid”, as depicted in FIG. 6A was prepared as described in Example 1 and used to transiently transfect 293 cells. 293-6E cells were grown in serum-free FreeStyle™ 293 Expression Medium (Life Technologies, Carlsbad, CA, USA). Cells were maintained in Erlenmeyer flasks (Corning Inc., Acton, MA) at 37 °C with 5% CO ₂ on an orbital shaker (VWR Scientific, Chester, PA). One day before transfection, cells were seeded at the appropriate density in Corning Erlenmeyer flasks. On the day of transfection, DNA and transfection reagent were mixed in the optimal ratio and then added to the flask containing cells ready for transfection. The recombinant plasmid encoding the target protein was transiently transfected into a 293-6E cell culture suspension. Cell density and viability at day 2, day 4 and day 5 after transfection are shown in Table 13 . Cell culture supernatant samples collected on day 2, day 4, and day 5 were used to assess protein expression. Cell culture supernatant collected at day 6 posttransfection was used for purification.

[00615]

Table 13: Cell Density and Viability of ceTTX-IgG1-Adu in 293-6E Cells Sample Density (x10 ⁶ cells/ml) Viability (%) U8948CK300-1, day 2 1.80 95.36 U8948CK300-1, day 4 2.36 83.98 U8948CK300-1, day 5 2.20 72.28

[00616] Antibody Expression and Purification: To assess protein expression levels, cell culture supernatants were collected on days 2, 4, and 5 posttransfection and analyzed by SDS-PAGE and Western blotting (data not shown). To purify the protein, the cell culture broth was centrifuged and the supernatant was filtered. The filtered supernatant was loaded onto a prepacked 1 mL Monofinity A Resin column (GenScript) at a rate of 1.0 mL/min, followed by washing and elution with appropriate buffers. The eluted fractions were pooled and the buffer was exchanged to phosphate-buffered saline (PBS), pH 7.2. Purified protein was analyzed by SDS-PAGE ( Figure 8A ), Western blotting ( Figure 8B ), and gel permeation HPLC (SEC-HPLC) (data not shown) using standard protocols for measuring molecular weight, yield, and purity.

[00617] In general, aducanumab was expressed from a cDNA vector produced from the plasmid pFBdual-ccDNA-aducanumab in 293-6E cells grown in suspension culture. The antibody was expressed at levels determined by SDS-PAGE analysis ( Fig. 8A ). After one-step purification, the antibody was detected with calculated molecular weights of ca. 55 kDa and approx. 25 kDa under reducing conditions and ca. 150 kDa under non-reducing conditions ( Figure 8B ). These data indicate that the heavy and light chains of the antibody self-assemble into a full-length antibody in this system.

EXAMPLE 7: Large-Scale Production of Recombinant Antibody in vitro

[00618] A cccDNA plasmid containing the aducanumab heavy chain and light chain coding sequences can be prepared as previously described in Examples 1 and 5 and transfected into a large-scale suspension culture, for example, FreeStyle ^TM 293-F cells (R790-07, Life Technologies), which have been adapted for growth under serum-free conditions. Adapted for suspension and serum-free culture, FreeStyleTM293-F cells (Life Technologies) are cultured in FreeStyleTM293 expression medium (Life Technologies) in sterile 125 ml Erlenmeyer flasks with vented stoppers (Sigma) at densities in the range of 1-5×10 ⁵ viable cells/ml , while rotating at 135 rpm on the orbital shaker platform. Each 125 mL flask containing 30 mL of cells at a concentration of 1 x 10 ⁶ viable cells/mL was transfected with Fugene6 cccDNA transfection reagent (Fugene6:ccDNA 3:1) according to the manufacturer's instructions. 24 hours after transfection, selection reagent (50 mg/ml) is added to the cells and the cells are maintained in selection mode for 2 weeks at densities in the range of 2-5×10 ⁵ viable cells/ml, followed by expansion in the flask for a 1 L shaker (Sigma), a 1 L centrifuge beaker (Sigma), or a 5 L WAVE bioreactor (GE Healthcare). Samples are collected every 48 hours and IgG expression levels are determined by anti-human IgG ELISA. At peak expression, culture supernatants were collected, centrifuged at 1000 × g for 15 min, passed through 0.45 μm (mm) filters (Sartorius), and stored at 4°C with 0.1% sodium azide (Sigma) until use. Antibodies can be purified by affinity chromatography on a 5 mL HiTrap Protein-G HP column (GE Healthcare) using the ÄKTA Prime system (GE Healthcare) and 0.2 µm filter buffers. For example, the column is equilibrated with 10 column volumes (CV) of phosphate saline (PBS) wash buffer (pH 7.0) and loaded with supernatant at a flow rate of 2 ml/min, followed by 10 column volumes of wash buffer. The antibody is eluted with 0.2 M glycine buffer (pH 2.3) and 2.5 ml fractions are collected in tubes containing 0.5 ml of 1 M Tris-HCl pH 8.6 for neutralization.

EXAMPLE 8: Preparation of cccDNA vector expressing full-length aducanumab and expression of antibody from ccDNA in vitro

[00619] A cccDNA plasmid or a ccDNA vector that expresses aducanumab or GFP was prepared as described in Examples 1 and 5 using the ccDNA plasmid Adu-Full-IgG1 (plasmid pFBdual-ccDNA-adecanumab or ccDNA-IgG plasmid) ( FIG. 6A ) prepared according to Example 1 . The yields of the resulting and purified ccDNA vector (referred to as “ccDNA IgG”) or ccDNA plasmid were determined based on UV absorbance at 260 nm. The cDNA vector or cDNA plasmid was transfected into HEK293T cells using Lipofectamine™ 3000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After 72 hours, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, and whole cell supernatant was collected and concentrated using filters (Amicon). The resulting antibody production was analyzed by Western blotting. Cell supernatants and lysate samples were normalized for concentration so that equal amounts of protein were loaded. The prepared samples were boiled at 70 °C in a thermoblock for 10 minutes and then placed on ice. Samples were run on an SDS-PAGE gel at 200 V for 32 minutes and then transferred to a nitrocellulose membrane using standard techniques. Commercially available human IgG (Abcam, Sigma) was used as a positive control. The membrane was blocked using blocking buffer (Odyssey) at room temperature for 30 minutes. Protein bands were visualized by staining the membrane with Ponceau S for 5 minutes, followed by washing with distilled water and destaining with TBST (Tris-buffered solution with NaCl and Tween 20). The membrane was then probed overnight at 4°C with gentle mixing with horseradish peroxidase-conjugated anti-human IgG primary antibody (Genscript) diluted 1:5000 in blocking buffer. The blot was then washed with TBST three times for 5 minutes each time and developed using an ECL kit (SuperSignal West Femto substrate) and visualized using a gel imaging system (Syngene Box Mini).

[00620] Transfection efficiency using the Lipofectamine™ 3000 transfection procedure was assessed by analyzing the fluorescence of 293T cells transfected with ccDNA-GFP plasmid (Figure 9A, top panel) or ccDNA vector-GFP (Figure 9A, bottom panel). Both samples had significant fluorescence as seen in FIG. 9A , indicating successful transfection in both cases. Despite the significant protein content in each sample ( Fig. 9B , bottom panel), the presence of expressed aducanumab was detected only in samples from cells transfected with cccDNA-IgG plasmid or ccDNA-IgG constructs ( Fig. 9B , top panel, observed as severe, and light chains). This indicates that aducanumab is expressed by the cDNA vector in 293T cells.

EXAMPLE 9: Confirmation of the identity of the expressed antibody

[00621] Plasmids containing a nucleic acid of interest encoding aducanumab were prepared for expression in human embryonic kidney cells (HEK293-6E). HEK293-6E cells were grown in serum-free medium (FreeStyle™ 293 Expression Medium, Thermo Fisher Scientific). Cells were maintained in Erlenmeyer flasks at 37 °C with 5% CO ₂ on an orbital shaker. The day before transfection, cells were seeded at the appropriate density in Erlenmeyer flasks. On the day of transfection, DNA and transfection reagent were mixed in the optimal ratio and then added to the flask containing cells ready for transfection. The recombinant plasmid encoding the target protein was transiently transfected into suspension cell cultures of HEK293-6E. Cell culture supernatants collected on day 6 were used for purification.

[00622] The cell culture broth was centrifuged, filtered, and loaded onto an affinity column (Monofinity A Resin™ prepacked column, GenScript) at an appropriate flow rate. After washing and elution, the eluted fractions were pooled and the buffer was replaced with final composition buffer. The purified protein was analyzed by (a) SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, (b) Western blotting using goat anti-human IgG-HRP (GenScript) and goat anti-human kappa-HRP as primary antibodies, with chemiluminescence detection, and (c) size exclusion chromatography using high performance liquid chromatography TSKgel G3000SWxl (Tosoh Bioscience) to determine molecular weight and purity. Protein concentration was determined by absorbance at A260/280. The results are presented in Fig. 10A . HPLC analysis revealed a single peak ( Figure 10A ). When samples of this protein were run on an SDS-PAGE gel ( Figure 8A ), one band was observed under non-reducing conditions (lane 2), while two bands were observed under reducing conditions (lane 1). This is consistent with the proposal that the protein is an antibody in which the light chain and heavy chain subunits remain disulfide-linked under non-reducing conditions and migrate as a single band, but under reducing conditions separate into separate subunits, migrating as two bands. Western blotting showed a similar banding pattern ( Figure 8B ) and further confirmed the presence of the antibody as detection was with primary antibodies specific for human IgG and human kappa light chain.

[00623] The ability of purified aducanumab to recognize its ligand amyloid beta (1-42) (“Abeta”) was assessed by ELISA. Amyloid aggregated in vitro and adhered to the plate, followed by exposure to aducanumab or control antibodies at various concentrations and for various periods of time, followed by colorimetric exposure. Synthetic Aβ1-42 peptide (AnaSpec, Fremont, CA, USA) was reconstituted in hexafluoroisopropanol at a concentration of 1 mg/mL, air-dried, and concentrated in vacuo to form a film. The film was dissolved in DMSO and AB42 oligomers, and AB42 fibrils were prepared by diluting the DMSO-reduced monomer in phosphate-buffered saline (PBS) at a concentration of 100 μg/ml and incubating at 37 ^° C. for at least 3 days and 1 week, respectively. ELISA plates were coated with aggregated amyloid.

[00624] Briefly, Abeta was prepared, Western blotted, and stained according to the methods described by Stine et al., Methods Mol. Biol. (2011) 670: 13-32. Equivalent amounts of Abeta were run on the gel in each of lanes 3, 4, and 5. After transfer to nitrocellulose, each lane was separated and probed with different primary antibodies: purified aducanumab described herein in lane 3, purified anti-amyloid beta antibody (17- 24) (BioLegend, clone 4G8) in lane 4, and purified anti-amyloid beta (1-16) antibody (BioLegend, clone 6E10) in lane 5. After washing, each sample was probed with goat anti-human IgG-HRP (lane 3) (GenScript) or goat anti-mouse IgG-HRP (lanes 4 and 5) (GenScript) as a secondary antibody and developed with HRP substrate. The results are presented in Fig. 10B . Plasmid-expressed aducanumab bound to nitrocellulose-immobilized Abeta monomer, as did two other anti-Abeta antibodies, indicating that purified aducanumab was able to recognize its ligand as expected.

Example 10 Antibody Expression in vivo in wild-type mice.

[00625] A cccDNA vector with a wild-type left ITR and a truncated mutant right ITR and with a transgenic region encoding aducanumab heavy chain and light chain, each under the control of its own EF1 promoter, was prepared and purified as described above in Examples 1 and 5 ( "cDNA-IgG vector"). The cccDNA-IgG vector or the control cccDNA vector containing the luciferase transgene under the control of the liver-specific hAAT promoter was administered to male C57bl/6J mice at approximately 6 weeks of age. Non-encapsulated cccDNA vectors were administered at a dose of 0.005 mg per animal (4 animals per group) by hydrodynamic intravenous injection through the lateral tail vein in a volume of 2.2 ml. Blood samples were collected from each treated animal on days 3, 7, 14, 21, and the final study day 28. The presence of expressed aducanumab in serum samples was measured by ELISA using a polyclonal anti-human immunoglobulin antibody that recognizes human antibodies of any specificity, commercially available available Discovery Human/NHP IgG kit, according to the manufacturer's instructions (Meso Scale Discovery).

[00626] As shown in FIG. 11 , human antibodies were readily detected on days 3 and 7 in serum samples from mice receiving the cccDNA-IgG vector, but were not observed in mice receiving cccDNA expressing luciferase rather than the human antibody. The maximum serum expression level observed at these two time points for this particular vector was approximately 500 ng/ml.

Example 11 Antibody Expression in vivo in a mouse model of Alzheimer's disease

[00627] A cccDNA vector with aducanumab heavy chain and light chain coding sequences can be prepared as previously described in Examples 1 and 5 . The cccDNA vector composition will be formulated with lipid nanoparticles (LNPs) and administered to Tg2576 mice (Kawarabayashi et al., J. Neurosci 21(2):372-381 (2001)) and normal mice. LNPs with cDNA vectors are administered to the corresponding mice in doses from 0.3 to 5 mg/kg in a volume of 1.2 ml. Each dose should be administered by hydrodynamic intravenous administration or, for example, intraperitoneal injection. Administration to normal mice serves as a control and can also be used to determine the presence and amount of aducanumab monoclonal antibody (mAb). Both in vitro and ex vivo binding assays (amyloid aggregate ELISA and brain section immunohistochemistry (IHC)) will be performed, respectively, as described herein and in Example 6 .

[00628] Following an acute dose, for example a single dose of LNP-TTX-Adu, plasma and brain cccDNA concentrations will be determined by ELISA at various time points, for example, 10, 20, 30, 40, 50, 100 and 200 days or more, etc. More specifically, frozen brains are homogenized in 10 volumes (10 ml/g wet tissue) of a solution containing 50 mM NaCl, 0.2% diethylamine (DEA), with protease inhibitors, and sonicated for approximately 15-20 s on ice. Samples were centrifuged at 100,000 g for 30 minutes at 4°C and the supernatant was saved as the DEA-extracted amyloid β soluble fraction. The remaining pellet was resuspended in 10 volumes of 5 M guanidine hydrochloride (Gu-HCl), sonicated and centrifuged as described above. The resulting supernatant is retained as the guanidine-extracted insoluble amyloid β (Αβ) fraction, and the remaining precipitate is discarded. To determine antibody concentration in plasma and brain, 96-well microplates (Nunc Maxisorp, Corning Costar) are coated with Αβ(1-42) peptide (Αβ42) at a concentration of 5 μg/ml in cold coating buffer overnight at 4° C. Plasma samples or DHEA-extracted brain homogenates from (i.e., detergent-free) brains are diluted to final working concentrations and incubated for 2 hours at room temperature. Binding is detected using a reporter antibody, such as horseradish peroxidase (HRP)-conjugated goat anti-human polyclonal antibody (Jackson ImmunoResearch), followed by measurement of HRP activity using the substrate TMB (tetramethylbenzidine). Concentrations are determined by comparison with a calibration curve obtained using purified antibodies. Alternatively, the presence of antibody (Ab) in plasma is confirmed by Western blotting as described in

[00629] In vivo binding of the antibody to Αβ deposits after a single dose of Tg2576 mice can also be determined using a biotinylated anti-human secondary antibody and can be compared with pan-Αβ 5F3 antibody staining performed ex vivo on a serial section as a positive control. Tissues are formalin fixed and paraffin embedded, sections are 5 μm (μπι) deparaffinized, and high temperature antigen retrieval with EDTA borate (Ventana CC1) can be used. The tissues can then be coated with goat anti-human secondary antibody at a dilution of 1:500 and the tissues stained with hematoxylin.

[00630] Systemically administered LNP-TTX-Adu cccDNA is expected to be expressed in plasma and brain and bind to parenchymal amyloid plaques with high affinity.

EXAMPLE 12: Analysis of Amyloid Load Reduction Following Dosing of cccDNA

[00631] The effectiveness of cDNA-expressed aducanumab in reducing amyloid load will be assessed following chronic administration to Tg2576 transgenic mice. For example, doses ranging from 0.3, 1, 3, 10 to 30 mg/kg, etc., or phosphate buffered saline (PBS) will be administered on a predetermined time frame, such as weekly or monthly. Plasma and brain drug levels will be measured by ELISA as described above in Example 8. Plasma samples are collected 24-72 hours after the last dose and antibody levels are measured to determine the dose-response relationship.

[00632] The total brain amyloid load in the cortex and hippocampus will be determined by immunohistochemistry. More specifically, the brain is dissected and fixed by immersion in 10% neutral buffered formalin for 48-72 hours. The fixed brain is then processed and embedded in a horizontal orientation. Each block is sectioned until the hippocampus is identified, after which 300 consecutive 5-μm sections are obtained (3 sections per slide). Every 14th slide is stained for both 6E10 and thioflavin-S (approximately 1 section every 225 μm). Immunohistochemistry to detect brain amyloid would use, for example, mouse monoclonal antibody against Αβ 1-16 (Clone 6E10, Covance, Princeton, NJ) as the primary antibody at a dilution of 1:750, and the Ultramap anti-mouse alkaline phosphatase kit (Ventana Medical Systems, Tucson, Arizona) and perform quantitation using the ^VISIOPHARM® application program as described herein. Slides are pretreated with 88% formic acid and then placed on a Ventana Discovery XT immunostainer. Slides were counterstained with Ventana hematoxylin (Ventana Medical Systems, Tucson, AZ), coverslipped, and air-dried overnight.

[00633] After 6E10 immunostaining, slides were scanned using an Aperio XT Whole Slide Imaging System (Aperio Technologies, Inc., Vista, CA) at 20× magnification, according to the manufacturer's instructions. Digital images can then be viewed and manually annotated as individual masks and then analyzed using an algorithm written using VISIOPHARM ^® software. The algorithm determines the area of the annotated hippocampus or cortex and the areas of parenchymal and vascular amyloid in these anatomical regions at 10x virtual magnification. The application program is taught, for example, on 50 slides. Slides are also stained with thioflavin-S (Thio-S) as described in (Bussiere et al., Am J Pathol 165:987-995 (2004)) and mounted in ^{VECTASHIELD®} mounting medium with DAPI (4,6-diamidino-2 -phenylindole) (Vector Laboratories Burlingame, CA). After thioflavin-S staining, the slides were analyzed and scanned using an imaging system, such as the Aperio FL Whole Slide Fluorescence Imaging System (Aperio Technologies, Inc., Vista, CA) at 20× magnification, according to the manufacturer's instructions. As with 6E10, the hippocampus or cortex is annotated manually as individual masks and then analyzed using an algorithm written in VISIOPHARM ^® software and tailored for fluorescence. The algorithm determines the area of the hippocampus and cortex, as well as the areas of parenchymal and vascular amyloid in these anatomical regions at 10x virtual magnification. The application program can be taught in 10 slides. The total area occupied by stained deposits is expected to be significantly reduced compared to the phosphate-buffered saline (PBS) control.

EXAMPLE 13: Expression of Antibody and Fc Fusion Protein from ccDNA in vitro

[00634] To evaluate the ability of cccDNA to express antibodies and immunoglobulin-like molecules other than aducanumab, the experiments described in Example 8 were repeated with two additional ccDNA constructs—one encoding the light and heavy chains of bevacizumab (an antibody that specifically binds to vascular endothelial growth factor (VEGF)) in a gene cassette, and another encoding the Fc fusion protein aflibercept (human IgG1 Fc fused to the VEGF-binding regions of the extracellular domains of human VEGF receptors 1 and 2). The results of Western blot analysis on unreduced samples are presented in Fig. 12 (two images of the same blot are shown with different exposure times - 6 seconds and 12 seconds). Aducanumab was again expressed in cells transfected with ccDNA-Adu plasmid and ccDNA-Adu vector as previously described in Example 8 (lanes 5 and 7). Bevacizumab was similarly expressed in cells transfected with the cccDNA-bevacizumab vector (lane 11). Aflibercept was also expressed in cells transfected with the cccDNA-aflibercept vector (lane 9). These results demonstrate that the cellular expression of various IgG and immunoglobulin-like molecules from cccDNA constructs is possible.

LINKS TO SOURCES

[00635] All publications and references, including, without limitation, patents and patent applications referenced in this specification and the examples provided herein, are hereby incorporated by reference in their entirety as if each individual publication or reference were specifically and individually and expressly stated to be incorporated by reference into this document. Any patent application to which this application claims priority is also incorporated herein by reference in the manner described above for publications and references.

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LIST OF SEQUENCES

<110> GENERATION BIO INC.

<120> NON-VIRAL DNA VECTORS AND THEIR APPLICATION FOR PRODUCTION

ANTIBODIES

AND FUSION PROTEINS [11]

<130> 080170-091100-WOPT

<140>

<141>

<150> 62/680,087

<151> 2018-06-04

<150> 62/680,092

<151> 2018-06-04

<150> 62/630,670

<151> 2018-02-14

<150> 62/630,676

<151> 2018-02-14

<160> 190

<170> Patent In version 3.5

<210> 1

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 1

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

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ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc

120

gagcgcgcag ctgcctgcag g

141

<210> 2

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

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cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca

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actccatcac taggggttcc t

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<210> 3

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

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<400> 3

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc

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tgcctgcagg

130

<210> 4

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 4

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct

130

<210> 5

<211> 143

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 5

ttgcccactc cctctctgcg cgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc

60

agacggcaga ggtctcctct gccggcccca ccgagcgagc gacgcgcgca gagagggagt

120

gggcaactcc atcactaggg taa

143

<210> 6

<211> 144

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 6

ttggccactc cctctatgcg cactcgctcg ctcggtgggg cctggcgacc aaaggtcgcc

60

agacggacgt gggtttccac gtccggcccc accgagcgag cgagtgcgca tagagggagt

120

ggccaactcc atcactagag gtat

144

<210> 7

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 7

ttggccactc cctctatgcg cgctcgctca ctcactcggc cctggagacc aaaggtctcc

60

agactgccgg cctctggccg gcagggccga gtgagtgagc gagcgcgcat agagggagtg

120

gccaact

127

<210> 8

<211> 166

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 8

tcccccctgt cgcgttcgct cgctcgctgg ctcgtttggg ggggcgacgg ccagaggggcc

60

gtcgtctggc agctctttga gctgccaccc ccccaaacga gccagcgagc gagcgaacgc

120

gacagggggg agagtgccac actctcaagc aagggggttt tgtaag

166

<210> 9

<211> 144

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 9

ttgcccactc cctctaatgc gcgctcgctc gctcggtggg gcctgcggac caaaggtccg

60

cagacggcag aggtctcctc tgccggcccc accgagcgag cgagcgcgca tagagggagt

120

gggcaactcc atcactaggg gtat

144

<210> 10

<211> 143

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 10

ttaccctagt gatggagttg cccactccct ctctgcgcgc gtcgctcgct cggtggggcc

60

ggcagaggag acctctgccg tctgcggacc tttggtccgc aggccccacc gagcgagcga

120

gcgcgcagag agggagtggg caa

143

<210> 11

<211> 144

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 11

atacctctag tgatggagtt ggccactccc tctatgcgca ctcgctcgct cggtggggcc

60

ggacgtggaa acccacgtcc gtctggcgac ctttggtcgc caggccccac cgagcgagcg

120

agtgcgcata gagggagtgg ccaa

144

<210> 12

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 12

agttggccac attagctatg cgcgctcgct cactcactcg gccctggaga ccaaaggtct

60

ccagactgcc ggcctctggc cggcagggcc gagtgagtga gcgagcgcgc atagagggag

120

tggccaa

127

<210> 13

<211> 166

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 13

cttacaaaac ccccttgctt gagagtgtgg cactctcccc cctgtcgcgt tcgctcgctc

60

gctggctcgt ttgggggggt ggcagctcaa agagctgcca gacgacggcc ctctggccgt

120

cgccccccca aacgagccag cgagcgagcg aacgcgacag ggggga

166

<210> 14

<211> 144

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 14

atacccctag tgatggagtt gcccactccc tctatgcgcg ctcgctcgct cggtggggcc

60

ggcagaggag acctctgccg tctgcggacc tttggtccgc aggccccacc gagcgagcga

120

gcgcgcatta gagggagtgg gcaa

144

<210> 15

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 15

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

cgcacgcccg ggtttcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg

120

<210> 16

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 16

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg

122

<210> 17

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 17

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gcgcctcagt gagcgagcga gcgcgcagct

120

gcctgcagg

129

<210> 18

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 18

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ctttgcctca gtgagcgagc gagcgcgcag ctgcctgcag g

101

<210> 19

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 19

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgaca aagtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga

120

gcgcgcagct gcctgcagg

139

<210> 20

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 20

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgaaa atcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc

120

gcgcagctgc ctgcagg

137

<210> 21

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 21

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgaaa cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc

120

gcagctgcct gcagg

135

<210> 22

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 22

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcaaag cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc

120

aggctgcctgc agg

133

<210> 23

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 23

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gtttcccggg cggcctcagt gagcgagcga

120

gcgcgcagct gcctgcagg

139

<210> 24

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 24

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg tttccgggcg gcctcagtga gcgagcgagc

120

gcgcagctgc ctgcagg

137

<210> 25

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 25

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgt ttcgggcggc ctcagtgagc gagcgagcgc

120

gcagctgcct gcagg

135

<210> 26

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 26

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgccctt tgggcggcct cagtgagcga gcgagcgcgc

120

aggctgcctgc agg

133

<210> 27

<211> 131

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 27

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgccttt ggcggcctca gtgagcgagc gagcgcgcag

120

ctgcctgcag g

131

<210> 28

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 28

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgctttg cggcctcagt gagcgagcga gcgcgcagct

120

gcctgcagg

129

<210> 29

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 29

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgtttcg gcctcagtga gcgagcgagc gcgcagctgc

120

ctgcagg

127

<210> 30

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 30

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg

122

<210> 31

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 31

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc

120

tgcctgcagg

130

<210> 32

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 32

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcg

60

cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct

120

<210> 33

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 33

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgt cgggcgacct ttggtcgccc

60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc

120

ct

122

<210> 34

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 34

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc

120

ct

122

<210> 35

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 35

cctgcaggca gctgcgcgct cgctcgctca ctgaggcgcc cgggcgtcgg gcgacctttg

60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta

120

ggggttcct

129

<210> 36

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 36

cctgcaggca gctgcgcgct cgctcgctca ctgaggcaaa gcctcagtga gcgagcgagc

60

gcgcagagag ggagtggcca actccatcac taggggttcc t

101

<210> 37

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 37

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacttt gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac

120

tccatcacta ggggttcct

139

<210> 38

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 38

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgatttt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc

120

catcactagg ggttcct

137

<210> 39

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 39

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgtttcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttcct

135

<210> 40

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 40

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggctttgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc

120

actaggggtt cct

133

<210> 41

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 41

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtcgg

60

gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac

120

tccatcacta ggggttcct

139

<210> 42

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 42

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccggaaaccg ggcgtcgggc

60

gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc

120

catcactagg ggttcct

137

<210> 43

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 43

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgaaacggg cgtcgggcga

60

cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttcct

135

<210> 44

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 44

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccaaagggcg tcgggcgacc

60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc

120

actaggggtt cct

133

<210> 45

<211> 131

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 45

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc caaaggcgtc gggcgacctt

60

tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac

120

tagggggttcct

131

<210> 46

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 46

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc aaagcgtcgg gcgacctttg

60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta

120

ggggttcct

129

<210> 47

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 47

cctgcaggca gctgcgcgct cgctcgctca ctgaggccga aacgtcgggc gacctttggt

60

cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg

120

ggttcct

127

<210> 48

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 48

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg

122

<210> 49

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 49

cgatcgttcg at

12

<210> 50

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 50

atcgaaccat cg

12

<210> 51

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 51

atcgaacgat cg

12

<210> 52

<211> 165

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 52

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc

120

gagcgcgcag agagggagtg gccaactcca tcactagggg ttcct

165

<210> 53

<211> 140

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 53

cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc

60

cggggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg

120

cgcagagaga tcactagggg

140

<210> 54

<211> 91

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 54

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg

60

tcgcccggcc tcagtgagcg agcgagcgcg c

91

<210> 55

<211> 91

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 55

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc

60

ccgggcggcc tcagtgagcg agcgagcgcg c

91

<210> 56

<211> 10808

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 56

aaagtagccg aagatgacgg tttgtcacat ggagttggca ggatgtttga ttaaaaacat

60

aacaggaaga aaaatgcccc gctgtgggcg gacaaaatag ttgggaactg ggaggggtgg

120

aaatggagtt tttaaggatt atttagggaa gagtgacaaa atagatggga actgggtgta

180

gcgtcgtaag ctaatacgaa aattaaaaat gacaaaatag tttggaacta gatttcactt

240

atctggttcg gatctcctag gcggccggcc cctgcaggca gctgcgcgct cgctcgctca

300

ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga

360

gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa

420

tgattaaccc gccatgctac ttatctacgt agccatgcgc ggccgcggcc tgaaataacc

480

tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg

540

tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg

600

catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt

660

atgcaaagca tgcatctcaa ttagtcagca accatagtcc cactagtgga gccgagagta

720

attcatacaa aaggagggat cgccttcgca aggggagagc cccaggaccg tccctaaatt

780

ctcacagacc caaatccctg tagccgcccc acgacagcgc gaggagcatg cgctcagggc

840

tgagcgcggg gagagcagag cacacaagct catagaccct ggtcgtgggg gggaggaccg

900

gggagctggc gcggggcaaa ctgggaaagc ggtgtcgtgt gctggctccg ccctcttccc

960

gagggtgggg gagaacggta tataagtgcg gcagtcgcct tggacgttct ttttcgcaac

1020

gggtttgccg tcagaacgca ggtgaggggc gggtgtggct tccgcgggcc gccgagctgg

1080

aggtcctgct ccgagcgggc cgggccccgc tgtcgtcggc ggggattagc tgcgagcatt

1140

cccgcttcga gttgcgggcg gcgcgggagg cagagtgcga ggcctagcgg caaccccgta

1200

gcctcgcctc gtgtccggct tgaggcctag cgtggtgtcc gcgccgccgc cgcgtgctac

1260

tccggccgca ctctggtctt tttttttttt gttgttgttg ccctgctgcc ttcgattgcc

1320

gttcagcaat aggggctaac aaagggaggg tgcggggctt gctcgcccgg agcccggaga

1380

ggtcatggtt ggggaggaat ggagggacag gagtggcggc tggggcccgc ccgccttcgg

1440

agcacatgtc cgacgccacc tggatggggc gaggcctggg gtttttcccg aagcaaccag

1500

gctggggtta gcgtgccgag gccatgtggc cccagcaccc ggcacgatct ggcttggcgg

1560

cgccgcgttg ccctgcctcc ctaactaggg tgaggccatc ccgtccggca ccagttgcgt

1620

gcgtggaaag atggccgctc ccgggccctg ttgcaaggag ctcaaaatgg aggacgcggc

1680

agcccggtgg agcgggcggg tgagtcaccc acacaaagga agagggcctg gtccctcacc

1740

ggctgctgct tcctgtgacc ccgtggtcct atcggccgca atagtcacct cgggcttttg

1800

agcacggcta gtcgcggcgg ggggagggga tgtaatggcg ttggagtttg ttcacatttg

1860

gtgggtggag actagtcagg ccagcctggc gctggaagtc atttttggaa tttgtcccct

1920

tgagttttga gcggagctaa ttctcgggct tcttagcggt tcaaaggtat cttttaaacc

1980

cttttttagg tgttgtgaaa accaccgcta attcaaagca accggtatgg actggacctg

2040

gcgaattctt ttccttgtag cagcggctac tggtgcacat tcattggttg agtcaggcgg

2100

tggggtagta cagcctggcc ggtcccttag gctctcctgc gccgcgtccg ggttcgcgtt

2160

tagctcctac gggatgcact gggttagaca ggctcctgga aagggactgg aatgggttgc

2220

agtcatttgg tttgacggga ccaaaaaata ctatacagat tccgttaagg ggaggtttac

2280

aatcagtaga gataatagta aaaacacgtt gtatcttcaa atgaatactc tcagagccga

2340

agatacagca gtatattact gcgcccgcga tcggggaata ggcgctcgga ggggacccta

2400

ctatatggat gtgtggggta aaggtacgac cgtgactgtg agctctgcgt ccaccaaagg

2460

tccaagtgtt tttcccctcg ccccttcaag caagtcaacc tcaggcggta ctgctgcttt

2520

gggatgtctg gttaaagatt actttccaga gcctgtgact gtgagttgga attcaggcgc

2580

tcttaccagc ggtgttcaca cattccccgc agtcttgcaa tcctctggtt tgtattccct

2640

gagtagcgta gtcacggtgc ctagcagtag cctcgggaca caaacgtaca tttgcaatgt

2700

gaatcacaaa cccagtaata ccaaggttga taagcgggtc gaacctaaat cctgtgacaa

2760

gacccacaca tgtcctccgt gcccggctcc cgagctgctg ggcgggccgt ccgtattcct

2820

gtttccccct aaaccgaaag acactttgat gatatcacgg acacccgagg ttacgtgtgt

2880

ggtcgtcgat gtttctcacg aggaccccga agtcaaattc aattggtacg tggatggtgt

2940

tgaggttcac aacgccaaaa ctaaaccccg cgaggagcag tacaattcaa cctatagggt

3000

tgtgagtgtc cttactgtcc tccatcaaga ttggctgaac ggcaaagaat ataaatgcaa

3060

agtctcaaac aaagctctgc cagccccgat agaaaagacc atctccaaag ctaaggggca

3120

acccagagaa cctcaagtgt acaccctccc tccatcaagg gaggagatga cgaaaaatca

3180

agtaagcttg acctgtcttg taaaagggtt ctacccaagt gatatagctg tagaatggga

3240

gagtaatggg cagcccgaga ataattacaa aacgacaccg cccgtgctgg actcagacgg

3300

tagctttttc ctctacagca aacttactgt agataaaagt aggtggcagc agggaaatgt

3360

tttttcctgt tcagtcatgc atgaagcatt gcacaaccat tacacccaga agtctctcag

3420

tttgagcccg ggcaagtaat gaccagacat gataagatac attgatgagt ttggacaaac

3480

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt

3540

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat

3600

gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg

3660

tggtatggcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc

3720

cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat

3780

tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat

3840

catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat

3900

gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc

3960

gctattacca tgatgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac

4020

tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttgactagtg

4080

gagccgagag taattcatac aaaaggaggg atcgccttcg caaggggaga gcccagggac

4140

cgtccctaaa ttctcacaga cccaaatccc tgtagccgcc ccacgacagc gcgaggagca

4200

tgcgcccagg gctgagcgcg ggtagatcag agcacacaag ctcacagtcc ccggcggtgg

4260

ggggaggggc gcgctgagcg ggggccaggg agctggcgcg gggcaaactg ggaaagtggt

4320

gtcgtgtgct ggctccgccc tcttcccgag ggtgggggag aacggtatat aagtgcggta

4380

gtcgccttgg acgttctttt tcgcaacggg tttgccgtca gaacgcaggt gagtggcggg

4440

tgtggcttcc gcgggccccg gagctggagc cctgctctga gcgggccggg ctgatatgcg

4500

agtgtcgtcc gcagggttta gctgtgagca ttcccacttc gagtggcggg cggtgcgggg

4560

gtgagagtgc gaggcctagc ggcaaccccg tagcctcgcc tcgtgtccgg cttgaggcct

4620

agcgtggtgt ccgccgccgc gtgccactcc ggccgcacta tgcgtttttt gtccttgctg

4680

ccctcgattg ccttccagca gcatgggcta acaaagggag ggtgtggggc tcactcttaa

4740

ggagcccatg aagcttacgt tggataggaa tggaagggca ggaggggcga ctggggcccg

4800

cccgccttcg gagcacatgt ccgacgccac ctggatgggg cgaggcctgt ggctttccga

4860

agcaatcggg cgtgagttta gcctacctgg gccatgtggc cctagcactg ggcacggtct

4920

ggcctggcgg tgccgcgttc ccttgcctcc caacaagggt gaggccgtcc cgcccggcac

4980

cagttgcttg cgcggaaaga tggccgctcc cggggccctg ttgcaaggag ctcaaaatgg

5040

aggacgcggc agcccggtgg agcgggcggg tgagtcaccc acacaaagga agagggcctt

5100

gcccctcgcc ggccgctgct tcctgtgacc ccgtggtcta tcggccgcat agtcacctcg

5160

ggcttctctt gagcaccgct cgtcgcggcg gggggagggg atctaatggc gttggagttt

5220

gttcacattt ggtgggtgga gactagtcag gccagcctgg cgctggaagt cattcttgga

5280

atttgcccct ttgagtttgg agcgaggcta attctcaagc ctcttagcgg ttcaaaggta

5340

ttttctaaac ccgtttccag gtgttgtgaa agccaccgct aattcaaagc aatccggaat

5400

gcttccaagt cagttgatag ggtttttgct gctgtgggtg cctgcttcaa gaggtattca

5460

gatgactcaa tctccttctt ctctgtcagc cagcgtaggt gatcgcgtca ccatcacctg

5520

tcgagcctca cagtccatt ctagctatct caattggtat cagcagaagc caggaaaagc

5580

ccctaaactt ctcatatacg ccgcctctag tctccaaagt ggggtgccca gtcggttcag

5640

tggctccgga tctgggactg atttcacttt gactatatct agtctccagc ctgaggattt

5700

tgctacatat tattgtcaac aatcctactc tacacccctg acgttcggtg gcggaacgaa

5760

ggttgaaata aagaggactg tggccgcccc atcagttttc attttcccgc cgtctgatga

5820

acaactcaaa agcggcacag catctgtagt ctgccttttg aacaactttt atccacggga

5880

ggcaaaagtg caatggaaag tggataatgc tctgcaaagt ggtaacagcc aagaaagtgt

5940

aaccgaacag gattccaaag actcaaccta ttccctcagt tccacactca cactgtctaa

6000

ggctgattac gagaagcata aggtttacgc ctgtgaagtt acgcaccaag gactctctag

6060

tccagttact aaaagcttta atcgggggga atgttagtga tgtgccttct agttgccagc

6120

catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg

6180

tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc

6240

tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg

6300

ctggggatgc ggtgggctct atggcggcgc gccgcatggc tacgtagata agtagcatgg

6360

cgggttaatc attaactaca cctgcaggag gaacccctag tgatggagtt ggccactccc

6420

tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc

6480

ggcctcagtg agcgagcgag cgcgcagctg cctgcaggtc gacgcatgcg gtaccgggag

6540

atgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac

6600

ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg

6660

ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg gaccaatacg

6720

cccgcgtttc ttccttttcc ccaccccaac ccccaagttc gggtgaaggc ccagggctcg

6780

cagccaacgt cggggcggca agccctgcca tagccactac gggtacgtag gccaaccact

6840

agaactatag ctagagtcct gggcgaacaa acgatgctcg ccttccagaa aaccgaggat

6900

gcgaaccact tcatccgggg tcagcaccac cggcaagcgc cgcgacggcc gaggtctacc

6960

gatctcctga agccagggca gatccgtgca cagcaccttg ccgtagaaga acagcaaggc

7020

cgccaatgcc tgacgatgcg tggagaccga aaccttgcgc tcgttcgcca gccaggacag

7080

aaatgcctcg acttcgctgc tgcccaaggt tgccgggtga cgcacaccgt ggaaacggat

7140

gaaggcacga acccagttga cataagcctg ttcggttcgt aaactgtaat gcaagtagcg

7200

tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt

7260

ggcggttttc atggcttgtt atgactgttt ttttgtacag tctatgcctc gggcatccaa

7320

gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac

7380

gcagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta ggtggctcaa

7440

gtatgggcat cattcgcaca tgtaggctcg gccctgacca agtcaaatcc atgcgggctg

7500

ctcttgatct tttcggtcgt gagttcggag acgtagccac ctactcccaa catcagccgg

7560

actccgatta cctcgggaac ttgctccgta gtaagacatt catcgcgctt gctgccttcg

7620

accaagaagc ggttgttggc gctctcgcgg cttacgttct gcccaggttt gagcagccgc

7680

gtagtgagat ctatatctat gatctcgcag tctccggcga gcaccggagg cagggcattg

7740

ccaccgcgct catcaatctc ctcaagcatg aggccaacgc gcttggtgct tatgtgatct

7800

acgtgcaagc agattacggt gacgatcccg cagtggctct ctatacaaag ttgggcatac

7860

gggaagaagt gatgcacttt gatatcgacc caagtaccgc cacctaacaa ttcgttcaag

7920

ccgagatcgg cttcccggcc gcggagttgt tcggtaaatt gtcacaacgc cgcgaatata

7980

gtctttacca tgcccttggc cacgcccctc tttaatacga cgggcaattt gcacttcaga

8040

aaatgaagag tttgctttag ccataacaaa agtccagtat gctttttcac agcataactg

8100

gactgatttc agtttacaac tattctgtct agtttaagac tttattgtca tagtttagat

8160

ctattttgtt cagtttaaga ctttattgtc cgcccacacc cgcttacgca gggcatccat

8220

ttattactca accgtaaccg attttgccag gttacgcggc tggtctgcgg tgtgaaatac

8280

cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg

8340

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa

8400

tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc

8460

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc

8520

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat

8580

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc

8640

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct

8700

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg

8760

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc

8820

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga

8880

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa

8940

ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta

9000

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc

9060

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg

9120

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga

9180

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg

9240

agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct

9300

gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg

9360

agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc

9420

cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa

9480

ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc

9540

cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt

9600

cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc

9660

ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt

9720

tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc

9780

catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt

9840

gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata

9900

gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga

9960

tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag

10020

catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa

10080

aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt

10140

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga

10200

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa

10260

acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc

10320

aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga

10380

gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag

10440

ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt

10500

ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta

10560

gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag

10620

cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg

10680

cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcaa ataagcgttg

10740

atattcagtc aattacaaac attaataacg aagagatgac agaaaaattt tcattctgtg

10800

acagagaa

10808

<210> 57

<211> 453

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Any amino acid

<400> 57

Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Phe Asp Gly Thr Lys Lys Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Ile Gly Ala Arg Arg Gly Pro Tyr Tyr Met Asp

100 105 110

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly

450

<210> 58

<211> 214

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 59

<211> 1310

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 59

ggagccgaga gtaattcata caaaaggagg gatcgccttc gcaaggggag agcccaggga

60

ccgtccctaa attctcacag acccaaatcc ctgtagccgc cccacgacag cgcgaggagc

120

atgcgctcag ggctgagcgc ggggagagca gagcacacaa gctcatagac cctggtcgtg

180

ggggggagga ccggggagct ggcgcggggc aaactgggaa agcggtgtcg tgtgctggct

240

ccgccctctt cccgagggtg ggggagaacg gtatataagt gcggcagtcg ccttggacgt

300

tctttttcgc aacgggtttg ccgtcagaac gcaggtgagg ggcgggtgtg gcttccgcgg

360

gccgccgagc tggaggtcct gctccgagcg ggccggggccc cgctgtcgtc ggcggggatt

420

agctgcgagc attcccgctt cgagttgcgg gcggcgcggg aggcagagtg cgaggcctag

480

cggcaacccc gtagcctcgc ctcgtgtccg gcttgaggcc tagcgtggtg tccgcgccgc

540

cgccgcgtgc tactccggcc gcactctggt cttttttttt tttgttgttg ttgccctgct

600

gccttcgatt gccgttcagc aataggggct aacaaaggga gggtgcgggg cttgctcgcc

660

cggagcccgg agaggtcatg gttggggagg aatggaggga caggagtggc ggctggggcc

720

cgcccgcctt cggagcacat gtccgacgcc acctggatgg ggcgaggcct ggggtttttc

780

ccgaagcaac caggctgggg ttagcgtgcc gaggccatgt ggccccagca cccggcacga

840

tctggcttgg cggcgccgcg ttgccctgcc tccctaacta gggtgaggcc atcccgtccg

900

gcaccagttg cgtgcgtgga aagatggccg ctcccggggcc ctgttgcaag gagctcaaaa

960

tggaggacgc ggcagcccgg tggagcgggc gggtgagtca cccacacaaa ggaagagggc

1020

ctggtccctc accggctgct gcttcctgtg accccgtggt cctatcggcc gcaatagtca

1080

cctcgggctt ttgagcacgg ctagtcgcgg cggggggagg ggatgtaatg gcgttggagt

1140

ttgttcacat ttggtgggtg gagactagtc aggccagcct ggcgctggaa gtcatttttg

1200

gaatttgtcc ccttgagttt tgagcggagc taattctcgg gcttcttagc ggttcaaagg

1260

tatcttttaa accctttttt aggtgttgtg aaaaccaccg ctaattcaaa

1310

<210> 60

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 60

gcgcgctcgc tcgctc

16

<210> 61

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 61

ggttga

6

<210> 62

<211> 4

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 62

agtt

4

<210> 63

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 63

ggttgg

6

<210> 64

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 64

agttgg

6

<210> 65

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 65

agttga

6

<210> 66

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 66

rrttrr

6

<210> 67

<211> 581

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 67

gagcatctta ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat

60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt

120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc

180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt

240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct

300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt

360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac

420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct

480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca

540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c

581

<210> 68

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 68

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct

60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct

120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg

180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggc

225

<210> 69

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 69

actgaggc

8

<210> 70

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 70

gcctcagt

8

<210> 71

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 71

gagcgagcga gcgcgc

16

<210> 72

<211> 1923

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 72

tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta

60

ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc

120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg

180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc

240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat

300

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc

360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga

420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg

480

gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg

540

cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta

600

attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg

660

gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg

720

cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg

780

aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg

840

ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc

900

gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt

960

ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc gggggggagc

1020

ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc

1080

ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg

1140

aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag

1200

gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc

1260

tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg

1320

gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg

1380

ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc

1440

ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg

1500

taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct

1560

gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg

1620

aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc

1680

cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg

1740

ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tttagccttc

1800

ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcatttgtcg

1860

acagaattcc tcgaagatcc gaaggggttc aagcttggca ttccggtact gttggtaaag

1920

cca

1923

<210> 73

<211> 1272

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 73

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc

60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc

120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc

180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc

240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt

300

ggtttaggta gtgtgagagg gtccgggttc aaaaccactt gctgggtggg gagtcgtcag

360

taagtggcta tgccccgacc ccgaagcctg tttccccatc tgtacaatgg aaatgataaa

420

gacgcccatc tgatagggtt tttgtggcaa ataaacattt ggtttttttg ttttgttttg

480

ttttgttttt tgagatggag gtttgctctg tcgcccaggc tggagtgcag tgacacaatc

540

tcatctcacc acaaccttcc cctgcctcag cctcccaagt agctgggatt acaagcatgt

600

gccaccacac ctggctaatt ttctattttt agtagagacg ggtttctcca tgttggtcag

660

cctcagcctc ccaagtaact gggattacag gcctgtgcca ccacacccgg ctaatttttt

720

ctatttttga cagggacggg gtttcaccat gttggtcagg ctggtctaga ggtaccggat

780

cttgctacca gtggaacagc cactaaggat tctgcagtga gagcagaggg ccagctaagt

840

ggtactctcc cagagactgt ctgactcacg ccaccccctc caccttggac acaggacgct

900

gtggtttctg agccaggtac aatgactcct ttcggtaagt gcagtggaag ctgtacactg

960

cccaggcaaa gcgtccgggc agcgtaggcg ggcgactcag atcccagcca gtggacttag

1020

cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc

1080

ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag

1140

cttcaggcac caccactgac ctgggacagt gaatccggac tctaaggtaa atataaaatt

1200

tttaagtgta taatgtgtta aactactgat tctaattgtt tctctctttt agattccaac

1260

ctttggaact ga

1272

<210> 74

<211> 1177

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 74

ggctcagagg ctcagaggca cacaggagtt tctgggctca ccctgccccc ttccaacccc

60

tcagttccca tcctccagca gctgtttgtg tgctgcctct gaagtccaca ctgaacaaac

120

ttcagcctac tcatgtccct aaaatgggca aacattgcaa gcagcaaaca gcaaacacac

180

agccctccct gcctgctgac cttggagctg gggcagaggt cagagacctc tctgggccca

240

tgccacctcc aacatccact cgaccccttg gaatttcggt ggagaggagc agaggttgtc

300

ctggcgtggt ttaggtagtg tgagagggtc cgggttcaaa accacttgct gggtggggag

360

tcgtcagtaa gtggctatgc cccgaccccg aagcctgttt ccccatctgt acaatggaaa

420

tgataaagac gcccatctga tagggttttt gtggcaaata aacatttggt ttttttgttt

480

tgttttgttt tgttttttga gatggaggtt tgctctgtcg cccaggctgg agtgcagtga

540

cacaatctca tctcaccaca accttcccct gcctcagcct cccaagtagc tgggattaca

600

agcatgtgcc accacacctg gctaattttc tatttttagt agagacgggt ttctccatgt

660

tggtcagcct cagcctccca agtaactggg attacaggcc tgtgccacca cacccggcta

720

attttttcta tttttgacag ggacggggtt tcaccatgtt ggtcaggctg gtctagaggt

780

accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag cagaggcca

840

gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac cttggacaca

900

ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca gtggaagctg

960

tacactgccc aggcaaagcg tccgggcagc gtaggcgggc gactcagatc ccagccagtg

1020

gacttagccc ctgtttgctc ctccgataac tggggtgacc ttggttaata ttcaccagca

1080

gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca gggccctgtc

1140

tcctcagctt caggcaccac cactgacctg ggacagt

1177

<210> 75

<211> 547

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 75

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc

60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag

180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag

240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct

300

gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt

360

gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca

420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa

480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg

540

gaactga

547

<210> 76

<211> 556

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 76

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc

60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg tggtttaggt

180

agtgtgagag gggaatgact cctttcggta agtgcagtgg aagctgtaca ctgcccaggc

240

aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact tagcccctgt

300

ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc

360

ccctctggat ccactgctta aatacggacg aggacactcg agggccctgt ctcctcagct

420

tcaggcacca ccactgacct gggacagtga atccggacat cgattctaag gtaaatataa

480

aatttttaag tgtataattt gttaaactac tgattctaat tgtttctctc ttttagattc

540

caacctttgg aactga

556

<210> 77

<211> 1179

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 77

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg

60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt

120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca

180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc

240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt

300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg

360

gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg

420

cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct

480

ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg

540

caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc

600

gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga

660

gcgcggccac cgagaatcgg acggggggtag tctcaagctg gccggcctgc tctggtgcct

720

ggtctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca

780

gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg

840

acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg

900

tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat

960

tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg

1020

gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa

1080

ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca

1140

gtggttcaaa gtttttttct tccatttcag gtgtcgtga

1179

<210> 78

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 78

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt

60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc

120

tgcggcgcgc gcagcacctt t

141

<210> 79

<211> 317

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 79

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt

60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa

180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag

240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag

300

gcctaggctt ttgcaaa

317

<210> 80

<211> 241

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 80

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag

60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga

120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat

180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga

240

c

241

<210> 81

<211> 215

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 81

gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa

60

cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc

120

tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg

180

gatttgggaa tcgtataaga actgtatgag accac

215

<210> 82

<211> 546

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 82

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc

60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag

180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag

240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct

300

gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt

360

gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca

420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa

480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg

540

gaactg

546

<210> 83

<211> 576

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 83

tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa

60

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata

120

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag

180

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc

240

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta

300

tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg

360

cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt

420

ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca

480

aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag

540

gtctatataa gcagagctgg tttagtgaac cgtcag

576

<210> 84

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 84

ataaacgata acgccgttgg tggcgtgagg catgtaaaag gttacatcat tatcttgttc

60

gccatccggt tggtataaat agacgttcat gttggttttt gtttcagttg caagttggct

120

gcggcgcgcg cagcaccttt gcggccatct

150

<210> 85

<211> 1313

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 85

ggagccgaga gtaattcata caaaaggagg gatcgccttc gcaaggggag agcccaggga

60

ccgtccctaa attctcacag acccaaatcc ctgtagccgc cccacgacag cgcgaggagc

120

atgcgcccag ggctgagcgc gggtagatca gagcacacaa gctcacagtc cccggcggtg

180

gggggagggg cgcgctgagc gggggccagg gagctggcgc ggggcaaact gggaaagtgg

240

tgtcgtgtgc tggctccgcc ctcttcccga gggtggggga gaacggtata taagtgcggt

300

agtcgccttg gacgttcttt ttcgcaacgg gtttgccgtc agaacgcagg tgagtggcgg

360

gtgtggcttc cgcgggcccc ggagctggag ccctgctctg agcgggccgg gctgatatgc

420

gagtgtcgtc cgcagggttt agctgtgagc attcccactt cgagtggcgg gcggtgcggg

480

ggtgagagtg cgaggcctag cggcaacccc gtagcctcgc ctcgtgtccg gcttgaggcc

540

tagcgtggtg tccgccgccg cgtgccactc cggccgcact atgcgttttt tgtccttgct

600

gccctcgatt gccttccagc agcatgggct aacaaaggga gggtgtgggg ctcactctta

660

aggagcccat gaagcttacg ttggatagga atggaagggc aggaggggcg actggggccc

720

gcccgccttc ggagcacatg tccgacgcca cctggatggg gcgaggcctg tggctttccg

780

aagcaatcgg gcgtgagttt agcctacctg ggccatgtgg ccctagcact gggcacggtc

840

tggcctggcg gtgccgcgtt cccttgcctc ccaacaaggg tgaggccgtc ccgcccggca

900

ccagttgctt gcgcggaaag atggccgctc ccggggccct gttgcaagga gctcaaaatg

960

gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg aagagggcct

1020

tgcccctcgc cggccgctgc ttcctgtgac cccgtggtct atcggccgca tagtcacctc

1080

gggcttctct tgagcaccgc tcgtcgcggc ggggggaggg gatctaatgg cgttggagtt

1140

tgttcacatt tggtgggtgg agactagtca ggccagcctg gcgctggaag tcattcttgg

1200

aatttgcccc tttgagtttg gagcgaggct aattctcaag cctcttagcg gttcaaaggt

1260

attttctaaa cccgtttcca ggtgttgtga aagccaccgc taattcaaag caa

1313

<210> 86

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 86

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta

60

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag

120

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt

180

tttaaagcaa gtaaaacctc tacaaatgtg gta

213

<210> 87

<211> 7

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 87

Pro Lys Lys Lys Arg Lys Val

15

<210> 88

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 88

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 89

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 89

Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala

1 5 10 15

Ser Arg Gly

<210> 90

<211> 7

<212>PRT

<213> Monkey virus 40

<400> 90

Pro Lys Lys Lys Arg Lys Val

15

<210> 91

<211> 21

<212> DNA

<213> Monkey virus 40

<400> 91

cccaagaaga agaggaaggt g

21

<210> 92

<211> 16

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

Bipartite NLS sequence of nucleoplasmin

<400> 92

Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1 5 10 15

<210> 93

<211> 9

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

NLS sequence C-myc

<400> 93

Pro Ala Ala Lys Arg Val Lys Leu Asp

15

<210> 94

<211> 11

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

NLS sequence C-myc

<400> 94

Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro

1 5 10

<210> 95

<211> 38

<212>PRT

<213> Homo sapiens

<400> 95

Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly

1 5 10 15

Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro

20 25 30

Arg Asn Gln Gly Gly Tyr

35

<210> 96

<211> 42

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

IBB domain from the importin-alpha sequence

<400> 96

Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu

1 5 10 15

Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys

20 25 30

Asp Glu Gln Ile Leu Lys Arg Arg Asn Val

35 40

<210> 97

<211> 8

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

Fibroids T protein sequence

<400> 97

Val Ser Arg Lys Arg Pro Arg Pro

15

<210> 98

<211> 8

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

Fibroids T protein sequence

<400> 98

Pro Pro Lys Lys Ala Arg Glu Asp

15

<210> 99

<211> 8

<212>PRT

<213> Homo sapiens

<400> 99

Pro Gln Pro Lys Lys Lys Pro Leu

15

<210> 100

<211> 12

<212>PRT

<213> Mus musculus

<400> 100

Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro

1 5 10

<210> 101

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 101

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtgcgcct cagtgagcga

60

gcgagcgcgc

70

<210> 102

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 102

gcgcgctcgc tcgctcactg aggcgcacgc ccgggtttcc cgggcggcct cagtgagcga

60

gcgagcgcgc

70

<210> 103

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 103

gcgcgctcgc tcgctcactg aggccgtcgg gcgacctttg gtcgcccggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 104

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 104

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 105

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 105

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 106

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 106

gcgcgctcgc tcgctcactg aggccgacgc ccgggctttg cccgggcggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 107

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 107

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg ctttgcccgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 108

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 108

gcgcgctcgc tcgctcactg aggccgggca aagcccgacg cccgggcttt gcccgggcgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 109

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 109

gcgcgctcgc tcgctcactg aggccgaaac gtcgggcgac ctttggtcgc ccggcctcag

60

tgagcgagcg agcgcgc

77

<210> 110

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 110

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgtt tcggcctcag

60

tgagcgagcg agcgcgc

77

<210> 111

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 111

gcgcgctcgc tcgctcactg aggcaaagcc tcagtgagcg agcgagcgcg c

51

<210> 112

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 112

gcgcgctcgc tcgctcactg aggctttgcc tcagtgagcg agcgagcgcg c

51

<210> 113

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 113

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct

60

cagtgagcga gcgagcgcgc

80

<210> 114

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 114

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 115

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 115

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgc

79

<210> 116

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 116

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcgcctc

60

agtgagcgag cgagcgcgc

79

<210> 117

<211> 5

<212>PRT

<213> Influenza virus

<400> 117

Asp Arg Leu Arg Arg

15

<210> 118

<211> 7

<212>PRT

<213> Influenza virus

<400> 118

Pro Lys Gln Lys Lys Arg Lys

15

<210> 119

<211> 10

<212>PRT

<213> Hepatitis delta virus

<400> 119

Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu

1 5 10

<210> 120

<211> 10

<212>PRT

<213> Mus musculus

<400> 120

Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg

1 5 10

<210> 121

<211> 20

<212>PRT

<213> Homo sapiens

<400> 121

Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys

1 5 10 15

Lys Ser Lys Lys

20

<210> 122

<211> 17

<212>PRT

<213> Homo sapiens

<400> 122

Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys

1 5 10 15

Lys

<210> 123

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 123

gtttaaac

8

<210> 124

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 124

ttaattaa

8

<210> 125

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 125

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt

60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc

120

tgcggcgcgc gcagcacctt t

141

<210> 126

<211> 317

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 126

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt

60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa

180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag

240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag

300

gcctaggctt ttgcaaa

317

<210> 127

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 127

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 128

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 128

gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac

60

<210> 129

<211> 1260

<212> DNA

<213> Adeno-associated virus-2

<400> 129

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag

60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag

120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg

180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc

300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg

360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt

420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc

480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa

540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg

600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg

660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag

720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc

780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag

840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc

900

aactacgcag acaggtacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg

960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga

1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc

1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataaatgatt

1200

taaatcaggt atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga

1260

<210> 130

<211> 1932

<212> DNA

<213> Adeno-associated virus-2

<400> 130

atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc

60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat

120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag

180

cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg

240

caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg

300

aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt

360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc

420

gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa

480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg

540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag

600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact

660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag

720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg

780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc

840

cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg gatttataaa

900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc

960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag

1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc

1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg

1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc

1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc

1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg

1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag

1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg

1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca

1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg

1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg

1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc

1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt

1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg

1800

cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa

1860

caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga

1920

cactctctctga

1932

<210> 131

<211> 1876

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 131

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg

60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt

120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga

180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct

240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac

300

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat

360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac

420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt

480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag

540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc

600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag

660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac

720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc

780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac

840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg

900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct

960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac

1020

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt

1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg

1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag

1200

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca

1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga

1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt

1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc

1500

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca

1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc

1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc

1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat

1800

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg

1860

catctttgaa caataa

1876

<210> 132

<211> 1194

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 132

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag

60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag

120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg

180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc

300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg

360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt

420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc

480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa

540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg

600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg

660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag

720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc

780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag

840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc

900

aactacgcag accgctacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg

960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga

1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc

1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataa

1194

<210> 133

<211> 1876

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 133

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg

60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt

120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga

180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct

240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac

300

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat

360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac

420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt

480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag

540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc

600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag

660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac

720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc

780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac

840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg

900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct

960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac

1020

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt

1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg

1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag

1200

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca

1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga

1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt

1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc

1500

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca

1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc

1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc

1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat

1800

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg

1860

catctttgaa caataa

1876

<210> 134

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 134

ctaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg c

51

<210> 135

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 135

ctaggactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct

60

cagtc

65

<210> 136

<211> 67

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 136

ggactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag

60

tcctgca

67

<210> 137

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 137

gtgcgggcga ccaaaggtcg cccgacgccc gggcgcactc a

41

<210> 138

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 138

ggactgaggc cgggcgacca aaggtcgccc gacgcccggg cggcctcagt cctgca

56

<210> 139

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 139

ctaggactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtc

54

<210> 140

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 140

ggactgaggc cgggcgacca aaggtcgccc gacggcctca gtcctgca

48

<210> 141

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 141

ctaggactga ggccgtcggg cgacctttgg tcgcccggcc tcagtc

46

<210> 142

<211> 67

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 142

ggactgaggc ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcgcctcag

60

tcctgca

67

<210> 143

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 143

atacctaggc acgcgtgtta ctagttatta atagtaatca attacgg

47

<210> 144

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 144

atacctaggg gccgcacgcg tgttactag

29

<210> 145

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 145

atacactcag tgcctgcagg cacgtggtcc ggagatccag ac

42

<210> 146

<211> 3754

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 146

cctaggtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct

60

tgtagttaat gattaacccg ccatgctact tatcgcggcc gctcaatatt ggccattagc

120

catattattc attggttata tagcataaat caatattggc tattggccat tgcatacgtt

180

gtatctatat cataatatgt acatttatat tggctcatgt ccaatatgac cgccatgttg

240

gcattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc

300

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa

360

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac

420

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca

480

agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg

540

gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt

600

agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc

660

ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat

720

gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg

780

cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc

840

ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg

900

agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc

960

cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc

1020

cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa

1080

gccttgaggg gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc

1140

gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct

1200

gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg

1260

gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt

1320

gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac

1380

ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt

1440

ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg

1500

gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc

1560

ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagaggcg

1620

cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc

1680

ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag

1740

ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg

1800

cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg

1860

accggcggct ctagagcctc tgctaaccat gttttagcct tcttcttttt cctacagctc

1920

ctgggcaacg tgctggttat tgtgctgtct catcatttgt cgacagaatt cctcgaagat

1980

ccgaaggggt tcaagcttgg cattccggta ctgttggtaa agccagttta aacgccgcca

2040

ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg

2100

acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct

2160

acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca

2220

ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga

2280

agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct

2340

tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc

2400

tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc

2460

acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga

2520

acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg

2580

ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc

2640

actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg

2700

tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt

2760

aattaattaa gagcatctta ccgccatta ttcccatatt tgttctgttt ttcttgattt

2820

gggtatacat ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg

2880

atatgtaatt actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg

2940

ttatttacgc tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac

3000

tgatattctt aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct

3060

gtatctagct attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt

3120

gctgtctctt ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt

3180

gtttgctgac gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg

3240

gactttcgct ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg

3300

ctgctggaca ggggctaggt tgctgggcac tgataattcc gtggtgttgt ctgtgccttc

3360

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc

3420

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg

3480

tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa

3540

tagcaggcat gctggggatg cggtgggctc tatggctcta gagcatggct acgtagataa

3600

gtagcatggc gggttaatca ttaactacac ctgcagcagg aacccctagt gatggagttg

3660

gccactccct ctctgcgcgc tcgctcgctc cctgcaggac tgaggccggg cgaccaaagg

3720

tcgcccgacg cccgggcggc ctcagtcctg cagg

3754

<210> 147

<211> 8418

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 147

ggcagctgcg cgctcgctcg ctcacctagg ccgcccgggc aaagcccggg cgtcgggcga

60

cctttggtcg cccggcctag gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatcg cggccgctca

180

atattggcca ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg

240

gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct catgtccaat

300

atgaccgcca tgttggcatt gattattgac tagttattaa tagtaatcaa ttacggggtc

360

attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc

420

tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt

480

aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca

540

cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg

600

taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca

660

gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt

720

cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt

780

attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg

840

gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc

900

gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag

960

cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg

1020

cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg

1080

acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc

1140

tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg ggggagcggc

1200

tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggccc gcgctgcccg

1260

gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg

1320

ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct

1380

gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt

1440

aacccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg

1500

ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg

1560

gtgccgggcg gggcggggcc gcctcggggcc ggggagggct cgggggaggg gcgcggcggc

1620

ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa

1680

tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg

1740

aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag

1800

gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag

1860

cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc

1920

ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgtttt agccttcttc

1980

tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca tttgtcgaca

2040

gaattcctcg aagatccgaa ggggttcaag cttggcattc cggtactgtt ggtaaagcca

2100

gtttaaacgc cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca

2160

tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgaggcg

2220

agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc

2280

ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct

2340

accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc

2400

aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt

2460

tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg

2520

gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg

2580

ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg

2640

gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc

2700

tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga

2760

agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg

2820

acgagctgta caagtaatta attaagagca tcttaccgcc atttattccc atatttgttc

2880

tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca

2940

tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt

3000

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat

3060

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc

3120

tgctttatag cctctgtatc tagctattgc ttcccgtacg gctttcgttt tctcctcctt

3180

gtataaatcc tggttgctgt ctcttttaga ggagttgtgg cccgttgtcc gtcaacgtgg

3240

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg

3300

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc

3360

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt

3420

gttgtctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt

3480

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca

3540

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga

3600

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctctagagca

3660

tggctacgta gataagtagc atggcgggtt aatcattaac tacacctgca gcaggaaccc

3720

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctccctgc aggactgagg

3780

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tcctgcaggg agcgagcgag

3840

cgcgcagctg cctgcacggg cgcgccggta ccgggagatg ggggaggcta actgaaacac

3900

ggaagggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa

3960

cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct

4020

gtcgataccc caccgagacc ccattgggac caatacgccc gcgtttcttc cttttcccca

4080

ccccaacccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaagc

4140

cctgccatag ccactacggg tacgtaggcc aaccactaga actatagcta gagtcctggg

4200

cgaacaaacg atgctcgcct tccagaaaac cgaggatgcg aaccacttca tccggggtca

4260

gcaccaccgg caagcgccgc gacggccgag gtctaccgat ctcctgaagc cagggcagat

4320

ccgtgcacag caccttgccg tagaagaaca gcaaggccgc caatgcctga cgatgcgtgg

4380

agaccgaaac cttgcgctcg ttcgccagcc aggacagaaa tgcctcgact tcgctgctgc

4440

ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagttgacat

4500

aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc aactggtcca

4560

gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg

4620

actgtttttt tgtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg

4680

gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagcaacga tgttacgcag

4740

cagggcagtc gccctaaaac aaagttaggt ggctcaagta tgggcatcat tcgcacatgt

4800

aggctcggcc ctgaccaagt caaatccatg cgggctgctc ttgatctttt cggtcgtgag

4860

ttcggagacg tagccaccta ctcccaacat cagccggact ccgattacct cgggaacttg

4920

ctccgtagta agacattcat cgcgcttgct gccttcgacc aagaagcggt tgttggcgct

4980

ctcgcggctt acgttctgcc caggtttgag cagccgcgta gtgagatcta tatctatgat

5040

ctcgcagtct ccggcgagca ccggaggcag ggcattgcca ccgcgctcat caatctcctc

5100

aagcatgagg ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga ttacggtgac

5160

gatcccgcag tggctctcta tacaaagttg ggcatacggg aagaagtgat gcactttgat

5220

atcgacccaa gtaccgccac ctaacaattc gttcaagccg agatcggctt cccggccgcg

5280

gagttgttcg gtaaattgtc acaacgccgc gaatatagtc tttaccatgc ccttggccac

5340

gcccctcttt aatacgacgg gcaatttgca cttcagaaaa tgaagagttt gctttagcca

5400

taacaaaagt ccagtatgct ttttcacagc ataactggac tgatttcagt ttacaactat

5460

tctgtctagt ttaagacttt attgtcatag tttagatcta ttttgttcag tttaagactt

5520

tattgtccgc ccacacccgc ttacgcaggg catccattta ttactcaacc gtaaccgatt

5580

ttgccaggtt acgcggctgg tctgcggtgt gaaataccgc acagatgcgt aaggagaaaa

5640

taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg

5700

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg

5760

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag

5820

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga

5880

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct

5940

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc

6000

tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg

6060

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc

6120

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca

6180

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag

6240

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct

6300

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc

6360

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga

6420

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca

6480

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat

6540

taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac

6600

caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt

6660

gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt

6720

gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag

6780

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct

6840

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt

6900

gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc

6960

tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt

7020

agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg

7080

gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg

7140

actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct

7200

tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc

7260

attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt

7320

tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt

7380

tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg

7440

aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat

7500

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg

7560

cgcacatttc cccgaaaagt gccacctgaa attgtaaacg ttaatatttt gttaaaattc

7620

gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc

7680

ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag

7740

agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc

7800

gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa

7860

gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg

7920

aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt

7980

gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacaggc

8040

gcgtcccatt cgccattcag gctgcaaata agcgttgata ttcagtcaat tacaaacatt

8100

aataacgaag agatgacaga aaaattttca ttctgtgaca gagaaaaagt agccgaagat

8160

gacggtttgt cacatggagt tggcaggatg tttgattaaa aacataacag gaagaaaaat

8220

gccccgctgt gggcggacaa aatagttggg aactgggagg ggtggaaatg gagtttttaa

8280

ggattattta gggaagagtg acaaaataga tgggaactgg gtgtagcgtc gtaagctaat

8340

acgaaaatta aaaatgacaa aatagtttgg aactagatt cacttatctg gttcggatct

8400

cctagtgagc tccctgca

8418

<210> 148

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 148

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct

60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct

120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg

180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggc

225

<210> 149

<211> 1177

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 149

ggctcagagg ctcagaggca cacaggagtt tctgggctca ccctgccccc ttccaacccc

60

tcagttccca tcctccagca gctgtttgtg tgctgcctct gaagtccaca ctgaacaaac

120

ttcagcctac tcatgtccct aaaatgggca aacattgcaa gcagcaaaca gcaaacacac

180

agccctccct gcctgctgac cttggagctg gggcagaggt cagagacctc tctgggccca

240

tgccacctcc aacatccact cgaccccttg gaatttcggt ggagaggagc agaggttgtc

300

ctggcgtggt ttaggtagtg tgagagggtc cgggttcaaa accacttgct gggtggggag

360

tcgtcagtaa gtggctatgc cccgaccccg aagcctgttt ccccatctgt acaatggaaa

420

tgataaagac gcccatctga tagggttttt gtggcaaata aacatttggt ttttttgttt

480

tgttttgttt tgttttttga gatggaggtt tgctctgtcg cccaggctgg agtgcagtga

540

cacaatctca tctcaccaca accttcccct gcctcagcct cccaagtagc tgggattaca

600

agcatgtgcc accacacctg gctaattttc tatttttagt agagacgggt ttctccatgt

660

tggtcagcct cagcctccca agtaactggg attacaggcc tgtgccacca cacccggcta

720

attttttcta tttttgacag ggacggggtt tcaccatgtt ggtcaggctg gtctagaggt

780

accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag cagaggcca

840

gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac cttggacaca

900

ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca gtggaagctg

960

tacactgccc aggcaaagcg tccgggcagc gtaggcgggc gactcagatc ccagccagtg

1020

gacttagccc ctgtttgctc ctccgataac tggggtgacc ttggttaata ttcaccagca

1080

gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca gggccctgtc

1140

tcctcagctt caggcaccac cactgacctg ggacagt

1177

<210> 150

<211> 1326

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 150

ctgcagggcc cactagtgga gccgagagta attcatacaa aaggagggat cgccttcgca

60

aggggagagc cccaggaccg tccctaaatt ctcacagacc caaatccctg tagccgcccc

120

acgacagcgc gaggagcatg cgcccagggc tgagcgcggg tagatcagag cacacaagct

180

cacagtcccc ggcggtgggg ggaggggcgc gctgagcggg ggccagggag ctggcgcggg

240

gcaaactggg aaagtggtgt cgtgtgctgg ctccgccctc ttcccgaggg tggggggagaa

300

cggtatataa gtgcggtagt cgccttggac gttctttttc gcaacgggtt tgccgtcaga

360

acgcaggtga gtggcgggtg tggcttccgc gggccccgga gctggagccc tgctctgagc

420

gggccgggct gatatgcgag tgtcgtccgc agggtttagc tgtgagcatt cccacttcga

480

gtggcgggcg gtgcgggggt gagagtgcga ggcctagcgg caaccccgta gcctcgcctc

540

gtgtccggct tgaggcctag cgtggtgtcc gccgccgcgt gccactccgg ccgcactatg

600

cgttttttgt ccttgctgcc ctcgattgcc ttccagcagc atgggctaac aaagggaggg

660

tgtggggctc actcttaagg agcccatgaa gcttacgttg gataggaatg gaagggcagg

720

aggggcgact ggggcccgcc cgccttcgga gcacatgtcc gacgccacct ggatggggcg

780

aggcctgtgg ctttccgaag caatcgggcg tgagtttagc ctacctgggc catgtggccc

840

tagcactggg cacggtctgg cctggcggtg ccgcgttccc ttgcctccca acaagggtga

900

ggccgtcccg cccggcacca gttgcttgcg cggaaagatg gccgctcccg gggccctgtt

960

gcaaggagct caaaatggag gacgcggcag cccggtggag cgggcgggtg agtcaccac

1020

acaaaggaag agggccttgc ccctcgccgg ccgctgcttc ctgtgacccc gtggtctatc

1080

ggccgcatag tcacctcggg cttctcttga gcaccgctcg tcgcggcggg gggaggggat

1140

ctaatggcgt tggagtttgt tcacatttgg tgggtggaga ctagtcaggc cagcctggcg

1200

ctggaagtca ttcttggaat ttgccccttt gagtttggag cgaggctaat tctcaagcct

1260

cttagcggtt caaaggtatt ttctaaaccc gtttccaggt gttgtgaaag ccaccgctaa

1320

ttcaaa

1326

<210> 151

<211> 573

<212> DNA

<213> Mus musculus

<400> 151

gtaagagttt tatgtttttt catctctgct tgtatttttc tagtaatgga agcctggtat

60

tttaaaatag ttaaattttc ctttagtgct gatttctaga ttattattac tgttgttgtt

120

gttattattg tcattatttg catctgagaa cccttaggtg gttatattat tgatatattt

180

ttggtatctt tgatgacaat aatgggggat tttgaaagct tagctttaaa tttcttttaa

240

ttaaaaaaaa atgctaggca gaatgactca aattacgttg gatacagttg aatttattac

300

ggtctcatag ggcctgcctg ctcgaccatg ctatactaaa aattaaaagt gtgtgttact

360

aattttataa atggagtttc catttatatt tacctttatt tcttatttac cattgtctta

420

gtagatattt acaaacatga cagaaacact aaatcttgag tttgaatgca cagatataaa

480

cacttaacgg gttttaaaaa taataatgtt ggtgaaaaaa tataactttg agtgtagcag

540

agaggaacca ttgccacctt cagattttcc tgt

573

<210> 152

<211> 1993

<212> DNA

<213> Mus musculus

<400> 152

acgatcggga actggcatct tcagggagta gcttaggtca gtgaagagaa gaacaaaaag

60

cagcatatta cagttagttg tcttcatcaa tctttaaata tgttgtgtgg tttttctctc

120

cctgtttcca cagacaagag tgagatcgcc catcggtata atgatttggg agaacaacat

180

ttcaaaggcc tgtaagttat aatgctgaaa gcccacttaa tatttctggt agtattagtt

240

aaagttttaa aacacctttt tccaccttga gtgtgagaat tgtagagcag tgctgtccag

300

tagaaatgtg tgcattgaca gaaagactgt ggatctgtgc tgagcaatgt ggcagccaga

360

gatcacaagg ctatcaagca ctttgcacat ggcaagtgta actgagaagc acacattcaa

420

ataatagtta attttaattg aatgtatcta gccatgtgtg gctagtagct cctttcctgg

480

agagagaatc tggagcccac atctaacttg ttaagtctgg aatcttattt tttatttctg

540

gaaaggtcta tgaactatag ttttgggggc agctcactta ctaactttta atgcaataag

600

atctcatggt atcttgagaa cattattttg tctctttgta gtactgaaac cttatacatg

660

tgaagtaagg ggtctatact taagtcacat ctccaacctt agtaatgttt taatgtagta

720

aaaaaatgag taattaattt atttttagaa ggtcaatagt atcatgtatt ccaaataaca

780

gaggtatatg gttagaaaag aaacaattca aaggacttat ataatatcta gccttgacaa

840

tgaataaatt tagagagtag tttgcctgtt tgcctcatgt tcataaatct attgacacat

900

atgtgcatct gcacttcagc atggtagaag tccatattcc tttgcttgga aaggcaggtg

960

ttcccattac gcctcagaga atagctgacg ggaagaggct ttctagatag ttgtatgaaa

1020

gatatacaaa atctcgcagg tatacacagg catgatttgc tggttgggag agccacttgc

1080

ctcatactga ggtttttgtg tctgcttttc agagtcctga ttgccttttc ccagtatctc

1140

cagaaatgct catacgatga gcatgccaaa ttagtgcagg aagtaacaga ctttgcaaag

1200

acgtgtgttg ccgatgagtc tgccgccaac tgtgacaaat cccttgtgag taccttctga

1260

ttttgtggat ctactttcct gctttctgga actctgtttc aaagccaatc atgactccat

1320

cacttaaggc cccgggaaca ctgtggcaga gggcagcaga gagattgata aagccaggt

1380

gatgggaatt ttctgtggga ctccatttca tagtaattgc agaagctaca atacactcaa

1440

aaagtctcac cacatgactg cccaaatggg agcttgacag tgacagtgac agtagatatg

1500

ccaaagtgga tgagggaaag accacaagag ctaaaccctg taaaaagaac tgtaggcaac

1560

taaggaatgc agagagaaga agttgccttg gaagagcata ccaactgcct ctccaatacc

1620

aatggtcatc cctaaaacat acgtatgaat aacatgcaga ctaagcaggc tacatttagg

1680

aatatacatg tatttacata aatgtatatg catgtaacaa caatgaatga aaactgaggt

1740

catggatctg aaagagagca agggggctta catgagagg tttggaggga ggggttggag

1800

ggagggaggt attattcttt agttttacag ggaacgtagt aaaaacatag gcttctccca

1860

aaggagcaga gcccatgagg agctgtgcaa ggttccccag cttgatttta cctgctcctc

1920

aaattccctt gatttgtttt tattataatg actttactcc tagcttttag tgtcagatag

1980

aaaacatgga agg

1993

<210> 153

<211> 1350

<212> DNA

<213> Homo sapiens

<400> 153

taggaggctg aggcaggagg atcgcttgag cccaggagtt cgagaccagc ctgggcaaca

60

tagtgtgatc ttgtatctat aaaaataaac aaaattagct tggtgtggtg gcgcctgtag

120

tccccagcca cttggagggg tgaggtgaga ggattgcttg agcccgggat ggtccaggct

180

gcagtgagcc atgatcgtgc cactgcactc cagcctgggc gacagagtga gaccctgtct

240

cacaacaaca acaacaacaa caaaaaggct gagctgcacc atgcttgacc cagtttctta

300

aaattgttgt caaagcttca ttcactccat ggtgctatag agcacaagat tttatttggt

360

gagatggtgc tttcatgaat tcccccaaca gagccaagct ctccatctag tggacaggga

420

agctagcagc aaaccttccc ttcactacaa aacttcattg cttggccaaa aagagagtta

480

attcaatgta gacatctatg taggcaatta aaaacctatt gatgtataaa acagtttgca

540

ttcatggagg gcaactaaat acattctagg actttataaa agatcacttt ttatttatgc

600

acagggtgga acaagatgga ttatcaagtg tcaagtccaa tctatgacat caattattat

660

acatcggagc cctgccaaaa aatcaatgtg aagcaaatcg cagcccgcct cctgcctccg

720

ctctactcac tggtgttcat ctttggtttt gtgggcaaca tgctggtcat cctcatcctg

780

ataaactgca aaaggctgaa gagcatgact gacatctacc tgctcaacct ggccatctct

840

gacctgtttt tccttcttac tgtccccttc tgggctcact atgctgccgc ccagtgggac

900

tttggaaata caatgtgtca actcttgaca gggctctatt ttataggctt cttctctgga

960

atcttcttca tcatcctcct gacaatcgat aggtacctgg ctgtcgtcca tgctgtgttt

1020

gctttaaaag ccaggacggt cacctttggg gtggtgacaa gtgtgatcac ttgggtggtg

1080

gctgtgtttg cgtctctccc aggaatcatc tttaccagat ctcaaaaaga aggtcttcat

1140

tacacctgca gctctcattt tccatacagt cagtatcaat tctggaagaa tttccagaca

1200

ttaaagatag tcatcttggg gctggtcctg ccgctgcttg tcatggtcat ctgctactcg

1260

ggaatcctaa aaactctgct tcggtgtcga aatgagaaga agaggcacag ggctgtgagg

1320

cttatcttca ccatcatgat tgtttatttt

1350

<210> 154

<211> 1223

<212> DNA

<213> Homo sapiens

<400> 154

tgacagagac tcttgggatg acgcactgct gcatcaaccc catcatctat gcctttgtcg

60

gggagaagtt cagaaactac ctcttagtct tcttccaaaa gcacattgcc aaacgcttct

120

gcaaatgctg ttctattttc cagcaagagg ctcccgagcg agcaagctca gtttacaccc

180

gatccactgg ggagcaggaa atatctgtgg gcttgtgaca cggactcaag tgggctggtg

240

acccagtcag agttgtgcac atggcttagt tttcatacac agcctgggct gggggtgggg

300

tggggagaggt cttttttaaa aggaagttac tgttatagag ggtctaagat tcatccatt

360

atttggcatc tgtttaaagt agattagatc ttttaagccc atcaattata gaaagccaaa

420

tcaaaatatg ttgatgaaaa atagcaacct ttttatctcc ccttcacatg catcaagtta

480

ttgacaaact ctcccttcac tccgaaagtt ccttatgtat atttaaaaga aagcctcaga

540

gaattgctga ttcttgagtt tagtgatctg aacagaaata ccaaaattat ttcagaaatg

600

tacaactttt tacctagtac aaggcaacat ataggttgta aatgtgttta aaacaggtct

660

ttgtcttgct atggggagaa aagacatgaa tatgattagt aaagaaatga cacttttcat

720

gtgtgatttc ccctccaagg tatggttaat aagtttcact gacttagaac caggcgagag

780

acttgtggcc tgggagagct ggggaagctt cttaaatgag aaggaatttg agttggatca

840

tctattgctg gcaaagacag aagcctcact gcaagcactg catgggcaag cttggctgta

900

gaaggagaca gagctggttg ggaagacatg gggaggaagg acaaggctag atcatgaaga

960

accttgacgg cattgctccg tctaagtcat gagctgagca ggggagatcct ggttggtgtt

1020

gcagaaggtt tactctgtgg ccaaaggagg gtcaggaagg atgagcattt agggcaagga

1080

gaccaccaac agccctcagg tcagggtgag gatggcctct gctaagctca aggcgtgagg

1140

atgggaagga gggaggtatt cgtaaggatg ggaaggaggg aggtattcgt gcagcatatg

1200

aggatgcaga gtcagcagaa ctg

1223

<210> 155

<211> 215

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 155

gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa

60

cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc

120

tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg

180

gatttgggaa tcttataagt tctgtatgag accac

215

<210> 156

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 156

cctgcaggca gctgcgcgct cgctcgctca cctaggccgc ccgggcaaag cccgggcgtc

60

gggcgacctt tggtcgcccg gcctaggtga gcgagcgagc gcgcagagag ggagtggcca

120

actccatcac taggggttcc t

141

<210> 157

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 157

gcgcgctcgc tcgctcacc

19

<210> 158

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 158

ctaggtgagc gagcgagcgc gc

22

<210> 159

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 159

cctgcaggac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg

60

cctcagtcct gcagg

75

<210> 160

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 160

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagt

60

gcgggcgacc aaaggtcgcc cgacgcccgg gcgcactcag tgagcgagcg agcgcgcagc

120

tgcctgcagg

130

<210> 161

<211> 142

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 161

cctgcaggca gctgcgcgct cgctcgctcc ctaggactga ggccgcccgg gcgtcgggcg

60

acctttggtc gcccggcctc agtcctaggg agcgagcgag cgcgcagaga gggagtggcc

120

aactccatca ctaggggttc ct

142

<210> 162

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 162

gcgcgctcgc tcgctcactg agtgcgggcg accaaaggtc gcccgacgcc cgggcgcact

60

cagtgagcga gcgagcgcgc

80

<210> 163

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 163

gcgcgctcgc tcgctcactg a

21

<210> 164

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 164

gtgagcgagc gagcgcgc

18

<210> 165

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 165

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgactttgtc

60

gcccggcctc agtgagcgag cgagcgcgc

89

<210> 166

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 166

gcgcgctcgc tcgctcactg aggccgggcg acaaagtcgc ccgacgcccg ggctttgccc

60

gggcggcctc agtgagcgag cgagcgcgc

89

<210> 167

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 167

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgattttcgc

60

ccggcctcag tgagcgagcg agcgcgc

87

<210> 168

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 168

gcgcgctcgc tcgctcactg aggccgggcg aaaatcgccc gacgcccggg ctttgcccgg

60

gcggcctcag tgagcgagcg agcgcgc

87

<210> 169

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 169

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgtttcgccc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 170

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 170

gcgcgctcgc tcgctcactg aggccgggcg aaacgcccga cgcccgggct ttgcccgggc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 171

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 171

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtcgggcg acctttggtc

60

gcccggcctc agtgagcgag cgagcgcgc

89

<210> 172

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 172

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggtttccc

60

gggcggcctc agtgagcgag cgagcgcgc

89

<210> 173

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 173

gcgcgctcgc tcgctcactg aggccgcccg gaaaccgggc gtcgggcgac ctttggtcgc

60

ccggcctcag tgagcgagcg agcgcgc

87

<210> 174

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 174

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cggtttccgg

60

gcggcctcag tgagcgagcg agcgcgc

87

<210> 175

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 175

gcgcgctcgc tcgctcactg aggccgcccg aaacgggcgt cgggcgacct ttggtcgccc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 176

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 176

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgtttcgggc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 177

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 177

gcgcgctcgc tcgctcactg aggccgccca aagggcgtcg ggcgaccttt ggtcgcccgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 178

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 178

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc ctttgggcgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 179

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 179

gcgcgctcgc tcgctcactg aggccgccaa aggcgtcggg cgacctttgg tcgcccggcc

60

tcagtgagcg agcgagcgcg c

81

<210> 180

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 180

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc tttggcggcc

60

tcagtgagcg agcgagcgcg c

81

<210> 181

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 181

gcgcgctcgc tcgctcactg aggccgcaaa gcgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgc

79

<210> 182

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 182

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgct ttgcggcctc

60

agtgagcgag cgagcgcgc

79

<210> 183

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 183

ctgcgcgctc gctcgctcac tgaggccgaa acgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgca g

81

<210> 184

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 184

ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg tttcggcctc

60

agtgagcgag cgagcgcgca g

81

<210> 185

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 185

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 186

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 186

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 187

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 187

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgc

79

<210> 188

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 188

ggagtcaaag ttctgtttgc cctgatctgc atcgctgtgg ccgaggcc

48

<210> 189

<211> 99

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 189

attcatacca acttgaagaa aaagttcagc ctcttcatcc tggtctttct cctgttcgca

60

gtcatctgtg tttggaagaa agggagcgac tatgaggcc

99

<210> 190

<211> 588

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polynucleotide

<400> 190

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt

60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc

120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag

180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac

240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc

300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc

360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca

420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt

480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg

540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaacc

588

<---

Claims (61)

1. A non-capsid closed-end DNA vector (ccDNA) for delivery of a heterologous nucleotide sequence, containing: a first and a second heterologous nucleotide sequence between flanking inverted terminal repeats (ITRs), wherein the first heterologous nucleotide sequence encodes an antibody heavy chain variable region and the second heterologous nucleotide sequence encodes an antibody light chain variable region, and wherein the ccDNA comprises an expression cassette comprising a dual promoter system, wherein different promoters are used to express the heavy chain variable region and the light chain variable region of the antibody. 2. cccDNA vector according to claim 1, characterized in that said antibody is a full-length antibody. 3. cccDNA vector according to claim 1, characterized in that said antibody is a Fab, Fab', single domain antibody or single chain antibody (scFv). 4. cccDNA vector according to claim 3, characterized in that said antibody is a single-domain antibody or a single-chain antibody. 5. cccDNA vector according to claim 4, characterized in that said first heterologous nucleotide sequence additionally encodes a heavy chain constant region or part thereof and said second heterologous nucleotide sequence additionally encodes a light chain constant region or part thereof. 6. cccDNA vector according to claim 5, characterized in that said first heterologous nucleotide sequence and/or said second heterologous nucleotide sequence further encodes a secretory leader sequence preceding the heavy chain variable region and/or the light chain variable region. 7. cccDNA vector according to any one of paragraphs. 1-6, characterized in that: said antibody is a human or humanized antibody; said antibody is an IgG, IgA, IgD, IgM or IgE antibody; and/or said antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. 8. cccDNA vector according to any one of paragraphs. 1-7, characterized in that said antibody binds to at least one target selected from the group consisting of the following: Transferrin receptor, Integrin alpha-4, EGFR, CD25 (IL-2 receptor alpha chain), TNF-alpha , CTLA4, IgE, C5, IL-6R, BAFF (BLyS), VEGF-A, P-selectin, HER2, PCSK9, PD-L1, oxLDL, Dabigatran, Parathyroid hormone-related protein (PTHrP), RANKL, NGF, PD1 receptor , Alpha4beta7 integrin, Integrin alpha4beta7, IL17a, CSa, PD1, PDGFR-beta, TiKA, TrkA, Amyloid Beta, Activin, CD33, CD19, IL-6, NGF, CD20, GFRa3, CORP, CGRP, Annexin IV or phospholipid; and (b) complement inhibitor, Nav 1.7, Factor D, Rh antigen, TGF-beta, Alpha-synuclein, Bacillus anthracis protective antigen, ANGPTL-3, B7RP1, BAFF, BDCA2, C1, CD22, CD30, CD38, CD52 , CD319 (SLAMF7), Clostridia toxins A and B, Fel d1, GDF8, Glycolipid GD2, Glycoprotein IIb/IIa receptor, GM-CSF, IFN-alpha receptor, IG2-kappa, IL-1-beta, IL-3R, IL -5, IL-5R (CD125), IL-17 receptor, IL-23, IL-31, IL-33, Influenza A, Integrin alphaIIbbeta3, Kallikrein, LAG-3, LEPR, LINGO-1, Middle East respiratory coronavirus, Myostatin , NGF, PSMA, RSV F protein, Sclerostin, SOST, Staphylococcus aureus toxin, Tau protein, TFPI, Thymic stromal lymphoprotein, Alpha3beta7/alphaEbeta7, CD20/CD3, Factor IX/factor X, IL-4/ IL-13, IL-12/IL-23, IL-33/STR2, Psl/PcrV, VEGF-A/Ang2, Activin receptor type 2AIgG-Fc fusion protein; IL-17RA, PD-L1, IL-4Rα, CD20, PD-L1, IL-6R, IL-23 p19, CD22, IL-5Rα, Factor IXa, X; CD4, FGF23, IL-23 p19, von Willebrand factor, CGRP receptor, CGRP, Sclerostin, CCR4; CD62 (aka P-selectin), Rh D, Plasma Kallikrein, Amyloid, MASP-2, C5, Thymic stromal lymphopoietin, IL-13, Il-23 p19, Interferon receptor (IFN) α, β, ω type 1 , Integrin receptor α4-β7/αE-β7, Mucosal cell adhesion molecule addressin-1, IL-6, CD19, IL-6R, IFN γ, CCR5, Amyloid β, CGRP, NGF (nerve growth factor), IGF-1R , Complement factor D, VEGF-A; CD45, CD38, CD20, CD19, 4–1BB (CD137), EpCAM, PD-1, HER2, MMP-9, CTLA-4, NGcGM3, PD-1, Fibronectin Extradomain B and Endoglin. 9. cccDNA vector according to any one of paragraphs. 1-8, characterized in that said antibody is selected from the group consisting of the following: Abaloparatide, TYMLOS®, abciximab, REOPRO®, adalimumab, HUMIRA®, aducanumab, aflibercept, EYLEA®, alemtuzumab, CAMPATH, LEMTRADA®, alirocumab, PRALUENT ®, anifrolumab, atezolizumab, TECENTRIQ®, atlizumab, avelumab, BAVENCIO®, basiliximab, SIMULECT®, bapinezumab, belimumab, BENLYSTA®, benralizumab, FASENRA®, bevacizumab, AVASTIN®, bezlotoxumab, ZINPLAVA®, blinatumomab, BLINCY TO®, blosozumab, bococizumab, brentuximab vedotin, ADCETRIS®, brodalumab, SILIQ®, LUMICEF®, KYNTHEUM®, brolucizumab, burosumab, burosumab-twza, CRYSVITA®, canakinumab, ILARIS®, caplacizumab, Capromab penetide, PROSTASCINT®, Cemiplimab, certolizumab pegol , CIMZIA® , cetuximab, ERBITUX®, concizumab, crenezumab, MEDI1814, daclizumab, ZENAPAX®, ZINBRYTA®, daratumumab, DARZALEX®, denosumab, PROLIA®, XGEVA®, dinutuximab, UNITUXIN®, dinutuximab beta, ISQUETTE®, Domagrozumab, RG6206, dupil umab, DUPIXENT®, durvalumab, IMFINZI®, Ecallantide, KALBITOR®, eculizumab, SOLIRIS®, elotuzumab, EMPLICITI®, emicizumab, emicizumab-kxwh, HEMLIBRA®, evinacumab, erenumab, erenumab-aooe, AIMOVIG®, evolocumab, REPATHA®, fasinumab, fremanezumab, Fulraintin, 4D4, AMG-403, lN.142160443, Fulranumab; 4D4, AMG-403, JNJ-, REGN 2477, BIMAGRUMAB®, garetosmab, galcanezumab, gantenerumab, gemtuzumab, gemtuzumab ozogamicin, Mylotarg®, golimumab, SIMPONI®, SIMPONI ARIA®, guselkumab, TREMFYA®, ibalizumab, ibalizumab- uiyk, TROGARZO ®, ibritumomab, ibritumomab tiuxetan, ZEVALIN®, idarucizumab, PRAXBIND®, Inclacumab, LC1004-002, R04905417, inebilizumab, infliximab, REMICADE®, inotuzumab, inotuzumab ozogamicin, BESPONSA®, Orencia, ocrelizumab, O CREVUS®, ipilimumab, YERVOY®, ixekizumab, TALTZ®, Mavrilimumab, mepolizumab, NUCALA®, Mirikizumab, Mogamulizumab, POTELIGEO®, Namilumab, natalizumab, TYSABRI®, necitumumab, PORTRAZZA®, Nemolizumab, nivolumab, OPDIVO®, obiltoxaximab, ANTHIM®, obinutuzumab, G AZYVA®, ocrelizumab, OCREVUS®, Ofatumumab, ARZERRA®, 3299N, Olaratumab, LARTRUVO®, Omalizumab, XOLAIR®, Opininumab, Orticumab, BI-204, MLDL-1278A, R7418, Lambrolizumab, Fab238, Lampalizumab, Lanadelumab, Ligelizumab, Lodelcizumab, Pali vizumab, SYNAGIS® , panitumumab, VECTIBIX®, pembrolizumab, KEYTRUDA®, pertuzumab, PERJETA®, KADCYLA®, REGN3918, pexelizumab, Prezalumab, ralpancizumab, ramucirumab, CYRAMZA®, ranezumab, ranibizumab, LUCENTIS®, raxibacumab, reslizumab, CINQAIR®, roledumab, DMATRIA® , romosozumab, rituximab, RITUXAN®, satralizumab, sarilumab, KEVZARA®, secukinumab, COSENTYX®, solenezumab, siltuximab, SYLVANT®, Suvratoxumab, tadocizumab, Tezepelumab, tildrakizumab, tildrakizumab-asmn, ILUMYA®, tocilizumab b, ACTEMRA®, trastuzumab, HERCEPTIN ®, Trevogrumab, ustekinumab, STELARA®, vedolizumab, ENTYVIO®, BNJ378, BNJ383, BXhVH5VLl, GBR, VH5(P60A, T62 S)VLl, BAN2401, LY3002813, H4H2364S; 01, cluster headache; BXhVHl®, HUVHWOV®, H1Hl015B, DOM23h-271-12, BIIB054, RG7935, VAY736, BIIB059, BIVV009, REGN1908, REGN1909, SHP647, FENSENRA®, GS3511294, REGN3500, MEDI3 506, GSK3772847 (formally CNTO 716), MEDI8852, SHP643, DX-2930, REGN3767, REGN3048, REGN3049, REGN3050, REGN3051, 42160443, MEDI8897, SP0232, BIIB092, RG6100, NN7415; REGN1979, SAR156597, AMG 282, RG6149, MEDI3902, RG7716; ACE-011, SOTATERCEPT®; Crizanlizumab, NEOD001, OMS721, Ravulizumab (ALXN1210), Tralokinumab, Risankizumab, Etrolizumab, Olokizumab, Emapalumab, PRO-140, PA14, Eptinezumab, Tanezumab, Teprotumumab; I-131-BC8, Iomab-B (Iomab-B), Isatuximab, Ublituximab, XMAB-5574, MOR208, Utomilumab, Oportuzumab Monatox, Camrelizumab, Margetuximab, Andecaliximab, Tremelimumab, Racotumomab, IBI308, BGB-A317, BCD-100, PDR001, L19IL2/L19TNF and Carotuximab. 10. cccDNA vector according to any one of paragraphs. 1-9, characterized in that said cDNA vector contains one or more poly(A) sites. 11. cccDNA vector according to any one of paragraphs. 1-10, characterized in that at least one heterologous nucleotide sequence is a cDNA. 12. cccDNA vector according to any one of paragraphs. 1-11, characterized in that at least one or both of said flanking ITRs contain a functional end resolution site and a Rep binding site. 13. cccDNA vector according to any one of paragraphs. 1-12, characterized in that one or both of said flanking ITRs are taken from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV). 14. cccDNA vector according to any one of paragraphs. 1-13, characterized in that: said flanking ITRs are symmetric or asymmetric; said flanking ITRs are symmetrical or substantially symmetrical; said flanking ITRs are asymmetric; or one or both of the flanking ITRs are wild type or both of the ITRs are wild type. 15. cccDNA vector according to any one of paragraphs. 1-14, characterized in that said flanking ITRs are taken from different viral serotypes. 16. cccDNA vector according to any one of paragraphs. 1-15, characterized in that said flanking ITRs are selected from the group consisting of pairs of viral serotypes given in the Table: 17. cccDNA vector according to any one of paragraphs. 1-16, wherein one or both of said flanking ITRs contain a sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 5-48. 18. cccDNA vector according to any one of paragraphs. 1-17, wherein at least one of said flanking ITRs is altered relative to the wild-type AAV ITR sequence by deletion, addition or substitution that affects the overall three-dimensional conformation of said ITR. 19. cccDNA vector according to any one of paragraphs. 1-18, characterized in that one or both of said flanking ITRs are derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. 20. cccDNA vector according to any one of paragraphs. 1-19, characterized in that one or both of said flanking ITRs are synthetic. 21. cccDNA vector according to any one of paragraphs. 1-20, wherein one or both of the flanking ITRs are not wild-type ITRs, or both of the flanking ITRs are not wild-type. 22. cccDNA vector according to any one of paragraphs. 1-21, characterized in that one or both of said flanking ITRs is modified by deletion, insertion and/or substitution in at least one of the regions of said ITRs selected from the group consisting of A, A', B, B', C , C', D and D'. 23. The cccDNA vector of claim 22, wherein said deletion, insertion and/or substitution results in the removal of all or part of the stem-loop structure typically formed by regions A, A', B, B', C or C' . 24. cccDNA vector according to any one of paragraphs. 1-23, characterized in that: one or both of said flanking ITRs is modified by deletion, insertion and/or substitution that removes all or part of the stem-loop structure formed by regions B and B'; one or both of said flanking ITRs is modified by deletion, insertion and/or substitution that removes all or part of the stem-loop structure formed by regions C and C'; or one or both of said flanking ITRs is modified by deletion, insertion and/or substitution that removes part of the stem-loop structure typically formed by regions B and B' and/or part of the stem-loop structure formed by regions C and C'. 25. cccDNA vector according to any one of paragraphs. 1-24, characterized in that one or both of said flanking ITRs are modified by deletion, insertion and/or substitution that results in the removal of part of the stem-loop structure formed by regions B and B' and/or part of the stem-loop structure, formed by regions C and C'. 26. cccDNA vector according to any one of paragraphs. 1-25, characterized in that: one or both of said flanking ITRs contains a single stem-loop structure in a region that, in a wild-type ITR, contains a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'; one or both of said flanking ITRs contains one stem and two loops in a region which, in a wild-type ITR, contains a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'; or one or both of said flanking ITRs contains one stem and one loop in a region which, in a wild-type ITR, contains a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'. 27. cccDNA vector according to any one of paragraphs. 1-26, characterized in that both of said flanking ITRs are changed in such a way as to result in an overall three-dimensional symmetry in which said flanking ITRs are inverted relative to each other. 28. cccDNA vector according to any one of paragraphs. 1-27, characterized in that said cDNA vector contains at least one regulatory switch. 29. cccDNA vector according to claim 28, characterized in that at least one regulatory switch is selected from the group consisting of a binary regulatory switch, a small molecule regulatory switch, a password regulatory switch, a nucleic acid-based regulatory switch, a post-transcriptional regulatory switch switch, radiation-controlled or ultrasound-controlled regulatory switch, hypoxia-mediated regulatory switch, inflammatory response regulatory switch, shift-activated regulatory switch and "safety switch". 30. A method for expressing an antibody in a cell, comprising bringing the cell into contact with a cDNA vector according to any one of claims. 1-29. 31. The method according to claim 30, characterized in that the contacting cell is a eukaryotic cell and/or a cell that is in vitro or in vivo. 32. Method according to any one of paragraphs. 30, 31, characterized in that at least one heterologous nucleotide sequence is codon-optimized for expression in a eukaryotic cell. 33. Method according to any one of paragraphs. 30-32, characterized in that said antibody is secreted from a cell or in which said antibody is retained in a cell. 34. A method of treating a subject with a therapeutic antibody, comprising administering to the subject a cccDNA vector according to any one of claims. 1-29, characterized in that said antibody is a therapeutic antibody. 35. The method of claim 34, wherein the subject has a disease or disorder selected from the group consisting of cancer, autoimmune disease, neurodegenerative disorder, hypercholesterolemia, acute organ rejection, multiple sclerosis, postmenopausal osteoporosis, skin disease, asthma and hemophilia . 36. The method according to claim 34, characterized in that said cancer is selected from the group consisting of solid tumor, soft tissue sarcoma, lymphoma and leukemia; said autoimmune disease is selected from the group consisting of rheumatoid arthritis and Crohn's disease; said skin disease is selected from psoriasis and atopic dermatitis; and said neurodegenerative disorder is Alzheimer's disease. 37. Pharmaceutical composition for the expression of at least one antibody, containing the specified cDNA vector according to any one of paragraphs. 1-29. 38. A cell producing a cDNA vector according to any one of claims. 1-29. 39. A composition for the expression of at least one antibody, containing a cDNA vector according to any one of paragraphs. 1-29 and lipid. 40. The composition according to claim 39, characterized in that the lipid is a lipid nanoparticle (LNP). 41. A kit for the expression of at least one antibody containing the specified cDNA vector according to any one of paragraphs. 1-29, or the specified composition according to claim 39 or 40, or the specified cell according to claim 38. 42. A method for producing an antibody, comprising culturing said cell according to claim 38 under conditions suitable for producing said antibody. 43. The method of claim 42, further comprising isolating said antibody.