Nonviral DNA vector for expressing factor IX therapeutic agents and its use

Abstract

Providing a nonviral DNA vector for expressing factor IX therapeutic agents and its use. [Solution] This application describes a ceDNA vector having a linear and continuous structure for the delivery and expression of a transgene. The ceDNA vector comprises an expression cassette in which two ITR sequences are adjacent, and the expression cassette encodes a transgene encoding a FIX protein. Some ceDNA vectors further comprise a cis-regulatory element containing a regulatory switch. Methods and cell lines for in vitro, ex vivo, and reliable gene expression of the FIX protein using ceDNA vectors are further provided herein. Methods and compositions comprising ceDNA vectors useful for the expression of the FIX protein in cells, tissues, or subjects, as well as methods for treating diseases with the ceDNA vector expressing the FIX protein, are also provided herein.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 62 / 993,857, filed on 24 March 2020, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing, which has been filed electronically in ASCII format and is incorporated herein by whole reference. The ASCII copy thereof, created on 22 March 2021, is named 131698-06520_SL.txt and has a size of 394,694 bytes.

[0003] This disclosure relates to the field of gene therapy, including the production of nonviral vectors for expressing transgenes or isolated polynucleotides in subjects or cells. The disclosure also relates to nucleic acid constructs, promoters, vectors, and host cells containing polynucleotides, as well as methods for delivering exogenous DNA sequences to target cells, tissues, organs, or organisms. For example, the disclosure provides a method for expressing FIX from cells using a nonviral ceDNA vector, for example, a method for expressing the FIX therapeutic protein for the treatment of a subject with hemophilia B. The methods and compositions can be used, for example, for the purpose of treating a disease by expressing the FIX protein in cells or tissues of a subject requiring treatment. [Background technology]

[0004] Gene therapy aims to improve clinical outcomes for patients suffering from either genetic mutations or acquired diseases caused by abnormalities in the gene expression profile. Gene therapy includes the treatment or prevention of medical conditions resulting from defective genes or abnormal gene regulation or expression, such as underexpression or overexpression, which can lead to disorders, diseases, malignancies, etc. For example, diseases or disorders caused by defective genes can be treated, prevented, or improved by delivery of repair genetic material to the patient, or by bringing about therapeutic expression of the genetic material in the patient, for example, by modifying or silencing the defective gene using repair genetic material in the patient.

[0005] The basis of gene therapy is supplying an active gene product (sometimes called a transgene) to a transcription cassette, which may result in, for example, a positive gain-of-function effect, a negative loss-of-function effect, or other outcomes. Such outcomes may result from the expression of activating antibodies or fusion proteins or inhibitory (neutralizing) antibodies or fusion proteins. Gene therapy can also be used to treat diseases or malignancies caused by other factors. Human monogenetic disorders, which are disorders caused by mutations in a single gene, can be treated by the delivery and expression of the normal gene to target cells. The delivery and expression of repair genes in the patient's target cells can be carried out through many methods, including the use of engineered viruses and viral gene delivery vectors. Among the many virus-derived vectors available (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated virus (rAAV) is gaining popularity as a versatile vector in gene therapy.

[0006] Adeno-associated viruses (AAVs) belong to the Parvoviridae family and more specifically to the Depend Parvovirus genus. AAV-derived vectors (i.e., rAVV or AAV vectors) are attractive for delivering genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons; (ii) they reduce the host cell response to viral infection, such as the interferon-mediated response, by lacking viral structural genes; (iii) wild-type viruses are considered non-pathological in humans; (iv) in contrast to wild-type AAV which can be incorporated into the host cell genome, replication-deficient AAV vectors lack replication (rep) genes and generally persist as episomes, thus limiting the risk of insertional mutations or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered relatively poor immunogens and therefore do not induce a significant immune response (see ii), thus obtaining vector DNA of therapeutic transgenes and potentially long-term sustained expression.

[0007] However, there are several significant drawbacks to using AAV particles as gene delivery vectors. One major drawback associated with rAAV is its limited viral packaging ability for heterologous DNA of approximately 4.5 kb (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), which consequently limits the use of AAV vectors to a protein coding capacity of less than 150,000 Da. A second drawback is that, as a result of outbreaks of wild-type AAV infection in populations, rAAV gene therapy candidates must be screened for the presence of neutralizing antibodies that eliminate the vector from patients. A third drawback concerns capsid immunogenicity, which prevents re-administration to patients who were not excluded from initial treatment. The immune system in patients may stimulate the production of high-titer anti-AAV antibodies that interfere with future treatments in response to a vector that acts effectively as a "booster" shot. Several recent reports have shown a relationship with immunogenicity in high-dose situations. Given that single-stranded AAV DNA must be converted to double-stranded DNA before heterologous gene expression can occur, another significant drawback is the relatively late onset of AAV-mediated gene expression.

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

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

[0010] Hemophilia B has a significant unmet need for disease-modifying therapies. Current therapies are burdensome and require frequent intravenous (IV) administration. Firstly, these factor IX injections do not provide continuous delivery of the factor at trough levels that enable bleeding episodes. Secondly, there are no approved gene therapies for hemophilia B, and AAV-based therapies are unusable for 25-40% of patients due to existing antibodies. AAV can only be administered once, and the resulting factor IX levels may not be high enough to be effective or may be abnormal, and dose levels cannot be titrated. Thirdly, some hemophilia B patients cannot utilize these therapies due to the development of neutralizing antibodies against these exogenous artificial coagulation factors.

[0011] Therefore, technologies that enable the expression of therapeutic FIX proteins in cells, tissues, or subjects are needed in this field for the treatment of hemophilia B. [Overview of the Initiative] [Means for solving the problem]

[0012] Simple explanation The techniques described herein relate to methods and compositions for the treatment of hemophilia B by expression of factor IX (FIX) protein from a capsid-free (e.g., nonviral) DNA vector having a covalent closed end (referred herein to as “closed-end DNA vector” or “ceDNA vector”), wherein the ceDNA vector comprises the FIX nucleic acid sequence or a codon-optimized version thereof. These ceDNA vectors can be used to produce FIX protein for therapeutic, monitoring, and diagnostic purposes. The application of a ceDNA vector expressing FIX to a subject for the treatment of hemophilia B is useful because it (i) provides disease-modifying levels of the FIX enzyme, is minimally invasive in delivery, is reproducible and administered to exert an effect, has a rapid onset of therapeutic effect, results in sustained expression of the modified FIX enzyme in the liver, restores the coagulation cascade, and / or is titrable to achieve appropriate pharmacological levels of the defective enzyme.

[0013] In some embodiments, a ceDNA vector expressing FIX is optionally present in liposomal nanoparticle formulations (LNPs) for the treatment of hemophilia B. The ceDNA LNP formulations described herein may provide one or more benefits, including providing disease-modifying levels of the FIX protein, being minimally invasive in delivery, being reproducible and administered to exert an effect, having a rapid onset of therapeutic effect typically within days of therapeutic intervention, having sustained expression of modified FIX levels in circulation, being titrable to achieve appropriate pharmacological levels of the defective coagulation factor, and / or providing treatment for other types of hemophilia, including factor VII deficiency.

[0014] Accordingly, this disclosure relates to a capsid-free (e.g., nonviral) DNA vector having a covalent closed end containing a gene encoding FIX (referred to herein as a “closed-end DNA vector” or “ceDNA vector”) for enabling the expression of a FIX therapeutic protein in cells. In one embodiment, the gene encoding FIX is a heterologous gene.

[0015] The ceDNA vectors for the expression of FIX protein production described herein are capsid-free linear double-stranded DNA molecules formed from a continuous strand of complementary DNA having covalently closed ends (linear, continuous, and non-capsidized structures), comprising 5' inverted end repeat (ITR) sequences and 3' ITR sequences, wherein the 5' ITR and 3' ITR may have the same symmetric three-dimensional configuration with respect to each other (i.e., symmetric or substantially symmetric), or alternatively, the 5' ITR and 3' ITR may have different three-dimensional configurations with respect to each other (i.e., asymmetric ITRs). Furthermore, the ITRs may originate from the same or different serotypes. In some embodiments, the ceDNA vectors may contain ITR sequences having a symmetric three-dimensional configuration such that their structures are the same shape in geometric space or have the same A, C-C' and B-B' loops in three-dimensional space (i.e., they are the same or mirror images of each other). In some embodiments, one ITR may originate from one AAV serotype, while the other ITR may originate from a different AAV serotype.

[0016] Accordingly, some aspects of the techniques described herein relate to ceDNA vectors for improved protein expression and / or production of the above-mentioned FIX protein, comprising an ITR sequence adjacent to a nucleic acid sequence including any open reading frame sequence contained in the FIX nucleic acid sequence disclosed in Table 1 or any ceDNA sequence disclosed in Table 12, wherein the ITR sequence is selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (ITR) (e.g., an asymmetric modified ITR); (ii) two modified ITRs (e.g., asymmetric modified ITRs) where the mod-ITR pair has a different three-dimensional spatial configuration with respect to each other; or (iii) a symmetric or substantially symmetric WT-WT ITR pair where each WT-ITR has the same three-dimensional spatial configuration; or (iv) a symmetric or substantially symmetric modified ITR pair where each mod-ITR has the same three-dimensional spatial configuration. The ceDNA vectors disclosed herein can be produced in eukaryotic cells and therefore may be free from prokaryotic DNA modification and bacterial endotoxin contamination in insect cells.

[0017] The methods and compositions described herein relate in part to the discovery of nonviral capsid-free DNA vectors (ceDNA vectors) having covalent closed ends that can be used to express at least one FIX protein, or two or more FIX proteins, from cells including, but not limited to, liver cells.

[0018] In one embodiment, a DNA vector (e.g., a ceDNA vector) comprising at least one nucleic acid sequence is provided herein, wherein the nucleic acid sequence encodes a transgene operably ligated to a promoter positioned between two different AAV inverted terminal repeat sequences (ITRs), one of which comprises a functional AAV terminal degradation site and a Rep binding site, and the other comprising a deletion, insertion, or substitution with respect to the other ITR, the transgene encodes a FIX protein, and the DNA, when digested with a restriction enzyme having a single recognition site on the DNA vector, has the presence of a characteristic band of linear, continuous DNA compared to a linear, discontinuous DNA control when analyzed on a non-denaturing gel. Other embodiments include the delivery of the FIX protein by in vivo expression from the ceDNA vector described herein, and further, the treatment of hemophilia B using a ceDNA vector encoding the FIX protein. Cells comprising a ceDNA vector encoding the FIX protein described herein are also contemplated herein.

[0019] Aspects of this disclosure relate to methods for producing ceDNA vectors useful for the production of FIX protein in cells as described herein. Other embodiments relate to ceDNA vectors produced by methods provided herein. In one embodiment, a capsid-free (e.g., nonviral) DNA vector (ceDNA vector) for the production of FIX protein is obtained from a plasmid (referred to herein as a “ceDNA-plasmid”) containing a polynucleotide expression construct template comprising, in this order, a first 5' inverted end repeat (e.g., AAV ITR), a nucleic acid sequence, and a 3' ITR (e.g., AAV ITR), the 5' ITR and 3' ITR may be asymmetric or symmetric with respect to each other (e.g., WT-ITR or modified symmetric ITR), as defined herein.

[0020] The ceDNA vectors for the expression of the FIX protein disclosed herein can be obtained by several means which will be known to the expert by reading this disclosure. For example, the polynucleotide expression construct templates used to generate the ceDNA vectors of this disclosure may be ceDNA-plasmids, ceDNA-bacmids, and / or ceDNA-baculoviruses. In one embodiment, a ceDNA-plasmid includes restriction cloning sites operably positioned between ITRs (e.g., SEQ ID NOs. 123 and / or 124) into which an expression cassette containing a promoter operably linked to a transgene, e.g., the nucleic acid encoding FIX, can be inserted. In some embodiments, the ceDNA vector for the expression of the FIX protein is produced from a polynucleotide template (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses) containing symmetric or asymmetric ITRs (modified or WT ITRs).

[0021] In a suitable host cell, for example in the presence of Rep, a polynucleotide template having at least two ITRs replicates to produce a ceDNA vector expressing the FIX protein. ceDNA vector production involves two steps: first, the excision ("rescue") of the template from the template scaffold (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.) via the Rep protein; and second, Rep-mediated replication of the excised ceDNA vector. Rep proteins and Rep-binding sites for various AAV serotypes are well known to those skilled in the art. Those skilled in the art understand that, based on at least one functional ITR, a Rep protein is selected from a serotype that binds to and replicates a nucleic acid sequence. For example, if the replicable ITR is derived from AAV serotype 2, the corresponding Rep is derived from an AAV serotype that cooperates with that serotype, such as AAV2. ITRs cooperate with AAV2 or AAV4 Rep but not with AAV5 Rep. During replication, the covalent closed-end ceDNA vector continues to accumulate in accepting cells, and the ceDNA vector is sufficiently stable over long periods, preferably under standard replication conditions and in the presence of Rep proteins, accumulating in amounts such as at least 1 pg / cell, preferably at least 2 pg / cell, preferably at least 3 pg / cell, more preferably at least 4 pg / cell, and even more preferably at least 5 pg / cell.

[0022] Thus, one aspect of the present disclosure is a process for producing a ceDNA vector for the expression of such FIX protein, comprising: a) incubating a population of host cells (e.g., insect cells) containing a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus) in the absence of a viral capsid coding sequence, under conditions effective to induce the production of the ceDNA vector in the host cells and for a time sufficient therefor, wherein the host cells are incubated in the presence of a Rep protein, and b) harvesting and isolating the ceDNA vector from the host cells. The presence of the Rep protein induces the replication of the vector polynucleotide having a modified ITR to produce a ceDNA vector for the expression of FIX protein in the host cells. However, viral particles (e.g., AAV virions) are not expressed. Thus, there is no virion forced size limitation.

[0023] The presence of a ceDNA vector useful for the expression of FIX protein isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme having a single recognition site on the ceDNA vector and analyzing the digested DNA material on a denaturing gel and a non - denaturing gel to confirm the presence of a characteristic linear and continuous DNA band as compared to linear and discontinuous DNA.

[0024] Also provided herein is a method for expressing a FIX protein having therapeutic use in a cell or subject using a ceDNA vector. Such FIX protein can be used for the treatment of hemophilia B. Thus, a method for the treatment of hemophilia B is provided herein, which includes administering a ceDNA vector encoding a therapeutic FIX protein to a subject that needs it.

[0025] In some embodiments, one aspect of the technology described herein relates to a nonviral capsid-free DNA vector (ceDNA vector) having a covalent closed end, wherein the ceDNA vector comprises at least one nucleic acid sequence operably positioned between two ITRs, the ITR sequences of which may be asymmetric, symmetric, or substantially symmetric, as these terms are defined herein, the at least one of the ITRs comprising a functional end degradation site (trs) and a Rep binding site, the nucleic acid sequence optionally encoding a transgene (e.g., a FIX protein), and the vector is not present in a viral capsid.

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

[0027] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by referring to exemplary embodiments of the present disclosure depicted in the accompanying drawings. However, the accompanying drawings only illustrate typical embodiments of the present disclosure and should not be considered limiting in scope, as the present disclosure may allow for other equally valid embodiments. [Brief explanation of the drawing]

[0028] [Figure 1A] An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein, including an asymmetric ITR, is shown. In this embodiment, the exemplary ceDNA vector comprises an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding the FIX transgene may be inserted at the cloning site (R3 / R4) between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs)—a wild-type AAV2 ITR upstream (5' end) and a modified ITR downstream (3' end) of the expression cassette, so that the two ITRs flanking the expression cassette are asymmetric with respect to each other. [Figure 1B]An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein is shown, comprising an asymmetric ITR having an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding the FIX transgene can be inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs)—a modified ITR upstream (5' end) and a wild-type ITR downstream (3' end) of the expression cassette. [Figure 1C] An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein is shown, comprising an asymmetric ITR having an expression cassette containing an enhancer / promoter, a FIX transgene, a post-transcription element (WPRE), and a polyA signal. An open reading frame (ORF) allows for the insertion of the FIX transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two asymmetric inverted terminal repeats (ITRs), a modified ITR upstream (5' end) and a modified ITR downstream (3' end) of the expression cassette, where both the 5'ITR and 3'ITR are modified ITRs but have different modifications (i.e., do not have the same modifications). [Figure 1D] An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein is shown, comprising a symmetric modified ITR or substantially symmetric modified ITR as defined herein, having an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding the FIX transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is adjacent to two modified inverted terminal repeats (ITRs) in which the 5' modified ITR and 3' modified ITR are symmetric or substantially symmetric. [Figure 1E]An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein is shown, comprising a symmetric modified ITR or substantially symmetric modified ITR as defined herein, having an expression cassette containing an enhancer / promoter, a transgene, a post-transcription element (WPRE), and a polyA signal. An open reading frame (ORF) allows for the insertion of a transgene (e.g., FIX) into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs) in which the 5' modified ITR and 3' modified ITR are symmetric or substantially symmetric. [Figure 1F] An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein is shown, comprising a symmetric WT-ITR or substantially symmetric WT-ITR as defined herein, having an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding the transgene (e.g., FIX) is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is adjacent to two wild-type inverted-end repeats (WT-ITRs) where the 5'WT-ITR and 3'WT ITR are symmetric or substantially symmetric. [Figure 1G] An exemplary structure of a ceDNA vector for the expression of the FIX protein disclosed herein is shown, comprising a symmetrically modified ITR or substantially symmetrically modified ITR as defined herein, having an expression cassette containing an enhancer / promoter, a transgene (e.g., FIX), a post-transcription element (WPRE), and a polyA signal. An open reading frame (ORF) allows for the insertion of the transgene (e.g., FIX) into the cloning site between the CAG promoter and the WPRE. The expression cassette is adjacent to two wild-type inverted-end repeats (WT-ITRs), the 5'WT-ITR and 3'WT-ITR being symmetric or substantially symmetric. [Figure 2A]The T-shaped stem-loop structure of the wild-type left ITR of AAV2 (SEQ ID NO: 52) is provided, with the identification of A-A', B-B', C-C' arms and two Rep-binding sites (RBE and RBE'), and also showing terminal degradation sites (trs). RBE contains a series of four bitetramers thought to interact with either Rep78 or Rep68. In addition, RBE' is also thought to interact with Rep complexes assembled on wild-type or mutant ITRs in the construct. The D and D' regions contain transcription factor binding sites and other conserved structures. [Figure 2B] The proposed Rep-catalyzed nicking and ligation activity in the wild-type left ITR (SEQ ID NO: 53) is shown, including the T-shaped stem-loop structure of the wild-type left ITR of AAV2, which has identified A-A', B-B', and C-C' arms and two Rep-binding sites (RBE and RBE'), as well as the D and D' regions containing terminal degradation sites (trs), several transcription factor binding sites, and other conserved structures. [Figure 3A] This document provides the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the A-A' arm, and the C-C' and B-B' arms of the wild-type left AAV2 ITR (SEQ ID NO: 54). [Figure 3B] An exemplary mutant ITR (also called a modified ITR) sequence for the left ITR is shown. The primary structure (left) and predicted secondary structure (right) of the RBE portion of the A-A' arm, C arm, and B-B' arm of the exemplary mutant left ITR (ITR-1, left) (SEQ ID NO: 113) are shown. [Figure 3C] The primary structure (left) and secondary structure (right) of the RBE-containing portions of the A-A' loop, as well as the B-B' and C-C' arms, of the wild-type right AAV2 ITR (Sequence ID 55) are shown. [Figure 3D]An exemplary right-modified ITR is shown. The primary structure (left) and predicted secondary structure (right) of the RBE-containing portions of the A-A' arm, and the B-B' and C arms, of an exemplary mutant left ITR (ITR-1, right) (SEQ ID NO: 114) are shown. Any combination of left and right ITRs (e.g., AAV2 ITR or other viral serotypes or synthetic ITRs) can be used as taught herein. Each of the polynucleotide sequences in Figures 3A-3D refers to a sequence used in a plasmid or bacmid / baculovirus genome used to produce the ceDNA described herein. The corresponding ceDNA secondary structure, inferred from the ceDNA vector configuration in the plasmid or bacmid / baculovirus genome and the predicted Gibbs free energy value, is also included in each of Figures 3A-3D. [Figure 4A] Figure 4B is a schematic diagram showing the upstream process for producing baculovirus-infected insect cells (BIIC) that are useful for producing the ceDNA vector for the expression of FIX disclosed herein, in the process described herein. [Figure 4B] This is a schematic diagram illustrating an exemplary method for ceDNA production. [Figure 4C] This paper describes the biochemical methods and processes for confirming ceDNA vector production. [Figure 4D]Figure 4B is a schematic diagram illustrating the process for identifying the presence of ceDNA in DNA extracted from a cell pellet obtained during the ceDNA production process. Figure 4D shows schematic expected bands of exemplary ceDNA subjected to electrophoresis on either an undenatured or denatured gel, either uncut or digested with restriction endonucleases. The leftmost schematic diagram is of an undenatured gel, showing multiple bands in its double-stranded and uncut form, suggesting that ceDNA exists in at least monomeric and dimeric states, appearing as faster-moving smaller monomers and slower-moving dimers twice the size of the monomers. The second schematic diagram from the left shows that when ceDNA is cut with restriction endonucleases, the original bands disappear, and faster-moving (e.g., smaller) bands appear, corresponding to the expected fragment size remaining after cleavage. Under denatured conditions, the original double-stranded DNA is single-stranded, and because the complementary strands are covalently linked, it moves as a species twice as large as that observed on an undenatured gel. Therefore, in the second schematic diagram from the right, the digested ceDNA exhibits a binding distribution similar to that observed on the undenatured gel, but the bands migrate as fragments twice the size of the undenatured gel counterpart. The schematic diagram on the far right shows that uncut ceDNA under denatured conditions migrates as a single-stranded, ring-opened fragment, and therefore the observed bands are twice the size of those observed under undenatured conditions where the ring is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecule based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in the denatured state) or the number of base pairs (e.g., for double-stranded molecules observed in the undenatured state), depending on the context. Figure 4E shows DNA with a discontinuous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on a ceDNA vector, producing two DNA fragments of different sizes (1kb and 2kb) under both neutral and denatured conditions. Figure 4E also shows ceDNA with a linear and continuous structure.The ceDNA vector can be cleaved by restriction endonucleases, producing two DNA fragments that move as 1kb and 2kb under neutral conditions, but under denaturing conditions, the strand remains connected and produces a single strand that moves as 2kb and 4kb. [Figure 4E]Figure 4B is a schematic diagram illustrating the process for identifying the presence of ceDNA in DNA extracted from a cell pellet obtained during the ceDNA production process. Figure 4D shows schematic expected bands of exemplary ceDNA subjected to electrophoresis on either an undenatured or denatured gel, either uncut or digested with restriction endonucleases. The leftmost schematic diagram is of an undenatured gel, showing multiple bands in its double-stranded and uncut form, suggesting that ceDNA exists in at least monomeric and dimeric states, appearing as faster-moving smaller monomers and slower-moving dimers twice the size of the monomers. The second schematic diagram from the left shows that when ceDNA is cut with restriction endonucleases, the original bands disappear, and faster-moving (e.g., smaller) bands appear, corresponding to the expected fragment size remaining after cleavage. Under denatured conditions, the original double-stranded DNA is single-stranded, and because the complementary strands are covalently linked, it moves as a species twice as large as that observed on an undenatured gel. Therefore, in the second schematic diagram from the right, the digested ceDNA exhibits a binding distribution similar to that observed on the undenatured gel, but the bands migrate as fragments twice the size of the undenatured gel counterpart. The schematic diagram on the far right shows that uncut ceDNA under denatured conditions migrates as a single-stranded, ring-opened fragment, and therefore the observed bands are twice the size of those observed under undenatured conditions where the ring is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecule based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in the denatured state) or the number of base pairs (e.g., for double-stranded molecules observed in the undenatured state), depending on the context. Figure 4E shows DNA with a discontinuous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on a ceDNA vector, producing two DNA fragments of different sizes (1kb and 2kb) under both neutral and denatured conditions. Figure 4E also shows ceDNA with a linear and continuous structure.The ceDNA vector can be cleaved by restriction endonucleases, producing two DNA fragments that move as 1kb and 2kb under neutral conditions, but under denaturing conditions, the strand remains connected and produces a single strand that moves as 2kb and 4kb. [Figure 5] This is an illustrative figure of denatured gel flow examples of ceDNA vectors with or without endonuclease digestion (EcoRI for ceDNA constructs 1 and 2, BamH1 for ceDNA constructs 3 and 4, SpeI for ceDNA constructs 5 and 6, and XhoI for ceDNA vectors 7 and 8). Constructs 1-8 are described in Example 1 of International Application PCT PCT / US18 / 49996, which is incorporated herein by reference in its entirety. The size of the bands highlighted with asterisks was determined and is shown below the figure. [Figure 6] The results of the experiment described in Example 7 are shown, specifically the IVIS images obtained from mice treated with LNP-PolyC control (leftmost mouse) and four mice treated with LNP-ceDNA-luciferase (all mice except the leftmost mouse). The four ceDNA-treated mice showed significant fluorescence in regions including the liver. [Figure 7] This image shows the results of the experiment described in Example 8. The dark spots indicate the presence of proteins derived from the expressed ceDNA transgene, demonstrating the association of the administered LNP-ceDNA with hepatocytes. [Figure 8A]Figure 8A shows the results of the eye study described in Example 9. Figure 8A shows representative IVIS images from the eyes of rats injected with JetPEI®-ceDNA-luciferase (top left) and the uninjected eyes of the same rats (top right), or from the eyes of rats injected with plasmid-luciferase DNA (bottom left) and the uninjected eyes of the same rats (bottom right). Figure 8B shows graphs of the mean radiance observed in the treated eyes or the corresponding untreated eyes in each treatment group. Rats treated with ceDNA demonstrated significant fluorescence (and therefore luciferase transgene expression) over 99 days, in clear contrast to rats treated with plasmid-luciferase, which showed minimal relative fluorescence (and therefore luciferase transgene expression). [Figure 8B] Figure 8A shows the results of the eye study described in Example 9. Figure 8A shows representative IVIS images from the eyes of rats injected with JetPEI®-ceDNA-luciferase (top left) and the uninjected eyes of the same rats (top right), or from the eyes of rats injected with plasmid-luciferase DNA (bottom left) and the uninjected eyes of the same rats (bottom right). Figure 8B shows graphs of the mean radiance observed in the treated eyes or the corresponding untreated eyes in each treatment group. Rats treated with ceDNA demonstrated significant fluorescence (and therefore luciferase transgene expression) over 99 days, in clear contrast to rats treated with plasmid-luciferase, which showed minimal relative fluorescence (and therefore luciferase transgene expression). [Figure 9A] The results of the ceDNA persistence and re-dosing study in Rag2 mice described in Example 10 are shown. Figure 9A shows a graph of total flux over time observed in wild-type c57bl / 6 mice or Rag2 mice treated with LNP-ceDNA-Luc. Figure 9B provides a graph showing the effect of re-dosing on the expression level of the luciferase transgene in Rag2 mice, resulting in a stable increase in expression observed after re-dosing (arrows indicate the time of re-dosing). [Figure 9B]The results of the ceDNA persistence and re-dosing study in Rag2 mice described in Example 10 are shown. Figure 9A shows a graph of total flux over time observed in wild-type c57bl / 6 mice or Rag2 mice treated with LNP-ceDNA-Luc. Figure 9B provides a graph showing the effect of re-dosing on the expression level of the luciferase transgene in Rag2 mice, resulting in a stable increase in expression observed after re-dosing (arrows indicate the time of re-dosing). [Figure 10] We provide data from the ceDNA luciferase expression study in treated mice described in Example 11, showing the total flux in each group of mice over the duration of the study. High levels of unmethylated CpG correlated with a decrease in total flux observed in mice over time, while the use of a liver-specific promoter correlated with the robust and stable expression of the transgene from the ceDNA vector for at least 77 days. [Figure 11A] This shows the hydrodynamic delivery of ceDNA vectors expressing FIX. Figure 11A shows the FIX expression levels in serum samples from mice at days 3 and 7 after hydrodynamic injection of two different ceDNA vectors expressing FIX (LPS1-FIX-v1, LPS1-FIX-v2) or a control ceDNA vector (ceDNA expressing only luciferase) (shown as the vehicle). Both of these FIX ceDNA vectors showed FIX expression. Figure 11B shows the FIX expression levels in serum samples from mice over a 28-day period after hydrodynamic injection of two different ceDNA vectors expressing FIX (LPS1-FIX-v1; LPS1-FIX-v2) or the vehicle-control ceDNA vector (expressing only luciferase). [Figure 11B]This shows the hydrodynamic delivery of ceDNA vectors expressing FIX. Figure 11A shows the FIX expression levels in serum samples from mice at days 3 and 7 after hydrodynamic injection of two different ceDNA vectors expressing FIX (LPS1-FIX-v1, LPS1-FIX-v2) or a control ceDNA vector (ceDNA expressing only luciferase) (shown as the vehicle). Both of these FIX ceDNA vectors showed FIX expression. Figure 11B shows the FIX expression levels in serum samples from mice over a 28-day period after hydrodynamic injection of two different ceDNA vectors expressing FIX (LPS1-FIX-v1; LPS1-FIX-v2) or the vehicle-control ceDNA vector (expressing only luciferase). [Figure 12] The plasma concentration of factor IX in mice that were injected with LNP-formulated FIX ceDNA construct (2.0 mg / kg) on ​​day 0 and day 36, and orally administered ruxolitinib 300 mg / kg on day -2, day -1, day 0, day 1, and day 36 is shown. [Figure 13A] Figure 13A shows FIX expression in male CD-1 mice treated with ceDNA-FIX constructs (ceDNA-FIX v1, ceDNA-FIX2109, or ceDNA-FIX2112) containing codon-optimized human FIX sequences. Figure 13A shows human FIX expression levels measured on days 3 and 7 in CD-1 mice treated with 1 μg of ceDNA-FIX v1, ceDNA-FIX2109, or ceDNA-FIX2112 by hydrodynamic delivery. Figure 13B shows human FIX expression levels measured on days 3 and 7 in CD-1 mice treated with 10 μg of ceDNA-FIX v1, ceDNA-FIX2109, or ceDNA-FIX2112 by hydrodynamic delivery. [Figure 13B]Figure 13A shows FIX expression in male CD-1 mice treated with ceDNA-FIX constructs (ceDNA-FIX v1, ceDNA-FIX2109, or ceDNA-FIX2112) containing codon-optimized human FIX sequences. Figure 13A shows human FIX expression levels measured on days 3 and 7 in CD-1 mice treated with 1 μg of ceDNA-FIX v1, ceDNA-FIX2109, or ceDNA-FIX2112 by hydrodynamic delivery. Figure 13B shows human FIX expression levels measured on days 3 and 7 in CD-1 mice treated with 10 μg of ceDNA-FIX v1, ceDNA-FIX2109, or ceDNA-FIX2112 by hydrodynamic delivery. [Modes for carrying out the invention]

[0029] A method for treating hemophilia B is provided herein using a ceDNA vector comprising one or more nucleic acids encoding the FIX therapeutic protein or a fragment thereof. A ceDNA vector for the expression of the FIX protein described herein, comprising one or more nucleic acids encoding the FIX protein, is also provided herein. In some embodiments, the expression of the FIX protein may involve the secretion of the therapeutic protein from the cell on which it is expressed. Alternatively, in some embodiments, the expressed FIX protein can act or function (e.g., exert its effect) within the cell on which it is expressed. In some embodiments, the ceDNA vector expresses the FIX protein in the liver, the muscle of the subject (e.g., skeletal muscle), or other body part, which may act as a depot for the production and secretion of the FIX therapeutic protein into many systemic compartments.

[0030] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art in which this disclosure pertains. This disclosure is not limited to, but may vary, the specific methodologies, protocols, and reagents described herein. The terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of this disclosure as defined by the claims. For definitions of general terms in immunology and molecular biology, see The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6 th Edition, published by Lippincott Williams&Wilkins, Philadelphia, PA, USA (2013), Knipe, DMand Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference,published by VCH Publishers,Inc.,1995(ISBN 1-56081-569-8)、Immunology by Werner Luttmann,published by Elsevier,2006、Janeway‘s Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor&Francis Limited,2014(ISBN 0815345305,9780815345305)、Lewin’s Genes XI,published by Jones&Bartlett Publishers,2014(ISBN -1449659055)、Michael Richard Green and Joseph Sambrook,Molecular Cloning:A Laboratory Manual,4 thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414), Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X), Laboratory Methods in Enzymology: DNA, Jon Lorsch(ed.) Elsevier,2013(ISBN 0124199542), Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,2014(ISBN 047150338X,9780471503385),Current Protocols in Protein Science(CPPS),John E.Coligan(ed.), John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan This can be found in M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), and all of this content is incorporated herein by reference in its entirety.

[0031] As used herein, the terms “heterogeneic nucleic acid sequence” and “transgene” are interchangeable and refer to nucleic acids of interest (other than the nucleic acid encoding a capsid polypeptide) that can be incorporated into the ceDNA vector disclosed herein and thereby delivered and expressed. According to some embodiments, the term “heterogeneic nucleic acid” is intended to refer to nucleic acids (or transgenes) that are not present in, not expressed by, or derived from the contacting cell or subject.

[0032] As used herein, the terms “expression cassette” and “transcription cassette” refer to a linear stretch of nucleic acid that is interchangeable and operably ligated to one or more promoters or other regulatory sequences sufficient to orient the transcription of the transgene, but does not contain a capsid-coding sequence, other vector sequences, or inverted terminal repeat regions. An expression cassette may additionally include one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0033] The terms “polynucleotide” and “nucleic acid,” as used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, these terms include single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically modified, biochemically modified, unnatural, or derivatized nucleotide bases. “Oligonilocyte” generally refers to polynucleotides of about 5 to about 100 nucleotides in single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of oligonucleotides. Oligonucleotides are also known as “oligomers” or “oligos” and can be isolated from genes or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the embodiments described.

[0034] DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA double helix, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalent closed DNA vectors), closed-end linear double helix (CELiD or ceDNA), doggybone (dbDNA™) DNA, dumbbell-shaped DNA, minimally immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-natural, and possess similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholino), phosphoramides, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, locked nucleic acids (LNA®), and peptide nucleic acids (PNA). Unless otherwise specified, this term encompasses nucleic acids containing known analogs of naturally occurring nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0035] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via the phosphate group.

[0036] "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0037] As used herein, the term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, modified to contain a nucleic acid segment in a manner that would otherwise not be naturally occurring, or is synthetic. The term “nucleic acid construct” is synonymous with the term “expression cassette” if the nucleic acid construct contains a regulatory sequence necessary for the expression of the coding sequence of this disclosure. An “expression cassette” includes a DNA coding sequence operably ligated to a promoter.

[0038] "Hybridizable," "complementary," or "substantially complementary" means that a nucleic acid (e.g., RNA) contains a sequence of nucleotides that allows it to "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, i.e., to bind noncovalently, i.e., to form Watson-Crick base pairs and / or G / U base pairs. As is known in the art, standard Watson-Crick base pairings include 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 a guanine (G) base can pair with uracil (U) for hybridization between two RNA molecules (e.g., dsRNA). For example, G / U base pairing, in the context of tRNA anticodon base pairing with codons in mRNA, partially contributes to the degeneracy (i.e., redundancy) of the genetic code. In the context of this disclosure, guanine (G) in the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule is considered complementary to uracil (U), and vice versa. Therefore, if a G / U base pair can be constructed at a given nucleotide position in the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule, that position is not considered complementary, but rather complementary.

[0039] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0040] A DNA sequence that "codes" a specific FIX protein is a DNA nucleic acid sequence that is transcribed into a specific RNA and / or protein. A DNA polynucleotide may code for RNA (mRNA) that is translated into a protein, or it may code for RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA-targeted RNA, also called "non-coding" RNA or "ncRNA").

[0041] As used herein, the term “fusion protein” refers to a polypeptide comprising protein domains from at least two different proteins. For example, a fusion protein may comprise (i) a FIX or fragment thereof, and (ii) at least one skew gene (GOI) protein. Fusion proteins as incorporated herein include, but are not limited to, antibodies or FIX proteins, such as the Fc or antigen-binding fragment of an antibody fused to the extracellular domain of a receptor, ligand, enzyme, or peptide. The FIX protein or fragment thereof that is part of a fusion protein may be a monospecific antibody or a bispecific or multispecific antibody.

[0042] As used herein, the terms “genome-safe harbor gene” or “safe harbor gene” refer to a gene or locus into which a nucleic acid sequence can be inserted so that the sequence can be integrated and function in a predictable manner (e.g., express a protein of interest) without significant adverse effects on endogenous gene activity or without promoting cancer. In some embodiments, a safe harbor gene is also a locus or gene into which the inserted nucleic acid sequence can be expressed more efficiently and at higher levels than a non-safe harbor site.

[0043] As used herein, the term “gene delivery” means the process by which foreign DNA is introduced into host cells for use in gene therapy.

[0044] As used herein, the term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that includes at least one minimally required replication origin and a region containing a palindromic hairpin structure. Rep-binding sequences (“RBS”) (also referred to as RBEs (Rep-binding elements)) and terminal degradation sites (“TRS”) together constitute a “minimumly required replication origin,” and therefore, a TR includes at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically referred to as “inverted terminal repeats” or “ITRs,” respectively. In a viral context, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found, TRs that are not reverse complements over their entire length can still perform the conventional function of an ITR, and therefore, the term ITR is used herein to refer to TRs in a ceDNA genome or ceDNA vector that can mediate replication of a ceDNA vector. It will be understood by those skilled in the art that three or more ITRs or asymmetric ITR pairs may be present in the composite ceDNA vector configuration. The ITRs may be AAV ITRs or non-AAV ITRs, or may be derived from AAV ITRs or non-AAV ITRs. For example, the ITRs may be derived from the Parvoviridae family, which includes parvoviruses and dependent viruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as the origin of SV40 replication, may be used as an ITR, which may be further modified by cleavage, substitution, deletion, insertion, and / or addition. The Parvoviridae viruses consist of two subfamilies: the Parvovirinae, which infects vertebrates, and the Densovirinae, which infects invertebrates. Depend parvoviruses include a family of adeno-associated viruses (AAVs) that can replicate in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep.For convenience, in this specification, the ITR located 5' (upstream) of the expression cassette in the ceDNA vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' (downstream) of the expression cassette in the ceDNA vector is referred to as the "3' ITR" or "right ITR."

[0045] "Wild-type ITR" or "WT-ITR" refers to a sequence of a naturally occurring ITR sequence in AAV or other dependent viruses that retains, for example, Rep-binding activity and Rep-nicking ability. The nucleic acid sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring canonical sequence due to genetic coding or drift degeneracy, and therefore the WT-ITR sequences included herein include WT-ITR sequences resulting from naturally occurring changes (e.g., replication errors) that occur during the production process.

[0046] As used herein, the terms “substantially symmetric WT-ITR” or “substantially symmetric WT-ITR pair” refer to a pair of WT-ITRs in a single ceDNA genome or ceDNA vector, both of which are wild-type ITRs having reverse complementary sequences over their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, as long as the changes do not affect the sequence properties and the overall three-dimensional structure. In some embodiments, the deviating nucleotides represent a conserved sequence change. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the canonical sequence (e.g., measured using BLAST with default settings) and has a three-dimensional spatial configuration that is symmetric with respect to other WT-ITRs such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric WT-ITRs have the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetrical WT-ITR can be functionally confirmed as WT by determining that it possesses a manipulable Rep-binding site (RBE or RBE') and terminal degradation sites (trs) that pair with the appropriate Rep protein. Optionally, other functions, including transgene expression under tolerable conditions, can be tested.

[0047] As used herein, the terms “modified ITR,” “mod-ITR,” or “mutant ITR” are interchangeable herein and refer to an ITR having mutations in at least one nucleotide compared to a WT-ITR from the same serotype. Mutations may result in changes to one or more of the A, C, C', B, and B' regions of the ITR compared to the three-dimensional spatial configuration of a WT-ITR from the same serotype, and may result in changes to the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space).

[0048] As used herein, the term “asymmetric ITR,” also referred to as “asymmetric ITR pair,” refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are not reverse complements over their entire length. As a non-limiting example, an asymmetric ITR pair does not have a three-dimensional spatial configuration symmetric to its congener ITRs, such that their three-dimensional structures have different shapes in geometric space. In other words, an asymmetric ITR pair has a different overall geometric structure, i.e., a different configuration of their A, C-C', and B-B' loops in three-dimensional space (for example, one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to a congener ITR). Sequence differences between two ITRs may result from one or more nucleotide additions, deletions, cleavages, or point mutations. In one embodiment, one ITR in an asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR as defined herein (e.g., a non-wild-type or synthetic ITR sequence). In another embodiment, neither ITR in an asymmetric ITR pair is a wild-type AAV array, and the two ITRs are modified ITRs having different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR in an asymmetric ITR pair may have a short C-C' arm, and the other ITR may have a different modification (e.g., a single arm, or a short B-B' arm) so that they have a different three-dimensional spatial configuration compared to a congeneral asymmetric mod-ITR.

[0049] As used herein, the term “symmetric ITR” refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are mutated or modified relative to the wild-type dependvirus ITR sequence and are reverse complements over their entire length. Neither ITR is a wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and their sequences may differ from the wild-type ITR due to nucleotide additions, deletions, substitutions, cleavages, or point mutations. For convenience, in this specification, an ITR located 5' (upstream) of the expression cassette in a ceDNA vector is referred to as the “5' ITR” or “left ITR,” and an ITR located 3' (downstream) of the expression cassette in a ceDNA vector is referred to as the “3' ITR” or “right ITR.”

[0050] As used herein, the terms “substantially symmetric modified ITR” or “substantially symmetric mod-ITR pair” refer to a pair of modified ITRs in a single ceDNA genome or ceDNA vector, both having reverse complementary sequences over their entire length. For example, a modified ITR can be considered substantially symmetric even if it has some nucleotide sequences that deviate from the reverse complementary sequence, as long as the changes do not affect its properties and overall shape. As a non-limiting example, sequences have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) with respect to the canonical sequence, and have a three-dimensional spatial configuration that is symmetric with respect to their congenerated modified ITRs, such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops configured in three-dimensional space. In some embodiments, ITRs from a mod-ITR pair may have different reverse complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial configuration. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., 5'ITR) in a mod-ITR pair may originate from one serotype, and the other ITR (e.g., 3'ITR) may originate from a different serotype, but both may have the same corresponding mutations (e.g., if the 5'ITR has a deletion in the C region, the modified 3'ITR of the same family from the different serotype may have a deletion at the corresponding position in the C' region), thereby the modified ITR pair having the same symmetrical three-dimensional spatial configuration. In such embodiments, each ITR in a modified ITR pair may originate from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected in the corresponding position of a congener ITR of a different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) insofar as differences in nucleotide sequences between the ITRs do not affect their properties or overall shape, and they have substantially the same shape in three-dimensional space.As a non-limiting example, mod-ITRs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% alignment identity with a canonical mod-ITR, as determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and have a symmetric three-dimensional spatial configuration such that their three-dimensional structures have the same shape in geometric space. Substantially symmetric mod-ITR pairs have the same A, C-C', and B-B' loops in three-dimensional space. For example, if a modified ITR of a substantially symmetric mod-ITR pair has a C-C' arm deletion, then a related mod-ITR has a corresponding C-C' loop deletion and a similar three-dimensional structure of the remaining A and B-B' loops with the same shape in geometric space.

[0051] The term "adjacent" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in sequence ABC, B is adjacent to A and C. The same is true for arrangement AxBxC. Therefore, an adjacent sequence may precede or follow the adjacent sequence, but it does not need to be continuous with or immediately next to the adjacent sequence. In one embodiment, the term adjacent refers to terminal repeats at each end of a linear double-stranded ceDNA vector.

[0052] As used herein, the term “ceDNA genome” refers to an expression cassette further incorporating at least one inverted terminal repeat region. The ceDNA genome may further include one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0053] As used herein, the term “ceDNA spacer region” refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. In some embodiments, a ceDNA spacer region holds two functional elements at a desired distance for optimal functionality. In some embodiments, a ceDNA spacer region provides or enhances the genetic stability of a ceDNA genome, for example, in a plasmid or baculovirus. In some embodiments, a ceDNA spacer region facilitates easy genetic manipulation of a ceDNA genome by providing a convenient location for cloning sites, etc. For example, in certain embodiments, cis-acting factors can be separated by positioning oligonucleotides “polylinkers” containing several restriction endonuclease sites, or non-open reading frame sequences designed to not have known protein (e.g., transcription factor) binding sites, in the ceDNA genome, for example, by inserting 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc., between terminal degradation sites and upstream transcriptional regulatory elements. Similarly, a spacer can be incorporated between a polyadenylation signal sequence and a 3' terminal degradation site.

[0054] As used herein, “Rep binding site,” “Rep binding element,” “RBE,” and “RBS” are interchangeable and refer to a binding site for a Rep protein (e.g., AAV Rep78 or AAV Rep68), which, upon binding by the Rep protein, enables the Rep protein to perform its site-specific endonuclease activity on the sequence incorporating the RBS. The RBS sequence and its reverse complement together form a single RBS. The RBS sequence is known in the art and includes, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), which is the RBS sequence identified in AAV2. Any known RBS sequence may be used in embodiments of this disclosure, including other known AAV RBS sequences and other naturally known or synthetic RBS sequences. While not bound by theory, the nuclease domain of Rep proteins is thought to bind to the double-stranded nucleic acid sequence GCTC, and therefore the two known AAV Rep proteins are thought to directly bind to the double-stranded oligonucleotide, 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO: 60), and assemble stably. In addition, soluble aggregate conformation isomers (i.e., an unspecified number of interrelated Rep proteins) dissociate and bind to oligonucleotides containing the Rep binding site. Each Rep protein interacts with both nitrogenous bases and phosphodiester backbone on each strand. Interactions with nitrogenous bases provide sequence specificity, while interactions with phosphodiester backbone are non-sequence specific or low-sequence specific and stabilize the protein-DNA complex.

[0055] As used herein, the terms “terminal degradation site” and “TRS” are interchangeable herein and refer to a region where Rep forms a tyrosine-phosphodiester bond with a 5'-thymidine that produces a 3'OH that serves as a substrate for DNA elongation via cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may be involved in coordination ligation reactions. In some embodiments, the TRS comprises at least a non-basepair thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled at least partially by its distance from the RBS within the same molecule. When the receptor substrate is a complementary ITR, the resulting product is an intermolecular double helix. The TRS sequence includes 5'-GGTTGA-3' (SEQ ID NO: 61), a hexanucleotide sequence known in the art and identified, for example, in AAV2. Any known TRS sequence, including other known AAV TRS sequences, other naturally known 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), can be used in embodiments of this disclosure.

[0056] As used herein, the term “ceDNA” refers to capsid-free closed-end linear double-stranded (ds) double-stranded DNA for synthesis or other nonviral gene transfer. A detailed description of ceDNA is found in the international application PCT / US2017 / 020828, filed on 3 March 2017, which is expressly incorporated herein by reference in its entirety. Certain methods for producing ceDNA containing various inverted-end repeat (ITR) sequences and configurations using cell-based methods are found in Example 1 of international applications PCT / US18 / 49996, filed on 7 September 2018, and PCT / US2018 / 064242, filed on 6 December 2018, each of which is expressly incorporated herein by reference in its entirety. Certain methods for producing synthetic ceDNA vectors, including various ITR sequences and configurations, are described, for example, in International Application PCT / US2019 / 14122, filed on January 18, 2019, which is incorporated herein by reference in its entirety. As used herein, the terms “ceDNA vector” and “ceDNA” are used interchangeably to refer to a closed-end DNA vector comprising at least one terminal palindrom. In some embodiments, the ceDNA comprises two covalent closed ends.

[0057] As used herein, the term "ceDNA-plasmid" refers to a plasmid containing a ceDNA genome as an intermolecular double helix.

[0058] As used herein, the term “ceDNA-bacmid” refers to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular double helix that can be propagated as a plasmid in E. coli, thereby enabling it to function as a baculovirus shuttle vector.

[0059] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains the ceDNA genome as an intermolecular double helix within its baculovirus genome.

[0060] As used herein, the terms “ceDNA-baculovirus-infected insect cells” and “ceDNA-BIIC” are interchangeable and refer to invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with ceDNA-baculovirus.

[0061] As used herein, the term “closed-end DNA vector” refers to a capsid-free DNA vector having at least one covalent closed end and having at least a portion of the vector having an intramolecular double-strand structure.

[0062] As defined herein, “reporter” refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal such as fluorescence, color, or luminescence. The reporter protein coding sequence encodes a protein whose presence in a cell or organism is readily observable. For example, a fluorescent protein causes a cell to fluoresce when excited with light of a particular wavelength, a luciferase catalyzes a reaction in a cell that produces light, and an enzyme such as β-galactosidase converts a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP), and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0063] As used herein, the term “effector protein” refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide, or more appropriately, as a cell-killing polypeptide, such as a toxin, or as a drug that makes a cell more susceptible to toxicity with or without a selected drug. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. Examples of effector proteins include, but are not limited to, restriction endonucleases (whether genomic or extrachromosomal factors) that target host cell DNA sequences, proteases that target polypeptides necessary for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. In some embodiments, the expression of an effector protein controlled by a synthetic biological circuit described herein may be involved as a factor in another synthetic biological circuit, thereby extending the range and complexity of the responsiveness of the biological circuit system.

[0064] Transcriptional regulators refer to transcriptional activators and repressors that activate or repress the transcription of genes of interest, such as FIX. A promoter is a region of nucleic acid that initiates the transcription of a particular gene. Transcriptional activators typically bind near the transcriptional promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to the transcriptional promoter and sterically interfere with RNA polymerase-mediated transcription initiation. Other transcriptional regulators may function as either activators or repressors depending on their binding site, as well as cellular and environmental conditions. Non-limiting examples of the transcriptional regulator class include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine-zipper proteins.

[0065] As used herein, “repressor protein” or “inducer protein” is a protein that binds to a regulatory sequence element and represses or activates the transcription of a sequence operably linked to the regulatory sequence element, respectively. Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins described herein are, for example, modules in the form of separable DNA-binding and input agent-binding, or responsive elements or domains.

[0066] As used herein, “carrier” includes all kinds of solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retardants, 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. Complementary active ingredients may also be incorporated into the composition. The term “pharmaceutically acceptable” means molecular entities and compositions that, when administered to a host, do not produce a toxic, allergic, or similarly undesirable reaction.

[0067] As used herein, “input agent-responsive domain” is a domain of a transcription factor that binds to a condition or input agent, or otherwise reacts to a condition or input agent such that a linked DNA-binding fusion domain becomes reactive to the presence of that condition or input. In one embodiment, the presence of a condition or input results in a conformational change of the input agent-responsive domain or the protein to which it fuses, thereby modifying the transcriptional regulatory activity of the transcription factor.

[0068] The term "in vivo" refers to an assay or process that takes place in or within an organism, such as a multicellular animal. In some embodiments described herein, a method or use may be said to take place "in vivo" when a single-celled organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses carried out using living cells with intact membranes outside of a multicellular animal or plant, such as, in particular, explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells). The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and may refer to introducing a programmable synthetic biological circuit into a non-cellular system, such as a medium that does not contain cells or cell lines, such as cell extracts.

[0069] As used herein, the term “promoter” refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inductive, repressive, tissue-specific, or any combination thereof. A promoter is a regulatory region of a nucleic acid sequence, which controls the initiation and rate of the rest of the transcription of the nucleic acid sequence. Promoters can also contain gene elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments of the aspects described herein, a promoter can drive the expression of a transcription factor that modifies the expression of the promoter itself. Within a promoter sequence, a transcription start site, as well as a protein-binding domain involved in RNA polymerase binding, will be found. Eukaryotic promoters often, though not necessarily, contain “TATA” and “CAT” boxes. Various promoters, including inductive promoters, can be used to drive the expression of a transgene in the ceDNA vector disclosed herein. The promoter sequence may be bound at its 3' end by a transcription start site and extends upstream (5' orientation) to contain the minimum number of bases or elements required to initiate transcription at a detectable level above the background.

[0070] As used herein, the term “enhancer” refers to a cis-acting regulatory sequence (e.g., 10 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase the transcriptional activation of a nucleic acid sequence. Enhancers may be located up to 1,000,000 base pairs upstream or downstream of the gene start site they regulate. Enhancers may be located within the intronic or exonic regions of unrelated genes.

[0071] A promoter can be said to drive the expression or transcription of the nucleic acid sequence it regulates. The terms “operatably linked,” “operatably positioned,” “operatably linked,” “controlled,” and “transcriptionally controlled” indicate that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence and is regulated to control the transcription initiation and / or expression of that sequence. As used herein, “inverted promoter” refers to a promoter in which the nucleic acid sequence is in reverse orientation, so that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments to modulate the state of the switch. In addition, in various embodiments, the promoter can be used in conjunction with an enhancer.

[0072] A promoter may be naturally associated with a gene or sequence, and can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon of a given gene or sequence. Such a promoter may be referred to as “endogenous.” Similarly, in some embodiments, an enhancer may be naturally associated with a nucleic acid sequence, located either downstream or upstream of its sequence.

[0073] In some embodiments, the coding nucleic acid segment is positioned under the control of a “recombinant promoter” or a “heterogeneous promoter,” both of which refer to promoters not typically associated with an encoded nucleic acid sequence operably linked in its natural environment. Recombinant or heterogeneous enhancers refer to enhancers not typically 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., they may include different elements of different transcriptional regulatory regions and / or mutations that alter expression through genetic engineering methods known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, with respect to the synthetic biological circuits and modules disclosed herein (see, for example, U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, it is intended that regulatory sequences that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts may be used in a similar manner.

[0074] Where otherwise described herein, “inducible promoter” is characterized by initiating or enhancing transcriptional activity in the presence of, being influenced by, or being contacted by an inducer or inducer. Where otherwise defined herein, “inducer” or “inducer” may be endogenous or typically exogenous compounds or proteins administered in a manner that is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducer, i.e., a chemical, compound, or protein, may itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducer protein expressed by another component or module) and 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 repressors. Examples of inducible promoters include, but are not limited to, tetracycline, metallothione, ecdysone, mammalian viruses (e.g., late adenovirus promoters and long terminal repeats of mouse mammary tumor virus (MMTV-LTR)), and other steroid-responsive promoters, rapamycin-responsive promoters, etc.

[0075] As used interchangeably herein, the terms “DNA regulatory sequence,” “regulatory element,” and “regulatory element” refer to transcriptional and translational regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, and proteolytic signals, which provide and / or regulate the transcription of non-coding sequences (e.g., DNA-targeted RNA) or coding sequences (e.g., site-directed modified polypeptides or Cas9 / Csn1 polypeptides) and / or regulate the translation of encoded polypeptides.

[0076] As used herein, the term “open reading frame (ORF)” is intended to refer to a sequence of several nucleotide triplets that can be translated into a peptide or protein. An open reading frame preferably includes a start codon, i.e., a combination of three subsequent nucleotides that typically encode the amino acid methionine (ATG), at its 5' end, and also includes a subsequent region that is typically a multiple of three nucleotides in length. An ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). Typically, this is the only stop codon in an open reading frame. Thus, in the context of the present invention, an open reading frame is a nucleotide sequence consisting of several nucleotides that can be divided by three, preferably beginning with a start codon (e.g., ATG) and preferably ending with a stop codon (e.g., TAA, TGA, or TAG). An open reading frame may be isolated or incorporated into a longer nucleic acid sequence, such as the ceDNA vector described herein.

[0077] "Operatively linked" refers to a parallel relationship in which components described in this way are related in a way that allows them to function as intended. For example, if a promoter affects its transcription or expression, the promoter is operationally linked to the coding sequence. An "expression cassette" contains a DNA sequence operationally linked to a promoter or other regulatory sequence sufficient to direct the transcription of the transgene in a ceDNA vector. Preferred promoters include, for example, tissue-specific promoters. Promoters may also be of AAV origin.

[0078] As used herein, the term “Subject” refers to a human or animal to which a treatment, including prophylactic treatment with a ceDNA vector according to this disclosure, is provided. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals, but are not limited to these. Examples of primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, avian species such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject may be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, for example, the subject is in the womb. Preferably, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be advantageously used as subjects representing animal models of diseases and disorders. In addition, the methods and compositions described herein may be used in domesticated animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, for example, Caucasian (white), Asian, African, Black, African American, Afro-European, Latin American, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already receiving treatment. In some embodiments, the subject is an embryo, fetus, neonatal, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human neonatal, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or non-human primate embryo. In some embodiments, the subject is a human embryo.

[0079] As used herein, the term “host cell” includes any cell type that is susceptible to transformation, transfection, transduction, etc., by the nucleic acid constructs or ceDNA expression vectors of this disclosure. In non-limiting examples, host cells include isolated primary cells, pluripotent stem cells, CD34 + The host cell may be any of the following: a cell, an induced pluripotent stem cell, or one of several immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be a tissue, organ, or an in situ or in vivo cell in an organism.

[0080] The term “exogenous” refers to a substance present in a cell other than its natural source. As used herein, “exogenous” may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide introduced into a biological system such as a cell or organism by a human-involved process, where it is not normally observed and it is desirable to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” may refer to a nucleic acid or polypeptide introduced into a biological system such as a cell or organism by a human-involved process, where it is observed in relatively small amounts and it is desirable to increase the amount of nucleic acid or polypeptide in the cell or organism, for example, to result in ectopic expression or levels. In contrast, the term “endogenous” refers to a substance that is natural to a biological system or cell.

[0081] The term "sequence identity" refers to the relationship between two nucleotide sequences. For the purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined by the Needleman-Wunsch algorithm (Needleman) as 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. This is determined using (and Wunsch, 1970, above). The optional parameters used are a gap-open penalty of 10, a gap-expand penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows: (identical deoxyribonucleotides × 100) / alignment length (total number of gaps in the alignment). The alignment length is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0082] As used herein, the terms “homology” or “homology” are defined as the percentage of nucleotide residues that are identical to the nucleotide residues of the corresponding sequence on a target chromosome after the sequences have been aligned as necessary and gaps have been introduced to achieve the maximum possible sequence identity percentage. Alignment for the purpose of determining the nucleotide sequence homology percentage can be achieved in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum possible alignment over the entire length of the sequences being compared. In some embodiments, for example, a nucleic acid sequence (e.g., DNA sequence) of a homology arm is considered "homologous" if the sequence is identical to the corresponding unmodified or unedited nucleic acid sequence (e.g., genome sequence) of the host cell by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more.

[0083] As used herein, the term “heterogeneous” means a nucleotide or polypeptide sequence not found in naturally occurring nucleic acids or proteins, respectively. Heterogeneous nucleic acid sequences can be ligated (e.g., by genetic engineering) to naturally occurring nucleic acid sequences (or their variants) to produce chimeric nucleotide sequences encoding chimeric polypeptides. Heterogeneous nucleic acid sequences can be ligated (e.g., by genetic engineering) to variant polypeptides to produce nucleic acid sequences encoding fusion variant polypeptides. Alternatively, the term “heterogeneous” may refer to nucleic acid sequences not naturally occurring in cells or subjects.

[0084] A “vector” or “expression vector” is a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., “insertion,” can be ligated to result in the replication of the ligated segment in a cell. A vector may be a nucleic acid construct designed for delivery to a host cell or for movement between different host cells. As used herein, a vector may be viral or nonviral in its origin and / or final form, but for the purposes of this disclosure, “vector” generally refers to a ceDNA vector, as the term is used herein. The term “vector” encompasses any gene element that can replicate and transfer a gene sequence to a cell, in conjunction with appropriate regulatory elements. In some embodiments, a vector may be an expression vector or a recombinant vector.

[0085] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not always, heterogeneous to the cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems so that it can be maintained in two organisms, e.g., human cells in the case of expression, and a prokaryotic host in the case of cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, as appropriate, secreted proteins, including but not limited to transcription, transcriptional processing, translation, and protein folding, modification, and processing. “Expression products” include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means a nucleic acid sequence (DNA) that is transcribed to RNA in vitro or in vivo when operably ligated to an appropriate regulatory sequence. Genes may or may not include regions before and after the coding region, such as the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0086] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence, or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is an episome. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in a target in high-copy-number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal incorporation.

[0087] As used herein, the term “hereditary disorder” refers to a disorder caused, directly or indirectly, partially or completely, by one or more abnormalities in the genome, particularly a condition present from birth. The abnormality may be a mutation, insertion, or deletion. The abnormality may affect the coding sequence or regulatory sequence of a gene. According to some embodiments, the hereditary disorder is the result of a mutation in the FIX gene. According to some embodiments, the hereditary disorder is the result of reduced FIX protein expression. According to some embodiments, the hereditary disorder is hemophilia. According to some embodiments, hemophilia is hemophilia B.

[0088] As used herein, “coagulation factor IX (fIX; FIX)” is intended to refer to a vitamin K-dependent protein necessary for efficient blood clotting, which functions in coagulation as an activator of factor X. A blood fIX concentration of approximately 1–5 μg / ml is considered within the normal range. FIX deficiency is associated with hemophilia B, and severe cases occur when the FIX concentration is less than approximately 1% of the normal FIX concentration (i.e., less than approximately 0.01–0.05 μg of FIX per ml of blood).

[0089] As used herein, the terms “administer,” “to administer,” and variations thereof refer to the introduction of a composition or drug (e.g., ceDNA as described herein) into a subject, including the simultaneous and sequential introduction of one or more compositions or drugs. “Administer” may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administer” also encompasses in vitro and ex vivo treatments. Introduction of a composition or drug into a subject may be by any preferred route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration may include self-administration and administration by another person. Administration may be carried out by any preferred route. A preferred route of administration allows the composition or drug to perform its intended function. For example, if the preferred route is intravenous, the composition is administered by introducing the composition or drug into the vein of the subject.

[0090] As used herein, the terms “nucleic acid therapy,” “therapeutic nucleic acid,” and “TNA” are interchangeable and refer to any modality of therapy that uses nucleic acid as the active ingredient of a therapeutic agent for treating a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-exclusive examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutic agents include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone® DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped minimal DNA vectors ("dumbbell DNA"). According to some embodiments, the therapeutic nucleic acid is ceDNA.

[0091] As used herein, the term “immunosuppressant” refers to a group of small molecules, monoclonal antibodies, or polypeptide antagonists that inhibit protein kinases such as tyrosine kinases.

[0092] As used herein, the term “therapeutic effect” refers to the outcome of treatment, which is deemed desirable and beneficial. A therapeutic effect may include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. A therapeutic effect may also include, directly or indirectly, the prevention, reduction, or elimination of disease progression.

[0093] For any therapeutic agent described herein, the effective therapeutic dose can first be determined from preliminary in vitro studies and / or animal models. The effective therapeutic dose can also be determined from human data. The dose applied can be adjusted based on the relative bioavailability and potency of the compound administered. Adjusting the dose to achieve maximum potency based on the above methods and other well-known methods is within the capabilities of those skilled in the art. General principles for determining therapeutic efficacy, as can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), which are incorporated herein by reference, are summarized below.

[0094] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve the desired level of therapeutic effect while minimizing unacceptable side effects. Plasma drug concentrations can be measured, and in situations related to therapeutic concentrations, additional guidance on dosage adjustments can be obtained.

[0095] As used herein, the terms “to treat,” “to treat,” and / or “treatment” include, to substantially inhibit, delay, or reverse the progression of a condition, substantially improve the clinical symptoms of a condition, or substantially prevent the appearance of the clinical symptoms of a condition, or to obtain a beneficial or desirable clinical outcome. To treat further means to achieve one or more of the following: (a) reducing the severity of the disability; (b) limiting the onset of symptoms characteristic of the disability being treated; (c) limiting the exacerbation of symptoms characteristic of the disability being treated; (d) limiting the recurrence of the disability in patients who previously had the disability; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic with respect to the disability.

[0096] Beneficial or desired clinical outcomes, such as pharmacological and / or physiological effects, include, but are not limited to, preventing the onset of a disease, disorder, or condition (preventive treatment) in subjects who may be predisposed to the disease, disorder, or condition but have not yet experienced or exhibited symptoms of the disease; alleviating symptoms of the disease, disorder, or condition; reducing the severity of the disease, disorder, or condition; stabilizing the disease, disorder, or condition (i.e., preventing exacerbation); preventing the spread of the disease, disorder, or condition; slowing or delaying the progression of the disease, disorder, or condition; improving or reducing the disease, disorder, or condition; and combinations thereof; as well as extending survival compared to the survival expected without treatment.

[0097] As used herein, the terms “increase,” “enhance,” “raise” (and similar terms) generally refer to an action that directly or indirectly increases concentration, level, function, activity, or behavior relative to natural, predicted, or average conditions, or to control conditions.

[0098] As used herein, the terms “suppress,” “reduce,” “interfere,” “inhibit,” and / or “reduce” (and similar terms) generally refer to the act of directly or indirectly reducing concentration, level, function, activity, or behavior to natural, expected, or average, or to control conditions.

[0099] As used herein, “control” is intended to refer to a reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with hemophilia. In yet another embodiment, the control is a historical control or standard reference value or range of values ​​(such as a group of hemophilia A patients with known prognosis or outcome, or a group of samples representing baseline or normal values, or previously tested control samples). The difference between the test sample and the control may be an increase or a decrease. The difference may be a qualitative or quantitative difference, for example, a statistically significant difference. In some examples, the difference is an increase or decrease of at least about 5%, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% compared to the control.

[0100] As used herein, the terms “comprising” or “comprises” are used in reference to a composition, method, and each of its components that are essential to the method or composition, but are open to the inclusion of unspecified elements, whether essential or not.

[0101] As used herein, the term “essentially derived from” refers to elements necessary for a given embodiment. The term allows for the presence of elements that do not materially affect the basic, novel, or functional features of that embodiment. The use of “includes” indicates inclusion rather than limitation.

[0102] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not enumerated in the description of the embodiments.

[0103] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context otherwise clearly indicates. Thus, for example, a reference to “method” includes one or more methods and / or steps of the type described herein and / or which would be apparent to those skilled in the art by reading this disclosure, etc. Similarly, the word “or” is intended to include “and” unless the context otherwise clearly indicates. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. The abbreviation “e.g. (eg)” is derived from the Latin “exempli gratia” and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g. (eg)” is synonymous with “for example.”

[0104] Unless otherwise indicated in the examples of operation or elsewhere, all numbers representing quantities of components or reaction conditions used herein should be understood in all cases to be modified by the term “approximately.” The term “approximately” used in relation to percentages may mean ±1%. This disclosure is described in further detail by the following examples, but the scope of this disclosure should not be limited thereto.

[0105] The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limitation. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or removal, the specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0106] In some embodiments of the embodiments described herein, the disclosures described herein do not relate to human cloning processes, processes for correcting the genetic identity of human germline cells, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, or processes for correcting the genetic identity of animals resulting from such processes.

[0107] Other terms are defined within the descriptions of various aspects of this disclosure.

[0108] All patents and other publications cited throughout this application, including references to literature, issued patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of explaining and disclosing methodologies described in such publications that may be used, for example, in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment by the inventor that there is no prior right to such disclosure, either because of or for any other reason. All statements regarding dates or expressions regarding the content of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or content of these documents.

[0109] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. While specific embodiments and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be apparent to those skilled in the art. For example, while the steps or functions of a method are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as needed. The various embodiments described herein may be combined to provide further embodiments. The aspects of the disclosure may be modified as needed to provide further embodiments of the disclosure using the compositions, functions, and concepts of the above-mentioned references and applications. Furthermore, several modifications to the protein structure may be made without affecting the type or amount of biological or chemical action, taking into consideration the equivalence of biological function. These and other modifications may be made in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0110] Certain elements of any of the embodiments described above can be combined with or replaced by elements of other embodiments. Furthermore, while advantages related to certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments necessarily need to demonstrate such advantages in order to be within the scope of the present disclosure.

[0111] The techniques described herein are further illustrated by the following examples and should not be construed as further limiting them. This disclosure is not limited to, and may be modified to include, specific methodologies, protocols, reagents, etc., described herein. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of this disclosure as defined by the claims.

[0112] II. Expression of FIX protein from a ceDNA vector The techniques described herein generally concern the expression and / or production of FIX protein in cells from nonviral DNA vectors, such as the ceDNA vectors described herein. CeDNA vectors for FIX protein expression are described herein in the section titled “ceDNA Vectors in General.” In particular, ceDNA vectors for FIX protein expression include a pair of ITRs (e.g., symmetric or asymmetric as described herein) and a nucleic acid encoding the FIX protein described herein, operably linked between the ITR pair to a promoter or regulatory sequence. A clear advantage of ceDNA vectors for FIX protein expression over conventional AAV vectors, and even lentiviral vectors, is that there are no size constraints on the nucleic acid sequence encoding the desired protein. Thus, even a FIX protein as short as 6.8 kb can be expressed from a single ceDNA vector. Therefore, the ceDNA vectors described herein can be used to express therapeutic FIX protein in subjects that require it, such as subjects with hemophilia B.

[0113] As can be understood, the ceDNA vector technologies described herein can be adapted to any level of complexity or can be used in a modular manner in which the expression of different components of the FIX protein can be controlled independently. For example, the ceDNA vector technologies designed herein may be as simple as expressing a single gene sequence (e.g., a FIX protein) using a single ceDNA vector, or as complex as using multiple ceDNA vectors, each expressing multiple FIX proteins or associated cofactors or accessory proteins, each independently controlled by a different promoter. The following embodiments are specifically contemplated herein and can be adapted as needed by those skilled in the art.

[0114] In one embodiment, a single ceDNA vector can be used to express a single component of the FIX protein. Alternatively, a single ceDNA vector can be used to express multiple components (e.g., at least two) of the FIX protein under the control of a single promoter (e.g., a strong promoter), with the optional use of an IRES sequence to ensure proper expression of each component, e.g., a cofactor or accessory protein.

[0115] In other embodiments described herein, a single ceDNA vector is intended to contain at least two inserts (e.g., expressing a heavy chain or a light chain), where the expression of each insert is under the control of its own promoter. The promoter may include multiple copies of the same promoter, multiple different promoters, or any combination thereof. As those skilled in the art will understand, it is often desirable to express the components of the FIX protein at different expression levels, and thus control the stoichiometry of the individual components being expressed, in order to ensure efficient folding and assembly of the FIX protein in cells.

[0116] Further variations of ceDNA vector technology can be conceived by those skilled in the art or adapted from conventional methods of protein production using vectors.

[0117] A. Expression of factor IX (FIX) In some embodiments, the transgene encoding the FIX protein may also encode a secretory sequence, thereby directing the FIX protein to the Golgi apparatus and endoplasmic reticulum, and as the FIX protein passes through the ER and exits the cell, it is folded into the correct three-dimensional structure by chaperone molecules. Exemplary secretory sequences include VH-02 (SEQ ID NO: 88) and VK-A26 (SEQ ID NO: 89) and Ig κThis includes, but is not limited to, a signal sequence (SEQ ID NO: 126), a Gluc secretion signal (SEQ ID NO: 188) that enables the tagged protein to be secreted from the cytosol, and a TMD-ST secretion sequence (SEQ ID NO: 189) that directs the tagged protein towards the Golgi apparatus.

[0118] The expression of the FIX protein can also be fine-tuned using a control switch, thereby ensuring that the FIX protein is expressed as needed (including, but not limited to, at a desired expression level or amount), or alternatively, in the presence or absence of specific signals, including cellular signaling events. For example, as described herein, the expression of the FIX protein from a ceDNA vector can be turned on or off when certain conditions occur, as described in the section titled “Control Switches” herein.

[0119] For example, and for illustrative purposes only, the FIX protein can be used to halt undesirable reactions, such as the production of excessively high levels of the FIX protein. The FIX gene may contain a signal peptide marker to deliver the FIX protein to the desired cells. However, in any situation, it may be desirable to regulate the expression of the FIX protein. ceDNA vectors readily accommodate the use of regulatory switches.

[0120] A clear advantage of ceDNA vectors over conventional AAV vectors, and even lentiviral vectors, is that there are no size constraints on the nucleic acid sequence encoding the FIX protein. Therefore, the full-length FIX, and even any cofactor or evaluation protein, can be expressed from a single ceDNA vector. Furthermore, depending on the required atomic chemistry, multiple segments of the same FIX protein can be expressed, the same or different promoters can be used, and the expression of each region can be fine-tuned using control switches. For example, as shown in the examples, a ceDNA vector containing a dual promoter system can be used, thereby using different promoters for each domain of the FIX protein. The use of a ceDNA plasmid to produce the FIX protein may involve unique combinations of promoters for the expression of the domains of the FIX protein, resulting in the appropriate ratio of each domain for the formation of a functional FIX protein. Therefore, in some embodiments, ceDNA vectors can be used to express different regions of the FIX protein separately (e.g., under the control of different promoters).

[0121] In another embodiment, the FIX protein expressed from the ceDNA vector further includes additional functions such as fluorescence, enzymatic activity, secretory signaling, or immune cell activator.

[0122] In some embodiments, the ceDNA encoding the FIX protein may further include, for example, a linker domain. As used herein, “linker domain” refers to an oligo or polypeptide region of about 2 to 100 amino acids in length that links together any of the domains / regions of the FIX protein described herein. In some embodiments, the linker may include or be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers may be used if it is desirable that two adjacent domains not sterically interfere with each other. The linker may be cleavable or incleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or their functional equivalents, and combinations thereof. The linker may be a linker region that is T2A derived from the Thosea asigna virus.

[0123] For example, it is well within the capabilities of those skilled in the art to take a known and / or publicly available protein sequence, such as FIX, and reverse-engineer the cDNA sequence to encode such a protein. The cDNA can then be codon-optimized to match the intended host cell and inserted into a ceDNA vector as described herein.

[0124] B.FIX protein expressing ceDNA vector A ceDNA vector for the expression of a FIX protein having one or more sequences encoding the desired FIX may include regulatory sequences such as promoters, secretory signals, polyA regions, and enhancers. At a minimum, the ceDNA vector contains one or more nucleic acid sequences encoding the FIX protein.

[0125] To achieve highly efficient and precise assembly of FIX proteins, in some embodiments, the FIX protein is specifically intended to include an endoplasmic reticulum (ER) reader sequence that directs it to the ER, causing protein folding to occur. For example, a sequence that directs the expressed protein to the ER for folding.

[0126] In some embodiments, a cellular or extracellular localization signal (e.g., secretory signal, nuclear localization signal, mitochondrial localization signal, etc.) is included in the ceDNA vector to direct the secretion or desired intracellular localization of the FIX protein, thereby enabling the FIX protein to bind to an intracellular target (e.g., an intrabody) or an extracellular target.

[0127] In some embodiments, the ceDNA vectors for the expression of FIX proteins described herein enable the assembly and expression of any desired FIX protein in a modular manner. As used herein, the term “module” refers to an element in a ceDNA expression plasmid that can be easily removed from the construct. For example, the modular elements of a ceDNA-generating plasmid include a unique pair of restriction sites adjacent to each element in the construct, enabling the exclusive manipulation of individual elements (see, for example, Figures 1A–1G). Thus, the ceDNA vector platform can enable the expression and assembly of any desired FIX protein configuration. In various embodiments, ceDNA plasmid vectors are provided herein that can reduce and / or minimize the amount of manipulation required to assemble a desired ceDNA vector encoding a FIX protein.

[0128] Exemplary FIX protein expressed by a C.ceDNA vector In particular, the ceDNA vectors for the expression of the FIX protein disclosed herein may, but are not limited to, encoding the FIX protein, as well as its variants and / or active fragments, for use in the treatment, prevention, and / or improvement of one or more symptoms of hemophilia B. In one embodiment, hemophilia B is human hemophilia B.

[0129] (i) FIX therapeutic protein and its fragments Essentially, any version of the FIX therapeutic protein or its fragments (e.g., functional fragments) can be encoded by a ceDNA vector and expressed in and out of a ceDNA vector, as described herein. Those skilled in the art will understand that the FIX therapeutic protein includes all splice variants and orthologues of the FIX protein. The FIX therapeutic protein includes the intact molecule and its fragments (e.g., functional fragments).

[0130] Factor IX (FIX) Factor IX (or Christmas factor) (EC3.4.21.22) is a serine protease of the coagulation system and belongs to the S1 peptidase family. Deficiency of this protein causes hemophilia B. Factor IX is produced as an inactive precursor called zymogen. After being processed to remove the signal peptide and glycosylated, it is cleaved by factor XIa (of the contact pathway) or factor VIIa (of the tissue factor pathway) to produce a double-stranded form in which the chains are linked by disulfide crosslinks. When activated into factor IXa, in the presence of Ca2+, membrane phospholipids, and factor VIII cofactor, it hydrolyzes one arginine-isoleucine bond of factor X to form factor Xa. Factor IX is vitamin K-dependent. Factor IX is inhibited by antithrombin.

[0131] The factor IX gene or protein may also be referred to as F9, coagulation factor IX, plasma thromboplastin component, plasma thrombogenesis component, Christmas factor, EC3.4.21.22, PTC, Christmas disease, factor IX F9, hemophilia B, factor IX, EC3.4.21, factor IX, F9 P22, THPH8, HEMB, FIX, or P19.

[0132] The human FIX gene is located on the X chromosome, has eight exons, and spans 33.5 kb. Factor IX is produced in the liver, and the inactive precursor protein is processed in the endoplasmic reticulum and Golgi apparatus, where it undergoes multiple post-translational modifications before being secreted into the bloodstream during the proteolytic cleavage of the propeptide.

[0133] Factor IX mRNA expression primarily occurs in the liver. Additional tissues, including bone marrow, whole blood, lymph nodes, thymus, brain, cerebral cortex, cerebellum, retina, spinal cord, tibial nerve, heart, arteries, smooth muscle, skeletal muscle, small intestine, colon, adipocytes, kidneys, lungs, spleen, stomach, esophagus, bladder, pancreas, thyroid gland, salivary glands, adrenal glands, pituitary gland, breast, skin, ovaries, uterus, placenta, prostate, and thymus, may also express factor IX mRNA.

[0134] Factor IX protein is primarily expressed in serum, plasma, and monocytes. However, it can also be detected in tissues throughout the body, including, but not limited to, the tonsils, bone marrow mesenchymal stem cells, spinal cord, heart, colonic muscle, oral epithelium, esophagus, stomach, cardia, colon, rectum, liver, fetal liver, kidney, spleen, synovial fluid, vitreous fluid, salivary glands, thyroid gland, adrenal gland, breast, pancreas, islets of Langerhans, gallbladder, prostate, urine, bladder, skin, placenta, uterus, cervix, ovaries, testes, and seminal vesicles.

[0135] There are at least two known mRNA variants, each encoding one protein isoform of factor IX. Variant 1 represents a longer transcript and encodes the longer isoform 1. Variant 2 lacks an alternative in-frame exon in the 5' coding region compared to variant 1. This encodes the shorter isoform 2 than isoform 1. Isoform 2 may undergo proteolytic degradation similar to isoform 1. Representative sequence identifiers for mRNA variants 1 and 2, as well as protein isoforms 1 and 2, are shown below: Homo sapiens coagulation factor IX (F9), transcription variant 1, mRNA (NCBI reference sequence: NM_000133.3) 1386 bp (SEQ ID NO: 377) Homo sapiens coagulation factor IX isoform 1 preproprotein (NCBI reference sequence: NP_000124.1) 461 amino acids (SEQ ID NO: 378) Homo sapiens coagulation factor IX (F9), transcription variant 2, mRNA (NCBI reference sequence: NM_001313913.1) 2688 bp (SEQ ID NO: 379) Homo sapiens coagulation factor IX isoform 2 precursor (NCBI reference sequence: NP_001300842.1) 423 amino acids (SEQ ID NO: 380)

[0136] A clear advantage of ceDNA vectors over conventional AAV vectors, and even lentiviral vectors, is that there are no size constraints on the nucleic acid sequence encoding the desired protein. Therefore, multiple full-length FIX therapeutic proteins can be expressed from a single ceDNA vector.

[0137] Expression of FIX therapeutic proteins or fragments thereof from ceDNA vectors can be achieved both spatially and temporally using one or more inductive or repressive promoters, including regulatory switches, as known in the art or as described herein.

[0138] In one embodiment, the FIX therapeutic protein is a “therapeutic protein variant,” which refers to a FIX therapeutic protein having a modified amino acid sequence, composition, or structure compared to the corresponding native FIX therapeutic protein. In one embodiment, FIX is a functional version (e.g., wild type). For example, it may also be useful to express mutant versions of the FIX protein, such as point mutations or deletion mutations leading to hemophilia B, in order to evaluate animal models of disease and / or drugs for hemophilia B. To generate disease models, delivery of mutant or modified FIX proteins to cell or animal model systems can be performed. Such cell or animal models can be used for research and / or drug screening. The FIX therapeutic protein expressed from a ceDNA vector may further include sequences / moments that confer additional functionality, such as fluorescence, enzymatic activity, or secretory signaling. In one embodiment, the FIX therapeutic protein variant includes a non-native tag sequence (e.g., an immunotag) for identification to enable it to be distinguished from the endogenous FIX therapeutic protein in recipient host cells.

[0139] For example, it is well within the capabilities of those skilled in the art to take a known and / or publicly available protein sequence of a FIX therapeutic protein and reverse-engineer the cDNA sequence to encode such a protein. The cDNA can then be codon-optimized to match the intended host cell and inserted into a ceDNA vector as described herein.

[0140] In one embodiment, the FIX therapeutic protein coding sequence may be derived from an existing host cell or cell line, for example, by reverse transcribing mRNA obtained from a host and amplifying the sequence using PCR.

[0141] (ii) FIX therapeutic protein expressing a ceDNA vector A ceDNA vector having one or more sequences encoding a desired FIX therapeutic protein may include regulatory sequences such as a promoter (see, e.g., Table 7), a secretory signal, a poly-A region (see, e.g., Table 10), and an enhancer (see, e.g., Table 8). At a minimum, the ceDNA vector contains one or more nucleic acid sequences encoding a FIX therapeutic protein or a functional fragment thereof. Exemplary cassette insertions for generating a ceDNA vector encoding a FIX therapeutic protein are shown in Figures 1A–1G. In one embodiment, the ceDNA vector contains a FIX sequence listed in Table 1 of this specification. [Table 1-1] [Table 1-2]

[0142] (iii) FIX therapeutic protein and its use for the treatment of hemophilia B Using the ceDNA vector described herein, therapeutic FIX protein can be delivered for the treatment of hemophilia B associated with inappropriate expression of FIX protein and / or mutations within FIX protein.

[0143] Any desired FIX therapeutic protein can be expressed using the ceDNA vectors described herein. Exemplary therapeutic FIX therapeutic proteins include, but are not limited to, any FIX proteins expressed by the sequences shown in Table 1 herein.

[0144] In one embodiment, the expressed FIX therapeutic protein is functional in treating hemophilia B. In some embodiments, the FIX therapeutic protein does not elicit an immune system response.

[0145] In another embodiment, a chimeric protein can be generated using a ceDNA vector encoding the FIX therapeutic protein or a fragment thereof (e.g., a functional fragment). Therefore, it is specifically intended herein that a ceDNA vector expressing a chimeric protein can be administered to any one or more tissues selected from, for example, the liver, kidney, gallbladder, prostate, and adrenal gland. In some embodiments, when a ceDNA vector expressing FIX is administered to an infant or to an intrauterine target, the ceDNA vector expressing FIX can be administered to any one or more tissues selected from the liver, adrenal gland, heart, intestine, lung, and stomach, or to its liver stem cell precursor, for in vivo or ex vivo treatment of hemophilia B.

[0146] hemophilia B Hemophilia B is a blood clotting disorder characterized by a genetic mutation in the factor IX gene, leading to increased bruising and bleeding, and a deficiency of factor IX. Hemophilia B is inherited as an X-linked recessive trait. Current treatments to prevent bleeding in patients with hemophilia B include intravenous infusion and / or transfusion of factor IX.

[0147] Many complications exist in the treatment of hemophilia B. In children, easily accessible intravenous ports may be inserted to minimize frequent traumatic intravenous cannula insertions. However, these ports become useless due to high infection rates and the risk of thrombus formation at the catheter tip. Viral infections can be common in hemophilia patients due to frequent blood transfusions, which expose patients to the risk of bloodborne infections such as HIV, hepatitis B, and hepatitis C. Prion infections can also be transmitted through blood transfusions.

[0148] In some cases, mutations in the promoter region of the FIX gene result in a less severe form of hemophilia B, Leiden, characterized by a near-complete absence of FIX in childhood and a steady increase in endogenous FIX levels to near-normal levels during adolescence.

[0149] Coagulation cascade Coagulation, also known as thrombus formation, is the process by which blood changes from a liquid to a gel to form a blood clot. It potentially leads to hemostasis, cessation of blood loss from damaged blood vessels, and subsequent repair. The mechanism of coagulation involves the activation, adhesion, and aggregation of platelets, in addition to the deposition and maturation of fibrin. Coagulation disorders are disease conditions that can result in bleeding (hemorrhage or bruising) or obstructive coagulation (thrombosis).

[0150] Coagulation begins almost instantaneously after damage to a blood vessel, damaging the endothelium that lines the inside of the vessel. Exposure of blood to the subendothelial space initiates two processes: platelet changes and exposure of subendothelial tissue factor to plasma factor VII, which ultimately leads to fibrin formation. Platelets immediately form a thrombus at the site of injury, which is called primary hemostasis. Secondary hemostasis occurs simultaneously: additional coagulation factors or thrombus-forming factors beyond factor VII (including factor VIII) react in a complex cascade to form fibrin chains and strengthen the platelet thrombus.

[0151] The secondary hemostasis coagulation cascade has two initial pathways that lead to fibrin formation. These are the contact activation pathway (also known as the endogenous pathway) and the tissue factor pathway (also known as the exogenous pathway), both of which trigger the same basic reactions that produce fibrin. The primary pathway for initiating blood coagulation is the tissue factor (exogenous) pathway. This pathway is a series of reactions in which the serine protease zymogen (inactive enzyme precursor) and its glycoprotein cofactor are activated to become active components, which then catalyze the next reactions in the cascade, ultimately resulting in cross-linked fibrin. Coagulation factors are generally denoted by Roman numerals, with lowercase letters added to indicate the active form.

[0152] Coagulation factors are generally serine proteases (enzymes) that act by cleaving downstream proteins. Tissue factors FV, ​​FVIII, and FXIII are exceptions. FV and FVIII are glycoproteins, while factor XIII is a transglutaminase. Coagulation factors circulate as inactive zymogen. Therefore, the coagulation cascade is classically divided into three pathways. Both the tissue factor and contact-activated pathways activate the "final common pathway" of factor X, thrombin, and fibrin.

[0153] The primary role of the tissue factor (exogenous) pathway is to generate a "thrombin burst," a process in which thrombin, the most important component of the coagulation cascade in terms of its role in feedback activation, is released very rapidly. FVIIa circulates in greater quantities than any other activated coagulation factor. This process involves the following steps:

[0154] Step 1: After blood vessel damage, FVII leaves the circulation and comes into contact with tissue factor (TF) expressed on cells containing tissue factor (interstitial fibroblasts and leukocytes), forming an activated complex (TF-FVIIa).

[0155] Step 2: TF-FVIIa activates FIX and FX.

[0156] Step 3: FVII itself is activated by thrombin, FXIa, FXII, and FXa.

[0157] Step 4: Activation of FX by TF-FVIIa (to form FXa) is inhibited almost immediately by tissue factor pathway inhibitors (TFPIs).

[0158] Step 5: FXa and its cofactor FVa form a prothrombinase complex that activates prothrombin into thrombin.

[0159] Step 6: Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which forms a complex with FIX), and activates FVIII to release it from binding to von Willebrand factor (vWF).

[0160] Step 7: FVIIIa is a cofactor of FIXa, and together they form a "tenase" complex that activates FX, thereby continuing this cycle.

[0161] The contact-activated (endogenous) pathway begins with the formation of a primary complex on collagen by high molecular weight kininogen (HMWK), prekallikrein, and FXII (Hagemann factor). Prekallikrein is converted to kallikrein, and FXII becomes FXIIa. FXIIa converts FXI to FXIa. Factor XIa activates FIX, which, together with its cofactor FVIIIa, forms a tenase complex, activating FX to FXa. The small role of the contact-activated pathway in the initiation of thrombus formation can be explained by the fact that patients with severe deficiencies of FXII, HMWK, and prekallikrein do not have bleeding disorders. Instead, the contact-activated system is more involved in inflammation and innate immunity.

[0162] The final common pathway shared by endogenous and extrinsic coagulation pathways involves the conversion of prothrombin to thrombin and fibrinogen to fibrin. Thrombin has a wide range of functions beyond just the conversion of fibrinogen to fibrin, a component of hemostatic thrombi. Furthermore, it is the most important platelet activator, and moreover, it activates factor VIII and factor V, as well as their inhibitor protein C (in the presence of thrombomodulin), and activates factor XIII, which forms covalent bonds that crosslink fibrin polymers formed from activated monomers.

[0163] Following activation by the contact factor or tissue factor pathway, the coagulation cascade is maintained in a thrombogenic state by the continuous activation of FVIII and FIX, forming tenase complexes, until it is downregulated by the anticoagulant pathway.

[0164] The method involves administering an effective amount of a composition comprising a ceDNA vector encoding a FIX therapeutic protein or a fragment thereof (e.g., a functional fragment) to a subject, as described herein. As will be understood by those skilled in the art, the term “effective amount” means the amount of the administered ceDNA composition that results in the expression of the protein in a “therapeutic effective amount” for the treatment of a disease or disorder.

[0165] The dosage range for a composition containing a ceDNA vector encoding a FIX therapeutic protein or a fragment thereof (e.g., a functional fragment) depends on the potency (e.g., promoter efficiency) and includes an amount sufficient to produce the desired effect, e.g., expression of the desired FIX therapeutic protein, for the treatment of hemophilia B. The dosage should not be so high as to cause unacceptable adverse side effects. In general, the dosage will vary depending on the specific properties of the ceDNA vector, its expression efficiency, and the patient's age, condition, and sex. The dosage can be determined by those skilled in the art, and unlike conventional AAV vectors, the ceDNA vector does not contain an immune-activating capsid protein that prevents repeated dosing, and can therefore be adjusted by the individual physician in cases of complications.

[0166] The administration of the ceDNA compositions described herein can be repeated for a limited period. In some embodiments, doses are given periodically or by pulsed administration. In preferred embodiments, the doses listed above are administered over several months. The duration of treatment depends on the clinical progress of the subject and their response to treatment. Time-dependent booster therapy is intended. Furthermore, the level of expression can be titrated as the subject grows.

[0167] The FIX therapeutic protein can be expressed in subjects for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 12 months / 1 year, at least 2 years, 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 longer. Long-term expression can be achieved by repeatedly administering the ceDNA vector described herein at predetermined or desired intervals.

[0168] As used herein, the term “therapeutic effective dose” refers to the amount of expressed FIX therapeutic protein or its functional fragment that is sufficient to produce a statistically significant measurable change in the expression of a disease biomarker or a reduction in a given disease symptom (see “Measurement of Effectiveness” below). Such an effective dose can be evaluated in clinical and animal studies of a given ceDNA composition.

[0169] The exact amount of ceDNA vector to be administered depends on the practitioner's judgment and is specific to each individual. There are also various regimens suitable for administration, but they are typically represented by an initial dose followed by repeated doses at one or more intervals with subsequent injections or other administrations. Alternatively, particularly for the treatment of acute illness / disorder, continuous intravenous infusion sufficient to maintain blood concentrations within the range specified for in vivo treatment is intended.

[0170] The agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), intracellularly, intratissuely, orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, or intracavitarially, and may be delivered by peristaltic means or by other known means by those skilled in the art, as necessary. The agents may be administered systemically, as necessary. They may also be administered intrauterine.

[0171] The effectiveness of a given treatment for hemophilia B can be determined by a skilled clinician. However, if one or all of the signs or symptoms of the disease or impairment are modified in a beneficial manner, or if other clinically acceptable symptoms or markers of the disease are improved or enhanced by at least 10% after treatment with, for example, a ceDNA vector encoding FIX or a functional fragment thereof, the treatment is considered “effective” as the term is used herein. Effectiveness can also be measured by the stabilization of the disease or the failure of the individual’s deterioration, which is assessed by the need for medical intervention (i.e., the cessation or at least slowing of disease progression). 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 humans or mammals), including: (1) inhibiting the disease, e.g., stopping or delaying the progression of the disease or disorder; (2) alleviating the disease, e.g., causing a reduction in symptoms; and (3) preventing or reducing the likelihood of the disease developing, or preventing secondary diseases / disorders associated with the disease, e.g., hepatic failure or renal failure. An effective dose for the treatment of a disease means an amount sufficient to produce an effective treatment for the disease when administered to a mammal in need, as the term is defined herein.

[0172] The efficacy of a drug can be determined by evaluating physical indicators specific to hemophilia B. Standard analytical methods for hemophilia B indicators are known in the art.

[0173] In some embodiments, the ceDNA vectors for the expression of the FIX protein disclosed herein may also encode cofactors or other polypeptides, sense or antisense oligonucleotides, or RNA (coding or non-coding, e.g., siRNA, shRNA, microRNA, antisense counterparts that can be used in combination with the FIX protein expressed from ceDNA (e.g., antagoMiR)). Additionally, the expression cassette containing the sequence encoding the FIX protein may also include exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, e.g., β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0174] In one embodiment, the ceDNA vector contains a nucleic acid sequence for expressing a therapeutic FIX protein that is functional for the treatment of hemophilia B. In a preferred embodiment, the therapeutic FIX protein does not elicit an immune system response unless desired.

[0175] III. General ceDNA vectors for use in the production of FIX therapeutic proteins Embodiments of this disclosure are based on methods and compositions comprising a closed-end linear double-stranded (ceDNA) vector capable of expressing a FIX transgene. In some embodiments, the transgene is a sequence encoding a FIX protein. The ceDNA vectors for FIX protein expression described herein are not limited by size, thereby enabling the expression of all components necessary for transgene expression from, for example, a single vector. The ceDNA vectors for FIX protein expression are preferably double-stranded over at least a portion of the molecule, such as an expression cassette, e.g., self-complementary (e.g., ceDNA is not a double-stranded cyclic molecule). The ceDNA vector has a covalent closed end and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37°C for at least 1 hour.

[0176] Generally, the ceDNA vectors for the expression of FIX proteins disclosed herein have, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleic acid sequence of interest (e.g., an expression cassette described herein), and a second AAV The ITR sequence is selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR); (ii) two modified ITRs (e.g., asymmetric modified ITRs) where the mod-ITR pair has different three-dimensional spatial configurations with respect to each other; (iii) a symmetric or substantially symmetric WT-WT ITR pair where each WT-ITR has the same three-dimensional spatial configuration; or (iv) a symmetric or substantially symmetric modified ITR pair where each mod-ITR has the same three-dimensional spatial configuration.

[0177] Methods and compositions comprising a ceDNA vector for the production of FIX protein are included herein, but are not limited to, delivery systems, which may further include, but are not limited to, liposome nanoparticle delivery systems. Non-limiting exemplary liposome nanoparticle systems included for use are disclosed herein. In some embodiments, this disclosure provides lipid nanoparticles comprising ceDNA and ionized lipids. For example, lipid nanoparticle formulations prepared and loaded using process-obtained ceDNA are disclosed in International Application No. PCT / US2018 / 050042, filed on 7 September 2018, and are incorporated herein.

[0178] The ceDNA vectors for the expression of FIX proteins disclosed herein do not have the packaging constraints imposed by the limited space within the viral capsid. The ceDNA vectors represent a variable eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors, in contrast to the encapsulated AAV genome. This allows for the insertion of regulatory elements, such as the regulatory switches, large transgenes, and multiple transgenes disclosed herein.

[0179] Figures 1A–1E show schematic diagrams of non-restrictive exemplary ceDNA vectors for the expression of the FIX protein, or sequences of corresponding ceDNA plasmids. The ceDNA vector for the expression of the FIX protein does not contain a capsid and can be obtained from a plasmid encoding a first ITR, an expression cassette containing the transgene, and a second ITR in that order. The expression cassette may contain one or more regulatory sequences that enable and / or control the expression of the transgene. For example, the expression cassette may contain one or more of the following in this order: enhancer / promoter, ORF reporter (transgene), post-transcriptional regulatory element (e.g., WPRE), and polyadenylation and termination signals (e.g., BGH polyA).

[0180] The expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir-regulatory elements, post-transcriptional regulatory elements, tissue and cell type-specific promoters, and enhancers. In some embodiments, the ITR may act as a promoter for a transgene, e.g., a FIX protein. In some embodiments, the ceDNA vector may include additional components for regulating the expression of the transgene, e.g., regulatory switches for controlling and regulating the expression of a FIX protein, as described in the section entitled “Regulatory Switches” herein, and, if desired, a regulatory switch that is a kill switch enabling controlled cell death of the cell containing the ceDNA vector.

[0181] The expression cassette may include more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range of about 4,000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette may include transgenes in the range of 500 to 50,000 nucleotides in length. In some embodiments, the expression cassette may include transgenes in the range of 500 to 75,000 nucleotides in length. In some embodiments, the expression cassette may include transgenes in the range of 500 to 10,000 nucleotides in length. In some embodiments, the expression cassette may include transgenes in the range of 1,000 to 10,000 nucleotides in length. In some embodiments, the expression cassette may include transgenes in the range of 500 to 5,000 nucleotides in length. ceDNA vectors do not have the size limitations of capsidized AAV vectors, and therefore, the delivery of large-sized expression cassettes can result in efficient transgene expression. In some embodiments, ceDNA vectors lack prokaryotic cell-specific methylation.

[0182] A ceDNA expression cassette may include, for example, an expressible exogenous sequence (e.g., an open reading frame) or transgene encoding a protein that is absent, inactive, or insufficiently active in the recipient, or a gene encoding a protein having a desired biological or therapeutic effect. A transgene may encode a gene product capable of functioning to correct the expression of a defective gene or transcript. In principle, an expression cassette may include any gene encoding a protein, polypeptide, or RNA that is reduced or absent due to mutation, or any gene that, when overexpression is considered to be within the scope of this disclosure, would produce a therapeutic effect.

[0183] The expression cassette may contain any transgene (e.g., encoding a FIX protein), for example, a FIX protein useful for treating hemophilia B in a subject, i.e., a therapeutic FIX protein. The ceDNA vector can be used alone or in combination with exogenous genes and nucleic acid sequences, including nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miR, etc.), and viral sequences in the subject's genome, such as HIV virus sequences, to deliver and express any FIX protein of interest in a subject. Preferably, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or for immunogenic polypeptides. In certain embodiments, the ceDNA vector is useful for expressing any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNA (coding or non-coding, e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)), antibodies, fusion proteins, or any combination thereof.

[0184] Expression cassettes may also encode polypeptides, sense or antisense oligonucleotides, or RNA (coding or non-coding, e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). Expression cassettes may include exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, e.g., β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0185] The expression cassette, the expression construct of the ceDNA vector for the expression of the FIX protein described herein, can be codon-optimized for target host cells. As used herein, the terms “optimized codon” or “codon optimization” refer to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a significant number of codons in an unmodified sequence (e.g., a prokaryotic sequence) with codons that are more or most frequently used in the genes of a target vertebrate, e.g., mouse or human, for enhanced expression in that vertebrate's cells. Different species exhibit a particular bias towards certain codons of certain amino acids. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons are, for example, those in the GENE of Aptagen. This can be determined using the FORGE® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database. In some embodiments, the nucleic acid encoding the FIX protein is optimized for human expression and / or is human FIX or a functional fragment thereof, as known in the art.

[0186] As disclosed herein, the transgene expressed by a ceDNA vector for the expression of the FIX protein encodes the FIX protein. Many structural features of ceDNA vectors for FIX protein expression exist that differ from plasmid-based expression vectors. A ceDNA vector may have one or more of the following features: deletion of original (i.e., uninserted) bacterial DNA; deletion of prokaryotic cell replication origin; presence of a hairpin-forming ITR sequence that is self-contained (i.e., requires no sequences other than two ITRs containing rep-binding and terminal degradation sites (RBS and TRS), and no exogenous sequences between ITRs); and absence of bacterial-type DNA methylation, or any other methylation that is actually considered abnormal by the mammalian host. Generally, it is preferable that the vector does not contain any prokaryotic cell DNA, although some prokaryotic cell DNA may be inserted as exogenous sequences in the promoter or enhancer region, as an example, in non-limiting cases. Another important characteristic that distinguishes ceDNA vectors from plasmid expression vectors is that ceDNA vectors are single-stranded linear DNA with closed ends, while plasmids are always double-stranded DNA.

[0187] The ceDNA vectors for the expression of the FIX protein produced by the methods provided herein preferably have a linear and continuous structure rather than a discontinuous structure, as determined by restriction enzyme digestion assay (Figure 4D). A linear and continuous structure is considered to be more stable against attack by cellular endonucleases and less likely to undergo recombination and mutagenesis. Therefore, linear and continuous ceDNA vectors are preferred embodiments. Continuous linear single-stranded intramolecular double-stranded ceDNA vectors may have a covalently bonded terminal without a sequence encoding the AAV capsid protein. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are bacterial circular double-stranded nucleic acid molecules. While the complementary strand of a plasmid can separate following denaturation to produce two nucleic acid molecules, conversely, a ceDNA vector, although having a complementary strand, is a single DNA molecule and therefore remains a single molecule even upon denaturation. In some embodiments, the ceDNA vectors described herein, unlike plasmids, may be produced without prokaryotic cell-type DNA base methylation. Therefore, ceDNA vectors and ceDNA plasmids differ in both their structure (particularly linear-to-cyclic) and the methods used to produce and purify these different objects (see below), as well as in their DNA methylation, with ceDNA plasmids being prokaryotic and ceDNA vectors being eukaryotic.

[0188] There are several advantages to using ceDNA vectors for the expression of the FIX protein described herein over plasmid-based expression vectors, including, but are not limited to: 1) Plasmids contain bacterial DNA sequences and are subjected to prokaryotic cell-specific methylation, e.g., 6-methyladenosine and 5-methylcytosine methylation, whereas capsid-free AAV vector sequences are of eukaryotic origin and do not undergo prokaryotic cell-specific methylation, resulting in capsid-free AAV vectors being less likely to induce inflammatory and immune responses compared to plasmids. 2) Plasmids require the presence of resistance genes during the production process, whereas ceDNA vectors do not. 3) Circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors can contain a viral cis-element, i.e., an ITR, that confers resistance to nucleases, and can be designed to be targeted and delivered to the nucleus. We hypothesize that the minimal defining elements essential to ITR function are the Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' in the case of AAV2 (SEQ ID NO: 60)) and the terminal degradation site (TRS, 5'-AGTTGG-3' in the case of AAV2 (SEQ ID NO: 64)), in addition to a variable palindromic sequence that enables hairpin formation. 4) The ceDNA vector does not have the over-presentation of CpG dinucleotides often found in prokaryotic plasmids that bind to members of the Toll-like family of receptors and induce T cell-mediated immune responses. In contrast, transduction with the capsid-free AAV vectors disclosed herein can efficiently target cell and tissue types that are difficult to transduce with conventional AAV virions using a variety of delivery reagents.

[0189] IV. Inverted terminal repeat (ITR) As disclosed herein, a ceDNA vector for the expression of a FIX protein comprises a transgene or nucleic acid sequence positioned between two inverted end repeat (ITR) sequences, and the ITR sequences may be asymmetric ITR pairs or symmetric or substantially symmetric ITR pairs, as these terms are defined herein. The ceDNA vectors disclosed herein may comprise ITR sequences selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted end repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITRs) where the mod-ITR pairs have different three-dimensional spatial configurations with respect to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair where each WT-ITR has the same three-dimensional spatial configuration, and the methods disclosed herein may further comprise, but are not limited to, delivery systems such as liposome nanoparticle delivery systems.

[0190] In some embodiments, the ITR sequence may be derived from viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infects vertebrates, and Densovirinae, which infects insects. Parvovirinae (referred to as parvoviruses) includes the genus Dependovirus, whose members, under most conditions, require co-infection with a helper virus such as an adenovirus or herpesvirus for reproductive infection. The Dependovirus family includes adeno-associated viruses (AAVs) that typically infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., cattle, dog, horse, and sheep adeno-associated viruses). Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FIELDS VIROLOGY (3rd Ed. 1996).

[0191] ITRs are illustrated in the specification, and the example herein is AAV2 WT-ITR, but those skilled in the art will recognize that ITRs can be used from any known parvovirus, e.g., dependent viruses, e.g., AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes; e.g., NCBI:NC002077;NC001401;NC001729;NC001829;NC006152;NC006260;NC006261), chimeric ITRs, or ITRs from any synthetic AAV. In some embodiments, AAV can infect warm-blooded animals, such as birds (AAAV), cattle (BAAV), dogs, horses, and sheep adeno-associated viruses. In some embodiments, ITRs are derived from B19 parvovirus (GenBank accession number NC000883), mouse microvirus (MVM) (GenBank accession number NC001510), goose parvovirus (GenBank accession number NC001701), and snake parvovirus 1 (GenBank accession number NC006148). In some embodiments, as discussed herein, 5'WT-ITR may be derived from one serotype, and 3'WT-ITR may be derived from different serotypes.

[0192] Experts will know that ITR sequences have a general structure of a double-stranded Holiday junction, typically a T-shaped or Y-shaped hairpin structure (see, e.g., Figures 2A and 3A), and that each WT-ITR is formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), as well as a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). See, for example, the structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6) and described in Grimm et al., J. Virology, 2006;80(1);426-439, Yan et al., J. Virology, 2005;364-379, and Duan et al., Virology 1999;261;8-14. Those skilled in the art can readily determine the WT-ITR sequence from any AAV serotype for use in a ceDNA vector or ceDNA plasmid based on the exemplary AAV2 ITR sequences provided herein. For example, see the sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006;80(1);426-439. This shows the identity percentage of the left ITR of AAV2 to 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%).

[0193] A. Symmetric ITR pair In some embodiments, the ceDNA vector for the expression of the FIX protein described herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleic acid sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, where the first ITR (5'ITR) and the second ITR (3'ITR) are symmetric or substantially symmetric with respect to each other, i.e., the ceDNA vector may contain ITR sequences having a symmetric three-dimensional spatial configuration, thereby their structures having the same shape in geometric space or having the same A, C-C', B-B' loops in three-dimensional space. In such embodiments, the symmetric ITR pair, or substantially symmetric ITR pair, may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair may have one or more modifications from the wild-type ITR and have the same sequence that is inversely complementary (inverted) to each other. In alternative embodiments, the modified ITR pairs are substantially symmetric as defined herein, i.e., the modified ITR pairs may have different arrangements but may have corresponding or the same symmetric three-dimensional shapes.

[0194] (i) Wild-type ITR In some embodiments, a symmetric or substantially symmetric ITR is wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences but do not necessarily have to be WT-ITRs of the same AAV serotype. In some embodiments, one WT-ITR may originate from one AAV serotype, and the other WT-ITR may originate from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they may have one or more conserved nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0195] Accordingly, as disclosed herein, a ceDNA vector contains a transgene or nucleic acid sequence positioned between two adjacent wild-type inverted end repeat (WT-ITR) sequences that are inversely complementary (inverted) to each other, or alternatively substantially symmetric to each other. That is, the WT-ITR pair has a symmetric three-dimensional spatial configuration. In some embodiments, the wild-type ITR sequence (e.g., AAV WT-ITR) includes a functional Rep-binding site (RBS, e.g., 5'-GCGCGCTCGCTCGCTC-3' in the case of AAV2, SEQ ID NO: 60) and a functional end-degradation site (TRS, e.g., 5'-AGTT-3', SEQ ID NO: 62).

[0196] In one embodiment, a ceDNA vector for the expression of the FIX protein can be obtained from a vector polynucleotide encoding a nucleic acid operably positioned between two WT inverted terminal repeat sequences (WT-ITRs) (e.g., AAV WT-ITRs). That is, both ITRs have wild-type sequences but do not necessarily have to be WT-ITRs of the same AAV serotype. In some embodiments, one WT-ITR may originate from one AAV serotype, and the other WT-ITR may originate from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they can have one or more conserved nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration. In some embodiments, the 5'WT-ITR originates from one AAV serotype, and the 3'WT-ITR originates 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 symmetric. In some embodiments, the 5'WT-ITR and 3'WT-ITR originate from the same AAV serotype.

[0197] WT ITRs are well known. In one embodiment, two ITRs are derived from the same AAV2 serotype. In certain embodiments, WTs from other serotypes can be used. There are several homologous serotypes, e.g., AAV2, AAV4, AAV6, and AAV8. In one embodiment, closely homologous ITRs (e.g., ITRs with similar loop structures) can be used. In another embodiment, a wider variety of AAV WT ITRs, e.g., AAV2 and AAV5, can be used, and in yet another embodiment, a substantially WT ITR can be used, i.e., it has the basic loop structure of a WT but has some conserved nucleotide changes that do not alter or affect its properties. When using WT-ITRs derived from the same viral serotype, one or more regulatory sequences can be used in addition. In certain embodiments, the regulatory sequence is a regulatory switch that allows for the regulation of ceDNA activity, e.g., the expression of the encoded FIX protein.

[0198] In some embodiments, one aspect of the technique described herein relates to a ceDNA vector for the expression of a FIX protein, wherein the ceDNA vector comprises at least one nucleic acid sequence encoding a FIX protein operably positioned between two wild-type inverted end repeat sequences (WT-ITRs), the WT-ITRs may originate from the same serotype, different serotypes, or be substantially symmetric to one another (i.e., they have a symmetric three-dimensional spatial configuration such that their structures are the same shape in geometric space, or they have the same A, C-C', and B-B' loops in three-dimensional space). In some embodiments, the symmetric WT-ITRs include a functional end degradation site and a Rep-binding site. In some embodiments, the nucleic acid sequence encodes a transgene, and the vector is not a viral capsid.

[0199] In some embodiments, the WT-ITRs are the same but are inverse complements of each other. For example, the sequence AACG of the 5'ITR may be CGTT (i.e., inverse complement) of the 3'ITR at the corresponding site. In one embodiment, the 5'WT-ITR sense strand contains the sequence ATCGATCG, and the corresponding 3'WT-ITR sense strand is [ka] (i.e., the reverse complement of ATCGATCG). In some embodiments, the WT-ITR ceDNA further includes terminal degradation sites and replication protein-binding sites (RPS) (sometimes referred to as replication protein-binding sites), such as Rep-binding sites.

[0200] Exemplary WT-ITR sequences for use in ceDNA vectors for the expression of FIX proteins containing WT-ITR are shown in Table 2 of this specification, which shows pairs of WT-ITR (5'WT-ITR and 3'WT-ITR).

[0201] As an exemplary embodiment, the Disclosure provides a ceDNA vector for the expression of a FIX protein comprising a promoter operably linked to a transgene (e.g., a nucleic acid sequence), with or without a regulatory switch, wherein the ceDNA lacks a capsid protein and is produced from a ceDNA plasmid encoding a WT-ITR (see, e.g., Figures 1F-1G), each WT-ITR having the same number of intramolecularly doubled base pairs in its hairpin secondary configuration (preferably excluding any deletion of AAA or TTT terminal loops in this configuration compared to these reference sequences), and (b) identified as ceDNA using the assay for identification of ceDNA by agarose gel electrophoresis under undenatured and denatured conditions, as in Example 1.

[0202] In some embodiments, adjacent WT-ITRs are substantially symmetrical to one another. In this embodiment, the 5'WT-ITR may originate from one serotype of AAV, and the 3'WT-ITR may originate from a different serotype of AAV, such that the WT-ITRs are not identical reverse complements. For example, the 5'WT-ITR may originate from AAV2, and the 3'WT-ITR may originate from a different serotype (e.g., AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). In some embodiments, the WT-ITR can be selected from two different parvoviruses selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, goat parvovirus, tripe parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAVs. In some embodiments, such a combination of WT-ITRs is a combination of WT-ITRs from AAV2 and AAV6. In one embodiment, substantially symmetric WT-ITRs have the same symmetric three-dimensional spatial configuration when one is inverted with respect to the other ITR, being at least 90% identical, at least 95% identical, at least 96%...97%...98%...99%...99.5%, and all points in between. In some embodiments, WT-ITR pairs are substantially symmetric because they have a symmetric three-dimensional spatial configuration, for example, having the same three-dimensional configuration of arms A, C-C', B-B', and D. In one embodiment, a substantially symmetric WT-ITR pair is inverted with respect to the other and is at least 95% identical, at least 96%...97%...98%...99%...99.5%, and all points in between, with one WT-ITR holding the Rep-binding site (RBS) and terminal decomposition sites (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60).In some embodiments, substantially symmetrical WT-ITR pairs are inverted relative to each other and are at least 95% identical, at least 96%...97%...98%...99%...99.5%, and all points in between, with one WT-ITR retaining the Rep-binding site (RBS) and terminal decomposition site (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), in addition to a variable palindromic sequence that enables hairpin secondary structure formation. Homology can be determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool), BLASTN with default settings, etc.

[0203] In some embodiments, the structural elements of the ITR can be any structural elements involved in the functional interaction between the ITR and a large Rep protein (e.g., Rep78 or Rep68). In certain embodiments, the structural elements provide selectivity for the interaction between the ITR and the large Rep protein; that is, they determine, at least partially, which Rep protein functionally interacts with the ITR. In other embodiments, the structural elements physically interact with the large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleic acid sequence of the ITR, the space between two or more elements, or any combination of the above. In one embodiment, the structural elements are selected from the group consisting of A and A' arms, B and B' arms, C and C' arms, D arm, Rep binding sites (RBEs) and RBE' (i.e., complementary RBE sequences), and terminal degradation sites (trs).

[0204] As a simple example, Table 2 shows exemplary combinations of WT-ITR.

[0205] Table 2: Exemplary combinations of WT-ITRs from the same or different serotypes, or from different parvoviruses. The order shown does not indicate the ITR positions; for example, "AAV1, AAV2" indicates that the ceDNA may contain a WT-AAV1 ITR at the 5' position and a WT-AAV2 ITR at the 3' position, or vice versa, a WT-AAV2 ITR at the 5' position and a WT-AAV1 ITR at the 3' position. Abbreviations: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12); AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome (e.g., NCBI:NC002077;NC001401;NC00172) 9;NC001829;NC006152;NC006260;NC006261), ITRs derived from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV), dog, horse, and sheep AAV), ITRs derived from B19 parvovirus (GenBank accession number: NC000883), microviruses (MVMs) derived from mice (GenBank accession number NC001510); geese: goose parvovirus (GenBank accession number: NC001701); snakes: snake parvovirus 1 (GenBank accession number NC006148). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0206] As a simple example, Table 3 shows exemplary WT-ITR sequences from several different AAV serotypes. [Table 3]

[0207] In some embodiments, the nucleic acid sequence of the WT-ITR sequence may be modified (for example, by modifying one, two, three, four, or five or more nucleotides or any range thereof) such that the modification is a substitution of complementary nucleotides, for example, G in the case of C and vice versa, T in the case of A and vice versa.

[0208] In certain embodiments of this disclosure, the ceDNA vector for the expression of the FIX protein does not have a WT-ITR consisting of a nucleic acid sequence selected from any of SEQ ID NOs: 1, 2, 5-14. In alternative embodiments of this disclosure, if the ceDNA vector has a WT-ITR containing a nucleic acid sequence selected from any of SEQ ID NOs: 1, 2, 5-14, then the adjacent ITRs are also WT, and the ceDNA includes a regulatory switch, for example, as disclosed herein and in international application PCT / US18 / 49996 (see, for example, Table 11 of PCT / US18 / 49996, which is incorporated herein by reference in its entirety). In some embodiments, the ceDNA vector for the expression of the FIX protein includes a regulatory switch, as disclosed herein, and a selected WT-ITR having a nucleic acid sequence selected from any of the group consisting of SEQ ID NOs: 1, 2, 5-14.

[0209] The ceDNA vectors for the expression of FIX proteins described herein may include a WT-ITR structure that holds an operable RBE, trs, and RBE' moiety. Figures 2A and 2B, using wild-type ITR for illustrative purposes, illustrate one possible mechanism for the operation of the trs site within the wild-type ITR structural portion of the ceDNA vector. In some embodiments, the ceDNA vector for the expression of FIX proteins contains one or more functional WT-ITR polynucleotide sequences including a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' in the case of AAV2 (SEQ ID NO: 60)) and a terminal degradation site (TRS, 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one WT-ITR is functional. In alternative embodiments, the ceDNA vector for the expression of FIX proteins includes two WT-ITRs that are substantially symmetrical to each other, where at least one WT-ITR is functional and at least one WT-ITR is non-functional.

[0210] B. Common modified ITRs (mod-ITRs) in ceDNA vectors containing asymmetric or symmetric ITR pairs. As discussed herein, ceDNA vectors for the expression of FIX proteins may include symmetric ITR pairs or asymmetric ITR pairs. In either case, one or both ITRs may be modified ITRs, the difference being that in the first case (i.e., symmetric mod-ITR), the mod-ITRs have the same three-dimensional spatial configuration (i.e., the same A-A', C-C', and B-B' arm configurations), while in the second case (i.e., asymmetric mod-ITR), the mod-ITRs have different three-dimensional spatial configurations (i.e., different configurations of the A-A', C-C', and B-B' arms).

[0211] In some embodiments, a modified ITR is an ITR that is modified by deletion, insertion, and / or substitution compared to a wild-type ITR sequence (e.g., AAV ITR). In some embodiments, at least one of the ITRs in a ceDNA vector includes a functional Rep-binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' in the case of AAV2, SEQ ID NO: 60) and a functional end-degradation 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 different or modified ITR is not a wild-type ITR from a different serotype.

[0212] While specific modifications and mutations of ITRs are described in detail herein, in the context of ITRs, “modified,” “mutant,” or “altered” means that a nucleotide has been inserted, deleted, and / or substituted relative to the wild-type, reference, or original ITR sequence. A modified or mutant ITR may be an engineered ITR. As used herein, “engineered” means an embodiment that has been manipulated by human hands. For example, a polypeptide is considered “engineered” if at least one embodiment of the polypeptide, e.g., its sequence, has been manipulated by human hands to differ from its naturally occurring embodiment.

[0213] In some embodiments, mod-ITRs can be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITR does not contain an AAV base sequence. In yet another embodiment, the synthetic ITR preserves the above ITR structure but has a small amount of AAV source sequence or none at all. In some embodiments, the synthetic ITR may preferentially interact with wild-type Rep or Rep of a specific serotype, or, in some cases, may not be recognized by wild-type Rep but only by mutant Rep.

[0214] Those skilled in the art can determine the corresponding sequences in other serotypes by known means. For example, they can determine whether there is a change in the A, A', B, B', C, C', or D regions and determine the corresponding region in another serotype. The corresponding sequences can be determined using BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs in their default settings. This disclosure further provides populations containing mod-ITRs and multiple ceDNA vectors from combinations of different AAV serotypes. That is, one mod-ITR may originate from one AAV serotype, and other mod-ITRs may originate from different serotypes. While not bound by theory, in one embodiment, one ITR may originate from or be based on an AAV2 ITR sequence, and other ITRs in the ceDNA vector may originate from or be based on any one or more ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[0215] Any parvovirus ITR can be used for modification as an ITR or as a base ITR. Preferably, the parvovirus is a depend virus. More preferably, it is an AAV. The selected serotype may be based on the histological tropism of the serotype. AAV2 has broad histological tropism, AAV1 preferentially targets neurons and skeletal muscle, and AAV5 targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 preferentially targets skeletal muscle and lungs. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue. AAV9 preferentially targets liver, skeletal, and lung tissue. In one embodiment, the modified ITR is based on an AAV2 ITR.

[0216] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleic acid sequence of a structural element can be modified compared to the wild-type sequence of the ITR. In one embodiment, the structural elements of the ITR (e.g., A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from different parvoviruses. For example, the substitution structure may be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, goat parvovirus, tripe parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITR may be an AAV2 ITR, and the A or A' arm or RBE can be replaced with structural elements from the AAV5. In another embodiment, the ITR may be an AAV5 ITR, and the C or C' arm, RBE, and trs can be replaced with structural elements from the AAV2. In yet another embodiment, the AAV ITR may be an AAV5 ITR, and the B and B' arms can be replaced with AAV2 ITR B and B' arms.

[0217] As a mere example, Table 4 shows exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in a region of a modified ITR, where X represents a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any of the regions C and / or C' and / or B and / or B' results in at least one terminal loop containing three consecutive T nucleotides (i.e., TTT). For example, if the modification results in a single-arm ITR (e.g., a single C-C' arm or a single B-B' arm), a modified C-B' arm or C'-B arm, or a two-arm ITR having at least one cleaved arm (e.g., a cleaved C-C' arm and / or a cleaved B-B' arm), then at least one of the arms of the single-arm or two-arm ITR (one of which may be cleaved) holds three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In some embodiments, the cleaved C-C' arm and / or the cleaved B-B' arm have three consecutive T nucleotides (i.e., TTT) in the terminal loop. [Table 4]

[0218] In some embodiments, the mod-ITR for use in a ceDNA vector for the expression of the FIX protein comprises an asymmetric ITR pair or a symmetric mod-ITR pair disclosed herein, and may include any one of the modification combinations shown in Table 4, and at least one nucleotide modification in any 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 in the C or C' or B or B' region (e.g., deletion, insertion, and / or substitution) still preserves the terminal loop of the stem-loop. In some embodiments, any modification of at least one nucleotide between C and C' and / or between B and B' (e.g., deletion, insertion, and / or substitution) retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In alternative embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or between B and B' results in at least one terminal loop containing three consecutive A nucleotides (i.e., AAA). In some embodiments, the modified ITR for use herein may include any one of the modification combinations shown in Table 4, and at least one nucleotide modification (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, the modified ITR for use herein may include any one of the modification combinations shown in Table 4, and at least one modification (e.g., deletion, insertion, and / or substitution) in the A region. In some embodiments, the modified ITR for use herein may include any one of the modification combinations shown in Table 4, and at least one nucleotide modification (e.g., deletion, insertion, and / or substitution) in the A' region.In some embodiments, the modified ITR for use herein may include any one of the modification combinations shown in Table 4, and modification of at least one nucleotide in the A and / or A' region (e.g., deletion, insertion, and / or substitution). In some embodiments, the modified ITR for use herein may include any one of the modification combinations shown in Table 4, and modification of at least one nucleotide in the D region (e.g., deletion, insertion, and / or substitution).

[0219] In one embodiment, a modified structural element can be produced by modifying the nucleotide sequence of a structural element (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range thereof). In one embodiment, specific modifications to the ITR are exemplified herein (e.g., SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187), or shown in Figures 7A-7B of International Patent Application PCT / US2018 / 064242 filed on 6 December 2018 (e.g., SEQ ID NOs: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 of PCT / US2018 / 064242). In some embodiments, the ITR can be modified (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 thereof). In other embodiments, the ITR may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with one of the modified ITRs of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or with Tables 2-9 of International Patent Application PCT / US18 / 49996 (i.e., SEQ ID NOs: 110-112, 115-190, 200-468), which is incorporated herein by reference in its entirety, the RBE-containing sections of the A-A' arm and the C-C' and B-B' arms.

[0220] In some embodiments, a modified ITR may include, for example, the removal or deletion of all of a particular arm, e.g., all or part of the A-A' arm, or all or part of the B-B' arm, or all or part of the C-C' arm, or the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form the stem of a loop, as long as a final loop that caps the stem (e.g., a single arm) still exists (see, for example, ITR-21 in Figure 7A of PCT / US2018 / 064242 filed 6 December 2018). In some embodiments, a modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the B-B' arm. In some embodiments, the modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm (see, for example, ITR-1 in Figure 3B or ITR-45 in Figure 7A of International Patent Application PCT / US2018 / 064242 filed 6 December 2018). In some embodiments, the modified ITR may include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C-C' arm and the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the B-B' arm. Any combination of base pair removals can be conceived, for example, 6 base pairs in the C-C' arm and 2 base pairs in the B-B' arm may be removed. As an exemplary embodiment, Figure 3B shows an exemplary modified ITR having at least seven base pairs deleted from each of the C and C' regions, a nucleotide substitution in the loop between the C and C' regions, and the deletion of at least one base pair from each of the B and B' regions, such that the modified ITR includes two arms in which at least one arm (e.g., C-C') is cleaved. In some embodiments, the modified ITR also includes the deletion of at least one base pair from each of the B and B' regions, thereby cleaving the B-B' arm relative to the WT ITR.

[0221] In some embodiments, the modified ITR may have 1 to 50 nucleotide deletions (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) 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 may have 2 to 20 nucleotide deletions relative to the full-length wild-type ITR sequence.

[0222] In some embodiments, the modified ITR does not contain any nucleotide deletions in the RBE-containing portion of the A or A' region so as not to interfere with DNA replication (e.g., binding to RBE by Rep proteins or nicking at terminal degradation sites). In some embodiments, the modified ITR incorporated herein has one or more deletions in the B, B', C, and / or C regions described herein.

[0223] In some embodiments, a ceDNA vector for the expression of a FIX protein comprising a symmetric ITR pair or an asymmetric ITR pair comprises a regulatory switch disclosed herein and at least one selected modified ITR having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187.

[0224] In another embodiment, the structure of the structural element may be modified. For example, the structural element may have changes in the stem height and / or the number of nucleotides in the loop. For example, the stem height may be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. In one embodiment, the stem height may be about 5 to about 9 nucleotides and may functionally interact with Rep. In another embodiment, the stem height may be about 7 nucleotides and may functionally interact with Rep. In another embodiment, the loop may have 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0225] In another embodiment, the number of gagy-binding sites or gagy-related binding sites within the RBE or extended RBE may be increased or decreased. In one embodiment, the RBE or extended RBE may contain one, two, three, four, five, or six or more gagy-binding sites, or any range thereof. Each gagy-binding site may independently be an exact gagy sequence or a gagy-like sequence, as long as the sequence is sufficient to bind to the Rep protein.

[0226] In another embodiment, the functional interaction with the larger Rep protein can be altered by changing (e.g., increasing or decreasing) the space between two elements (but not limited to RBE and hairpin). For example, the space may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 or more nucleotides, or any range therein.

[0227] The ceDNA vectors for the expression of the FIX protein described herein may include an ITR structure modified with respect to the wild-type AAV2 ITR structure disclosed herein, but still retaining the operable RBE, trs, and RBE' portions. Figures 2A and 2B illustrate one possible mechanism for manipulating the trs site within the wild-type ITR structure of the ceDNA vector for the expression of the FIX protein. In some embodiments, the ceDNA vector for the expression of the FIX protein contains one or more functional ITR polynucleotide sequences including a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' in the case of AAV2 (SEQ ID NO: 60)) and a terminal degradation site (TRS, 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one ITR (wild-type or modified ITR) is functional. In alternative embodiments, the ceDNA vector for the expression of the FIX protein includes two modified ITRs that are different from or asymmetric, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0228] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vector for the expression of the FIX protein described herein has modifications within the loop arm, cleavage arm, or spacer. Exemplary arrangements of ITRs having modifications within loop arms, cutting arms, or spacers are listed in Table 2 (i.e., SEQ ID NOs. 135-190, 200-233), Table 3 (e.g., SEQ ID NOs. 234-263), Table 4 (e.g., SEQ ID NOs. 264-293), Table 5 (e.g., SEQ ID NOs. 294-318 as specified herein), Table 6 (e.g., SEQ ID NOs. 319-468), and Tables 7-9 (e.g., SEQ ID NOs. 101-110, 111-112, 115-134) or Table 10A or 10B (e.g., SEQ ID NOs. 9, 100, 469-483, 484-499) of International Patent Application No. PCT / US18 / 49996, which is incorporated herein by reference in its entirety.

[0229] In some embodiments, the modified ITRs for use in a ceDNA vector for the expression of FIX proteins, including asymmetric ITR pairs or symmetric mod-ITR pairs, are selected from any of those shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, and 10A-10B of International Patent Application No. PCT / US18 / 49996, which is incorporated herein in its entirety by reference, or in combination thereof.

[0230] Tables 5A and 5B show additional exemplary modified ITRs for use in ceDNA vectors for the expression of FIX proteins, each of the above classes including asymmetric ITR pairs or symmetric mod-ITR pairs. The predicted secondary structures of the right-modified ITRs in Table 5A are shown in Figure 7A of International Patent Application No. PCT / US2018 / 064242, filed on December 6, 2018, and the predicted secondary structures of the left-modified ITRs in Table 5B are shown in Figure 7B of International Patent Application No. PCT / US2018 / 064242, filed on December 6, 2018, which is incorporated herein by reference in its entirety.

[0231] Tables 5A and 5B list exemplary sequence numbers for right- and left-modified ITRs. [Table 5A] [Table 5B]

[0232] In one embodiment, the ceDNA vector for the expression of the FIX protein comprises a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleic acid sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, oriented from 5' to 3', wherein the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric, i.e., they have different three-dimensional spatial configurations. In exemplary embodiments, the first ITR may be a wild-type ITR and the second ITR may be a mutant or modified ITR, or vice versa, or the first ITR may be a mutant or modified ITR and the second ITR may be a wild-type ITR. In some embodiments, both the first and second ITRs are mod-ITRs but have different sequences or different modifications, and therefore are not the same modified ITRs but have different three-dimensional spatial configurations. In other words, ceDNA vectors using asymmetric ITRs may have different sequences and different three-dimensional shapes relative to each other if they contain ITRs in which any modification of one ITR relative to the WT-ITR is not reflected in the other ITR, or alternatively, if they have a pair of asymmetric ITRs in which the asymmetric ITRs are modified. Exemplary asymmetric ITRs for use in generating ceDNA plasmids in ceDNA vectors for FIX protein expression are shown in Tables 5A and 5B.

[0233] In alternative embodiments, the ceDNA vector for FIX protein expression includes two symmetric mod-ITRs. That is, both ITRs have the same sequence but are inverse complements (inverted) of each other. In some embodiments, the symmetric mod-ITR pair includes at least one or any combination of deletions, insertions, or substitutions compared to the wild-type ITR sequence from the same AAV serotype. Additions, deletions, or substitutions of symmetric ITRs are identical but are inverse complements of each other. For example, an insertion of three nucleotides into the C region of the 5' ITR is reflected in the insertion of three inverse complementary nucleotides into the corresponding section of the C' region of the 3' ITR. For illustrative purposes only, if the addition is AACG in the 5' ITR, the addition is CGTT in the 3' ITR at the corresponding site. For example, the sense strand of the 5' ITR is

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[0234] In alternative embodiments, the modified ITR pairs are substantially symmetric as defined herein; that is, modified ITR pairs may have different sequences but may have corresponding or the same symmetric three-dimensional shapes. For example, one modified ITR may originate from one serotype, and other modified ITRs may originate from different serotypes, but they have the same mutation (e.g., nucleotide insertion, deletion, or substitution) in the same region. In other words, for illustrative purposes only, a 5'mod-ITR may originate from AAV2 and have a deletion in the C region, and a 3'mod-ITR may originate from AAV5 and have a corresponding deletion in the C' region. Provided that the 5'mod-ITR and 3'mod-ITR have the same or symmetric three-dimensional spatial configuration, they are included in their use as modified ITR pairs as defined herein.

[0235] In some embodiments, substantially symmetric mod-ITR pairs have the same A, C-C' and B-B' loops in three-dimensional space. For example, if the modified ITR of a substantially symmetric mod-ITR pair has a C-C' arm absence, then the related mod-ITR has a corresponding C-C' loop absence and also has a similar three-dimensional structure of the remaining A and B-B' loops of the same shape in the geometric space of that related mod-ITR. As just one example, substantially symmetric ITRs may have symmetric spatial configurations such that their structures have the same shape in geometric space. This can occur, for example, when a GC pair is modified to, for example, a CG pair, or vice versa, or when an AT pair is modified to, for example, a TA pair, or vice versa. Therefore, [ka] Modified form 5'ITR, and [ka] The modified type 3'ITR as (i.e., [ka] Using the above exemplary example of the inverse complement (SEQ ID NO: 51), for example, 5'ITR is [ka] If the sequence (sequence number 50) was such that (the G in the adductor is modified by C), these modified ITRs are still symmetric, and the substantially symmetric 3'ITR is without modifying the T in the adductor by the corresponding a, [ka] It has the sequence (SEQ ID NO: 49). In some embodiments, the modified ITR pair has symmetric stereochemistry, so such modified ITRs are substantially symmetric.

[0236] Table 6 shows exemplary symmetrically modified ITR pairs (i.e., left-modified ITRs and symmetrically right-modified ITRs) for use in ceDNA vectors for FIX protein expression. The bold (red) portions of the sequences identify partial ITR sequences (i.e., A-A', C-C', and B-B' loop sequences), also shown in Figures 31A–46B. These exemplary modified ITRs may include the RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70), and the RBE' (i.e., complement for RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71). [Table 6]

[0237] In some embodiments, a ceDNA vector for the expression of a FIX protein containing an asymmetric ITR pair may include an ITR sequence or ITR subsequence shown in one or more of Tables 5A-5B herein, or an ITR having a modification corresponding to any of the modifications in the sequences shown in Figures 7A-7B of International Patent Application PCT / US2018 / 064242, filed on December 6, 2018, which is incorporated herein in its entirety, or in Tables 2, 3, 4, 5, 6, 7, 8, 9, or 10A-10B of International Patent Application PCT / US18 / 49996, filed on September 7, 2018, which is incorporated herein in its entirety by reference.

[0238] V. Exemplary ceDNA vector As described above, the Disclosure relates to recombinant ceDNA expression vectors and ceDNA vectors encoding a FIX protein comprising any one of the asymmetric ITR pairs, symmetric ITR pairs, or substantially symmetric ITR pairs. In certain embodiments, the Disclosure relates to a recombinant ceDNA vector for the expression of a FIX protein having adjacent ITR sequences and a transgene, wherein the ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other as defined herein, and the ceDNA further comprises a nucleic acid sequence of interest located between adjacent ITRs (e.g., an expression cassette containing the nucleic acid of a transgene), the nucleic acid molecule lacking a viral capsid protein coding sequence.

[0239] The ceDNA expression vector for the expression of the FIX protein may be any ceDNA vector that can be conveniently used in recombinant DNA procedures involving the nucleic acid sequences described herein, provided that at least one ITR is modified. The ceDNA vector for the expression of the FIX protein of this disclosure is compatible with the host cell into which the ceDNA vector is introduced. In certain embodiments, the ceDNA vector may be linear. In certain embodiments, the ceDNA vector may exist as an extrachromosomal entity. In certain embodiments, the ceDNA vector of this disclosure may contain elements that enable the integration of the donor sequence into the genome of the host cell. As used herein, “transgene,” “nucleic acid sequence,” and “heterogeneic nucleic acid sequence” are synonymous and encode the FIX protein as described herein.

[0240] Referring here to Figures 1A–1G, schematic diagrams of the functional components of two non-restrictive plasmids useful for constructing ceDNA vectors for FIX protein expression are shown. Figures 1A, 1B, 1D, and 1F show sequences of ceDNA vector constructs or corresponding ceDNA plasmids for FIX protein expression. The ceDNA vector can be obtained from a plasmid that does not contain a capsid and encodes a first ITR, an expressible transgene cassette, and a second ITR in that order, wherein the first and second ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. The ceDNA vector for FIX protein expression can be obtained from a plasmid that does not contain a capsid and encodes a first ITR, an expressible transgene (protein or nucleic acid), and a second ITR in that order, wherein the first and second ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. In some embodiments, the expressible transgene cassette optionally includes an enhancer / promoter, one or more homology arms, a donor sequence, a post-transcriptional regulatory element (e.g., WPRE, e.g., SEQ ID NO: 67), and polyadenylation and termination signals (e.g., BGH polyA, e.g., SEQ ID NO: 68).

[0241] Figure 5 is a gel confirming the production of ceDNA from multiple plasmid constructs using the method described in the examples. CeDNA is confirmed by the characteristic band pattern in the gel, as related to FIG. 4A above and as discussed in the examples.

[0242] A. Regulatory element. The ceDNA vector for the expression of the FIX protein described herein, which includes an asymmetric ITR pair or a symmetric ITR pair as defined herein, may further include a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type specific promoters, and enhancers. Exemplary promoters are listed in Table 7. Exemplary enhancers are listed in Table 8. In some embodiments, the ITR can act as a promoter for a transgene, such as the FIX protein. In some embodiments, the ceDNA vector for the expression of the FIX protein described herein includes additional components for regulating the expression of the transgene, such as a regulatory switch described herein, and regulates the expression of a kill switch that can kill cells containing the transgene or the ceDNA vector encoding its FIX protein. Regulatory elements including regulatory switches that can be used in the present disclosure are more fully discussed in International Patent Application No. PCT / US18 / 49996, which is hereby incorporated by reference in its entirety.

[0243] In some embodiments, the second nucleic acid sequence includes a regulatory sequence and a nucleic acid sequence encoding a nuclease. In certain embodiments, the gene regulatory sequence is operably ligated to the nucleic acid sequence encoding the nuclease. In certain embodiments, the regulatory sequence is suitable for controlling the expression of the nuclease in a host cell. In certain embodiments, the regulatory sequence includes a preferred promoter sequence that can direct the transcription of a gene operably ligated to a promoter sequence, such as the nucleic acid sequence encoding the nuclease of the Disclosure. In certain embodiments, the second nucleic acid sequence includes an intron sequence ligated to the 5' end of the nucleic acid sequence encoding the nuclease. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the potency of the promoter. In certain embodiments, the regulatory sequence includes an enhancer and a promoter, the second nucleic acid sequence includes an intron sequence upstream of the nucleic acid sequence encoding the nuclease, the intron includes one or more nuclease cleavage sites, and the promoter is operably ligated to the nucleic acid sequence encoding the nuclease.

[0244] ceDNA vectors for the expression of FIX protein, produced synthetically or using cell-based production methods as described in the examples herein, may further include specific combinations of cis-regulatory elements such as the WHP post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67) and BGH poly(A) (SEQ ID NO: 68). Suitable expression cassettes for use in expression constructs are not limited by packaging constraints imposed by the viral capsid.

[0245] (i) Promoter: Those skilled in the art will understand that the promoters used in the ceDNA vectors for the expression of the FIX protein disclosed herein should be appropriately tuned to the specific sequences they promote. Exemplary promoter sequence identifiers operably ligated to useful transgenes (e.g., FIX) in ceDNA vectors are disclosed in Table 7 herein. Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7

[0246] Expression cassettes for ceDNA vectors for FIX protein expression may include promoters that can influence cell specificity as well as overall expression levels, such as any of the promoters selected from Table 7. In the case of transgene expression, such as FIX protein expression, they may include highly active virus-derived early promoters. Expression cassettes may contain tissue-specific eukaryotic promoters to restrict transgene expression to specific cell types and reduce toxic effects and immune responses resulting from uncontrolled ectopic expression. In some embodiments, expression cassettes may include promoters or synthetic regulatory elements such as the CAG promoter (SEQ ID NO: 72). The CAG promoter includes (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and first intron of the chicken β-actin gene, and (iii) a splice acceptor of the rabbit β-globin gene. Alternatively, the expression cassette may contain an α-1-antitrypsin (AAT) promoter (SEQ ID NO: 73 or 74), a liver-specific (LP1) promoter (SEQ ID NO: 75 or 76), or a human elongation factor-1α (EF1a) promoter (e.g., SEQ ID NO: 77 or 78). In some embodiments, the expression cassette includes one or more constitutive promoters, e.g., a retroviral Roussarcoma virus (RSV) LTR promoter (optionally containing an RSV enhancer), or a cytomegalovirus (CMV) early promoter (optionally containing a CMV enhancer, e.g., SEQ ID NO: 79). Alternatively, an inducible promoter, an unvariable promoter for the transgene, a tissue-specific promoter, or various promoters known in the art may be used.

[0247] Suitable promoters, including those listed in Table 7 and those mentioned above, may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Expression can be driven by any RNA polymerase (e.g., pol I, pol II, pol III) using a suitable promoter. Exemplary promoters include the SV40 early promoter, the mouse mammary tumor virus long-terminal repeat (LTR) promoter, and the adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, e.g., CMV earliest promoter region (CMVIE), Roussarcoma virus (RSV) promoter, human U6 micronucleus 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.) Examples include, but are not limited to, the human H1 promoter (H1) (e.g., SEQ ID NO: 81 or SEQ ID NO: 155), the CAG promoter, and the human α1-anti-trypsin (HAAT) promoter (e.g., SEQ ID NO: 82). In certain embodiments, these promoters are modified at their downstream intron-containing ends to include one or more nuclease cleavage sites. In certain embodiments, the DNA containing the nuclease cleavage sites is foreign to the promoter DNA.

[0248] In one embodiment, the promoter used is the unvariable promoter of the gene encoding the therapeutic protein. The promoters and other regulatory sequences of each gene encoding the therapeutic protein are known and characterized. The promoter region used may further include one or more additional regulatory sequences (e.g., unvariable), such as enhancers (e.g., SEQ ID NOs: 79 and SEQ ID NOs: 83) containing the SV40 enhancer (SEQ ID NO: 126).

[0249] In some embodiments, the promoter may 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 natural or synthetic tissue-specific promoter, e.g., a liver-specific promoter, e.g., human α1-antitypesin (HAAT). In one embodiment, delivery to the liver may be achieved using an endogenous ApoE-specific target of the composition containing the ceDNA vector to hepatocytes via low-density lipoprotein (LDL) receptors present on the surface of hepatocytes.

[0250] Non-limiting examples of suitable promoters for use in accordance with this disclosure include any of the promoters listed in Table 7, or, for example, any of the CAG promoter (SEQ ID NO: 72), HAAT promoter (SEQ ID NO: 82), human EF1-α promoter (SEQ ID NO: 77), or EF1a promoter (SEQ ID NO: 78), IE2 promoter (e.g., SEQ ID NO: 84), and rat EF1-α promoter (SEQ ID NO: 85), mEF1 promoter (SEQ ID NO: 59), or 1E1 promoter fragment (SEQ ID NO: 125).

[0251] (ii) Enhancer In some embodiments, the ceDNA expressing FIX includes one or more enhancers. In some embodiments, the enhancer sequence is located at 5' of the promoter sequence. In some embodiments, the enhancer sequence is located at 3' of the promoter sequence. Exemplary enhancer sequence identifiers are listed in Table 8 of this specification. [Table 8]

[0252] (iii) Exemplary 5'UTR sequences and intron sequences In some embodiments, the ceDNA vector includes a 5'UTR sequence and / or an intron sequence located at 3' of the 5'ITR sequence. In some embodiments, the 5'UTR is located at 5' of the sequence encoding the transgene, e.g., the FIX protein. Exemplary 5'UTR sequence identifiers are listed in Table 9A. [Table 9A-1] [Table 9A-2]

[0253] (iv) 3'UTR sequence In some embodiments, the ceDNA vector includes a 3'UTR sequence located at 5' of the 3'ITR sequence. In some embodiments, the 3'UTR is located at 3' of the sequence encoding the transgene, e.g., the FIX protein. Exemplary 3'UTR sequence identifiers are listed in Table 9B. [Table 9B]

[0254] (v) Polyadenylated sequences: Sequences encoding polyadenylated sequences may be included in the ceDNA vector for FIX protein expression to stabilize the mRNA expressed from the ceDNA vector and to assist in nuclear transport and translation. In one embodiment, the ceDNA vector does not contain a polyadenylated sequence. In other embodiments, the ceDNA vector for FIX protein expression contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments, the polyadenylated sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range in between.

[0255] The expression cassette may include any polyadenylated sequence or a variation thereof known in the art. In some embodiments, the polyadenylated (poly-A) sequence is selected from any of those listed in Table 10. Other poly-A sequences commonly known in the art may also be used, including, but not limited to, naturally occurring sequences isolated from bovine BGHpA (e.g., SEQ ID NO: 68) or viral SV40pA (e.g., SEQ ID NO: 86), or synthetic sequences (e.g., SEQ ID NO: 87). Some expression cassettes may also include an SV40 late poly-A signal upstream enhancer (USE) sequence. In some embodiments, the USE sequence may be used in combination with SV40pA or a heterologous poly-A signal. The poly-A sequence is located at 3' of the transgene encoding the FIX protein.

[0256] The expression cassette may also include post-transcriptional elements to increase the expression of the transgene. In some embodiments, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67) is used to increase the expression of the transgene. Other post-transcriptional elements, such as those from the herpes simplex virus or hepatitis B virus (HBV) thymidine kinase genes, can be used. The secretory sequence may be ligated to the transgene, e.g., the VH-02 and VK-A26 sequences, e.g., SEQ ID NO: 88 and SEQ ID NO: 89. [Table 10]

[0257] (vi). Nuclear localization sequence In some embodiments, the ceDNA vector for the expression of the FIX protein contains one or more nuclear localization sequences (NLSs), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, one or more NLSs are located at or near the amino terminus, at or near the carboxyl terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and / or one or more NLSs at the carboxyl terminus). If two or more NLSs are present, each can be selected independently of the other NLSs, thereby allowing a single NLS to exist in two or more copies and / or in combination with one or more other NLSs present in one or more copies. Sequence identifiers of non-limiting examples of NLSs are shown in Table 11. [Table 11]

[0258] Additional components of the B.ceDNA vector The ceDNA vectors for the expression of the FIX protein of this disclosure may contain nucleotides encoding other components for gene expression. For example, to select a specific gene target event, a protective shRNA can be embedded in a microRNA and inserted into a recombinant ceDNA vector designed to site-specifically integrate into a highly active locus, such as the albumin locus. Such embodiments may provide a system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background, as described in Nygaard et al., A universal system to select gene-modified hepatocytes in vivo, Gene Therapy, June 8, 2016. The ceDNA vectors of this disclosure may contain one or more selectable markers that enable the selection of cells that have been transformed, transfected, transduced, etc. The selectable markers are genes whose products provide biocide or virus resistance, heavy metal resistance, protrophotrophy to nutritional requirements, NeoR, etc. In certain embodiments, a positive selectable marker is incorporated into a donor sequence such as NeoR. Negative selection markers can be incorporated downstream of the donor sequence; for example, the nucleic acid sequence HSV-tk encoding a negative selection marker can be incorporated into a nucleic acid construct downstream of the donor sequence.

[0259] C. Adjustment switch Molecular regulatory switches are those that, in response to a signal, produce a measurable change in state. Such regulatory switches can be usefully combined with ceDNA vectors for the expression of FIX proteins as described herein to control the output of FIX protein expression from the ceDNA vector. In some embodiments, ceDNA vectors for the expression of FIX proteins include regulatory switches that help fine-tune FIX protein expression. For example, they may serve as a biological containment function for the ceDNA vector. In some embodiments, the switch is an "on / off" switch designed to start or stop (i.e., shut down) FIX protein expression in the ceDNA vector in a controllable and regulated manner. In some embodiments, the switch may include a "kill switch" that, once activated, can instruct a cell containing the ceDNA vector to undergo programmed cell death. Exemplary regulatory switches included in the use of ceDNA vectors for the expression of FIX proteins can be used to regulate the expression of transgenes, which are discussed more fully in their entirety in International Application PCT / US18 / 49996, which is incorporated herein by reference.

[0260] (i) Binary control switch In some embodiments, a ceDNA vector for the expression of a FIX protein includes a regulatory switch that can help controllably adjust the expression of the FIX protein. For example, the expression cassette located between the ITRs of the ceDNA vector may additionally include a regulatory region operably linked to the nucleic acid sequence encoding the FIX protein, such as a promoter, cis-element, repressor, or enhancer, which is regulated by one or more cofactors or exogenous agents. As just one example, the regulatory region may be regulated by a small molecule switch or an inductive or repressive promoter. Non-limiting examples of inductive promoters include hormone-inducible or metal-inducible promoters. Other exemplary inductive promoter / enhancer elements include, but are not limited to, the RU486-inducible promoter, the ecdysone-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

[0261] (ii) Small molecule control switch Various small molecule-based regulatory switches known in the art can be combined with ceDNA vectors for the expression of FIX proteins disclosed herein to form ceDNA vectors controlled by the regulatory switches. In some embodiments, the regulatory switches are orthogonal ligand / nuclear receptor pairs, e.g., retinoid receptor variant / LG335 and GRQCIMFI (disclosed in Taylor, et al. BMC Biotechnology 10(2010):15, along with artificial promoters that control the expression of operablely linked transgenes), engineered steroid receptors, e.g., a modified progesterone receptor having a C-terminal cleavage that cannot bind to progesterone but binds to RU486 (mifepristone) (U.S. Patent No. 5,364,791), ecdysone receptors from Drosophila and their ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517), or Sando R 3 rdOne or a combination of switches controlled by the antibiotic trimethoprim (TMP) disclosed in Nat Methods. 2013, 10(11):1085-8 may be selected. In some embodiments, the regulatory switch for controlling the transgene expressed by the ceDNA vector is a prodrug-activating switch, such as those disclosed in U.S. Patents 8,771,679 and 6,339,070.

[0262] (iii) "Passcode" adjustment switch In some embodiments, the regulatory switch may be a “passcode switch” or “passcode circuit”. The passcode switch allows for fine-tuning of the control of transgene expression from a ceDNA vector when specific conditions occur. That is, a combination of conditions must exist for transgene expression and / or repression to occur. For example, at least conditions A and B must occur for transgene expression to occur. The passcode regulatory switch can be any number of conditions; for example, at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or more conditions must exist for transgene expression to occur. In some embodiments, at least 2 conditions (e.g., conditions A and B) must occur, and in some embodiments, at least 3 conditions must occur (e.g., A, B, and C or A, B, and D). Just as an example, for gene expression from a ceDNA having a passcode “ABC” regulatory switch to occur, conditions A, B, and C must be present. Conditions A, B, and C may be as follows: Condition A is the presence of a pathological condition or disease, condition B is a hormonal response, and condition C is a response to transgene expression. For example, when an transgene edits a defective EPO gene, condition A is the presence of chronic kidney disease (CKD), condition B occurs when the subject has hypoxia in the kidney, and condition C is that erythropoietin-producing cell (EPC) recruitment in the kidney is impaired, or alternatively, HIF-2 activation is impaired. Once oxygen levels increase or the desired EPO level is reached, the transgene is turned off again until one of the three conditions occurs, and then turned back on.

[0263] In some embodiments, the passcode modulating switch or “passcode circuit” incorporated for use in ceDNA vectors includes a hybrid transcription factor (TF) to extend the range and complexity of environmental signals used to define biological containment conditions. In contrast to a dead man switch that induces cell death in the presence of a predetermined condition, the “passcode circuit” allows cell survival or transgene expression in the presence of a specific “passcode” and can be readily reprogrammed to allow transgene expression and / or cell survival only when a given environmental condition or passcode is present.

[0264] Any and all combinations of the regulatory switches disclosed herein, such as small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulatory radiation control switches, hypoxia-mediated switches, and other regulatory switches known to those skilled in the art disclosed herein, can be used in the passcode regulatory switches disclosed herein. The regulatory switches included for use are also discussed in the review article Kis et al., JR Soc Interface. 12:20141000 (2015), summarized in Table 1 of Kis. In some embodiments, the regulatory switches for use in the passcode system can be selected from any or combinations thereof of the switches disclosed in Table 11 of International Patent Application PCT / US18 / 49996, which is incorporated herein in its entirety by reference.

[0265] (iv) Nucleoside regulatory switches for controlling transgene expression In some embodiments, the regulatory switches for controlling the expression of the FIX protein by ceDNA are based on nucleic acid-based regulatory mechanisms. Exemplary nucleic acid regulatory mechanisms are known in the art and are intended for use. For example, such mechanisms include riboswitches, such as those disclosed in US2009 / 0305253, US2008 / 0269258, US2017 / 0204477, WO2018 / 026762A1, U.S. Patent No. 9,222,093, and European Patent Application No. 288071, as well as those disclosed in the review by Villa JK et al., Microbiol Spectr. 2018 May;6(3). Metabolite-reactive transcriptional biosensors, such as those disclosed in WO2018 / 075486 and WO2017 / 147585, are also included. Other known mechanisms in the relevant art where use is anticipated include silencing of transgenes by siRNA or RNAi molecules (e.g., miR, shRNA). For example, a ceDNA vector may contain a regulatory switch encoding an RNAi molecule complementary to a portion of the transgene expressed by the ceDNA vector. If such RNAi is expressed when the transgene (e.g., FIX protein) is expressed by the ceDNA vector, it will be silenced by the complementary RNAi molecule; if RNAi is not expressed when the transgene is expressed by the ceDNA vector, the transgene (e.g., FIX protein) will not be silenced by RNAi.

[0266] In some embodiments, the regulatory switch is, for example, a tissue-specific autoinactivation regulatory switch disclosed in US2002 / 0022018, which intentionally switches off the transgene (e.g., FIX protein) at sites where transgene expression would otherwise be detrimental. In some embodiments, the regulatory switch is, for example, a recombinase reversible gene expression system disclosed in US2014 / 0127162 and U.S. Patent No. 8,324,436.

[0267] (v) Post-transcriptional and post-translational regulatory switches In some embodiments, the regulatory switch for controlling the expression of the FIX protein by a ceDNA vector is a post-transcriptional modification system. For example, such a regulatory switch is described in US2018 / 0119156, GB2011 / 07768, WO2001 / 064956A3, European Patent No. 2707487, and Beilstein et al., ACS. As disclosed in Synth.Biol.,2015,4(5),pp526-534 and Zhong et al.,Elife.2016 Nov2;5.pii:e18858, aptazyme riboswitches can be sensitive to tetracycline or theophylline. In some embodiments, it is assumed that those skilled in the art can encode both an inhibitory siRNA containing a transgene and a ligand-sensitive (off-switch) aptamer, the final result of which is a ligand-sensitive on-switch.

[0268] (vi) Other exemplary control switches Any known regulatory switch can be used in the ceDNA vector to control the expression of the FIX protein, including those triggered by environmental changes. An additional example is Suzuki et al., Scientific. Examples include, but are not limited to, the BOC method, genetic code extensions and non-physiological amino acids, and radiation-controlled or ultrasonically controlled on / off switches described in Reports8;10051(2018) (see, for example, Scott S et al., Gene Ther. 2000 Jul;7(13):1121-5, U.S. Patents 5,612,318, 5,571,797, 5,770,581, 5,817,636, and WO1999 / 025385A1). In some embodiments, the control switch is controlled by an embeddable system, for example, disclosed in U.S. Patent 7,840,263 and U.S.2007 / 0190028A1, and gene expression is controlled by one or more energy forms, including electromagnetic energy to activate a promoter operably linked to the transgene in a ceDNA vector.

[0269] In some embodiments, the regulatory switches intended for use in ceDNA vectors are hypoxia-mediated or stress-activated switches, such as those disclosed in WO1999 / 060142A2, U.S. Patents 5,834,306, 6,218,179, 6,709,858, US2015 / 0322410, Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD, and NRSE elements, and conditionally induceable silence elements (including, for example, hypoxia-responsive elements (HRE), inflammatory response elements (IRE), and shear stress-activated elements (SSAE) disclosed in U.S. Patent 9,394,526). Such embodiments are useful for turning on the expression of transgenes from ceDNA vectors in post-ischemic or ischemic tissue and / or tumors.

[0270] (vii). Kill switch Other embodiments described herein relate to ceDNA vectors for the expression of the FIX protein described herein, including a kill switch. The kill switches disclosed herein can cause cells containing the ceDNA vector to undergo programmed cell death as a means of killing or permanently removing the introduced ceDNA vector from the system of interest. It will be understood by those skilled in the art that the use of a kill switch in a ceDNA vector for the expression of the FIX protein is typically associated with the targeting of the ceDNA vector to a limited number of cells that the subject can tolerately lose, or to a cell type to which apoptosis is desirable (e.g., cancer cells). In all embodiments, the “kill switches” disclosed herein are designed to result in rapid and robust cell killing of cells containing the ceDNA vector in the absence of an input survival signal or other specified conditions. In other words, a kill switch encoded by a ceDNA vector for the expression of the FIX protein as described herein can restrict the cell survival of cells containing the ceDNA vector to an environment defined by a specific input signal. Such kill switches serve as a biological containment mechanism when it is desirable to remove the ceDNA vector for the expression of the FIX protein in the target, or to ensure that the encoded FIX protein is not expressed.

[0271] Other kill switches known to those skilled in the art include, for example, ceDNA vectors for the expression of FIX proteins as disclosed herein, disclosed in US2010 / 0175141, US2013 / 0009799, US2011 / 0172826, US2013 / 0109568, as well as their use in kill switches as disclosed in Jusiak et al, Reviews in Cell Biology and Molecular Medicine; 2014; 1-56, Kobayashi et al., PNAS, 2004; 101; 8419-9, Marchisio et al., Int. Journal of Biochem and Cell Biol., 2011; 43; 310-319, and Reinshagen et al., Science Translational Medicine, 2018, 11.

[0272] Therefore, in some embodiments, the ceDNA vector for the expression of the FIX protein may include a kill switch nucleic acid construct containing a nucleic acid encoding an effector toxin or a reporter protein, and the expression of the effector toxin (e.g., death protein) or reporter protein is controlled by predetermined conditions. For example, predetermined conditions may be the presence of environmental substances, such as exogenous substances, in which case the cell will be killed by default by expressing an effector toxin (e.g., death protein). In alternative embodiments, predetermined conditions may be the presence of two or more environmental substances, for example, the cell will survive only if two or more necessary exogenous substances are supplied, and if none are present, the cell containing the ceDNA vector will be killed.

[0273] In some embodiments, ceDNA vectors for FIX protein expression are modified to incorporate a kill switch that destroys cells containing the ceDNA vector, effectively terminating the in vivo expression of the transgene expressed by the ceDNA vector (e.g., FIX protein expression). Specifically, the ceDNA vector is further genetically engineered to express a switch protein that does not function in mammalian cells under normal physiological conditions. Only when a drug or environmental condition that specifically targets this switch protein is administered are cells expressing the switch protein destroyed, thereby terminating the expression of the therapeutic protein or peptide. For example, it has been reported that cells expressing HSV-thymidine kinase can be killed when administered 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, edited by You (2011), and Beltinger et al., Proc. Natl. Acad. Sci. USA 96(15):8699-8704 (1999). In some embodiments, the ceDNA vector may contain an siRNA kill switch called DISE (death induced by survival gene exclusion) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[0274] D. Exemplary ceDNA-FIX vector According to some embodiments, the exemplary ceDNA-FIX vector is selected from the ceDNA-FIX vectors shown in Table 12 below. According to some embodiments, the disclosure provides a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between adjacent reverse-terminal repeat sequences (ITRs), wherein the at least one nucleic acid sequence encodes at least one FIX protein, where the ceDNA vector is selected from the ceDNA-FIX vectors shown in Table 12. [Table 12]

[0275] According to some embodiments, the exemplary ceDNA vector is ceDNA-FIXv1, which includes SEQ ID NO: 404. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 404.

[0276] According to some embodiments, an exemplary ceDNA vector is ceDNA-FIX2109, which includes SEQ ID NO: 405. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 405.

[0277] According to some embodiments, an exemplary ceDNA vector is ceDNA-FIX2112, which includes SEQ ID NO: 406. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 406.

[0278] According to some embodiments, an exemplary ceDNA vector is ceDNA-FIX2113, which includes SEQ ID NO: 407. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 407.

[0279] According to some embodiments, an exemplary ceDNA vector is ceDNA-FIX2114, which includes SEQ ID NO: 408. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 408.

[0280] According to some embodiments, an exemplary ceDNA vector is ceDNA-FIX2115, which includes SEQ ID NO: 409. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 409.

[0281] According to some embodiments, an exemplary ceDNA vector is ceDNA-FIX2116, which includes SEQ ID NO: 410. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 410.

[0282] According to some embodiments, the Disclosure provides a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between adjacent reverse-terminal repeat sequences (ITRs), wherein the at least one nucleic acid sequence encodes at least one FIX protein, and the ceDNA vector comprises SEQ ID NO: 404. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 404.

[0283] According to some embodiments, the Disclosure provides a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between adjacent reverse-terminal repeat sequences (ITRs), wherein the at least one nucleic acid sequence encodes at least one FIX protein, and the ceDNA vector comprises SEQ ID NO: 405. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 405.

[0284] According to some embodiments, the Disclosure provides a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between adjacent reverse-terminal repeat sequences (ITRs), wherein the at least one nucleic acid sequence encodes at least one FIX protein, and the ceDNA vector comprises SEQ ID NO: 406. According to some embodiments, the ceDNA vector is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 406.

[0285] According to some embodiments, Sequence ID No. 404 comprises the following components, where the numbers indicate nucleic acid residues: 1..141=Left-ITR_v1 142..194=Spacer_Left-ITR_v1 195..524 = Enhancer 525..921=Promoter 922..950=5pUTR 951..1034=hFIX signal peptide 951..1037=CDS 951..3774=ExonIntron_ORF 1039..2476 = Intron 2476..3774=CDS "Translation 2476-3774" 3538..3540=R338L Padua mutation 3775..3862=3p UTR 3863..4090=Poly A 4091..4151=Spacer Right-ITR 4152..4281=Right-ITR

[0286] According to some embodiments, Sequence ID No. 405 comprises the following components, where the numbers indicate nucleic acid residues: 1..141=Left-ITR 142..183=Spacer_Left-ITR_v.2.1 184..255 = Serpine enhancer 184..837=3x Serpin-TTRe_PromoterSet 257..328 = Serpine enhancer 330..401 = Serpine Enhancer 562..745=MouseTTR 5pUTR 714..745=5pUTR 746..836 = MVM intron 838..845=PmeI_site 846..854 = Consensus Kozak 855..2240=FIX-cDNA-691_2 2241..2248=PacI site 2249..2829=WPRE_3pUTR 2830..3054=bGH 3055..3115=Spacer_Right-ITR_v1 3116..3245=Right-ITR_v1

[0287] According to some embodiments, Sequence ID No. 406 comprises the following components, where the numbers indicate nucleic acid residues: 1..141=Left-ITR_v1 142..183=Spacer_Left-ITR_v2.1 184..255 = Serpine enhancer 184..837=3x Serpin-TTRe_PromoterSet 257..328 = Serpine enhancer 330..401 = Serpine Enhancer 562..745=MouseTTR 5pUTR(NM_013697.5) 714..745=5pUTR 746..836 = MVM intron 838..845=PmeI_site 846..854=Consensus_Kozak 855..2240=FIX-cDNA-691_16 2241..2248=PacI_site 2249..2829=WPRE_3pUTR 2830..3054=bGH 3055..3115=Spacer_Right-ITR_v1 3116..3245=Right-ITR_v1

[0288] VI. Detailed method for producing ceDNA vectors A. General production Certain methods for producing ceDNA vectors for the expression of FIX proteins, including asymmetric ITR pairs or symmetric ITR pairs as defined herein, are described in Section IV of International Application PCT / US18 / 49996, filed on September 7, 2018, which is incorporated herein by reference in its entirety. In some embodiments, the ceDNA vectors for the expression of FIX proteins disclosed herein may be produced using insect cells as described herein. In alternative embodiments, the ceDNA vectors for the expression of FIX proteins disclosed herein may be produced synthetically, and in some embodiments by cell-free methods, as disclosed in International Application PCT / US19 / 14122, filed on January 18, 2019, which is incorporated herein by reference in its entirety.

[0289] As described herein, in one embodiment, a ceDNA vector for the expression of the FIX protein may be obtained by a process comprising, for example, a) incubating a population of host cells (e.g., insect cells) containing a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), wherein the host cells are incubating under conditions effective for inducing the production of the ceDNA vector in the host cells in the presence of the Rep protein, and for a sufficient time thereafter lacking the viral capsid coding sequence, and b) harvesting and isolating the ceDNA vector from the host cells. The presence of the Rep protein induces the replication of the vector polynucleotide having a modified ITR, thereby producing the ceDNA vector in the host cells. However, no viral particles (e.g., AAV virions) are expressed. Therefore, there are no size limitations, such as those naturally imposed in AAV or other virus-based vectors.

[0290] The presence of a ceDNA vector isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme having a single recognition site on the ceDNA vector, analyzing the digested DNA material on a non-denaturing gel, and confirming the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0291] In yet another aspect, the disclosure provides the use of a host cell line that stably incorporates a DNA vector polynucleotide expression template (ceDNA template) into its own genome in the production of a nonviral DNA vector, as described, for example, Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, the Rep is attached to the host cell at an MOI of about 3. If the host cell line is a mammalian cell line, e.g., HEK293 cells, the cell line may have a stably incorporated polynucleotide vector template, and the Rep protein can be introduced into the cell using a second vector, such as a herpesvirus, enabling the excision and amplification of ceDNA in the presence of the Rep and helper virus.

[0292] In one embodiment, the host cell used to construct a ceDNA vector for the expression of the FIX protein as described herein is an insect cell, and a baculovirus is used to deliver both the polynucleotide encoding the Rep protein and a nonviral DNA vector polynucleotide expression construct template of the ceDNA, as described in Figures 4A-4C and Example 1, for example. In some embodiments, the host cell is engineered to express the Rep protein.

[0293] Next, the ceDNA vector is harvested and isolated from host cells. The time for harvesting and collecting the ceDNA vector from cells as described herein may be selected and optimized to achieve high yield production of the ceDNA vector. For example, the harvesting time may be selected considering cell viability, cell morphology, cell proliferation, etc. In one embodiment, cells are harvested after sufficient time has elapsed since baculovirus infection for proliferation and production of the ceDNA vector, but before most of the cells begin to die due to the toxicity of the baculovirus. The DNA vector may be isolated using a plasmid purification kit such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation may also be adapted for DNA vectors. In general, any nucleic acid purification method may be employed.

[0294] DNA vectors can be purified by any means known to those skilled in the art for the purification of DNA. In one embodiment, the ceDNA vector is purified as a DNA molecule. In another embodiment, the ceDNA vector is purified as an exosome or microparticle.

[0295] The presence of a ceDNA vector for FIX protein expression can be confirmed by digesting the vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector, and by using gel electrophoresis to analyze both the digested and undigested DNA material to confirm the presence of characteristic linear and continuous DNA compared to linear and discontinuous DNA. Figures 4C and 4D show one embodiment for identifying the presence of a closed-end ceDNA vector produced by the process described herein.

[0296] According to some embodiments, ceDNA is produced synthetically in a cell-free environment.

[0297] B.ceDNA plasmid A ceDNA plasmid is a plasmid used for the late production of a ceDNA vector for the expression of a FIX protein. In some embodiments, a ceDNA plasmid can be constructed using known techniques that provide, as operably linked components in the transcription direction, at least (1) a modified 5'ITR sequence, (2) an expression cassette containing a cis-regulatory element, e.g., a promoter, inducible promoter, regulatory switch, enhancer, etc., and (3) a modified 3'ITR sequence (the 3'ITR sequence is asymmetric with respect to the 5'ITR sequence). In some embodiments, the expression cassette flanked by the ITRs includes a cloning site for introducing an exogenous sequence. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0298] In one embodiment, the ceDNA vector for the expression of the FIX protein is obtained from a plasmid referred to herein as a “ceDNA-plasmid” encoding a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette containing a transgene, and a mutant or modified AAV ITR in this order, the ceDNA-plasmid lacking an AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes a first (or 5') modified or mutant AAV ITR, an expression cassette containing a transgene, and a second (or 3') modified AAV ITR in this order, the ceDNA-plasmid lacking an AAV capsid protein coding sequence, and the 5' and 3' ITRs being symmetrical with respect to each other. In an alternative embodiment, the ceDNA plasmid encodes a first (or 5') modified or mutant AAV ITR, an expression cassette containing the transgene, and a second (or 3') mutant or modified AAV ITR in this order, wherein the ceDNA plasmid lacks an AAV capsid protein coding sequence, and the 5' and 3' modified ITRs have the same modifications (i.e., they are inversely complementary or symmetrical to each other).

[0299] In further embodiments, the ceDNA-plasmid system lacks a viral capsid protein-coding sequence (i.e., it lacks not only the AAV capsid gene but also the capsid genes of other viruses). Additionally, in certain embodiments, the ceDNA-plasmid also lacks an AAV Rep protein-coding sequence. Thus, in preferred embodiments, the ceDNA-plasmid lacks a variable palindromic sequence that enables hairpin formation, in addition to the functional AAV cap and AAV rep genes (GG-3' in the case of AAV2).

[0300] The ceDNA plasmids of this disclosure can be generated using native nucleic acid sequences of the genomes of any AAV serotype known in the art. In one embodiment, the ceDNA plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes. For example, NCBI:NC NC 002077, NC 001401, NC001729, NC001829, NC006152, NC 006260, NC 006261, and Kotin and Smith, The Springer Index of Viruses, available at URLs maintained by Springer (www web address: oesys.springer.de / viruses / database / mkchapter.asp?virID=42.04) (Note - References to URLs or databases refer to the content of URLs or databases as of the effective filing date of this application). In certain embodiments, the ceDNA-plasmid backbone is derived from an AAV2 genome. In another particular embodiment, the ceDNA-plasmid backbone is a synthetic backbone genetically engineered to be contained in the 5' and 3' ITRs derived from one of these AAV genomes.

[0301] A ceDNA plasmid may optionally contain a selectable or selective marker for use in establishing a ceDNA vector-producing cell line. In one embodiment, the selective marker may be inserted downstream of the 3' ITR sequence (i.e., 3'). In another embodiment, the selective marker may be inserted upstream of the 5' ITR sequence (i.e., 5'). Suitable selective markers include, for example, those that confer drug resistance. Selective markers may include, for example, the blasticidine S resistance gene, kanamycin, or geneticin. In a preferred embodiment, the drug selective marker is the blasticidine S resistance gene.

[0302] Exemplary ceDNA (e.g., rAAV0) vectors for the expression of FIX proteins are produced from rAAV plasmids. Methods for producing rAAV vectors may include (a) providing host cells with such rAAV plasmids, where both the host cells and the plasmid lack a capsid protein-coding gene; (b) culturing the host cells under conditions that enable the production of a ceDNA genome; and (c) collecting the cells and isolating the AAV genome produced from the cells.

[0303] Exemplary method for constructing a ceDNA vector from a C.ceDNA plasmid Methods for producing capsid-free ceDNA vectors for the expression of FIX proteins, particularly methods having a sufficiently high yield to provide vectors for in vivo experiments, are also provided herein.

[0304] In some embodiments, a method for producing a ceDNA vector for the expression of the FIX protein includes: (1) introducing a nucleic acid construct comprising an expression cassette and two symmetric ITR sequences into a host cell (e.g., Sf9 cell); (2) optionally establishing a clonal cell line, for example, by using a selection marker present on a plasmid; (3) introducing a Rep-coding gene into the insect cell (either by transfection or infection with a baculovirus carrying the gene); and (4) collecting the cells and purifying the ceDNA vector. The nucleic acid construct comprising the expression cassette and two ITR sequences described above for the production of the ceDNA vector may be in the form of a ceDNA plasmid, or a bacmid or baculovirus produced by the ceDNA plasmid as described below. The nucleic acid construct may be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

[0305] D. Cell line The host cell lines used in the production of ceDNA vectors for the expression of FIX protein may include insect cell lines derived from Spodoptera frugiperda (Sf9, Sf21, etc.) or Trichoplusia ni cells, or other eukaryotic cell lines including other invertebrate, vertebrate, or mammalian cells. Other cell lines known to those skilled in the art, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes, and mature and immature dendritic cells may also be used. The host cell lines may be transfected for stable expression of ceDNA plasmids for high-yield ceDNA vector production.

[0306] ceDNA plasmids can be introduced into Sf9 cells by transient transfection using reagents known in the art (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation). Alternatively, stable Sf9 cell lines can be established that stably incorporate ceDNA plasmids into their genomes. Such stable cell lines can be established by incorporating a selection marker into the ceDNA plasmid described above. If the ceDNA plasmid used to transfect the cell line contains a selection marker such as an antibiotic, cells transfected with the ceDNA plasmid and incorporating the ceDNA plasmid DNA into their genomes can be selected by adding the antibiotic to the cell growth medium. Resistant clones of the cells can then be isolated and propagated by single-cell dilution or colony transfer techniques.

[0307] Isolate and purify the E. ceDNA vector. Examples of the process for obtaining and isolating ceDNA vectors are described in Figures 4A–4E and in the specific examples below. The ceDNA vectors for the expression of FIX protein disclosed herein are obtained from producer cells expressing AAV Rep protein and can be further transformed with ceDNA plasmids, ceDNA bacmids, or ceDNA baculoviruses. Plasmids useful for producing ceDNA vectors include plasmids encoding FIX protein or plasmids encoding one or more REP proteins.

[0308] In one embodiment, the polynucleotide encodes an AAV Rep protein (Rep78 or 68) delivered to a producer cell in a plasmid (Rep-plasmid), bacmid (Rep-bacmid), or baculovirus (Rep-baculovirus). The Rep-plasmid, Rep-bacmid, and Rep-baculovirus can be produced by the above method.

[0309] A method for producing a ceDNA vector for the expression of the FIX protein is described herein. The expression construct used to generate the ceDNA vector for the expression of the FIX protein described herein may be a plasmid (e.g., ceDNA-plasmid), a bacmid (e.g., ceDNA-bacmid), and / or a baculovirus (e.g., ceDNA-baculovirus). As just one example, a ceDNA vector may be generated from cells co-infected with ceDNA-baculovirus and Rep-baculovirus. The Rep protein produced from the Rep-baculovirus can replicate the ceDNA-baculovirus to generate a ceDNA vector. Alternatively, a ceDNA vector for the expression of the FIX protein may be generated from cells stably transfected with a construct containing a sequence encoding the AAV Rep protein (Rep78 / 52) delivered in a Rep-plasmid, Rep-bacmid, or Rep-baculovirus. ceDNA-baculoviruses can be transiently transfected into cells, replicated by Rep proteins, and produce ceDNA vectors.

[0310] Bacmids (e.g., ceDNA-bacmids) can be transfected into permissive insect cells such as Sf9, Sf21, Tni (Trichoplusia ni) cells, and High Five cells to produce ceDNA-baculoviruses, which are recombinant baculoviruses containing a symmetric ITR and an expression cassette. The ceDNA-baculoviruses can be reinfected into insect cells to obtain the next generation of recombinant baculoviruses. Optionally, this step can be repeated once or multiple times to produce larger quantities of recombinant baculoviruses.

[0311] The time for harvesting and collecting the ceDNA vector for the expression of the FIX protein described herein from cells may be selected and optimized to achieve high yield production of the ceDNA vector. For example, the harvesting time may be selected considering cell viability, cell morphology, cell proliferation, etc. Typically, cells may be harvested after sufficient time has elapsed since baculovirus infection for the production of the ceDNA vector (e.g., the ceDNA vector), but before the majority of cells begin to die due to the virulence of the virus. The ceDNA vector may be isolated from Sf9 cells using a plasmid purification kit such as the Qiagen ENDO-FREE PLASMID® kit. Other methods developed for plasmid isolation may also be adapted for the ceDNA vector. In general, any nucleic acid purification method known in the art, as well as commercially available DNA extraction kits, may be employed.

[0312] Alternatively, purification can be implemented by subjecting the cell pellet to an alkaline lysis process, centrifuging the resulting lysate, and performing chromatographic separation. As one non-limiting example, this process can be carried out by loading the supernatant onto an ion-exchange column that holds nucleic acids (e.g., SARTOBIND Q®), then eluting (e.g., with 1.2 M NaCl solution), and performing further chromatographic purification on a gel filtration column (e.g., 6-speed flow GE). The capsid-free AAV vector is then recovered, for example, by precipitation.

[0313] In some embodiments, the ceDNA vector for FIX protein expression may also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein / nucleic acid cargo via the shedding of membrane microvesicles (Cocucci et al, 2009, EP10306226.1). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles), both of which contain proteins and RNA as cargo. Microvesicles are generated from direct budding of the plasma membrane, and exosomes are released into the extracellular environment upon fusion of multivesicle endosomes with the plasma membrane. Thus, microvesicles and / or exosomes containing ceDNA vectors may be isolated from cells transduced with a ceDNA plasmid or a bacmid or baculovirus produced by a ceDNA plasmid.

[0314] Microvesicles can be isolated by filtering or ultracentrifugation of the culture medium at 20,000 x g and exosome centrifugation at 100,000 x g. The optimal duration of ultracentrifugation can be determined experimentally and will depend on the specific cell type from which the vesicles are isolated. Preferably, the culture medium is first cleared by slow centrifugation (e.g., 2000 x g for 5-20 minutes) and subjected to spin concentration using, for example, an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified via FACS or MACS by using specific antibodies that recognize specific surface antigens present on the microvesicles and exosomes. Other 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. During purification, the vesicles are washed with, for example, phosphate-buffered saline. One advantage of using microvesicles or exosomes to deliver ceDNA-containing vesicles is that these vesicles can be targeted to various cell types by being incorporated onto their membrane proteins, which are recognized by specific receptors on each cell type. (See also EP10306226)

[0315] Another aspect of the disclosure herein relates to a method for purifying ceDNA vectors from host cell lines that stably incorporate ceDNA constructs into their own genomes. In one embodiment, the ceDNA vector is purified as a DNA molecule. In another embodiment, the ceDNA vector is purified as an exosome or microparticle.

[0316] Figure 5 of international application PCT / US18 / 49996 shows a gel confirming the production of ceDNA from multiple ceDNA-plasmid constructs using the method described in the Examples. The ceDNA is identified by a characteristic band pattern in the gel, as discussed with respect to Figure 4D of the Examples.

[0317] VII. Pharmaceutical Compositions In another embodiment, a pharmaceutical composition is provided, comprising a ceDNA vector for the expression of the FIX protein described herein, and a pharmaceutically acceptable carrier or diluent.

[0318] The ceDNA vectors for the expression of FIX protein disclosed herein may be incorporated into a pharmaceutical composition suitable for administration to a target for in vivo delivery to cells, tissues, or organs of interest. Typically, the pharmaceutical composition comprises the ceDNA vector disclosed herein and a pharmaceutically acceptable carrier. For example, the ceDNA vectors for the expression of FIX protein described herein may be incorporated into a pharmaceutical composition suitable for a desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via hyperbaric intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also intended. Pharmaceutical compositions for therapeutic purposes may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high concentrations of the ceDNA vector. The sterile injection solution can be prepared by incorporating the required amount of ceDNA vector compound into a suitable buffer, along with one or a combination of the components listed above, as needed, and then by filtration sterilization containing the ceDNA vector, thereby formulating the transgene in the nucleic acid to be delivered to recipient cells, resulting in the therapeutic expression of the transgene or donor sequence therein. This composition may also contain a pharmaceutically acceptable carrier.

[0319] A pharmaceutically active composition containing a ceDNA vector for the expression of the FIX protein can be formulated to deliver a transgene for various purposes to cells, for example, a target cell.

[0320] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under manufacturing and storage conditions. Compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. Sterile injectable solutions may be prepared by filtration sterilization, incorporating the required amount of ceDNA vector compound in a suitable buffer, along with one or a combination of the components listed above, as needed.

[0321] The ceDNA vectors for the expression of FIX protein disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intraamniotic, subarachnoid, intracranial, intraarterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac), intrahepatic, intrarenal, intracerebral), subarachnoid, intrabladder, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, interstitial, intraacular, and intravitreous), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via hyperbaric intravenous or intraarterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also intended.

[0322] In some embodiments, the methods provided herein include delivering one or more ceDNA vectors for the expression of the FIX protein disclosed herein to a host cell. The methods also provide cells produced by such methods, and organisms (such as animals, plants, or fungi) that include or are produced from such cells. Methods of nucleic acid delivery may include lipofection, nucleofection, microinjection, bioristic, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, and drug-enhanced uptake in DNA. Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0323] Various techniques and methods for delivering nucleic acids to cells are known in the art. For example, nucleic acids such as ceDNA for the expression of FIX proteins can be formulated in lipid nanoparticles (LNPs), lipidoids, liposomes, lipoplexes, or core-shell nanoparticles. Typically, LNPs consist of a nucleic acid molecule (e.g., ceDNA), one or more ionized or cationic lipids (or salts thereof), one or more nonionic or neutral lipids (e.g., phospholipids), a molecule to prevent aggregation (e.g., PEG or PEG-lipid conjugates), and optionally a sterol (e.g., cholesterol).

[0324] Another method for delivering nucleic acids, such as ceDNA for the expression of FIX proteins, into cells is by conjugating the nucleic acid with a ligand that is internalized by the cell. For example, the ligand may bind to a receptor on the cell surface and be internalized via endocytosis. The ligand may be covalently linked to a nucleotide in the nucleic acid. Exemplary conjugates for delivering nucleic acids into cells are described, for example, in WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515, and WO2017 / 177326.

[0325] Nucleic acids, such as ceDNA, for the expression of FIX proteins can also be delivered to cells by transfection. Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art, including TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS® P protein transfection reagent (New England Biolabs), CHARIOT® protein delivery reagent (Active Motif), PROTEOJUICE® protein transfection reagent (EMD Millipore), 293 fectin, LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000 (Thermo Fisher Scientific), LIPOFECTAMINE® (Thermo Fisher Scientific), LIPOFECTIN® (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN® (Thermo Fisher Scientific), and OLIGOFECTAMINE® (Thermo Fisher Scientific). Scientific), LIPOFECTACE(TM), FUGENE(TM)(Roche, Basel, Switzerland), FUGENE(TM) HD(Roche), TRANSFECTAM(TM)(Transfectam, Promega, Madison, Wis.), TFX-10(TM)(Promega), T FX-20(TM)(Promega), TFX-50(TM)(Promega), TRANSFECTIN(TM)(BioRad, Hercules,Calif.), SILENTFECT(TM)(Bio-Rad), Effectene(TM)(Qiagen,Valencia,Calif.), DC-chol(Avanti Polar Lipids), GENEPORTER(TM) (Gene Therapy Systems, San This includes, but is not limited to, products such as Diego, Calif., DHARMAFECT1(Trademark) (Dharmacon, Lafayette, Colo.), DHARMAFECT2(Trademark) (Dharmacon), DHARMAFECT3(Trademark) (Dharmacon), DHARMAFECT4(Trademark) (Dharmacon), ESCORT(Trademark)III (Sigma, St. Louis, Mo.), and ESCORT(Trademark)IV (Sigma Chemical Co.). Nucleic acids such as ceDNA can also be delivered to cells via microfluidics methods known to those skilled in the art.

[0326] The ceDNA vectors for the expression of FIX proteins described herein may also be administered directly to organisms for in vivo transduction of cells. Administration may be by any of the routes commonly used to ultimately bring the molecules into contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. Preferred methods for administering such nucleic acids are available and well known to those skilled in the art, and two or more routes may be used to administer a particular composition, although a particular route may often be more immediate and provide a more effective response than another.

[0327] The method for introducing a nucleic acid vector for the expression of FIX protein into a ceDNA vector disclosed herein can be delivered, for example, to hematopoietic stem cells by the method described in U.S. Patent No. 5,928,638.

[0328] The ceDNA vector for the expression of the FIX protein according to this disclosure may be attached to a liposome for delivery to a cell or target organ in question. A liposome is a vesicle having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of formulation development. They act by fusing with the cell membrane and rearranging their lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposome compositions for such delivery consist of compounds having phospholipids, particularly phosphatidylcholine, but these compositions may also contain other lipids. Exemplary liposomes and liposome formulations containing, but not limited to, polyethylene glycol (PEG) functional groups containing compounds are disclosed in International Application PCT / US2018 / 050042, filed on 7 September 2018, and International Application PCT / US2018 / 064242, filed on 6 December 2018, see, for example, the section titled “Pharmaceutical Formulations”.

[0329] ceDNA vectors can be delivered in vitro or in vivo using various delivery methods known in the art or modifications thereof. For example, in some embodiments, ceDNA vectors for the expression of FIX proteins are delivered by creating transient penetrations of the cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy, thereby facilitating DNA entry into targeted cells. For example, ceDNA vectors may be delivered by compressing cells through a size-limited channel or by transiently disrupting the cell membrane by other means known in the art. In some cases, only the ceDNA vector is injected directly as naked DNA into any one or more tissues selected from the liver, kidney, gallbladder, prostate, adrenal gland, heart, intestine, lung, and stomach, skin, thyroid, cardiac muscle, or skeletal muscle. In some cases, ceDNA vectors are delivered by gene guns. Gold or tungsten spherical particles (1-3 μm in diameter) coated with capsid-free AAV vectors can be accelerated at high speed by pressurized gas and penetrate into target tissue cells.

[0330] This specification specifically envisions compositions comprising a ceDNA vector for the expression of FIX protein and a pharmaceutically acceptable carrier. In some embodiments, the ceDNA vector is formulated in a lipid delivery system, e.g., liposomes as described herein. In some embodiments, such compositions are administered by any route desired by a skilled practitioner. The compositions may be administered to a subject by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, via oral administration, intrapleural, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intra-articular, or combinations thereof. For veterinary use, the compositions may be administered as formulations that are suitably acceptable according to common veterinary practice. A veterinarian can easily determine the most appropriate administration plan and route for a particular animal. The compositions may be administered by conventional syringes, needle-free injection devices, "microprojectile bombardment guns," or by other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0331] In some cases, the ceDNA vector for FIX protein expression is delivered by hydrodynamic injection, a simple and highly efficient method for the direct intracellular delivery of any water-soluble compounds and particles to the skeletal muscle of the viscera and limbs.

[0332] In some cases, ceDNA vectors for FIX protein expression are delivered by ultrasound by creating nanoscopic pores in the membrane, facilitating intracellular delivery of DNA particles to visceral or tumor cells; therefore, plasmid DNA size and concentration play a significant role in the efficiency of this system. In other cases, ceDNA vectors are delivered by magnetofection using a magnetic field, enriching nucleic acid-containing particles in target cells.

[0333] In some cases, chemical delivery systems may be used by employing nanomer complexes, for example, those involving the compression of negatively charged nucleic acids by cationic liposomes / micelles or polycationic nanomer particles belonging to cationic polymers. Cationic lipids used in delivery methods include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol derivative compounds, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, and other cationic polymers), and lipid-polymer hybrids.

[0334] A. Exosome: In some embodiments, the ceDNA vector for the expression of the FIX protein disclosed herein is delivered by being packaged in exosomes. Exosomes are small membrane vesicles of endocytosis origin that are released into the extracellular environment following fusion of a polyspleen with the plasma membrane. Their surface consists of a lipid bilayer from the cell membrane of a donor cell, and they contain cytosol from the cell that produced the exosome and exhibit membrane proteins from the parent cell on their surface. Exosomes are produced by a variety of cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). In some embodiments, exosomes having diameters of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, and 50 nm to 100 nm are envisioned for use. Exosomes can be isolated for delivery to target cells either by using their donor cells or by introducing specific nucleic acids into them. Exosomes containing the capsid-free AAV vector of this disclosure can be produced using various approaches known in the art.

[0335] B. Fine particles / nanoparticles In some embodiments, the ceDNA vector for the expression of the FIX protein disclosed herein is delivered by lipid nanoparticles. Generally, lipid nanoparticles include, for example, ionized aminolipids (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and coating lipids (polyethylene glycol-dimyristoylglycerol, PEG-DMG), as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507.

[0336] In some embodiments, the lipid nanoparticles have an average diameter of about 10 to about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of about 10 to about 300 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of about 25 to about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising multiple lipid nanoparticles) has a size distribution, with an average size (e.g., diameter) of about 70 nm to about 200 nm, and more typically, an average size of about 100 nm or less.

[0337] Various lipid nanoparticles known in the art can be used to deliver the ceDNA vector for the expression of the FIX protein disclosed herein. For example, various delivery methods using lipid nanoparticles are described in U.S. Patents 9,404,127, 9,006,417, and 9,518,272.

[0338] In some embodiments, the ceDNA vector for the expression of the FIX protein disclosed herein is delivered by gold nanoparticles. Generally, nucleic acids can be covalently bound to gold nanoparticles or non-covalently bound to gold nanoparticles (e.g., by charge-charge interactions), as described, for example, by Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol.Ther. 22(6); 1075-1083. In some embodiments, the gold nanoparticle-nucleic acid conjugate is produced using, for example, the method described in U.S. Patent No. 6,812,334.

[0339] C. Conjugate In some embodiments, the ceDNA vector for the expression of the FIX protein disclosed herein is conjugated (e.g., covalently conjugated to a drug that increases cellular uptake). A “drug that increases cellular uptake” is a molecule that facilitates the transport of nucleic acids across lipid membranes. For example, nucleic acids can be conjugated to lipophilic compounds (e.g., cholesterol, tocopherol, etc.), transcellular peptides (CPPs) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine). Further examples of drugs that increase cellular uptake are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[0340] In some embodiments, the ceDNA vectors for the expression of the FIX protein disclosed herein are compounded with a polymer (e.g., a polymer molecule) or a folate molecule (e.g., a folic acid molecule). Generally, the delivery of nucleic acids compounded with polymers is known in the art, as described, for example, in WO2000 / 34343 and WO2008 / 022309. In some embodiments, the ceDNA vectors for the expression of the FIX protein disclosed herein are compounded with a poly(amide) polymer, as described, for example, in U.S. Patent No. 8,987,377. In some embodiments, the nucleic acids described herein are compounded with a folic acid molecule, as described in U.S. Patent No. 8,507,455.

[0341] In some embodiments, the ceDNA vector for the expression of the FIX protein disclosed herein is compounded with a carbohydrate, for example, as described by U.S. Patent No. 8,450,467.

[0342] D. Nanocapsules Alternatively, nanocapsule formulations of ceDNA vectors for the expression of the FIX protein disclosed herein may be used. Nanocapsules can generally capture substances in a stable and regenerative manner. To avoid side effects resulting from intracellular polymer overload, such fine particles (approximately 0.1 μm in size) should be designed to be degradable in vivo using polymers. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are intended for use.

[0343] E. liposomes The ceDNA vector for the expression of the FIX protein according to this disclosure may be attached to a liposome for delivery to a cell or target organ in question. A liposome is a vesicle having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of formulation development. They act by fusing to the cell membrane and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery consist of compounds having phospholipids, particularly phosphatidylcholine, but these compositions may also contain other lipids.

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

[0345] F. Exemplary liposome and lipid nanoparticle (LNP) compositions The ceDNA vector for the expression of the FIX protein according to this disclosure can be attached to liposomes for delivery to cells, for example, cells requiring the expression of a transgene. Liposomes are vesicles having at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of formulation development. They act by fusing with the cell membrane and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery consist of compounds having phospholipids, particularly phosphatidylcholine, but these compositions may also contain other lipids.

[0346] Lipid nanoparticles (LNPs) containing ceDNA vectors are disclosed in International Application No. PCT / US2018 / 050042, filed on 7 September 2018, and International Application No. PCT / US2018 / 064242, filed on 6 December 2018, both of which are incorporated herein by reference and are intended for use in methods and compositions for ceDNA vectors for the expression of FIX proteins disclosed herein.

[0347] In some embodiments, the disclosure provides liposomal formulations comprising one or more compounds having polyethylene glycol (PEG) functional groups (so-called "PEGylated compounds") that can reduce immunogenicity / antigenicity, provide hydrophilicity and hydrophobicity to the compound, and reduce the frequency of administration. Alternatively, the liposomal formulation simply comprises a polyethylene glycol (PEG) polymer as an additional component. In such embodiments, the molecular weight of PEG or the PEG functional group may be 62 Da to about 5,000 Da.

[0348] In some embodiments, the disclosure provides liposomal formulations that will deliver APIs having extended-release or controlled-release profiles over a period of several hours to several weeks. In some relevant embodiments, the liposomal formulation may comprise an aqueous chamber bound by a lipid bilayer. In other relevant embodiments, the liposomal formulation encapsulates an API having components that undergo physical migration at high temperatures, releasing the API over a period of several hours to several weeks.

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

[0350] In some embodiments, the present disclosure relates to N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol) conjugate lipids, HSPC (hydrogenated soy phosphatidylcholine), PEG (polyethylene glycol), DSPE (distearoyl-sn-glycero-phosphoethanolamine), DSPC (distearoyl phosphatidylcholine), DOPC (dioleoyl phosphatidylcholine), DPPG (dipalmitoyl phosphatidylglycerol), EPC (egg phosphatidylcholine), DOPS (dioleoyl phosphatidyl glycerol) The present invention provides a liposome formulation containing one or more lipids selected from phatidylserine, POPC (palmitoyloleoylphosphatidylcholine), SM (sphingomyelin), MPEG (methoxypolyethylene glycol), DMPC (dimiristoylphosphatidylcholine), DMPG (dimiristoylphosphatidylglycerol), DSPG (distearoylphosphatidylglycerol), DEPC (dioleoylphosphatidylcholine), DOPE (dioleoyl-sn-glycero-phosphoethanolamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphatidylcholine), or any combination thereof.

[0351] In some embodiments, the Disclosure provides liposomal formulations containing phospholipids, cholesterol, and PEGylated lipids in a molar ratio of 56:38:5. In some embodiments, the total lipid content of the liposomal formulation is 2 to 16 mg / mL. In some embodiments, the Disclosure provides liposomal formulations containing lipids containing phosphatidylcholine functional groups, lipids containing ethanolamine functional groups, and PEGylated lipids. In some embodiments, the Disclosure provides liposomal formulations containing lipids containing phosphatidylcholine functional groups, lipids containing ethanolamine functional groups, and PEGylated lipids in a molar ratio of 3:0.015:2, respectively. In some embodiments, the Disclosure provides liposomal formulations containing lipids containing phosphatidylcholine functional groups, cholesterol, and PEGylated lipids. In some embodiments, the Disclosure provides liposomal formulations containing lipids containing phosphatidylcholine functional groups and cholesterol. In some embodiments, the PEGylated lipid is PEG-2000-DSPE. In some embodiments, the Disclosure provides liposomal formulations comprising DPPG, soybean PC, MPEG-DSPE lipid conjugate, and cholesterol.

[0352] In some embodiments, the disclosure provides liposomal formulations comprising one or more lipids containing a phosphatidylcholine functional group and one or more lipids containing an ethanolamine functional group. In some embodiments, the disclosure provides liposomal formulations comprising one or more lipids containing a phosphatidylcholine functional group, lipids containing an ethanolamine functional group, and sterols, such as cholesterol. In some embodiments, the liposomal formulations comprise DOPC / DEPC and DOPE.

[0353] In some embodiments, the present disclosure provides liposomal formulations further comprising one or more pharmaceutically active ingredients, such as sucrose and / or glycine.

[0354] In some embodiments, the Disclosure provides liposome formulations which are either single-lamellar or multi-lamellar structures. In some embodiments, the Disclosure provides liposome formulations which include multicellular particles and / or foam-based particles. In some embodiments, the Disclosure provides liposome formulations which are larger in size relative to typical nanoparticles, with a size of about 150–250 nm. In some embodiments, the liposome formulation is a lyophilized powder. In some embodiments, the Disclosure provides liposomal formulations prepared and packed with ceDNA vectors disclosed or described herein by adding a weak base to a mixture having isolated ceDNA outside the liposomes. This addition increases the pH outside the liposomes to approximately 7.3, delivering the API into the liposomes. In some embodiments, the Disclosure provides liposomal formulations having an acidic pH inside the liposomes. In such cases, the pH inside the liposomes may be 4 to 6.9, more preferably 6.5. In other embodiments, the Disclosure provides liposomal formulations prepared by using intraliposomal drug stabilization techniques. In such cases, polymers or nonpolymers with high charge anions and intraliposomal scavengers, such as polyphosphates or sucrose octasulfate, are utilized.

[0355] In some embodiments, the present disclosure provides lipid nanoparticles comprising ceDNA and ionized lipids. For example, lipid nanoparticle formulations prepared and loaded using process-obtained ceDNA are disclosed in International Application PCT / US2018 / 050042, filed on 7 September 2018, and are incorporated herein. This can be achieved by high-energy mixing of ethanol lipids and aqueous ceDNA at a low pH, which protonates the ionized lipids and provides a favorable energy for ceDNA / lipid association and nucleation of the particles. The particles can be further stabilized by aqueous dilution and removal of organic solvents. The particles can be concentrated to a desired level.

[0356] Generally, lipid nanoparticles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 60:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) may be in the range of about 1:1 to about 60:1, about 1:1 to about 55:1, about 1:1 to about 50:1, about 1:1 to about 45:1, about 1:1 to about 40:1, about 1:1 to about 35:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, and about 30:1 to about 60:1. According to some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with ceDNA (mass or weight) relative to a total lipid ratio of about 60:1. According to some embodiments, lipid particles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 30:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) may be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. By adjusting the amounts of lipid and ceDNA, a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher, can be provided. Generally, the total lipid content of the lipid particle formulation may be in the range of about 5 mg / mL to about 30 mg / mL.

[0357] Ionized lipids are typically used to condense nucleic acid cargoes, such as ceDNA, at low pH and to drive membrane association and fusion. Generally, ionized lipids are lipids containing at least one amino group that is positively charged or protonated under acidic conditions, for example, pH 6.5 or lower. Ionized lipids are also referred to herein as cationic lipids.

[0358] Exemplary ionized lipids are described in International PCT Patent Publications 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, 2017 / 075531, 2017 / 117528, 2011 / 022460, 2013 / 148541, 2013 / 116126, and 20 11 / 153120, 2012 / 044638, 2012 / 054365, 2011 / 090965, 2013 / 016058, 2012 / 162210, 2008 / 042973, 2010 / 129709, 2010 / 1 No. 44740, No. 2012 / 099755, No. 2013 / 049328, No. 2013 / 086322, No. 2013 / 086 No. 373, No. 2011 / 071860, No. 2009 / 132131, No. 2010 / 048536, No. 2010 / 088537 No. 2010 / 054401, No. 2010 / 054406, No. 2010 / 054405, No. 2010 / 054384, Same No. 2012 / 016184, Same No. 2009 / 086558, Same No. 2010 / 042877, Same No. 2011 / 000106, Same No. 2011 / 000107, 2005 / 120152, 2011 / 141705, 2013 / 126803, 20 06 / 007712, 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / U.S. Patent Publications 127060, 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, as well as U.S. Patent Publications 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, and 2013 / 0178541,Same No. 2013 / 0303587, No. 2015 / 0141678, No. 2015 / 0239926, No. 2016 / 0376224, No. 2017 / 0119904, No. 2012 / 014989 No. 4, No. 2015 / 0057373, No. 2013 / 0090372, No. 2013 / 0274523, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 0023 No. 673, No. 2012 / 0128760, No. 2010 / 0324120, No. 2014 / 0200257, No. 2015 / 0203446, No. 2018 / 0005363, No. 2014 / 0 308304, 2013 / 0338210, 2012 / 0101148, 2012 / 0027796, 2012 / 0058144, 2013 / 0323269, 2011 / 0117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 0083780, 2013 / 0123338, 2 015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 0022649, 2010 / 0130588, This is described in issues 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, all of which are incorporated herein by reference in their entirety.

[0359] In some embodiments, the ionized lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3) having the following structure. [ka]

[0360] The lipid DLin-MC3-DMA 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.

[0361] In some embodiments, the ionized lipid is lipid ATX-002, as described in WO2015 / 074085 (the contents of which are incorporated herein by reference in their entirety).

[0362] In some embodiments, the ionized lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (compound 32), as described in WO2012 / 040184 (the contents of which are incorporated herein by reference in their entirety).

[0363] In some embodiments, the ionized lipid is compound 6 or compound 22, as described in WO2015 / 199952 (the contents of which are incorporated herein by reference in their entirety).

[0364] Without limitation, ionized lipids may constitute 20-90% (mol) of the total lipids present in the lipid nanoparticles. For example, the molar content of ionized lipids may be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, ionized lipids constitute approximately 50 mol% to approximately 90 mol% of the total lipids present in the lipid nanoparticles.

[0365] In some embodiments, lipid nanoparticles may further contain noncationic lipids. Examples of nonionic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Therefore, noncationic lipids can be neutral, uncharged, zwitterionic, or anionic lipids. Noncationic lipids are typically used to enhance membrane fusion properties.

[0366] Exemplary noncationic lipids intended for use in the methods and compositions disclosed herein are described in International Patent Application No. PCT / US2018 / 050042, filed on 7 September 2018, and PCT / US2018 / 064242, filed on 6 December 2018. Exemplary noncationic lipids are described in International Patent Publication No. 2017 / 099823 and U.S. Patent Publication No. 2018 / 0028664, both of which are incorporated herein by reference in their entirety.

[0367] Noncationic lipids may constitute 0-30% (mol) of the total lipids present in the lipid nanoparticles. For example, the noncationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipids present in the lipid nanoparticles. In various embodiments, the molar ratio of ionized lipids to neutral lipids is in the range of approximately 2:1 to approximately 8:1.

[0368] In some embodiments, the lipid nanoparticles are completely free of phospholipids. In some embodiments, the lipid nanoparticles may further contain components such as sterols to provide membrane integrity.

[0369] One exemplary sterol that may be used in lipid nanoparticles is cholesterol and its derivatives. Exemplary cholesterol derivatives are described in International Patent Application No. 2009 / 127060 and U.S. Patent Publication No. 2010 / 0130588, both of which are incorporated herein by reference in their entirety.

[0370] Components that provide membrane integrity, such as sterols, can constitute 0-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, such components constitute 20-50% (mol) or 30-40% (mol) of the total lipid content of the lipid nanoparticles.

[0371] In some embodiments, the lipid nanoparticles may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit aggregation of the lipid nanoparticles and / or provide steric stabilization. Exemplary complex lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymeric lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the complex lipid molecule is a PEG-lipid conjugate, e.g., (methoxypolyethylene glycol)-complex lipid. Examples of PEG-lipid conjugates include PEG-diacylglycerol (DAG) (e.g., l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG)), PEG-dialkoxypropylcarbam, N-(carbonyl-methoxy) Examples include, but are not limited to, polyethylene glycol (2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5,885,613, US6,287,591, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904, all of which are incorporated herein by reference in their entirety.

[0372] In some embodiments, PEG-lipids are compounds as defined in US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, PEG-lipids are disclosed in US2015 / 0376115 or US2016 / 0376224, the contents of both of which are incorporated herein by reference in their entirety.

[0373] PEG-DAA conjugates may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. PEG-lipids may be PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamide-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG -DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether) and 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] may be one or more of these. In some examples, the PEG-lipid may be selected from the group consisting of PEG-DMG and 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0374] Lipids conjugated with molecules other than PEG can also be used as a substitute for PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer-lipid (CPL) conjugates can be used as a substitute for or in addition to PEG-lipids. Exemplary complex lipids, namely PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in International Patent Application Publications No. 1996 / 010392, No. 1998 / 051278, No. 2002 / 087541, No. 2005 / 026372, and No. 2008 / 147438. Issues 2009 / 086558, 2012 / 000104, 2017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 006282 U.S. Patent Application Publication Nos. 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, and 2016 / 031745 These are described in U.S. Patent Nos. 8, 2013 / 0303587, 2013 / 0303587, 2011 / 0123453, and U.S. Patents Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, all of which are incorporated herein by reference in their entirety.

[0375] In some embodiments, one or more additional compounds may be therapeutic agents. The therapeutic agent may be selected from any class suitable for the therapeutic purpose. In other words, the therapeutic agent may be selected according to the desired therapeutic purpose and biological action. For example, if the ceDNA in the LNP is useful for treating hemophilia B, the additional compound may be an antihemophilia B agent (e.g., a chemotherapeutic agent, or other hemophilia B therapies, including but not limited to small molecules or antibodies). In another embodiment, if the LNP containing ceDNA is useful for treating an infection, the additional compound may be an antimicrobial agent (e.g., an antibiotic or antiviral compound). In yet another embodiment, if the LNP containing ceDNA is useful for treating an immune disease or disorder, the additional compound may be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulant, or a compound that modulates one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as ceDNA encoding different proteins or different compounds (e.g., therapeutic agents), may be used in the compositions and methods of the present disclosure.

[0376] In some embodiments, the additional compound is an immunomodulator. For example, the additional compound is an immunosuppressant. Immunosuppressants described herein include protein kinase inhibitors (PKIs), such as tyrosine kinase inhibitors (TKIs), and include, but are not limited to, small molecule compounds, biologics (such as monoclonal antibodies), and large polypeptide molecules that inhibit the activity of, for example, IFN signaling and production pathways, or other forms of antagonists that can reduce the expression of target proteins in immune response pathways. It should be understood that this disclosure intends to explore the use of any therapeutic modality that can act, for example, as an antagonist of IFN signaling and production pathways that modulate the immune response.

[0377] Immunosuppressants are protein kinase inhibitors, a broad class of compounds that inhibit the activity of protein kinases, and can be used in combination with any nucleic acid therapy that induces an immune response (innate and / or adaptive) in host cells or subjects suffering from genetic diseases. Tyrosine kinases regulate various cellular functions, including cell proliferation (e.g., IFN signaling and production and epidermal growth factor ("EGFRs" such as ERBB1, ERBB2 / HER2, ERBB3 / HER3, ERBB4 / HER4)). They are major signal transducers and activators that act downstream of multiple cytokines, growth factors, and hormones, thereby modulating immune responses. For example, when a particular ligand binds to its homologous receptor, conformational changes result in receptor oligomerization and activation of receptor-associated JAKs. JAKs autophosphorylate and transphosphorylate each other, phosphorylating the receptor chain and providing a docking site for STAT molecules. Subsequently, STAT undergoes phosphorylation via JAK, dimerizes, and translocates to the nucleus, where it regulates the transcription of target genes involved in immune responses (e.g., interferon-α, interferon-β, interferon-γ, TNFα, IL-2, IL-6, IL-18, etc.).

[0378] In some embodiments, the immunosuppressant is an antagonist of Jak1, Jak2, Jak3, Stat, Tyk2, c-MET, EGFR, c-KIT, BTK, ALK, ABL, SRC, ROS1, Syk, MEK, ATM, NRF2, Flt3, fms / CSF1R, FDGFR, RON, IGF1R, EPHA2, EPHA3, VEGF, or VEGFR. In some embodiments, the immunosuppressant is an antagonist of tyrosine kinase. In one embodiment, the immunosuppressant is an antagonist of Jak1. In another embodiment, the immunosuppressant is an antagonist of Jak2. In one embodiment, the immunosuppressant is an antagonist of Jak3. In yet another embodiment, the immunosuppressant is an antagonist of Tyk2. In yet another embodiment, the immunosuppressant is an antagonist of EGFR. In one embodiment, the immunosuppressant is an antagonist of ALK. In yet another embodiment, the immunosuppressant is a Syk antagonist.

[0379] In one embodiment, the immunosuppressant is a small molecule antagonist. In another embodiment, the immunosuppressant is an antibody that binds to a protein kinase target. In yet another embodiment, the immunosuppressant is an antibody that binds to a tyrosine kinase. In yet another embodiment, the immunosuppressant is a monoclonal antibody against a protein kinase. In yet another embodiment, the immunosuppressant is a monoclonal antibody against a tyrosine kinase. In yet another embodiment, the immunosuppressant is a monoclonal antibody against a target selected from the group consisting of Jak1, Jak2, Jak3, Stat, Tyk2, c-MET, EGFR, c-KIT, BTK, ALK, ABL, SRC, ROS1, Syk, MEK, ATM, NRF2, Flt3, fms / CSF1R, FDGFR, RON, IGF1R, EPHA2, EPHA3, VEGF, and VEGFR. In yet another embodiment, the immunosuppressant is a polypeptide having binding affinity to a protein kinase. In yet another embodiment, the immunosuppressant is a nucleic acid such as RNAi or antisense oligonucleotide that attenuates the expression of Jak1, Jak2, Jak3, Stat, Tyk2, c-MET, EGFR, c-KIT, BTK, ALK, ABL, SRC, ROS1, Syk, MEK, ATM, NRF2, Flt3, fms / CSF1R, FDGFR, RON, IGF1R, EPHA2, EPHA3, VEGF, or VEGFR.

[0380] In some embodiments, inhibition of a protein kinase, such as tyrosine kinase, can be achieved by using a small molecule that binds to the ATP pocket of a given protein kinase and blocks it from catalyzing the phosphorylation of the target protein. Therefore, in some embodiments, the immunosuppressant may be a small molecule antagonist of the protein kinase. Non-limiting examples of immunosuppressive protein kinase antagonists include imatinib mesylate (GLEEVEC®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), sunitinib (SUTNET®), dasatinib (SPRCEL®), acalabrutinib, alectinib, axitinib, baricitinib, afatinib, bosutinib, brigatinib, cabozantinib, and celdu. Examples include latinib, ceritinib, cobimetinib, crizotinib, dacomitinib, dasatinib, erlotinib, imatinib, fostamatinib, gefitinib, AG-1478, lapatinib, lorlatinib, TAK-659, ruxolitinib, osimertinib, pazopanib, pegaptanib, pegaptinib, regorafenib, saracatinib, tofacitinib, BMS-986165, vandetinib, vemurafenib, or pharmaceutically acceptable salts thereof.

[0381] In some embodiments, the immunosuppressant may be a small molecule antagonist of tyrosine kinase and is selected from the group consisting of baricitinib, afatinib, brigatinib, celduratinib, ceritinib, cobimetinib, dacomitinib, dasatinib, osimertinib, fostamatinib, salakatinib, TAK-659, ruxolitinib, BMS-986165, tofacitinib, and pharmaceutically acceptable salts thereof.

[0382] In some embodiments, the TKI is selected from the group consisting of sunitinib, imatinib, sorafenib, dasatinib, entprestinib, fostamatinib, TAK-659, ruxolitinib, baricitinib, BMS-986165, tofacitinib, and pharmaceutically acceptable salts thereof.

[0383] In some embodiments, the TKI is selected from the group consisting of fostamatinib, ruxolitinib, BMS-986165, and pharmaceutically acceptable salts thereof.

[0384] In one embodiment, the TKI is ruxolitinib or ruxolitinib phosphate.

[0385] In some embodiments, TKIs may selectively inhibit one or more kinases, or target multiple kinases in the same pathway. For example, ruxolitinib and baricitinib may inhibit Jak1 and Jak2. Lorlatinib may inhibit ROS1 and ALK. Dasatinib may inhibit Alb, Src, and c-Kit. Brigatinib, genfitinib, erlotinib, AG-1478, and lapatinib may inhibit EGFR. Criditinib may inhibit both ALK and c-Met. Fostamatinib and celduritinib may selectively inhibit Syk. Salakatinib may inhibit Src and Abl. In some embodiments, the TKI is a Jak1 inhibitor. In some embodiments, the TKI is a Jak2 inhibitor. In some embodiments, the TKI is an inhibitor of both Jak1 and Jak2. In some embodiments, the TKI is an EGFR inhibitor. In some embodiments, the TKI is an ALK inhibitor. In some embodiments, the TKI is a Syk inhibitor.

[0386] Protein kinase activity in immune response pathways can also be inhibited by biologics such as monoclonal antibodies against protein kinases. These therapeutics exert their effects by preventing the activation of receptor protein kinases and can bind to cell surface antigens with high specificity. Some monoclonal antibodies target receptor protein kinases that play a role in inhibiting protein kinases involved in DNA that sense immune response signaling pathways. Trastuzumab and bevacizumab are non-exclusive examples of such monoclonal antibodies.

[0387] In some embodiments, the biological agent that functions to suppress an undesirable immune response to TNA is a monoclonal antibody selected from the group consisting of ad-trastuzumab emtansine, cetuximab, CetuGEX®, cictumumab, darotuzumab, durigotumab, erzumaxomab, futuximab, ganitumab, iculkumab, margetuximab, nalunatumab, necitumumab, nimotuzumab (h-R3), oraratumab, onarutuzumab, panitumumab, pertuzumab, ranibizumab, ramucirumab, cerivantuzumab, tanibirumab, teprotumumab, trasGEX®, trastuzumab, and zatuximab. In one embodiment, the monoclonal antibody is trastuzumab....

Claims

[Claim 1] A capsid-free closed-end DNA (ceDNA) vector, A ceDNA vector comprising at least one nucleic acid sequence between adjacent reverse terminal repeat sequences (ITRs), wherein the at least one nucleic acid sequence encodes at least one FIX protein.

Images