Compositions and methods for adeno-associated (AAV) virus dnase expression

Abstract

The invention relates to gene therapy, and more specifically, to AAV gene therapy vectors containing a novel chimeric deoxyribonuclease (DNase) protein transgene and methods of treating ailments such as cancer and neurodegeneration.

Description

[0001] COMPOSITIONS AND METHODS FOR ADENO-ASSOCIATED (AAV) VIRUS DNASE EXPRESSION BACKGROUND

[0002] Major advances in gene therapy have been achieved by using viruses to deliver therapeutic genetic material. Adeno-Associated Virus (AAV) is a small, naturally occurring, non-pathogenic virus belonging to the Dependovirus genus of the Parvovirldae. Despite not causing disease, AAV is known to be able to infect humans and other primates and is prevalent in human populations. AAVs can infect a broad range of different cell types (e g., cells of the central nervous system, heart, kidney, liver, lung, pancreas, retinal pigment epithelium or photoreceptor cells, or skeletal muscle cells). Twelve serotypes of the virus (e g., AAV2, AAV5. AAV6, etc.) exhibiting different tissue infection capabilities ("tropisms") have been identified. Because of its ability to effectively transduce non-dividing cells, AAV has attracted attention as a highly effective viral vector for gene therapy.

[0003] AAV is a single-stranded DNA virus that is composed of approximately 4,800 nucleotides. The viral genome may be described as having a 5’ half and a 3’ half which together comprise the genes that encode the virus’ proteins. The 5‘ half of the AAV genome comprises the AAV rep gene, which, through the use of multiple reading frames, staggered initiating promoters (P5, P19, and P40) and alternate splicing, encodes four non-structural Rep proteins (Rep40. Rep52. Rep68 and Rep78) that are required for viral transcription control and replication and for the packaging of viral genomes into the viral capsule. In the presence of viral proteins (such as Ad proteins), the P5 promoter becomes activated and mediates the transcription of Rep68 and Rep78 proteins, which are involved in transcriptional control, in latency, in rescue, and in viral DNA replication and thus function as master controllers of the AAV life cycle.

[0004] Expression of the Rep68 and Rep78 proteins activates the P19 promoter, which is responsible for the transcription of the Rep40 and Rep52 proteins. The 3’ half of the AAV genome comprises the AAV capsid gene (cap), which encodes three capsid proteins (VP): VP1, VP2 and VP3. The three capsid proteins are translated from a single mRNA transcript that is controlled by a single promoter (P40 in case of AAV2). The 3’ half of the AAV genome also comprises the AAP gene, which encodes the AAV assembly-activating protein (AAP). Sixty VP monomers (comprising approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3) self-assemble around the AAV genome to form the icosahedral protein shell (capsid) of the mature viral particle. The AAV AAP protein is believed to be required for stabilizing and transporting newly produced VP proteins from cytoplasm into the cell nucleus. The 3’ half of the AAV genome also comprises the AAV X gene, which is believed to encode a protein that supports genome replication.

[0005] AAV is an inherently defective virus, lacking the capacity to perform at least two critical functions: the ability to initiate the synthesis of viral-specific products and the ability to assemble such products to form the icosahedral protein shell (capsid) of the mature infectious viral particle. It thus requires co-infecting “helper” virus, such as adenovirus (Ad), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus or human papillomavirus to provide the viral-associated (VA) RNA that is not encoded by genes of the AAV genome. Such VA RNA is not translated but plays a role in regulating the translation of other viral genes. Similarly, the AAV genome does not include genes that encode the viral proteins, Ela, Elb, E2a, and E4; thus, these proteins must also be provided by a co-infecting “helper” virus. The Ela protein greatly stimulates viral gene transcription during the productive infection. The Elb protein blocks apoptosis in adenovirus-infected cells, and thus allows productive infection to proceed. The E2a protein plays a role in the elongation phase of viral strand displacement replication by unwinding the template and enhancing the initiation of transcription. The E4 protein has been shown to affect transgene persistence, vector toxicity and immunogenicity.

[0006] AAV viruses infect both dividing and non-dividing cells and persist as circular episomal molecules or can be integrated into the DNA of a host cell at specific chromosomic loci (Adeno-Associated Virus Integration Sites or AAVS). AAV remains latent in such infected cells unless a helper virus is present to provide the functions needed for AAV replication and maturation.

[0007] In light of AAV properties, recombinantly modified versions of AAV (rAAV) have found substantial utility as vectors for gene therapy. rAAV are typically produced using circular plasmids (“rAAV plasmid vector”). The AAV rep and cap genes are typically deleted from such constructs and replaced with a promoter, a |3- globin intron, a cloning site into which a therapeutic gene of choice (transgene) has been inserted, and a poly-adenylation (“poly A”) site. The inverted terminal repeated sequences (ITR) of the rAAV are, however, retained, so that the transgene expression cassette of the rAAV plasmid vector is flanked by AAV ITR sequences. Thus, in the 5’ to 3’ direction, the rAAV comprises a 5’ ITR. the transgene expression cassette of the rAAV, and a 3’ ITR. rAAV have been used to deliver a transgene to patients suffering from any of a multitude of genetic diseases (e.g., hereditary lipoprotein lipase deficiency (LPLD), Leber's congenital amaurosis (LCA), aromatic L-amino acid decarboxylase deficiency (AADC), choroideremia and hemophilia), and have utility in new clinical modalities, such as Crispr / Cas9. rAAV vectors have been, or are currently in use, in 332 phase I / II / III clinical trials.

[0008] Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA, which were expelled from activated neutrophils. NETs have been implicated as key players into the pathogenesis of an increasingly large number of human diseases including cancer, acute organ injury, kidney disease, GVH disease, stroke, thrombosis, diabetes, atherosclerosis, sepsis, eclampsia, infertility, coagulopathies and neurodegeneration. Endogenous deoxyribonuclease I (DNase I) and deoxyribonuclease 1L3 (DNase IL3) enzymes activity is heavily suppressed in diseases accompanied by intensive NETs formation. It was discovered that DNase I and DNase IL3 can effectively degrade established NETs, thereby abolishing their pathogenic effects. Deoxyribonuclease enzyme is thus a useful therapeutic compound to treat pathologic conditions related to increased amount of circulating cell-free DNA and neutrophil extracellular traps.

[0009] The poor pharmacokinetic properties of natural deoxyribonuclease enzymes limit their therapeutic efficacy due to an inability7to maintain meaningful DNA hydrolytic activity7in blood. Industrial applicability of natural deoxyribonuclease enzymes is also limited because the quantities of enzyme required to maintain meaningful DNA hydrolytic activity in blood makes such treatment non-compliant for the patient and economically unfeasible. To overcome this unmet need in the art, it was proposed to use recombinant adeno-associated virus (rAAV) expression vectors comprising a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity for treatment of various diseases and conditions accompanied with intravascular and extravascular accumulation of cell free DNA (cfDNA). Such use has been described in US 2019-0241908-Al, US Patent No. 11,046,943 and WO 2017 / 019876, which are incorporated herein.

[0010] While there has been substantial progress in the use of adeno-associated virus (AAV) gene therapy vectors in real world clinical settings, setbacks related to vector toxicity7and immunogenicity still represent major challenges. In many cases, these two issues appear to be inextricably linked. Immunogenicity7of AAV vectors is thought to cause or exacerbate some of the more serious adverse events associated wi th AAV gene therapy, such as hepatotoxicity and thrombotic microangiopathy (TMA). Moreover, these adverse events tend to be correlated with vector dosage, increasing in both prevalence and severity with higher doses resulting sometime in the death of study subjects. An in-depth analysis of morbidity and mortality related to AAV gene therapies by the FDA Cellular, Tissue, and Gene Therapies Advisory Committee concluded that morbidity' and mortality was mostly observed at AAV doses equal or higher 13

[0011] than 5x 10 gc / kg. The reported treatment-emergent serious adverse events includes acute liver injury, thrombocytopenia, TMA anemia, complement activation, acute kidney injury, cardiopulmonary insufficiency, gastrointestinal infection, cardiac injury, liver failure, sepsis and death. Not surprisingly, high vector doses are also associated with increased immunogenicity7, leading to a vicious cycle when vector doses of 1x10 gc / kg or higher are required for efficacy (see, e.g., Takashi et al., Expert Opinion on Biological Therapy, 22:9, 1067-1071, 2022).

[0012] Available preclinical data show that AAV DNase I vector gene therapy is effective in 14

[0013] preclinical models of cancer when used in 1x10 gc / kg dose ( Xia, Y., et al., AAV-mediated gene transfer of DNase I in the liver of mice w ith colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response. Mol. Oncol.. 14: 2920-2935, 2020) and almost lacking efficacy when applied in lower doses (Melanie Herre, Neutrophil extracellular traps as potential therapeutic targets to prevent tumor-induced organ failure and metastasis, Digital Comprehensive Summaries of Uppsala Dissertations, https: / / uu diva- pc>riai!org / sm 3h / gei / di a2:j 787322 / F U LLTEXTO 1. pdf). AAV DNase I vector gene therapy is not effective for treatment of preclinical models of autoimmune disease (systemic lupus erythematosus) when applied at 1x10 gc / kg dose ( Amina Ahmad et al.. Conversion of the Liver into a Biofactory for DNasel Using Adeno-Associated Virus Vector Gene Transfer Reduces Neutrophil Extracellular Traps in a Model of Systemic Lupus Erythematosus. Human Gene Therapy. May 2022.560-571). That means that upon translation to the clinical development the existing AAV DNase I vectors need to be applied to patients in doses carrying out substantial risks of serious adverse events associated with AAV gene therapy.

[0014] Thus there is a need to develop more efficient recombinant AAV DNase vectors which can be applied in clinical settings in safe low ( less than 1 xlO^ gc / kg) and ultralow ( less than 1 xlO12gc / kg) doses while maintaining the efficacy to treat diseases and conditions associated with increased levels of cfDNA in a subject's blood and tissues. There are several approaches under development aiming to reduce clinical AAV / rAAV vector dose with a view to improve the safety of AAV / rAAV gene therapies while maintain efficacy. For example: increasing the ratio of full / empty capsids, the use of capsids with increased transduction efficiency, provision of more powerful promoter and enhancer designs, engineering of more catalytically effective transgenes as exemplified by the Padua variant used in hemophilia B. However, such approaches cany’ inherent risks. For example, promoter and enhancer redesign can present risks of neoplastic transformation. Capsid mutations present risks of hepatotoxicity, immunogenicity and immunotoxicity.

[0015] The present invention includes recombinant adeno-associated virus (rAAV) expression vectors that overcome the limitations of those conventionally used. The vectors can include a capsid protein and a nucleic acid with a promoter operably linked to a nucleotide sequence encoding an chimeric enzyme which has a deoxyribonuclease (DNase) activity. The enzyme can include at least two chorionic gonadotropin carboxy terminal peptides attached to the amino terminus or carboxy terminus. The rAAV expression vector can be used to treat diseases and conditions associated with increased levels of cfDNA in the blood and tissues (e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.). The vector is effective when given in low doses (e.g., below 1.0 xlO^ GC / kg) and ultralow doses (e.g., below 1.0 xlO12GC / kg).

[0016] Further it was unexpectedly discovered the chimeric DNase enzy me expressed by cells transduced with recombinant AAV DNase vectors of present invention exhibit superior pharmacologic properties as compared with wild type DNase enzyme. The chimeric DNase enzyme exhibits superior targeting of NETs and provides enhanced cleavage of NETs DNA backbone.

[0017] SUMMARY OF THE INVENTION

[0018] The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description. A full appreciation of the various aspects of the embodiments disclosed herein can be gained by taking into consideration the entire specification, claims, drawings, and abstract as a whole.

[0019] The invention includes AVV and rAAV gene therapy vectors comprising deoxyribonuclease (DNase) transgene for delivery and expression of an enzy me which has a deoxyribonuclease (DNase) activity. The novel methods and approaches of the present invention provide clinically safe and enhanced clearance of cell-free DNA (cfDNA) accumulated within the vasculature or accumulated extra-vascularly.

[0020] Also known in the art as AAV and rAAV expression vectors, the gene therapy vectors of the present invention can include a capsid protein and a nucleic acid segment. In aspects, a promoter is operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity7. In aspects, the enzyme contains at least two chorionic gonadotropin carboxy terminal peptides (CTP).

[0021] In aspects, the CTP (carboxy -terminal peptide) is derived from the naturally occurring 28 carboxy-terminal residues of human chorionic gonadotropin (hCG). The CTP has 28 amino acids and the capacity for glycosylation at four to six O- linked sugar chains, which are all at serine residue.

[0022] In one aspect, the chorionic gonadotropin carboxy terminal peptides are attached to the amino terminus of the expressed enzyme. In one aspect, the chorionic gonadotropin carboxy terminal peptides are attached to the carboxy terminus of the expressed enzy me.

[0023] In aspects the enzyme which has a deoxyribonuclease (DNase) activity and contains at least two chorionic gonadotropin carboxy terminal peptides (CTP) provide superior targeting of NETs and enhanced enzy matic cleavage of NETs DNA backbone.

[0024] The present invention also contemplates several capsid proteins to encapsulate the AAV and rAAV vector for therapeutic delivery. In one aspect of the invention, capsid protein LK03 is preferable as a non-limiting example. Other capsid proteins such as AAV2 / 6. AAVhu37, AAV5, AAVrhlO, AAVS3, SparklOO, AAV8. and all other capsid proteins suitable for the therapeutic purposes of this invention that are known in the art are inherently contemplated.

[0025] The present invention further contemplates the use of any adenoassociated virus suitable as a vector for gene therapy delivery, gene therapy expression, and enhanced clearance of cell-free DNA accumulated either intravascularly or extravascular.

[0026] The present invention alleviates an unmet need in the art for delivering effective therapy at relatively low rAAV dosages. The gene therapy vector of the present invention allow dosing of rAAV in the range between lxlO1Ogc / kg IxlO13gc / kg in a human setting to achieve sustainable and pharmacologically sufficient DNA hydrolytic activity in blood and other biological fluids; thereby avoiding use of high vector doses, and greatly reducing the incidence of treatment-emergent serious adverse events.

[0027] The present invention further contemplates a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding CTP-modified human hyperactive actin resistant deoxyribonuclease I enzyme. It can include three CTP molecules attached to the C-terminal and / or C-terminal. The amino acid sequence of the manufactured (CTP)-modified human hyperactive actin resistant deoxyribonuclease I enzyme is set forth in the sequence listings provided herein. In certain embodiments, the amino acid sequence is that set forth in SEQ ID NO: 1. However, other embodiments contemplate alternative sequences including: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and any other amino acid sequence known in the art as suitable for the therapeutic and / or prophylactic purposes of the present invention.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 depicts structure of DNase I chimeric genes CTP1, CTP2 and CTP3.

[0030] FIG. 2A is a map of the pAAV-ApoEHCR-hAATp-hDnasel-CTPl-WPRE- Xinact vector.

[0031] FIG. 2B is a map of the pAAV-ApoE HCR-hAATp-hDNaseI-CTP2-WPRE Xinact vector.

[0032] FIG. 2C is a map of the pAAV-APOE HCR-hAATp-hDnaseI-CTP3-WPRE- Xinact vector.

[0033] FIG.3 shows the levels of DNase enzymatic activity in supernatants of HuH-7 and HepG2 cells at 8h after transfection with AAV-hDNAse I CTP1, AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors.

[0034] FIG. 4 is Western blot photograph confirming presence of DNasel protein in supernatants of HepG2 cells at 48h after transfection with AAV-hDNAse I CTP1, AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors.

[0035] FIG.5 shows mice serum DNasel activity levels at 3, 10, and 21 days post injection of AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors in comparison with same AAV vectors carrying hyperactive and wild type DNase I transgenes. FIG.6 is Western blot photograph confirming presence of DNasel protein in serum of mice injected with AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors.

[0036] FIG. 7 shows long-term effect of NET-rich supernatant on enzymatic activity of human recombinant DNasel or CTP Dnasel variants. Enzymes were incubated with NET-rich supernatant for 6 h before adding genomic DNA for 30 min.

[0037] FIG. 8. Quantification of genomic DNA digestion in DNA rich plasma. Genomic DNA was digested by human recombinant DNasel or CTP Dnasel variants. Enzymes and DNA were incubated in DNA rich plasma over a 3h time period, as indicated.

[0038] FIG. 9. Fluorescent imaging of beads conjugated with recombinant Dnasel and CTP Dnasel variants after incubation with NETs.

[0039] DEFINITIONS

[0040] Reference in this specification to "one embodiment / aspect" or "an embodiment / aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the disclosure. The use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places in the specification are not necessarily all referring to the same embodiment / aspect, nor are separate or alternative embodiments / aspects mutually exclusive of other embodiments / aspects. Moreover, various features are described which may be exhibited by some embodiments / aspects and not by others. Similarly, various requirements are described which may be requirements for some embodiments / aspects but not other embodiments / aspects. Embodiment and aspect can in certain instances be used interchangeably.

[0041] The terms used in this specification generally have their ordinary' meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain temis that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. It will be appreciated that the same thing can be said in more than one way.

[0042] As used herein, the transitional phrase '‘consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising.” Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0043] The term “an enzyme which has a deoxyribonuclease (DNase) activity” is used herein to refer to an enzyme capable of hydrolytic cleavage of phosphodiester linkages in the DNA backbone.

[0044] As used herein, the terms “deoxyribonuclease” and “DNase” are used to refer to any enzy me that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone.

[0045] A vector for use in gene therapy can include a virus. In an embodiment, a virus is a retrovirus, herpes simplex virus or an adenovirus.

[0046] The term “subject” or "patient" refers to any single animal, more preferably a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. Most preferably, the patient herein is a human. In an embodiment, a “subject” of diagnosis or treatment is a prokaryotic or a eukaryotic cell, a tissue culture, a tissue, or an animal, e.g., a mammal, including a human.

[0047] The term " AAV" refers to adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subty pes and both naturally occurring and recombinant forms, except where required otherwise. Adeno-associated virus (AAV), a member of the Parvovirus family, is a small nonenveloped, icosahedral virus with single-stranded linear DNA genomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus, Dependovirus, because the virus was discovered as a contaminant in purified adenovirus stocks. AAV's life cycle includes a latent phase at which AAV genomes, after infection, are site specifically integrated into host chromosomes and an infectious phase in which, following either adenovirus or herpes simplex virus infection, the integrated genomes are subsequently rescued, replicated, and packaged into infectious viruses. The properties of non-pathogenicity, broad host range of infectivity, including non-dividing cells, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer. There are twelve AAV serotypes, with AAV1, AAV2. AAV4, AAV5 and AAV8. There are also different variants of AAVs, including chimerics or psuedotypes, haploids, polyploids and self-complimentary.

[0048] The term "cell free DNA" or “cfDNA” refers to free DNA molecules (e.g., of 25 nucleotides or longer) that are not contained within any intact cells and circulates in body fluids. cfDNA can be measured in human blood (e.g., human serum or plasma). cfDNA is believed to be released during normal cell functions, such as secretion and export in exosomes, as well as during cell death programs, such as NETosis, apoptosis and necrosis.

[0049] The term “neutrophil extracellular traps” or “NETs” refers to net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils or other blood cells. In addition to their key role in the neutrophil innate immune response. NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, aseptic inflammation and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, neurodegeneration and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Endogenous deoxyribonuclease I (DNase I) and deoxyribonuclease 1L3 (DNase IL3) enzy mes activity is heavily suppressed in diseases accompanied by intensive NETs formation. It was recently discovered that DNase I and DNase IL3 can effectively degrade established NETs, thereby abolishing their pathogenic effect.

[0050] The terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. " Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (i.e., arresting its development) and (c) relieving the disease, (i.e., causing regression of the disease).

[0051] Tre term “chimeric protein” refers to proteins created through the joining of two or more genes which are originally coded for separate or same proteins. Translation of this chimeric gene results in a single polypeptide with functional properties derived from each of the original proteins.

[0052] In an embodiment, “an effective amount” refers to the amount of the defined component sufficient to achieve the desired therapeutic result. In an embodiment, that result can be effective cancer treatment.

[0053] A "treatment effective" amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, a "treatment effective" amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0054] In an embodiment, as used herein, the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and / or may be therapeutic in terms of amelioration of the symptoms of the disease or infection, or a partial or complete cure for a disorder and / or adverse effect attributable to the disorder.

[0055] As used herein, the term "recombinant" refers to polypeptides or polynucleotides that do not exist naturally and which may be created by combining polynucleotides or polypeptides in arrangements that would not normally occur together. The term can refer to a polypeptide produced through a biological host, selected from a mammalian expression system, an insect cell expression system, a yeast expression system, and a bacterial expression system.

[0056] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term " AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV -2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9),, AAV-Rh74, and AAVRhlO. avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. " Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc. Hybrid AAVs include AAV1-13, AAV2 / 1, 2 / 2, 2 / 5, 2 / 6, 2 / 8, 2 / 9, DJ; retro, PHP. In an embodiment, a chimeric capsid protein as used herein means an AAV capsid protein that has been modified by substitutions in one or more (e.g., 2. 3, 4, 5. 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to wild type, as well as insertions and / or deletions of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to wild type. In some embodiments, complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wild type domain, functional region, epitope, etc. of a different AAV serotype, in any combination, to produce a chimeric capsid protein of this invention. Production of a chimeric capsid protein can be carried out according to protocols well known in the art and a large number of chimeric capsid proteins are described in the literature as well as herein that can be included in the capsid of this invention. In another embodiment, the AAV capsid of this invention can be AAV AAV2 / 8 / 9, HAAV82, H-AAV92, H-AAV82G9, AAV2 / 83: 1, AAV2 / 8 1:1, AAV2 / 8 1:3, or AAV8 / 9.

[0057] In an embodiment, nonlimiting examples of AAV capsid proteins that can be included in the capsid of this invention in any combination w ith other capsid proteins described herein and / or with other capsid proteins now known or later developed, include LK3, LK01-19, AAV-DJ, OligOOl, rAAV2-retro, AAV-LiC. AAVOKeral, AAV-Kera2, AAV-Kera3, AAV 7m8, AAV1.9. AAVr3.45, AAV clone 32. AAV clone 83, AAV-U87R7-C5. AAV ShH13, AAV ShH19, AAV Ll-12, AAV HAE-1, AAV HAE-2, AAV variant ShHIO, AAV2.5T, AAV LS1-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV2 Rec 1-4, AAV8BP2, AAV-B1, PHP. B, AAV9.45, AAV9.61, AAV9.47, AAVM41, AAV2 displayed peptides, AAV2-GMN, AAV9-peptide displayed. AAV8 and AAV9 peptide displayed, AAVpo2.1, AAVpo4. AAVpo5, AAVpo6, AAV rh, AAV Hu, AAV-Go.l, AAV-mo.l, BAAV, AAAV, AAV8 K137R, AAV Anc80L65, AAV2G9, AAV2265 insertion-AAV2 / 265D, AAV2.5, AAV3 SASTG, AAV2i8, AAV8G9, AAV2 tyrosine mutants AAV2 Y-F, AAV8 Y-F. AAV9 Y-F, AAV6 Y-F, AAV6.2 and any combination thereof. As a nonlimiting example, the AAV capsid proteins and virus capsids of this invention can be chimeric in that they can comprise all or a portion of a capsid subunit from another virus, optionally another parvovirus or AAV. In another embodiment, an AAV is a haploid AAV, for instance, as disclosed in U. S. Patent No. 11,702,676, which is incorporated herein.

[0058] An "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence

[0059]

[0060] of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one. and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0061] An " AAV virus" or " AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-tjpe AAV) and an encapsulated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle. As used herein, the term “promoter’" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “tissue specific” promoter may be preferentially active in specific types of tissues or cells.

[0062] The term “liver-specific promoter” is used herein to refer to a promoter which is predominantly or exclusively active in a liver cell (e.g., hepatocyte) and directs / initiates transcription in the liver to a substantially greater extent than in other tissues and organs. In this context, the term “predominantly"’ means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in liver cells. The ratio of liver expression to non-liver expression can vary between different liver-specific promoters. In some embodiments, a liver-specific promoter may preferentially direct / initiate transcription in a particular liver cell type (e.g., hepatocytes, Kupffer cells, endothelial cells, etc.). Some liver-specific promoters useful in the expression cassettes of the invention include at least one, typically several, hepatic nuclear factor binding sites. Liver-specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters. Some non-limiting examples of hepatic promoters useful in the expression cassettes of the invention include: albumin promoter (Alb), human alpha-1 anti-trypsin (hAAT) promoter, thyroxine binding globulin (TBG), Apolipoprotein E hepatic control region promoter. Apolipoprotein A-II (AP0A2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter Pl, murine transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, lecithin-cholesterol acyl transferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, and mouse prealbumin gene promoter. Non-limiting examples of liver-specific promoters include, e.g., albumin promoter (Alb), human alpha- 1 anti-trypsin (hAAT) promoter, thyroxine binding globulin (TBG) promoter, Apolipoprotein E hepatic control region promoter, Apolipoprotein A-II (AP0A2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, Liver-specific IGF-II promoter Pl, murine transthyretin (MTTR) promoter, the alpha-fetoprotein (AFP) promoter, a thy roid hormone-binding globulin promoter, an alcohol dehydrogenase promoter, the factor VIII (FVIII) promoter, a HBV basic core promoter (BCP) and PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an AAT promoter combined with the mouse albumin gene enhancer (Ealb) element, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, TBG, TBG51, TTR HLP. an apolipoprotein H (ApoH) promoter, the transferrin promoter, a transthyretin promoter, an alpha-fibrinogen and beta-fibrinogen promoters, an alpha 1 -anti chymotry psin promoter, an alpha 2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, promoters of the complement proteins (e.g., Clq, Clr. C2, C3, C4, C5, C6, C8, C9, complement Factor I, and Factor H), C3 complement activator and the [alpha] 1-acid glycoprotein promoter. Additional tissue-specific promoters may be found in the Tissue-Specific Promoter Database, TiProD (Nucleic Acids Research, J4: D104-D107 (2006).

[0063] The term “liver-specific expression” as used herein refers to a predominant or exclusive expression in the liver, i.e., expression to a substantially greater extent than in other tissues and organs

[0064] The virus vectors of the invention can further be "targeted" virus vectors (e.g., having a directed tropism) and / or a "hybrid" parvovirus (i.e., in which the viral TRs and viral capsid are from different parvoviruses) as described in international patent publication WO 00 / 28004 and Chao et al.. (2000) Molecular Therapy 2:619.

[0065] The virus vectors of the invention can further be duplexed parvovirus particles as described in international patent publication WO 01 / 92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, double stranded (duplex) genomes can be packaged into the virus capsids of the invention. Further, the viral capsid or genomic elements can contain other modifications, including insertions, deletions and / or substitutions.

[0066] A “chimeric” capsid protein as used herein means an AAV capsid protein that has been modified by substitutions in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc) amino acid residues in the amino acid sequence of the capsid protein relative to wild type, as well as insertions and / or deletions of one or more (e.g., 2. 3, 4, 5. 6, 7, 8. 9, 10, etc) amino acid residues in the amino acid sequence relative to wild type. In some embodiments, complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wildtype domain, functional region, epitope, etc. of a different AAV serotype, in any combination, to produce a chimeric capsid protein of this invention. Production of a chimeric capsid protein can be carried out according to protocols well known in the art and a large number of chimeric capsid proteins are described in the literature as well as herein that can be included in the capsid of this invention.

[0067] The term "variant" as used herein includes modifications or chemical equivalents of the amino acid and nucleotide sequences disclosed herein that perform substantially the same function as the proteins or nucleic acid molecules disclosed herein in substantially the same way. For example, variants of proteins disclosed herein include, without limitation, conservative amino acid substitutions. Variants of proteins disclosed herein also include additions and deletions to the proteins disclosed herein. In addition, variant peptides and variant nucleotide sequences include analogs and chemical derivatives thereof.

[0068] " Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG. which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0069] The amino acid substitutions may be conservative or non-conservative. A "conservative amino acid substitution", as used herein, is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Ala / Pro, Lys / Arg. Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly, in both directions. Amino acid exchanges in proteins and peptides, which do not generally alter the activity of the proteins or peptides, are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).

[0070] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double- stranded form.

[0071] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

[0072] A “transgene” refers to any gene that has been transferred from one organism to another.

[0073] " Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0074] The term "control element" or "control sequence" refers to a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulator}' sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.

[0075] The term "operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues betw een the promoter and coding region so long as this functional relationship is maintained.

[0076] The term "substantial homology" or "substantial similarity," when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homolog}' is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0077] The term "expression vector" refers to a vector comprising a region which encodes a polypeptide of interest and is used for effecting the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0078] As used herein, the term "gene delivery’* refers to a process by which foreign DNA is transferred to host cells for applications of gene therapy

[0079] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.

[0080] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.

[0081] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). The term “ailment” refers to a disease, illness or medical condition. An ailment can be, associated with increased levels of circulating cfDNA. Examples of ailments include cancers, neurodegeneration, muscular dystrophy, amyotrophic lateral sclerosis (ALS), inflammatory disorders and inflammation, autoimmune disorders, autoimmune deficiencies, diseases and disorders of the cardiovascular system, integumentary' system, skeletal system, respiratory system, lymphatic system, endocrine system, digestive system, genetic disorders and genetic deficiencies.

[0082] The term “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, etc., including solid tumors, kidney, breast, lung, kidney, bladder, urinary tract, urethra, penis, vulva, vagina, cervical, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer. Additional cancers include, for example, Hodgkin's Disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary’ thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.

[0083] The term "serotype" is a distinction with respect to an AAV having a capsid which is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to the AAV as compared to other AAV.

[0084] In accordance with the present invention there may be employed conventional pharmacology and molecular biology techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein “Sambrook et al.. 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); among others.

[0085]

[0086] DETAILED DESCRIPTION OF THE INVENTION

[0087] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology as claimed. Additional features and advantages of the subject technology are set forth in the description below, and in part will be apparent from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

[0088] Applicant has demonstrated that systemic administration of DNase protein into a patient's circulation can treat ailments that are associated with increased levels of cfDNA in the blood. These ailments include, for example, cancers (e.g., carcinomas, sarcomas, lymphomas, melanoma; see, e.g., U. S. Pat. Nos. 7,612,032; 8,710,012; 9,248,166), development of somatic mosaicism (see, e.g., U. S. Pat. App. Pub. No. US20170056482), side effects associated with a chemotherapy or a radiation therapy (see, e.g., U. S. Pat. App. Pub. No. US20170100463), neurodegenerative diseases (see, e.g., Int. App. Pub. No.

[0089] W02016 / 190780), infections (see, e.g., U. S. Pat. Nos. 8,431,123 and 9,072,733), diabetes (see, e.g., U. S. Pat. No. 8,388,951), atherosclerosis (see, e.g., U. S. Pat. No. 8,388,951), stroke (see, e.g., U. S. Pat. No. 8,796,004), angina (see, e.g., U. S. Pat. No. 8,796,004), ischemia (see, e.g., U. S. Pat. No. 8,796.004), kidney damage (see, e.g., U. S. Pat. No. 9,770,492), delayed-type hypersensitivity reactions such as, e.g.. graft-versus- host disease [GVHD]) (see, e.g.. U. S. Pat. No. 8,535,663), reduction of fertility (see, e.g., U. S. Pat. No. 8,91,151), agespecific sperm motility' impairment (see, e.g., U. S. Pat. No. 8,871,200), and aging (see, e.g., U. S. Pat. App. Pub. No. US20150110769).

[0090] The present invention provides vectors and methods for systemic delivery of a DNase into a patient's circulation to treat ailments associated with increased levels of cfDNA in the blood to treat such diseases or conditions. Accordingly, embodiments include recombinant adeno-associated virus (rAAV) expression vectors. In aspects, the vectors include a capsid protein and a nucleic acid segment with a promoter operably linked to a sequence encoding (CTP)-modified human hyperactive actin resistant deoxyribonuclease I enzy me which has a deoxyribonuclease (DNase) activity. In aspects, the CTP (carboxy -terminal peptide) is derived from the naturally occurring 28 carboxy -terminal residues of human chorionic gonadotropin (hCG). This relatively conserved peptide (the sequence identity to its monkey and rat homologues is ~79% and ~74%, respectively) has been shown to provide hCG with the required longevity to maintain pregnancy (see, e.g., Matzuk, et al., Endocrinology 1990, 126, 376-383.). CTP has 28 amino acids and the capacity for glycosylation at four to six O-linked sugar chains, which are all at serine residue.

[0091] In embodiments, the vectors include a nucleotide sequence that encodes an enzyme which has a deoxyribonuclease (DNase) activity. The enzyme can contain at least two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the amino terminus or carboxy terminus of the enzyme. When expressed in the liver, such deoxyribonuclease enzy me provides unexpectedly high levels of DNA hydrolytic activity in blood over the time. This activity is likely due to a markedly increased distribution phase and reduced clearance of CTP modified enzyme following secretion to blood circulation relative to the unmodified DNases. Such remarkable pharmacokinetic properties allow dosing of rAAV in the ranges described without adverse events associated with high vector doses.

[0092] Each CTP molecule typically carries four serine-linked oligosaccharides. The invention is further based on the unexpected finding that upon injection of an rAAV the chimeric DNase expressed in the liver has improved the pharmacologic properties including increased binding to NETs and augmented enzymatic cleavage of NETs.

[0093] Another embodiment is a recombinant adeno-associated virus (rAAV) expression vector that includes (i) a capsid protein and (ii) a nucleic acid with a promoter operably linked to a nucleotide sequence encoding (CTP)-modified human hyperactive actin resistant deoxyribonuclease I enzyme which has a deoxyribonuclease (DNase) activity. The hyperactive actin resistant deoxy ribonuclease I can include three CTP molecules attached in tandem on its C- terminal end. In aspects, the amino acid sequence of the manufactured (CTP)- modified human hyperactive actin resistant deoxyribonuclease I enzyme is set forth in SEQ ID NO: 1.

[0094] Another embodiment is a recombinant adeno-associated virus (rAAV) expression vector that includes (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding (CTP)-modified human hyperactive actin resistant deoxyribonuclease I enzyme which has a deoxyribonuclease (DNase) activity. The said hyperactive actin-resistant deoxyribonuclease I can include two CTP molecules attached in tandem on its C- terminal end. In aspects, the amino acid sequence of the manufactured (CTP)- modified human hyperactive actin resistant deoxyribonuclease I enzy me is set forth in SEQ ID NO: 2.

[0095] Another embodiment is a recombinant adeno-associated virus (rAAV) expression vector. The vector can include (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding (CTP)- modified human hyperactive actin resistant deoxyribonuclease I enzyme which has a deoxyribonuclease (DNase) activity. The hyperactive actin resistant deoxyribonuclease I can include two CTP molecules attached in tandem on its C- terminal end and one CTP molecule attached on its N-terminal end. In aspects, the amino acid sequence of the manufactured (CTP)-modified human hyperactive actin resistant deoxyribonuclease I enzyme is set forth in SEQ ID NO: 3.

[0096] In aspects, the amino acid sequence of the (CTP)-modified human hyperactive actin resistant deoxyribonuclease I enzyme is set forth in one of the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9.

[0097] Another embodiment is a recombinant adeno-associated virus (rAAV) expression vector that includes (i) a capsid protein and (ii) a nucleic acid with a promoter operably linked to a nucleotide sequence encoding CTP-modified human hyperactive actin resistant deoxyribonuclease I enzy me that has three CTP molecules attached in different order to N terminal and / or to the C-terminal end. Upon expression of the rAAV in liver cells, the CTP modified hyperactive actin resistant deoxyribonuclease I enzy me has a high sialic acid content and has improved the pharmacologic properties including increased binding to NETs and augmented enzymatic cleavage of NETs.

[0098] The vectors have improved pharmacologic properties over conventional vectors. Specifically,

[0099] 11 14

[0100] dosing of rAAV can be achieved in the range between 1x10 gc / kg 1x10 gc / kg in humans to achieve sustainable and pharmacologically sufficient DNA hydrolytic activity in blood. This avoids the necessity' of use of high vector doses that can lead to toxicity' and adverse events

[0101]

[0102] Another embodiment is a method of treating a subject having disease or condition accompanied by intravascular or extravascular accumulation of extracellular DNA. The method can include administering a therapeutically effective amount of recombinant adeno-associated virus (rAAV) expression vector. The rAAV expression vector can include (i) a capsid protein and (ii) a nucleic acid with a promoter operably linked to a nucleotide sequence encoding CTP-modified human hyperactive actin resistant deoxyribonuclease I enzyme that has three CTP molecules attached in different order to N terminal and / or to the C-terminal end.

[0103] Another embodiment is a method of treating a subject having disease or condition accompanied by intravascular or extravascular accumulation of extracellular DNA. The method can include administering a therapeutically effective amount of recombinant adeno-associated virus (rAAV) expression vector. The rAAV expression vector can include (i) a capsid protein and (ii) a nucleic acid with a promoter operably linked to a nucleotide sequence encoding CTP-modified human hyperactive actin resistant deoxyribonuclease I enzyme that has three CTP molecules attached in different order to N terminal and / or to the C-terminal end. Upon expression of the rAAV in liver cells, the CTP modified hyperactive actin resistant deoxyribonuclease I enzyme molecule equally effective expressed in livers of males and females.

[0104] Carboxy Terminal Peptide Variations

[0105] In one embodiment, the carboxy terminal peptide (CTP) has the amino acid sequence from amino acid 112 to position 145 of human chorionic gonadotrophin, as set forth in SEQ ID NO: 10. In another embodiment, the CTP sequence has the amino acid sequence from amino acid 118 to position 145 of human chorionic gonadotropin, as set forth in SEQ ID NO: 11. In another embodiment, the CTP sequence also commences from any position between positions 112 - 118 and terminates at position 145 of human chorionic gonadotrophin. In some embodiments, the CTP sequence peptide is 28, 29, 30, 31, 32, 33 or 34 amino acids long and commences at position 112, 113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.

[0106] In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1 - 5 conservative amino acid substitutions as described in U. S. Patent No. 5,712,122. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1 conservative amino acid substitution. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 2 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by conservative amino acid substitutions. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 70% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 80% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 90% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 95% homologous to the native CTP amino acid sequence or a peptide thereof.

[0107] In one embodiment, at least one of the chorionic gonadotrophin CTP amino acid sequences is truncated. In another embodiment, both of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, 2 of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, 2 or more of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, all of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, all but one of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, all but two of the chorionic gonadotrophin amino acid sequences are truncated. In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 12. In one embodiment, the truncated CTP comprises the first 11 amino acids of SEQ ID NO: 12. In one embodiment, the truncated CTP comprises the first 12 amino acids of SEQ ID NO: 12. In one embodiment, the truncated CTP comprises the first 13 amino acids of SEQ ID NO: 12. In one embodiment, the truncated CTP comprises the first 14 amino acids of SEQ ID NO: 12. In one embodiment, the truncated CTP comprises the first 15 amino acids of SEQ ID NO: 12. In one embodiment, the truncated CTP comprises the first 16 amino acids of SEQ ID NO: 12. In an embodiment, a CTP disclosed herein may have a length of, e.g.. about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13. about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, or about 75 amino acids. In another embodiment, a CTP disclosed herein may have a length of, e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20. at least 25. at least 30. at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or at least 75 amino acids. In yet another embodiment, a CTP disclosed herein may have a length of, e.g., at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, or at most 75 amino acids.

[0108] In an embodiment, a CTP disclosed herein may have a length of, e.g., between about 7 to about 10 amino acids, about 7 to about 12 amino acids, about 7 to about 15 amino acids, about 7 to about 18 amino acids, about 7 to about 20 amino acids, about 7 to about 25 amino acids, about 7 to about 30 amino acids, about 7 to about 35 amino acids, about 7 to about 40 amino acids, about 7 to about 45 amino acids, about 7 to about 50 amino acids, about 7 to about 55 amino acids, about 7 to about 60 amino acids, about 7 to about 65 amino acids, about 7 to about 70 amino acids, about 7 to about 75 amino acids, about 10 to about 12 amino acids, about 10 to about 15 amino acids, about 10 to about 18 amino acids, about 10 to about 20 amino acids, about 10 to about 25 amino acids, about 10 to about 30 amino acids, about 10 to about 35 amino acids, about 10 to about 40 amino acids, about 10 to about 45 amino acids, about 10 to about 50 amino acids, about 10 to about 55 amino acids, about 10 to about 60 amino acids, about 10 to about 65 amino acids, about 10 to about 70 amino acids, about 10 to about 75 amino acids, about 12 to about 15 amino acids, about 12 to about 18 amino acids, about 12 to about 20 amino acids, about 12 to about 25 amino acids, about 12 to about 30 amino acids, about 12 to about 35 amino acids, about 12 to about 40 amino acids, about 12 to about 45 amino acids, about 12 to about 50 amino acids, about 12 to about 55 amino acids, about 12 to about 60 amino acids, about 12 to about 65 amino acids, about 12 to about 70 amino acids, about 12 to about 75 amino acids, about 15 to about 18 amino acids, about 15 to about 20 amino acids, about 15 to about 25 amino acids, about 15 to about 30 amino acids, about 15 to about 35 amino acids, about 15 to about 40 amino acids, about 15 to about 45 amino acids, about 15 to about 50 amino acids, about 15 to about 55 amino acids, about 15 to about 60 amino acids, about 15 to about 65 amino acids, about 15 to about 70 amino acids, about 15 to about 75 amino acids, about 18 to about 20 amino acids, about 18 to about 25 amino acids, about 18 to about 30 amino acids, about 18 to about 35 amino acids, about 18 to about 40 amino acids, about 18 to about 45 amino acids, about 18 to about 50 amino acids, about 18 to about 55 amino acids, about 18 to about 60 amino acids, about 18 to about 65 amino acids, about 18 to about 70 amino acids, about 18 to about 75 ammo acids, about 20 to about 25 amino acids, about 20 to about 30 amino acids, about 20 to about 35 amino acids, about 20 to about 40 amino acids, about 20 to about 45 amino acids, about 20 to about 50 amino acids, about 20 to about 55 amino acids, about 20 to about 60 amino acids, about 20 to about 65 amino acids, about 20 to about 70 amino acids, about 20 to about 75 amino acids, about 25 to about 30 amino acids, about 25 to about 35 amino acids, about 25 to about 40 amino acids, about 25 to about 45 amino acids, about 25 to about 50 amino acids, about 25 to about 55 amino acids, about 25 to about 60 amino acids, about 25 to about 65 amino acids, about 25 to about 70 amino acids, about 25 to about 75 amino acids, about 30 to about 35 amino acids, about 30 to about 40 amino acids, about 30 to about 45 ammo acids, about 30 to about 50 amino acids, about 30 to about 55 amino acids, about 30 to about 60 amino acids, about 30 to about 65 amino acids, about 30 to about 70 amino acids, about 30 to about 75 amino acids, about 35 to about 40 amino acids, about 35 to about 45 amino acids, about 35 to about 50 amino acids, about 35 to about 55 amino acids, about 35 to about 60 amino acids, about 35 to about 65 amino acids, about 35 to about 70 amino acids, about 35 to about 75 amino acids, about 40 to about 45 amino acids, about 40 to about 50 amino acids, about 40 to about 55 amino acids, about 40 to about 60 amino acids, about 40 to about 65 amino acids, about 40 to about 70 amino acids, about 40 to about 75 amino acids, about 45 to about 50 amino acids, about 45 to about 55 amino acids, about 45 to about 60 amino acids, about 45 to about 65 amino acids, about 45 to about 70 amino acids, about 45 to about 75 amino acids, about 50 to about 55 amino acids, about 50 to about 60 amino acids, about 50 to about 65 amino acids, about 50 to about 70 amino acids, or about 50 to about 75 amino acids.

[0109] Constructs

[0110] Figure 1 depicts the structure of a DNase I, wherein the DNase comprises a DNase and a chimeric gene. The depiction of a chimeric gene wherein chimeric includes, comprises or constitutes a DNase and a CTP1, CTP2 and CTP3. Figure 2 depicts a genomic map comprising, constituting or including a pAAV-ApoEHCR-hAATp-hDnasel-CTPl-WPRE- Xinact vector.

[0111] Figure 2B depicts a genomic map of apAAV-ApoE HCR-hAATp-hDNaseI-CTP2-WPRE Xinact vector.

[0112] Figure 2C depicts a genomic map of apAAV-APOE HCR-hAATp-hDnaseI-CTP3-WPRE-Xinact vector.

[0113] DNase Variations

[0114] Specific non-limiting examples of enzymes which have a DNase activity that can be used in the compositions and methods of the invention include DNase I, DNase X, DNase γ, DNaselLl, DNaselL2, DNase 1L3, DNase II (e.g., DNase Ila, DNase lip), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof. If the enzyme which has a DNase activity is DNase I, various mutants weakening actin-binding may be used. Specific non-limiting examples of residues in wild-type recombinant human DNase I (SEQ ID NO: 4) that can be mutated include: Gln-9, Glu-13, Thr-14, His-44. Asp-53, Tyr-65, Val-66, Val-67, Glu- 69, Asn-74. and Ala-114. In various embodiments, the Ala-114 mutation is used. For example, in human DNase I hyperactive mutant comprising the sequence of SEQ ID NO: 5, the Ala-114 residue is mutated. Complementary residues in other DNases may also be mutated. Specific non-limiting examples of mutations in wild- ty pe human recombinant DNAse I include H44C, H44N, L45C. V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S. D58T, Y65A, Y65E. Y65R. Y65C, V66N, V67E. V67K. V67C, E69R, E69C, A114C, A114R, H44N: T46S, D53R: Y65A, D53R: E69R, H44A: D53R: Y65A, H44A: Y65A: E69R, H64N: V66S, H64N: V66T, Y65N: V67S, Y65N: V67T, V66N: S68T, V67N: E69S, V67N: E69T, S68N: P70S, S68N: P70T, S94N: Y96S, S94N: Y96T. Various DNase mutants for increasing DNase activity may be used. Specific non-limiting examples of mutations in wild- type human recombinant DNAse I include, e.g., Gln-9, Glu-13, Thr-14, His-44, Asp- 53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, and Ala-114. Specific non-limiting examples of mutations for increasing the activity of wild-type human recombinant DNase I include Q9R. E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F. For example, a combination of the Q9R. E13R, N74K. and Al 14F mutations may be used, with such combination found at least in the hyperactive DNase I mutant comprising the sequence of SEQ ID NO: 5. WO2021168413 (Al) and W02020076817(A1) describes certain mutants and derivatives of DNaselL2, and DNase 1L3 enzymes suitable for use in the present invention.

[0115] AAV Capsid Variations

[0116] AAV vectors disclosed herein may be derived from any AAV serotype, including combinations of serotypes (e.g., "pseudotyped ' AAV) or from various genomes (e.g., singlestranded or self-complementary). A ‘‘serotype” is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such crossreactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences). Non-limiting examples of AAV serotypes which can be used to develop the AAV expression vectors of the invention include, e.g., AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO (as disclosed, e.g., in U. S. Patent No. 9,790,472, International Patent Application Pub. No.

[0117] WO2017 / 180857 and WO2017 / 180861), AAVLK03. AAVKP1 (as disclosed, e.g.. in Wang et al., Mol. Then, 2015, 23(12):1877-1887), AAVhu37 (as disclosed, e g., in Int. Pat. Appl. Pub. No. WO2017180857), AAVrh64Rl (as disclosed, e.g., in Int. Pat. Appl. Pub. No.

[0118] WO2017180857), Anc80 (based on a predicted ancestor of serotypes AAV1, AAV2. AAV8 and AAV9; see Zinn et al., Cell Rep., 2015, 12(67): 1056-1068), avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Recently, a number of putative new AAV serotypes and clades have been identified (see, e.g., Gao et al, (2004) J. Virology 78:6381-6388; Moris et al, (2004) Virology 33-:375-383). The genomic sequences of the various serotypes of AAV and the autonomous parvoviruses, as well as the sequences of the terminal repeats, Rep proteins, and capsid subunits are know n in the art, by way of example, Srivistava et al, (1983) J. Virology 45:555; Chiorini et al, (1998) J. Virology 71:6823; Chiorini et al, (1999) J. Virology 73:1309; Bantel-Schaal et al, (1999) J. Virology 73:939; Xiao et al, (1999) J. Virology 73:3994; Muramatsu et al, (1996) Virology 221: 208; Shade et al, (1986) J. Virol. 58:921; Gao et al, (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al, (2004) Virology 33-: 375- 383; GenBank Accession number U89790; GenBank Accession number J01901; GenBank Accession number AF043303; GenBank Accession number AF085716; GenBank Accession number NC 006152; GenBank Accession number Y18065; GenBank Accession number NC 006260; GenBank Accession number NC_006261; International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U. S. Pat. No. 6,156,303.

[0119] Dosing

[0120] In an embodiment, a dose of DNase or DNase-CTP may be in the range of, e.g., about 1 pg to about 10 pg, about 1 pg to about 20 pg, about 1 pg to about 30 gig, about 1 pg to about 40 pg, about 1 pg to about 50 pg. about 1 pg to about 60 pg. about 1 pg to about 70 pg, about 1 pg to about 80 pg, about 1 pg to about 90 pg, about 1 pg to about 100 p, about 1 pg to about 1 10 pg, about 1 pg to about 120 pg, about 1 pg to about 130 pg, about 1 pg to about 140 pg, about 1 pg to about 150 pg, about 5 pg to about 10 pg, about 5 pg to about 20 pg, about 5 pg to about 30 pg. about 5 pg to about 40 pg. about 5 pg to about 50 pg. about 5 pg to about 60 pg, about 5 pg to about 70 pg, about 5 pg to about 80 pg, about 5 pg to about 90 pg, about 5 pg to about 100 pg, about 5 pg to about 1 10 pg, about 5 pg to about 120 pg, about 5 pg to about 130 pg, about 5 pg to about 140 ig, about 5 pg to about 150 pg, about 10 pg to about 20 pg, about 10 pg to about 30 pg, about 10 pg to about 40 pg. about 10 to about 50 pg. about 10 to about 60 pg, about 10 pg to about 70 pg, about 10 pg to about 80 pg, about 10 pg to about 90 pg, about 10 pg to about 100 pg, about 10 pg to about 1 10 pg, about 10 pg to about 120 pg, about 10 pg to about 130 pg, about 10 pg to about 140 v, about 10 pg to about 150 pg, about 10 pg to about 175 pg, about 10 pg to about 200 pg, about 10 pg to about 225 pg, about 10 pg to about 250 pg. about 25 pg to about 50 [ig. about 25 [ig to about 75 pi, about 25 [ig to about 100 [ig, about 25 p<; to about 125 [ig, about 25 pg to about 150 pg, about 25 pg to about 175 pg, about 25 pg to about 200 pg, about 25 pg to about 225 pg, about 25 pg to about 250 pg, about 50 pg to about 75 pg, about 50 pg to about 100 pg, about 50 pg to about 125 pg, about 50 pg to about 150 pg. about 50 pg to about 175 pg, about 50 pg to about 200 pg, about 50 pg to about 225 pg, about 50 pg to about 250 pg, about 75 pg to about 100 pg, about 75 pg to about 125 pg, about 75 pg to about 150 pg, about 75 pg to about 175 pg, about 75 pg to about 200 pg, about 75 pg to about 225 pg, or about 75 pg to about 250 [ig. In an embodiment, a dose of DNase or DNase-CTP may be in the range of. e.g., about 100 pg to about 125 pg, about 100 pg to about 150 pg, about 100 pg to about 175 pg. about 100 pg to about 200 pg, about 100 pg to about 225 pg, about 100 pg to about 250 pg, about 100 pg to about 275 pg, about 100 pg to about 300 pg, about 100 pg to about 325 pg, about 100 pg to about 350 pg, about 100 pg to about 375 pg, about 100 pg to about 400 pg. about 100 pg to about 425 pig, about 100 ig to about 450 pig, about 100 pig to about 475 pig. about 100 pig to about 500 pig, about 100 pig to about 525 pig, about 100 pig to about 550 pig. about 100 pig to about 575 pig, about 100 pig to about 600 pig, about 125 pig to about 150 pig, about 125 pig to about 175 pig, about 125 pig to about 200 pig, about 125 pig to about 225 pig, about 125 pig to about 250 pig, about 125 pig to about 275 pig, about 125 pig to about 300 pig, about 125 pig to about 325 pig, about 125 pig to about 350 pig, about 125 pig to about 375 pig. about 125 pig to about 400 pig, about 125 pig to about 425 pig, about 125 pig to about 450 pig. about 125 pig to about 475 pig, about 125 pig to about 500 pig, about 125 pig to about 525 pig, about 125 pig to about 550 pig, about 125 pig to about 575 pig, about 125 pig to about 600 pig, about 150 pig to about 175 pig, about 150 pig to about 200 pig, about 150 pig to about 225 pig. about 150 pig to about 250 pig, about 150 pig to about 275 pig, about 150 pig to about 300 pig. about 150 pig to about 325 pig, about 150 pig to about 350 pig, about 150 pig to about 375 pig, about 150 pig to about 400 pig, about 150 pig to about 425 pig, about 150 pig to about 450 pig, about 150 pig to about 475 pig, about 150 pig to about 500 pig, about 150 pig to about 525 pig, about 150 pig to about 550 pig, about 150 pig to about 575 pig, about 150 pig to about 600 pig. about 200 pig to about 225 pig, about 200 pig to about 250 pig, about 200 pig to about 275 pig, about 200 pig to about 300 pig, about 200 pig to about 325 pig, about 200 pig to about 350 pig, about 200 pig to about 375 pig, about 200 pig to about 400 pig, about 200 pig to about 425 pig, about 200 pig to about 450 pig, about 200 pig to about 475 pig, about 200 pig to about 500 pig. about 200 pig to about 525 pig, about 200 pig to about 550 pig, about 200 pig to about 575 pig. about 200 pig to about 600 pig, about 200 pig to about 625 pig, about 200 pig to about 650 pig, about 200 pig to about 675 pig, about 200 pig to about 700 pig, about 200 pig to about 725 pig, about 200 pig to about 750 pig, about 200 pig to about 775 pig, about 200 pig to about 800 pig. about 200 pig to about 825 pig, about 200 pig to about 850 pig, about 200 pig to about 875 pig. about 200 pig to about 900 pig, about 200 pig to about 925 pig, about 200 pig to about 950 pig, about 200 pig to about 975 pig, or about 200 pig to about 1,000 pig.

[0121] In an embodiment, a dose of a therapeutic composition, including a DNase or DNase-CTP, may be administered to a patient at a dose of, e.g., about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg. about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 1 10 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg,

[0122]

[0123] about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, 260 mg, about 270 mg. about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg. about 620 mg, about 630 mg, about 640 mg, about 650 mg, 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg. about 850 mg, 860 mg, about 870 mg. about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, 960 mg, about 970 mg, about 980 mg, about 990 mg, or about 1,000 mg.

[0124] In an embodiment, a dose of a therapeutic composition, including a DNase or DNase-CTP, may be administered to a patient at a dose of, e.g., at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 6 mg, at least 7 mg, at least 8 mg, at least 9 mg, at least 10 mg, at least 15 mg. at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg. at least 50 mg. at least 55 mg. at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 1 10 mg, at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, at least 200 mg, at least 210 mg, at least 220 mg, at least 230 mg, at least 240 mg, at least 250 mg, 260 mg, at least 270 mg, at least 280 mg, at least 290 mg, at least 300 mg, at least 310 mg, at least 320 mg, at least 330 mg, at least 340 mg, at least 350 mg, 360 mg, at least 370 mg, at least 380 mg, at least 390 mg, at least 400 mg, at least 410 mg, at least 420 mg, at least 430 mg, at least 440 mg, at least 450 mg, 460 mg, at least 470 mg, at least 480 mg, at least 490 mg, at least 500 mg, at least 510 mg, at least 520 mg, at least 530 mg, at least 540 mg, at least 550 mg, 560 mg, at least 570 mg, at least 580 mg, at least 590 mg, at least 600 mg, at least 610 mg, at least 620 mg, at least 630 mg, at least 640 mg, at least 650 mg, 660 mg, at least 670 mg, at least 680 mg, at least 690 mg, at least 700 mg, at least 710 mg, at least 720 mg, at least 730 mg, at least 740 mg, at least 750 mg. 760 mg, at least 770 mg. at least 780 mg, at least 790 mg. at least 800 mg, at least 810 mg, at least 820 mg, at least 830 mg, at least 840 mg, at least 850 mg, 860 mg, at least 870 mg, at least 880 mg, at least 890 mg, at least 900 mg, at least 910 mg, at

[0125]

[0126] least 920 mg, at least 930 mg, at least 940 mg. at least 950 mg, 960 mg, at least 970 mg, at least 980 mg, at least 990 mg, or at least 1,000 mg.

[0127] In an embodiment, a dose of a therapeutic composition, including a DNase or DNase-CTP, may be administered to a patient at a dose of, e.g., at most 1 mg, at most 2 mg, at most 3 mg, at most 4 mg, at most 5 mg, at most 6 mg, at most 7 mg, at most 8 mg, at most 9 mg, at most 10 mg, at most 15 mg, at most 20 mg, at most 25 mg, at most 30 mg, at most 35 mg, at most 40 mg, at most 45 mg, at most 50 mg, at most 55 mg, at most 60 mg, at most 65 mg, at most 70 mg, at most 75 mg, at most 80 mg, at most 85 mg, at most 90 mg, at most 95 mg, at most 100 mg, at most 1 10 mg, at most 120 mg. at most 130 mg, at most 140 mg, at most 150 mg. at most 160 mg, at most 170 mg, at most 180 mg, at most 190 mg, at most 200 mg, at most 210 mg, at most 220 mg, at most 230 mg, at most 240 mg, at most 250 mg, 260 mg, at most 270 mg, at most 280 mg. at most 290 mg, at most 300 mg, at most 310 mg, at most 320 mg, at most 330 mg, at most 340 mg, at most 350 mg, 360 mg, at most 370 mg. at most 380 mg, at most 390 mg, at most 400 mg, at most 410 mg, at most 420 mg, at most 430 mg, at most 440 mg, at most 450 mg, 460 mg, at most 470 mg, at most 480 mg, at most 490 mg, at most 500 mg, at most 510 mg, at most 520 mg, at most 530 mg, at most 540 mg, at most 550 mg, 560 mg, at most 570 mg. at most 580 mg, at most 590 mg, at most 600 mg. at most 610 mg, at most 620 mg, at most 630 mg, at most 640 mg. at most 650 mg, 660 mg. at most 670 mg, at most 680 mg, at most 690 mg, at most 700 mg, at most 710 mg, at most 720 mg, at most 730 mg, at most 740 mg, at most 750 mg, 760 mg, at most 770 mg, at most 780 mg, at most 790 mg, at most 800 mg. at most 810 mg, at most 820 mg, at most 830 mg, at most 840 mg, at most 850 mg, 860 mg. at most 870 mg, at most 880 mg, at most 890 mg. at most 900 mg, at most 910 mg, at most 920 mg, at most 930 mg, at most 940 mg, at most 950 mg, 960 mg, at most 970 mg, at most 980 mg, at most 990 mg, or at most 1,000 mg.

[0128] In an embodiment, a dose of a therapeutic composition, including a DNase or DNase-CTP, may be administered to a patient in a range of, e.g., about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 30 mg, about 1 mg to about 40 mg, about 1 mg to about 50 mg, about 1 mg to about 60 mg, about 1 mg to about 70 mg, about 1 mg to about 80 mg, about 1 mg to about 90 mg, about 1 mg to about 100 mg, about 1 mg to about 1 10 mg, about 1 mg to about 120 mg, about 1 mg to about 130 mg, about 1 mg to about 140 mg, about 1 mg to about 150 mg, about 5 mg to about 10 mg, about 5 mg to about 20 mg, about 5 mg to about 30 mg, about 5 mg to about 40 mg, about 5 mg to about 50 mg, about 5 mg to about 60 mg,

[0129]

[0130] about 5 mg to about 70 mg, about 5 mg to about 80 mg, about 5 mg to about 90 mg, about 5 mg to about 100 mg. about 5 mg to about 1 10 mg, about 5 mg to about 120 mg, about 5 mg to about 130 mg, about 5 mg to about 140 mg, about 5 mg to about 150 mg, about 10 mg to about 20 mg, about 10 mg to about 30 mg, about 10 mg to about 40 mg, about 10 mg to about 50 mg, about 10 mg to about 60 mg, about 10 mg to about 70 mg, about 10 mg to about 80 mg, about 10 mg to about 90 mg, about 10 mg to about 100 mg, about 10 mg to about 1 10 mg, about 10 mg to about 120 mg. about 10 mg to about 130 mg. about 10 mg to about 140 mg, about 10 mg to about 150 mg, about 10 mg to about 175 mg, about 10 mg to about 200 mg, about 10 mg to about 225 mg, about 10 mg to about 250 mg, about 25 mg to about 50 mg, about 25 mg to about 75 mg, about 25 mg to about 100 mg, about 25 mg to about 125 mg, about 25 mg to about 150 mg. about 25 mg to about 175 mg. about 25 mg to about 200 mg, about 25 mg to about 225 mg, about 25 mg to about 250 mg, about 50 mg to about 75 mg, about 50 mg to about 100 mg, about 50 mg to about 125 mg, about 50 mg to about 150 mg, about 50 mg to about 175 mg, about 50 mg to about 200 mg, about 50 mg to about 225 mg, about 50 mg to about 250 mg. about 75 mg to about 100 mg, about 75 mg to about 125 mg, about 75 mg to about 150 mg, about 75 mg to about 175 mg, about 75 mg to about 200 mg, about 75 mg to about 225 mg, or about 75 mg to about 250 mg.

[0131] In an embodiment, an effective amount of a therapeutic compound, including a DNase and / or a DNase-CTP, disclosed herein may be in the range of, e.g., about 1 mg / kg / day to about 10 mg / kg / day, about 1 mg / kg / day to about 15 mg / kg / day, about 1 mg / kg / day to about 20 mg / kg / day. about 1 mg / kg / day to about 25 mg / kg / day, about 1 mg / kg / day to about 30 mg / kg / day. about 1 mg / kg / day to about 35 mg / kg / day, about 1 mg / kg / day to about 40 mg / kg / day, about 1 mg / kg / day to about 45 mg / kg / day, about 1 mg / kg / day to about 50 mg / kg / day, about 1 mg / kg / day to about 75 mg / kg / day, or about 1 mg / kg / day to about 100 mg / kg / day. In yet other aspects of this embodiment, an effective amount of a therapeutic compound disclosed herein may be in the range of, e.g.. about 5 mg / kg / day to about 10 mg / kg / day, about 5 mg / kg / day to about 15 mg / kg / day, about 5 mg / kg / day to about 20 mg / kg / day, about 5 mg / kg / day to about 25 mg / kg / day, about 5 mg / kg / day to about 30 mg / kg / day, about 5 mg / kg / day to about 35 mg / kg / day, about 5 mg / kg / day to about 40 mg / kg / day. about 5 mg / kg / day to about 45 mg / kg / day, about 5 mg / kg / day to about 50 mg / kg / day. about 5 mg / kg / day to about 75 mg / kg / day, or about 5 mg / kg / day to about 100 mg / kg / day. In another embodiment, a therapeutic range of times for administration of a dose of a therapeutic composition, including a DNase or DNase-CTP, is 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes. 27 minutes, 28 minutes, 29 minutes. 30 minutes, 35 minutes, 40 minutes, 45 minutes. 50 minutes, 55 minutes, 1 hour, 2 hours. 3 hours, 4 hours. 5 hours, 6 hours,7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or more hours. In a further embodiment, therapeutic range of times is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, at least 20 minutes, at least 21 minutes, at least 22 minutes, at least 23 minutes, at least 24 minutes, at least 25 minutes, at least 26 minutes, at least 27 minutes, at least 28 minutes, at least 29 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours or more hours.

[0132] In an embodiment, a therapeutic range of times for administration of a dose of a therapeutic composition, including a DNase or DNase-CTP, is no more than 1 minute, no more than 2 minutes, no more than 3 minutes, no more than 4 minutes, no more than 5 minutes, no more than 6 minutes, no more than 7 minutes, no more than 8 minutes, no more than 9 minutes, no more than 10 minutes, no more than 11 minutes, no more than 12 minutes, no more than 13 minutes, no more than 14 minutes, no more than 15 minutes, no more than 16 minutes, no more than 17 minutes, no more than 18 minutes, no more than 19 minutes, no more than 20 minutes, no more than 21 minutes, no more than 22 minutes, no more than 23 minutes, no more than 24 minutes, no more than 25 minutes, no more than 26 minutes, no more than 27 minutes, no more than 28 minutes, no more than 29 minutes, no more than 30 minutes, no more than 35 minutes, no more than 40 minutes, no more than 45 minutes, no more than 50 minutes, no more than 55 minutes, no more than 1 hour, no more than 2 hours, no more than 3 hours, no more than 4 hours, no more than 5 hours, no more than 6 hours, no more than 7 hours, no more than 8 hours, no more than 9 hours, no more than 10 hours, no more than 11 hours, no more than 12 hours or more hours.

[0133] In a further embodiment, a therapeutic range of times for administration of a dose of a therapeutic composition, including a DNase or DNase-CTP, is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours or more hours.

[0134] Therapeutic Effectiveness

[0135] A therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42 %. at least43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%. A therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease by no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%, no more than 14%, no more than 15%, no more than 16%, no more than 17%, no more than 18%, no more than 19%, no more than 20%, no more than 21%, no more than 22%, no more than 23%. no more than 24%, no more than 25%, no more than 26%, no more than 27%. no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%, no more than 33%, no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%, no more than 40%, no more than 41%, no more than 42 %, no more than43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, no more than 50%, no more than 51%, no more than 52%, no more than 53%, no more than 54%, no more than 55%, no more than 56%, no more than 57%, no more than 58%, no more than 59%, no more than 60%, no more than 61%, no more than 62%, no more than 63%, no more than 64%, no more than 65%. no more than 66%, no more than 67%, no more than 68%, no more than 69%, no more than 70%, no more than 71%, no more than 72%, no more than 73%, no more than 74%, no more than 75%, no more than 76%, no more than 77%, no more than 78%, no more than 79%, no more than 80%, no more than 81%, no more than 82%, no more than 83%, no more than 84%. no more than 85%. no more than 86%, no more than 87%, no more than 88%, no more than 89%, no more than 90%, no more than 91%, no more than 92%, no more than 93%, no more than 94%, no more than 95%, no more than 96%, no more than 97%, no more than 98%, no more than 99%, no more than 100%.

[0136] A therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%. about 10%, about 11%, about 12%, about 13%, about 14%. about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%. about 40%, about 41%, about 42 %. about 43%, about 44%, about 45%, about 46%. about 47%, about 48%, about 49%, about 50%. about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%. about 70%, about 71%, about 72%, about 73%, about 74%. about 75%, about 76%. about 77%, about 78%, about 79%, about 80%. about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%.

[0137] A therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease, wherein the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, a cardiovascular disease (e.g., atherosclerosis, stroke, angina, ischemia), kidney damage, delayed-type hypersensitivity reactions (e.g., graft- versus-host disease), reduction of fertility, age-specific sperm motility impairment and aging,. by at least 1%, at least 2%. at least 3%, at least 4%, at least 5%. at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%. at least 35%. at least 36%. at least 37%. at least 38%. at least 39%, at least 40%, at least 41%, at least 42 %, at least43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%. at least 63%. at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%.

[0138] A therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease, wherein the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, a cardiovascular disease (e.g., atherosclerosis, stroke, angina, ischemia), kidney damage, delayed-type hypersensitivity reactions (e.g., graft- versus-host disease), reduction of fertility, age-specific sperm motility impairment and aging, by no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%, no more than 14%, no more than 15%, no more than 16%, no more than 17%, no more than 18%. no more than 19%. no more than 20%, no more than 21%, no more than 22%, no more than 23%, no more than 24%, no more than 25%, no more than 26%, no more than 27%, no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%, no more than 33%. no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%, no more than 40%. no more than 41%. no more than 42 %, no more than43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, no more than 50%, no more than 51%, no more than 52%, no more than 53%, no more than 54%, no more than 55%, no more than 56%, no more than 57%, no more than 58%, no more than 59%, no more than 60%, no more than 61%, no more than 62%, no more than 63%, no more than 64%, no more than 65%, no more than 66%, no more than 67%, no more than 68%, no more than 69%, no more than 70%, no more than 71%, no more than 72%, no more than 73%, no more than 74%, no more than 75%. no more than 76%, no more than 77%, no more than 78%, no more than 79%, no more than 80%, no more than 81%, no more than 82%. no more than 83%. no more than 84%, no more than 85%, no more than 86%, no more than 87%, no more than 88%, no more than 89%, no more than 90%, no more than 91%, no more than 92%, no more than 93%, no more than 94%, no more than 95%, no more than 96%, no more than 97%, no more than 98%, no more than 99%, no more than 100%.

[0139] A therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease, wherein the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, a cardiovascular disease (e.g., atherosclerosis, stroke, angina, ischemia), kidney damage, delayed-type hypersensitivity reactions (e.g., graft- versus-host disease), reduction of fertility, age-specific sperm motility impairment and aging, by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%. about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%. about 23%. about 24%, about 25%, about 26%, about 27%. about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42 %, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%. about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%. about 61%, about 62%, about 63%, about 64%. about 65%. about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%. about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%. about 91%, about 92%, about 93%, about 94%. about 95%, about 96%, about 97%, about 98%, about 99%, about 100%.

[0140] Kit

[0141] In an embodiment, in instances in which each of the therapeutic compounds, including a DNase and / or a DNase-CT p, themselves are administered, without limitation, as individual or separate dosage forms (e.g., capsules tablets or liquid), the kit comprises, without limitation, each of a therapeutic compound making up the composition of the invention, along with instructions for use. In an additional embodiment, a therapeutic compound component, without limitation, may be packaged in any manner suitable for administration, so long as the packaging, when considered along with the instructions for administration, without limitation, clearly indicates the manner in which each of the therapeutic compound components is to be administered. In a further embodiment, each of a therapeutic compound or a combination of such therapeutic compounds may, without limitation, be combined into a single administrable dosage form such as a capsule, tablet, or other solid or liquid formulation. The therapeutic compound can be provided to an individual in a package. The package can be a container, for instance, without limitation, a bottle, a canister, a tube or other enclosed vessel. The package can also be a packet, such as a blister pack.

[0142] In an embodiment, the individual or separate dosage is in the form of a blister pack,. In an aspect of this embodiment, a blister pack is a term for several types of pre-formed plastic packaging used for small consumer goods, foods, and for pharmaceuticals. In a further embodiment, a blister pack is comprised of a cavity or pocket made from a formable web, usually a thermoformed plastic and typically includes a backing of paperboard or a lidding seal of aluminum foil or plastic. In a further embodiment, a blister that folds onto itself is a clamshell. In an aspect of this embodiment, a blister pack is commonly used as unit-dose packaging for pharmaceutical tablets, capsules or lozenges. In an embodiment, a blister pack can provide barrier protection for shelf life requirements, and a degree of tamper resistance and can be used for packing physician samples of therapeutic compound products or for Over The Counter (OTC) products, including for sale in a pharmacy.

[0143] In an embodiment, an individual is provided a treatment protocol wherein a pharmaceutical composition is administered on a periodic schedule, wherein the individual is informed by electronic notification that administration of the therapeutic compound is to occur at a set date or time or to administer the therapeutic compound so that the individual is reminded to take the therapeutic compound on a period schedule. In an aspect of this embodiment, the electronic notification is by email, text, instant messaging or by another electronic notification method. In an embodiment, an individual is informed to administer the therapeutic compound on a period schedule through receipt of a telephone call, postal mail, overnight express (including, without limitation, FedEx and UPS) or other method of notification.

[0144] EXAMPLES

[0145] The present invention is also described and demonstrated by way of the following examples.

[0146] Example 1. Cloning of AAV Vector: ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive) leader CTP-WPRE Xinact constructs

[0147] A cassette genes containing the C-Terminal peptide (CTP) of the beta subunit of hCG was fused to the coding sequence of human actin resistant DNase I at different sequences and locations. Three DNase CTP variants were constructed as illustrated in FIG.l. A Gibson assembly strategy7was used to generate a ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive) leader CTP-WPRE Xinact constructs. Gibson ds DNA fragments hDNasel (hyper) CTP1 (SEQ ID NO: 16), hDNasel (hyper) CTP2 (SEQ ID NO: 17) and hDNasel (hyper) CTP3(SEQ ID NO: 18) were generated. pAAV -ApoEHCR enhancer-hAAT promoter-hDNasel (wild type) -WPRE plasmid (SEQ ID NO: 15) was cut with BamHI and Nhel and 4698 bp fragment which is the plasmid backbone was gel extracted. Gibson assembly reaction (NEB Gibson Assembly Master mix E2611S) was set up and the mix of vector and different DNAse I-CTP fragments was incubated at 50°C for 15 min. Three assemblies, one for each CTP variant were made. The ligated constructs were transformed into SURE electrocompetent cells. pAAV-ApoEHCR-hAATp-hDnasel-CTPl-WPRE- Xinact vector, pAAV-ApoE HCR-hAATp-hDNaseI-CTP2-WPRE Xinact vector and pAAV-APOE HCR-hAATp-hDnaseI-CTP3-WPRE- Xinact vector plasmids were generated as illustrated in FIG.2A-2

[0148] Example 2. Manufacture of DNase I CTP AAV Vectors

[0149] The pAAV-ApoEHCR-hAATp-hDnasel-CTP 1 -WPRE-Xinact, p AAV-ApoEHCR-hAATp-hDNaseI-CTP2-WPRE Xinact and pAAV-APOE HCR-hAATp-hDnaseI-CTP3-WPRE-Xinact expression plasmids were manufactured by GenScript USA Inc. (Piscataway, NJ 08854, USA). The corresponding, AAV-hDNAse I CTP1, AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors were packaged into AAV serotypes LK03 or KPI by triple transfection of HEK-293 cells. HEK-293 cells were obtained from ATCC and cultured in Dulbecco’s medium Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (complete DMEM) and passaged using trypsin. Briefly, HEK-293 cells (American Type Culture Collection) were cultured in plastic culture dishes with complete medium (DMEM containing 10% FBS) and were passaged using try psin. Cells transfected with packaging constructs were collected 48 h post-transfection and subjected to four freeze / thaw cycles in a dry' ice / 100% ethanol bath and centrifuged at 3,000 g for 15 min. Supernatant was treated with benzonase at 50 U / mL at 37°C for 30 min and vector was precipitated using ammonium sulfate, purified using CsCl purification, dialyzed against 5% glycerol in phosphate- buffered saline (PBS), and stored at -80°C. The final viral titer was assigned by quantitative PCR (qPCR).

[0150] Example 3. Transfection of cell lines with DNase I CTP variants.

[0151] HuH-7 and HepG2 human ceils of liver origin were cultured in complete medium (DMEM containing 10% FBS) and passaged using trypsin. All lines were mycoplasma free. For transduction studies, cells were plated into 12-well plates in complete DMEM al 2 x 10’ cells per well and incubated ov emight at 37 °C in a humidified incubator with 5% CO2 The following morning, the AAV-hDNAse I CTP1. AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors stocks were diluted in 1 ml of complete DMEM and added to HuH-7 and HepG2 ceils at a multiplicity of infection (MOI) = 20,000. Same AAV vector containing unmodified hyperactive DNAse I transgene was used as the positive control. After a 48-h incubation, the

[0152]

[0153] cells supernatant were analysed for DNase activity as follows: DNA-agarose solution was prepared by adding 15 rnL of RED buffer [0.05M Tris-HCl (pH 7.2). 0.05M MgC12, 0.05 M CaCl2, and 0.15 M NaCl to 20 mL water and agarose was slowly dissolved into this solution with 2 mkl of RedSafe nucleic acid staining solution (iNtRON biotechnology) to make a 1% (w / v) agarose gel. Once the solution cooled to 60°C, 5 rnL of 2 mg / rnL calf thymus DNA (DI 501; Sigma- Aldrich. St. Louis, MO) was added and the solution poured into 15 cm round cell culture dish plates (Thermofisher). After the DNA-agarose solution was set, 1 mm diameter wells were cut into the gel using a glass pipette and suction apparatus. Samples were diluted appropriately in RED buffer and 2 mkl was added into each well. Double dilutions of DNasel standards (Sigma) were also prepared in RED buffer from 1 mkg / rnL to 15 ng / mL and loaded at 2 mkl per well. The gels were incubated overnight at 37°C, then overlayed with 0.05 M EDTA, and imaged under UV light on the Flour Chem 5500 (Alpha Innotech). For analyses, the diameter of the halo around each w ell, representative of the DNA digested, was measured. A standard curve generated using the DNasel standard was used to calculate DNasel concentrations.

[0154] Figure 3 shows the levels of DNase enzymatic activity in supernatants of HuH-7 and HepG2 cells at 48h after transfection. Data confirmed that all off AAV-hDNAse I CTP1. AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors induce expression and secretion of enzymatically active chimeric CTP-DNase enzy mes both in HuH-7 and HepG2 cells.

[0155] Expression of CTP-DNases was further confirmed by Western blot. Western blot was performed on supernatant protein concentrates to detect DNasel. After transfer, membranes were blocked with 5% milk / 1 • PBST for 1 h and then incubated with polyclonal rabbit antimouse DNasel (PA522017, dilution; Thermofisher) in 1% milk / 1 x PBST overnight at 4°C, followed by washing and incubation with goat anti-rabbit secondary antibody (sc-2004, 1:5,000; Santa Cruz Biotechnology') in 5% Milk / 1 ■ PBST for 1 h. at 4°C. Signals were detected using Supersignal West femto chemiluminescent substrate (Thermofisher) and visualized on the Fujifilm LAS4000 (Berthold). Blots were stripped using a solution containing glycine (25 mM), SDS (1.5% SDS w / v) dissolved in distilled water, washed and probed using antibodies to vinculin (V9131, dilution; Sigma- Aldrich), and imaged.

[0156] Figure 4 shows recognition of DNasel protein in supernatants of HepG2 cells at 48h after transfection with AAV-hDNAse I CTP1, AAV-hDNAse 1 CTP2 and AAV-hDNAse I CTP3

[0157]

[0158] vectors with anti-DNase I antibody. Molecular weight of recognized DNasel proteins corresponds to calculated molecular weights of CTP1 Dnasel, CTP2 Dnasel and CTP3 Dnasel chimeric proteins.

[0159] Example 4. Expression of DNase I CTP variants in vivo.

[0160] C57BL / 6 mice were obtained from the Animal Resource Centre (Caningvale, Western Australia). Mice were housed in standard boxes and received normal food and water ad libitum for the duration of the experiments. Animals were injected through the intravenous (i.v.) route at 8-12 weeks of age with lOOmL of the AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors after dilution in saline at 5x1012GC / kg vector dose. Same AAV vectors containing unmodified hyperactive DNAse I transgene and wild type DNAse I transgene were used as the controls. Blood was collected on days 7, 15 and 21 using the tail vein nicking into uncoated collection tubes (Becton Dickinson). Blood was left to clot for 30-40 min at room temperature, and then centrifuged at 3,000 g for 5 min. The serum was then taken and centrifuged again at 20,000 g for 5min to eliminate any residual red blood cells. Serum was aliquoted and stored at -80°C. DNase activity in serum was analyzed as specified in Example 3. Figure 5 shows mice serum DNasel activity levels at 3, 10, and 21 days post injection of AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors in comparison with same AAV vectors carrying hyperactive and wild type DNase I transgenes. Unexpectedly, despite relatively low enzymatic activity of CTP -DNases in cell culture supernatants upon in vitro transfection, mice injected with AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors show dramatic 7-15 fold (p<0.05) increase of DNase enzymatic activity in blood in comparison with mice injected with same AAV vectors containing unmodified hyperactive DNAse I transgene and wild type DNAse I transgene. Presence of CTP-DNases in serum of mice injected with AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 was further confirmed by Western blot. Western blot was performed on serum samples as specified in Example 3. Figure 6 shows recognition of DNasel protein in serum of mice injected with AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors with anti-DNase I antibody. Molecular weight of recognized DNasel proteins corresponds to calculated molecular weights of CTP1 Dnasel, CTP2 Dnasel and CTP3 Dnasel chimeric proteins..

[0161] Thus, AAV-hDNAse I CTP vectors of the invention has substantially improved pharmacologic properties: at least an order of magnitude less vector dose might be required to achieve sustainable and pharmacologically sufficient DNase enzymatic activity in patient blood in clinical settings. Such potential reduction of AAV vector clinical dose have major advantage in terms of clinical safety of AAV-DNase gene therapy.

[0162] Example 5. Performance of CTP1, CTP2 and CTP3 Dnase I in NET-rich supernatants

[0163] DNase I CTP variants were purified from supernatants of transduced cells by immuno-affinity chromatography. The anti-beta-hCG-CTP (Carboxy-Terminal-Peptide) monoclonal antibody (clone 7B11.5; catalog number 5-50028A-1. American Research Products) coupled to Sepharose 4B (1ml. Amersham Biosciences) was used for immuno purification of soluble Dnase CTP proteins. Cellular supernatants with CTP1, CTP2 and CTP3 Dnase I variants were collected at 60 h post transduction of HepG2 cells with corresponding AAV-hDNAse I CTP1, AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors (MOI's =20.000). Supernatants (50 ml) were run through the antibody column. The column was washed with 25 ml of Tris- saline buffer (lOrnM Tris, 150mM NaCl, pH 8.4) and the retained protein was eluted with TEA-saline buffer (50 mM triethylamine and 150 mM NaCl, pH 11.0) and neutralized with IM Tris buffer, pH 6.0. Protein solution was ultrafiltered and concentrated to ~1 ml in 0.001 M calcium chloride, 0.15 M sodium chloride using a Sartocon slice 200 cassette (Sartorius, Germany). Protein content was quantified using Pierce BCA Protein Assay Kit.

[0164] Neutrophil extracellular traps were prepared as described by L. Barrientos (Barrientos L. et al., (2013) An improved strategy to recover large fragments of functional human neutrophil extracellular traps. Front. Immunol. 4:166). Polymorphonuclear neutrophils were isolated from fresh buffy coats prepared from blood of healthy donors. First, leukocytes were separated from erythrocytes by sedimentation on a separating medium containing 5% Dextran T500® (Pharmacia, Uppsala, Sweden) in 0.9% saline. PMN were separated from mononuclear cells by Ficoll centrifugation (d = 1.077 g / L, Eurobio, Les Ulis, France). Contaminating erythrocytes were removed by hypotonic lysis, and PMN were resuspended in Hank’s buffered salt solution (HBSS) (Gibco Life Technologies, Saint Aubin, France) supplemented with 0.05% fetal calf serum (FCS) (PAA, Les Mureaux, France) and 10 nM Hepes (Gibco). Freshly isolated PMN were seeded in 12-well culture plates (1.5 x 106 cells / well) and stimulated with PMA (50 nM). After 3 h at 37°C in the presence of 5% CO2, each well was carefully washed tw ice w ith 1 mL PBS. The supernatant of each well was collected and centrifuged for 5 min at 300 x g at 4°C in order to remove whole cells and debris. DNA was quantified in the NET-rich supernatants using Quant-iT™ NETs are known to contain high levels of serine proteases such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3) being the reservoir of active proteases with substantial destructive proteolytic activities contributing to DNase protein proteolytic degradation. We had tested CTP-DNases enzymatic activity after extensive incubation in NET-rich supernatant. Wild type human recombinant Dnasel (Kevelt AS) and CTP-DNases were incubated for 6 h with NET-rich supernatant (0.2 pg / ml DNase and 0,15 pg / ml NET DNA) before adding genomic DNA (7.5 pg / ml of genomic DNA. D1234999-G01, AmsBio.

[0165] Subsequent DNA concentrations were measured via Quant-iT™ PicoGreen® dsDNA assay (Molecular Probes) after 30 min incubation. The experiment was repeated 3 times. The data is shown in FIG. 7. Following 30 min of incubation in vitro, DNA digestion was significantly greater with any of the 3 CTP-DNases than with human recombinant Dnasel which lost almost all enzy matic activity'. These results confirmed that in contrast to human recombinant DNasel CTP-DNases are more stable and can preserve their enzymatic activity in a NET-rich environment. The superior performance of CTP-DNase variants in NET reach environment constitute valuable pharmacologic advantage.

[0166] Example 6. Performance of CTP1, CTP2 and CTP3 Dnase I in DNA rich plasma.

[0167] We had tested CTP-DNases performance in DNA rich plasma. Plasma samples from patients with gastric cancer were sourced from Audubon Bioscience. Plasma samples were further spiked with genomic DNA (D1234999-G01, AmsBio) up to 30 pg / ml. Wild type human recombinant of Dnasel and CTP-DNases (0,2 pg / ml) were added to spiked plasma samples. Samples were incubated for 3 h. Genomic DNA digestion was tracked over a 3h time period using Quant-iT™ PicoGreen® dsDNA assay (Molecular Probes). All CTP-DNases provide substantially more potent cleavage of genomic DNA in comparison with wild type DNase I. CTP3 Dnasel provide the most potent cleavage of genomic DNA: at 3h of incubation genomic DNA content was 38% lower when compared to human recombinant DNasel (FIG. 8). Both CTP1 DNasel and CTP2 DNasel demonstrated greater efficacy than human recombinant DNasel in DNA rich plasma as well. It is known that catalytic activity of DNase I in blood rapidly decrease to undetectable levels when quantity of cell-free DNA in blood increases over 0,3-0, 5 ng / uL. For example quantity of cell-free DNA in blood in cancer patients can be as much as 1,0-10,0 ng / uL. Therefore, the superior performance of CTP-DNase variants in DNA rich plasma constitute valuable pharmacologic advantage. Example 7. Binding of CTP1, CTP2 and CTP3 Dnase I to NETs.

[0168] Wild type human recombinant Dnasel, CTP1 Dnasel, CTP2 Dnasel and CTP3 Dnasel were conjugated with cellulose beads as follows: cellulose beads (bead size of 100-250 micrometers, Sigma-Aldrich) were oxidized with sodium metaperiodate. Aqueous suspension of the beads (300mg, 0,5 mL) and NalO, (0.5 mmol) in 1 mL of water was shaken at room temperature for 4 h. The activated beads were collected and washed with 1 M sodium bicarbonate, 0.1 M hydrochloric acid and 20 mL of water. Wild type human recombinant Dnasel, CTP1 Dnasel, CTP2 Dnasel and CTP3 Dnasel were dialyzed and concentrated (1 mL; 0,05 mg / mL) in 0.1 M NaHCCh (pH 8). The solutions was incubated with oxidized beads (0,5 ml) at room temperature for 4 h with stirring. After the incubation, 1 M ethanolamine (1.5 mL) was added to the activated beads suspension (1,5 ml) to block the free CHO groups; the reaction continued for 1 h at room temperature. The resulting cellulose beads with immobilized wild type human recombinant Dnasel, CTP1 Dnasel. CTP2 Dnasel and CTP3 Dnasel were washed three times with TBS buffer to remove soluble protein contaminants. 500pL aliquots of the beads were spiked with NETs (0,05 pg / ml NET DNA) and incubated at 15°C for 10 min. After incubation samples were gently washed three times for 5 min each time in 10 mM PBS at 4°C. and the DNA w as stained with DNA specific DAPI stain ( Invitrogen) diluted in 10 mM PBS (1 pg / ml DAPI) for 5 min at room temperature. Specimens were visualized using a fluorescence microscope (Olympus BX60 Fluorescence Microscope) (FIG.9 ). Beads conjugated with CTP1 Dnasel, CTP2 Dnasel and CTP3 Dnasel proteins absorbs NETs as evidenced with intensive staining of the beads with DNA specific DAPI stain, while beads conjugated with wild type human recombinant Dnasel do not. These results confirmed that all CTP-DNases of the invention exhibits superior affinity to NETs compared to wild type DNase 1. After enzymatic desialylation of CTP3 Dnasel beads with sialidase (Sialidase AuA, E-S001, Ludger; 5 pL a(2-3,6,8,9) per aliquot, incubation for 1 h. at 37°C) CTP3 Dnasel beads lucking their capacity to absorb NETs. Active drug targeting strategy where ligands specifically deliver linked therapeutic biomolecules to site of their action is valuable approach increasing the efficacy and safety of therapeutic interventions. Thus CTP-DNases of the invention has improved pharmacologic properties through targeting of NETs.

[0169] Example 8 FIG.3 shows the levels of DNase enzymatic activity in supernatants of HuH-7 and HepG2 cells at48h after transfection with AAV-hDNAse I CTP1, AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors.

[0170] Example 9

[0171] FIG. 4 is Western blot photograph confirming presence of DNasel protein in supernatants of HepG2 cells at 48h after transfection with AAV-hDNAse I CTP1, AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors.

[0172] Example 10

[0173] FIG.5 shows mice serum DNasel activity levels at 3, 10, and 21 days post injection of AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors in comparison with same AAV vectors carrying hyperactive and wild type DNase I transgenes.

[0174] Example 11

[0175] FIG.6 is Western blot photograph confirming presence of DNasel protein in serum of mice injected with AAV-hDNAse I CTP2 and AAV-hDNAse I CTP3 vectors.

[0176] Example 12

[0177] FIG. 7 shows long-term effect of NET-rich supernatant on enzymatic activity of human recombinant DNasel or CTP Dnasel variants. Enzymes were incubated with NET-rich supernatant for 6 h before adding genomic DNA for 30 min.

[0178] Example 13

[0179] FIG. 8. Quantification of genomic DNA digestion in DNA rich plasma. Genomic DNA was digested by human recombinant DNasel or CTP Dnasel variants. Enzymes and DNA were incubated in DNA rich plasma over a 3h time period, as indicated.

[0180] Example 14

[0181] FIG. 9. Fluorescent imaging of beads conjugated with recombinant Dnasel and CTP Dnasel variants after incubation with NETs SEQUENCES

[0182] SEQ ID No. SEQUENCE SEQUENCE DESCRIPTION SEQ ID NO: Ammo acid sequence LKI AAFNIRTFGRTKMSN AT L V S YI VQIL SR YD1 AL VQE V 1 of hyperactive actin RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYK resistant human ERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREP Dnasel comprising FIVRFFSRFTEVREFAIVPLHAAPGDAV AEIDAL YDVYL three chorionic DVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLW gonadotropin carboxy TSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAV terminal peptides VPDS ALPFNFQAA YGL SDQLAQ Al SDH YP VE VMLK SS (CTP) attached to the SSK APPPSLPSPSRLPGPSDTPILPQS S S SK APPPSLP carboxy terminus SPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSD TPILPQ (CTP1)

[0183] SEQ ID NO: Amino acid sequence LKI AAFNIRTFGRTKMSNATL V SYI VQIL SR YDI AL VQE V 2 of hyperactive actin RDSI ILT AVG KLLDNLNQDAPDT YI I Y V VSEPLGRK S YK resistant human ERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREP Dnasel comprising FI VRFF SRFTE VREF Al VPLH AAPGD AV AEIDAL YD V YL two chorionic DVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLW gonadotropin carboxy TSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAV terminal peptides VPDS ALPFNFQAA YGL SDQLAQ Al SDH YPVE VMLK SS (CTP) attached to the SSK APPPSLPSPSRLPGPSDTPILPQS S S SK APPPSLP carboxy terminus SPSRLPGPSDTPILPQ

[0184] (CTP2)

[0185] SEQ ID NO: Amino acid sequence SSSSKAPPPSLPSPSRLPGPSDTPILPQLKIAAFNIRTF 3 of hyperactive actin GRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVG resistant human KLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRP Dnasel comprising one DQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFT chorionic EVREF Al VPLHAAPGDAV AEIDAL YD VYLDVQEKWGLE gonadotropin carboxy D VMLMGDFNAGC S YVRPSQW S SIRL WT SPTFQWLIP terminal peptide (CTP) DSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNF attached to the amino Q AA YGL SDQLAQ Al SDH YPVE VMLK S S S SK APPP SLP terminus and two SPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ chorionic

[0186] gonadotropin carboxy

[0187] terminal peptides

[0188] (CTP)attached to the

[0189] carboxy terminus

[0190] (CTP3)

[0191] SEQ ID NO: Amino acid sequence MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGRTKM 4 of hyperactive actin SNA'ILVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNL resistant human NQD APDT YH Y V V SEPLGRK S YKER YLF V YRPDQ V SA V Dnasel comprising DSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAI three chorionic VPLHAAPGDAV AEIDAL YD VYLDVQEKWGLEDVMLMG gonadotropin carboxy DFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA terminal peptides TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGL (CTP) attached to the SDQLAQAISDHYPVEVMLKSSSSKAPPPSLPSPSRLP carboxy terminus GPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ (CTP1) with leader SSSSKAPPPSLPSPSRLPGPSDTPILPQ

[0192] peptide

[0193] SEQ ID NO: Amino acid sequence MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGRTKM 5 of hyperactive actin SNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNL resistant human NQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAV Dnasel comprising DSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAI

[0194]

[0195] two chorionic VPLHAAPGDAV AEID AL YDVYLDVQEKWGLEDVMLMG gonadotropin carboxy DFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA terminal peptides TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGL (CTP) attached to the SDQLAQAISDHYPVEVMLKSSSSKAPPPSLPSPSRLP carboxy terminus GPSDTPTI, PO S S S SK APPPST. PSPSRI, PGPSDTPTI, PQ (CTP2) with leader

[0196] peptide

[0197] SEQ ID NO: Amino acid sequence MRGMKLLGALLALAALLQGAVSSSSSKAPPPSLPSPS 6 of hyperactive actin RI, PGPSDTPTI, PQI XI A AFNTRTFGRTKMSNATI. V S YT V resistant human QILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYH Dnasel comprising one YVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGC chorionic EPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGD gonadotropin carboxy AVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSY terminal peptide (CTP) VRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYD attached to the amino RIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAIS terminus and two DHYPVEVMLKSSSSKAPPPSLPSPSRLPGPSDTP1LPQ chorionic SSSSKAPPPSLPSPSRLPGPSDTPILPQ

[0198] gonadotropin carboxy

[0199] terminal peptides

[0200] (CTP) attached to the

[0201] carboxy terminus

[0202] (CTP3) with leader

[0203] peptide

[0204] SEQ ID NO: Nucleotide sequence AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACC 7 of ApoEHCR CTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAG enhancer-hAAT CAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGA promoter-hDNasel ACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCA (hyperactive)- leader AACATTGCAAGCAGCAAACAGCAAACACACAGCCCT CTP1-WPRE Xinact CCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTC expression cassete AGAGACCTCTCTGGGCCCATGCCACCTCCAACATC CACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCA GAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAG GGATCTTGCTACCAGTGGAACAGCCACTAAGGATTC TGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTC TCCCAGAGACTGTCTGACTCACGCCACCCCCTCCA CCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGG TACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTG TACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGG CGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCC CTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTT AATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTG'G yirCCACTGCTTAAATACGGACGAGGACAGGGCCCT GTCTCCTCAGCTTCAGGCACCACCACTGACCTGGG ACAGTGAATGCCGCCACCATGAGGGGCATGAAGCT GCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTG CAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAA CATCAGGACATTTGGGAGGACCAAGATGTCCAATGC CACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCG CTATGACATCGCCCTGGTCCAGGAGGTCAGAGACA GCCACCTGACTGCCGTGGGGAAGCTGCTGGACAAC CTCAATCAGGATGCACCAGACACCTATCACTACGTG GTCAGTGAGCCACTGGGACGGAAGAGCTATAAGGA GCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGT CTGCGGTGGACAGCTACTACTACGATGATGGCTGC GAGCCCTGCGGGAACGACACCTTCAACCGAGAGCC ATTCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGT CAGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCC CGGGGGACGCAGTAGCCGAGATCGACGCTCTCTAT GACGTCTACCTGGATGTCCAAGAGAAATGGGGCTT GGAGGACGTCATGTTGATGGGCGACTTCAATGCGG

[0205]

[0206] GCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCC ATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCT GATCCCCGACAGCGCTGACACCACAGCTACACCCA CGCACTGTGCCTATGACAGGATCGTGGTTGCAGGG ATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGC TCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAG TGACCAACTGGCCCAAGCCATCAGTGACCACTATCC AGTGGAGGTGATGCTGAAGTCC TCC AGT AGT AAG GCC CCA CCT CCC AGT TTG CCC AGT CCT TCC AGG TTG CCA GGG CCA AGT GAC ACC CCT ATT TTG CCA CAA TCA TCT AGC AGT AAA GCA CCA CCA CCT TCT CTT CCT TCT CCC AGC AGA TTG CCC GGA CCC TCT GAT ACT CCC A l A TTG CCC CAG TCC TCT TCT TCA AAG GCC CCT CCT CCC TCA CTT CCA TCT CCT AGC AGG CTT CCC GGA CCT TCA GAT ACA CCA ATC CTG CCT CAG TGAAGTGGCGGCCGCTCGAGCTAGCGGCCGCTCTA GAAGATAATCAACCTCTGGATTACAAAATTTGTGAAA GATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCAT GCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCT TGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTT GTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCA CTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGC ATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTC GCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATC GCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTC GGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCG GGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGT GTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTG CTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCC TTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTC CGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATC TCCCTTTGGGCCGCCTCCCCGCATCGGACTAG SEQ ID NO: Nucleotide sequence AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACC 8 of ApoEHCR CTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAG enlrancer-hAAT CAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGA promoter-hDNasel ACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCA (hyperactive)- leader AACATTGCAAGCAGCAAACAGCAAACACACAGCCCT CTP2-WPRE Xinact CCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTC expression cassete AGAGACCTCTCTGGGCCCATGCCACCTCCAACATC CACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCA GAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAG GGATCTTGCTACCAGTGGAACAGCCACTAAGGATTC TGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTC TCCCAGAGACTGTCTGACTCACGCCACCCCCTCCA CCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGG TACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTG TACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGG CGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCC CTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTT AATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGG ATCCACTGCTTAAATACGGACGAGGACAGGGCCCT GTCTCCTCAGCTTCAGGCACCACCACTGACCTGGG ACAGTGAATGCCGCCACCATGAGGGGCATGAAGCT GCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTG CAGGGGGCCGTGTCCCTGAAGATCGCAGCCTTCAA CATCAGGACATTTGGGAGGACCAAGATGTCCAATGC CACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCG CTATGACATCGCCCTGGTCCAGGAGGTCAGAGACA GCCACCTGACTGCCGTGGGGAAGCTGCTGGACAAC

[0207]

[0208] CTCAATCAGGATGCACCAGACACCTATCACTACGTG GTCAGTGAGCCACTGGGACGGAAGAGCTATAAGGA GCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGT CTGCGGTGGACAGCTACTACTACGATGATGGCTGC GAGCCCTGCGGGAACGACACCTTCAACCGAGAGCC ATTCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGT CAGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCC CGGGGGACGCAGTAGCCGAGATCGACGCTCTCTAT GACGTCTACCTGGATGTCCAAGAGAAATGGGGCTT GGAGGACGTCATGTTGATGGGCGACTTCAATGCGG GCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCC ATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCT GATCCCCGACAGCGCTGACACCACAGCTACACCCA CGCACTGTGCCTATGACAGGATCGTGGTTGCAGGG ATGCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGC TCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAG TGACCAACTGGCCCAAGCCATCAGTGACCACTATCC AGTGGAGGTGATGCTGAAGTCC AGC AGT AGT AAA GOT CCA CCC CCT TCC TTG CCA AGT CCC TCT CGG CTC CCA GGT CCA AGC GAT ACC CCC ATT CTG CCT CAA AGT AGT AGC AGC AAA GCA CCT CCT CCT AGT CTT CCA AGT CCA AGT CGA CTG CCA GGG CCA AGT GAC ACC CCC ATA TTG CCC CAG TGAAGTGGCGGCCGCTCGAGCTAGCGGCCGCTCTA GAAGATAATCAACCTCTGGATTACAAAATTTGTGAAA GATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCAT GCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCT TGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTT GTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCA CTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGC ATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTC GCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATC GCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTC GGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCG GGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGT GTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTG CTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCC TTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTC CGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATC TCCCTTTGGGCCGCCTCCCCGCATCGGACTAG SEQ ID NO: Nucleotide sequence AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCAC 9 of ApoEHCR CCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCC enhancer-hAAT

[0209] promoter-hDNasel AGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACAC (hyperactive)- leader TGAACAAACTTCAGCCTACTCATGTCCCTAAAATG CTP3-WPRE Xinact GGCAAACATTGCAAGCAGCAAACAGCAAACACAC expression cassete AGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCA GAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCC AACATCCACTCGACCCCTTGGAATTTCGGTGGAGA GGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTG TGAGAGGGATCTTGCTACCAGTGGAACAGCCACTA AGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAG TGGTACTCTCCCAGAGACTGTCTGACTCACGCCAC CCCCTCCACCTTGGACACAGGACGCTGTGGTTTCT GAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAG TGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGG GCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGT

[0210]

[0211] GGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGG

[0212]

Claims

GTGACCTTGGTTAATATTCACCAGCAGCCTCCCCC GTTGCCCCTCTGGATCCACTGCTTAAATACGGACG AGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACC ACCACTGACCTGGGACAGTGAATGCCGCCACCAT GAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCA CTGGCGGCCCTACTGCAGGGGGCCGTGTCC TCA AGT TCC TCC AAG GCA CCC CCA CCT AGT CTT CCTAGCCCCAGTCGCCTCCCCGGACCTTCAGACAC TCCTATTCTGCCACAACTGAAGATCGCAGCCTTCA ACATCAGGACATTTGGGAGGACCAAGATGTCCAAT GCCACCCTCGTCAGCTACATTGTGCAGATCCTGAG CCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAG ACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGA CAACCTCAATCAGGATGCACCAGACACCTATCACT ACGTGGTCAGTGAGCCACTGGGACGGAAGAGCTA TAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACC AGGTGTCTGCGGTGGACAGCTACTACTACGATGAT GGCTGCGAGCCCTGCGGGAACGACACCTTCAACC GAGAGCCATTCATTGTCAGGTTCTTCTCCCGGTTC ACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCA TGCGGCCCCGGGGGACGCAGTAGCCGAGATCGAC GCTCTCTATGACGTCTACCTGGATGTCCAAGAGAA ATGGGGCTTGGAGGACGTCATGTTGATGGGCGACT TCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAG TGGTCATCCATCCGCCTGTGGACAAGCCCCACCTT CCAGTGGCTGATCCCCGACAGCGCTGACACCACA GCTACACCCACGCACTGTGCCTATGACAGGATCGT GGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTC CCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCC TATGGCCTGAGTGACCAACTGGCCCAAGCCATCAG TGACCACTATCCAGTGGAGGTGATGCTGAAGTCAT CTAGTTCCAAGGCTCCTCCCCCTTCCTTGCCATCTC CATCCCGGTTGCCCGGTCCCTCAGACACCCCTATT CTTCCCCAAAGTTCCAGTTCTAAAGCTCCTCCCCC CAGTCTTCCTTCCCCTAGCAGGCTCCCCGGACCAT CCGACACTCCCATATTGCCTCAATGAAGTGGCGGC CGCTCGAGCTAGCGGCCGCTCTAGAAGATAATCAA CCTCTGGATTACAAAATTTGTGAAAGATTGACTGG TATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGA TACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATC CTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTT GCTGACGCAACCCCCACTGGTTGGGGCATTGCCAC CACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCC CCCTCCCTATTGCCACGGCGGAACTCATCGCCGCC TGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTT GGGCACTGACAATTCCGTGGTGTTGTCGGGGAAAT CATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCG CGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTC TTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTT TGGGCCGCCTCCCCGCATCGGACTAGSEQ ID NO: CTP amino acid Asp-Pro- Arg-Phe-Gln- Asp- Ser- Ser-Ser- Ser-Ly s- Ala-Pro- Pro-Pro- Ser- 10 sequence Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro- Ser-Asp-Thr-Pro-Ile-Leu- Pro-GlnSEQ ID NO: CTP amino acid Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser- Pro-Ser-Arg- 11 sequence Leu-Pro-Gly-Pro- Ser- Asp-Thr-Pro-Ile-Leu- Pro-GinSEQ ID NO: CTP amino acid Ser- Ser-Ser- Ser-Ly s- Ala-Pro-Pro-Pro-Ser-Leu-Pro- Ser- Pro- Ser- Arg 12 sequenceSEQ ID NO: Nucleotide sequence AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCC 14 of ApoEHCR CCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGT enhancer-hAAT GCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCA promoter-hDNasel TGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAA (hyperactive) -WERE ACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCA Xinact GAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCC ACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTG TCCTGGCGTGGTTTAGGTAGTGTGAGAGGGATCTTGCTACCA GTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGC CAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCA CCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCA GGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACA CTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTC AGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGAT AACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCC GTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAG GGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGA CAGTGAATGCCGCCACCATGAGGGGCATGAAGCTGCTGGGG GCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTC CCTGAAGATCGCAGCCTTCAACATCAGGACATTTGGGAGGAC CAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGAT CCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCAGAGA CAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCA ATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGC CACTGGGACGGAAGAGCTATAAGGAGCGCTACCTGTTCGTGT ACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTACTACTACG ATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGA GAGCCATTCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTC AGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCCCGGGGGAC GCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACCTGGAT GTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGG CGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTG GTCATCCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGGCT GATCCCCGACAGCGCTGACACCACAGCTACACCCACGCACT GTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAG GCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGG CTGCCTATGGCCTGAGTGACCAACTGGCCCAAGCCATCAGTG ACCACTATCCAGTGGAGGTGATGCTGAAGTGAAGTGGCGGC CGCTCGAGCTAGCGGCCGCTCTAGAAGATAATCAACCTCTGG ATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGT ATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTG TATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATC ATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAAT CCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATC TCCCTTTGGGCCGCCTCCCCGCATCGGACTAG SEQ ID NO: Nucleotide sequence AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCC 15 of ApoEHCR CCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTG enhancer-hAAT TGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTC promoter-hDNasel ATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCA wild type-WPRE AACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGC Xinact AGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACAT CCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGT TGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGATCTTGCTAC CAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGG GCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGC CACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGC CAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGT ACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGA CTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCC GATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCC CCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGA CAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTG GGACAGTGAATGCCGCCACCATGAGGGGCATGAAGCTGCTG GGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGT GTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGG AGACCAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGC AGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTC AGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAA CCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCA GTGAGCCACTGGGACGGAACAGCTATAAGGAGCGCTACCTG TTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTA CTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCT TCAACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCA CAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCATGCGGCCC CGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTC TACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCAT GTTGATGGGCGACTTCAATGCGGGCTGCAGCTATGTGAGAC CCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGCCCCACCT TCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACA CCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGAT GCTGCTCCGAGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTT TAACTTCCAGGCTGCCTATGGCCTGAGTGACCAACTGGCCCA AGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGT GAAGTGGCGGCCGCTCGAGCTAGCGGCCGCTCTAGAAGATA ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTA TTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGC TTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATG AGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGC ACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCC CTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG GTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCC TGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTAC GTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAT CGGACTAG(APOE HCR enhancer: bases 1-326: human alpha- 1 -antitrypsin promoter: bases 321-717: Kozak sequence: bases 718-726; human DNasel wild type with natural full correct leader sequence: bases 727- 1575; WPRE X protein inactivated: bases 1576-2212)SEQ ID NO: Nucleotide sequence TGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGGAT 16 of hDNasel (hyper) CCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTC CTP1 Gibson AGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGCCGC fragment CACCATGAGGGGCATGAAGCTGCTGGGGGCGCT GCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCTGA AGATCGCAGCCTTCAACATCAGGACATTTGGGAGGACCAAG ATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTG AGCCGCTATGACATCGCCCTGGTCCAGGAGGTCA GAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAAC CTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAG TGAGCCACTGGGACGGAAGAGCTATAAGGAGCGCTACCTGT TCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACA GCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGAC ACCTTCAACCGAGAGCCATTCATTGTCAGGTTCTTCTCCCGG TTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCATGCG GCCCCGGGGGACGCAGTAGCCGAGATCGACGCTC TCTATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTG GAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAG CTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGAC AAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCG CTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGG ATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCC GACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTG AGTGACCAACTGGCCCAAGCCATCAGTGACCACT ATCCAGTGGAGGTGATGCTGAAGTCC TCC AGP AGP AAG GCC CCA CCP CCC AGP PPG CCC AGP CCP PCC AGG PPG CCA GGG CCA AGP GAC ACC CCP APP PPG CCA CAA PCA f CP AGC AGP AAA GCA CCA CCA CCP PC f CPP CCP PC f CCC AGC AGA PPG CCC GGA CCC PCP GAP ACP CCC APA PPG CCC CAG PCC PCP PCP PCA AAG GCC CCP CCP CCC PCA CPP CCA PCP CCP AGC AGG CPP CCC GGA CCP PCA GAP ACA CCA APC CPG CCP CAGPGAAGPGGCGGCCGCPCGAGC7I4GCCGGCCGCPCPAGA AGAPAAPCAACCPCP GGAPPACAAAAPSEQ ID NO: Nucleotide sequence TGGTTAA TA TTCA CCA GCA GCCTCCCCCGTTGCCCCTCTGGGA T 17 of hDNasel (hyper) CCACPGCPPAAAPACGGACGAGGACAGGGCCCPGPCPCCPC CTP2 Gibson AGCPPCAGGCACCACCACPGACCPGGGACAGPGAAPGCCGC fragment CACCAPGAGGGGCAPGAAGCPGCPGGGGGCGCP GCPGGCACPGGCGGCCCPACPGCAGGGGGCCGPGPCCCPGA AGAPCGCAGCCPPCAACAPCAGGACAPPPGGGAGGACCAAG ATGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTG AGCCGCPAPGACAPCGCCCPGGPCCAGGAGGPCA GAGACAGCCACCPGACPGCCGPGGGGAAGCPGCPGGACAAC CPCAAPCAGGAPGCACCAGACACCPAPCACPACGPGGPCAG PGAGCCACPGGGACGGAAGAGCPAPAAGGAGCGCPACCPGP PCGPGPACAGGCCPGACCAGGPGPCPGCGGPGGACA GCPACPACPACGAPGAPGGCPGCGAGCCCPGCGGGAACGAC ACCPPCAACCGAGAGCCAPPCAPPGPCAGGPPCPPCPCCCGG PPCACAGAGGPCAGGGAGPPPGCCAPPGPPCCCCPGCAPGCG GCCCCGGGGGACGCAGPAGCCGAGAPCGACGCPCPCPAPGACGPCPACCPGGAPGPCCAAGAGAAAPGGGGCPPGGAGGACGTCATGTTGATGGGCGACTTCAATGCGGGCTGCAG CTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGAC AAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCG CTGACACCACAGCTACACCCACGCACTGTGCCTATGACAGG ATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCC GACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTG AGTGACCAACTGGCCCAAGCCATCAGTGACCACT ATCCAGTGGAGGTGATGCTGAAGTCC AGC AGT AGT AAA GCT CCA CCC CCT TCC TTG CCA AGT CCC TCT CGG CTC CCA GGT CCA AGC GAT ACC CCC ATT CTG CCT CAA AGT AGT AGC AGC AAA GCA CCT CCT CCT AGT CTT CCA AGT CCA AGT CGA CTG CCA GGG CCA AGT GAC ACC CCC ATA TTG CCC CAGTGAAGTGGCGGCCGCTCGAGC7NGC CGGCCGCTCTAGAAGATAATCAACCTCTGGATTACAAAAT SEQ ID NO: Nucleotide sequence 7GG77EL47G77CACCAGCAGCC7’CCCCCG77GCCCC7’C7GGG47’ 18 of hDNasel (hyper) CCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTC CTP3 Gibson AGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGCCGC fragment CACCATGAGGGGCATGAAGCTGCTGGGGGCGCT GCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCC TCA AGT TCC TCC AAG GCA CCC CCA CCT AGT CTT CCT AGC CCC AGT CGC CTC CCC GGA CCT TCA GAC ACT CCT ATT CTG CCA CAACTGAAGATCGCAGCCTTCAACATCAGGACATTTGGGAG GACCAAGATGTCCAATGCCACCCTCGTCAGCTACATTGTGCA GATCCTGAGCCGCTATGACATCGCCCTGGTCCAGGAGGTCA GAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAAC CTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAG TGAGCCACTGGGACGGAAGAGCTATAAGGAGCGCTACCTGT TCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTAC TACTACGATGATGGCTGCGAGCCCTGCGGGAA CGACACCTTCAACCGAGAGCCATTCATTGTCAGGTTCTTCTC CCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCTGCA TGCGGCCCCGGGGGACGCAGTA GCCGAGATCGACGCTCTCTATGACGTCTACCTGGATGTCCAA GAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTT CAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCAT CCATCCGCCTGTGGACAAGCCCCACCTTCCAGTGG CTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCA CTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCG AGGCGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCA GGCTGCCTATGGCCTGAGTGACCAACTGGCCCAA GCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGTC A TCT AGT TCC AAG GCT CCT CCC CCT TCC TTG CCA TCT CCA TCC CGG TTG CCC GGT CCC TCA GAC ACC CCT ATT CTT CCC CAA AGT TCC AGT TCT AAA GCT CCT CCC CCC AGT CTT CCT TCC CCT AGC AGG CTC CCC GGA CCA TCC GAC ACT CCC ATA TTG CCT CAATGAAGTGGCGGCCGCTCGAGC714GCCGGCCGCTCTAGAAGATAATCAACCTCTGGATTACAAAATClaims:

1. A recombinant adeno-associated virus (rAAV) expression vector comprising:

1. (i) a capsid protein and2. (ii) a nucleic acid segment,wherein the nucleic acid segment comprises a promoter operably linked to a nucleotide sequence encoding an enzyme with deoxyribonuclease (DNase) activity, wherein the enzyme comprises at least two chorionic gonadotropin carboxy terminal peptides (CTP).

2. The rAAV expression vector of claim 1, wherein the promoter is a liver-specific promoter.

3. The rAAV expression vector of claim 1, wherein the two chorionic gonadotropin carboxy terminal peptides (CTP) are attached to the amino terminus of the enzyme.

4. The rAAV expression vector of claim 1. wherein the two chorionic gonadotropin carboxy terminal peptides (CTP) are attached to the carboxy terminus of the enzyme.

5. The rAAV expression vector of claim 1. wherein the capsid protein mediates efficient and / or preferential targeting of the vector to the liver when administered in vivo.

6. The rAAV expression vector of claim 2. wherein the capsid protein mediates efficient and / or preferential targeting of the vector to the liver when administered in vivo.

7. The rAAV expression vector of claim 1. wherein the capsid protein is LK03.

8. The rAAV expression vector of claim 1, wherein the enzyme with DNase activity' is selected from the group consisting of DNase I, DNase X, DNase γ, DNaselLl. DNaselL2, DNase 1L3, DNase II. DNase Iia. DNase lip. Caspase-activated DNase (CAD), DNA fragmentation factor B, L DNase II, Endonuclease G (ENDOG), Granzyme B and mutants or derivatives thereof.

9. The rAAV expression vector of claim 1, wherein the enzyme with DNase activity is human DNase I or a mutant or derivative thereof.

10. The rAAV expression vector of claim 1, wherein the enzyme with DNase activity is comprised of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

11. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the rAAV expression vector of claim 1, wherein the disease or condition is accompanied by intravascular or exlravascular accumulation of extracellular DNA.

12. The method of claim 11, where the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, a cardiovascular disease (e.g.. atherosclerosis, stroke, angina, ischemia), kidney damage, delayed-type hypersensitivity reactions (e.g., graft- versus-host disease), reduction of fertility, age-specific sperm motility impairment and aging.

13. The method of claim 11, wherein the therapeutically effective dose of the rAAV is below 141.0 x1014GC / kg.

14. The method of claim 11, wherein the therapeutically effective dose of the rAAV is below 1.0 x1013GC / kg.

15. The method of claim 11, wherein the therapeutically effective dose of the rAAV is below 1.0 xlO12GC / kg.

16. The method of claim 11, wherein the therapeutically effective dose of the rAAV is below 1.0 x1011GC / kg.

17. The method of claim 11. wherein administering to the subject of the rAAV vector results in liver expression of DNase enzyme having at least one of the chorionic gonadotropin carboxy terminal peptides (CTP) glycosylated via glycosylation18. The method of claim 17, wherein the glycosylation comprises O-linked glycans.

19. The method of claim 17, wherein at least one molecule of sialic acid is added to a CTP-modified DNase amino acid sequence following glycosylation.

20. The method of claim 17, wherein about 1 to 120 sialic acid molecules are added in total per each CTP-modified DNase amino acid sequence.

21. The method of claim 1 and claim 17, wherein the CTP amino acid sequence is at least 70% homologous to SEQ ID NO: 10.

22. The method of claim 1 and claim 17, wherein the CTP amino acid sequence is at least 70% homologous to SEQ ID NO: 11.

23. The method of claim 17 wherein chimeric DNase molecule expressed in the liver shows increased binding to NETs and augmented enzymatic cleavage of NETs24. The method of claim 11, wherein, a CTP has a length of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18. about 19, about 20, about 25, about 30, about 35, about 40, about 45. about 50. about 55, about 60, about 65, about 70, or about 75 amino acids. In another embodiment, a CTP disclosed herein may have a length of, e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25. at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65. at least 70. or at least 75 amino acids. In yet another embodiment, a CTP disclosed herein may have a length of, e.g., at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30. at most 35. at most 40. at most 45. at most 50. at most 55. at most 60. at most 65. at most 70, or at most 75 amino acids.

25. The method of claim 11, wherein a CTP has alength of between about 7 to about 10 amino acids, about 7 to about 12 amino acids, about 7 to about 15 amino acids, about 7 to about 18 amino acids, about 7 to about 20 amino acids, about 7 to about 25 amino acids, about 7 to about 30 amino acids, about 7 to about 35 amino acids, about 7 to about 40 amino acids, about 7 to about 45 amino acids, about 7 to about 50 amino acids, about 7 to about 55 amino acids, about 7 to about 60 amino acids, about 7 to about 65 amino acids, about 7 toabout 70 amino acids, about 7 to about 75 amino acids, about 10 to about 12 amino acids, about 10 to about 15 amino acids, about 10 to about 18 amino acids, about 10 to about 20 amino acids, about 10 to about 25 amino acids, about 10 to about 30 amino acids, about 10 to about 35 amino acids, about 10 to about 40 amino acids, about 10 to about 45 amino acids, about 10 to about 50 amino acids, about 10 to about 55 amino acids, about 10 to about 60 amino acids, about 10 to about 65 amino acids, about 10 to about 70 amino acids, about 10 to about 75 amino acids, about 12 to about 15 amino acids, about 12 to about 18 amino acids, about 12 to about 20 amino acids, about 12 to about 25 amino acids, about 12 to about 30 amino acids, about 12 to about 35 amino acids, about 12 to about 40 amino acids, about 12 to about 45 amino acids, about 12 to about 50 amino acids, about 12 to about 55 amino acids, about 12 to about 60 amino acids, about 12 to about 65 amino acids, about 12 to about 70 amino acids, about 12 to about 75 amino acids, about 15 to about 18 amino acids, about 15 to about 20 amino acids, about 15 to about 25 amino acids, about 15 to about 30 amino acids, about 15 to about 35 amino acids, about 15 to about 40 amino acids, about 15 to about 45 amino acids, about 15 to about 50 amino acids, about 15 to about 55 amino acids, about 15 to about 60 amino acids, about 15 to about 65 amino acids, about 15 to about 70 amino acids, about 15 to about 75 amino acids, about 18 to about 20 amino acids, about 18 to about 25 amino acids, about 18 to about 30 amino acids, about 18 to about 35 amino acids, about 18 to about 40 amino acids, about 18 to about 45 amino acids, about 18 to about 50 amino acids, about 18 to about 55 amino acids, about 18 to about 60 amino acids, about 18 to about 65 amino acids, about 18 to about 70 amino acids, about 18 to about 75 amino acids, about 20 to about 25 amino acids, about 20 to about 30 amino acids, about 20 to about 35 amino acids, about 20 to about 40 amino acids, about 20 to about 45 amino acids, about 20 to about 50 amino acids, about 20 to about 55 amino acids, about 20 to about 60 amino acids, about 20 to about 65 amino acids, about 20 to about 70 amino acids, about 20 to about 75 amino acids, about 25 to about 30 amino acids, about 25 to about 35 amino acids, about 25 to about 40 amino acids, about 25 to about 45 amino acids, about 25 to about 50 amino acids, about 25 to about 55 amino acids, about 25 to about 60 amino acids, about 25 to about 65 amino acids, about 25 to about 70 amino acids, about 25 to about 75 amino acids, about 30 to about 35 amino acids, about 30 to about 40 amino acids, about 30 to about 45 amino acids, about 30 to about 50 amino acids, about 30 to about 55 amino acids, about 30 to about 60 amino acids, about 30 to about 65 amino acids, about 30 to about 70 amino acids, about 30 to about 75 amino acids, about 35 to about 40 amino acids, about 35 to about 45 amino acids, about 35 to about 50 amino acids, about 35 to about 55 amino acids, about 35 to about 60 amino acids.about 35 to about 65 amino acids, about 35 to about 70 amino acids, about 35 to about 75 amino acids, about 40 to about 45 amino acids, about 40 to about 50 amino acids, about 40 to about 55 amino acids, about 40 to about 60 amino acids, about 40 to about 65 amino acids, about 40 to about 70 amino acids, about 40 to about 75 amino acids, about 45 to about 50 amino acids, about 45 to about 55 amino acids, about 45 to about 60 amino acids, about 45 to about 65 amino acids, about 45 to about 70 amino acids, about 45 to about 75 amino acids, about 50 to about 55 amino acids, about 50 to about 60 amino acids, about 50 to about 65 amino acids, about 50 to about 70 amino acids, or about 50 to about 75 amino acids.

26. The method of claim 11, wherein the dose of DNase or DNase-CTP is about 1 gg to about 10 gg, about 1 gg to about 20 gg, about 1 gg to about 30 gg, about 1 gg to about 40 gg, about 1 gg to about 50 gg, about 1 gg to about 60 gg. about 1 gg to about 70 gg, about 1 gg to about 80 gg, about 1 gg to about 90 gg, about 1 gg to about 100 gg, about 1 gg to about 1 10 gg, about 1 gg to about 120 gg, about 1 gg to about 130 gg, about 1 gg to about 140 gg, about 1 gg to about 150 gg, about 5 gg to about 10 gg, about 5 gg to about 20 gg, about 5 gg to about 30 gg, about 5 gg to about 40 gg, about 5 gg to about 50 gg, about 5 gg to about 60 gg, about 5 gg to about 70 gg, about 5 gg to about 80 gg, about 5 gg to about 90 gg, about 5 gg to about 100 gg, about 5 gg to about 1 10 gg, about 5 gg to about 120 gg, about 5 gg to about 130 gg, about 5 gg to about 140 ig, about 5 gg to about 150 gg, about 10 gg to about 20 gg. about 10 gg to about 30 gg, about 10 gg to about 40 gg. about 10 to about 50 gg. about 10 to about 60 gg, about 10 gg to about 70 gg, about 10 gg to about 80 gg, about 10 gg to about 90 gg, about 10 gg to about 100 gg, about 10 gg to about 1 10 gg, about 10 gg to about 120 gg, about 10 gg to about 130 gg. about 10 gg to about 140 v, about 10 gg to about 150 gg, about 10 gg to about 175 gg, about 10 gg to about 200 gg, about 10 gg to about 225 gg, about 10 gg to about 250 gg, about 25 gg to about 50 [ig, about 25 [ig to about 75 gg, about 25 [ig to about 100 [ig, about 25 g to about 125 [ig, about 25 gg to about 150 gg, about 25 gg to about 175 gg, about 25 gg to about 200 gg, about 25 gg to about 225 gg, about 25 gg to about 250 gg, about 50 gg to about 75 gg, about 50 gg to about 100 gg, about 50 gg to about 125 gg. about 50 gg to about 150 gg. about 50 gg to about 175 gg, about 50 gg to about 200 gg, about 50 gg to about 225 gg, about 50 gg to about 250 gg, about 75 gg to about 100 gg, about 75 gg to about 125 gg, about 75 gg to about 150 gg, about 75 gg to about 175 gg, about 75 gg to about 200 gg, about 75 gg to about 225 gg, or about 75 gg to about 250 [ig. In an embodiment, a dose of DNase or DNase-CTP may be in the range of. e.g.. about 100 gg to about 125 gg, about 100 gg to about 150 gg, about 100 gg to about 175 gg, about 100 gg toabout 200 pg, about 100 ig to about 225 pig, about 100 pig to about 250 pig. about 100 pig to about 275 pig, about 100 pig to about 300 pig, about 100 pig to about 325 pig. about 100 pig to about 350 pig, about 100 pig to about 375 pig, about 100 pig to about 400 pig, about 100 pig to about 425 pig, about 100 pig to about 450 pig, about 100 pig to about 475 pig, about 100 pig to about 500 pig, about 100 pig to about 525 pig, about 100 pig to about 550 pig, about 100 pig to about 575 pig, about 100 pig to about 600 pig, about 125 pig to about 150 pig. about 125 pig to about 175 pig, about 125 pig to about 200 pig, about 125 pig to about 225 pig. about 125 pig to about 250 pig, about 125 pig to about 275 pig, about 125 pig to about 300 pig, about 125 pig to about 325 pig, about 125 pig to about 350 pig, about 125 pig to about 375 pig, about 125 pig to about 400 pig, about 125 pig to about 425 pig, about 125 pig to about 450 pig. about 125 pig to about 475 pig, about 125 pig to about 500 pig, about 125 pig to about 525 pig. about 125 pig to about 550 pig, about 125 pig to about 575 pig, about 125 pig to about 600 pig, about 150 pig to about 175 pig, about 150 pig to about 200 pig, about 150 pig to about 225 pig, about 150 pig to about 250 pig, about 150 pig to about 275 pig, about 150 pig to about 300 pig, about 150 pig to about 325 pig, about 150 pig to about 350 pig, about 150 pig to about 375 pig. about 150 pig to about 400 pig, about 150 pig to about 425 pig, about 150 pig to about 450 pig, about 150 pig to about 475 pig, about 150 pig to about 500 pig, about 150 pig to about 525 pig, about 150 pig to about 550 pig, about 150 pig to about 575 pig, about 150 pig to about 600 pig, about 200 pig to about 225 pig, about 200 pig to about 250 pig, about 200 pig to about 275 pig. about 200 pig to about 300 pig, about 200 pig to about 325 pig, about 200 pig to about 350 pig. about 200 pig to about 375 pig, about 200 pig to about 400 pig, about 200 pig to about 425 pig, about 200 pig to about 450 pig, about 200 pig to about 475 pig, about 200 pig to about 500 pig, about 200 pig to about 525 pig, about 200 pig to about 550 pig, about 200 pig to about 575 pig. about 200 pig to about 600 pig, about 200 pig to about 625 pig, about 200 pig to about 650 pig. about 200 pig to about 675 pig, about 200 pig to about 700 pig, about 200 pig to about 725 pig, about 200 pig to about 750 pig, about 200 pig to about 775 pig, about 200 pig to about 800 pig, about 200 pig to about 825 pig, about 200 pig to about 850 pig, about 200 pig to about 875 pig, about 200 pig to about 900 pig, about 200 pig to about 925 pig, about 200 pig to about 950 pig. about 200 pig to about 975 pig, or about 200 pig to about 1.000 pig.

27. The method of claim 11, wherein the dose of a therapeutic composition, including a DNase or DNase-CTP, is administered to a patient at a dose of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg. about 6 mg, about 7 mg. about 8 mg, about 9 mg. about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about45 mg, about 50 mg, about 55 mg, about 60 mg. about 65 mg, about 70 mg, about 75 mg, about 80 mg. about 85 mg. about 90 mg, about 95 mg, about 100 mg. about 1 10 mg. about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg. about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, 660 mg. about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, 960 mg, about 970 mg, about 980 mg, about 990 mg, or about 1,000 mg.

28. The method of claim 11, wherein the dose of a therapeutic composition, including a DNase or DNase-CTP, is administered to a patient at a dose of, at least 1 mg, at least 2 mg, at least 3 mg. at least 4 mg. at least 5 mg, at least 6 mg, at least 7 mg, at least 8 mg. at least 9 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 1 10 mg, at least 120 mg. at least 130 mg, at least 140 mg. at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, at least 200 mg, at least 210 mg, at least 220 mg, at least 230 mg, at least 240 mg, at least 250 mg, 260 mg, at least 270 mg, at least 280 mg, at least 290 mg, at least 300 mg, at least 310 mg, at least 320 mg, at least 330 mg, at least 340 mg, at least 350 mg, 360 mg, at least 370 mg, at least 380 mg, at least 390 mg. at least 400 mg, at least 410 mg. at least 420 mg, at least 430 mg. at least 440 mg, at least 450 mg, 460 mg, at least 470 mg, at least 480 mg, at least 490 mg, at least 500 mg, at least 510 mg, at least 520 mg, at least 530 mg, at least 540 mg, at least 550 mg, 560 mg, at least 570 mg, at least 580 mg, at least 590 mg, at least 600 mg, at least 610 mg, at least 620 mg, at least 630 mg, at least 640 mg, at least 650 mg, 660 mg, at least 670 mg, at least 680 mg, at least 690 mg, at least 700 mg, at least 710 mg, at least 720 mg, at least730 mg, at least 740 mg, at least 750 mg, 760 mg, at least 770 mg, at least 780 mg, at least 790 mg, at least 800 mg. at least 810 mg, at least 820 mg. at least 830 mg, at least 840 mg. at least 850 mg, 860 mg, at least 870 mg, at least 880 mg, at least 890 mg, at least 900 mg, at least 910 mg, at least 920 mg, at least 930 mg, at least 940 mg, at least 950 mg, 960 mg, at least 970 mg, at least 980 mg, at least 990 mg, or at least 1,000 mg.In an embodiment, a dose of a therapeutic composition, including a DNase or DNase-CTP. may be administered to a patient at a dose of, e.g., at most 1 mg, at most 2 mg. at most 3 mg, at most 4 mg, at most 5 mg, at most 6 mg, at most 7 mg, at most 8 mg, at most 9 mg, at most 10 mg, at most 15 mg, at most 20 mg, at most 25 mg, at most 30 mg, at most 35 mg, at most 40 mg, at most 45 mg, at most 50 mg, at most 55 mg, at most 60 mg, at most 65 mg, at most 70 mg, at most 75 mg, at most 80 mg, at most 85 mg, at most 90 mg, at most 95 mg, at most 100 mg, at most 1 10 mg, at most 120 mg, at most 130 mg, at most 140 mg, at most 150 mg, at most 160 mg, at most 170 mg, at most 180 mg, at most 190 mg, at most 200 mg, at most 210 mg, at most 220 mg, at most 230 mg, at most 240 mg, at most 250 mg, 260 mg, at most 270 mg, at most 280 mg. at most 290 mg, at most 300 mg, at most 310 mg. at most 320 mg, at most 330 mg, at most 340 mg, at most 350 mg, 360 mg, at most 370 mg, at most 380 mg, at most 390 mg, at most 400 mg, at most 410 mg, at most 420 mg, at most 430 mg, at most 440 mg, at most 450 mg, 460 mg, at most 470 mg, at most 480 mg, at most 490 mg, at most 500 mg, at most 510 mg. at most 520 mg, at most 530 mg, at most 540 mg, at most 550 mg, 560 mg, at most 570 mg. at most 580 mg, at most 590 mg, at most 600 mg. at most 610 mg, at most 620 mg, at most 630 mg, at most 640 mg, at most 650 mg, 660 mg, at most 670 mg, at most 680 mg, at most 690 mg, at most 700 mg, at most 710 mg, at most 720 mg, at most 730 mg, at most 740 mg. at most 750 mg, 760 mg, at most 770 mg, at most 780 mg, at most 790 mg, at most 800 mg. at most 810 mg, at most 820 mg, at most 830 mg. at most 840 mg, at most 850 mg, 860 mg, at most 870 mg, at most 880 mg, at most 890 mg, at most 900 mg, at most 910 mg, at most 920 mg, at most 930 mg, at most 940 mg, at most 950 mg, 960 mg, at most 970 mg, at most 980 mg, at most 990 mg, or at most 1,000 mg.

29. The method of claim 11. wherein the dose of a therapeutic composition, including a DNase or DNase-CTP, is administered to a patient in a range of, about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 30 mg, about 1 mg to about 40 mg, about 1 mg to about 50 mg, about 1 mg to about 60 mg, about 1 mg to about 70 mg, about 1 mg to about 80 mg. about 1 mg to about 90 mg, about 1 mg to about 100 mg, about 1 mg to about 1 10 mg, about 1 mg to about 120 mg, about 1 mg to about 130 mg, about 1 mg to about 140mg, about 1 mg to about 150 mg, about 5 mg to about 10 mg, about 5 mg to about 20 mg, about 5 mg to about 30 mg, about 5 mg to about 40 mg. about 5 mg to about 50 mg, about 5 mg to about 60 mg, about 5 mg to about 70 mg, about 5 mg to about 80 mg, about 5 mg to about 90 mg, about 5 mg to about 100 mg, about 5 mg to about 1 10 mg, about 5 mg to about 120 mg, about 5 mg to about 130 mg, about 5 mg to about 140 mg, about 5 mg to about 150 mg, about 10 mg to about 20 mg, about 10 mg to about 30 mg, about 10 mg to about 40 mg, about 10 mg to about 50 mg. about 10 mg to about 60 mg, about 10 mg to about 70 mg, about 10 mg to about 80 mg, about 10 mg to about 90 mg, about 10 mg to about 100 mg, about 10 mg to about 1 10 mg, about 10 mg to about 120 mg, about 10 mg to about 130 mg, about 10 mg to about 140 mg, about 10 mg to about 150 mg, about 10 mg to about 175 mg, about 10 mg to about 200 mg. about 10 mg to about 225 mg, about 10 mg to about 250 mg, about 25 mg to about 50 mg, about 25 mg to about 75 mg, about 25 mg to about 100 mg, about 25 mg to about 125 mg, about 25 mg to about 150 mg, about 25 mg to about 175 mg, about 25 mg to about 200 mg, about 25 mg to about 225 mg, about 25 mg to about 250 mg, about 50 mg to about 75 mg, about 50 mg to about 100 mg, about 50 mg to about 125 mg. about 50 mg to about 150 mg, about 50 mg to about 175 mg, about 50 mg to about 200 mg, about 50 mg to about 225 mg, about 50 mg to about 250 mg, about 75 mg to about 100 mg, about 75 mg to about 125 mg, about 75 mg to about 150 mg, about 75 mg to about 175 mg, about 75 mg to about 200 mg, about 75 mg to about 225 mg, or about 75 mg to about 250 mg.

30. The method of claim 11, wherein an effective amount of a therapeutic compound, including a DNase and / or a DNase-CTP, disclosed herein is in the range of, e.g., about 1 mg / kg / day to about 10 mg / kg / day, about 1 mg / kg / day to about 15 mg / kg / day, about 1 mg / kg / day to about 20 mg / kg / day. about 1 mg / kg / day to about 25 mg / kg / day, about 1 mg / kg / day to about 30 mg / kg / day, about 1 mg / kg / day to about 35 mg / kg / day, about 1 mg / kg / day to about 40 mg / kg / day, about 1 mg / kg / day to about 45 mg / kg / day, about 1 mg / kg / day to about 50 mg / kg / day, about 1 mg / kg / day to about 75 mg / kg / day, or about 1 mg / kg / day to about 100 mg / kg / day. In yet other aspects of this embodiment, an effective amount of a therapeutic compound disclosed herein may be in the range of. e.g., about 5 mg / kg / day to about 10 mg / kg / day, about 5 mg / kg / day to about 15 mg / kg / day, about 5 mg / kg / day to about 20 mg / kg / day, about 5 mg / kg / day to about 25 mg / kg / day, about 5 mg / kg / day to about 30 mg / kg / day, about 5 mg / kg / day to about 35 mg / kg / day, about 5 mg / kg / day to about 40 mg / kg / day. about 5 mg / kg / day to about 45 mg / kg / day, about 5mg / kg / day to about 50 mg / kg / day, about 5 mg / kg / day to about 7 mg / kg / day, or about 5 mg / kg / day to about 100 mg / kg / day.

31. The method of claim 11, wherein a therapeutic range of times for administration of a dose of a therapeutic composition, including a DNase or DNase-CTP, is 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes. 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes. 13 minutes, 14 minutes, 15 minutes. 16 minutes, 17 minutes. 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours. 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours. 9 hours, 10 hours, 11 hours, 12 hours or more hours. In a further embodiment, therapeutic range of times is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, at least 20 minutes, at least 21 minutes, at least 22 minutes, at least 23 minutes, at least 24 minutes, at least 25 minutes, at least 26 minutes, at least 27 minutes, at least 28 minutes, at least 29 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours or more hours.

32. The method of claim 11, wherein a therapeutic range of times for administration of a dose of a therapeutic composition, including a DNase or DNase-CTP, is no more than 1 minute, no more than 2 minutes, no more than 3 minutes, no more than 4 minutes, no more than 5 minutes, no more than 6 minutes, no more than 7 minutes, no more than 8 minutes, no more than 9 minutes, no more than 10 minutes, no more than 11 minutes, no more than 12 minutes, no more than 13 minutes, no more than 14 minutes, no more than 15 minutes, no more than 1 minutes, no more than 17 minutes, no more than 18 minutes, no more than 19 minutes, no more than 20 minutes, no more than 21 minutes, no more than 22 minutes, no more than 23 minutes, no more than 24 minutes, no more than 25 minutes, no more than 26 minutes, no more than 27 minutes, no more than 28 minutes, no more than 29 minutes, no more than 30 minutes, no more than 35 minutes, no more than 40 minutes, no more than 45 minutes, nomore than 50 minutes, no more than 55 minutes, no more than 1 hour, no more than 2 hours, no more than 3 hours, no more than 4 hours, no more than 5 hours, no more than 6 hours, no more than 7 hours, no more than 8 hours, no more than 9 hours, no more than 10 hours, no more than 11 hours, no more than 12 hours or more hours.

33. The method of claim 11, wherein a therapeutic range of times for administration of a dose of a therapeutic composition, including a DNase or DNase-CTP, is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours or more hours.

34. The method of claim 11, wherein a therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%. at least 6%, at least 7%, at least 8%, at least 9%. at least 10%. at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42 %, at least43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%.

35. The method of claim 11, wherein a therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease by no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%, no more than 14%, no more than 15%, no more than 16%, no more than 17%, no more than 18%, no more than 19%, no more than 20%, no more than 21%. no more than 22%, no more than 23%, no more than 24%, no more than 25%. no more than 26%, no more than 27%, no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%, no more than 33%, no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%, no more than 40%. no more than 41%. no more than 42 %, no more than43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, no more than 50%, no more than 51%, no more than 52%, no more than 53%, no more than 54%, no more than 55%, no more than 56%, no more than 57%, no more than 58%, no more than 59%, no more than 60%, no more than 61%, no more than 62%, no more than 63%. no more than 64%, no more than 65%, no more than 66%, no more than 67%, no more than 68%, no more than 69%, no more than 70%, no more than 71%, no more than 72%, no more than 73%, no more than 74%, no more than 75%, no more than 76%, no more than 77%, no more than 78%, no more than 79%, no more than 80%, no more than 81%, no more than 82%. no more than 83%. no more than 84%, no more than 85%, no more than 86%, no more than 87%, no more than 88%, no more than 89%, no more than 90%, no more than 91%, no more than 92%, no more than 93%, no more than 94%, no more than 95%, no more than 96%, no more than 97%, no more than 98%, no more than 99%, no more than 100%.

36. The method of claim 11, wherein a therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%. about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%. about 20%. about 21%, about 22%, about 23%, about 24%. about 25%. about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42 %, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%. about 50%. about 51%, about 52%, about 53%, about 54%. about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about64%, about 65%. about 66%, about 67%, about 68%, about 69%, about 70%. about 71%, about 72%. about 73%, about 74%, about 75%, about 76%. about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%.

37. The method of claim 11. wherein a therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease, wherein the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, a cardiovascular disease (e.g., atherosclerosis, stroke, angina, ischemia), kidney damage, delay ed-type hypersensitivity reactions (e.g., graft- versus-host disease), reduction of fertility, age-specific sperm motility impairment and aging,.by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%. at least 40%. at least 41%. at least 42 %. at least43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%.

38. The method of claim 11, wherein a therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease, wherein the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, acardiovascular disease (e.g., atherosclerosis, stroke, angina, ischemia), kidney damage. delay ed-type hypersensitivity reactions (e.g., graft- versus-host disease), reduction of fertility, age-specific sperm motility impairment and aging, by no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 11%, no more than 12%, no more than 13%. no more than 14%, no more than 15%, no more than 16%, no more than 17%. no more than 18%. no more than 19%, no more than 20%, no more than 21%, no more than 22%, no more than 23%, no more than 24%, no more than 25%, no more than 26%, no more than 27%, no more than 28%, no more than 29%, no more than 30%, no more than 31%, no more than 32%. no more than 33%, no more than 34%, no more than 35%, no more than 36%, no more than 37%, no more than 38%, no more than 39%. no more than 40%. no more than 41%, no more than 42 %, no more than43%, no more than 44%, no more than 45%, no more than 46%, no more than 47%, no more than 48%, no more than 49%, no more than 50%, no more than 51%, no more than 52%, no more than 53%, no more than 54%, no more than 55%, no more than 56%, no more than 57%, no more than 58%, no more than 59%, no more than 60%, no more than 61%, no more than 62%, no more than 63%, no more than 64%, no more than 65%, no more than 66%, no more than 67%, no more than 68%, no more than 69%, no more than 70%, no more than 71%, no more than 72%, no more than 73%, no more than 74%. no more than 75%, no more than 76%, no more than 77%, no more than 78%, no more than 79%, no more than 80%, no more than 81%. no more than 82%. no more than 83%, no more than 84%, no more than 85%, no more than 86%, no more than 87%, no more than 88%, no more than 89%, no more than 90%, no more than 91%, no more than 92%, no more than 93%, no more than 94%, no more than 95%, no more than 96%, no more than 97%, no more than 98%, no more than 99%, no more than 100%.

39. The method of claim 11, wherein a therapeutic of the present invention, including a DNase or DNase-CTP reduces the severity of a disease, wherein the disease or condition is one or more of cancer (e.g., carcinomas, sarcomas, lymphomas, melanoma), an autoimmune disease, development of somatic mosaicism, side effects associated with chemotherapy, immunotherapy, or radiation therapy, a neurodegenerative disease, an infection, diabetes, a cardiovascular disease (e.g., atherosclerosis, stroke, angina, ischemia), kidney damage, delay ed-type hypersensitivity' reactions (e.g., graft- versus-host disease), reduction of fertility', age-specific sperm motility impairment and aging, by about 1%, about 2%. about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%. about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%. about 21%. about 22%, about 23%, about 24%, about 25%. about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42 %, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%. about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%. about 59%, about 60%, about 61%, about 62%. about 63%. about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%. about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%. about 89%, about 90%, about 91%, about 92%. about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%.

40. The composition of claim 1, wherein the therapeutic compounds, including a DNase and / or a DNase-CTP, are administered, as individual or separate dosage forms.

41. The method of claim 40, wherein the dosage form is a capsule, a tablet or a liquid.41 A kit, wherein the kit comprises a therapeutic compound, along with instructions for use.

42. The kit of claim 41, wherein the kit is with the instructions for administration.

43. The method of claim 41, wherein a therapeutic compound or a combination of such therapeutic compounds are combined into a single administrable dosage form such as a capsule, tablet, or other solid or liquid formulation.

44. The kit of claim 41, wherein the kit is provided to an individual in a package.

45. The kit of claim 44, wherein the package is a container.

46. The kit of claim 45, wherein the container is a bottle, a canister, a blister pack, a tube or other enclosed vessel.

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