DNASE1-LIKE3 engineered for improved expression and therapeutic use

Abstract

The present disclosure provides methods for designing proteins (eg, DNase enzymes) for improved productivity, structural uniformity, and / or reduced immunogenicity.

Description

[Technical Field]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 425,106, filed November 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure provides, in part, DNase enzymes that have been engineered for increased productivity, structural uniformity, and / or reduced immunogenicity for therapeutic uses.

[0003] Description of electronically submitted XML files

[0001] This application contains a Sequence Listing. The Sequence Listing has been submitted electronically via EFS-Web as an XML file titled "NTR-015PC_119604-5015_sequence_listing.xml". The Sequence Listing is 85,383 bytes in size and was created on November 13, 2023. The Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Enzyme replacement therapy is a promising approach for treating various diseases and disorders. For example, inflammatory diseases such as systemic lupus erythematosus (SLE) could potentially be treated with DNase enzymes (Laukova et al., Deoxyribonucleases and Their Applications in Biomedicine, Biomolecules 2020;10(7):1036). Enzymes are produced in microbial hosts such as Pichia pastoris or in mammalian cells. However, in addition to other hurdles, not all proteins of interest are produced or secreted at sufficient titers in expression systems such as Pichia pastoris. For example, some studies have revealed unwanted secondary modifications, structural heterogeneity, and inefficient processing of N-terminal signal sequences. For example, Arbeitman et al., Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells.Sci Rep 2020;10:21779(2020);Reverter et al., Overexpression of Human Procarboxypeptidase A2 in Pichia pastoris and Detailed Characterization of Its Activation Pathway,Protein Chemistry and Structure|1998;273(6):3535-3541;Katla et al., Novel glycosylated human interferon alpha 2b expressed in glycoengineered Pichia pastoris and its biological activity:N-linked glycoengineering approach, Enzyme and Microbial Technology 2019;128: 49-58.Therefore, there is a need for the development of expression systems that allow for the production of recombinant therapeutic proteins with improved production titers, structural homogeneity, and / or reduced immunogenicity. Summary of the Invention

[0005] In various embodiments, the present disclosure provides recombinant proteins produced by secretion from expression systems in which the N-terminal signal sequence has been fully processed. In various embodiments, the present disclosure provides methods for making such proteins, including DNase enzymes, and using them therapeutically.

[0006] In various aspects and embodiments, the present disclosure is based, in part, on the discovery that adding a linker of four or more amino acids (e.g., at least five amino acids) between the secretion signal and the protein of interest can improve processing and expression titers from microbial expression systems and control post-translational modification profiles. The present disclosure is also based, in part, on the discovery that C-terminal modifications can improve the product homogeneity profile of proteins produced in expression systems.

[0007] In some aspects, the disclosure provides variants of DNASE1-LIKE3 (D1L3 variants) comprising an N-terminal extension. In some embodiments, the N-terminal extension comprises at least four amino acids. In some embodiments, the N-terminal extension is not subject to post-translational modification by the host cell and / or is non-immunogenic upon administration to a human or animal subject. In some embodiments, the D1L3 variant produced by the host cell comprises a D1L3 enzyme lacking a signal peptide and comprising an amino acid sequence having at least 80% sequence identity to amino acids 21-282 of SEQ ID NO:4 (isoform 1) or amino acids 21-252 of SEQ ID NO:5 (isoform 2).

[0008] In some embodiments, the D1L3 variant is produced in a host cell (such as, but not limited to, Pichia pastoris) by cleavage of the N-terminal signal peptide. Any signal peptide that allows for secretion of D1L3 in the desired expression system can be used. In some embodiments, the signal peptide is the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae (SEQ ID NO: 38).

[0009] In various embodiments, the N-terminal extension has a length ranging from 4 to about 18 amino acids. In various embodiments, the first N-terminal amino acid is not a Met residue or a Gly residue (which may be subject to post-translational modification as disclosed herein). In some embodiments, the last amino acid residue of the N-terminal extension is not a serine residue, which may increase the immunogenicity risk of D1L3 as disclosed herein. In some embodiments, the last amino acid residue of the N-terminal extension is not a polar or charged amino acid residue, e.g., an amino acid residue selected from Ser, Thr, Gln, Asn, Glu, Asp, Arg, His, and Lys. In some embodiments, the last amino acid residue of the N-terminal extension is a Gly residue. In some embodiments, the first amino acid residue of the N-terminal extension is not a Gly residue and the last amino acid residue of the N-terminal extension is a Gly residue. In some embodiments, the N-terminal extension is predominantly Gly residues (i.e., more than 50% Gly residues). In some embodiments, the linker comprises at least one cysteine ​​residue. For example, placing a cysteine ​​at the N-terminus allows for site-specific chemical attachment, such as polyethylene glycol or disulfide dimerization. In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of SGGGG (SEQ ID NO: 60). In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of CGGGG (SEQ ID NO: 74). In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of SGGSGGSGG (SEQ ID NO: 61). In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of SGGSGGSGGSGGSGGSGG (SEQ ID NO: 62).

[0010] In some embodiments, the N-terminal extension improves the expression titer of the D1L3 variant relative to the same D1L3 sequence lacking the N-terminal extension for a selected signal sequence and expression host, hi some embodiments, the signal peptide is completely removed from the D1L3 variant upon secretion from the host.

[0011] In some embodiments, the D1L3 variant further comprises a C-terminal extension that reduces heterogeneity. In some embodiments, the C-terminal extension is at least 2, or at least 3, or at least 4, or at least 5 amino acids in length. In some embodiments, the extension is a hydrophilic sequence of 2, 3, or 4 amino acids. In some embodiments, the C-terminal amino acid is lysine (Lys) or arginine (Arg). In some embodiments, the C-terminal extension comprises or consists of the amino acid sequence SSR, which in some embodiments can be used in D1L3 variants having a deletion of all or part of the C-terminal basic domain (described further herein). In some embodiments, the D1L3 variant comprises a C-terminal basic domain having the amino acid sequence SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75), or a modified version thereof. When these D1L3 enzymes are expressed in Pichia pastoris, the resulting polypeptide is cleaved after the SSR sequence at the beginning of the basic domain sequence. In yet another embodiment, D1L3 is encoded and expressed with a 20 amino acid deletion of the basic domain, thereby having the sequence SSR at the C-terminus.

[0012] In some embodiments, the D1L3 variant comprises a deletion of at least 3, or at least 5, or at least 8, or at least 10, or at least 12, or at least 15, or at least 18, or at least 20, or all 23 amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO: 4. In some embodiments, the D1L3 variant comprises a deletion of 20 amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO: 4, such that the D1L3 variant comprises a C-terminal extension of the sequence SSR.

[0013] In various embodiments, the D1L3 variant comprises an amino acid sequence having at least 70% sequence identity to D1L3 isoform 1 (SEQ ID NO: 4) or D1L3 isoform 2 (SEQ ID NO: 5) lacking the BD (i.e., at least 80% sequence identity to amino acids 21-282 of SEQ ID NO: 4 or amino acids 21-252 of SEQ ID NO: 5), wherein the D1L3 variant has one or more amino acids deleted from the BD. In various embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein, and / or the D1L3 variant comprises a C-terminal extension of any of the embodiments disclosed herein.

[0014] In some embodiments, the D1L3 variants have an amino acid substitution corresponding to C48 of SEQ ID NO: 4. These variants can remove an unpaired cysteine, thereby improving recombinant production and stability of the enzyme.

[0015] In some embodiments, the D1L3 variant includes one or more unpaired cysteines configured to dimerize and / or capable of dimerization, e.g., an unpaired cysteine ​​at position 48 relative to the amino acid sequence of SEQ ID NO: 4 (numbering excluding the signal peptide). In some embodiments, the present disclosure provides a D1L3 dimer according to the present disclosure that dimerizes via a disulfide bond at C48. In some embodiments, the D1L3 variant includes incorporation of a non-native cysteine ​​that may contribute to dimerization, including incorporation of a non-native cysteine ​​at the N-terminus. In such embodiments, the present disclosure provides a D1L3 dimer that can be used for treatment according to the present disclosure. In some embodiments, the D1L3 variant includes a substitution of the cysteine ​​at position 48 (C48) relative to SEQ ID NO: 4 to further control the dimerization position. For example, the mutation can be selected from C48A, C48G, and C48S. In some embodiments, the substitution of C48 (e.g., to C48A or C48G) increases the enzymatic ability (e.g., chromatin degradation) of the D1L3 enzyme. In some embodiments, the D1L3 variant has a C-terminal extension comprising or consisting of the amino acid sequence SSR and a C48A mutation with respect to SEQ ID NO: 4 (numbered without the signal peptide).

[0016] Exemplary D1L3 variants according to the present disclosure include SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:77, which lack a signal peptide and are fully processed by the host expression system.

[0017] In some embodiments, the D1L3 variant comprises a fusion or conjugation with a half-life extending moiety. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, the PEG is attached to the N-terminus. In some embodiments, the PEG polymer connects two D1L3 variant molecules via their N-terminus. In some embodiments, the PEG is attached to the C-terminus. In some embodiments, the PEG polymer connects two D1L3 variant molecules via their C-terminus.

[0018] In some embodiments, the D1L3 variant comprises a fusion or conjugation with a half-life extending moiety. In some embodiments, the half-life extending moiety is a fusion partner. In some embodiments, the fusion partner is selected from albumin, transferrin, Fc, or elastin-like protein, an XTEN sequence, or variants thereof. In some embodiments, the fusion partner is albumin. In some embodiments, the fusion partner is fused to the N-terminus (and via a linker sequence) of a mature D1L3 enzyme lacking a signal peptide. In some embodiments, the D1L3 variant comprises the amino acid sequence of SEQ ID NO: 76 or SEQ ID NO: 78, or a variant thereof having at least about 99% sequence identity thereto. In some embodiments, the N-terminal extension of any of the embodiments disclosed herein links the fusion partner to the N-terminus of the mature D1L3 enzyme. In some embodiments, the linker length is about 5 to about 50 amino acids, or about 10 to about 35 amino acids, or about 15 to about 35 amino acids in length. In some embodiments, the linker comprises the amino acid sequences S(GGS)4GSS (SEQ ID NO: 23), S(GGS)9GSS (SEQ ID NO: 24), and (GGS)9GS (SEQ ID NO: 25).

[0019] In some aspects, the present disclosure provides a method for expressing a D1L3 mutant described in any one of the embodiments disclosed herein. In some embodiments, the method includes introducing a genetic construct encoding a D1L3 mutant described in any one of the embodiments disclosed herein and including a signal peptide into a yeast cell, and recovering the D1L3 mutant. In some embodiments, the yeast cell is Pichia pastoris. In some embodiments, the signal peptide is the alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38) from Saccharomyces cerevisiae. In some embodiments, the fusion protein is synthesized with an N-terminal signal peptide. The signal peptide may be completely removed during secretion from the host cell. For expression in Pichia pastoris, the alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38) from Saccharomyces cerevisiae can be used for expression. These elements are cleaved during expression and are not present in the D1L3 mutant enzyme product.

[0020] In some aspects, the present disclosure provides an isolated polynucleotide encoding any of the D1L3 variants of the embodiments disclosed herein, which provides advantages for in vitro or in vivo expression. In some aspects, the present disclosure provides a polynucleotide that is mRNA or modified mRNA (mmRNA). In some aspects, the present disclosure provides a polynucleotide that is DNA.

[0021] In some aspects, the present disclosure provides a pharmaceutical composition comprising a D1L3 enzyme described herein, or a polynucleotide encoding the D1L3 enzyme, or a transfection or expression vector containing the same, or a cell containing the polynucleotide or vector, and a pharmaceutically acceptable carrier.

[0022] In some aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of a D1L3 variant described in any of the embodiments disclosed herein, a D1L3 variant produced according to a method described in any of the embodiments disclosed herein, a polynucleotide described in any of the embodiments disclosed herein, a vector described in any of the embodiments disclosed herein, or a host cell described in any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier.

[0023] In some embodiments, a composition comprises a D1L3 variant of any of the embodiments disclosed herein and a pharmaceutically acceptable carrier for parenteral administration. In some embodiments, the pharmaceutical composition is formulated for topical, parenteral, or pulmonary administration. In some embodiments, the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial, ocular, oral, sublingual, pulmonary, or transdermal administration.

[0024] In embodiments, the recombinant production method for the D1L3 enzyme variants uses a non-mammalian expression system, e.g., a eukaryotic non-mammalian expression system such as Pichia pastoris. In embodiments, the Pichia pastoris encodes a DNase enzyme with a native signal peptide that allows for secretion from the host cell. In embodiments, the expression system is a mammalian cell expression system, such as Chinese hamster ovary (CHO) cells. In embodiments, the recombinant production method for the D1L3 enzyme variants further comprises isolating and / or purifying the D1L3 enzyme and modifying the isolated and / or purified D1L3 enzyme. In embodiments, the modification comprises conjugating the isolated and / or purified D1L3 enzyme to a polymer (e.g., but not limited to, PEG). In embodiments, the polymer is attached to a specific site using a desired conjugation chemistry (e.g., but not limited to, maleimide chemistry).

[0025] In another aspect, the disclosure provides a method of treating a subject in need of extracellular chromatin degradation, extracellular trap (ET) degradation, and / or neutrophil extracellular trap (NET) degradation, comprising administering a therapeutically effective amount of a D1L3 enzyme or composition described herein.

[0026] In some aspects, the present disclosure provides an expression construct for improving processing of a polypeptide precursor in a host, the expression construct comprising a signal peptide fused to a polypeptide via a linker. In some embodiments, the linker is at least three amino acids in length. In some embodiments, the signal peptide is completely removed from the polypeptide. In some embodiments, the signal peptide is not removed from the polypeptide. In some embodiments, the signal peptide is incompletely processed in the host in the absence of the linker. In some embodiments, the polypeptide is or comprises an enzyme, cytokine, cytokine agonist, cytokine antagonist, hormone, hormone agonist, hormone antagonist, antibody or antigen-binding fragment thereof, antibody-like molecule or antigen-binding fragment thereof, antigen, vaccine component, fusion protein, or combination thereof.

[0027] Other aspects and embodiments of the present disclosure will become apparent from the following detailed description and examples. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the expression of D1L3 in Pichia pastoris using either the native secretion signal or the Saccharomyces cerevisiae alpha mating factor (αMF). A shows the N-terminus of D1L3 driven by the aMF secretion leader from Saccharomyces cerevisiae. B shows that the αMF-derived secretion signal results in glycosylation and no processing of the signal. [Figure 2]1 shows a structural model of the N-terminal native secretion signal from alpha mating factor attached to the D1L3 enzyme via a linker. Without being bound by theory, it is believed that the flexible linker positions the cleavage site of the alpha mating factor to allow efficient processing. [Figure 3] Mass spectrometry analysis of D1L3 enzyme mutants, including BDD_D1L3 enzyme (S283_S305del), generated by a construct containing alpha mating factor + GGGGS linker (SEQ ID NO: 58). These data indicated that the secretion signal was properly removed, but the protein contained a post-translational modification (presumably myristoylation) at the N-terminal glycine residue. [Figure 4] 1 shows the results of a titration experiment carried out to compare the chromatin degradation activity of the D1L3 mutant of SEQ ID NO: 63 with that of DNase 1 (D1, SEQ ID NO: 1). [Figure 5] A shows the structural heterogeneity of the D1L3 variant of SEQ ID NO: 63 as revealed by mass spectrometry. B shows the structural heterogeneity of the D1L3 variant of SEQ ID NO: 66 as revealed by mass spectrometry. [Figure 6] Western blot analysis using an anti-DNASE1L3 antibody of culture supernatants from hosts expressing four different DNASE1L3 mutants is shown. In all mutants, C48 was mutated to alanine or serine. Sample 1 (SEQ ID NO: 70) was a D1L3 mutant generated by a construct containing an alpha mating factor plus an SGGGG linker (SEQ ID NO: 60) and possessing a C48A substitution and a C-terminal extension with the sequence SSR. Samples 2 to 4 (SEQ ID NOs: 71 to 73, respectively) were generated by a construct containing an alpha mating factor plus a CGGGG linker (SEQ ID NO: 74) and possessing either a C-terminal extension with the sequence SSR or the wild-type C-terminal basic domain SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75). Dimers were detected in DNASE1L3 mutants containing an N-terminal cysteine. [Figure 7]Figure 1 shows Western blot analysis of two DNASE1L3 mutants, each characterized by the wild-type C-terminal amino acid sequence, i.e., SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75), or a modified C-terminal amino acid sequence, i.e., SSR. The mutants were expressed in Pichia pastoris using alpha mating factor as a signal sequence in combination with an N-terminal SGGGG linker (SEQ ID NO: 60). Western blot analysis of the supernatants did not detect the theoretical mass difference of 2.3 kDa between the two mutants. [Figure 8] High-molecular-weight (HMW) chromatin degradation assay comparing wild-type D1L3 and the C48 mutant. Wild-type D1L3 contains an unpaired cysteine ​​at position 48 (e.g., C48). The effect of the C48 amino acid substitution on enzyme activity was characterized using HMW chromatin (i.e., purified nuclei from HEK293 cells) incubated with an equal amount of the D1L3 mutant. After incubation, DNA was separated and its degradation visualized by agarose gel electrophoresis (AGE). Mutation of C48 to C48A or C48G was associated with increased enzyme activity. [Figure 9] Western blot analysis of Pichia pastoris supernatants to assess dimer formation of DNASE1L3 mutants. The mutant with unpaired C48 showed dimerization (a signal at approximately 60 kDa), whereas the mutant with mutated C48 did not. DETAILED DESCRIPTION OF THE INVENTION

[0029] In various embodiments, the present disclosure provides recombinant proteins (e.g., recombinant proteins for human or animal therapy, including but not limited to, DNase enzymes such as D1L3) produced by secretion from an expression system in which the N-terminal signal sequence is fully processed. In various embodiments, the present disclosure provides methods for making such proteins and using them therapeutically.

[0030] In various aspects and embodiments, the present disclosure is based, in part, on the discovery that adding a linker of four or more amino acids (e.g., at least five amino acids) between the secretion signal and the protein of interest can improve processing and expression titers from microbial expression systems and control post-translational modification profiles. The present invention is also based, in part, on the discovery that C-terminal modifications can improve the product homogeneity profile of proteins produced in expression systems.

[0031] In some aspects, the present disclosure provides a variant of DNASE1-LIKE3 (D1L3 variant) comprising an N-terminal extension. In some embodiments, the N-terminal extension comprises at least four amino acids. As used herein, the term "N-terminal extension" refers to an amino acid sequence that is not a secretory signal and therefore is not removed / processed upon secretion from the host. In some embodiments, the N-terminal extension is not subject to post-translational modification by the host and / or is non-immunogenic upon administration to a human or animal subject. In exemplary embodiments, the N-terminal extension is predominantly Gly residues (i.e., more than 50% Gly residues) and may have one or more Ser or Cys residues.

[0032] In some embodiments, the D1L3 variant produced by the host cell comprises a D1L3 enzyme lacking a signal peptide and comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to amino acids 21-282 of SEQ ID NO:4 (isoform 1) or amino acids 21-252 of SEQ ID NO:5 (isoform 2). When referring to sequence identity to SEQ ID NO:4 or 5, unless otherwise specified, the sequence refers to the mature enzyme lacking the signal peptide. Furthermore, unless otherwise specified, amino acid positions are numbered relative to the natural N-terminus of the enzyme excluding the signal peptide. Thus, for example, reference to sequence identity to the enzyme of SEQ ID NO:4 (human D1L3, isoform 1) refers to the percent identity to the mature enzyme having M21 at the N-terminus.

[0033] In some embodiments, the D1L3 variant is produced in a host cell (such as, but not limited to, Pichia pastoris) by cleavage of the N-terminal signal peptide. Any signal peptide that allows for secretion of D1L3 in the desired expression system can be used. In some embodiments, the signal peptide is the signal peptide of a naturally secreted protein. In some embodiments, the signal peptide is a chimeric or synthetic signal peptide that allows for protein secretion. In some embodiments, the signal peptide is a prokaryotic signal peptide. In some embodiments, the signal peptide is a microbial signal peptide. In some embodiments, the signal peptide is a eukaryotic signal peptide. In some embodiments, the signal peptide is a yeast signal peptide. In some embodiments, the signal peptide is a mammalian signal peptide. Suitable signal peptides for secretion are disclosed in U.S. Patent Nos. 5,580,758, 6,107,057, 7,741,075, 10,435,694, 11,306,127, 11,370,815, and U.S. Patent Application Publication Nos. 2007 / 0117186, 2010 / 0055125, and 2016 / 0168198, the disclosures of each of which are incorporated herein by reference.

[0034] In some embodiments, the signal peptide is selected from the E. coli OmpA signal peptide (SEQ ID NO: 56), the E. coli DsbA signal peptide (SEQ ID NO: 67), the E. coli ST-II signal peptide (SEQ ID NO: 68), the E. coli FimD signal peptide (SEQ ID NO: 55), the Salmonella enterica DsbA signal peptide (SEQ ID NO: 51), the synthetic Bordetella pertussis signal peptide (SEQ ID NO: 57), and synthetic signal peptide sequences (e.g., SEQ ID NOs: 49, 50, 52, 53, and 54).

[0035] In some embodiments, the signal peptide is selected from DNASE1L3 signal peptide (SEQ ID NO: 37), alpha mating factor (SEQ ID NO: 38), alpha mating factor presequence (SEQ ID NO: 39), human serum albumin signal peptide (SEQ ID NO: 40), bovine DNASE1 signal peptide (SEQ ID NO: 41), bovine DNASE1 signal peptide + Kex2 site (SEQ ID NO: 42), alpha amylase signal peptide (SEQ ID NO: 43), glucoamylase signal peptide (SEQ ID NO: 44), inulinase signal peptide (SEQ ID NO: 45), invertase signal peptide (SEQ ID NO: 46), killer protein signal peptide (SEQ ID NO: 47), and lysozyme signal peptide (SEQ ID NO: 48).

[0036] In some embodiments, the signal peptide is the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae (SEQ ID NO: 38).

[0037] In some embodiments, the N-terminal extension is at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15 amino acids in length. In various embodiments, the N-terminal extension has a length ranging from 4 to about 18 amino acids, or from 4 to about 12 amino acids, or from 4 to about 9 amino acids, or from 4 to about 7 amino acids. For example, the N-terminal extension can be 4, 5, 6, 7, 8, or 9 amino acids in length. In some embodiments, the N-terminal extension is a flexible or rigid sequence. In some embodiments, the first N-terminal amino acid is not a Met residue. In some embodiments, the first amino acid residue of the N-terminal extension is not a Gly residue, which may be subject to post-translational modifications as disclosed herein. In some embodiments, the last amino acid residue of the N-terminal extension is not a Ser residue, which may increase the risk of immunogenicity as disclosed herein. In some embodiments, the last amino acid residue of the N-terminal extension is not a polar or charged amino acid residue, e.g., an amino acid residue selected from Ser, Thr, Gln, Asn, Glu, Asp, Arg, His, and Lys. In some embodiments, the last amino acid residue of the N-terminal extension is an amino acid selected from Gly, Ala, and Val. In some embodiments, the last amino acid residue of the N-terminal extension is a Gly residue. In some embodiments, the first amino acid residue of the N-terminal extension is not a Gly residue, and the last amino acid residue of the N-terminal extension is a Gly residue. In some embodiments, the N-terminal extension is predominantly Gly residues (i.e., 50% or more Gly residues), consists essentially of Ser and Gly residues, or consists of Ser and Gly residues. In some embodiments, the linker comprises at least one cysteine ​​residue. For example, placing a cysteine ​​at the N-terminus allows for site-specific chemical attachment, such as polyethylene glycol or disulfide dimerization. In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence SGGGG (SEQ ID NO: 60).In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of CGGGG (SEQ ID NO: 74). In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of SGGSGGSGG (SEQ ID NO: 61). In some embodiments, the N-terminal extension comprises or consists of the amino acid sequence of SGGSGGSGGSGGSGGSGG (SEQ ID NO: 62). In some embodiments, the N-terminal extension comprises a protease cleavage site. In some embodiments, the N-terminal extension is cleavable by a coagulation pathway protease. In some embodiments, the protease is thrombin, or factor XII, or a neutrophil protease. In some embodiments, the protease is thrombin. In some embodiments, the protease cleavage site comprises the amino acid sequence LVPRG (SEQ ID NO: 64), e.g., the N-terminal extension is represented by the sequence SGGGGLVPRGSGGGG (SEQ ID NO: 65).

[0038] In some embodiments, the N-terminal extension does not include a consensus sequence for myristoylation. In some embodiments, the N-terminal extension does not include a consensus sequence for one or more of protein acetylation, propionylation, methylation, myristoylation, palmitoylation, ubiquitination, and a protease cleavage site to avoid undesired post-translational modifications. In another embodiment, the N-terminal extension includes a consensus sequence for one or more of protein acetylation, propionylation, methylation, myristoylation, palmitoylation, ubiquitination, and a protease cleavage site, if these modifications are desired. In some embodiments, the N-terminal extension includes an amino acid bearing a chemical group suitable for chemical conjugation, wherein the chemical group is selected from a thiol group, an amino group, an amide group, and a carboxyl group.

[0039] In some embodiments, the N-terminal extension is non-immunogenic. In some embodiments, the junction between the N-terminal extension and a sequence derived from a desired protein (such as, but not limited to, D1L3) is non-immunogenic as indicated by an in silico immunogenicity prediction algorithm (such as, but not limited to, Lonza Group AG's Epibase® in silico and in vitro immunogenicity platform). In some embodiments, the last amino acid residue of the N-terminal extension is not a Ser residue. In some embodiments, the last amino acid residue of the N-terminal extension is not a polar or charged amino acid residue selected from Ser, Thr, Gln, Asn, Glu, Asp, Arg, His, and Lys. In some embodiments, the last amino acid residue of the N-terminal extension is an amino acid selected from Gly, Ala, and Val. In some embodiments, the last amino acid residue of the N-terminal extension is a Gly residue.

[0040] In some embodiments, the N-terminal extension improves the expression titer of the D1L3 variant by at least 25%, or at least 50%, or at least 100%, or at least 150%, or at least 200%, or at least 250%, or at least 300%, or at least 350%, or at least 400%, or at least 500%, or more, for a selected signal sequence and expression host, compared to the same D1L3 sequence lacking the N-terminal extension. In some embodiments, the signal peptide is completely removed from the D1L3 variant upon secretion from the host.

[0041] In some embodiments, the D1L3 variant further comprises a C-terminal extension that reduces heterogeneity. In some embodiments, the C-terminal extension is at least 2, or at least 3, or at least 4, or at least 5 amino acids in length. In some embodiments, the extension is a hydrophilic sequence of 2, 3, or 4 amino acids. In some embodiments, the C-terminal amino acid is lysine (Lys) or arginine (Arg). In some embodiments, the C-terminal extension comprises or consists of the amino acid sequence SSR, which in some embodiments can be used for D1L3 variants having a deletion of all or part of the C-terminal basic domain (described further herein). In some embodiments, the D1L3 variant comprises a C-terminal basic domain having the amino acid sequence SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75), or a modified version thereof having 1 to 5 amino acid modifications independently selected from amino acid substitutions, deletions, and insertions. When these D1L3 enzymes are expressed in Pichia pastoris, the resulting polypeptide is cleaved after the SSR sequence that precedes the basic domain sequence.

[0042] In some embodiments, the present disclosure provides a DNASE1-LIKE3 mutant (D1L3 mutant) comprising a C-terminal extension as described above. In some embodiments, the C-terminal extension reduces the heterogeneity observed in D1L3 mutants lacking the basic domain. In some embodiments, the heterogeneity is caused by post-translational modification. In some embodiments, the present disclosure provides a D1L3 mutant comprising a previously described N-terminal extension and a heterogeneity-reducing C-terminal extension. In some embodiments, the C-terminal extension comprises or consists of the amino acid sequence SSR.

[0043] In some embodiments, the C-terminal extension comprises an amino acid having a chemical group suitable for chemical conjugation, the chemical group being selected from a thiol group, an amino group, an amide group, and a carboxyl group. In some embodiments, the C-terminal extension does not comprise a consensus sequence for one or more of protein acetylation, propionylation, methylation, myristoylation, palmitoylation, ubiquitination, and a protease cleavage site to avoid undesired post-translational modifications. In another embodiment, the C-terminal extension comprises a consensus sequence for one or more of protein acetylation, propionylation, methylation, myristoylation, palmitoylation, ubiquitination, and a protease cleavage site when post-translational modifications are desired. In some embodiments, the C-terminal extension is non-immunogenic. In some embodiments, the junction between the C-terminal extension and the D1L3-derived sequence is non-immunogenic.

[0044] In some embodiments, the D1L3 variant comprises a deletion of at least 3, or at least 5, or at least 8, or at least 10, or at least 12, or at least 15, or at least 18, or at least 20, or at least 21, or all 23 amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO: 4. In some embodiments, the D1L3 variant comprises a deletion of at least 3 amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO: 4, wherein the D1L3 variant comprises a C-terminal extension of any embodiment disclosed herein (e.g., having the amino acid sequence SSR). In such embodiments, the C-terminus with the SSR extension corresponds to a deletion of 20 amino acids in the basic domain.

[0045] D1L3 is characterized by a 23-amino acid C-terminal tail, defined by amino acids 283-305 of SEQ ID NO:4, known as the basic domain (BD) because it contains nine basic amino acids. The BD is unique to D1L3 and is absent in DNASE1 (D1). The BD contains a nuclear localization signal (NLS), which is thought to target the enzyme to the nucleus during apoptosis. The BD has also been widely believed to be important for the biological activity of D1L3 in the extracellular space; indeed, deletion of the C-terminal tail stimulates the chromatinase activity of D1L3. In some embodiments, the D1L3 variant comprises a BD having the sequence SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO:75), or a derivative thereof with a truncation of at least 3, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, or about 20 amino acids.

[0046] In some embodiments, the D1L3 variant comprises mutations (e.g., substitutions, insertions, or deletions) of three clusters of paired basic amino acids of unknown function, i.e., K291 / K292, R297 / K298 / K299, and K303 / R304, corresponding to SEQ ID NO: 4. In some embodiments, the D1L3 variant comprises a BD having the sequence SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75), or a derivative thereof having one to five amino acid modifications that alter the paired basic amino acids. In some embodiments, the D1L3 variant comprises a truncation of the BD that deletes at least one of the paired basic amino acids. In some embodiments, the D1L3 variant exhibits reduced proteolytic cleavage at these paired basic amino acids. In some embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein (including, but not limited to, for example, the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)), and / or the D1L3 variant comprises a C-terminal extension of any of the embodiments disclosed herein (including, but not limited to, for example, the amino acid sequence SSR). In some embodiments, the C-terminal extension is added in place of the C-terminal basic domain.

[0047] In various embodiments, the D1L3 variant comprises an amino acid sequence having at least 70% sequence identity to D1L3 isoform 1 (SEQ ID NO: 4) or D1L3 isoform 2 (SEQ ID NO: 5) lacking the BD (i.e., at least 80% sequence identity to amino acids 21-282 of SEQ ID NO: 4 or amino acids 21-252 of SEQ ID NO: 5), wherein the D1L3 variant has one or more amino acids deleted from the BD. Deletion of amino acids in the basic domain of D1L3 improves its chromatin degrading activity. Furthermore, increased deletion of the 23-amino acid BD directly correlates with increased chromatin degrading activity. In some embodiments, D1L3 variants with deletion of at least one amino acid, or at least three, or at least five, or at least eight, or at least nine, or at least 13, or at least 14, or at least 15, or at least 18, or about 20 C-terminal amino acids of the D1L3 basic domain have improved ability to degrade mononucleosomes. In some embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein (e.g., including but not limited to, including or consisting of the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)), and / or the D1L3 variant comprises a C-terminal extension of any of the embodiments disclosed herein (e.g., including but not limited to, having the amino acid sequence SSR).

[0048] In various embodiments, the deletion of amino acids from BD occurs at the C-terminus of BD. For example, a D1L3 variant can have at least 5 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant has at least 8 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 8 C-terminal amino acids of BD. In some embodiments, a D1L3 variant has at least 10 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 10 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 10 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 12 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 12 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 15 C-terminal amino acids of BD deleted. In some embodiments, a D1L3 variant is truncated by at least 15 C-terminal amino acids of BD deleted. In some embodiments, the D1L3 variant has at least 18 C-terminal amino acids of BD deleted. In some embodiments, the D1L3 variant has at least 18 C-terminal amino acids of BD truncated. In some embodiments, the D1L3 variant has at least 20 C-terminal amino acids of BD deleted. In some embodiments, the D1L3 variant has at least (or about) 20 C-terminal amino acids of BD deleted. In some embodiments, the D1L3 variant has at least 23 C-terminal amino acids of BD deleted. In some embodiments, the D1L3 variant has at least 23 C-terminal amino acids of BD deleted. In some embodiments, the D1L3 variant has at least the C-terminal serine of BD deleted. In some embodiments, the D1L3 variant has the C-terminal serine of BD truncated.In some embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein (including, but not limited to, for example, the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)), and / or the D1L3 variant comprises a C-terminal extension of any of the embodiments disclosed herein (including, but not limited to, for example, the amino acid sequence SSR).

[0049] Alternatively, the BD deletion (e.g., 3-23 amino acids) can occur anywhere in the BD and does not necessarily occur from the C-terminus of the BD. For example, in various embodiments, the D1L3 variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids deleted from the BD. In some embodiments, the D1L3 variant has at least 1, or at least 3, or at least 5, or at least 8, or at least 12, or at least 15, or at least 18, or at least 21 amino acids deleted from the BD. In some embodiments, the D1L3 variant has at least 1, or at least 3, or at least 5, or at least 8, or at least 12, or at least 15, or at least 18, or at least 20, or at least 21 amino acids truncated from the BD. These deletions can be independently selected from the N-terminal side of the BD, the C-terminal side of the BD, and within the BD. In some embodiments, the one or more amino acid deletions are within the NLS. In some embodiments, the deleted amino acid is a C-terminal serine of the BD. In some embodiments, the deletions are sufficient to remove all paired basic amino acids within the BD from the enzyme. In some embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein (including, but not limited to, the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)) and / or a C-terminal extension of any of the embodiments disclosed herein (including, but not limited to, the amino acid sequence SSR). In some embodiments, the C-terminal extension of the sequence SSR corresponds to a 20 amino acid truncation of the BD.

[0050] In addition to one or more amino acid deletions, the BD may also contain amino acid substitutions, which may further affect chromatin degradation activity. For example, a D1L3 variant may contain at least three amino acid deletions and one to twenty BD amino acid substitutions. In some embodiments, the BD contains at least three amino acid substitutions, or at least five amino acid substitutions, or at least ten amino acid substitutions. In some embodiments, at least two amino acid substitutions are present within the NLS of the BD. In some embodiments, one or more paired basic amino acids within the BD are substituted to prevent cleavage. In such embodiments, a more homogeneous enzyme may be expressed and secreted, for example, for recombinant enzyme production. In some embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein (e.g., including but not limited to, the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)) and / or a C-terminal extension of any of the embodiments disclosed herein (e.g., including but not limited to, the amino acid sequence SSR).

[0051] In some embodiments, the D1L3 variant has one or more additional amino acids deleted from the C-terminus in addition to the deletion of the BD. For example, the D1L3 variant can have an additional 1 to 50 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, or 1 to 5 amino acids deleted from the C-terminal amino acid of SEQ ID NO: 4 or SEQ ID NO: 5 in addition to the deletion of the BD. In some embodiments, the D1L3 variant comprises an N-terminal extension of any of the embodiments disclosed herein (including, but not limited to, the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)) and / or a C-terminal extension of any of the embodiments disclosed herein (including, but not limited to, the amino acid sequence SSR).

[0052] In some embodiments, partial or complete deletion of the BD as described can be followed by the addition of 1 to 10 amino acids, or 1 to 5 amino acids, to the C-terminus, which does not affect chromatin degrading activity. In some embodiments, the addition of an N-terminal extension of any of the embodiments disclosed herein (including, but not limited to, the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)) and / or a C-terminal extension of any of the embodiments disclosed herein (having, but not limited to, the amino acid sequence SSR) does not affect chromatin degrading activity.

[0053] In some embodiments, the D1L3 variant comprises a substitution of C68 and / or C194 (C48 and C174 when numbered without the signal peptide) with respect to SEQ ID NO: 4. In some embodiments, the mutation is selected from C68S, C68A, C68G, C194S, C194A, and C194G with respect to SEQ ID NO: 4. In some embodiments, the D1L3 variant comprises one or more mutations that confer resistance to proteolysis by one or more of plasmin, thrombin, trypsin, and proteases produced by mammalian and non-mammalian cell lines. In some embodiments, the D1L3 variant has one or more mutations of amino acid residues selected from K180, K200, K259, and R285 with respect to SEQ ID NO: 4. In some embodiments, the D1L3 variant has one or more mutations in amino acid residues selected from R22, R29, K45, K47, K74, R81, R92, K107, K176, R212, R226, R227, K250, K259, and K262 with respect to SEQ ID NO: 4.

[0054] In some embodiments, the D1L3 variant includes one or more unpaired cysteines configured and / or capable of dimerization, e.g., an unpaired cysteine ​​at position 48 relative to the amino acid sequence of SEQ ID NO: 4 (numbering excluding the signal peptide). In some embodiments, the present disclosure provides a D1L3 dimer according to the present disclosure that dimerizes via a disulfide bond at C48. In some embodiments, the D1L3 variant includes incorporation of a non-native cysteine ​​that may contribute to dimerization, including incorporation of a non-native cysteine ​​at the N-terminus. In such embodiments, the present disclosure provides a D1L3 dimer that can be used for treatment according to the present disclosure. In some embodiments, the D1L3 variant includes a substitution of the cysteine ​​at position 48 (C48) relative to SEQ ID NO: 4 to further control the dimerization position. For example, the mutation can be selected from C48A, C48G, and C48S. In some embodiments, substitution of C48 (e.g., to C48A or C48G) increases the enzymatic ability (such as chromatin degradation) of the D1L3 enzyme. In some embodiments, the D1L3 mutant has a C-terminal extension comprising or consisting of the amino acid sequence SSR and a C48A mutation with respect to SEQ ID NO: 4 (numbered without the signal peptide).

[0055] In some embodiments, the D1L3 variant comprises: (i) a D1L3 enzyme lacking a signal peptide and comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98, or at least 99% sequence identity to amino acids 21-282 of SEQ ID NO:4 or amino acids 21-252 of SEQ ID NO:5; (ii) a deletion of at least three amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO:4; and (iii) an N-terminal extension of at least four amino acids as described herein. In some embodiments, the D1L3 variant further comprises a substitution of C48 with respect to SEQ ID NO:4. In some embodiments, the mutation is selected from C48S or C48A with respect to SEQ ID NO:4 (numbered without the signal peptide). In some embodiments, the D1L3 variant further comprises a modification (e.g., without limitation, PEGylation) of C48 and / or C174 with respect to SEQ ID NO:4.

[0056] In some embodiments, the D1L3 variant comprises: (i) a D1L3 enzyme lacking a signal peptide and comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to amino acids 21-282 of SEQ ID NO:4 or amino acids 21-252 of SEQ ID NO:5; (ii) a deletion of at least three amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO:4; and (iii) a C-terminal extension consisting of the amino acid sequence SSR. In some embodiments, the D1L3 variant further comprises a substitution of C48 and / or C174 (numbered without the signal peptide) with respect to SEQ ID NO:4. In some embodiments, the mutation is selected from C48S or C48A with respect to SEQ ID NO:4. In some embodiments, the D1L3 variant further comprises a modification (e.g., without limitation, PEGylation) of C48 and / or C174 with respect to SEQ ID NO:4.

[0057] In some embodiments, the D1L3 variant comprises (i) a mature D1L3 enzyme lacking a signal peptide and comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to amino acids 21-282 of SEQ ID NO:4 or amino acids 21-252 of SEQ ID NO:5, (ii) a deletion of at least three amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO:4, (iii) an N-terminal extension described herein, and (iv) a C-terminal extension comprising the amino acid sequence SSR. In some embodiments, the D1L3 variant further comprises a substitution of C48 and / or C174 (numbered without the signal peptide) with respect to SEQ ID NO:4. In some embodiments, the mutation is selected from C48S and C48A with respect to SEQ ID NO:4. In some embodiments, the D1L3 variant further comprises a modification (for example, but not limited to, PEGylation) at C48 and / or C174 relative to SEQ ID NO:4.

[0058] In some embodiments, the D1L3 variant has one or more mutations of serine residues, such as those selected from S91C, S131C, and S253C, with respect to SEQ ID NO: 4 (numbering includes the signal peptide).

[0059] Exemplary D1L3 variants according to the present disclosure include SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:77, which lack a signal peptide and are fully processed by the host expression system. SEQ ID NO:63 utilizes an N-terminal extension of SEQ ID NO:60 to achieve signal peptide processing and contains a deletion of the complete basic domain. SEQ ID NO:66 further contains a C48S substitution and an SSR C-terminal extension (relative to SEQ ID NO:63). SEQ ID NO:70 exemplifies a C48A substitution (but otherwise has the sequence of SEQ ID NO:66). SEQ ID NO:71 is similar to SEQ ID NO:70 but contains a CGGGG (SEQ ID NO:74) linker before the signal peptide. SEQ ID NO:72 exemplifies a C48S substitution combined with a CGGGG (SEQ ID NO:74) linker. SEQ ID NO:73 exemplifies a C48S substitution combined with a CGGGG (SEQ ID NO:74) linker and the complete basic domain. When produced in Pichia pastoris, the polypeptide of SEQ ID NO:73 will have an SSR at the C-terminus (e.g., 20 amino acids of the basic domain are truncated). SEQ ID NO:77 employs a C-terminal extension (SSR) added after a C48A substitution, an SGGGG (SEQ ID NO:60) linker, and an additional 12 amino acid deletion (in addition to the deletion of the basic domain).

[0060] In some embodiments, the D1L3 variant comprises a fusion or conjugation with a half-life extending moiety. In some embodiments, the half-life extending moiety is a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, the PEG is attached to the N-terminus (optionally within an N-terminal extension of any of the embodiments disclosed herein, e.g., comprising the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74)). In some embodiments, the PEG polymer connects two D1L3 variant molecules via their N-terminus. In some embodiments, the PEG is attached to the C-terminus (optionally within an N-terminal extension of any of the embodiments disclosed herein, e.g., having the amino acid sequence SSR). In some embodiments, the D1L3 comprises a basic domain and the PEG is attached to the basic domain. In some embodiments, the PEG polymer connects two D1L3 variant molecules via their C-terminus.

[0061] In some embodiments, the PEG polymer is attached to one or more amino acids within positions corresponding to R95 to V126 of SEQ ID NO: 4. In some embodiments, the one or more PEGylated amino acids are selected from lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine, optionally, the one or more PEGylated amino acids are introduced by substitution of one or more amino acids between R95 and V126 relative to SEQ ID NO: 4.

[0062] In some embodiments, one or more amino acids are PEGylated by (a) PEGylation of lysine (Lys or K) via an amine bond, (b) PEGylation of glutamine via transglutaminase (TGase)-mediated enzymatic conjugation, and / or (c) PEGylation of cysteine ​​(Cys or C) via a thiol bond. In some embodiments, one or more PEGylated amino acids are conjugated to a PEG moiety independently selected from linear or branched PEGs having independently selected molecular weights ranging from about 2 kDa to about 60 kDa, or from about 5 kDa to about 30 kDa. PEGylation is disclosed in WO2019 / 036719 and WO2020 / 076817, both of which are incorporated herein by reference in their entireties.

[0063] In these embodiments, the PEG moiety provides half-life extension properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moiety is attached via maleimide chemistry, which can be performed under mild conditions. Other conjugation chemistries, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistries, are also known and can be used.

[0064] In some embodiments, the D1L3 variant comprises a fusion or conjugation with a half-life extending moiety. In some embodiments, the half-life extending moiety is a fusion partner. In some embodiments, the fusion partner is selected from albumin, transferrin, Fc, or elastin-like protein, an XTEN sequence, or variants thereof. In some embodiments, the fusion partner is albumin. In some embodiments, the fusion partner is human albumin comprising an amino acid sequence at least 80% identical to SEQ ID NO:26. In some embodiments, the human albumin comprises at least one of E505Q, T527M, and K573P substitutions with respect to SEQ ID NO:26. In some embodiments, the human albumin comprises at least two of E505Q, T527M, and K573P substitutions with respect to SEQ ID NO:26. In some embodiments, the human albumin comprises E505Q, T527M, and K573P substitutions with respect to SEQ ID NO:26. In some embodiments, the fusion partner is fused N-terminally to a mature D1L3 enzyme lacking a signal peptide (and via a linker sequence) and comprising an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to amino acids 21-282 of SEQ ID NO:4 or amino acids 21-252 of SEQ ID NO:5, with a deletion of at least three amino acids in the C-terminal basic domain (BD) defined by amino acids 283-305 of SEQ ID NO:4. In some embodiments, the D1L3 variant comprises the amino acid sequence of SEQ ID NO:76 or SEQ ID NO:78, or a variant thereof having at least about 99% sequence identity thereto. In some embodiments, the N-terminal extension of any of the embodiments disclosed herein links the fusion partner to the N-terminus of the mature D1L3 enzyme. In some embodiments, the fusion partner is fused to the mature D1L3 enzyme via a linker flanking the fusion partner and the mature D1L3 enzyme. In some embodiments, the N-terminal extension of any of the embodiments disclosed herein links the fusion partner to the N-terminus of the mature D1L3 enzyme.In some embodiments, the linker is flexible or rigid and / or comprises a protease cleavage site. In some embodiments, the linker is cleavable by a coagulation pathway protease. In some embodiments, the protease is factor XII or a neutrophil protease. In some embodiments, the protease is thrombin. In some embodiments, the protease cleavage site comprises the amino acid sequence LVPRG (SEQ ID NO: 64), such as a linker represented by the sequence SGGGGLVPRGSGGGG (SEQ ID NO: 65). In some embodiments, the linker is about 5 to about 50 amino acids in length, or about 10 to about 35 amino acids in length, or about 15 to about 35 amino acids in length. In some embodiments, the linker comprises the amino acid sequence S(GGS)4GSS (SEQ ID NO: 23), S(GGS)9GSS (SEQ ID NO: 24), or (GGS)9GS (SEQ ID NO: 25). In some embodiments, the linker comprises the sequence (GGGGS)5GGGG (SEQ ID NO:79), as shown by the fusion protein of SEQ ID NO:76.

[0065] In some embodiments, the D1L3 variant comprises a flexible linker between the D1L3 sequence and the half-life extending moiety. The flexible linker is composed primarily or entirely of small, non-polar or polar residues, such as Gly, Ser, and Thr. Exemplary flexible linkers include (Gly y Ser) n S z linkers, where y is 1-10 (e.g., 1-5), n is 1 to about 10, and z is 0 or 1. In some embodiments, n is 3 to about 8, or 3 to about 6. In exemplary embodiments, y is 2-4, and n is 3-8. These linkers are unstructured due to their flexibility. More rigid linkers include polyproline or polyPro-Ala motifs and alpha-helical linkers. An exemplary alpha-helical linker is A(EAAAK). nA, where n is as defined above (e.g., 1-10, or 3-6). Generally, the linker can be composed primarily of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences contain at least 10 amino acids and may range from 10 to about 50 amino acids, or from about 15 to about 40 amino acids, or from about 15 to about 35 amino acids. Exemplary linker designs are provided as SEQ ID NOs: 18-25.

[0066] In some embodiments, the D1L3 variant having a fusion partner includes a linker, the amino acid sequence of which is primarily, consists essentially of, or consists of glycine and serine residues. In some embodiments, the Ser:Gly ratio in the linker is about 1:1 to about 1:10, about 1:2 to about 1:6, or about 1:4, respectively. Exemplary linker sequences include or consist of S(GGS)4GSS (SEQ ID NO:23), S(GGS)9GSS (SEQ ID NO:24), and (GGS)9GS (SEQ ID NO:25). In some embodiments, the linker has at least 10 amino acids, or at least 15 amino acids, or at least 20 amino acids, or at least 25 amino acids, or at least 30 amino acids. For example, the linker length is 15-40 amino acids. In various embodiments, a longer linker of at least 15 amino acids improves titer upon expression in Pichia pastoris.

[0067] In some aspects, the present disclosure provides a method for expressing a DNase 1-like 3 mutant described in any one of the embodiments disclosed herein. In some embodiments, the method includes introducing a genetic construct encoding a DNase 1-like 3 mutant described in any one of the embodiments disclosed herein and including a signal peptide into a yeast cell, and recovering the DNase 1-like 3 mutant. In some embodiments, the yeast cell is Pichia pastoris. In some embodiments, the signal peptide is the alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38) from Saccharomyces cerevisiae. In some embodiments, the fusion protein is synthesized with an N-terminal signal peptide. The signal peptide may be completely removed during secretion from the host cell. For expression in Pichia pastoris, the alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38) from Saccharomyces cerevisiae can be used for expression. These elements are cleaved during expression and are not present in the D1L3 mutant enzyme product.

[0068] In some aspects, the present disclosure provides an isolated polynucleotide encoding any of the D1L3 variants of the embodiments disclosed herein, which provides advantages for in vitro or in vivo expression. In some aspects, the present disclosure provides a polynucleotide that is mRNA or modified mRNA (mmRNA). In some aspects, the present disclosure provides a polynucleotide that is DNA. In some aspects, the present disclosure provides a pharmaceutical composition comprising a D1L3 enzyme described herein, or a polynucleotide optionally encoding the D1L3 enzyme, or a transfection or expression vector comprising the same, or a cell comprising the polynucleotide or vector, and a pharmaceutically acceptable carrier.

[0069] In some embodiments, delivery of polynucleotides is used for therapy. The encoding polynucleotide can be delivered as an mRNA or DNA construct using known procedures (e.g., electroporation or cell squeezing) and / or vectors (including viral vectors). The mRNA polynucleotide can include known modifications (mRNA) to avoid activation of the innate immune system. See WO2014 / 028429, incorporated herein by reference in its entirety. In some embodiments, the polynucleotide is delivered to a cell in vitro and the cell is delivered to the subject. Cells include, for example, leukocytes (e.g., T cells, B cells, or macrophages), endothelial cells, epithelial cells, hepatocytes, fibroblasts, or stem cells (e.g., hematopoietic stem cells).

[0070] In some embodiments, polynucleotides used for therapeutic purposes are modified mRNA (mmRNA). In some embodiments, the mmRNA is administered to a subject in need of treatment. In some embodiments, cells are transformed in vitro or ex vivo with the modified mRNA (mmRNA), grown before or after transfection, and used for treatment (cell therapy). In some embodiments, the mmRNA may be uniformly modified along the entire length of the molecule. In other embodiments, the mmRNA may not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may be present at various positions within the nucleic acid. In some embodiments, nucleotide analogs or other modification(s) may be placed at any position(s) of the nucleic acid such that the function of the nucleic acid is not substantially diminished. In some embodiments, the mmRNA may include a 5'-end modification or a 3'-end modification.

[0071] In some embodiments, the mmRNA may contain at least about 5% modified nucleotides, or at least about 10% modified nucleotides, or at least about 20% modified nucleotides, or at least about 50% modified nucleotides, or at least about 80% modified nucleotides. In some embodiments, the mmRNA may include less than about 10% modified nucleotides, less than about 20% modified nucleotides, or less than about 50% modified nucleotides.

[0072] In some embodiments, the mmRNA may include polynucleotide modifications, such as, but not limited to, nucleoside modifications such as pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -Taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy -2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl- 1-Deaza-pseudoisocytidine, Zebularine, 5-Aza-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl-Cytidine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, 2-Aminopurine, 2,6-Diaminopurine, 7-Deaza-Adenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-Aminopurine, 7-Deaza-8-Aza-2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxyadenine, inosine, 1-methyl-inosine, wiosine, wibutosine, 7-deaza Suitable modifications include, but are not limited to, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof. Suitable modifications are disclosed in US20190060458, the contents of which are incorporated herein by reference in their entirety.

[0073] In some aspects, the present disclosure provides a vector for introducing a polynucleotide of any of the embodiments disclosed herein into a host cell. In some aspects, the present disclosure provides a host cell comprising a vector of any of the embodiments disclosed herein.

[0074] In some embodiments, the polynucleotide used in therapy is a DNA molecule encoding a wild-type D1L3 enzyme or any of the D1L3 variants disclosed herein (i.e., gene therapy). In some embodiments, cells are transformed in vitro or ex vivo with a DNA molecule encoding a wild-type D1L3 enzyme or any of the D1L3 variants disclosed herein, grown, and used in therapy (i.e., cell therapy). In some embodiments, the DNA molecule is a vector. A vector generally comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In some embodiments, the vector is a viral vector. Exemplary vectors include autonomously replicating plasmids or viruses (e.g., AAV vectors). This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0075] In some embodiments, polynucleotide or cell therapy can use an expression vector comprising a nucleic acid encoding a chromatinase (e.g., D1L3) operably linked to an expression control region that functions in a host cell. The expression control region can drive expression of the operably linked encoding nucleic acid such that the chromatinase is produced in a human cell transformed with the expression vector. Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that affect the expression of an operably linked nucleic acid. The expression control region of an expression vector can express the operably linked encoding nucleic acid in a human cell. In one embodiment, the expression control region confers controllable expression to the operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of the nucleic acid operably linked to the expression control region. Expression control regions that increase expression in response to a signal are often referred to as inducible. Expression control regions that decrease expression in response to a signal are often referred to as repressible. In various embodiments, expression of the chromatinase is inducible or repressible. Typically, the amount of increase or decrease conferred by the element is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0076] In some embodiments, the viral vector is an adeno-associated viral vector (AAV). In some embodiments, suitable AAV-based vectors of the present disclosure have very limited ability to induce an immune response in humans. The AAV genome is typically composed of positive- or negative-sense single-stranded deoxyribonucleic acid (ssDNA) and is approximately 4.7 kilobases in length. The AAV genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), rep and cap. In the development of AAV as a gene therapy vector, the rep and cap sequences have been removed from the vector DNA to eliminate the vector's integration ability. In some embodiments, a gene encoding a wild-type D1L3 enzyme or any of the D1L3 mutants disclosed herein, operably linked to a promoter, can be inserted between the inverted terminal repeats (ITRs). In some embodiments, AAV vectors containing a wild-type D1L3 enzyme or any of the mutant D1L3 enzymes disclosed herein can form concatemers in the nucleus after the single-stranded vector DNA is converted to double-stranded DNA by the host cell's DNA polymerase complex. Thus, in some embodiments, AAV vectors comprising wild-type D1L3 enzymes or any of the mutant D1L3s disclosed herein can form episomal concatemers in the nucleus of a host cell. In some embodiments, the concatemers can remain intact for the life of the host cell, even if the cell does not divide. In some embodiments, the concatemers can be lost as the cell divides.

[0077] In an exemplary embodiment, an AAV serotype 8 (AAV2 / 8) vector is used. In some embodiments, the recombinant AAV serotype used to deliver the polynucleotide is replication-defective, generally does not integrate into the host genome, and does not exhibit pathogenicity or immune response in human subjects. Any AAV vector can be used, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and combinations thereof. In some cases, the AAV contains an LTR of a heterologous serotype compared to the capsid serotype (e.g., an AAV2 ITR and an AAV5, AAV6, or AAV8 capsid).

[0078] Expression systems that function in human cells are well known in the art and include viral systems. Generally, a promoter that functions in human cells is any DNA sequence capable of binding mammalian RNA polymerase and initiating downstream (3') transcription of a coding sequence into mRNA. A promoter typically contains a transcription initiation region, located proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100-200 base pairs upstream of the TATA box. The upstream promoter element determines the rate at which transcription is initiated and can act in either direction. Of particular use as promoters are promoters derived from mammalian viral genes, as these genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter.

[0079] If necessary, gene delivery agents such as integration sequences can also be used. Numerous integration sequences are known in the art (see, for example, Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol., 335:667-678, 2004), Sleeping Beauty, transposases of the Mariner family, and components for integrating viruses such as AAV, retroviruses, and antivirals with components providing viral integration such as retroviral or lentiviral LTR sequences and AAV ITR sequences (Kootstra et al., J. Mol. Biol., 1999, 2004). (e.g., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Additionally, direct and targeted gene integration strategies, including CRISPR / CAS9, zinc finger, TALEN, and meganuclease gene editing technologies, may be used to insert the nucleic acid sequence.

[0080] Thus, in some embodiments, the present invention provides mammalian host cells (e.g., human host cells) and methods of production and use thereof. The host cells comprise a heterologous polynucleotide encoding a chromatinase enzyme (as described). Host cells delivered to a subject express and secrete the encoded chromatinase enzyme. These aspects circumvent the challenges of large-scale production of chromatinases, such as D1L3. Furthermore, expression and delivery of D1L3 through heterologous expression in leukocytes, such as T cells, B cells, macrophages, or fibroblasts, can partially localize D1L3 therapy to areas of inflammation or tissue destruction, cell apoptosis, or wound healing. Furthermore, because the circulating half-life of WT D1L3 is less than about 30 minutes, the cell therapies described herein, in some embodiments, provide sustained therapy with as few as one, two, three, or four treatments. In some embodiments, therapy is provided to a subject for treatment of cancer (e.g., leukemia) or viral infections, including infections of the lower respiratory tract. In some embodiments, the host cells are generated from cells of the subject to be treated or an HLA-matched donor, hi some embodiments, the cells are HLA-null or generated from HLA-matched source cells.

[0081] In some aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of a D1L3 variant described in any of the embodiments disclosed herein, a D1L3 variant produced according to a method described in any of the embodiments disclosed herein, a polynucleotide described in any of the embodiments disclosed herein, a vector described in any of the embodiments disclosed herein, or a host cell described in any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier.

[0082] In some embodiments, a composition comprises a D1L3 variant of any of the embodiments disclosed herein and a pharmaceutically acceptable carrier for parenteral administration. In some embodiments, the pharmaceutical composition is formulated for topical, parenteral, or pulmonary administration. In some embodiments, the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial, ocular, oral, sublingual, pulmonary, or transdermal administration.

[0083] In embodiments, the recombinant production method for the D1L3 enzyme variants uses a non-mammalian expression system, e.g., a eukaryotic non-mammalian expression system such as Pichia pastoris. In embodiments, the Pichia pastoris encodes a DNase enzyme with a native signal peptide that allows for secretion from the host cell. In embodiments, the expression system is a mammalian cell expression system, such as Chinese hamster ovary (CHO) cells. In embodiments, the recombinant production method for the D1L3 enzyme variants further comprises isolating and / or purifying the D1L3 enzyme and modifying the isolated and / or purified D1L3 enzyme. In embodiments, the modification comprises conjugating the isolated and / or purified D1L3 enzyme to a polymer (e.g., but not limited to, PEG). In embodiments, the polymer is attached to a specific site using a desired conjugation chemistry (e.g., but not limited to, maleimide chemistry).

[0084] In another aspect, the present disclosure provides a method of treating a subject in need of extracellular chromatin degradation, extracellular trap (ET) degradation, and / or neutrophil extracellular trap (NET) degradation. The method comprises administering a therapeutically effective amount of the D1L3 enzyme or composition described herein. Exemplary indications in which a subject requires extracellular chromatin degradation (including ET or NET degradation) are disclosed in PCT / US18 / 47084, the disclosure of which is incorporated herein by reference.

[0085] Neutrophils, the primary leukocytes in acute inflammation, produce neutrophil extracellular traps (NETs), which are lattices of high-molecular-weight chromatin filaments decorated with biologically active proteins and peptides, to immobilize bacteria within wounds. When NETs accumulate throughout the body, they harm tissues and organs due to their cytotoxic, proinflammatory, and prothrombotic properties. Indeed, NETs are frequently associated with inflammatory, ischemic, and autoimmune diseases, including systemic lupus erythematosus (SLE).

[0086] In various embodiments, the present invention provides methods for treating, preventing, or managing diseases or conditions characterized by the presence or accumulation of NETs. See Jimenez-Alcazar et al., "Host DNases prevent vascular occlusion by neutrophil extracellular traps." Science 358(6367):1202-1206 (2017). A variety of stimuli, which may contribute to inflammation and / or pathogenesis, can cause NETs. These stimuli include the potent mitogen phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), the calcium ionophore A23187, the antibiotic nigericin, which also acts as a potassium ionophore, fungi such as Candida albicans, bacteria such as Streptococcus agalactiae (group B streptococcus) and Klebsiella pneumoniae, and viruses such as SARS-CoV2. Leppkes et al. “Vascular occlusion by neutrophil extracellular traps in COVID-19.”EBioMedicine 58(2020)102925(2020);Claushuis et al., “Role of peptidylarginine deiminase 4 in neutrophil extracellular trap formation and host defense during Klebsiella pneumoniae-induced pneumonia-derived sepsis.”J Immunol.201:1241-1252(2018); and Kenny et al., “Diverse stimuli engage different neutrophil extracellular trap pathways.”Elife.6:e24437(2017).Diseases or conditions characterized by the presence or accumulation of NETs include, but are not limited to, chronic neutrophilia, neutrophil aggregation and / or leukostasis, thrombosis and vascular occlusion, ischemia-reperfusion injury, surgical and traumatic tissue injury, acute or chronic inflammatory responses or diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, systemic inflammation, inflammatory diseases of the airways, renal inflammatory diseases, inflammatory diseases associated with transplanted tissue or hematopoietic stem cell transplantation (e.g., graft-versus-host disease), inflammation due to viral infections (e.g., COVID-19), and cancers (including leukemia). In various embodiments, the present invention provides methods for treating complete or partial vascular or vessel obstruction involving extracellular chromatin and containing NETs.

[0087] In some embodiments, the method comprises administering a composition described herein to a subject. In some embodiments, the subject is at risk for vascular obstruction involving extracellular chromatin, including, inter alia, chromatin released by cancer cells and damaged endothelial cells. Thus, in exemplary embodiments, the subject is afflicted with cancer (e.g., leukemia or a solid tumor). In some embodiments, the subject has a hematological cancer selected from multiple myeloma (MM), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL). In some embodiments, the subject has metastatic cancer.

[0088] Subjects undergoing cancer treatment (including but not limited to T cell therapy) are at risk for tumor lysis syndrome and / or cytokine release syndrome, which occurs when tumor cells release their contents (including chromatin) into the bloodstream. Tumor lysis syndrome is a complication that occurs during cancer treatment, which simultaneously kills large numbers of tumor cells. Tumor lysis syndrome and / or cytokine release syndrome commonly occur after treatment for lymphoma and leukemia. In various embodiments, the therapeutic methods described herein treat, reduce, or prevent tumor lysis syndrome.

[0089] In yet other embodiments, the subject suffers from an inflammatory disease of the respiratory tract, such as the lower respiratory tract. Exemplary diseases include bacterial and viral infections. In various embodiments, the subject suffers from acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, or asthma. Exemplary viral infections include RSV and coronavirus infections (such as SARS, or SARS-CoV-2, e.g., COVID-19, and variants thereof).

[0090] In yet other embodiments, the subject has a disease or condition other than cancer. In various embodiments, the disease or condition is an autoimmune or immune condition such as one selected from systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriasis, inflammatory bowel disease, celiac disease, pernicious anemia, scleroderma, Graves' disease, Sjogren's syndrome, autoimmune hemolytic anemia (AIHA), myasthenia gravis, cryoglobulinemia, thrombotic thrombocytopenic purpura (TTP), allograft rejection (e.g., lung, kidney, heart, intestine, liver, pancreas, etc. transplant rejection), pemphigus vulgaris, vitiligo, Hashimoto's disease, Addison's disease, reactive arthritis, and type 1 diabetes.

[0091] In various embodiments, the subject has SLE. The discovery of NETs has led to speculation that neutrophils may be a major source of autoantigens (i.e., dsDNA, chromatin) in SLE (Brinkmann, et al. Neutrophil Extracellular Traps Kill Bacteria. Science, 303(5663):1532-1545(2004)). Indeed, autoantibodies such as anti-dsDNA antibodies, anti-histone antibodies, and anti-nucleosome antibodies bind to NETs and form pathological ICs. See, e.g., Hakkim, et al., Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis, Proceedings of the National Academy of Sciences 107:9813-9818(2010)). The accumulation of NET-ICs activates adaptive immune cells, breaking immune tolerance and producing autoantibodies against NET components, creating a vicious cycle of inflammation and autoimmunity. See, for example, Gupta and Kaplan, "The role of neutrophils and NETosis in autoimmune and renal diseases." Nat Rev Nephrol. 12(7):402-13 (2016). Therefore, reducing NET accumulation could break this vicious cycle and represent an attractive therapeutic strategy for SLE.

[0092] In various embodiments, the present invention relates to treating a disease or condition characterized by a deficiency in D1L3 or a deficiency in D1. In some cases, the subject has a mutation (e.g., a loss-of-function mutation) in the Dnase1l3 gene or the Dnase1 gene. The patient may develop an autoimmune disease such as systemic lupus erythematosus (SLE), lupus nephritis, scleroderma or systemic sclerosis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and urticaria vasculitis. In some cases, the subject has an acquired inhibitor of D1 (e.g., anti-DNase1 antibodies and actin) and / or D1L3 (e.g., anti-DNase1l3 antibodies). The subject may also suffer from an autoimmune or inflammatory disease (e.g., SLE, systemic sclerosis). In some embodiments, the subject is treated with a D1L3 variant having a half-life extending moiety.

[0093] In various embodiments, the subject suffers from or is at risk for developing NETs that obstruct the ductal system. For example, the D1L3 enzyme or composition disclosed herein can be administered to a subject to treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye, Stevens-Johnson syndrome, blocked vas deferens, or kidney disease. For example, in various embodiments, a D1L3 variant that does not contain a half-life extending moiety is administered in the form of eye drops to a subject with dry eye disease.

[0094] In various embodiments, the subject has or is at risk of NET accumulation on an endothelial surface (e.g., surgical adhesions), skin (e.g., wounds / scars), or synovial joints (e.g., gout and arthritis, e.g., rheumatoid arthritis). The D1L3 enzymes and compositions described herein can be administered to a subject to treat conditions characterized by the accumulation of NETs on endothelial surfaces, such as, but not limited to, surgical adhesions.

[0095] Other diseases and conditions associated with NETs that the D1L3 enzyme or compositions disclosed herein can be used to treat or prevent include ANCA-associated vasculitis, asthma, chronic obstructive pulmonary disease, neutrophilic dermatitis, dermatomyositis, burns, cellulitis, meningitis, encephalitis, otitis media, pharyngitis, tonsillitis, pneumonia, endocarditis, cystitis, pyelonephritis, appendicitis, cholecystitis, pancreatitis, uveitis, keratitis, disseminated intravascular coagulation, acute kidney injury, acute respiratory distress syndrome, liver shock, hepatorenal syndrome, myocardial infarction, stroke, ischemic bowel syndrome, limb ischemia, testicular torsion, preeclampsia, eclampsia, and solid organ transplantation (e.g., kidney, heart, liver, and / or lung transplantation). Additionally, the D1L3 enzyme or compositions disclosed herein can be used to prevent scarring or contracture in individuals at risk of scarring or contracture, such as those who have suffered a surgical incision, laceration, or burn, for example, by topical application to the skin.

[0096] In various embodiments, a D1L3 variant of the present disclosure (e.g., one that does not include a half-life extending moiety) is administered to a subject suffering from or at risk of ischemic stroke. In various embodiments, the subject may further receive treatment with tissue plasminogen activator (tPA).

[0097] In various embodiments, the subject has a disease or condition that is being or has been treated with wild-type DNase, including D1 and streptodornase, including thrombosis, stroke, sepsis, lung injury, arteriosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary transplant failure, limb ischemia-reperfusion, kidney injury, blood clotting, alum-induced inflammation, hepatorenal injury, pleural effusion, hemothorax, intrabiliary thrombus, post-emphysematous anemia, ulcers, otorhinolaryngological disease, oral infection, minor trauma, and adverse events. These include sinusitis, post-rhinoplasty, infertility, bladder catheterization, wound irrigation, skin reaction testing, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartagener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary ciliary dyskinesia, bronchiolitis, empyema, pleural infection, cancer, dry eye, lower respiratory tract infection, chronic hematoma, Alzheimer's disease, and obstructive pulmonary disease.

[0098] In various embodiments, the subject has a loss-of-function mutation in one or both D1L3 genes and exhibits symptoms of SLE or may even be clinically diagnosed with SLE. In various embodiments, the composition is administered no more than about once a week, or no more than about once every two or three weeks, or no more than about once a month.

[0099] In some aspects, the present disclosure provides an expression construct for improving processing of a polypeptide precursor in a host, the expression construct comprising a signal peptide fused to the polypeptide via a linker. In some embodiments, the linker is at least three amino acids in length. In some embodiments, the signal peptide is completely removed from the polypeptide. In some embodiments, the signal peptide is not removed from the polypeptide. In some embodiments, the signal peptide is incompletely processed in the host in the absence of the linker.

[0100] In some embodiments, the polypeptide is or comprises an enzyme, cytokine, cytokine agonist, cytokine antagonist, hormone, hormone agonist, hormone antagonist, antibody or antigen-binding fragment thereof, antibody-like molecule or antigen-binding fragment thereof, antigen, vaccine component, fusion protein, or combination thereof. In some embodiments, the enzyme is selected from DNASE1 (D1), DNASE1-LIKE1 (D1L1), DNASE1-LIKE2 (D1L2), DNASE1-LIKE3 isoform 1 (D1L3), DNASE1-LIKE3 isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B), or variants thereof. In some embodiments, the cytokine is selected from IL-1, IL-2, IL-5, IL-6, IL-10 and IL-13, IL-12, CXCL8 (formerly known as IL-18), interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β), TNF-α, G-CSF, and GM-CSF.

[0101] In some embodiments, the hormone is selected from adrenocorticotropic hormone (ACTH), adropin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), exenatide, gastrin, ghrelin, glucagon, GLP-1, growth hormone, GIP, EPO, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, and vasoactive intestinal peptide (VIP). In some embodiments, the hormone or polypeptide comprises a half-life extending moiety, such as an albumin or Fc fusion described herein. In some embodiments, the fusion occurs at the C-terminus.

[0102] In some embodiments, the linker is at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15 or more amino acids in length. In some embodiments, the first amino acid residue of the N-terminal extension is not a GIy residue. In some embodiments, the last amino acid residue of the N-terminal extension is not a Ser residue. In some embodiments, the last amino acid residue of the N-terminal extension is not a polar or charged amino acid residue selected from Ser, Thr, Gln, Asn, Glu, Asp, Arg, His, and Lys. In some embodiments, the last amino acid residue of the N-terminal extension is an amino acid selected from GIy, Ala, and Val. In some embodiments, the last amino acid residue of the N-terminal extension is a GIy residue. In some embodiments, the first amino acid residue of the N-terminal extension is not a GIy residue and the last amino acid residue of the N-terminal extension is a GIy residue. In some embodiments, the N-terminal extension is predominantly, consists essentially of, or consists of Ser and Gly residues. In some embodiments, the linker comprises glycine, cysteine, and serine residues. In some embodiments, the linker comprises the amino acid sequence of SGGGG (SEQ ID NO: 60). In some embodiments, the linker comprises the amino acid sequence of CGGGG (SEQ ID NO: 74). In some embodiments, the linker comprises the amino acid sequence of SGGSGGSGG (SEQ ID NO: 61). In some embodiments, the linker comprises the amino acid sequence of SGGSGGSGGSGGSGGSGG (SEQ ID NO: 62). In some embodiments, the linker comprises the amino acid sequence of LVPRG (SEQ ID NO: 64). In some embodiments, the linker comprises the amino acid sequence of SGGGGLVPRGSGGGG (SEQ ID NO: 65).

[0103] In some embodiments, the signal peptide is the signal peptide of a naturally secreted protein. In some embodiments, the signal peptide is a chimeric or synthetic signal peptide that enables protein secretion. In some embodiments, the signal peptide is a prokaryotic signal peptide. In some embodiments, the signal peptide is a microbial signal peptide. In some embodiments, the signal peptide is a eukaryotic signal peptide. In some embodiments, the signal peptide is a yeast signal peptide. In some embodiments, the signal peptide is a mammalian signal peptide. Signal peptides suitable for secretion are disclosed in U.S. Patent Nos. 5,580,758, 6,107,057, 7,741,075, 10,435,694, 11,306,127, 11,370,815, and U.S. Patent Application Publication Nos. 2007 / 0117186, 2010 / 0055125, and 2016 / 0168198, the disclosures of each of which are incorporated herein by reference.

[0104] In some embodiments, the N-terminal extension and / or the junction between the N-terminal extension and the sequence derived from the polypeptide are non-immunogenic. In some embodiments, the last amino acid of the N-terminal extension is not a polar, charged, or aromatic amino acid. In some embodiments, the last two, or last three, or last four amino acids of the N-terminal extension are not polar, charged, and / or aromatic amino acids. In some embodiments, the N-terminal residue is not methionine (Met). In some embodiments, the last amino acid of the N-terminal extension is Gly or Ala. In some embodiments, the N-terminal extension comprises an amino acid bearing a chemical group suitable for chemical conjugation, the chemical group being selected from a thiol group, an amino group, an amide group, and a carboxyl group. In some embodiments, the chemical modification is site-specific PEGylation, glycosylation, etc. In some embodiments, the signal peptide is selected from DNASE1L3 (SEQ ID NO: 37), alpha mating factor (SEQ ID NO: 38), alpha mating factor presequence (SEQ ID NO: 39), human serum albumin (SEQ ID NO: 40), bovine DNASE1 (SEQ ID NO: 41), bovine DNASE1+Kex2 site (SEQ ID NO: 42), alpha amylase (SEQ ID NO: 43), glucoamylase signal peptide (SEQ ID NO: 44), inulinase (SEQ ID NO: 45), invertase (SEQ ID NO: 46), killer protein (SEQ ID NO: 47), and lysozyme (SEQ ID NO: 48).

[0105] In some embodiments, the signal peptide is selected from E. coli OmpA signal peptide (SEQ ID NO:56), E. coli DsbA signal peptide (SEQ ID NO:67), E. coli ST-II signal peptide (SEQ ID NO:68), E. coli FimD signal peptide (SEQ ID NO:55), Salmonella enterica DsbA signal peptide (SEQ ID NO:51), a synthetic Bordetella pertussis signal peptide (SEQ ID NO:57), a synthetic signal peptide sequence (e.g., SEQ ID NOs:49, 50, 52, 53, and 54). In some embodiments, the host is yeast or a cell line selected from a mammalian cell line and an insect cell line. In some embodiments, the host is Pichia pastoris. In some embodiments, the signal peptide is the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae (SEQ ID NO:38).

[0106] Other aspects and embodiments of the present invention will become apparent from the following examples. [Example]

[0107] Example 1. Optimization of N-terminal secretory signal peptide Expression of the D1L3 enzyme and D1L3-albumin fusion protein in Pichia pastoris is disclosed in PCT International Application Publication No. WO2020076817, which is incorporated herein by reference in its entirety. Briefly, the alpha mating factor (aMF) prepro-secretory leader (SEQ ID NO: 38) from Saccharomyces cerevisiae was used as the N-terminal secretory signal peptide. See Figure 1A. aMF is a general and powerful tool for heterologous protein expression in Pichia pastoris. As shown in Figure 1B, the combination of aMF with human D1L3 unexpectedly resulted in the non-processing of aMF and concomitant glycosylation.

[0108] In Figure 1B, D1L3 was properly processed when its N-terminus was guided by its native secretory signal peptide, but the native signal peptide of D1L3 resulted in a 3.5-fold decrease in expression titer compared to aMF.

[0109] A series of secretory signal peptides, including those of serum albumin, alpha amylase, glycoamylase, inulinase, invertase, killer protein, lysozyme, and bovine DNASE1, were screened for expression in Pichia pastoris. Additionally, a variant of the bovine DNASE1 secretory signal peptide containing a Kex2 cleavage site was tested. See U.S. Patent No. 7,118,901, the entire contents of which are incorporated herein by reference. Experiments also included the signal peptide of human DNASE1L3, as well as two versions of the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae.

[0110] Briefly, plasmids were synthesized using cDNAs encoding various signal peptides (SEQ ID NOS: 37-48) at the N-terminus of human DNASE1L3. The plasmids were transformed into Pichia pastoris cells by electroporation. Clonal cells were cultured, and the supernatants were analyzed by microfluidic capillary electrophoresis (mCE) ​​to characterize target protein expression. As shown in Table 1, no or low levels of DNASE1L3 expression were detected with all secretory signal peptides tested. The highest titer was observed with aMF (SEQ ID NOS: 38), whereas no DNASE1L3 levels were detected with SEQ ID NOS: 41 and 43-46. [Table 1]

[0111] Structural analysis of DNASE1L3 suggested that reduced access of the alpha mating factor cleavage enzyme Kex2 to the consensus cleavage sequence LEKR inhibits correct processing to mature DNASE1L3.

[0112] A novel secretory signal peptide was designed (Figure 2) containing the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae (SEQ ID NO: 38) and a linker sequence that facilitates access to the Kex2 cleavage site.

[0113] In pilot studies, flexible glycine-serine linker compositions ranging in length from 1 to 15 amino acids were tested. The alpha mating factor (αMF) prepro-secretory leader (SEQ ID NO: 38)-linker sequence from Saccharomyces cerevisiae was ligated to a DNASE1L3 mutant with a basic domain deletion. As shown in Table 2, a significant increase in expression titer was observed starting with a linker length of 5 amino acids. [Table 2]

[0114] DNASE1L3 was purified from the culture supernatant using affinity chromatography. Mass spectrometry analysis showed complete processing of the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae (SEQ ID NO: 38) and confirmed the predicted N-terminal amino acid sequence.

[0115] Unexpectedly, a mass shift of 210 Da was observed, as shown in Figure 3, which is believed to be due to myristoylation of the N-terminal glycine residue. A new set of linker sequences featuring an N-terminal serine residue was designed. Linker lengths ranged from 1 to 18 amino acids. As shown in Table 3, a significant increase in expression titer was observed when the linker length was 3 amino acids or longer. [Table 3]

[0116] As shown in Table 3, a significant increase in expression titer was observed starting from three amino acids. However, mCE and Western blot analysis suggested that a linker length of three amino acids resulted in incomplete processing of the Saccharomyces cerevisiae alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38). Use of a five-amino acid linker with the sequence SGGGG (SEQ ID NO: 60) resulted in increased expression levels and complete processing of the alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38) from Saccharomyces cerevisiae. All linkers greater than five amino acids in length, including linkers with the sequences SGGSGGSGG (SEQ ID NO: 61), SGGSGGSGSS (SEQ ID NO: 69), and SGGSGGSGGSGGSGGSGG (SEQ ID NO: 62), allowed both increased expression levels and complete processing of the alpha mating factor (αMF) prepro secretory leader.

[0117] The potential immunogenicity of the linker itself and the linker-D1L3 junction (e.g., SGGSGGSGSS-MRICSFNVRS (SEQ ID NO: 79), SGGGG-MRICSFNVRS (SEQ ID NO: 80), and SGGSGGSGG-MRICSFNVRS (SEQ ID NO: 81)) was analyzed using an in silico immunogenicity risk prediction algorithm. This analysis indicated that the serine residue at the SEQ ID NO: 79 junction contributed to a potential dominant epitope, shown in bold and underlined. Other linkers with terminal Gly residues did not generate similar dominant epitopes. These results indicate, among other things, that linkers for D1L3 expression should not terminate in Ser residues and possibly other polar residues, and that linkers for D1L3 expression may terminate in Gly residues to reduce immunogenicity risk.

[0118] The BDD_D1L3 enzyme (S283_S305del, SEQ ID NO: 13) was generated using a construct containing an alpha mating factor plus an SGGGG linker (SEQ ID NO: 60), yielding SEQ ID NO: 63, and analyzed for enzymatic activity against chromatin substrates. Briefly, DNASE1 (D1) and SEQ ID NO: 63 were produced in Pichia pastoris. Enzyme activity in the culture supernatant was characterized using degradation of high molecular weight (HMW) chromatin (i.e., nuclei purified from HEK293 cells) as a readout. Briefly, HMW chromatin was incubated with an equal amount of D1 or SEQ ID NO: 63. After incubation, DNA was separated and visualized by agarose gel electrophoresis (AGE). As shown in Figure 4, unlike D1, D1L3 S283_S305del, which has an N-terminal SGGGG, was observed to specifically and efficiently decompose HMW chromatin (approximately 50-300,000,000 base pairs) into nucleosomes (approximately 180 base pairs), the basic unit of the chromatin fiber, whereas no such effect was observed in samples containing D1. These results suggest that the N-terminal extension does not affect the enzymatic activity of the D1L3 enzyme.

[0119] Example 2. C-terminal optimization SEQ ID NO:63 was analyzed by mass spectrometry. As shown in Figure 5A, the resulting protein elution chromatogram contained a left shoulder relative to the main elution peak, indicating sample heterogeneity. To address sample heterogeneity, various derivatives of SEQ ID NO:63 were analyzed. A derivative with the S283_S305delins SSR mutation (i.e., a modified C-terminus containing a deletion of the basic domain and characterized by the addition of three amino acids at the C-terminus (SSR, SEQ ID NO:66)) was found to not produce sample heterogeneity (Figure 5B). These results suggest that the C-terminal extension eliminates the source of the observed heterogeneity. Enzymes containing N- and / or C-terminal extensions do not affect the enzymatic activity of the D1L3 enzyme.

[0120] These results were supported by studies of two DNASE1L3 mutants, each characterized by a wild-type C-terminal amino acid sequence, i.e., SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75), or a modified C-terminal amino acid sequence, i.e., SSR. The mutants were expressed in Pichia pastoris using alpha mating factor as a signal sequence in combination with an N-terminal SGGGG linker (SEQ ID NO: 60). Western blot analysis of the supernatants did not detect the theoretical mass difference of 2.3 kDa between the two mutants (e.g., as shown in Figure 7). Furthermore, intact mass analysis showed that the secreted DNASE1L3 mutants had identical masses, consistent with the theoretical mass of the DNASE1L3 mutant with the modified C-terminal amino acid sequence, i.e., SSR. In summary, these data suggest that the wild-type C-terminal amino acid sequence, i.e., SSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 75), is cleaved by Pichia pastoris during secretion to yield the modified C-terminal amino acid sequence, i.e., SSR. Pichia pastoris contains processing enzymes, such as Kex1 and Kex2, which have human counterparts, such as homologs of furin. Processing of the DNASE1L3 C-terminus is predicted to occur naturally during secretion in humans as well.

[0121] Example 3: Cysteine ​​Mutations Wild-type D1L3 contains an unpaired cysteine ​​at position 48 (relative to the mature protein sequence) (e.g., C48). Mutation of this cysteine ​​stabilizes D1L3 and prevents cross-linking to plasma proteins. We tested the effect of various amino acid substitutions at C48 on enzymatic activity. Enzymatic activity was characterized using digestion of high-molecular-weight (HMW) chromatin (i.e., nuclei purified from HEK293 cells). Briefly, HMW chromatin was incubated with an equal amount of D1L3 mutants. After incubation, DNA was separated and visualized by agarose gel electrophoresis (AGE). As shown in Figure 8, mutation of C48A or C48G was associated with increased enzymatic activity compared to the D1L3 mutants. These results suggest that removal of the unpaired cysteine ​​increases the enzymatic activity of the D1L3 enzyme.

[0122] Example 4. Dimerization of DNASE1L3 via unpaired cysteines Structural analysis of DNASE1L3 revealed that the unpaired C48 is located on the surface of the molecule. Western blot analysis of Pichia pastoris supernatants revealed that dimerization was observed in DNASE1L3 mutants with an unpaired C48, but not in mutants with mutated C48 (e.g., as shown in Figure 9). These data suggest that dimerization of DNASE1L3 may exist physiologically.

[0123] The possibility of inserting a new cysteine ​​into a D1L3 mutant with a mutated C48, for example to enable site-specific PEGylation, was tested. The BDD-D1L3 enzyme (A286_S305del) and wild-type D1L3 were generated using a construct containing an alpha mating factor plus a CGGGG linker (SEQ ID NO: 74). Four samples were compared. Sample 1 (SEQ ID NO: 70) was generated using a construct containing an alpha mating factor plus a SGGGG linker and contained a D1L3 mutant with a C48A substitution and a C-terminal extension with the sequence SSR. Samples 2-4 (SEQ ID NOs: 71-73, respectively) were generated using a construct containing an alpha mating factor plus a CGGGG linker (SEQ ID NO: 74) and contained D1L3 mutants with a C48A / S substitution and a C-terminal extension. Dimers were detected in the DNASE1L3 mutant containing the N-terminal cysteine. Analysis of the supernatant using anti-DNASE1L3 Western blotting under non-reducing conditions revealed dimerized BDD-D1L3 mutants in addition to D1L3 monomers (e.g., shown in Figure 6). No dimers were observed under reducing conditions or with constructs containing alpha mating factor + SGGGG linker. The data confirm that introduction of the CGGGG linker enables dimerization via a disulfide bridge at the N-terminal cysteine ​​residue. Similarly, N-terminal mutations to cysteine ​​residues, etc., allow for improved functionality, such as site-specific PEGylation.

[0124] array Wild-type human DNASES

[0125] SEQ ID NO: 1 [ka]

[0126] SEQ ID NO: 2 [ka]

[0127] SEQ ID NO: 3 [ka]

[0128] SEQ ID NO:4 [ka]

[0129] SEQ ID NO:5 [ka]

[0130] SEQ ID NO:6 [ka]

[0131] SEQ ID NO:7 [ka]

[0132] C-terminal deletion mutant of human DNASE1L3

[0133] SEQ ID NO:8 [ka]

[0134] SEQ ID NO:9 [ka]

[0135] SEQ ID NO: 10 [ka]

[0136] SEQ ID NO: 11 [ka]

[0137] SEQ ID NO: 12 [ka]

[0138] SEQ ID NO: 13 [ka]

[0139] SEQ ID NO: 14 [ka]

[0140] SEQ ID NO: 15 [ka]

[0141] SEQ ID NO: 16 [ka]

[0142] SEQ ID NO: 17 [ka]

[0143] SEQ ID NO: 63 [ka]

[0144] SEQ ID NO: 66 [ka]

[0145] SEQ ID NO: 70 [ka]

[0146] SEQ ID NO: 71 [ka]

[0147] SEQ ID NO:72 [ka]

[0148] SEQ ID NO: 73 [ka]

[0149] SEQ ID NO:77 [ka]

[0150] Linker sequence

[0151] SEQ ID NO: 18 GGGGS

[0152] SEQ ID NO: 19 GGGGSGGGGSGGGGS

[0153] SEQ ID NO: 20 APAPAPAPAPAPAP

[0154] SEQ ID NO: 21 AEAAAKEAAAKA

[0155] SEQ ID NO: 22 SGGSGSS

[0156] SEQ ID NO: 23 SGGSGGSGGSGGSGSS

[0157] SEQ ID NO: 24 SGGSGGSGGSGGSGGSGGSGGSGGSGGSGSS

[0158] SEQ ID NO: 25 GGSGGSGGSGGSGGSGGSGGSGGSGGSGS

[0159] Other arrays

[0160] SEQ ID NO: 26 Human serum albumin (mature protein): DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0161] SEQ ID NO: 27 Human factor XI: MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDDVHCHFFTYATRQF PSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVFCHSSFYHDTDFLGEELDIVAAK SHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPTQRHLCGGSIIGNQWILTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIII HDQYKMAESGYDIALLKLETTVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGGKDACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILEKTQAV

[0162] SEQ ID NO: 28 Human prekallikrein: MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTNNIRCQFFSYATQTFHK AEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQ ETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVS EGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA

[0163] Activatable linker sequence SEQ ID NO: 29 FXIIa-sensitive linker (Factor XI peptide): CTTKIKPRIVGGTASVRGEWPWQVT

[0164] SEQ ID NO: 30 FXIIa-sensitive linker GGGGSPRIGGGGS

[0165] SEQ ID NO: 31 FXIIa-sensitive linker (prekallikrein peptide): VCTTKTSTRIVGGTNSSWGEWPWQVS

[0166] SEQ ID NO: 32 FXIIa-sensitive linker (prekallikrein peptide): STRIVGG

[0167] SEQ ID NO: 64 Thrombin-sensitive linker 1: LVPRG

[0168] SEQ ID NO: 65 Thrombin-sensitive linker 2: SGGGGLVPRGSGGGG

[0169] BD-deleted D1L3 fusion protein SEQ ID NO: 33 [ka]

[0170] SEQ ID NO: 34 [ka]

[0171] SEQ ID NO:76 [ka]

[0172] SEQ ID NO:78 [ka]

[0173] Wild-type D1L3 fusion protein

[0174] SEQ ID NO: 35 [ka]

[0175] SEQ ID NO: 36 [ka]

[0176] signal peptide

[0177] SEQ ID NO: 37 DNASE1L3 MSRELAPLLLLLLSIHSALA

[0178] SEQ ID NO: 38 Alpha mating factor MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR

[0179] SEQ ID NO: 39 Alpha mating factor presequence EFETMRFPSIFTAVLFAASSALA

[0180] SEQ ID NO: 40 human serum albumin MKWVTFISLLFLFSSAYS

[0181] SEQ ID NO: 41 Bovine DNASE1 MRGTRLMGLLLALAGLLQLGLS

[0182] SEQ ID NO: 42 Bovine DNASE1+Kex2 site MRGTRLMGLLLALAGLLQLGLSLEKR

[0183] SEQ ID NO: 43 Alpha amylase EFETMRFPSIFTAVLFAASSALA

[0184] SEQ ID NO: 44 Glucoamylase signal peptide EFETMSFRSLLALSGLVCSGLA

[0185] SEQ ID NO: 45 Inulinase EFETMKLAYSLLLPLAGVSA

[0186] SEQ ID NO: 46 Invertase EFETMLLQAFLFLLAGFAAKISA

[0187] SEQ ID NO: 47 Killer Protein EFETMTKPTQVLVRSVSILFFITLLHLVVA

[0188] SEQ ID NO: 48 Lysozyme EFETMLGKNDPMCLVLVLLGLTALLGICQG

[0189] SEQ ID NO: 49 Synthetic E. coli signal peptide MKKNIAFLLALMFVFSIATNAYA

[0190] SEQ ID NO:50 Synthetic E. coli signal peptide MKKNIAFLLAIMFVFSIATNAYA

[0191] SEQ ID NO:51 Salmonella enterica DsbA signal peptide MKKIWLALAGIVLAFSASA

[0192] SEQ ID NO:52 Synthetic E. coli signal peptide MKKIWLALAGLVLAFSAYA

[0193] SEQ ID NO:53 Synthetic E. coli signal peptide MKKNIAFLLAAMFVFSIATNAYA

[0194] SEQ ID NO:54 Synthetic E. coli signal peptide MKKNILFLLLLMFVFSIATNAYA

[0195] SEQ ID NO: 55 E. coli FimD signal peptide MMTKIKLLMLIIFYLIISASAHA

[0196] SEQ ID NO:56 E. coli OmpA signal peptide MKKRARAIAIAVALAGFATVAHA

[0197] SEQ ID NO:57 Signal peptide from Bordetella pertussis MKKWFVAAGIGAGLLMLSSAA

[0198] SEQ ID NO: 67 E. coli DsbA signal peptide KKIWLALAGLVLAFSASA

[0199] SEQ ID NO: 68 E. coli ST-II signal peptide MKKNIAFLLASMFVFSIATNAYA

[0200] A linker that separates the signal peptide from the secreted protein

[0201] SEQ ID NO:58 GGGGS

[0202] SEQ ID NO:59 GGGGSGGGGSGGGGS

[0203] SEQ ID NO: 60 SGGGG

[0204] SEQ ID NO:74 CGGGG

[0205] SEQ ID NO: 61 SGGSGGSGG

[0206] SEQ ID NO: 62 SGGSGGSGGSGGSGGSGG

[0207] SEQ ID NO: 69 SGGSGGSGSS

[0208] C-terminal basic domain SEQ ID NO: 75 SSRAFTNSKKSVTLRKKTKSKRS

Claims

1. A variant of DNASE1-LIKE3 comprising an N-terminal extension of at least 4 amino acids and up to about 18 amino acids relative to SEQ ID NO:4 (D1L3 variant).

2. 2. The D1L3 variant of claim 1, wherein the N-terminal amino acid is not Met.

3. 3. The D1L3 mutant of claim 1 or 2, wherein the N-terminal extension comprises predominantly Gly residues.

4. 4. The D1L3 variant of any one of claims 1 to 3, wherein the D1L3 variant comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to amino acids 21 to 282 of SEQ ID NO:4 or amino acids 21 to 252 of SEQ ID NO:

5.

5. 5. The D1L3 variant of any one of claims 1 to 4, wherein the D1L3 variant is generated by cleavage of an N-terminal signal peptide selected from SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:

48.

6. 6. The D1L3 variant of claim 5, wherein the signal peptide is the Saccharomyces cerevisiae alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38).

7. 7. The D1L3 variant of any one of claims 1 to 6, wherein the N-terminal extension is at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15 amino acids in length.

8. 8. The D1L3 variant of claim 7, wherein the N-terminal extension consists essentially of Ser and Gly residues or consists of Ser and Gly residues.

9. 9. The D1L3 variant of claim 8, wherein the N-terminal extension and / or the junction between the N-terminal extension and the D1L3-derived sequence is non-immunogenic.

10. 10. The D1L3 variant of any one of claims 7 to 9, wherein the N-terminal extension comprises an amino acid having a chemical group suitable for chemical conjugation, the chemical group being selected from a thiol group, an amino group, an amide group, and a carboxyl group.

11. 11. The D1L3 variant of any one of claims 7 to 10, wherein the N-terminal extension has the amino acid sequence SGGGG (SEQ ID NO: 60) or CGGGG (SEQ ID NO: 74).

12. The D1L3 mutant of any one of claims 1 to 11, wherein the N-terminal extension does not include a consensus sequence for myristoylation.

13. 13. The D1L3 mutant of any one of claims 1 to 12, further comprising a C-terminal extension that reduces heterogeneity.

14. 14. The D1L3 variant of claim 13, wherein the C-terminal extension is at least 2, or at least 3, or at least 4, or at least 5 amino acids in length.

15. The D1L3 mutant according to any one of claims 1 to 14, wherein the C-terminal amino acid is Lys or Arg.

16. 16. The D1L3 variant according to any one of claims 13 to 15, wherein the C-terminal extension comprises or consists of the amino acid sequence SSR.

17. 17. The D1L3 mutant of any one of claims 1 to 16, wherein the D1L3 mutant comprises a basic domain.

18. 18. The D1L3 variant of any one of claims 1 to 17, wherein the D1L3 variant comprises a deletion of at least 3, or at least 5, or at least 8, or at least 10, or at least 12, or at least 15, or at least 18, or at least 20, or all 23 amino acids of the C-terminal basic domain (BD) defined by amino acids 283 to 305 of SEQ ID NO:

4.

19. 19. The D1L3 mutant of any one of claims 1 to 18, wherein the D1L3 mutant is configured to form a dimer via an unpaired Cys, and the unpaired Cys is optionally C48 with respect to SEQ ID NO:

4.

20. 19. The D1L3 variant of any one of claims 1 to 18, wherein the D1L3 variant comprises a substitution of C48 and / or C174 with respect to SEQ ID NO:

4.

21. 21. The D1L3 mutant of claim 20, wherein the mutation is selected from C48S, C48G, C48A, C174S, C174G and C174A with respect to SEQ ID NO:

4.

22. 22. The D1L3 variant of claim 21, wherein the substitution is C48A with respect to SEQ ID NO:

4.

23. 23. The D1L3 variant according to any one of claims 1 to 22, wherein the C-terminal extension comprises or consists of the amino acid sequence SSR.

24. 24. The D1L3 variant of any one of claims 1 to 23, comprising one or more mutations that result in resistance to proteolysis by one or more of plasmin, thrombin, trypsin, and proteases produced by mammalian and non-mammalian cell lines.

25. The D1L3 mutant of claim 24, wherein the D1L3 mutant has one or more mutations of amino acid residues selected from K180, K200, K259, and R285 with respect to SEQ ID NO:

4.

26. 26. The D1L3 mutant of claim 24 or 25, wherein the D1L3 mutant has one or more mutations in amino acid residues selected from R22, R29, K45, K47, K74, R81, R92, K107, K176, R212, R226, R227, K250, K259, and K262 with respect to SEQ ID NO:

4.

27. 27. The D1L3 mutant according to claim 25 or claim 26, wherein the D1L3 mutant has one or more mutations of amino acid residues selected from S91C, S131C, and S253C with respect to SEQ ID NO:

4.

28. 2. The D1L3 variant of claim 1, having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO:

77.

29. 29. The D1L3 variant of claim 28, having the amino acid sequence of SEQ ID NO:

70.

30. 30. The D1L3 variant of any one of claims 1 to 29, wherein the D1L3 variant comprises a fusion or conjugation to a half-life extending moiety.

31. 31. The D1L3 variant of claim 30, wherein the half-life extending moiety is a polymer.

32. 32. The D1L3 variant of claim 31, wherein the polymer is polyethylene glycol (PEG).

33. 33. The D1L3 variant of claim 32, wherein the PEG polymer is attached to the N-terminus.

34. 34. The D1L3 variant of claim 33, wherein the PEG polymer links two D1L3 variant molecules through their N-termini.

35. 34. The D1L3 variant of any one of claims 31 to 33, wherein a PEG polymer is attached to one or more amino acids within positions corresponding to R95 to V126 of SEQ ID NO:

4.

36. 36. The D1L3 variant of claim 35, wherein the one or more PEGylated amino acids are selected from lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine, and optionally, the one or more PEGylated amino acids are introduced by substitution of one or more amino acids between R95 and V126 relative to SEQ ID NO:

4.

37. 37. The D1L3 variant of claim 36, wherein the one or more amino acids are: (a) PEGylation of lysine (Lys or K) via an amine bond (b) PEGylation of glutamine by enzymatic conjugation via transglutaminase (TGase), and / or (c) PEGylation of cysteine ​​(Cys or C) via thiol linkage; The D1L3 variant is PEGylated by

38. 38. The D1L3 variant of any one of claims 32-37, wherein one or more PEGylated amino acids are conjugated to a PEG moiety independently selected from linear or branched PEGs having molecular weights ranging from about 2 kDa to about 60 kDa, or from about 5 kDa to about 30 kDa.

39. 31. The D1L3 variant of claim 30, wherein the half-life extending moiety is a fusion partner.

40. 40. The D1L3 variant of claim 39, wherein the fusion partner is selected from albumin, transferrin, Fc, or elastin-like proteins, XTEN sequences, or variants thereof.

41. 41. The D1L3 variant of claim 40, wherein the fusion partner is albumin.

42. 42. The D1L3 variant of claim 41, wherein the fusion partner is human albumin comprising an amino acid sequence at least 80% identical to SEQ ID NO:

26.

43. 43. The D1L3 variant of claim 42, wherein the human albumin comprises E505Q, T527M, K573P substitutions with respect to SEQ ID NO:

26.

44. 44. The D1L3 variant of any one of claims 39 to 43, wherein the fusion partner is fused N-terminally to the mature D1L3 enzyme.

45. 45. The D1L3 variant of any one of claims 39 to 44, wherein the fusion partner is fused to the mature D1L3 enzyme via a linker flanking the fusion partner and the mature D1L3 enzyme.

46. 46. ​​The D1L3 variant of claim 45, wherein the linker is about 5 to about 50 amino acids in length, or about 10 to about 35 amino acids in length, or about 15 to 35 amino acids in length.

47. 47. The D1L3 mutation of claim 46, wherein the linker has the amino acid sequence S(GGS) ４ GSS (SEQ ID NO: 23), S(GGS) ９ GSS (SEQ ID NO: 24), and (GGS) ９ The D1L3 variant, comprising GS (SEQ ID NO: 25).

48. 48. The D1L3 variant of claim 46 or 47, wherein the D1L3 variant comprises the amino acid sequence of SEQ ID NO: 76 or SEQ ID NO:

78.

49. 47. The D1L3 variant of claim 45 or 46, wherein the linker is a flexible or rigid linker and / or comprises a protease cleavage site.

50. 50. The D1L3 variant of claim 49, wherein the linker is cleavable by a coagulation pathway protease.

51. 51. The D1L3 variant of claim 50, wherein the protease is factor XII or a neutrophil protease.

52. 52. The D1L3 variant of claim 51, wherein the protease is thrombin.

53. 52. The D1L3 variant of claim 50 or 51, wherein the protease cleavage site comprises the amino acid sequence LVPRG (SEQ ID NO: 64), and optionally SGGGGLVPRGSGGGG (SEQ ID NO: 65).

54. A method for expressing the DNase 1-like3 mutant according to any one of claims 1 to 53, comprising: introducing a gene construct encoding the DNase 1-like 3 mutant containing a signal peptide into a yeast cell; and recovering the DNase 1-like 3 mutant; The method comprising:

55. 55. The method of claim 54, wherein the yeast cell is Pichia pastoris.

56. 56. The method of claim 54 or claim 55, wherein the signal peptide is the alpha mating factor (αMF) prepro secretory leader from Saccharomyces cerevisiae (SEQ ID NO: 38).

57. 54. An isolated polynucleotide encoding the D1L3 variant of any one of claims 1 to 53.

58. 58. The isolated polynucleotide of claim 57, wherein the polynucleotide is mRNA or modified mRNA (mmRNA).

59. 59. The isolated polynucleotide of claim 58, wherein the polynucleotide is DNA.

60. A vector for introducing the polynucleotide according to any one of claims 57 to 59 into a host cell.

61. 61. A host cell comprising the vector of claim 60.

62. A pharmaceutical composition comprising an effective amount of a D1L3 variant described in any one of claims 1 to 53, or the D1L3 variant produced according to the method of any one of claims 54 to 56, or a polynucleotide described in any one of claims 57 to 59, or a vector described in claim 60, or a host cell described in claim 61, and a pharmaceutically acceptable carrier.

63. 63. A pharmaceutical composition according to claim 62, comprising a D1L3 variant according to any one of claims 1 to 53 and a pharmaceutically acceptable carrier for parenteral administration.

64. 63. The pharmaceutical composition of claim 62, formulated for ophthalmic or pulmonary administration.

65. 64. The pharmaceutical composition of claim 63, formulated for intradermal, intramuscular, intravenous, subcutaneous, or intra-arterial administration.

66. 66. A method of treating a subject in need of extracellular chromatin degradation, comprising administering to a subject in need thereof the pharmaceutical composition of any one of claims 63 to 65.

67. 67. The method of claim 66, wherein the subject is in need of extracellular trap (ET) degradation and / or neutrophil extracellular trap (NET) degradation.

68. 68. The method of claim 66 or 67, wherein the subject has a loss-of-function mutation in one or both D1L3 genes.

69. 69. The method of any one of claims 67 or 68, wherein the subject has SLE.

70. 70. The method of any one of claims 66-69, wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukostasis, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, acute or chronic inflammatory response or disease, autoimmune disease, cardiovascular disease, metabolic disease, systemic inflammation, respiratory inflammatory disease, renal inflammatory disease, inflammatory disease associated with transplant tissue, and cancer.

71. 70. The method of any one of claims 66-69, wherein the subject has or is at risk of having a NET that obstructs the ductal system, the condition being any selected from pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye, blocked vas deferens, and kidney disease.

72. 72. The method of any one of claims 66 to 71, wherein the subject has or is at risk of having NETs that accumulate on the endothelial surface.

73. An expression construct for improving processing of a polypeptide precursor in a host, said expression construct comprising a signal peptide fused to said polypeptide via a linker at least 3 amino acids in length.

74. 74. The expression construct of claim 73, wherein the signal peptide is incompletely processed in the host in the absence of the linker.

75. 75. The expression construct of claim 73 or claim 74, wherein the polypeptide is selected from an enzyme, a cytokine, a hormone, an antibody or antigen-binding fragment thereof, an antibody-like molecule or antigen-binding fragment thereof, an antigen, a component of a vaccine, a fusion protein, and combinations thereof.

76. 76. The expression construct of claim 75, wherein the enzyme is selected from DNASE1 (D1), DNASE1-LIKE1 (D1L1), DNASE1-LIKE2 (D1L2), DNASE1-LIKE3 isoform 1 (D1L3), DNASE1-LIKE3 isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B), or a variant thereof.

77. 77. The expression construct of any one of claims 73 to 76, wherein the linker is at least 5 amino acids in length, at least 9 amino acids in length, or at least 12 amino acids in length and is predominantly serine and glycine residues.

78. 78. An expression construct according to any one of claims 73 to 77, wherein the N-terminal extension and / or the junction between the N-terminal extension and the D1L3-derived sequence is non-immunogenic.

79. 79. The expression construct of any one of claims 73 to 78, wherein the N-terminal extension comprises an amino acid having a chemical group suitable for chemical conjugation, the chemical group being selected from a thiol group, an amino group, an amide group, and a carboxyl group.

80. 80. The expression construct of any one of claims 73 to 79, wherein the signal peptide is selected from DNASE1L3 (SEQ ID NO:37), alpha mating factor (SEQ ID NO:38), alpha mating factor presequence (SEQ ID NO:39), human serum albumin (SEQ ID NO:40), bovine DNASE1 (SEQ ID NO:41), bovine DNASE1+Kex2 site (SEQ ID NO:42), alpha amylase (SEQ ID NO:43), glucoamylase signal peptide (SEQ ID NO:44), inulinase (SEQ ID NO:45), invertase (SEQ ID NO:46), killer protein (SEQ ID NO:47), and lysozyme (SEQ ID NO:48).

81. 81. The expression construct of any one of claims 73 to 80, wherein the host is a yeast or a cell line selected from a mammalian cell line and an insect cell line.

82. 82. The expression construct of claim 81, wherein the host is Pichia pastoris.

83. 83. The expression construct of claim 82, wherein the signal peptide is the Saccharomyces cerevisiae alpha mating factor (αMF) prepro secretory leader (SEQ ID NO: 38).

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