ENGINEERED DNASE ENZYMES AND USE IN THERAPY
The present invention provides engineered DNase proteins (including DNase1-like 3 and DNase1) that are useful for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and/or release. In some aspects, the invention provides compositions and methods for preventing or treating vascular occlusion involving NETs. As demonstrated herein, NETs participate in a non-canonical mechanism for vascular occlusion, which is not dependent on fibrin or platelets.
This application claims the benefit of, and priority to, US Application Nos. 62/611,166 filed Dec. 28, 2017 and 62/547,220 filed Aug. 18, 2017, each of which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTIONInflammation is an essential host response to control invading microbes and heal damaged tissues. Uncontrolled and persistent inflammation causes tissue injury in a plethora of inflammatory disorders. Neutrophils are the predominant leukocytes in acute inflammation. During infections neutrophils generate neutrophil extracellular traps (NETs), lattices of DNA-filaments decorated with toxic histones and enzymes that immobilize and neutralize bacteria. The relevance of NETs in host defense is illustrated by the fact that extracellular DNases serve as virulence factors in several pathogenic bacteria. However, inappropriately released NETs may harm host cells due to their cytotoxic, proinflammatory, and prothrombotic activity. Indeed, NETs are frequently associated with inflammatory or ischemic organ damage and the therapeutic infusion of DNases limits host injury in various animal models.
Extracellular trap (ET) formation is not limited to neutrophils as other leukocytes, namely monocytes, macrophages, basophils, eosinophils, and mast cells release ETs as well. Furthermore, cancer cells, including acute promyelocytic leukemia (APL) cells, and injured endothelial cells can expose DNA-filaments, which have ET-like features.
How the host degrades NETs in vivo to limit tissue damage during episodes of inflammation is poorly understood. DNase1 (D1) in serum has been shown to digest the DNA-backbone of NETs in vitro. Other extracellular DNases have been identified, including DNase1-like 3 (D1L3) in circulation, which can digest extracellular microparticle-associated chromatin.
D1 and D1L3 form along with DNase1-like 1 (D1L1) and DNase1-like 2 (D1L2) the DNase1-protein family, a group of homologous secreted DNase enzymes that are expressed in humans and provide drug candidates for NET-associated diseases. However, the physical, enzymatic, and pharmacokinetic properties of these enzymes are not ideal for therapy.
The present invention provides engineered DNases, including DNase1-like 3 and DNase1, for therapy, including for preventing or treating vascular occlusions that can result from release of NETs during acute inflammatory episodes.
SUMMARY OF THE INVENTIONThe present invention provides engineered DNase proteins (including DNase1-like 3 and DNase1) that are useful for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and/or release. In some aspects, the invention provides compositions and methods for preventing or treating vascular occlusion involving NETs. As demonstrated herein, NETs participate in a non-canonical mechanism for vascular occlusion, which is not dependent on fibrin and platelets.
In some aspects, the invention provides DNase1-like 3 (D1L3) variants engineered to have physical, pharmacodynamic, and/or enzymatic properties more suitable for therapy, for example, to reduce or prevent NET accumulation in a subject. In some embodiments, the D1L3 enzyme has advantages in manufacturing, providing for production of the recombinant enzyme suitable for use in therapy. In various embodiments, the invention provides a recombinant D1L3 variant comprising one or more amino acid alterations resulting in one or more glycosylations, inactivation of a nuclear localization signal, and/or deletion of all or part of a C-terminal tail.
In various embodiments, the D1L3 protein variant has increased protein stability, increased resistance towards inhibition by proteases, increased bioavailability, and substantially the same or better DNA and/or chromatin and/or NET-degrading activity (in vitro or in vivo) as compared to wild-type D1L3 protein. In various embodiments, the D1L3 variant comprises one or more of the following properties relative to wild type D1L3 (e.g., SEQ ID NO:2): the same or substantially the same (or higher) protein-free DNA (naked DNA) degradation activity, the same or substantially the same (or higher) chromosomal DNA (chromatin) degradation activity, protease resistance, an increased circulatory half-life, and higher production levels with in vitro expression systems (e.g. Chinese hamster ovary cells and/or Pichia pastoris).
In some embodiments, the D1L3 variant includes one or a plurality of block substitutions from human DNase1 (SEQ ID NO:1), described herein as building block substitutions.
In some aspects, the invention provides DNase1 (D1) variants engineered to have physical, pharmacodynamic, and/or enzymatic properties more suitable for therapy, for example, to reduce or prevent NET accumulation in a subject. In some embodiments, the D1 enzyme has advantages in manufacturing, providing for production of the recombinant enzyme suitable for use in therapy. In various embodiments, the engineered D1 variant comprises an amino acid sequence that is at least 80% identical to the mature enzyme of amino acid sequence of SEQ ID NO:1, with one or more amino acid substitutions, additions (including insertions), or deletions resulting in one or more of a mutated DNA binding site, a mutated chromatin binding site, a mutated actin binding site, a mutated glycosylation site, addition of a nuclear localization signal (e.g., having similarity or identity to NLS1 or NLS2 of D1L3), and/or a C-terminal domain similar to or identical to the C-terminal tail of D1L3.
In various embodiments, the D1 variant is engineered to comprise one or more of the following characteristics relative to the wild type enzyme: the same or higher protein-free DNA (naked DNA) degradation activity, higher chromosomal DNA (chromatin) degradation activity, similar or improved protease resistance, actin resistance, similar or improved penetration from blood into urine and/or bile, and higher production levels in vitro expression systems (e.g. Chinese hamster ovary cells and/or Pichia pastoris).
D1 variants described herein may have a combination of point mutations, including addition of cationic residues, and/or may comprise one or more block substitutions from D1L3 (SEQ ID NO:2). Such block substitutions are described herein, and termed “building block substitutions.”
In some aspects, the invention provides pharmaceutical compositions comprising the D1L3 and/or D1 variants, or polynucleotides or vectors encoding the same, and a pharmaceutically acceptable carrier. In various embodiments, the composition is formulated for parenteral or pulmonary administration. In some embodiments, the composition is formulated for intravenous, intraarterial, intraperitoneal, intraarticular, intramuscular, topical, or subcutaneous administration. In some embodiments, the composition comprises both D1L3 and D1, which are each optionally engineered variants described herein. In some embodiments, the composition comprises a chimeric protein containing block sequences from both D1 and D1L3. Exemplary block substitutions between D1 and D1L3 are shown in
In still other aspects, the invention provides a process for DNase enzyme engineering, by producing chimeric sequences. For example, in these aspects the method comprises providing a protein-protein alignment of donor and recipient DNase enzymes; identifying variable amino acid sequences for transfer (“building block”). The variable amino acid or amino acids are flanked by one or more conserved amino acids in the donor and recipient DNase enzymes (upstream and downstream of the building block). These building blocks can be swapped between receipient and donor proteins. The chimeric enzyme is then produced recombinantly.
In still other aspects, the invention provides a method of making a pharmaceutical composition for reducing or preventing neutrophil extracellular trap (NET) accumulation. In these embodiments, the invention employs a genetically modified mouse deficient in D1 and D1L3 activity, and heterologous expression of G-CSF polynucleotide (e.g., in hepatocyte cells) or inducing a sustained endogenous G-CSF expression (e.g. via repetitive administration of microbial compounds). This mouse model accumulates NETs and rapidly develops NET-related vascular occlusions. In these embodiments, the invention comprises administering a candidate NET inhibitor or candidate DNase enzyme (including a D1L3 or D1 variant in accordance with this disclosure) to the genetically-modified mouse, and selecting a NET inhibitor or DNase enzyme that reduces the accumulation of NETs. The selected inhibitor or enzyme is formulated for administration to a human patient.
In some aspects, the invention provides a method for treating a subject in need of neutrophil extracellular trap (NET) degradation. The method comprises administering a therapeutically effective amount of a D1L3 and/or D1, and/or variants thereof according to this disclosure. The D1L3 or D1 variant may be administered as pharmaceutical compositions comprising the recombinant protein, or in some embodiments the encoding DNA or RNA, or in some embodiments, vectors comprising the same.
In various embodiments, the present invention pertains to the treatment of diseases or conditions characterized by the presence or accumulation of NETs. Such diseases or conditions include, but are not limited to, diseases associated with chronic neutrophilia (e.g., an increase in the number of neutrophils), neutrophil aggregation and leukostasis, thrombosis and vascular occlusion (e.g. sickle cell disease), ischemia-reperfusion injury (e.g. midgut volvulus, testicular torsion, limb ischemia reperfusion, vital organ ischemia-reperfusion, organ transplantation), surgical and traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease (e.g. systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, vasculitis, systemic sclerosis), cardiovascular disease (e.g., myocardial infarction, stroke, atherosclerosis, venous thromboembolism, including thrombolytic therapy), metabolic disease (e.g., diabetes), systemic inflammation (e.g., systemic inflammatory response syndrome (SIRS), sepsis, septic shock, disseminated intravascular coagulation (DIC), and thrombotic microangiopathy (TMA)), inflammatory diseases of the respiratory tract (e.g. cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), smoke induced lung injury, transfusion induced lung injury (TRALI), acute respiratory distress syndrome (ARDS), and asthma, atelectasis, bronchitis, empyema), renal inflammatory diseases (acute and chronic kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), inflammatory diseases related to transplated tissue (e.g. graft-versus-host disease) and cancer (e.g. leukemia, tumor metastasis, and solid tumors).
In some embodiments, the subject has or is at risk of a ductal occlusion in a ductal system. Non-limiting examples of a ductal system or an organ or tissue containing a ductal system include bile duct, tear duct, lactiferous duct, cystic duct, hepatic duct, ejaculatory duct, parotid duct, submandicular duct, major sublingual duct, bartholin's duct, cerebral aqueduct, pancreas, mammary gland, vas deferens, ureter, urinary bladder, gallbladder, and liver. As such, the present invention is useful for treating a subject who has pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, an obstruction of the vas deferens, or renal disease.
In other embodiments, the subject has or is at risk of NETs accumulating on endothelial surfaces (e.g., surgical adhesions), the skin (e.g., wounds/scarring, uclers), or in synovial joints (e.g., gout, arthritis). For instance, NETs may contribute to surgical adhesions, e.g., after an invasive medical procedure. The present invention can be administered during surgery to prevent or inhibit the formation of surgical adhesions. In some instances, D1, D1L3 (including variants thereof), or the combination of D1 and D1L3 (or variants thereof) as outlined herein can be administered topically to the skin to prevent or treat wounds and/or scarring. In other instances, D1, D1L3, or variants, or the combinations thereof as outlined herein can be administered to synovial joints to prevent or treat gout and arthritis.
In some embodiments, the subject has or is at risk of a vascular occlusion comprising NETs.
In various embodiments, the present invention pertains to the treatment of diseases that are treatable with DNase enzyme, including D1 and streptodornase. Such diseases or conditions include thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia reperfusion, kidney injury, blood clotting, alum-induced inflammation, hepatorenal injury, pleural exudations, hemotorax, intrabiliary blood clots, post pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, post-operative rhinoplasties, infertility, bladder catheter, wound cleaning, skin reaction test, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary cilliary dyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyes disease, lower respiratory tract infections, chronic hematomas, Alzheimer's disease, and obstructive pulmonary disease.
In still other embodiments, the invention provides a method for recombinant production of D1 or D1L3, or variants thereof, using a non-mammlian expression system, such as Pichia pastoris. In some embodiments, the Pichia pastoris encodes the DNase enzyme with the native signal peptide allowing for secretion from host cells.
Other objects, embodiments and advantages of the present invention are apparent in the Detailed Description below.
The present invention provides engineered DNase proteins, members of the DNase1-protein family (including DNase1-like 3 and DNase1) that are useful for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and/or release. In some aspects, the invention provides compositions and methods for preventing or treating vascular occlusion involving NETs. As demonstrated herein, NETs participate in a non-canonical mechanism for vascular occlusion, which is not dependent on fibrin and platelets.
As used herein, the term “neutrophil extracellular trap” or “NET” refers to any extracellular trap (“ET”) comprising extracellular DNA formed by cells such as, but not limited to, neutrophils, monocytes, macrophages, basophils, eosinophils, mast cells, cancer cells, injured cells (e.g., injured endothelial cells), and the like. Unless the context indicates otherwise, the terms NET and ET are used interchangeably herein.
There are two extracellular DNase enzymes found in the circulation of humans and mice, DNase1 (D1) and DNase1-like 3 (D1L3). These enzymes can be deficient in their ability and mechanism to degrade NETs in vitro and in vivo, and are found at relatively low concentrations in humans and mice. The natural enzymes do not have ideal physical, enzymatic, and/or pharmacodynamic properties for recombinant therapy, and particularly for systemic therapy.
D1 and D1L3 belong, along with DNase1-like 1 (D1L1) and DNase1-like 2 (D1L2), to the DNase1-protein family. D1 and D1L3 are expressed in a variety of species including, humans, primates, and rodents. In humans and mice, D1 and D1L3 show a protein similarity of 49-52%. However, despite their homology, D1 and D1L3 differ in cellular origin, sensitivity towards inhibitors, and substrate affinity. D1 preferentially cleaves protein-free DNA (e.g. bacterial DNA, plasmid DNA), whereas D1L3 targets chromatin, the complex of DNA and histones, which is commonly found in the nucleus of eukaryotic cells. D1 activity is inhibited upon binding to monomeric actin and sensitive to physiological salt concentrations. In addition, D1 is glycosylated at N40 (corresponds to N18 in the mature enzyme without signal peptide) and N128 (N106), which makes the enzyme resistant to inactivation by serum proteases. In contrast, D1L3 lacks glycosylation and actin-binding sites, which causes its susceptibility towards several proteases and resistance towards actin, respectively.
In some aspects, the invention provides D1L3 variants engineered to have physical, pharmacodynamic, and/or enzymatic properties more suitable for therapy, for example, to reduce or prevent NET accumulation in a subject. In various embodiments, the invention provides a recombinant D1L3 protein variant comprising: one or more glycosylations, one or more amino acid alterations resulting in increased substrate affinity, inactivation of a nuclear localization signal, deletion of all or part of a C-terminal tail, pegylation, and fusion to a carrier protein or moiety. In various embodiments, the D1L3 variant has increased protein stability, increased protease-resistance, increased bioavailability, increased half-life in circulation, and/or substantially the same or better DNA and/or chromatin and/or NET-degrading activity, higher production levels with in vitro expression systems (e.g. Chinese hamster ovary cells and/or Pichia pastoris) as compared to wild-type D1L3 protein of SEQ ID NO:2.
In various embodiments, the D1L3 variant comprises an amino acid sequence with at least 80% sequence identity to the enzyme defined by the amino acid sequence of SEQ ID NO:2, with one or more amino acid modifications with respect to SEQ ID NO:2 as described herein. In some embodiments, D1L3 variant comprises an amino acid sequence with at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity with with the DNase enzyme defined by SEQ ID NO:2.
As used herein, when referring to sequence identity with wild-type DNase enzymes, and unless stated otherwise, sequences refer to mature enzymes lacking the signal peptide. Further, unless stated otherwise, amino acid positions are numbered with respect to the full translated DNase sequence, including signal peptide, for clarity. Accordingly, for example, reference to sequence identity to the enzyme of SEQ ID NO:2 refers to a percent identity with the mature enzyme having M21 at the N-terminus. Similarly, reference to sequence identity to the enzyme of SEQ ID NO:1 (human D1) refers to a percent identity with the mature enzyme having L23 at the N-terminus.
The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments as known in the art. Such alignments can be carried out with several art-known algorithms, such as with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80). Exemplary algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410. When utilizing BLAST programs, the default parameters of the respective programs are used.
In some embodiments, the recombinant D1L3 protein variant comprises one or more glycosylation consensus sequences, e.g., NX(T/S). The human wild type enzyme (SEQ ID NO:2) does not contain a consensus glycosylation sequence. However, engineering a glycosylation consensus sequence into D1L3 can provide for glycosylation (and protease resistance) without substantial impact on the enzyme activity. In some embodiments, the glycosylation consensus sequence includes a D38N substitution and an N40T or N40S substitution, and/or an A127N substitution and a V129T or V129S substitution from the wild type sequence (e.g., SEQ ID NO:2). In some embodiments, the recombinant D1L3 enzyme has one, two, three, or four glycosylation sites. The consensus sequence can be introduced into the protein at a location that does not affect its enzymatic activity, and in some embodiments is located near portions of the enzyme that are important for maintaining structure of the enzyme (e.g., such as within amino acids 25 to 289, and in some embodiments, within amino acids 24 to 150).
In some embodiments, the invention provides a DNase enzyme comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the enzyme defined by SEQ ID NO:2, wherein the enzyme has a substitution of asparagine at the position corresponding to position 38 and/or position 127 of SEQ ID NO:2, and the N38 and/or N127 is glycosylated. In some embodiments, the substitution at position 38 and/or 127 is made via a building block substitution from SEQ ID NO:1 as described herein.
In various embodiments, D1L3 variants having one or more glycosylations exhibit increased resistance to serum protease degradation and/or an increased serum half-life compared to wild-type D1L3 protein (SEQ ID NO:2).
In some embodiments, D1L3 protein variant comprises an inactivation of a nuclear localization signal (NLS). D1L3 features two nuclear localization sites (NLS1, NLS2), which both target the enzyme to the nucleus during apoptosis. Indeed, D1L3 is required for fragmentation of nuclear DNA in apoptotic and necrotic cells in vivo. NLS1 is located near the N-terminus (about amino acid positions 80 to 96 with respect to SEQ ID NO: 2). NLS2 (amino acid positions 291 to 304 with respect to SEQ ID NO: 2) is embedded within a C-terminal tail (amino acid positions 283 to 305 with respect to SEQ ID NO: 2) that is unique to D1L3 and not present in D1. The C-terminal tail is thought to be required for D1L3 to degrade different substrates of extracellular DNA, namely lipid-encapsulated DNA and chromatin within apoptotic bodies. Lipids encapsulate DNA during transfections. In brief, cDNA, protein-free DNA, and cationic lipids form a complex that penetrates through the plasma membrane of target cells. D1L3 interferes with transfections and the C-terminal tail is critical for this function. Physiological substrates of D1L3 are apoptotic bodies, extracellular lipid vesicles filled with chromatin from apoptotic cells. The C-terminal tail enables D1L3 to penetrate through lipid membranes of apoptotic bodies and degrade the chromatin load. The C-terminal tail is also thought to be required for degradation of lipid-free, extracellular chromatin by D1L3. Thus, the C-terminal tail, a molecular feature unique to D1L3, appears to be responsible for the distinct substrate affinities of D1 and D1L3.
Inactivation of one or more NLSs enhances the availability and/or activity of the enzyme in the circulation. In some embodiments, the NLS inactivation is by deletion of all or part of NLS1 and/or NLS2. In some embodiments, the NLS is inactivated by substitution and/or deletion of amino acids within NLS1 and/or NLS2. In some embodiments, NLS2 is deleted, entirely or partially. In some embodiments, the D1L3 protein variant comprises a deletion of all or part of the C-terminal tail, and/or substitution of one or more amino acids in the C-terminal tail.
In certain embodiments, the D1L3 variant contains one or more, e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions (e.g., insertions), or deletions in the NLS1 domain. The NLS2 domain or a portion thereof may be deleted. In certain embodiments, the D1L3 protein variant contains one or more, e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions, or deletions in the C-terminal tail domain. The C-terminal tail domain or a portion thereof may be deleted. In various embodiments, the D1L3 protein variant contains one or more, e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions, or deletions in the NLS2 domain which is located in the C-terminal tail domain. The NLS2 domain or a portion thereof can be deleted in the D1L3 protein variant.
In various aspects, the invention provides a protein engineering technology that is based on a transfer of a single amino acid or multiple-adjacent amino acids, termed “building block”, between two members of a protein family, such as DNase1-protein family members to generate enzymatically active variants of DNase1-protein family members (including variant of D1 and D1L3). A “building block” is defined by amino acids that are variable between two or more members of the DNase1-protein family. These variable amino acids are flanked by amino acids that are conserved between two or more members of the DNase1-protein family (“anchors”). The variable single amino acid or multiple contiguous amino acids (“building blocks”) are exchanged between members of the DNase1-protein family by implanting them between conserved single amino acid or multiple contiguous amino acids (“anchors”).
This approach is referred to herein as “building-block protein engineering.” Where three or more amino acids are transferred, up to ⅓ of the amino acids may be further substituted. For example, where where three or six amino acids are transferred as a building block, one or up to two resides may be further substituted, respectively. In some embodiments, four or more amino acids are transferred as a building block substitution, and up to 25% of the transferred amino acids are further substituted, e.g., with conservative or non-conservative amino acid modifications. For example, where four, eight, or twelve amino acids are transferred, one, two, or three amino acids (respectively) may be further substituted in the building block substitution.
In various aspects (including in connection with embodiments described above), the D1L3 variant comprises at least one building block substitution from D1.
Exemplary building block substitutions from D1 include one or more of:
substitution of 1-20 (MSRELAPLLLLLLSIHSALA) of SEQ ID NO:2 with 1-22 (MRGMKLLGALLALAALLQGAVS) from SEQ ID NO:1; substitution of 21-25 (MRICS) of SEQ ID NO:2 with 23-27 (LKIAA) from SEQ ID NO:1; substitution of 28-30 (VRS) of SEQ ID NO:2 with 30-32 (IQT) from SEQ ID NO:1; substitution of 33-34 (ES) of SEQ ID NO:2 with 35-36 (ET) from SEQ ID NO:1; substitution of 36-45 (QEDKNAMDVI) of SEQ ID NO:2 with 38-47 (MSNATLVSYI) from SEQ ID NO:1; substitution of 47-51 (KVIK) of SEQ ID NO:2 with 49-53 (QILS) from SEQ ID NO:1; substitution of 52 (C) of SEQ ID NO:2 with 54 (Y) from SEQ ID NO:1; substitution of 54-58 (IILVM) of SEQ ID NO:2 with 56-60 (IALVQ) from SEQ ID NO:1; substitution of 60-61 (IK) of SEQ ID NO:2 with 62-63 (VR) from SEQ ID NO:1; substitution of 63-70 (SNNRICPI) of SEQ ID NO:2 with 65-72 (SHLTAVGK) from SEQ ID NO:1; substitution of 72-74 (MEK) of SEQ ID NO:2 with 74-76 (LDN) from SEQ ID NO:1; substitution of 77-84 (RNSRRGIT) of SEQ ID NO:2 with 79-84 (QDAPDT) from SEQ ID NO:1; substitution of 86 (N) of SEQ ID NO:2 with 86 (H) from SEQ ID NO:1; substitution of 88-89 (VI) of SEQ ID NO:2 with 88-89 (VV) from SEQ ID NO:1; substitution of 91-92 (SR) of SEQ ID NO:2 with 91-92 (EP) from SEQ ID NO:1; substitution of 96-97 (NT) of SEQ ID NO:2 with 96-97 (NS) from SEQ ID NO:1; substitution of 101 (Q) of SEQ ID NO:2 with 101 (R) from SEQ ID NO:1; substitution of 103 (A) of SEQ ID NO:2 with 103 (L) from SEQ ID NO:1; substitution of 105 (L) of SEQ ID NO:2 with 105 (V) from SEQ ID NO:1; substitution of 107-110 (KEKL) of SEQ ID NO:2 with 107-110 (RPDQ) from SEQ ID NO:1; substitution of 113-116 (VKRS) of SEQ ID NO:2 with 113-116 (AVDS) from SEQ ID NO:1; substitution of 118 (H) of SEQ ID NO:2 with 118 (Y) from SEQ ID NO:1; substitution of 120 (H) of SEQ ID NO:2 with 120 (D) from SEQ ID NO:1; substitution of 122-127 (YQDGDA) of SEQ ID NO:2 with 122-128 (GCEPCGN) from SEQ ID NO:1; substitution of 129 (V) of SEQ ID NO:2 with 130 (T) from SEQ ID NO:1; substitution of 131 (S) of SEQ ID NO:2 with 132 (N) from SEQ ID NO:1; substitution of 135-136 (FV) of SEQ ID NO:2 with 136-137 (AIV) from SEQ ID NO:1; substitution of 138 (W) of SEQ ID NO:2 with 139 (R) from SEQ ID NO:1; substitution of 140-143 (QSPH) of SEQ ID NO:2 with 141-144 (FSRF) from SEQ ID NO:1; substitution of 145-148 (AVKD) of SEQ ID NO:2 with 146-149 (EVRE) from SEQ ID NO:1; substitution of 150 (V) of SEQ ID NO:2 with 151 (A) from SEQ ID NO:1; substitution of 152 (I) of SEQ ID NO:2 with 153 (V) from SEQ ID NO:1; substitution of 156-157 (TT) of SEQ ID NO:2 with 157-158 (AA) from SEQ ID NO:1; substitution of 159-161 (ETS) of SEQ ID NO:2 with 160-162 (GDA) from SEQ ID NO:1; substitution of 163 (K) of SEQ ID NO:2 with 164 (A) from SEQ ID NO:1; substitution of 167 (E) of SEQ ID NO:2 with 168 (A) from SEQ ID NO:1; substitution of 169-170 (VE) of SEQ ID NO:2 with 170-171 (YD) from SEQ ID NO:1; substitution of 173 (T) of SEQ ID NO:2 with 174 (L) from SEQ ID NO:1; substitution of 176-178 (KHR) of SEQ ID NO:2 with 177-179 (QEK) from SEQ ID NO:1; substitution of 180-181 (KA) of SEQ ID NO:2 with 181-182 (GL) from SEQ ID NO:1; substitution of 183-186 (NFIF) of SEQ ID NO:2 with 184-187 (DVML) from SEQ ID NO:1; substitution of 198-201 (PKKA) of SEQ ID NO:2 with 199-202 (RPSQ) from SEQ ID NO:1; substitution of 203-204 (KN) of SEQ ID NO:2 with 204-205 (SS) from SEQ ID NO:1; substitution of 208 (R) of SEQ ID NO:2 with 209 (W) from SEQ ID NO:1; substitution of 210 (D) of SEQ ID NO:2 with 211 (S) from SEQ ID NO:1; substitution of 212 (R) of SEQ ID NO:2 with 213 (T) from SEQ ID NO:1; substitution of 214 (V) of SEQ ID NO:2 with 215 (Q) from SEQ ID NO:1; substitution of 218 (G) of SEQ ID NO:2 with 219 (P) from SEQ ID NO:1; substitution of 220-221 (QE) of SEQ ID NO:2 with 221-222 (SA) from SEQ ID NO:1; substitution of 225-228 (VKKS) of SEQ ID NO:2 with 226-228 (ATP) from SEQ ID NO:1; substitution of 230 (N) of SEQ ID NO:2 with 230 (H) from SEQ ID NO:1; substitution of 238-240 (LRG) of SEQ ID NO:2 with 238-240 (VAG) from SEQ ID NO:1; substitution of 241-246 (QEIVSS) of SEQ ID NO:2 with 241-246 (MLLRGA) from SEQ ID NO:1; substitution of 250 (K) of SEQ ID NO:2 with 250 (D) from SEQ ID NO:1; substitution of 252-254 (NSV) of SEQ ID NO:2 with 252-254 (ALP) from SEQ ID NO:1; substitution of 256 (D) of SEQ ID NO:2 with 256 (N) from SEQ ID NO:1; substitution of 259-260 (KA) of SEQ ID NO:2 with 259-260 (AA) from SEQ ID NO:1; substitution of 262 (K) of SEQ ID NO:2 with 262 (G) from SEQ ID NO:1; substitution of 264-267 (TEEE) of SEQ ID NO:2 with 264-267 (SDQL) from SEQ ID NO:1; substitution of 269-271 (LDV) of SEQ ID NO:2 with 269-271 (QAI) from SEQ ID NO:1;
substitution of 275 (F) from SEQ ID NO:2 with 275 (Y) from SEQ ID NO:1; substitution of 279-280 (FK) of SEQ ID NO:2 with 279-280 (VM) from SEQ ID NO:1; and
substitution of 282-305 (QSSRAFTNSKKSVTLRKKTKSKRS) of SEQ ID NO:2 with 282 (K) from SEQ ID NO:1.
These building block substitutions from D1 result in variants of D1L3 which feature one or more of the following mutations: M1_A20delinsMRGMKLLGALLALAALLQGAVS, M21_S25delinsLKIAA, V28_S30delinsIQT, E33_S34delinsET, Q36_I45delinsMSNATLVSYI, K47_K50delinsQILS, C52Y, I54_M58delinsIALVQ, I60_K61delinsVR, S63_I70delinsSHLTAVGK, M72_K74delinsLDN, R77_T84delinsQDAPDT, N86H, V88_I89delinsVV, S91_R92delinsEP, N96_T97delinsNS, Q101R, A103L, L105V, K107_L110delinsRPDQ, V113_S116delinsAVDS, H118Y, H120D, Y122_A127delinsGCEPCGN, V129T, S131N, 135F_I36VdelinsAI, W138R, Q140_H143delinsFSRF, A145_D148delinsAVKD, V150A, I152A, T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD, T173L, K176_R178delinsQEK, K180_A181delinsGL, N183_F186delinsDVML, P198_A20ldelinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delinsSA, V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259_A260delinsAA, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, Q282_S305delinsK.
The term “delins” refers to a deletion between and including two indicated amino acids, with an insertion of an amino acid or sequence of amino acids at the site of the deletion. For example, the notation E91_P92delinsSR means that the amino acids from E91 to P92 are deleted and the amino acids SR are inserted at the site of the deletion (e.g., the resulting amino acid sequence will have S91 and R92).
The term “ins” refers to an insertion of amino acids between two indicated amino acids. For example, the notation E91_P92insSR means that the amino acids SR are inserted between E91 and P92, resulting in the sequence E91, S92, R93, and P94.
Multiple mutations in one enyzme are separared by “/” e.g., T227K/A136F or R199_Q202delinsPKKA/S204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE.
In some embodiments, the D1L3 variant comprises one or more adjacent building block substitutions. For example, the D1L3 variant may comprise with respect to SEQ ID NO:2 the mutation F275Y/F279_K280delinsVM/Q282_S305delinsK (SEQ ID NO: 17). In some embodiments, the D1L3 variant may comprise with respect to SEQ ID NO:2 the mutation Q36_I45delinsMSNATLVSYI (SEQ ID NO: 19). In some embodiments, the D1L3 variant may comprise with respect to SEQ ID NO:2 the mutation Y122_A127delinsGCEPCGNN129T/S131N (SEQ ID NO: 20).
In an exemplary embodiment, the D1L3 variant comprises 2 or 3 substitutions selected from the mutations with respect to SEQ ID NO:2: Q36_I45delinsMSNATLVSYI, Y122_A127delinsGCEPCGNN129T/S131N, F275Y/F279_K280delinsVM/Q282_S305delinsK.
Thus, in some embodiments, the invention provides a DNase enzyme comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the enzyme defined by SEQ ID NO:2, and comprises at least one building block substitution from the amino acid sequence of SEQ ID NO:1. Building block substitutions between SEQ ID NOS: 1 and 2 are illustrated and numbered in
Exemplary D1L3 variants include those comprising the amino acid sequence of SEQ ID Nos: 17 to 20. The invention in some embodiments, includes derivatives having from 1 to 10 (e.g., 1 to 5) amino acid insertions, deletions, or substitutions with respect to any one of SEQ ID NOS:17 to 20. In some embodiments, the D1L3 variant comprises one or more additional block amino acid substitutions from a homologous DNase (e.g., from D1). Such block substitutions may replace at least 2, at least 3, at least 4, or at least 5 amino acids. In some embodiments, the block substitutions replace from 2 to 20 amino acids, such as from 3 to 15 amino acids, or from 3 to 10 amino acids, with the comparable building block from the homologous DNase.
The engineered variants of D1L3 protein may comprise one or more additional amino acid substitutions, additions (insertions), deletions, or truncations in the amino acid sequence of human D1L3 (SEQ ID NO: 2). Amino acid substitutions may include conservative and/or non-conservative substitutions.
For example, “conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. “Conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices. As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
In some embodiments, the D1L3 protein variant comprises an N-terminal or C-terminal fusion to a half-life extending moiety, such as albumin, transferrin, an Fc, or elastin-like protein. See U.S. Pat. No. 9,458,218, which is hereby incorporated by reference in its entirety. In some embodiments, the D1L3 is dimerized by an immunoglobulin hinge region. For example, the engineered enzymes described herein may also include an Fc-fusion domain (e.g. a hinge and CH2 domains and CH3 domains of an immunoglobulin). In other cases, the engineered D1L3 protein is fused to albumin, e.g., human albumin or a fragment thereof. See WO 2015/066550; U.S. Pat. No. 9,221,896, which are hereby incorporated by reference in its entirety. Albumin can be joined to at the N-terminus or the C-terminus of the engineered D1 protein, and may optionally comprise an amino acid linker. In some embodiments, D1L3 and D1 are together dimerized by an Fc hinge region, creating a dimeric molecule with synergistic functional properties for degrading NETs.
In some embodiments, the recombinant D1L3 protein variant comprises one or more polyethylene glycol (PEG) moieties, which may be conjugated at one or more of positions or the C-terminus. In some embodiments, the native amino acid at that position is substituted with an amino acid having a side chain suitable for crosslinking with hydrophilic moieties, to facilitate linkage of the hydrophilic moiety to the peptide. In other embodiments, an amino acid modified to comprise a hydrophilic group is added to the peptide at the C-terminus. The PEG chain(s) may have a molecular weight in the range of about 500 to about 40,000 Daltons. In some embodiments, the PEG chain(s) have a molecular weight in the range of about 500 to about 5,000 Daltons. In some embodiments, the PEG chain(s) have a molecular weight of about 10,000 to about 20,000 Daltons.
In various embodiments, the D1L3 protein variant has increased protein stability, increased serum availability, and substantially the same or better NET-degrading activity (as determined in vitro or in vivo) as compared to wild-type D1L3 protein. In various embodiments, the D1L3 variant comprises one or more of the following properties relative to wild type D1L3: higher protein-free DNA (naked DNA) degradation activity, the same or substantially the same (e.g., at least 50%) chromosomal DNA degradation activity, protease resistance, an increased half-life, and higher production levels with in vitro expression systems (e.g. Chinese hamster ovary cells and/or Pichia pastoris).
DNA fragmentation can be measured using methods known to those skilled in the art such as gel electrophoresis and DNase activity assays. Methods for quantifying NETs, NET degradation, and DNA fragmentation activity are described in, for example, Hakkim et al., Proc. Natl. Acad. Sci USA, 2010, 107(21):9813-9818; Napirei et al. Biochem J, 2005, 389: 355-364. Fuchs et al., J Cell Biol, 2007, 176(2):231-241, Sisirak et al. Cell, 2016, 166:88-101, and Jiménez-Alcázar et al. J Thromb Haemost, 2015, 13, 732-742.
In some aspects, the invention provides DNase1 (D1) variants engineered to have physical, pharmacodynamic, and/or enzymatic properties more suitable for therapy, for example, to reduce or prevent NET accumulation in a subject. In some embodiments, the variant is more amenable to recombinant expression, thereby providing considerable manufacturing advantages. In various embodiments, the engineered D1 variant comprises an amino acid sequence that is at least 80% identical to the mature enzyme defined by the amino acid sequence of SEQ ID NO:1, with one or more amino acid substitutions, additions, or deletions resulting in one or more of a mutated DNA binding site, addition of a chromatin binding site, a mutated actin binding site, mutation of a glycosylation site, addition of a nuclear localization signal (e.g., having similarity or identity to NLS1 or NLS2 of D1L3), and/or a C-terminal domain similar to the C-terminal tail of D1L3. The D1 variant comprises an amino acid sequence with at least 80% identity to the amino acid sequence of the enzyme defined by SEQ ID NO:1, with one or more amino acid modifications with respect to SEQ ID NO:1 as described herein. In some embodiments, D1L3 variant comprises an amino acid sequence with at least 90%, or at least 95%, or at least 97%, or at least 98% identity with the enzyme of SEQ ID NO:1.
In various embodiments, the D1 variant comprises one or more amino acid substitutions, additions, deletions, or truncations in the amino acid sequence of human D1 (SEQ ID NO: 1). In some embodiments, the amino acid substitutions, and may include conservative and/or non-conservative substitutions as described. For example, the D1 variant may have from 1 to 20 (or from 1 to 10, or from 1 to 5) amino acid substitutions, deletions, or insertions with respect to the enzyme of SEQ ID NO:1.
In some embodiments, the D1 variant comprises one or more additional block amino acid substitutions from a homologous DNase (e.g., from D1L3). Such block substitutions may each replace from 1 to 24 amino acids, such as from 3 to 15 amino acids, or from 3 to 10 amino acids, from the homologous DNase. In some embodiments, the D1 variant comprises from 2 to 5 block substitutions from the homologous DNase (e.g., D1L3). In some embodiments 2 or 3 contiguous building block substitutions are transferred.
In various embodiments, the D1 variant is engineered to comprise one or more of the following characteristics relative to the wild type enzyme: the substantially the same or higher protein-free DNA (naked DNA) degradation activity (e.g., at least 50% or better), higher chromosomal DNA degradation activity, similar or improved protease resistance, actin resistance, an increased serum half-life, improved penetetration from blood into urine or bile, or a combination of such properties.
In some embodiments, the engineered D1 enzyme comprises an amino acid sequence comprising at least 50% (or at least 70%, 80%, 90%) sequence identity to the C-terminal domain of wild-type D1L3 protein (e.g., amino acids 283 to 305 of SEQ ID NO:2).
In some embodiments, the D1 enzyme comprises an amino acid sequence having at least 50% or at least 75% sequence identity to the NLS1 of wild-type D1L3 (amino acids 35 to 51 of SEQ ID NO:2), a nuclear localization signal 2 (NLS2) having substantial sequence identity to the NLS2 of wild-type D1L3 protein (amino acids 296 to 304 of SEQ ID NO:2).
In various aspects, the recombinant D1 variant comprises one or more building block substitutions selected from: substitution of 1-22 (MRGMKLLGALLALAALLQGAVS) from SEQ ID NO:1 with 1-20 (MSRELAPLLL LLLSIHSALA) from SEQ ID NO:2; substitution of 23-27 (LKIAA) from SEQ ID NO:1 with 21-25 (MRICS) from SEQ ID NO:2; substitution of 30-32 (IQT) from SEQ ID NO:1 with 28-30 (VRS) from SEQ ID NO:2; substitution of 35-36 (ET) from SEQ ID NO:1 with 33-34 (ES) from SEQ ID NO:2; substitution of 38-47 (MSNATLVSYI) from SEQ ID NO:1 with 36-45 (QEDKNAMDVI) from SEQ ID NO:2; substitution of 49-52 (QILS) from SEQ ID NO:1 with 47-50 (KVIK) from SEQ ID NO:2; substitution of 54 (Y) from SEQ ID NO:1 with 52 (C) from SEQ ID NO:2; substitution of 56-60 (IALVQ) from SEQ ID NO:1 with 54-58 (IILVM) from SEQ ID NO:2; substitution of 62-63 (VR) from SEQ ID NO:1 with 60-61 (IK) from SEQ ID NO:2; substitution of 65-72 (SHLTAVGK) from SEQ ID NO:1 with 63-70 (SNNRICPI) from SEQ ID NO:2; substitution of 74-76 (LDN) from SEQ ID NO:1 with 72-74 (MEK) from SEQ ID NO:2; substitution of 79-84 (QDAPDT) from SEQ ID NO:1 with 77-84 (RNSRRGIT) from SEQ ID NO:2; substitution of 86 (H) from SEQ ID NO:1 with 86 (N) from SEQ ID NO:2; substitution of 88-89 (VV) from SEQ ID NO:1 with 88-89 (VI) from SEQ ID NO:2; substitution of 91-92 (EP) from SEQ ID NO:1 with 91-92 (SR) from SEQ ID NO:2; substitution of 96-97 (NS) from SEQ ID NO:1 with 96-97 (NT) from SEQ ID NO:2; substitution of 101 (R) from SEQ ID NO:1 with 101 (Q) from SEQ ID NO:2; substitution of 103 (L) from SEQ ID NO:1 with 103 (A) from SEQ ID NO:2; substitution of 105 (V) from SEQ ID NO:1 with 105 (L) from SEQ ID NO:2; substitution of 107-110 (RPDQ) from SEQ ID NO:1 with 107-110 (KEKL) from SEQ ID NO:2; substitution of 113-116 (AVDS) from SEQ ID NO:1 with 113-116 (VKRS) from SEQ ID NO:2; substitution of 118 (Y) from SEQ ID NO:1 with 118 (H) from SEQ ID NO:2, substitution of 120 (D) from SEQ ID NO:1 with 120 (H) from SEQ ID NO:2; substitution of 122-128 (GCEPCGN) from SEQ ID NO:1 with 122-127 (YQDGDA) from SEQ ID NO:2, substitution of 130 (TF) from SEQ ID NO:1 with 129 (V) from SEQ ID NO:2; substitution of 132 (N) from SEQ ID NO:1 with 131 (S) from SEQ ID NO:2; substitution of 136-137 (AI) from SEQ ID NO:1 with 135-136 (FV) from SEQ ID NO:2; substitution of 139 (R) from SEQ ID NO:1 with 138 (W) from SEQ ID NO:2; substitution of 141-144 (FSRF) from SEQ ID NO:1 with 140-143 (QSPH) from SEQ ID NO:2; substitution of 146-149 (EVRE) from SEQ ID NO:1 with 145-148 (AVKD) from SEQ ID NO:2; substitution of 151 (A) from SEQ ID NO:1 with 150 (V) from SEQ ID NO:2; substitution of 153 (V) from SEQ ID NO:1 with 152 (I) from SEQ ID NO:2; substitution of 157-158 (AA) from SEQ ID NO:1 with 156-157 (TT) from SEQ ID NO:2; substitution of 160-162 (GDA) from SEQ ID NO:1 with 159-161 (ETS) from SEQ ID NO:2; substitution of 164 (A) from SEQ ID NO:1 with 163 (K) from SEQ ID NO:2; substitution of 168 (A) from SEQ ID NO:1 with 167 (E) from SEQ ID NO:2; substitution of 170-171 (YD) from SEQ ID NO:1 with 169-171 (VE) from SEQ ID NO:2; substitution of 174 (L) from SEQ ID NO:1 with 173 (T) from SEQ ID NO:2; substitution of 177-179 (QEK) from SEQ ID NO:1 with 176-178 (KHR) from SEQ ID NO:2; substitution of 181-182 (GL) from SEQ ID NO:1 with 180-181 (KA) from SEQ ID NO:2; substitution of 184-187 (DVML) from SEQ ID NO:1 with 183-187 (NFIF) from SEQ ID NO:2; substitution of 199-202 (RPSQ) from SEQ ID NO:1 with 198-201 (PKKA) from SEQ ID NO:2; substitution of 204-205 (SS) from SEQ ID NO:1 with 203-204 (KN) from SEQ ID NO:2; substitution of 209 (W) from SEQ ID NO:1 with 208 (R) from SEQ ID NO:2; substitution of 211 (S) from SEQ ID NO:1 with 210 (D) from SEQ ID NO:2; substitution of 213 (T) from SEQ ID NO:1 with 212 (R) from SEQ ID NO:2; substitution of 215 (Q) from SEQ ID NO:1 with 214 (V) from SEQ ID NO:2; substitution of 219 (P) from SEQ ID NO:1 with 218 (G) from SEQ ID NO:2; substitution of 221-222 (SA) from SEQ ID NO:1 with 220-221 (QE) from SEQ ID NO:2; substitution of 226-228 (ATP) from SEQ ID NO:1 with 225-228 (VKKS) from SEQ ID NO:2; substitution of 230 (H) from SEQ ID NO:1 with 230(N) from SEQ ID NO:2; substitution of 238-239 (VA) from SEQ ID NO:1 with 238-239 (LR) from SEQ ID NO:2; substitution of 241-246 (MLLRGA) from SEQ ID NO:1 with 241-246 (QEIVSS) from SEQ ID NO:2; substitution of 250-251 (D) from SEQ ID NO:1 with 250-251 (K) from SEQ ID NO:2; substitution of 252-254 (ALP) from SEQ ID NO:1 with 252-254 (NSV) from SEQ ID NO:2; substitution of 256 (N) from SEQ ID NO:1 with 256 (D) from SEQ ID NO:2; substitution of 259-260 (AA) from SEQ ID NO:1 with 259-260 (KA) from SEQ ID NO:2; substitution of 262 (G) from SEQ ID NO:1 with 262 (K) from SEQ ID NO:2; substitution of 264-267 (SDQ) from SEQ ID NO:1 with 264-267 (TEEE) from SEQ ID NO:2; substitution of 269-271 (QAI) from SEQ ID NO:1 with 269-271 (LDV) from SEQ ID NO:2; substitution of 275 (Y) from SEQ ID NO:1 with 275 (F) from SEQ ID NO:2; substitution of 279-280 (VM) from SEQ ID NO:1 with 279-280 (FK) from SEQ ID NO:2; and substitution of 282 (K) from SEQ ID NO:1 with 282-305 (QSSRAFTNSKKSVTLRKKTKSKRS) from SEQ ID NO:2.
These building block substitutions from D1L3 results in variants of D1, which feature one or more of the following mutations: 1M_S22delinsMSRELAPLLLLLLSIHSALA, L23_A27delinsMRICS, 130 T32delinsVRS, E35 T36delinsES, M38 I47delinsQEDKNAMDVI, Q49 S52delinsKVIK, Y54C, 156 Q60delinsIILVM, V62_R63delinsIK, S65_K72delinsSNNRICPI, L74_N76delinsMEK, Q79 T84delinsRNSRRGIT, H86N, V88_V89delinsVI, E91_P92delinsSR, N96 S97delinsNT, R101Q, L103A, V105L, R107_Q110delinsKEKL, A113_S116delinsVKRS, Y118H, D120H, G122_N128delinsYQDGDA, T1305, N132S, A136_I137delinsFV, R139W, F141_F144delinsQSPH, E146_E149delinsAVKD, A151V, V153I, A157_A158delinsTT, G160_A162delinsETS, A164K, A168E, Y170 D171delinsVE, L174T, Q177_K179delinsKHR, G181_L182delinsKA, D184_L187delinsNFIF, R199_Q202delinsPKKA, S204_S205delinsKN, W209R, S211D, T213R, Q215V, P219G, S221_A222delinsQE, A226_P228delinsVKKS, H230N, V238_A239delinsLR, M241_A246delinsQEIVSS, D250K, A252_P254delinsNSV, N256D, A259_A260delinsKA, G262K, S264_L267delinsTEEE, Q269 I271delinsLDV, Y275F, V279_M280delinsFK, K282delinsQSSRAFTNSKKSVTLRKKTKSKRS.
In some embodiments, a recombinant D1 variant comprises the following modifications with respect to SEQ ID NO:1: Y275F, V279_M280delinsFK, and K282delinsQSSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 16). Such variants comprise a C-terminal tail, and display higher affinity for DNA-lipid complexes.
In some embodiments, a recombinant D1 variant comprises the following amino acid modifications with respect to SEQ ID NO:1: Q79 T84delinsRNSRRGIT (e.g., SEQ ID NO: 10); H86N, and/or, V88_V89delinsVI, and/or E91_P92delinsSR (e.g. SEQ ID NO: 11, which includes all three); A136_I137delinsFV (e.g., SEQ ID NO: 12); R199_Q202delinsPKKA, and/or S204_S205delinsKN, and/or W209R, and/or S211D, and/or T213R, and/or Q215V, and/or P219G, and/or S221_A222delinsQE (e.g., SEQ ID NO: 13, which includes all eight); and A226_P228delinsVKKS (e.g., SEQ ID NO: 14).
In some embodiments, where the D1 variant comprises the substitution H86N, the variant comprises at least one addition modification, such as a building block substitution or other point mutation described herein (such as the addition of a cationic amino acid or substitution that results in resistance to actin).
In some embodiments, the D1 variant comprises at least two adjacent, contiguous, building block substitutions that result in mutations with respect to SEQ ID NO:1 of: H86N/V88_V89delinsVI (e.g., SEQ ID NO: 11); R199_Q202delinsPKKA/S204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE (e.g., SEQ ID NO: 13); Y275F/V279_M280delinsFK/K282delinsQSSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 16).
In some embodiments, the invention provides a DNase enzyme comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the enzyme defined by SEQ ID NO:1, and comprising at least one building block substitution from the amino acid sequence of SEQ ID NO:2, where the DNase enzyme has increased chromatin-degrading activity as compared to the DNase enzyme of SEQ ID NO:1. In some embodiments, the enzyme comprises at least two adjacent building block substitutions from SEQ ID NO:2. Building block substitutions between D1 (SEQ ID NO:1) and D1L3 (SEQ ID NO:2) are illustrated in
In some embodiments, the enzyme comprises the following modification with respect to SEQ ID NO:1: Q79 T84delinsRNSRRGIT, and optionally comprises the amino acid sequence of SEQ ID NO: 10.
In some embodiments, the enzyme comprises the following modification with respect to SEQ ID NO:1: H86N/V88_V89delinsVI/E91_P92delinsSR, and optionally comprises the amino acid sequence of SEQ ID NO: 11.
In some embodiments, the enzyme comprises the following modifications with respect to SEQ ID NO:1: A136_I137delinsFV, and optionally comprises the amino acid sequence of SEQ ID NO: 12.
In some embodiments, the enzyme comprises two or more of the following modifications with respect to SEQ ID NO:1: R199_Q202delinsPKKA, and/or S204_S205delinsKN, and/or W209R, and/or S211D, and/or T213R, and/or Q215V, and/or P219G, and/or S221_A222delinsQE, and optionally comprises the amino acid sequence of SEQ ID NO: 13 (which comprises all eight).
In some embodiments, the enzyme comprises the following modifications with respect to SEQ ID NO:1: A226_P228delinsVKKS, and optionally comprises the amino acid sequence of SEQ ID NO: 14.
In some embodiments, the enzyme comprises two or more of the following modifications with respect to SEQ ID NO:1: Q79_T84delinsRNSRRGIT, and/or H86N, and/or V88_V89delinsVI, and/or E91_P92delinsSR, and/or A136_I137delinsFV, and/or R199_Q202delinsPKKA, and/or S204 _S205delinsKN, and/or W209R, and/or S211D, and/or T213R, and/or Q215V, and/or P219G, and/or S221_A222delinsQE, and/or A226_P228delinsVKKS, and optionally comprises the amino acid sequence of SEQ ID NO: 15.
In some embodiments, the enzyme comprises two or more the following modification with respect to SEQ ID NO:1:Y275F, and/or V279_M280delinsFK, and/or K282delinsQSSRAFTNSKKSVTLRKKTKSKRS, and optionally comprises the amino acid sequence of SEQ ID NO: 16.
Exemplary D1 variants include those comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. The invention in some embodiments, includes derivatives having from 1 to 10 (e.g., 1 to 5) amino acid insertions, deletions, or substitutions with respect to any one of SEQ ID NOS: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the D1 variant comprises one or more additional block amino acid substitutions from a homologous DNase (e.g., from D1L3). Such block substitutions may replace from 2 to 20 amino acids, such as from 3 to 15 amino acids, or from 3 to 10 amino acids of the D1 variant, with building block amino acids from the homologous DNase (e.g., D1L3, SEQ ID NO:2). Building block substitutions between D1 and D1L3 are illustrated in
In various embodiments, the enzyme exhibits one or more properties selected from: similar or higher affinity for protein-free DNA compared to the enzyme of SEQ ID NO:1, a higher chromatin degradation activity compared to the enzyme of SEQ ID NO:1, an enzymatic activity that is resistant to inhibition by actin compared to the enzyme of SEQ ID NO:1, and a combination thereof.
In some embodiments, the D1 variant comprises an N-terminal or C-terminal fusion to a half-life extending moiety, such as albumin, transferrin, an Fc, or elastin-like protein, as described for D1L3. In some embodiments, the D1 enzyme is dimerized by an immunoglobulin hinge region.
In various embodiments, the D1 variant exhibits one or more properties selected from: the same or higher protein-free DNA (naked DNA) degradation activity, higher chromosomal DNA (chromatin) degradation activity, similar or improved protease resistance, actin resistance, similar or improved penetration from blood into urine and/or bile, and higher production levels in vitro expression systems (e.g. Chinese hamster ovary cells and/or Pichia pastoris), and a combination thereof.
In various embodiments, the DNase enzyme comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the enzyme defined by SEQ ID NO:1, and comprises the substitution, insertion, or addition of one or more arginine and/or lysine residues, wherein the DNase enzyme has increased chromatin-degrading activity as compared to the enzyme of SEQ ID NO:1. In some embodiments, the one or more arginine and/or lysine residues are amino acid substitutions selected from substitutions at positions corresponding to positions 31, 41, 49, 52, 68, 76, 79, 81, 92, 109, 114, 115, 164, 177, 181, 200, 201, 204, 209, 213, 227, 239, 250, 259, 262, and 280 of SEQ ID NO:1. Exemplary modifications at these positions include Q31R, A41K, Q49R, S52K, T68R, N76K, Q79R, D80_A81insR, A81R, P92R, D109K, V114K, D115R, A164K, Q177K, G181K, P200K, S201K, 5204R, W209R, T213R, A226_T227insK, T227K, A239R, D250K, A259K, G262K, and K280M. In various embodiments, the enzyme comprises substitution and/or insertion of at least two, or at least three, or at least four, or at least five, or at least six arginine and/or lysine residues. In some embodiments, DNase enzyme comprises an arginine or lysine at one or more positions corresponding to positions 31, 49, 76, 79, 92, 109, 164, 177, 200, 201, 204, 209, 213, and 262 of SEQ ID NO:1, which are optionally selected from Q31R, Q49R, N76K, Q79R, P92R, D109K, A164K, Q177K, P200K, S201K, 5204R, W209R, T213R, W209R, and G262K. These positions/substitutions can independently provide better activity, including activity for degrading chromatin.
In various embodiments, if the enzyme comprises a substitution of Q31R or T227K, the enzyme comprises at least two amino acid modifications selected from Q31R, A41K, Q49R, S52K, T68R, N76K, Q79R, D80A8linsR, A81R, P92R, D109K, V114K, D115R, A164K, Q177K, G181K, P200K, S201K, 5204R, W209R, T213R, A226_T227insK, T227K, A239R, D250K, A259K, G262K, and K280M. In some embodiments, the amino acid substitutions include Q31R and T227K with respect to SEQ ID NO:1.
In some embodiments, the DNase enzyme has an arginine or lysine at positions corresponding to positions 31, 49, 79, and 209 of SEQ ID NO:1, and may optionally comprise the following amino acid substitutions: Q31R, Q49K, Q79R, and W209R. These positions harbor an arginine or lysine in murine D1, but not wild-type human D1, and can transfer chromatin-degrading activity to human D1.
In some embodiments, the DNase enzyme has an arginine or lysine at positions corresponding to positions 31, 49, 79, 177, 209, and 262 of SEQ ID NO:1, and may optionally comprise the amino acid substitutions Q31R, Q49K, Q79R, A136F, Q177K, W209R, G262K. Four of these positions harbor an arginine or lysine in rat D1, but not wild-type human D1, and together can transfer chromatin degrading activity to human D1.
In some embodiments, the DNase enzyme has a lysine or arginine residue at positions corresponding to positions 31 and 227 of SEQ ID NO:1.
In some embodiments, the DNase enzyme has a lysine or arginine at the position corresponding to position 164 of SEQ ID NO:1, which is optionally lysine (A164K).
In various embodiments, the engineered D1 protein contains one or more amino acid modifications (e.g., substitutions, additions, and deletions) in the actin binding site (e.g. substitution of A136, such as A136F), and thereby hindering or preventing actin binding. As such, the engineered D1 protein can be actin-resistant or substantially actin-resistant. See Ulmer et al., PNAS USA Vol. 93, pp 8225-8229 (1996). Thus, in various embodiments, the DNase enzyme comprises an amino acid substitution at position 136 with respect to SEQ ID NO:1, and which is optionally A136F. Mutations to D1 providing for actin resistance, such as mutations at position 136 (e.g., A136F, or substitution of another bulky, hydrophobic, cyclic or aromatic amino acid at this position), may be combined with any other mutation described herein, including addition of cationic residues or building block substitutions.
In some embodiments, the D1 variant is resistant to actin-inhibition and capable of degrading chromatin. For example, the D1 variant may comprise the following amino acid substitutions: Q31R, T227K, and A136F with respect to SEQ ID NO:1. In some embodiments, the DNase enzyme comprises the following amino acid substitutions: A164K and A136F.
In some embodiments, the DNase enzyme comprises a glycosylation consensus sequence that comprises a N40S substitution and/or an N128S substitution with respect to SEQ ID NO:1. For example, in some embodiments the enzyme comprises the amino acid substitutions: Q31R, N405, A136F, N128S, and T227K with respect to SEQ ID NO:1.
In various embodiments, the D1 variant contains one or more amino acid modifications (e.g., substitutions, additions, insertions, and deletions) in the glycosylation sites, and thereby increasing affinity towards lipid encapsulated DNA. As such, the engineered D1 protein can degrade lipid encapsulated DNA in transfection reactions. See Wilber et al., Mol Ther 6, 35-42 (2002).
In various embodiments, the D1 variant is glycosylated, or may be non-glycosylated particularly where long circulating half-life is not needed. The amino acid alterations described herein can be made in the context of glycosylated or non-glycosylated variants.
In some embodiments, D1 variant is resistant to actin-inhibition and capable of degrading chromatin within lipid vesciles. Exemplary D1 variants comprise one or more amino acid modifications selected from: Q31R, T227K, A136F, N40D or N40A, and N128D or N128S, with amino acids numbered according to SEQ ID NO:1. Such variants may lack glycosylation sites, which are beneficial from a manufacturing persepctive.
In some embodiments, the D1 variant is resistant to actin-inhibition and capable of degrading chromatin within lipid vesicles, and comprises amino acid modifications Q31R, T227K, A136F, N40D, and N128D with respect to SEQ ID NO:1.
Other modifications to D1 can be as described in Pan et al. J Biol Chem, 1998, 273, 11701-11708 and in US 2014/0199329, the entire disclosure of which is hereby incorporated by reference in its entirety.
The engineered D1 variant can have an increased or extended serum half-life compared to the wild-type D1 protein (SEQ ID NO:1). In some embodiments, the engineered D1 comprises an Fc-fusion domain (e.g. a hinge region, and/or and CH2 domains and CH3 domains of an immunoglobulin). In some embodiments, the engineered D1 is fused to albumin, e.g., human albumin or a fragment thereof. Albumin can be joined to at the N-terminus or the C-terminus of the engineered D1 enzyme, optionally through an amino acid a linker.
In some aspects, the invention provides pharmaceutical compositions comprising the recombinant D1L3 and/or D1 variants, or polynucleotides or vectors encoding the same, and a pharmaceutically acceptable carrier. In various embodiments, the recombinant D1L3 protein variant is formulated for parenteral or pulmonary administration. In some embodiments, the composition is formulated for intravenous, intraarterial, intraperitoneal, intraarticular, intramuscular, topical, or subcutaneous administration, or other route described herein. In some embodiments, the composition comprises both D1L3 and D1, which are each optionally engineered variants described herein. In some embodiments, the recombinant D1L3 variant and the recombinant D1 variant are formulated for parenteral or pulmonary administration.
The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial, oral, sublingual, pulmonary, or transdermal. In some embodiments, the administering is effected orally or by parenteral injection or infusion. In some embodiments, the route of administration is topical, including as eye drops or mouth wash.
In still other aspects, the invention provides a method of making a pharmaceutical composition for reducing or preventing neutrophil extracellular trap (NET) accumulation. In these embodiments, the invention employs a genetically modified mouse deficient in D1 and D1L3 activity, and heterologous expression of a G-CSF polynucleotide (e.g., in hepatocyte cells) or induction of a sustained endogenous G-CSF expression (e.g., via repetitive administration of microbial compounds). This mouse model accumulates NETs and rapidly develops NET-related vascular occlusions. In these embodiments, the invention comprises administering a candidate NET inhibitor or candidate DNase enzyme (including a D1L3 or D1 variant in accordance with this disclosure) to the genetically-modified mouse, and selecting a NET inhibitor or DNase enzyme that reduces the accumulation of NETs. The selected inhibitor or enzyme is formulated (as described) for administration to a human patient.
One skilled in the art recognizes standard methods for generating double knockout Dnase1−/−Dnase1l3−/− mice. Detailed descriptions can be found in, for example, European Application No. EP 17152528.0.
In some aspects, the invention provides a method for treating a subject in need of neutrophil extracellular trap (NET) degradation. The method comprises administering a therapeutically effective amount of D1L3 and/or D1 (which may include variants described herein, or in some embodiments one or more wild type enzymes, including for D1L3 and D1 combination therapy) according to this disclosure. The D1L3 or D1 may be administered as pharmaceutical compositions comprising the recombinant protein, or in some embodiments comprising the encoding DNA or RNA or vectors comprising the same.
In some embodiments, the method comprises administering a therapeutically effective amount of a D1L3 or variant thereof and a D1 or variant thereof. The D1L3 or variant thereof, and the D1 or variant thereof, may be administered in a single pharmaceutical composition or separate pharmaceutical compositions. In some embodiments, the ratio of D1L3 or variant thereof to D1 or variant thereof is in the range of 100:1 to 1:100 by mass, or 10:1 to 1:10 by mass, and is optionally about 1:1.
In some embodiments, the subject exhibits impaired NET degradation and/or exhibits pathological NET accumulation. In some embodiments, the subject has a chronic or acute inflammatory disorder. In some embodiments, the subject has an acute or chronic infection.
In various embodiments, the present invention pertains to the treatment of diseases or conditions characterized by the presence or accumulation of NETs. Such diseases or conditions include, but are not limited to, diseases associated with chronic neutrophilia (e.g., an increase in the number of neutrophils), neutrophil aggregation and leukostasis, thrombosis and vascular occlusion (e.g. sickle cell disease), ischemia-reperfusion injury (e.g. midgut volvulus, testicular torsion, limb ischemia reperfusion, vital organ ischemia-reperfusion, organ transplantation), surgical and traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease (e.g. systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, vasculitis, systemic sclerosis), cardiovascular disease (e.g., myocardial infarction, stroke, atherosclerosis, venous thromboembolism, including thrombolytic therapy), metabolic disease (e.g., diabetes), systemic inflammation (e.g., systemic inflammatory response syndrome (SIRS), sepsis, septic shock, disseminated intravascular coagulation (DIC), and thrombotic microangiopathy (TMA)), inflammatory diseases of the respiratory tract (e.g. cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), smoke induced lung injury, transfusion induced lung injury (TRALI), acute respiratory distress syndrome (ARDS), and asthma, atelectasis, bronchitis, empyema), renal inflammatory diseases (acute and chronic kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), inflammatory diseases related to transplated tissue (e.g. graft-versus-host disease) and cancer (e.g. leukemia, tumor metastasis, and solid tumors).
In some embodiments, the subject has or is at risk of NETs occluding ductural systems. The present invention can be administered to a subject to treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, obstructions of vas deferens, or renal diseases. In such embodiments, the method comprises administering a therapeutically effective amount of a D1L3 or variant thereof, and/or a therapeutically effective amount of D1 or a variant thereof. For example, the subject may be administered an enzyme comprising an amino acid sequence having 80% or more sequence identity with the enzyme defined by SEQ ID NO:2, and an enzyme comprising an amino acid sequence having 80% or more sequence identity with the enzyme defined by SEQ ID NO:1. In some embodiments, the enzymes comprise the amino acid sequence of the enzyme defined by SEQ ID NO:2 and the amino acid sequence of the enzyme defined by SEQ ID NO:1.
In some embodiments, the ductal system is bile duct, tear duct, lactiferous duct, cystic duct, hepatic duct, ejaculatory duct, parotid duct, submandicular duct, major sublingual duct, bartholin's duct, cerebral aqueduct, pancreas, mammary gland, vas deferens, ureter, urinary bladder, gallbladder, and liver. For example, the subject may have pancreatitis, cholangitis (e.g., primary sclerosing cholangitis), conjunctivitis, mastitis, dry eye disease, an obstruction of the vas deferens, or renal disease. In some embodiments, the DNase enzyme is administered by intravenous, intraarterial, or intraperitoneal administration. In various embodiments, the DNase when applied, for example, intravenously, will be present in enzymatically active form in various ductal systems, such as in bile fluid.
In some embodiments, the subject has or is at risk of NETs accumulating on endothelial surfaces (e.g. surgical adhesions), the skin (e.g. wounds/scarring), or in synovial joints (e.g. gout and arthritis). The present invention can be administered to a subject to treat a condition characterized by an accumulation of NETs on an endothelial surface such as, but not limited to, a surgical adhesion. In various embodiments, the present invention can be administered to a subject to treat a condition characterized by an accumulation of NETs on skin such as, but not limited to, wounds and scars. In certain embodiments, the present invention can be administered to a subject to treat a condition characterized by an accumulation of NETs in a synovial joint such as, but not limited to, gout and arthritis.
In some embodiments, the subject has or is at risk of a vascular occlusion comprising NETs. In such embodiments, the method comprises administering a therapeutically effective amount of a D1L3 or variant thereof, and/or a therapeutically effective amount of D1 or a variant thereof. For example, the subject may be administered an enzyme comprising an amino acid sequence having 80% or more sequence identity with the enzyme defined by SEQ ID NO:2. In some embodiments, the enzyme comprises the amino acid sequence of the enzyme defined by SEQ ID NO:2. In some embodiments, the subject is further administered an enzyme comprising an amino acid sequence having 80% or more sequence identity with the enzyme defined by SEQ ID NO:1, such as the enzyme defined by SEQ ID NO:1.
In some embodiments, the subject has a condition relating to NETs as described in WO 2016/118476 and U.S. Pat. No. 9,642,822, which are hereby incorporated by reference in its entirety.
In various embodiments, the subject has a disease that is or has been treated with wild-type DNases, including D1 and streptodomase. Such diseases or conditions include thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia reperfusion, kidney injury, blood clotting, alum-induced inflammation, hepatorenal injury, pleural exudations, hemotorax, intrabiliary blood clots, post pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, post-operative rhinoplasties, infertility, bladder catheter, wound cleaning, skin reaction test, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary cilliary dyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyes disease, lower respiratory tract infections, chronic hematomas, Alzheimer's disease, and obstructive pulmonary disease.
In certain embodiments, the present invention pertains to the treatment of diseases or conditions characterized by deficiency of D1, deficiency of D1L3, and deficiency of D1 and D1L3. In some cases, the subject has a mutation in the Dnase1 and/or the Dnase1l3 gene. Such subjects can also have an autoimmune disease (e.g., SLE, systemic sclerosis) or an inflammatory disease. In some cases, the subject has an acquired inhibitor of D1(e.g., anti-DNase1-antibody and actin) and/or the D1L3 (e.g., anti-DNase1l3-antibody). Such subjects can also have an autoimmune disease (e.g., SLE, systemic sclerosis) or an inflammatory disease (e.g., sepsis and ischemia-reperfusion injury).
In some embodiments, the subject has cystic fibrosis, and the DNase composition is administered by pulmonary delivery.
In various embodiments, for example, where the subject is at risk of, or exhibits symptoms of, NET-related vascular occlusion, the protein or composition (e.g., comprising D1L3, D1, or a variant of D1L3 or D1, including variants described herein) may be administered intravenously, intramuscularly, subcutaneously, or intraarterially.
In various embodiments, the subject is monitored for the NET-degrading activity of blood, plasma, or serum. For example, the subject may be monitored for about one week to about four weeks, to reduce the risk of vascular or ductal occlusion by NETs, or NET-related damage to organs. In some embodiments, the subject may receive from one to four administrations over the course of from one week to one month (or 1-2 weeks), as needed to prevent or reduce intravascular NET accumulation (including NET-related intravascular occlusions) during an acute infection or inflammatory event. In other embodiments, the subject may receive continuous infusion over the course of from one hour to one month (or 1-2 weeks). In some cases, the subject may receive at least one, e.g., 1, 2, 3, 4 or more infusions daily, for example (without limitation), in the case of critical illness such as sepsis, stroke, or myocardial infarction.
The treatment of disease or conditions relating to NETs may involve administration of a D1 variant, a D1L3 variant, or a combination comprising a D1 variant and a D1L3 variant. In some embodiments, two or more of variants of the D1 protein described herein are administered. In some embodiments, two or more of variants of the D1L3 protein described herein are administered.
The present D1 protein variants and D1L3 protein variants can be administered as a prophylactic therapy, such as before the onset of one or more symptoms of the disease or condition.
As used herein, “treatment” or “treating” or “treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. In other embodiments, “treatment” or “treating” or “treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease (e.g., an individual who carries a genetic marker for a disease such as lupus).
In the methods of the invention, therapy is used to provide a positive therapeutic response with respect to a disease or condition. By “positive therapeutic response” is intended an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition. For example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) a reduction in neutrophil extracellular traps; (2) a reduction in intravascular clots; (3) a reduction in ductural clots; (4) a reduction of accumulation of NETs in synovial joints; (5) an increase in the degradation of neutrophil extracellular traps; (6) an increase in degradation of extracellular protein-free DNA; (7) an increase in degradation of extracellular chromosomal DNA or protein-bound DNA; (8) an increase in the degradation of neutrophil extracellular traps in the presence of autoantibodies; (9) a reduction in tissue/organ inflammation; (10) a reduction in tissue/organ injury; (11) a reduction in tissue/organ atrophy; (12) an increased patient survival rate; and (13) some relief from one or more symptoms associated with the disease or condition.
Positive therapeutic responses in any given disease or condition can be determined by standardized response criteria specific to that disease or condition. Clinical response can be assessed for changes in vascular occlusion and the presence of NETs using screening techniques such as magnetic resonance imaging (MRI) scan, ultrasound scan, histology, and counting of NETs in the circulation. In addition to these positive therapeutic responses, the subject undergoing therapy may experience the beneficial effect of an improvement in the symptoms associated with the disease.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
In various embodiments, the present invention provides an expression vector, comprising a nucleic acid encoding a wild type or D1L3 or D1 variant described herein, which can find use for genetic therapy. Genetic therapy can be useful for chronic illness such as a genetic deficiency of D1L3 and/or D1 (such as a deleterious mutation). In various embodiments, the expression vector comprises DNA or RNA. In various embodiments, the expression vector is a mammalian expression vector.
In some embodiments, expression vectors comprise a nucleic acid encoding the protein variants operably linked to an expression control region, or complement thereof, that is functional in a host cell (e.g., prokaryotic, eukaryotic, or mammalian cell). The expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent 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 influence expression of an operably linked nucleic acid. An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
Expression systems functional in human cells are well known in the art, and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and typically a TATA box 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 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral 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 the CMV promoter.
Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., 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 (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric fusion proteins including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.
All cited references are herein expressly incorporated by reference in their entirety.
EXAMPLES Example 1: Host DNases Prevent Vascular Occlusion by Neutrophil Extracellular TrapsPlatelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a non-canonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), antimicrobial DNA-fibers released by activated neutrophils. We show that two DNases, DNase1 and DNase1-like 3, degrade NETs in circulation during episodes of murine inflammation. In the absence of both DNases, intravascular NETs form clots that obstruct blood vessels and cause organ damage. Vascular occlusions in patients with severe bacterial infections are associated with a defect to degrade NETs ex vivo and the formation of intravascular NET-clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of NETs in inflammation.
Inflammation is an essential host response to control invading microbes and heal damaged tissues (1). Uncontrolled and persistent inflammation causes tissue injury in a plethora of inflammatory disorders. Neutrophils are the predominant leukocytes in acute inflammation. During infections neutrophils generate extracellular traps (NETs), lattices of DNA-filaments decorated with toxic histones and enzymes that immobilize and neutralize bacteria (2). The relevance of NETs in host defense is illustrated by the fact that extracellular DNases serve as virulence factors in several pathogenic bacteria (3, 4).
However, inappropriately released NETs may harm host cells due to their cytotoxic, proinflammatory, and prothrombotic activity (5-7). Indeed, NETs are frequently associated with inflammatory or ischemic organ damage and the therapeutic infusion of DNases limits host injury in various animal models (8, 9).
How the host degrades NETs in vivo to limit tissue damage during episodes of inflammation is poorly understood. Earlier work has shown that DNase1 in serum digests the DNA-backbone of NETs in vitro (10). We analyzed serum from wild type mice by zymography and detected two enzymatically active DNases, DNase1 and DNase1-like 3 [(DNase1l3) (
To test the requirement of DNase1 and DNase1l3 for NET-degradation in vivo, we aimed to chronically stimulate wild type, DNase1−/−, DNase1l3−/−, and DNase1/DNase1l3−/− mice with the granulocyte-colony stimulating factor (G-CSF). G-CSF triggers neutrophilia, a hallmark of acute inflammation, and stimulates a subpopulation of neutrophils to spontaneously release NETs ex vivo (14). We cloned the cDNA of CSF3, which encodes G-CSF, into the hepatocyte-specific expression plasmid. Hydrodynamic injection of wild type mice with the CSF3-plasmid resulted in chronically elevated levels of G-CSF in plasma (
Next, we stably expressed CSF3 in the liver of DNase1−/−, DNase1l3−/−, and DNase1/DNase1l3−/− mice. Mice with a single deficiency in DNase1 or DNase1l3 did not show signs of distress (not shown), whereas all mice with a combined deficiency died within 6 days after CSF3-injection (
The histological analysis of DNase1/DNase1l3−/− mice with chronic neutrophilia showed intravascular hematoxylin-positive clots with entrapped erythrocytes that fully or partially occluded blood vessels in lungs, liver, and kidneys (
The formation of NET-clots in DNase1/DNase1l3−/− mice is associated with features of infection-induced thrombotic microangiopathies (TMAs) and disseminated intravascular coagulation in patients, including schistocytes, hemolytic anemia, and organ failure due to vascular occlusions. We analyzed plasma from TMA patients with hemolytic-uremic syndrome due to an infection with Shiga toxin-producing Escherichia coli [STEC-HUS, (16)]. Sepsis and septic shock was a frequent complication in these patients (17). NETs generated in vitro remained intact after exposure to patient plasma collected in the acute disease state, whereas plasma from patients in remission degraded NETs (
Large aggregates of NETs are reportedly formed in the synovial fluid and pancreatic ducts of patients (21,22), but have not yet been described in other tissues. Therefore, we aimed to identify intravascular aggregates of NETs in patients with severe inflammatory diseases. We screened lung tissue collected at autopsy from patients with acute respiratory distress syndrome and/or sepsis (
Septicemia is a potent and rapid trigger of intravascular NET-formation in mice (7). We therefore hypothesized that a defect in NET-degradation may aggravate the disease. Indeed, we observed that mice with a combined deficiency of DNase1 and DNase1l3, but not wild type mice, were highly susceptible to low doses lipopolysaccharide and heat-killed E. coli (
In summary, while platelets and fibrin form hemostatic clots and pathological thrombi (23), our data introduces NET-clots as a non-canonical mechanism for vascular occlusion in inflammatory states. Similar to fibrin strands, NETs are ultra-large and stable molecules (6). At high concentrations, such as found in chronic neutrophilia or septicemia, intravascular NETs may form clots, which are sufficient in size to obstruct blood vessels and thus cause damage to erythrocytes and organs. To maintain blood and tissue integrity during inflammation, the host independently expresses DNase1 and DNase1l3 as a dual protection system against intravascular NETs. However, acquired and genetic defects in these host factors may delay the degradation of NETs and thus precipitate disease. Acquired defects may involve DNase1-inhibition by monomeric actin externalized from damaged tissue and inactivation of DNase1l3 by serum proteases (11). Mutations in DNase1 and DNase1l3 have been identified in patients and are associated with systemic lupus erythematosus (SLE), a systemic autoimmune disease (24,25). Interestingly, DNase1 and DNase1l3-deficient mice spontaneously develop SLE-like disease with age (13,26). NETs are composed of prominent autoantigens and neutrophils from SLE patients have an increased capacity to release NETs (27). A reduced clearance capacity may increase the half-life NETs and thus promote the autoimmune disease (10,27). In conclusion, defects in host DNases may bring forward the deleterious effects of NETs in a plethora of inflammatory diseases.
Consequently, DNase1- and DNase1l3-mediated NET-degradation provides attractive therapeutic opportunity for patients suffering from inflammatory conditions. To test whether DNase1 and DNase1l3 degrade NETs by the same or via distinct mechanisms, we cross-linked proteins in NETs using fixative. Fixed NETs were efficiently degraded by DNase1l3−/− sera, but were resistant to degradation by DNase1−/− sera (
Finally, we compared the activity of DNase1 and DNase1l3 in the circulation of mice to humans. To discriminate DNase1 and DNase1l3 activity, we generated antibodies against human DNase1 (α-hDNase1), which block DNase1 activity in plasma from normal healthy donors (NHD) (
Materials and Methods
Patient Plasma
Citrated plasma samples were obtained from patients during the STEC-HUS outbreak 2011 in Germany (16). The criteria for inclusion in our study were positive identification of STEC/enterohemorrhagic E. coli or bloody diarrhea, platelet count≤150×109/L or decreased by ≥25% in 1 week, evidence of hemolysis (LDH above the normal limit, haptoglobin below the normal limit, or the presence of schistocytes) and acute kidney injury stage ≥1.
Human Tissue
Human tissues were fixed at room temperature overnight in 5% phosphate-buffered formalin. All tissues were collected at autopsy and originally submitted for diagnostic purposes to us and studied in accordance with national ethical principles. The cause of death of the patients is indicated in
Mice
All mice were on a C57BL/6 genetic background. We crossed previously described DNase1−/− and DNase1-like 3−/− mice (26,28) to generate DNase1/DNase1l3-/−mice. This strain of DNase1−/− mice has been recently reported to contain an off-target mutation in the DNase1 overlapping gene Trap1/Hsp75, which encodes a mitochondrial chaperone (29). We therefore included an alternative DNase1−/− strain (30) to generate DNase1/DNase1l3−/− mice for control experiments (
Detection of DNase1 by Denaturing Polyacrylamide Gel Electrophoresis Zymography (DPZ)
We performed DPZ, as previously described with modifications (19). In brief, sodium dodecyl sulfate (SDS)-polyacrylamide gels were prepared with 4% (v/v) stacking gels without DNA and 10% (v/v) resolving gels containing 200 μg/ml of salmon testes DNA (Sigma-Aldrich, Germany). For the detection of DNase1, 0.5 μl of murine serum were mixed with 14.5 μl of water and 5 μl SDS gel-loading buffer (BioRad, Germany). The mixture was boiled for 5 minutes and loaded onto the gels. Electrophoresis was carried out at 120 V using Tris/glycine electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.7). After electrophoresis, proteins were refolded by incubating the gels overnight at 37° C. in a refolding buffer containing 5% (w/v) milk powder, 10 mM Tris/HCl pH 7.8, 3 mM CaCl2, 3 mM MgCl2, 100 U/mL penicillin, and 100 μg/mL streptomycin. The gels were transferred to a refolding buffer without milk powder and incubated for additional 24 hours at 37° C. DNA was labeled fluorescently with 1×SYBR Safe (Thermo Scientific, Germany) and fluorescent images of gels were recorded using a fluorescence scanner (Molecular Imager FX, BioRad, Germany).
Detection of DNase1l3 by DPZ
For the detection of DNase1l3, 2 μl of serum were mixed with 12 μl of water, 5 μl SDS gelloading buffer, and 1 μl of beta-mercaptoethanol (BME, Sigma-Aldrich). BME reduces the disulfide bridges of DNase1, which causes its inactivation (11). The mixture was boiled for 5 minutes and loaded onto the gels. Electrophoresis was carried out as described for DNase1. SDS was removed by washing the gels with 10 mM Tris/HCl pH 7.8 for 30 minutes at 50° C. twice. The proteins were refolded by incubating the gels for 48 hours at 37° C. in a refolding buffer containing 10 mM Tris/HCl pH 7.8, 1 mM BME, 100 U/mL penicillin and 100 μg/mL streptomycin. The gels were then incubated for additional 48 hours at 37° C. in refolding buffer supplemented with 3 mM CaCl2 and 3 mM MnCl2. The addition of Mn2+ is required to enable efficient degradation of protein-free DNA by DNase1l3 (11). DNA was labeled and imaged as described for DNase1.
Detection of Total DNase Activity by Single Radial Enzyme Diffusion (SRED) Assay
To measure total DNase activity, we dissolved 55 μg/ml DNA from salmon testes in a buffer containing Mn2+ (20 mM Tris-HCl pH 7.8, 10 mM MnCl2, 2 mM CaCl2, and 2×SYBR Safe). The DNA solution was heated at 50° C. for 10 minutes and mixed with an equal volume of 2% ultra-pure agarose (Thermo Scientific). The mixture was poured into plastic trays and stored at room temperature until solidification. Two μl of murine serum were loaded into wells of 1.0 mm diameter. Gels were incubated for 4 hours at 37° C. in a humid chamber. The DNA fluorescence of the gels was recorded with a fluorescence scanner.
In Vitro NET-Degradation Assay
NET-degradation was analyzed as previously described (19). Purified human neutrophils in serum-free DMEM were seeded to sterile 96-well plates (Falcon, BD Technologies, Germany) coated with 0.001% polylysine (Sigma) at a concentration of 5×104 cells per well. To induce NET-formation, neutrophils were activated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 4 h at 37° C. with 5% CO2 and humidity. We added phosphate buffered saline (PBS) and stored the 96-well plates overnight at 4° C. NETs were washed with PBS, treated for 5 minutes with PBS containing 0.5% Triton X-100, and washed with PBS again. NETs were incubated with 10% murine serum or 10% citrated human plasma supplemented with 10 μM PPACK (Santa Cruz, Heidelberg, Germany) in HBSS with divalent cations (HBSS+; Thermo Scientific). NETs were allowed to be degraded for 3 hours at 37° C. with 5% CO2 and humidity. We discarded supernatants and stopped NET-degradation by adding 2% PFA in PBS for 1 hour at room temperature. PFA was discarded and non-degraded NETs were labeled fluorescently by adding 2 μM of the fluorescent DNA dye SytoxGreen (Thermo Scientific) in PBS. Images of fluorescently stained NETs were acquired with an inverted fluorescence microscope (Axiovert 200M, Zeiss, Germany). We quantified nondegraded NETs by recording the fluorescence intensity with a plate reader (Excitation: 485 nm; Emission: 535 nm) or by measuring the area coverage of NETs in microscopy pictures using ImageJ software (NIH, USA).
Preparation of In Vivo Expression Vectors
We used the pLIVE plasmid (Minis Bio, USA) to express proteins in mice. The vector enables a long-lasting and hepatocyte-specific expression of proteins. We generated pLIVE plasmids with murine DNase1, DNase1l3, and CSF3. For murine DNase1, a PCR of the cDNA (Genbank Accession Number NM010061) was performed using the pair of primers DNase1-F 5′-GTCGACATGCGGTACACAGG (SEQ ID NO:27) and DNase1-R 5′-CTCGAGTCAGATTTTTCTGAGTGTCA (SEQ ID NO:28) containing SalI and XhoI restriction sites. For DNase1l3, a PCR of the cDNA (Genbank Accession Number AF047355) was performed using the pair of primers DNase1l3-F 5′-GAAGTCCCAGGAATTCAAAGATGT (SEQ ID NO:29) and DNase1l3-R 5′-GCGTGATACCCGGGAGCGATTG (SEQ ID NO:30) containing BamHI and SacI restriction sites. Both cDNAs were cloned using the T4 ligase (New England Biolabs, Germany) into the multi-cloning site (MCS) of the pLIVE vector, which was predigested with the appropriate enzymes. The pLIVE vector containing DNase1l3 was subjected to site-directed mutagenesis with the pair of primers mutDNase1l3-F 5′-AGTCGACTCCCGGCCACCATGTCCCTGCA (SEQ ID NO:31) and its complementary mutDNase1113-R 5′-TGCAGGGACATGGTGGCCGGGAGTCGACT (SEQ ID NO:32) in order to match the consensus Kozak sequence and optimize protein expression. The generation of the pLIVE vector containing the cDNA of CSF3 was outsourced (Eurofins Genomics, Germany). The cDNA of CSF3 (Genbank Accession Number BC120761) was inserted in the MCS between restriction sites SalI and XhoI. Sequence of all the generated vectors was confirmed by double stranded DNA sequencing. As a control we used the parental pLIVE plasmid without additional inserts. All plasmids were purified using PureLink HiPure Plasmid Maxiprep Kit and potential contaminations of endotoxin were removed using High Capacity Endotoxin Removal Spin Columns (both Thermo Scientific).
In Vitro Expression of Murine DNase1 and DNase1l3
Plasmids containing the cDNA for DNase1 or DNase1l3, described elsewhere were transfected into Human Embryonic Kidney (HEK) cells with Lipofectamine 3000 (Thermo Scientific) in serum-free conditions with DMEM media (Thermo Scientific) supplemented with 10% KnockOut serum replacement (Thermo Scientific). After 72 hours, supernatants were collected, centrifuged at 500×g for 10 minutes, sterile filtered, and concentrated by ultracentrifugation with 3K columns (Amicon Ultra, Millipore, Darmstadt, Germany).
In Vivo Gene Expression
The pLIVE-plasmids containing DNase1, DNase1l3, CSF3, or empty control plasmids were administered to mice via hydrodynamic tail vein injection. In brief, 50 μg of plasmid were diluted in 0.9% saline in a volume equivalent to 10% of the body mass of the mouse. Mice were anaesthetized with isoflurane and the plasmid solution was then injected intravenously over 5 to 8 seconds via the tail vein. In rare cases, mice did not fully recover from the injection within the first 24 hours and these animals were excluded from the study. For coexpression studies, 50 μg of the CSF3-plasmid were mixed with 50 μg of the empty control plasmid, or the plasmids containing DNase1 and DNase1l3. The solution containing both plasmids was administered via hydrodynamic tail vein injection.
Chronic Neutrophilia
Female mice at 4 weeks or 8-12 weeks of age were injected with 50 μg of the pLIVE-plasmid containing CSF3 to induce G-CSF overexpression and neutrophilia. Mice were monitored every 8 hours during the first week after injection, and daily afterwards. Temperature was measured in the perianal area by a contactless infrared-thermometer (Etekcity, Germany). For survival studies, mice were euthanized and scored as “non-surviving” if the animals showed signs of distress (no spontaneous movement, closed eyes, occasional gasping). In all cases these signs of distress were accompanied with a rapidly progressing and severe hypothermia, defined as decrease in body temperature of ≥4° C. compared to the body temperature before the plasmid injection, and with hematuria. Non-hypothermic mice did not show any signs of distress and were euthanized at the end of the experiment. For organ, blood, and urine collection, we analyzed four groups of three D1/D1L3−/− mice. Mice in each group were injected with a mixture of CSF3 with empty plasma, DNase1, or DNase1l3. Each group of mice was euthanized for biosample collection, when the first animal of the group showed signs of distress, severe hypothermia, and hematuria. This time point was defined as “exitus” and occurred within 3 to 6 days after the injection.
Platelet Depletion
Mice received an intraperitoneal injection of 2 μg/g platelet-depleting antibody or isotype control antibody (both Emfret, Germany). This treatment depletes >95% of platelets from circulation. The treatment was started 24 hours after the injection of CSF3 and repeated every 48 hours until completion of the experiment.
Thrombin Inhibition
Powdered dabigatran etexilate (Pradaxa, Boehringer Ingelheim, Germany) was mixed with normal chow powder (Altromin, Germany) at a dose of 40 mg/g. Pellets were prepared by mixing the powder with distilled water and allowed to dry at room temperature. The feeding of dabigtran was started 24 hours after the injection of CSF3. WT mice fed with the dabigratan diet for 1 day showed a 6.48±1.19-fold (Mean±SD, N=4) increased activated partial thromboplastin time when compared to mice receiving normal chow without dabigratan (Student t-test; P<0.001).
Preparation of Bacteria for Septicemia
Escherichia coli (XEN 14, Perkin Elmer) was grown overnight in lysogeny broth media containing 50 μg/ml kanamycin. Bacteria were pelleted by centrifuging at 4000×g for 10 minutes, washed with and resuspended in PBS. Aliquots of 1.5×109 bacteria/ml were incubated at 70° C. for 15 minutes to heat-kill the bacteria. Aliquots stored at −20° C. until further use.
Septicemia
Male mice at 8-15 weeks of age received three daily intraperitoneal injections of 1 μg/g of LPS from Salmonella enterica serotype thyphimurium (Sigma-Aldrich) in 0.9% saline. Along with the third LPS injection, mice received an intravenous injection of 1.5×107 heat-killed E. coli/g. The shown survival time indicates the time after the injection of E. coli. Blood and organs were collected at the time of euthanasia. Insufficient biosamples were obtained from two animals (1×D1/D1L3−/−+Ctrl, 1×D1/D1L3−/− +D1L3) to perform the complete analysis shown in
Murine Blood, Plasma, and Tissue Collection
Blood was collected by submandibular puncture or by the retroorbital sinus. Blood was collected into 200 μl serum tubes (Monovette; Sarstedt, Germany) and 200 μl EDTA tubes (GK 150, KABE labortechnik, Germany). Plasma was obtained by collecting the supernatant of blood after centrifugation at 3000×g for 15 minutes. Serum was obtained by allowing the blood to clot for 1 hour at room temperature followed by a centrifugation for 15 minutes at 3000×g. Serum and plasma samples were stored in aliquots at −20° C. until further use. For organ analysis, mice were perfused by intracardiac infusion of PBS. Organs were collected and fixed for 24 hours in 4% PFA at 4° C. Fixed organs were embedded in paraffin.
Analysis of Blood and Plasma
LDH, AST, ALT, creatinine and BUN in plasma were quantified by using standardized kits (Biotron Diagnostics, CA, USA) following the manufacturer instructions. Mouse G-CSF was quantified with a Quantikine ELISA Kit (R&D, United Kingdom). Hemoglobin in EDTA blood was quantified by an automated hemocytometer (Idexx ProCyte Dx Hematology Analyzer, Netherlands). To quantify neutrophils by FACS, whole blood was incubated on ice for 15 minutes with 0.2 μg of phycoerythrine-labelled anti-mouse CD11b (M1/70, Biolegend, Germany) and 0.5 μg of Alexa Fluor 488 anti-mouse Ly6G (1AB, Biolegend, Germany). The blood was then diluted with 0.5 ml PBS and analyzed with a FACSCalibur (BD Bioscience, USA). Blood smears of EDTA-anticoagulated blood were prepared on polylysine-coated slides (Hecht Assistant, Germany). After air-drying were incubated for 1 minute in methanol supplemented with 1 μM SytoxGreen on dry ice or stained with Giemsa using commercial kit (In Vitro Diagnostikum, Germany).
Immunofluorescence Stainings
Paraffin-embedded sections were de-waxed, rehydrated, and subjected to antigen retrieval for 25 minutes at 100° C. in citrate buffer (10 mM sodium citrate, 0.1% Tween, pH 6). Thereafter, sections were blocked for 30 minutes with 2.5% normal goat serum (Vector, United Kingdom) followed by incubation with a mouse-on-mouse blocking kit (Vector) for one hour. The sections were then incubated over night at 4° C. with 2 μg/ml of the primary antibody against MPO (A0398, Agilent, Germany), CRAMP (PA-CRLP-100, Innovagen, Sweden), citrullinated histone 3 (ab5103, Abcam, United Kingdom), fibrin [clone 59D8], or the complex of histone H2A, H2B, and DNA to detect chromatin (2). Sections were incubated with anti-rabbit and anti-mouse IgG antibodies conjugated with AlexaFluor488 or AlexaFluor555 (all Thermo Scientific) for 1 hour. After washing, DNA was labeled with 1 μg/ml DAPI for 2 minutes. Autofluorescence was quenched by a 25-minute incubation with Sudan Black (0.1% in 70% ethanol), and sections were mounted with Fluoromount G (Southern Biotech, USA). Images of fluorescently labeled sections were acquired with an inverted fluorescence microscope (Axiovert 200M, Zeiss, Germany) or a confocal microscope (TCS SPS, Leica, Germany). Images of NET-clots were quantified using software (ImageJ). A vWF or fibrin staining area of less than 3% of NET-clots was considered negative.
Immunohistochemistry
After antigen retrieval, deparaffinized sections were blocked for 30 minutes with 2.5% normal horse serum (Vector). The sections were incubated over night at 4° C. with 2 μg/ml of the primary antibody against neutrophil elastase (ab68672, Abcam). After washing, sections were stained using the anti-rabbit IgG-AP kit, (ImmPRESS, Vector) according to manufacturer's instruction. Sections were counterstained with hemalum (Merck, Germany) and mounted with Neo-Mount media (Merck). Images of stained sections were acquired with an inverted microscope (Axiovert 200M, Zeiss).
Generation of NET-Clots In Vitro
Purified human neutrophils were seeded to tissue culture plates at 3×107 cells/ml in DMEM supplemented with 2% BSA. The cells were activated with 0.1 μM PMA for 4 hours at 37° C., 5% CO2 with humidity and rotating conditions (300 rpm). We used unstimulated neutrophils and neutrophils activated with PMA in the presence 10 U/ml recombinant human DNase1 (dornase alpha, Roche, Germany) as controls. NET-clots were fixed with 2% PFA overnight at 4° C. Fixed NET-clots were embedded in paraffin and sections were stained for NETs as described above.
Statistical Evaluation
Data are shown as mean±standard deviation. Statistical analysis was performed using Prism Software (GraphPad, USA) and included Student's t-test, one-way and two-way ANOVA followed by Bonferroni's multiple comparison test, and Log-rank test. Results were considered significant at P<0.05.
Movies
Chronic neutrophilia is tolerated in wild type mice. A movie generated shows the wild type mice one week after the injection of a CSF3-expression plasmid (CSF3) or a control plasmid lacking CSF3 (Ctrl). Both animals show normal behavior and no signs of distress.
Chronic neutrophilia causes distress in mice lacking DNase1 and DNase1l3. The movie shows DNase1/DNase1l3−/− mice co-expressing CSF3 with a control plasmid (CSF3/Ctrl), with DNase1 (CSF3/D1,), or with DNase1l3 (CSF3/D1L3). Mice expressing circulatory DNases, DNase1 or DNase1l3, show normal behavior. Mice lacking circulatory DNases (CSF3/Ctrl) show signs of distress (no spontaneous movement, closed eyes, occasional gasping).
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Uncontrolled inflammation causes a plethora of conditions (1). Neutrophil extracellular traps (NETs) are DNA filaments decorated with toxic proteins (2). NETs protect against microbial infection, but excessive NET formation triggers and exacerbates inflammatory and autoimmune diseases (3). DNase1 (D1), a DNA-degrading enzyme found in blood, has been widely used to disarm NETs in vitro and in vivo. Indeed, infusion of recombinant human D1, an FDA-approved drug (dornase alpha) for cystic fibrosis (CF), prevents the pathological effects of NETs in various disease models (3), supporting the use of D1 for prophylactic therapy.
DNase1-Like 3 is Novel Drug Candidate for NET-Associated DiseasesIn Example 1, we found that in addition to D1, DNase1-like 3 (D1L3) also degrades NETs. Both enzymes maintain blood and tissue homeostasis during inflammatory responses. In brief, we showed that NETs are generated within blood vessels during neutrophilia, a hallmark of inflammation, under sterile as well as infectious conditions. D1 and D1L3 cleave NETs and thus prevent the aggregation of DNA-filaments of NETs to vascular clots. In the absence of D1 and D1L3, clots of NETs obstruct blood flow and cause organ damage and death. NET-clots are formed independently of platelets or fibrin and are therefore resistant to conventional anti-thrombotic therapy. Thus, we uncovered clots of NETs as a novel mechanism for vascular occlusion and D1L3 a new prophylactic therapy against NETs (see Example 1).
D1 and D1L3 belong, along with DNase1-like 1 (D1L1) and DNase1-like 2 (D1L2), to the DNase1-protein family (4). D1 and D1L3 are expressed in a variety of species including, humans, primates, and rodents. D1 is predominantly expressed in the gastrointestinal tract and exocrine glands (5), whereas hematopoietic cells, namely macrophages and dendritic cells produce D1L3 (6, 7). We found both DNases at low concentrations in serum of mice and humans (Example 1,
D1 was first discovered in body fluids of the gastrointestinal tract and urine, taking part in the digestion of DNA from food intake. We observed in the course of this disclosure that systemically administered DNase1 (e.g. through intraperitoneal or intravenous injection) has a relatively short half-life in circulation and is secreted in an enzymatically active form into the urine and, unexpectedly into bile. Its secretion into the bile fluid opens up the opportuntity to target extracellular DNA and/or NETs in the biliary duct via systemic DNase1 therapy.
D1L3 was first described in the context of programmed cell death (14). D1L3 features two nuclear localization sites (NLS1, NLS2). NLS2 is embedded within a C-terminal tail that is unique to D1L3 and not present in D1. Both NLS target the enzyme to the nucleus during apoptosis (14). Indeed, intracellular D1L3 is required for fragmentation of nuclear DNA within apoptotic and necrotic cells in vivo (15). D1L3 requires its C-terminal tail to degrade extracellular DNA, namely lipid-encapsulated DNA, such as found in transfections, and chromatin within apoptotic bodies (7, 10). In transfections, cDNA and cationic lipids form a complex that penetrates through the plasma membrane of target cells. D1L3 interferes with transfections and the C-terminal tail is critical for this function (10). Apoptotic bodies, lipid vesicles filled with chromatin from apoptotic cells, are physiological substrates of D1L3 (7). Here, the C-terminal tail enables D1L3 to penetrate through lipid membranes of apoptotic bodies and degrade the chromatin load. Importantly, the C-terminal tail is also required for the degradation of lipid-free extracellular chromatin by D1L3 (7). The C-terminal tail is believed to be critical for the functions of D1L3 in the extracellular space.
While hD1 is effectively targeting NETs in animal models, mainly mice, it has shown limited or no therapeutic effect in clinical trials of CF (16) and emphysema (17), respectively. NETs are formed in CF (18), indicating that wild-type hD1 is not optimal for targeting NETs in patients. Hyperactive variants have been generated to circumvent the therapeutic limitations of D1, but their clinical manufacturing is challenging (12). Notably, NET-DNA contains histones and is structurally organized like chromatin (2). Thus, we speculated that the efficient degradation of chromatin is required for an optimal therapy against NETs.
A combinational therapy of hD1 and tissue plasminogen activator (tPA, alteplase) of emphysema patients has been described (17). The data are in line with our hypothesis because tPA enables D1 to degrade chromatin in vitro (19). U.S. Pat. No. 9,642,822, which is hereby incorporated by reference, further discloses combination therapy with tPA and D1 for therapeutic targeting of NETs (20). However, treatment with tPA is commonly associated with an increased risk of bleeding due to its fibrinolytic activity (17). D1L3 may provide a safer strategy for targeting NETs without putting patients at risk for bleeding, but a therapy with D1L3 protein has not been explored.
Engineering of DNase1 and DNase1-Like 3 Variants Through Amino Acid TransferIn this Example we generated variants of D1 and D1L3 that are engineered for therapy against extracellular chromatin (including NETs). To design variants, we hypothesized that the capacity of human D1L3 to degrade chromatin is encoded by individual amino acids and/or amino acid sequences, which are absent in human D1. Furthermore, we speculated that transferring these individual amino acids and/or an amino acid sequence from human D1L3 to D1 generates hyperactive D1 variants with increased chromatin-degrading activity. Similarly, enzymatic properties (e.g. glycosylation) can be transferred from D1 to D1L3. To test our hypothesis, we employed conventional amino acid mutations and developed a novel building-block (BB) technology to engineer variants of DNases from the DNase1-protein family.
To produce variants of D1 and D1L3, we used in vitro expression systems [e.g. Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, Pichia pastoris]. Enzymatic activity was characterized using the degradation of high-molecular weight (HMW)-DNA as a readout (
Selected D1 and D1L3 variants were further tested to degrade intravascular NETs, using an in vivo-expression system for members of the D1-protein family. See PCT/EP2018/051444, which is hereby incorporated by reference in its entirety. The system is based on two components, (a) an expression plasmid that enables the stable in vivo transfection of hepatocytes with cDNA of D1, D1L3, and variants thereof in recipient mice, and (b) Dnase1−/−Dnasell3−/− deficient mice, which are characterized by the absence of DNase activity in serum. In brief, the cDNA of candidate DNases is cloned into the hepatic expression vector. Next, the vector is co-expressed in Dnase1−/−Dnase1l3−/− deficient mice along with the cDNA of murine Csf3, which encodes for the granulocyte colony-stimulating factor (G-CSF). The expression of Csf3 induces neutrophilia with concomitant intravascular NET-formation, which in turn causes the aggregation of undigested NETs within blood vessels and death in Dnase1−/−Dnase1l3−/− mice. To identify drug candidates, we screened for D1, D1L3, and variants thereof that efficiently degrade NETs and thus prevent morbidity and mortality in this model.
Results Engineering DNase1 Variants Through Conventional Amino Acid SubstitutionsOur central hypothesis is that the capacity of human D1L3 to degrade chromatin is mediated by amino acids that are not present in D1. Sequence alignment showed that 44% of the amino acids in human D1 and human D1L3 are identical (
D1L3 is a cationic protein with an isoelectric point of 9.35, whereas D1 and the other members are more basic proteins. We therefore speculated that the cationic nature of D1L3 enables the enzyme to degrade chromatin. Through sequence alignment, we identified 37 arginine or lysine residues in the variable amino acids, which are absent in D1 (
Twenty-six of these additional arginine or lysine residues in D1L3 correspond to the following amino acid positions in D1 (SEQ ID NO: 1): 31 (position 9 with the signal peptide), 41 (19), 49 (40), 52 (30), 68 (46), 76 (54), 79 (57), 81 (59), 92 (70), 109 (87), 114 (92), 115 (93), 164 (142), 177 (155), 181 (159), 200 (178), 201 (179), 204 (182), 209 (187), 213 (191), 227 (205), 239 (217), 250 (228), 259 (237), 262 (240), and 280 (260). In addition, D1L3 features 3 sites of insertions of arginine or lysine residues: R80, K226 and a C-terminal tail (after amino acid position 282 in SEQ ID NO: 2), which features 3 arginine and 6 lysine residues.
We aimed to transfer the arginine and lysine residues that are part of the core body of D1L3 to D1 by introducing the following mutations into wild-type D1 (
In this Example, we were able to generate D1 variants that contain 23 of the 28 amino acid substitutions. We expressed the cDNA of these D1-variants in HEK293 cells and analyzed the dsDNA and chromatin degrading activity in the culture supernatants. We observed that the following amino acid modifications partially increased the chromatin degrading activity, while no major differences in dsDNA degradation were observed (
Several attempts have been made to generate hyperactive variants of D1 (6, 12, 13, 21-29). We found 184 mutations, which altered 82 of the 282 amino acids of wild-type human D1 (
We designed a new D1-variant that carries the combination of Q31R/T227K/A136F (SEQ ID NO: 7) and compared its enzymatic activity to wild-type D1 (SEQ ID NO: 1), as well as to D1-variants carrying single mutations in Q31R, T227K, A136F, or a combined mutation in Q31R and T227K (SEQ ID NO: 8). We characterized the dsDNA and chromatin-degrading activity in the culture supernatants of transfected HEK293 cells. We observed no major differences in dsDNA-degrading activity with D1-variants carrying single or combinational mutations of Q31R and T227K (
D1L3 features three sites that contain additional amino acids: the C-terminal tail starting after Q282 (NH2—SSRAFTNSKKSVTLRKKTKSKRS—COOH) (SEQ ID NO:33), and at two sites within the enzyme at S79/R80 and at K226 (
We conceptualized a building block-technology to transfer enzymatic properties from one member of the D1-protein family to another. The following cardinal steps characterize the technology (
-
- (1) Provide protein-protein alignment of donor and recipient DNase
- (2) Identify variable amino acid or amino acid sequence for transfer (building block)
- (3) Identify conserved amino acids in donor and recipient DNase that are located up and downstream of building block (anchors), respectively.
- (4) Replace cDNA encoding for building block between C- and N-anchors in recipient DNase, with cDNA between the anchors in donor DNase.
- (5) Synthesize cDNA of chimeric DNase, followed by in vitro/in vivo expression into a recipient organism that is preferably deficient in both donor and recipient DNase (e.g. CHO cells or Dnase1−/−Dnase1l3−/− mice).
A multiple-species alignment of D1 and D1L3 from human, mouse, rat, and chimpanzee (
The transfer of these building blocks from D1L3 into D1 generates D1-variants with the following mutations (
The following D1L3-variants are generated if the building blocks are transferred from D1 to D1L3 (
Next, we conceptualized a sequential approach to engineer D1-variants with D1L3 activity that starts with the transfer of multiple adjacent building blocks (clusters), continues with the transfer of individual building blocks, and ends with a transfer of individual amino acids or the combination of multiple building blocks into new chimeric enzymes (
To test our method, we designed a total of 19 D1-variants comprising either individual building blocks or clusters of building block cluster from D1L3 (
These D1-variants feature the following amino acid mutations: 1M_S22delinsMSRELAPLLLLLLSIHSALA, L23_A27delinsMRICS/I30_T32delinsVRS/E35_T36delinsES, M38_I47delinsQEDKNAMDVI, Q49_S52delinsKVIK/Y54C/I56_Q60delinsIILVM, V62_R63delinsIK/S65_K72delinsSNNRICPI/L74_N76delinsMEK, Q79_T84delinsRNSRRGIT, H86N/V88_V89delinsVI/E91_P92delinsSR, N96_S97delinsNT/R101Q/L103A/V105L, R107_Q10delinsKEKL/A113_S116delinsVKRS/Y118H/D120H, G122_N128delinsYQDGDA/T130S/N132S, A136_I137delinsFV, R139W/F141_F144delinsQSPH/E146_E149delinsAVKD/A151V/V153I/A157_A158d elinsTT/G160_A162delinsETS/A164K, A168E/Y170_D171delinsVE/L174T, Q177_K179delinsKHR/G181_L182delinsKA/D184_L187delinsNFIF, R199_Q202delinsPKKA/5204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE, A226_P228delinsVKKS, H230N/V238_A239delinsLR/M241_A246delinsQEIVSS/D250K/A252_P254delinsNS V, N256D/A259_A260delinsKA/G262K/S264_L267delinsTEEE/Q269_I271delinsLDV, and Y275F/V279_M280delinsFK/K282delinsQSSRAFTNSKKSVTLRKKTKSKRS.
Next, we cloned the cDNA into an expression vector, which was transfected into HEK293 cells. Analysis of the cell supernatants showed dsDNA degradation by all samples (
Thus, replacement of these amino acid sequences with the respective building blocks from D1L3, which do not interact with actin, likely generates actin-resistant variants of D1. We now combined BB 11, 14, 26, 41-19 in one novel D1-variant (SEQ ID NO: 15).
We observed that the combination of these gain-of-function BBs increased the chromatin degrading of the D1 variant to levels of wild-type D1L3 (
The BB cluster 60 to 62 of D1L3 includes the amino acid from its C-terminal tail (
Serum proteases, in particular plasmin, degrade D1L3, but not D1 (9). The sensitivity of D1L3 towards protease activity would limit its use in clinical practice. Glycosylated proteins display an increased protection against proteases (32). Using software analysis, we could not identify a known consensus sequence for glycosylation in human and murine D1L3. Software analysis of human D1 revealed 2 glycosylation sites, as observed both in silico and in vitro (9). We hypothesized that glycosylated variants of D1L3 may increase the half-life in circulation due to an improved resistance towards serum proteases. A glycosylation site requires a minimal consensus sequence of N-X-T/S. D1 contain two known N-glycosylation sites, N40 (N18 without signal peptide) and N128 [(N106 without signal peptide, (9)]. The amino acids of the D1-glycosylation sites N40-X-T42 and N128-X-T130 correspond to the D38-X-N40 and A127-X-V129 in D1L3. Thus, a glycosylated version of D1L3 needs to feature the amino acids mutations D38N, N40T, A127N, and V129T (see SEQ ID NO: 18). Instead of using conventional point mutations to insert these amino acid substitutions, we used BB technology to transfer the D1-glysylation sites N40-X-T42, which lies within BB4 (MSNATLVSY in SEQ ID NO: 1), and N128-X-T130, which lies within the BB cluster 23-25 (GCEPCGNDTFN in SEQ ID NO: 1), from D1 to D1L3 (
Therapy with DNase1-Like 3 and a Variant Thereof
Finally, we tested the therapeutic effects of D1L3 in vivo. Histological analysis of vascular clots of NETs show dense aggregates of chromatin (
We further explored the therapeutic application of D1L3V in a model of ischemia-reperfusion injury (
Nearly 70% of all protein therapeutics are produced using Chinese Hamster Ovary (CHO) (36). Indeed, wild-type DNase1 (dornase alpha) is produced in CHO cells. Despite significant advantages, cell line development and large-scale production in CHO still remains a huge challenge due a considerable degree of variability and no reliable methods for predicting or modeling cell growth characteristics (36). Importantly, CHO cells were not able to stably produce hyperactive variants of D1, which prevented their clinical manufacturing (12). The manufacturing properties of D1L3 are unknown. Thus, we developed stable CHO cell lines producing wild-type D1 (SEQ ID NO: 1), a hyperactive D1 variant (SEQ ID NO: 7), wild-type D1L3 (SEQ ID NO: 2) and a hyperactive D1L3 variant (SEQ ID NO: 17). We cultured the cell lines in bioreactors and observed a stable protein expression with wild-type D1 and the D1 variant, with the latter having strongly increased expression levels (
Due to high cell densities in large-scale bioreactors, cells compete for resources and undergo cellular stress leading to increased cell death called apoptosis, a cell death program used by multi-cellular organisms, including mammals (37). D1L3 targets DNA in apoptotic bodies (7). We speculated that the low levels of D1L3 are due to scavenging of the enzyme by these apoptotic vesicles. To overcome these limitations, we tested the production of D1, D1L3 and their variants using the microbial expression system, Pichia pastoris, an established, FDA approved and Generally Recognized As Safe (GRAS) organism with protein secretory abilities (38, 39). A key limitation of this approach is that proteins produced by P. pastoris are often hyperglycosylated and may result in enhanced immunogenicity (40). The composition of D1L3 and its variant are non-glycosylated and optimal for expression in P. pastoris. We mutated the glycosylation sites in D1 and its variant at N40S and N128S respectively to generate deglycosylated proteins (SEQ ID NO: 21, 23). Protein expression in P. pastoris is generally achieved by coupling proteins of interest to secretory signal peptides of various origins. We omitted this step to avoid the generation of immunogenic chimeric proteins. We speculated that the native signal peptide of D1 and/or D1L3 is recognized by P. pastoris. After codon-optimization was performed, we expressed the cDNA in P. pastoris (SEQ ID NO: 22, 24-26). We achieved expression of D1 and the D1 variant (not shown). Importantly, this approach furthermore enabled the expression of D1L3 and the D1L3 variant, with the latter showing increased expression levels (
Two DNA-degrading enzymes have been widely used therapeutically in experimental models and patients. Streptodornase, a secreted DNase from Streptococcus spp., has been tested in disease models of skin homograft, bladder clots, wound debridement, meningitis. DNase1 (including pancreatic DNase1) has been used therapeutically in experimental models of cancer, cystic fibrosis, lupus, thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia reperfusion, kidney injury, blood clotting, asthma, al urn-induced inflammation, hepatorenal injury. Streptodornase has also been used to treat patients with pleural exudations, hemotorax, intrabiliary blood clots, post pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, post-operative rhinoplasties, infertility, bladder catheter, wound cleaning, skin reaction test. Importantly, DNase1 has been tested therapeutically in patients with pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchi ectasis, lupus, primary cilliary dyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyes disease, lower respiratory tract infections, chronic hematomas, Alzheimer's disease, and obstructive pulmonary disease. In this example, we identified D1L3, variants thereof, as well as D1 variants as novel candidates for a DNase therapy (
To measure chromatin degrading activity, we mixed samples with nuclei isolated from HEK293 (1,500,000-3,000,000 per reaction), 20 mM Tris-HCl pH 7.4, 2 mM MgCl2, 2 mM CaCl2), 50 mM NaCl and protease inhibitors. After incubating the samples for 1 hour at 37° C., they were heated up to 65° C. for 10 minutes. We proceeded with DNA isolation using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's protocol. The eluent was then loaded onto an agarose gel and the DNA fragments were separated by gel electrophoresis. The DNA fluorescence of the gels was recorded with a fluorescence scanner.
Further methods are described in Example 1.
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Claims
1. A DNase 1-like 3 (D1L3) variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 2, with one or more modifications as compared to SEQ ID NO:2 selected from: a mutated DNA binding site or mutated chromatin binding site, a glycosylation site, inactivation of a nuclear localization signal, and deletion of all or part of a C-terminal tail.
2-5. (canceled)
6. The D1L3 variant of claim 1, wherein the variant comprises an inactivated nuclear localization signal (NLS).
7. The D1L3 variant of claim 6, wherein an NLS is inactivated by deletion of all or part of NLS1 and/or NLS2 of SEQ ID NO:2.
8. The D1L3 variant of claim 6, wherein an NLS is inactivated by substitution and/or deletion of amino acids within NLS1 and/or NLS2.
9-23. (canceled)
24. A pharmaceutical composition comprising the D1L3 variant of claim 1, and a pharmaceutically acceptable carrier.
25-32. (canceled)
33. A DNase1 (D1) variant comprising an amino acid sequence that is at least 80% identical to the enzyme of SEQ ID NO:1, and having one or more modifications with respect to SEQ ID NO:1 that are selected from: one or more mutated DNA binding sites or mutated chromatin binding sites, a mutated actin binding site, a mutated glycosylation site, addition of a nuclear localization signal, addition of a C-terminal tail having at least 50% sequence identity to the C-terminal tail of wild-type D1L3 protein of SEQ ID NO:2; and/or an N-terminal or C-terminal fusion to a half-life extending moiety.
34-40. (canceled)
41. A DNase enzyme comprising an amino acid sequence that is at least 80% identical to the enzyme of SEQ ID NO:1, and comprising the substitution, insertion, or addition of one or more arginine and/or lysine residues, wherein the DNase enzyme has increased chromatin-degrading activity as compared to the enzyme of SEQ ID NO:1.
42-77. (canceled)
78. A pharmaceutical composition comprising the enzyme of claim 1, and a pharmaceutically acceptable carrier.
79-85. (canceled)
86. A method for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation and/or neutrophil extracellular trap (NET) degradation, the method comprising administering a therapeutically effective amount of the D1L3 variant according to claim 1.
87. The method of claim 86, wherein upon treatment the subject exhibits extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation and/or neutrophil extracellular trap (NET) degradation.
88. The method of claim 86, wherein the subject has a chronic or acute inflammatory disorder.
89. (canceled)
90. The method of claim 86, wherein subject has or is at risk of a vascular occlusion comprising NETs.
91. The method of claim 86, wherein the subject has chronic neutrophilia (e.g., an increase in the number of neutrophils), neutrophil aggregation and leukostasis, thrombosis and vascular occlusion (e.g. sickle cell disease), ischemia-reperfusion injury (e.g. midgut volvulus, testicular torsion, limb ischemia reperfusion, vital organ ischemia-reperfusion, organ transplantation), surgical and traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease (e.g. systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, vasculitis, systemic sclerosis), cardiovascular disease (e.g., myocardial infarction, stroke, atherosclerosis, venous thromboembolism, including thrombolytic therapy), metabolic disease (e.g., diabetes), systemic inflammation (e.g., systemic inflammatory response syndrome (SIRS), sepsis, septic shock, disseminated intravascular coagulation (DIC), and thrombotic microangiopathy (TMA)), inflammatory diseases of the respiratory tract (e.g. cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), smoke induced lung injury, transfusion induced lung injury (TRALI), acute respiratory distress syndrome (ARDS), and asthma, atelectasis, bronchitis, empyema), renal inflammatory diseases (acute and chronic kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), inflammatory diseases related to transplated tissue (e.g. graft-versus-host disease) and cancer (e.g. leukemia, tumor metastasis, and solid tumors).
92. (canceled)
93. The method of claim 86, wherein the subject has or is at risk of a ductal occlusion in a ductal system.
94-97. (canceled)
98. The method of claim 86, wherein the subject has or is at risk of NETs accumulating on endothelial surfaces (e.g., surgical adhesions), the skin (e.g., wounds/scarring, uclers), or in synovial joints (e.g., gout, arthritis).
99-102. (canceled)
103. The method of claim 86, wherein the subject has thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia reperfusion, kidney injury, blood clotting, alum-induced inflammation, hepatorenal injury, pleural exudations, hemotorax, intrabiliary blood clots, post pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, post-operative rhinoplasties, infertility, bladder catheter, wound cleaning, skin reaction test, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary cilliary dyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyes disease, lower respiratory tract infections, chronic hematomas, Alzheimer's disease, and obstructive pulmonary disease.
104. The method of claim 86, wherein the subject further exhibits a coronary artery disease.
105. (canceled)
106. The method of claim 86, comprising monitoring the NET-degrading activity of blood, plasma, or serum from the subject.
107. A method for treating a subject exhibiting or at risk of intravascular occlusion involving NETs, the method comprising administering a therapeutically effective amount of a D1L3 or variant thereof, and/or a therapeutically effective amount of a D1 or a variant thereof.
108-109. (canceled)
110. A method for treating a subject that has or is at risk of a ductal occlusion in a ductal system, comprising administering a therapeutically effective amount of a D1L3 or variant thereof, and/or a therapeutically effective amount of D1 or a variant thereof.
111-155. (canceled)
Type: Application
Filed: Mar 5, 2021
Publication Date: Sep 9, 2021
Inventors: Tobias A. FUCHS (Wellesley, MA), Miguel JIMÉNEZ-ALCÁZAR (Madrid), Josephine GÖBEL (Reinbek), Hanna ENGLERT (Hamburg)
Application Number: 17/193,455
