Biopharmaceuticals – Daniel Batlle, Jan Wysocki, Northwestern University

Abstract for “Active low molecular-weight variants of angiotensin conversion enzyme 2 (ACE2)”

“Disclosed are variants ACE2, pharmaceutical compositions containing the variants ACE2, and treatment methods to reduce Angiotensin II (1-8 plasma levels) and/or increase Angiotensin (1-7 plasma levels) in a subject who is in need of such variants. The disclosed variants may include polypeptide fragments from ACE2 that have ACE2 activity to convert AngII (1-8) to Ang(1-7). The disclosed treatment methods are suitable for patients with chronic and non-diabetic kidney disease.

Background for “Active low molecular-weight variants of angiotensin conversion enzyme 2 (ACE2)”

The invention relates to Angiotensin converting enzyme 2(ACE2) and variants thereof for reducing plasma Angiotensin II (1-8), and/or increasing plasma Angiotensin (1-7). The disclosed variants may include fragments ACE2 that have ACE2 biological activity to convert AngII (1-8 to Ang (1) and/or for increasing plasma Angiotensin (1-7). This lower molecular mass allows the fragments to pass through the glomerulus. These variants of ACE2 could be used to treat conditions such as chronic kidney disease and acute kidney failure, non-diabetic and diabetic chronic kidney disease and its prevention, glomerulonephritis and its complications, scleroderma, severe hypertension and its skin, pulmonary, renal and hypertensive complications and malignant hypertension.

“Activation (RAS) of the renin-angiotensinsystem (RAS), plays a significant role in the pathogenesis and progression of CKD to ESRD1-3. The RAS can also be activated in acute renal failure4-7. New approaches are needed to combat RAS overactivity. These should be more effective than the current approaches that rely primarily on blocking Ang II formation and blocking the action of Ang II. Our group has been a pioneer in the development of therapies that promote the degradation of Ang II8-13. ACE2 has an important biological effect. It converts AngII (1-8), to Ang (1-7). This process tends to lower AngII (-8), and thus prevents any potentially harmful effects of the peptide. Ang(1-7), which is also formed by Ang II (1-8) cleavage, has tissue protective properties that are generally different to those of AngII (1-6). There is growing evidence that Ang (1-7) may have many therapeutic uses. This also highlights the importance of Ang (1-7) forming enzymes being potential therapeutic targets. They have the dual benefit of degrading Ang II as well as forming Ang (1-7)”.

“Years back, we and others purified and made murine ACE2 to avoid the immunogenicity14 we saw in our first studies. We used human ACE2 to treat hypertension in mice induced with AngII infusions13. In recent studies we examined the kidney effects of murine recombinant ACE2 given to mice with streptozotocin-induced diabetic kidney disease. (See Wysocki et al., Angiotensin-converting enzyme 2 amplification limited to circulation does not protect mice from development of diabetic nephropathy,? Kidney Int. 2016 Dec. 4. Pii: S0085-2538 (16), 30565-8. The content of this document is incorporated by reference in its entirety. This study used two approaches to amplify circulating ACE2: intraperitoneal daily injectables for four weeks and ACE2 gene delivery15. Minicircles were used to deliver ACE2 and resulted in a sustained, long-term and significant increase in serum ACE2 activity. Also, the ability to metabolize an acute Ang II load (1-8) was enhanced. Minicircle ACE2 was pretreated in mice with STZ-induced diabetics. The plasma ACE2 levels increased significantly and was associated with a greater than 100-fold increase of serum ACE2 activity. Minicircle ACE2 didn’t cause any changes in the urinary ACE2 activity in diabetic mice, however. As compared to untreated controls, albuminuria, glomerular cellularity, glomerular cellularity, and glomerular size were all similar in minicircle ACE2-treated mice to a similar degree. Despite months of high plasma ACE2 levels, a significant augmentation of ACE2 confined only to the circulation did not improve the glomerular lesion and hyperfiltration that are characteristic of early diabetes kidney disease. These results highlight the importance of targeting kidneys rather than the circulatory system for early stage diabetic kidney disease. Recombinant ACE2’s large molecular structure makes it difficult to filter by a normal glomerulus. This is a critical time to intervene to stop disease progression. However, in more severe glomerular renal disease we were able to demonstrate that infused rACE2 could be recovered from the urine10. It is not possible to reverse advanced kidney disease or reverse fibrosis at this stage. We designed shorter forms (ACE2) to overcome this limitation. They are more effective in treating kidney disease and allow for better tissue penetration to other organs like the heart and lungs.

“Based on our findings, we have developed forms of ACE2 with a shorter molecular structure that can be delivered to the renal before the development of marked alterations to glomerular permeability. They are also more easily delivered to the kidney in all cases. ACE2 is usually seen as a 110 kD protein that is not filterable in the kidney. It is found in the urine as a shedding product of the renal apical tubeular membrane where ACE2 expression is high9-11, 16. Our ACE2 proteins are active and have a lower molecular weight. They retain their full activity and therapeutic potential when the goal of increasing ACE2 activity is to not only increase systemic circulation just like the human recombinant intact ACE2. However, they are also unique in that they can be delivered to the kidney via glomerular filtration. This makes them more useful for treating kidney disease and tissue penetration to other organs.

“We have demonstrated that reducing the size of ACE2 makes it easier to filter through the glomerular barriers in mild increases in glomerular permeability such as in acute kidney injury, early stages of diabetes kidney disease (i.e. microalbuminuric phase. Our goal is to create a shorter form of ACE2 that can be delivered to the kidneys to combat kidney disease. This is a unique approach and complements current AT1 blockers and ACE inhibitors. Enhancing the degradation of Ang II has the unique advantage of leading to the formation Ang 1-7, a renoprotective peptide. It is also a more natural physiologic strategy than blocking the formation or activity of Ang II and its receptors, as done with current agents. The short ACE2 fragments can be used to increase tubular reabsorption and kidney uptake. Fc (constant fragment human IgG), DIII domain human serum albumin, and lysozyme are all examples of fusion polypeptides. All of these polypeptides were shown to be absorbed by receptor-mediated endocytosis on the kidney tubules’ apical surfaces. This application covers the subject matter.

“Disclosed are variants ACE2, pharmaceutical compositions containing the variants ACE2, and treatment methods to reduce Angiotensin II (1-8 plasma levels) and/or increase Angiotensin (1-7 plasma levels) in a subject who is in need of such variants. Polypeptide fragments from ACE2 that have ACE2 activity may be disclosed. These polypeptide fragments can convert AngII (1-8), to Ang(1-7). Preferably, the molecular weight of ACE2 polypeptides should be low enough that they can pass through the glomerulus to reach the kidney. Some embodiments of the polypeptide fragments may have a molecular mass less than 70 kD. We have studied A1-619, a compound we call 69 kD, and 1-605, a compound we call 1-605, with molecular masses of 65 kD to 60 kD to 55 kD or 50 kD. The subject receives the variant of ACE2 and a pharmaceutic formulation containing the variant of ACE2 in a suitable pharmaceutical carrier. The disclosed treatment methods are suitable for subjects who have or are at risk of developing non-diabetic and diabetic chronic kidney disease, acute kidney failure and its prevention and chronic kidney disease.

“The invention described herein uses several definitions as shown below and throughout the application.”

“As used herein and in the claims, the singular forms of?a? ?an,? ?an,? If the context requires otherwise, plural forms should be used. The term “a polypeptide fragment” could be used as an example. Should the term?a polypeptide fragment be understood to refer to?one or more of these fragments? Unless the context explicitly indicates otherwise. The term “plurality” is used herein. It can also be translated as “two or more”.

“As used herein, ?about?, ?approximately,? ?substantially,? ?substantially? They will be easily understood by people of ordinary skill in art. However, this will depend on the context in which it is used. If the meaning of the term is not understood by persons of ordinary skill in art, they will be referred to as?about? ?approximately’ and?about? It will be interpreted as a plus or minus 10% of that term, and?substantially? Significantly and plus or minus 10% of the particular term. “significantly” means more than 10% plus or minus of the specific term.”

“As used herein the terms ‘include? and?including? “The terms?include? and?including? have the same meaning as the terms?comprise? The same meanings of?including? and?comprise? are given to these terms. ?comprising. The terms ‘comprise? and?comprising? The terms?comprise? and?comprising are interchangeable. Should be read as?open? Transitional terms allow for the addition of additional components beyond those cited in the claims. The terms “consist of” and “consisting of” are interchangeable. The terms?consist? und?consisting? should be interpreted as being?closed? Should be understood as being ‘closed’ Transitional terms that prohibit the addition of other components than those cited in the claims are not permitted. “Consisting essentially of” is a term that refers to the inclusion of additional components. “Consisting essentially of” should be understood to mean partially closed, allowing for the addition of only those components that do no fundamentally alter the subject matter of the claimed claim.

“Subject” as used in this document. “Subject” may be interchangeable with the term “patient?” Or?individual? It may also include an ‘animal? an?animal?, and in particular a mammal. Mammalian subjects can include humans, other primates, domestic animals and farm animals.

“The disclosed compositions, methods, and kits can be used to treat a subject who is in need of them. A “subject in need of it?” This term refers to a subject who is at risk of developing certain diseases or disorders, such as non-diabetic and diabetic chronic kidney disease, acute kidney failure and its prevention and chronic kidney disease, acute kidney disease and its prevention, chronic renal disease and its prevention, chronic renal disease and its prevention, acute stroke, glomerulonephritis and its skin, pulmonary hypertension and its complications, malignant hypertension and renovascular hypertension secondary renal artery stensis, idiopathic, heart fibrosis and remodeling, cardiac fibrosisisisisisis and left ventricular hypertrophy, left ventricular hypertrophies, right vhyprophy and left ventricular hypertrophy and left acute.

“The terms “amino acid” and “amino sequence?” “Amino acid” and “amino acids sequence?” Refer to an oligopeptide or peptide or polypeptide or protein sequence (which terms can be interchangeably used), or a fragment thereof, as well as to naturally occurring and synthetic molecules. Where is?amino acids sequence? The sequence of naturally occurring proteins molecules, also known as the?amino acids sequence, is what is being referred to? Similar terms do not limit the amino acids sequence to the entire native amino acid sequence of the recited protein molecules.

“The amino acids sequences contemplated herein could include one or more amino- acid substitutions relative to the reference sequence. A variant polypeptide could include conservative and/or non-conservative amino acid substitutions relative the reference polypeptide. ?Conservative amino acid substitutions? These substitutions are those that are expected to have the least impact on the properties of the reference protein. Conservative amino acid substitutions preserve the structure and function of the reference protein. Below is a table that lists some examples of conservative amino acid substitutions.

“Original Conservative\nResidue Substitution\nAla Gly, Ser\nArg His, Lys\nAsn Asp, Gln, His\nAsp Asn, Glu\nCys Ala, Ser\nGln Asn, Glu, His\nGlu Asp, Gln, His\nGly Ala\nHis Asn, Arg, Gln, Glu\nIle Leu, Val\nLeu Ile, Val\nLys Arg, Gln, Glu\nMet Leu, Ile\nPhe His, Met, Leu, Trp, Tyr\nSer Cys, Thr\nThr Ser, Val\nTrp Phe, Tyr\nTyr His, Phe, Trp\nVal Ile, Leu, Thr”

“Conservative amino acids substitutions usually maintain one or more of the following: (a) The structure of the polypeptide-backbone in the area where the substitution is made, such as a beta or alpha helical conformation; (b) The charge or hydrophobicity at the site of the substitution and/or (c), the bulk of side chains. The structure of non-conservative amino acids substitutions is generally not maintained.

“The disclosed peptides can include an N-terminal esterification (e.g. a phosphoester modification) or a pegylation (e.g. to increase plasma stability (e.g. Resistance to exopeptidases and/or lower immunogenicity.

“A ?deletion? A?deletion? refers to any change in a reference sequence of amino acids (e.g. SEQ ID No:1 or SEQ ID NO:2) that causes the loss of one or more amino acids. A deletion is a change in a reference amino acid sequence (e.g. SEQ ID NO:1 or SEQ ID NO:2) that results in the loss of at least 1, 2, 4, 5, 10, 20, 100, 200, or 200 amino acid residues. An internal or terminal deletion can be included in a deletion. A “variant” A?variant? may be a sequence that is not the reference polypeptide. SEQID NO:3 (amino acid 1-619) & SEQID NO:4 (amino acid 1-605) both include C-terminal deletions relative SEQID NO:1 (amino acid 1-805).

“The words?”insertion? “The words?insertion? and?addition?” refer to changes in an amino acid sequence that result in one or more amino acids being added. The addition and insertion are changes in amino acid sequences that result in the addition or subtraction of amino acids. A insertion or an addition can refer to 1, 2, 4, 5, 10, 20, 40, 50, 70, 80 or 90 amino acid residues, or a range of amino acids bounded within any of these values (e.g. an insertion of 5-10 amino acid). Variant: A?variant? may be a modification or addition to a reference sequence of polypeptides.

“A ?fusion polypeptide? A?fusion polypeptide is a polypeptide that has at least one of its amino acids sequences at either the N-terminus or the C-terminus. Variant: A?variant? may be a sequence of reference polypeptides that includes a fusion protein.

“A ?fragment? A fragment is an amino acid sequence that is shorter than the reference sequence but is identical in sequence. A fragment can be as long as the length of the reference sequence but may contain at most one amino acid residue. A fragment could contain from 5 to 1000 consecutive amino acid residues of a reference sequence, for example. A fragment may contain at least 5, 10, 20, 25, 30, 40 or 50, 60 or 70, 80 or 90, 100 or 150, 250 or 500 contiguous amino acid residues. Or a fragment can comprise no more that 5, 10, 20, 25, 30, 40 or 50, 60 or 70, 80 or 90, 100 or 150, 250 or 500 contiguous amin acid residues. Some fragments may be preferred from particular regions of a molecule. Fragments are defined as a molecule that is at least one-third of its full length. The entire length of the polypeptide is included. Variant is the full length polypeptide. A?variant? may contain a fragment from a reference sequence. SEQID NO:3 (amino acid 1-619), and SEQID NO:4 (amino acid 1-605) contain fragments from the reference sequence SEQID NO:1 (amino acid 1-805).

“?Homology? “?Homology?” refers to sequence similarity, or, interchangeably: sequence identity, between two or three polypeptide sequences. Methods in the art, including those described herein, can be used to determine homology, sequence similarity, or percentage sequence identity.

“The phrases ‘percent identity? “Percent identity” and “% identity”, are two different phrases. Referring to polypeptide sequencing, the percentage of residue matches between at most two sequences of polypeptides aligned according to a standard algorithm. There are many methods for aligning polypeptide sequences. Some alignment methods take into account conservative amino acid substitutions. These conservative substitutions, which are explained in greater detail below, preserve the charge as well as the hydrophobicity at substitution sites. This preserves the structure and function of the polypeptide. As per the art, it is possible to determine percent identity for amino acids sequences. (See, e.g., U.S. Pat. No. No. 7,396,664, is included herein by reference in its entirety. The National Center for Biotechnology Information’s Basic Local Alignment Search Tool is a collection of widely used and freely accessible sequence comparison algorithms. (Altschul S. F. et. al. (1990) J. Mol. Biol. 215:403 403 410), which can be obtained from many sources including the NCBI in Bethesda (Md.) at its website. The BLAST software suite also includes sequence analysis programs such as?blastp. It is used to align an amino acid sequence with others from a variety databases.

“Percentage identity can be measured over the entire length of a defined polypeptide, such as a sequence number. Or it may be measured over a smaller length, such as the length of a segment taken from a larger sequence.

“In some embodiments, the term “variant” may be used. A?variant? may be a polypeptide that has at least 20% sequence identity with the specific sequence. It can also refer to a sequence of polypeptides that is longer than one of the sequences using blastp with?BLAST2 Sequences? The National Center for Biotechnology Information has a tool that can be used. (See Tatiana A. Tatusova and Thomas L. Madden (1999), “Blast 2 sequences?” a new tool to compare protein and nucleotide sequencing?, FEMS Microbiol. Lett. 174:247-250). This pair of polypeptides could show, for instance, at minimum 20%, 30 percent, at most 40%, and at the least 60%, a least 70%, a least 90%, a least 90%, a least 91% or more sequence identity over a defined length of one polypeptide or range of percentage identities bounded with any of these values (e.g. range of percentage identity between 80-99 %).”).

“The methods of treatment and pharmaceutical composition disclosed use and/or include angiotensin converting protein 2 (ACE2), or variants thereof, such as fragments. The National Center for Biotechnology Information of National Institutes of Health has the nucleotide sequence for the human ACE2 genes. NC_000023.11 is the location of human ACE2 genes (15494525 ). . . 15602069, complement). ACE2, isoform 1 is a transmembrane proteins that is first expressed as a precursor polypeptide with the amino acid sequence (SEQID NO:1). The following amino acid sequence is found in the homolog to ACE2 by Mus musculus:

“Amino acids 1-17” are a leader peptide that is cleaved form mature ACE2. Amino acids 18-740 are extracellular. Amino acids 741-761 form a helical transmembrane sequence. Cytoplasmic amino acids 762-805 exist. The natural variants of ACE2 that are considered herein include N638S and K26R. Herein are also considered natural isoforms for ACE2: isoform 2 with the following differences relative ACE2 isoform 1; F555L and???556-805. These variants of ACE2 may include fragments of ACE2.

“Fusion polypeptides from ACE2 and variants thereof are described in this document. A fusion polypeptide may contain the amino sequence of ACE2 (or a variant thereof) or a combination thereof (e.g. the amino sequence of a fragment from ACE2 fused to a heterologous sequence of amino acids). The preferred heterologous sequence of amino acids increases plasma half-life.

“The disclosed fusion polypeptides can contain the amino acids sequence of ACE2 (or a variant thereof) or a combination of them (e.g. the amino sequence of a fragment from ACE2 fused directly with a heterologous sequence or fused through a linker sequence. The suitable linker sequences can include amino acid sequences of 5,6, 7, 8, 9, 10, 11, 13, 14, or 15 amin acids, or any range that is bounded within these values (e.g. a linker of 5-15 amin acids). Some embodiments only contain glycine or serine residues in the linker sequence.

“Fusion polypeptides described herein include the amino acids sequence of ACE2 (or a variant thereof) fused to an antibody’s amino acid sequence or one or more fragments. For example, the Fc portion (constant fragment from human IgG) is preferably devoid of its hinge area to prevent dimerization (e.g. SEQ ID No:6). Fc can be fused with short ACE2 (e.g. SEQID NO:6) or monomeric CH3 Fc derivative (e.g. SEQID NO:7 or NO:8) to enable delivery via a functional FcRn dependent transport pathway in lung. This allows for more efficient administration of lung fibrosis treatment. The fusion polypeptides described herein also include the amino acids sequence of ACE2 and a variant thereof fused with serum albumin, or a fragment thereof. For example, domain III of human serumalbumin or a fragment thereof (e.g. SEQ ID NO.9). The amino acid sequence of ACE2 and a variant thereof fused with streptococcal proteins G or a fragment thereof, such as the Cterminal albumin binding domain (3 (ABD3)) of streptococcal proteins G (e.g. ABD3 strain G148 or ABD035 derivative (SEQID NO:5) are examples of fusion polypeptides. (See, e.g., Nilvebrant et al., Comput. Struct. Biotechnol. J. J.

“Fusion polypeptide described herein may include an amino acid tag sequence. This sequence may be used for purifying or identifying the polypeptide. Histidine tag sequences that contain 5-10 histidine residues are suitable.

“ACE2 is a carboxypeptidase that catalyzes angiotensin I’s conversion to angiotensin 9 (EC: 3.4.17.23). It also catalyzes angiotensin II (1-8), to angiotensin (1), which is a vasodilator. ACE2 also hydrolyzes apelin-13 and dynorphin-13. ACE2 is also the cellular receptor of sudden acute respiratory syndrome (SARS), coronavirus/SARSCoV, and human coronaviruses NL63/HCoV?NL63. These variants of ACE2 may include fragments of ACE2.

“ACE2 exhibits molecular functions that may include: carboxypeptidase activity, endopeptidase activity, glycoprotein binding activity, metallocarboxypeptidase activity, virus receptor binding activity, and zinc ion binding activity. These variants of ACE2 include fragments of ACE2. They all have at least one, cleavage Angiotensin II. However, they likely all possess all the molecular or enzymatic functions.

“Key structure features for ACE2 may include the following: helix, amino acids positions 53-77; 78-82; 79-82; helix, amino acids positions 134-141?helix, amino positions 453-460?helix and 465-460?turn? amino positions 539-541?helix, amino positions 559-562?helix and 575-578 respectively?helix and 576-574 respectively?beta strand and 607-609 respectively?helix These structural features may be present in variants of ACE2 described herein.

“ACE2 could include one or more of these amino acid modifications: amino acids 53, 90, N-linked glycosylation, amino acid position 103, N-linked glycosylation amino acid positions 133. ?141?disulfide bonds; amino acids position 322 and 344. N-linked glycosylation, amino acid positions 344. ?361?disulfide bonds; amino acids position 322?N-linked glycosylation, amino acid positions 344?N-linked glycosylation?N?linked glycosylation amino acid positions 531? ?542?disulfide bond; amino acid position 432?N-linked glysylation; amino acids positions 530?N?linked glycosylation? These variants of ACE2 may include fragments of ACE2 or may contain one or more of the amino acid modifications of ACE2 or may not contain these amino acids.

“ACE2 regulates biological process that can include angiotensin metabolism processes, angiotensin maturation and angiotensin-mediated drinking behaviors processes. These variants of ACE2 may regulate or fail to regulate any of the biological processes.

“The disclosed ACE2 variants could include an N-terminal methionine residue that is not found in the native amino acids for ACE2. The ACE2 variants may contain an N-terminal deletion in comparison to full-length ACE2. Further, it may be possible to modify the amino acids to include an Nterminal methionine.

“The disclosed ACE2 variations may contain an amino acid sequence or modified amino acids or other non-naturally occurring amino acids. This would make it impossible to say that the disclosed ACE2 varieties are naturally occurring. The disclosed ACE2 variants may be modified in some embodiments. This modification can be selected from the following groups: acylation and acetylation; formylation; lipolylation; myristoylation; palmitoylation; alkylations; isoprenylations. amidation. Modifications to an amino acid of the disclosed polypeptides can be made. The modifications may be at the N-terminus or C-terminus, depending on whether they are acetylations, acylations, or amidation. Modifications may increase the stability and/or resistance to proteolysis of polypeptides.

The ACE2 variants described may be modified to substitute a natural amino acids residue with an unnatural amino acid. Unnatural amino acids can include, but not be limited to, an amino acid with a D-configuration or an N-methyl?-amino, a non-proteogenic constrained amino acid or a?-amino.

“The ACE2 variants disclosed may be modified to improve plasma stability. The disclosed peptides could be modified to make them resistant to peptidases. Modifications to the disclosed peptides can be made to replace an amino bond between two amino acid with a non-amide one. The carbonyl moiety in the amide bond can be replaced with CH2 (i.e. to create a reduced amino-bond:?CH2-NH?)). An endothiopeptide (?C(S?NH)) can be used to replace the amide bond. A phosphonamide (?P(O?OH?NH)) is another possible non-amide substitute bond. The NH-amide bond can also be exchanged with O (depsipeptide),?CO?O S (thioester),?CO?S? or CH2 (ketomethylene) You can modify the peptide bond as follows: retroinverso bond?NH?CO? ), methyleneoxy bond (?CH2?) ), methylene-oxy bond (?CH2?) ), thiomethylene bond (?CH2??S? ), carbabond (?CH2?CH2????????????????????????????CH2?CH2??????CHOH.CH2????CH2????????CH2??????????CH2?CHOH?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH3?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH4?CH2?CH2?CH2?CHOH2?CH2?CH2?CH2?CH2?CH2?CH2?CHOH2?CH2?CH2?CH2? ”

“The disclosed ACE2 variants could include a non-naturally occurring C-terminal or N-terminal modification. The N-terminal end of disclosed peptides could be modified to include an N acylation or N-pyroglutamate modification (e.g. as a blocking modification). You can modify the C-terminal of disclosed peptides to include a C?amidation. To increase plasma stability and resistance to exopeptidases, the disclosed peptides can be conjugated with carbohydrate chains (e.g. via glycosylation to glucose or xylose).

“The variants described herein of ACE2 may be further modified. The polypeptide fragment of ACE2 could be modified in order to increase plasma half-life and/or enhance delivery to a target (e.g. the kidneys, lungs, heart, etc.). Some embodiments of the polypeptide fragment are covalently attached with a polyethylene glycol-polymer. Other embodiments allow the polypeptide fragment to be covalently attached to a polyethylene glycol polymer. Preferably the half-life of the polypeptide fragments of the described methods of treatment or pharmaceutical compositions is at least 6 hours, 12hrs, 3 days and 4 days, 5 day, 6 day, 6 week, 2 week, 3 days, 4 week, 5 days, 6 week, 6 week, 2 week, 3 week, 4 week, 5 days, 6 week, 6 week, 2 week, 3 week, 4 week, or more. The art has many strategies to increase plasma half-life for peptides and protein drugs. (See Werle and colleagues,?Strategies for improving plasma half-life time of peptides and protein drugs. Amino Acids 2006, 30(4):351-67. The content of which is incorporated by reference herein in its entirety.

“Pharmaceutical Compositions”

“The compositions described herein could include pharmaceutical compositions containing the presently disclosed bacteria toxins, and formulated to be administered to a subject who requires them.” These compositions can be prepared and/or administered in doses and using techniques that are well-known to medical professionals. This includes factors such as age, gender, weight, and current condition.

“The compositions can include pharmaceutical solutions that contain carriers, diluents and excipients as well as surfactants. The compositions can also include preservatives, such as anti-microbial and anti-bacterial agents like benzalkonium salt. Buffering agents may also be included in the compositions to maintain the pH between 6.5-7.5.

The compositions can be used therapeutically. The compositions may be administered therapeutically to patients in an amount that is sufficient to produce a therapeutic effect.

“Novel Active Short-ACE2 Fragments”

“The inventors discovered novel fragments full-length ACE2?molecular mass about 110 kD. They have a shorter molecular weight (less that 70 kD) and have high enzymatic activities. These fragments of ACE2 could be used in pharmaceutical compositions and treatment methods. The disclosed methods can be used in some cases to lower AngII (1-8) levels in a subject who is in need. There are many other substrates that can be cleaved using the novel ACE2 segments. The subject could be a pharmaceutical composition that contains a fragment of angiotensin-converting enzyme 2 (ACE2, SEQ ID NO:1) in a suitable pharmaceutical vehicle. The disclosed treatment methods are suitable for subjects with or at risk of developing diabetes and non-diabetic kidney disease, acute kidney failure and its prevention and chronic kidney disease. The disclosed pharmaceutical compositions can be administered in any manner, including intravenous injection and subcutaneously. Patients could also inject the contents themselves at home. Inhalation is another method of administering the disclosed pharmaceutical compositions. This could be useful for treating idiopathic lung fibrosis or other conditions.

“The ACE2 activity of the polypeptide fragment of ACE2 is for converting AngII (1-8) to Ang(1-7).” The polypeptide fragment of ACE2 may be delivered more efficiently to the kidneys than full-length ACE2.

“In general, the polypeptide of ACE2 is of low molecular weight so that it can be filtered through glomerulus and delivered directly to the kidney. Some embodiments have a polypeptide fragment with a molecular mass of less than 70 kD.

“The disclosed polypeptide fragments may contain a deletion relative full-length ACE2 (SEQID NO:1). A deletion may be made from either an N-terminal or C-terminal portion of the disclosed polypeptide fragments. This deletion could be relative to full-length ACE2(SEQ ID NO. 1). In some cases, the disclosed polypeptide fragments might also include an internal deletion. The deletion could remove approximately 1, 2, 3, 4, 5, 15, 20, 25, 50. 100, 150, 200 or more full-length ACE amino acids. Some embodiments may result in the deletion of one or more glycosylation site. As such, polypeptides fragments from ACE2 may be less well glycosylated that full-length ACE2. This further reduces the molecular weights of polypeptides fragments of ACE2.

“ILLUSTRATIVE EMBODIMENTS”

“The following embodiments should not be taken to limit the scope and benefits of the claimed subject matter.

“Embodiment 1”

“A variant angiotensin converting enzyme 2, (ACE2, SEQID NO: 1), the variant ACE2 with ACE2 activity and a lower molecular weight than 70 kD.”

“Embodiment 2”

“The variant ACE2 of embodiment 1, in which the variant of ACE2 contains an N-terminal deletion or a C-terminal deletion or both relative to full-length ACE2 [SEQ ID No:1], for example, SEQ ID NOT:3 or SEQID NO:4.

“Embodiment 3”

“The embodiment 2 variant of ACE2 where the deletion removes a glycosylation spot present in full-length ACE2.”

“Embodiment 4”

“The variant of ACE2 in any of the above embodiments, wherein ACE2’s molecular weight is less than 60 kD.”

“Embodiment 5”

“The variant of ACE2 in any of the above embodiments, wherein ACE2 is more active than full-length ACE2 (SEQID NO:1) to convert AngII (1-8) to Ang(1-7).

“Embodiment 6”

“The variant of ACE2 in any of the above embodiments, wherein it is a truncated version of ACE2 with a plasma half-life of at least 6 hours or 12 hours, 1 week, 2 days 3 days, 4 weeks, 5 days and 6 days, or more.”

“Embodiment 7”

“A fusion protein that contains the variant of ACE2 from any of the above embodiments, such a truncated version, fused with a heterologous sequence of amino acids that increases the plasma half-life of this variant of ACE2.”

“Embodiment 8”

“The embodiment 7 fusion protein, where the fusion proteins has a half life in plasma of at minimum of 6 hours, 12hrs, 1 day 2 days 3 days 4 days 5 days 6 days 6 days 6 days one week two weeks three weeks four weeks or longer.

“Embodiment 9”

“The embodiment 7 or 8 fusion protein, wherein the heterologous amin acid sequence consists of an amino acids sequence chosen from the group consisting (i) of an amino Acid sequence of an antibody Fc or a fraction thereof; (ii), an amino Acid sequence of serum albumin (e.g. SEQID NO:6); (ii), an amino A sequence of streptococcal proteins G or a portion thereof such as the amino sequence of C-terminalalbumin binding domain 3 (ABD3) (ABD3) of streptococcalprotein G) of streptococcalprotein G’s (e.

“Embodiment 10”

“The fusion protein in any of the embodiments 7-9 also includes a linker amino sequence between the variant ACE2 or the heterologous sequence (e.g. a linker sequence comprising 5-15 amino acids selected glycine or serine).

“Embodiment 11”

“The fusion protein in any of the embodiments 7-10 further comprises an amino acid tag sequence, such as an amino sequence consisting of 5-10 histidine residues.”

“Embodiment 12”

“A conjugate containing the variant of ACE2 from any of the embodiments 1-6 (e.g. a truncated version of ACE2), or the fusion proteins of any one of embodiments 7-11. In which the variant or fusion protein is covalently linked to a polyethylene glycol-polymer,

“Embodiment 13”

“A conjugate containing the variant of ACE2 in any of the embodiments 1-6, or the fusion proteins of any embodiments 7-11. In which the variant or fusion protein is bound to a nanoparticle such as a biogel or a polymer-coated Nanobin or gold nanoparticles,

“Embodiment 14”

“The claim 12 or 13 conjugate, wherein the conjugate has at least a half-life in plasma of at minimum of 6 hours, 12hrs, 3 days and 4 days, 5 day, 6 day, one week, two weeks, three weeks, or four weeks or more.”

“Embodiment 15”

“A pharmaceutical composition that includes: (i) any one of the above embodiments reciting variants ACE2, fusion protein or conjugates thereof; (ii) a suitable drug carrier.”

“Embodiment 16”

“A method to reduce AngII (1-8) levels or increase Ang(1-77) levels in a subject who is in need thereof. The method includes administering the pharmaceutical composition of embodiment 15.

“Embodiment 17”

“The method according to embodiment 16 wherein the subject is suffering from a condition that includes diabetic or non-diabetic acute kidney disease, acute kidney failure and its prevention and chronic kidney disease, severe hypertension, chronic kidney disease and its prevention, acute kidney disease, chronic kidney disease, chronic renal failure and its treatment, chronic kidney disease, severe kidney disease, glomerulonephritis, scleroderma, scleroderma, pulmonary hypertension, malignant hypertension and renoesis, cardiac fibrosis and remodeling.

“Embodiment 18”

“The embodiment 16 or 17 where the pharmaceutical composition can be administered intravenously, subcutaneously, or pulmonarily (e.g. via inhalation with an inhaler, nebulizer, or inhaler).

“EXAMPLES”

“The following examples should not be taken to limit the scope and use of the claimed subject matter.

“Example 1?”Novel Active Ace2 Fragments

“Introduction & Aims”

“Activation and progression of diabetic kidney disease (DKD), is influenced in large part by the renin-angiotensin (RAS). The RAS can be overactive in several situations, including malignant hypertension, diabetic kidney disease systemic, systemic scleroderma and idiopathic lung fibrosis. Angiotensin Converting Enzyme 2 is a transmembrane monocarboxypeptidase which converts Angiotensin II (AngII), to Angiotensin (Ang (1-7), (Ang (1-7)). ACE2 can lower AngII levels and prevent or reduce the negative effects of excessive amounts of this peptide. Ang-1-7, which is formed by AngII cleavage, and works on its own receptor, has tissue protection functions that are generally different from those of AngII. It can also be used in conjunction with AngII to lower AngII. Our lab was able, during the previous funding period to produce mouse recombinant ACE2 (“mrACE2”) as a way of circumventing the immunogenicity human ACE2 when it was given to mice. We investigated the kidney effects of systemic administrations of mrACE2 via either daily injections mrACE2 and DNA minicircle delivery. The result was a significant and sustained increase in plasma ACE2 activity, which conferred an enhanced ability to metabolize acute AngII loads. Streptozotocin-induced DKD in mice was caused by Streptozotocin minicircle gene ACE2 administration or ACE2 delivery to the rACE2 receptor. There was no increase in urinary ACE2 activity, albuminuria and glomerular mesangial growth, glomerular cellularity, and glomerular hypertrophy. A profound augmentation of ACE2, confined only to the circulation, did not improve the glomerular lesion and hyperfiltration that were characteristic of STZ-induced DKD. Targeting the circulatory RAS with a marked ACE2 amplification fails to alleviate DKD. This is due to the fact that the systemic RA is not active and the blood pressure has not increased in the STZ model. Our preliminary data from a mouse renin transgenic mouse model of systemic AngII excess show that rACE2 can be used therapeutically to reduce RAS activity. We found that rACE2 was fused with an Fctag to prolong its duration of action, which lowered plasma AngII levels and markedly reduced hypertension and albuminuria.

“ACE2’s large molecular weight (?110 kDa), renders it unfilterable by a normal glomerulus, or early forms of DKD. This explains why there was no significant therapeutic benefit in the STZ model. These ACE inhibitors are effective in this model as well as other DKD models. This is due to the small molecules being easily filtered, which are capable of suppressing local kidney ACE. As seen in the col4A3/?? model, there was a marked increase in glomerularpermeability. We were able to demonstrate that infused rACE2 in a mouse model of Alport disease or CKD can be filtered, as it is easily extracted from the urine. It is not possible to reverse kidney alterations or reverse fibrosis at this stage. We are interested in developing and testing new forms recombinant ACE2 that have a reduced molecular structure to pass a normal kidney glomerular filtration filter barrier. The observation of a particular ACE2 species in human and mouse urine led to the design of a new ACE2 biologic. This shorter form of ACE2 is 75 kD and we found it to be more active than the intact 110 kDa ACE2 protein. To facilitate kidney delivery via glomerular filtration, we have decreased the molecular weight of ACE2 with the primary goal of treating DKD kidney RAS excess activity. We have created ACE2 truncates with a lower molecular weight (1-619 (71 kD) and 1-605 (69) kD), which can be used for kidney delivery via glomerular filtration. We also plan to attach a carrier tag on the shortest ACE2 proteins that maintain high enzymatic activities to prolong its biological half-life and facilitate its continued use. Enhancing the degradation AngII with rACE2 has the unique advantage of concurrently forming Ang 1-7, a renoprotective peptide. This is a natural physiologic approach to AngII blockage. We believe that ACE2, which continuously degrades AngII formation when used with ACE inhibitors will reduce the effectiveness of the traditional RAS blockers. These are the aims of this work: (1) To produce the shortest human and murine ACE2 protein fragments that are highly enzymatically active and deliverable to the kidney via glomerular filtration; (2) To evaluate the renoprotective effect of short rACE2 truncates on murine models early DKD. (3) To increase the duration of action using protein fusion technology and study their renoprotective effect in murine models of DKD with and without an ACE inhibitor Our ultimate goal is to create enzymatically active, shorter ACE2 proteins that have a longer half-life and are more effective in fighting DKD than existing RAS inhibitors. These shorter ACE2 proteins will also be tested in other conditions, such as systemic scleroderma and malignant hypertension, cardiac fibrosis, and cardiac hypertrophy.

“Summary Of Work To Date”

“Our research has been focused on ACE2 amplification to increase Ang II degradation to treat kidney disease. A podocyte-specific transgenic mice was created by Dr. Kevin Burns, our collaborator. It was used as a proof-of-concept to study the effects of glomerular ACE2 expression on STZ-induced DKD4. The podocyte-specific transgenic mouse showed a slight increase in ACE2 expression (2-5 fold) within the glomeruli. This relatively small increase in glomerulus-restricted ACE2 activity was nevertheless sufficient to confer significant renoprotection based on reduction of albuminuria and of mesangial expansion in the STZ model of DKD4. As a way to amplify endogenous ACE2 we performed studies using a small molecular compound (1-[(2-dimethylamino) ethylamine]-4-(hydroxyethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one) (XNT) that was initially described to be an ACE2 activator. XNT, however, exhibited its effects on AngII induced hypotension in ACE2KO mice to our surprise. This indicates that it acts by a mechanism other than ACE25. Moreover, LC-MS/MS results showed that XNT did no alter plasma Ang II or Ang (1-7), nor Ang (1)-5) levels. However, rACE2, which was used as a positive control, significantly increased Ang (1-7 and Ang (5-7) levels due to enhanced Ang II degrad5

“Because we couldn’t use XNT and DIZE (another presumed ACE2 activator), for the purpose robust and clear cut ACE2 amplification and because of the toxic nature these compounds, our mouse recombinant ACE26-7 was developed. Ex-vivo experiments were conducted to examine the effects of our mouse (mrACE2) upon angiotensin peptides dynamics within the physiological plasma environment. We used LC-MS/MS to simultaneously measure 10 angiotensin peptides7-8. Then, we administered mouse rACE2 chronically to diabetic and control mice via daily intramuscular injections. Or, we used mini-circles technology to deliver ACE2 directly to the mice9-10. Minicircle DNA delivery is more resistant to gene silencing than lentiviral delivery and so offers a promising platform for gene replacement strategies. The intact mouse ACE2 cDNA was cloned to a circular expression cassette. FVB mice were then injected with the resulting ACE2 miniaturecircle using an i.v. hydrodynamic approach. The minicircle injected ACE2 to mice was monitored for several weeks for blood pressure, serum ACE2 activity, and plasma Ang II levels. After several months of monitoring, Ang II was infused immediately. Compared to vehicle-treated mice, the plasma Ang II levels in mice that were treated with ACE2 had a significantly lower increase. Next, we infected mice with STZ with ACE2 pretreated via minicircle delivery. Despite an expected rise in serum ACE2 activity over 26 weeks, the development of albuminuria was not stopped.

Summary for “Active low molecular-weight variants of angiotensin conversion enzyme 2 (ACE2)”

The invention relates to Angiotensin converting enzyme 2(ACE2) and variants thereof for reducing plasma Angiotensin II (1-8), and/or increasing plasma Angiotensin (1-7). The disclosed variants may include fragments ACE2 that have ACE2 biological activity to convert AngII (1-8 to Ang (1) and/or for increasing plasma Angiotensin (1-7). This lower molecular mass allows the fragments to pass through the glomerulus. These variants of ACE2 could be used to treat conditions such as chronic kidney disease and acute kidney failure, non-diabetic and diabetic chronic kidney disease and its prevention, glomerulonephritis and its complications, scleroderma, severe hypertension and its skin, pulmonary, renal and hypertensive complications and malignant hypertension.

“Activation (RAS) of the renin-angiotensinsystem (RAS), plays a significant role in the pathogenesis and progression of CKD to ESRD1-3. The RAS can also be activated in acute renal failure4-7. New approaches are needed to combat RAS overactivity. These should be more effective than the current approaches that rely primarily on blocking Ang II formation and blocking the action of Ang II. Our group has been a pioneer in the development of therapies that promote the degradation of Ang II8-13. ACE2 has an important biological effect. It converts AngII (1-8), to Ang (1-7). This process tends to lower AngII (-8), and thus prevents any potentially harmful effects of the peptide. Ang(1-7), which is also formed by Ang II (1-8) cleavage, has tissue protective properties that are generally different to those of AngII (1-6). There is growing evidence that Ang (1-7) may have many therapeutic uses. This also highlights the importance of Ang (1-7) forming enzymes being potential therapeutic targets. They have the dual benefit of degrading Ang II as well as forming Ang (1-7)”.

“Years back, we and others purified and made murine ACE2 to avoid the immunogenicity14 we saw in our first studies. We used human ACE2 to treat hypertension in mice induced with AngII infusions13. In recent studies we examined the kidney effects of murine recombinant ACE2 given to mice with streptozotocin-induced diabetic kidney disease. (See Wysocki et al., Angiotensin-converting enzyme 2 amplification limited to circulation does not protect mice from development of diabetic nephropathy,? Kidney Int. 2016 Dec. 4. Pii: S0085-2538 (16), 30565-8. The content of this document is incorporated by reference in its entirety. This study used two approaches to amplify circulating ACE2: intraperitoneal daily injectables for four weeks and ACE2 gene delivery15. Minicircles were used to deliver ACE2 and resulted in a sustained, long-term and significant increase in serum ACE2 activity. Also, the ability to metabolize an acute Ang II load (1-8) was enhanced. Minicircle ACE2 was pretreated in mice with STZ-induced diabetics. The plasma ACE2 levels increased significantly and was associated with a greater than 100-fold increase of serum ACE2 activity. Minicircle ACE2 didn’t cause any changes in the urinary ACE2 activity in diabetic mice, however. As compared to untreated controls, albuminuria, glomerular cellularity, glomerular cellularity, and glomerular size were all similar in minicircle ACE2-treated mice to a similar degree. Despite months of high plasma ACE2 levels, a significant augmentation of ACE2 confined only to the circulation did not improve the glomerular lesion and hyperfiltration that are characteristic of early diabetes kidney disease. These results highlight the importance of targeting kidneys rather than the circulatory system for early stage diabetic kidney disease. Recombinant ACE2’s large molecular structure makes it difficult to filter by a normal glomerulus. This is a critical time to intervene to stop disease progression. However, in more severe glomerular renal disease we were able to demonstrate that infused rACE2 could be recovered from the urine10. It is not possible to reverse advanced kidney disease or reverse fibrosis at this stage. We designed shorter forms (ACE2) to overcome this limitation. They are more effective in treating kidney disease and allow for better tissue penetration to other organs like the heart and lungs.

“Based on our findings, we have developed forms of ACE2 with a shorter molecular structure that can be delivered to the renal before the development of marked alterations to glomerular permeability. They are also more easily delivered to the kidney in all cases. ACE2 is usually seen as a 110 kD protein that is not filterable in the kidney. It is found in the urine as a shedding product of the renal apical tubeular membrane where ACE2 expression is high9-11, 16. Our ACE2 proteins are active and have a lower molecular weight. They retain their full activity and therapeutic potential when the goal of increasing ACE2 activity is to not only increase systemic circulation just like the human recombinant intact ACE2. However, they are also unique in that they can be delivered to the kidney via glomerular filtration. This makes them more useful for treating kidney disease and tissue penetration to other organs.

“We have demonstrated that reducing the size of ACE2 makes it easier to filter through the glomerular barriers in mild increases in glomerular permeability such as in acute kidney injury, early stages of diabetes kidney disease (i.e. microalbuminuric phase. Our goal is to create a shorter form of ACE2 that can be delivered to the kidneys to combat kidney disease. This is a unique approach and complements current AT1 blockers and ACE inhibitors. Enhancing the degradation of Ang II has the unique advantage of leading to the formation Ang 1-7, a renoprotective peptide. It is also a more natural physiologic strategy than blocking the formation or activity of Ang II and its receptors, as done with current agents. The short ACE2 fragments can be used to increase tubular reabsorption and kidney uptake. Fc (constant fragment human IgG), DIII domain human serum albumin, and lysozyme are all examples of fusion polypeptides. All of these polypeptides were shown to be absorbed by receptor-mediated endocytosis on the kidney tubules’ apical surfaces. This application covers the subject matter.

“Disclosed are variants ACE2, pharmaceutical compositions containing the variants ACE2, and treatment methods to reduce Angiotensin II (1-8 plasma levels) and/or increase Angiotensin (1-7 plasma levels) in a subject who is in need of such variants. Polypeptide fragments from ACE2 that have ACE2 activity may be disclosed. These polypeptide fragments can convert AngII (1-8), to Ang(1-7). Preferably, the molecular weight of ACE2 polypeptides should be low enough that they can pass through the glomerulus to reach the kidney. Some embodiments of the polypeptide fragments may have a molecular mass less than 70 kD. We have studied A1-619, a compound we call 69 kD, and 1-605, a compound we call 1-605, with molecular masses of 65 kD to 60 kD to 55 kD or 50 kD. The subject receives the variant of ACE2 and a pharmaceutic formulation containing the variant of ACE2 in a suitable pharmaceutical carrier. The disclosed treatment methods are suitable for subjects who have or are at risk of developing non-diabetic and diabetic chronic kidney disease, acute kidney failure and its prevention and chronic kidney disease.

“The invention described herein uses several definitions as shown below and throughout the application.”

“As used herein and in the claims, the singular forms of?a? ?an,? ?an,? If the context requires otherwise, plural forms should be used. The term “a polypeptide fragment” could be used as an example. Should the term?a polypeptide fragment be understood to refer to?one or more of these fragments? Unless the context explicitly indicates otherwise. The term “plurality” is used herein. It can also be translated as “two or more”.

“As used herein, ?about?, ?approximately,? ?substantially,? ?substantially? They will be easily understood by people of ordinary skill in art. However, this will depend on the context in which it is used. If the meaning of the term is not understood by persons of ordinary skill in art, they will be referred to as?about? ?approximately’ and?about? It will be interpreted as a plus or minus 10% of that term, and?substantially? Significantly and plus or minus 10% of the particular term. “significantly” means more than 10% plus or minus of the specific term.”

“As used herein the terms ‘include? and?including? “The terms?include? and?including? have the same meaning as the terms?comprise? The same meanings of?including? and?comprise? are given to these terms. ?comprising. The terms ‘comprise? and?comprising? The terms?comprise? and?comprising are interchangeable. Should be read as?open? Transitional terms allow for the addition of additional components beyond those cited in the claims. The terms “consist of” and “consisting of” are interchangeable. The terms?consist? und?consisting? should be interpreted as being?closed? Should be understood as being ‘closed’ Transitional terms that prohibit the addition of other components than those cited in the claims are not permitted. “Consisting essentially of” is a term that refers to the inclusion of additional components. “Consisting essentially of” should be understood to mean partially closed, allowing for the addition of only those components that do no fundamentally alter the subject matter of the claimed claim.

“Subject” as used in this document. “Subject” may be interchangeable with the term “patient?” Or?individual? It may also include an ‘animal? an?animal?, and in particular a mammal. Mammalian subjects can include humans, other primates, domestic animals and farm animals.

“The disclosed compositions, methods, and kits can be used to treat a subject who is in need of them. A “subject in need of it?” This term refers to a subject who is at risk of developing certain diseases or disorders, such as non-diabetic and diabetic chronic kidney disease, acute kidney failure and its prevention and chronic kidney disease, acute kidney disease and its prevention, chronic renal disease and its prevention, chronic renal disease and its prevention, acute stroke, glomerulonephritis and its skin, pulmonary hypertension and its complications, malignant hypertension and renovascular hypertension secondary renal artery stensis, idiopathic, heart fibrosis and remodeling, cardiac fibrosisisisisisis and left ventricular hypertrophy, left ventricular hypertrophies, right vhyprophy and left ventricular hypertrophy and left acute.

“The terms “amino acid” and “amino sequence?” “Amino acid” and “amino acids sequence?” Refer to an oligopeptide or peptide or polypeptide or protein sequence (which terms can be interchangeably used), or a fragment thereof, as well as to naturally occurring and synthetic molecules. Where is?amino acids sequence? The sequence of naturally occurring proteins molecules, also known as the?amino acids sequence, is what is being referred to? Similar terms do not limit the amino acids sequence to the entire native amino acid sequence of the recited protein molecules.

“The amino acids sequences contemplated herein could include one or more amino- acid substitutions relative to the reference sequence. A variant polypeptide could include conservative and/or non-conservative amino acid substitutions relative the reference polypeptide. ?Conservative amino acid substitutions? These substitutions are those that are expected to have the least impact on the properties of the reference protein. Conservative amino acid substitutions preserve the structure and function of the reference protein. Below is a table that lists some examples of conservative amino acid substitutions.

“Original Conservative\nResidue Substitution\nAla Gly, Ser\nArg His, Lys\nAsn Asp, Gln, His\nAsp Asn, Glu\nCys Ala, Ser\nGln Asn, Glu, His\nGlu Asp, Gln, His\nGly Ala\nHis Asn, Arg, Gln, Glu\nIle Leu, Val\nLeu Ile, Val\nLys Arg, Gln, Glu\nMet Leu, Ile\nPhe His, Met, Leu, Trp, Tyr\nSer Cys, Thr\nThr Ser, Val\nTrp Phe, Tyr\nTyr His, Phe, Trp\nVal Ile, Leu, Thr”

“Conservative amino acids substitutions usually maintain one or more of the following: (a) The structure of the polypeptide-backbone in the area where the substitution is made, such as a beta or alpha helical conformation; (b) The charge or hydrophobicity at the site of the substitution and/or (c), the bulk of side chains. The structure of non-conservative amino acids substitutions is generally not maintained.

“The disclosed peptides can include an N-terminal esterification (e.g. a phosphoester modification) or a pegylation (e.g. to increase plasma stability (e.g. Resistance to exopeptidases and/or lower immunogenicity.

“A ?deletion? A?deletion? refers to any change in a reference sequence of amino acids (e.g. SEQ ID No:1 or SEQ ID NO:2) that causes the loss of one or more amino acids. A deletion is a change in a reference amino acid sequence (e.g. SEQ ID NO:1 or SEQ ID NO:2) that results in the loss of at least 1, 2, 4, 5, 10, 20, 100, 200, or 200 amino acid residues. An internal or terminal deletion can be included in a deletion. A “variant” A?variant? may be a sequence that is not the reference polypeptide. SEQID NO:3 (amino acid 1-619) & SEQID NO:4 (amino acid 1-605) both include C-terminal deletions relative SEQID NO:1 (amino acid 1-805).

“The words?”insertion? “The words?insertion? and?addition?” refer to changes in an amino acid sequence that result in one or more amino acids being added. The addition and insertion are changes in amino acid sequences that result in the addition or subtraction of amino acids. A insertion or an addition can refer to 1, 2, 4, 5, 10, 20, 40, 50, 70, 80 or 90 amino acid residues, or a range of amino acids bounded within any of these values (e.g. an insertion of 5-10 amino acid). Variant: A?variant? may be a modification or addition to a reference sequence of polypeptides.

“A ?fusion polypeptide? A?fusion polypeptide is a polypeptide that has at least one of its amino acids sequences at either the N-terminus or the C-terminus. Variant: A?variant? may be a sequence of reference polypeptides that includes a fusion protein.

“A ?fragment? A fragment is an amino acid sequence that is shorter than the reference sequence but is identical in sequence. A fragment can be as long as the length of the reference sequence but may contain at most one amino acid residue. A fragment could contain from 5 to 1000 consecutive amino acid residues of a reference sequence, for example. A fragment may contain at least 5, 10, 20, 25, 30, 40 or 50, 60 or 70, 80 or 90, 100 or 150, 250 or 500 contiguous amino acid residues. Or a fragment can comprise no more that 5, 10, 20, 25, 30, 40 or 50, 60 or 70, 80 or 90, 100 or 150, 250 or 500 contiguous amin acid residues. Some fragments may be preferred from particular regions of a molecule. Fragments are defined as a molecule that is at least one-third of its full length. The entire length of the polypeptide is included. Variant is the full length polypeptide. A?variant? may contain a fragment from a reference sequence. SEQID NO:3 (amino acid 1-619), and SEQID NO:4 (amino acid 1-605) contain fragments from the reference sequence SEQID NO:1 (amino acid 1-805).

“?Homology? “?Homology?” refers to sequence similarity, or, interchangeably: sequence identity, between two or three polypeptide sequences. Methods in the art, including those described herein, can be used to determine homology, sequence similarity, or percentage sequence identity.

“The phrases ‘percent identity? “Percent identity” and “% identity”, are two different phrases. Referring to polypeptide sequencing, the percentage of residue matches between at most two sequences of polypeptides aligned according to a standard algorithm. There are many methods for aligning polypeptide sequences. Some alignment methods take into account conservative amino acid substitutions. These conservative substitutions, which are explained in greater detail below, preserve the charge as well as the hydrophobicity at substitution sites. This preserves the structure and function of the polypeptide. As per the art, it is possible to determine percent identity for amino acids sequences. (See, e.g., U.S. Pat. No. No. 7,396,664, is included herein by reference in its entirety. The National Center for Biotechnology Information’s Basic Local Alignment Search Tool is a collection of widely used and freely accessible sequence comparison algorithms. (Altschul S. F. et. al. (1990) J. Mol. Biol. 215:403 403 410), which can be obtained from many sources including the NCBI in Bethesda (Md.) at its website. The BLAST software suite also includes sequence analysis programs such as?blastp. It is used to align an amino acid sequence with others from a variety databases.

“Percentage identity can be measured over the entire length of a defined polypeptide, such as a sequence number. Or it may be measured over a smaller length, such as the length of a segment taken from a larger sequence.

“In some embodiments, the term “variant” may be used. A?variant? may be a polypeptide that has at least 20% sequence identity with the specific sequence. It can also refer to a sequence of polypeptides that is longer than one of the sequences using blastp with?BLAST2 Sequences? The National Center for Biotechnology Information has a tool that can be used. (See Tatiana A. Tatusova and Thomas L. Madden (1999), “Blast 2 sequences?” a new tool to compare protein and nucleotide sequencing?, FEMS Microbiol. Lett. 174:247-250). This pair of polypeptides could show, for instance, at minimum 20%, 30 percent, at most 40%, and at the least 60%, a least 70%, a least 90%, a least 90%, a least 91% or more sequence identity over a defined length of one polypeptide or range of percentage identities bounded with any of these values (e.g. range of percentage identity between 80-99 %).”).

“The methods of treatment and pharmaceutical composition disclosed use and/or include angiotensin converting protein 2 (ACE2), or variants thereof, such as fragments. The National Center for Biotechnology Information of National Institutes of Health has the nucleotide sequence for the human ACE2 genes. NC_000023.11 is the location of human ACE2 genes (15494525 ). . . 15602069, complement). ACE2, isoform 1 is a transmembrane proteins that is first expressed as a precursor polypeptide with the amino acid sequence (SEQID NO:1). The following amino acid sequence is found in the homolog to ACE2 by Mus musculus:

“Amino acids 1-17” are a leader peptide that is cleaved form mature ACE2. Amino acids 18-740 are extracellular. Amino acids 741-761 form a helical transmembrane sequence. Cytoplasmic amino acids 762-805 exist. The natural variants of ACE2 that are considered herein include N638S and K26R. Herein are also considered natural isoforms for ACE2: isoform 2 with the following differences relative ACE2 isoform 1; F555L and???556-805. These variants of ACE2 may include fragments of ACE2.

“Fusion polypeptides from ACE2 and variants thereof are described in this document. A fusion polypeptide may contain the amino sequence of ACE2 (or a variant thereof) or a combination thereof (e.g. the amino sequence of a fragment from ACE2 fused to a heterologous sequence of amino acids). The preferred heterologous sequence of amino acids increases plasma half-life.

“The disclosed fusion polypeptides can contain the amino acids sequence of ACE2 (or a variant thereof) or a combination of them (e.g. the amino sequence of a fragment from ACE2 fused directly with a heterologous sequence or fused through a linker sequence. The suitable linker sequences can include amino acid sequences of 5,6, 7, 8, 9, 10, 11, 13, 14, or 15 amin acids, or any range that is bounded within these values (e.g. a linker of 5-15 amin acids). Some embodiments only contain glycine or serine residues in the linker sequence.

“Fusion polypeptides described herein include the amino acids sequence of ACE2 (or a variant thereof) fused to an antibody’s amino acid sequence or one or more fragments. For example, the Fc portion (constant fragment from human IgG) is preferably devoid of its hinge area to prevent dimerization (e.g. SEQ ID No:6). Fc can be fused with short ACE2 (e.g. SEQID NO:6) or monomeric CH3 Fc derivative (e.g. SEQID NO:7 or NO:8) to enable delivery via a functional FcRn dependent transport pathway in lung. This allows for more efficient administration of lung fibrosis treatment. The fusion polypeptides described herein also include the amino acids sequence of ACE2 and a variant thereof fused with serum albumin, or a fragment thereof. For example, domain III of human serumalbumin or a fragment thereof (e.g. SEQ ID NO.9). The amino acid sequence of ACE2 and a variant thereof fused with streptococcal proteins G or a fragment thereof, such as the Cterminal albumin binding domain (3 (ABD3)) of streptococcal proteins G (e.g. ABD3 strain G148 or ABD035 derivative (SEQID NO:5) are examples of fusion polypeptides. (See, e.g., Nilvebrant et al., Comput. Struct. Biotechnol. J. J.

“Fusion polypeptide described herein may include an amino acid tag sequence. This sequence may be used for purifying or identifying the polypeptide. Histidine tag sequences that contain 5-10 histidine residues are suitable.

“ACE2 is a carboxypeptidase that catalyzes angiotensin I’s conversion to angiotensin 9 (EC: 3.4.17.23). It also catalyzes angiotensin II (1-8), to angiotensin (1), which is a vasodilator. ACE2 also hydrolyzes apelin-13 and dynorphin-13. ACE2 is also the cellular receptor of sudden acute respiratory syndrome (SARS), coronavirus/SARSCoV, and human coronaviruses NL63/HCoV?NL63. These variants of ACE2 may include fragments of ACE2.

“ACE2 exhibits molecular functions that may include: carboxypeptidase activity, endopeptidase activity, glycoprotein binding activity, metallocarboxypeptidase activity, virus receptor binding activity, and zinc ion binding activity. These variants of ACE2 include fragments of ACE2. They all have at least one, cleavage Angiotensin II. However, they likely all possess all the molecular or enzymatic functions.

“Key structure features for ACE2 may include the following: helix, amino acids positions 53-77; 78-82; 79-82; helix, amino acids positions 134-141?helix, amino positions 453-460?helix and 465-460?turn? amino positions 539-541?helix, amino positions 559-562?helix and 575-578 respectively?helix and 576-574 respectively?beta strand and 607-609 respectively?helix These structural features may be present in variants of ACE2 described herein.

“ACE2 could include one or more of these amino acid modifications: amino acids 53, 90, N-linked glycosylation, amino acid position 103, N-linked glycosylation amino acid positions 133. ?141?disulfide bonds; amino acids position 322 and 344. N-linked glycosylation, amino acid positions 344. ?361?disulfide bonds; amino acids position 322?N-linked glycosylation, amino acid positions 344?N-linked glycosylation?N?linked glycosylation amino acid positions 531? ?542?disulfide bond; amino acid position 432?N-linked glysylation; amino acids positions 530?N?linked glycosylation? These variants of ACE2 may include fragments of ACE2 or may contain one or more of the amino acid modifications of ACE2 or may not contain these amino acids.

“ACE2 regulates biological process that can include angiotensin metabolism processes, angiotensin maturation and angiotensin-mediated drinking behaviors processes. These variants of ACE2 may regulate or fail to regulate any of the biological processes.

“The disclosed ACE2 variants could include an N-terminal methionine residue that is not found in the native amino acids for ACE2. The ACE2 variants may contain an N-terminal deletion in comparison to full-length ACE2. Further, it may be possible to modify the amino acids to include an Nterminal methionine.

“The disclosed ACE2 variations may contain an amino acid sequence or modified amino acids or other non-naturally occurring amino acids. This would make it impossible to say that the disclosed ACE2 varieties are naturally occurring. The disclosed ACE2 variants may be modified in some embodiments. This modification can be selected from the following groups: acylation and acetylation; formylation; lipolylation; myristoylation; palmitoylation; alkylations; isoprenylations. amidation. Modifications to an amino acid of the disclosed polypeptides can be made. The modifications may be at the N-terminus or C-terminus, depending on whether they are acetylations, acylations, or amidation. Modifications may increase the stability and/or resistance to proteolysis of polypeptides.

The ACE2 variants described may be modified to substitute a natural amino acids residue with an unnatural amino acid. Unnatural amino acids can include, but not be limited to, an amino acid with a D-configuration or an N-methyl?-amino, a non-proteogenic constrained amino acid or a?-amino.

“The ACE2 variants disclosed may be modified to improve plasma stability. The disclosed peptides could be modified to make them resistant to peptidases. Modifications to the disclosed peptides can be made to replace an amino bond between two amino acid with a non-amide one. The carbonyl moiety in the amide bond can be replaced with CH2 (i.e. to create a reduced amino-bond:?CH2-NH?)). An endothiopeptide (?C(S?NH)) can be used to replace the amide bond. A phosphonamide (?P(O?OH?NH)) is another possible non-amide substitute bond. The NH-amide bond can also be exchanged with O (depsipeptide),?CO?O S (thioester),?CO?S? or CH2 (ketomethylene) You can modify the peptide bond as follows: retroinverso bond?NH?CO? ), methyleneoxy bond (?CH2?) ), methylene-oxy bond (?CH2?) ), thiomethylene bond (?CH2??S? ), carbabond (?CH2?CH2????????????????????????????CH2?CH2??????CHOH.CH2????CH2????????CH2??????????CH2?CHOH?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH3?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH2?CH4?CH2?CH2?CH2?CHOH2?CH2?CH2?CH2?CH2?CH2?CH2?CHOH2?CH2?CH2?CH2? ”

“The disclosed ACE2 variants could include a non-naturally occurring C-terminal or N-terminal modification. The N-terminal end of disclosed peptides could be modified to include an N acylation or N-pyroglutamate modification (e.g. as a blocking modification). You can modify the C-terminal of disclosed peptides to include a C?amidation. To increase plasma stability and resistance to exopeptidases, the disclosed peptides can be conjugated with carbohydrate chains (e.g. via glycosylation to glucose or xylose).

“The variants described herein of ACE2 may be further modified. The polypeptide fragment of ACE2 could be modified in order to increase plasma half-life and/or enhance delivery to a target (e.g. the kidneys, lungs, heart, etc.). Some embodiments of the polypeptide fragment are covalently attached with a polyethylene glycol-polymer. Other embodiments allow the polypeptide fragment to be covalently attached to a polyethylene glycol polymer. Preferably the half-life of the polypeptide fragments of the described methods of treatment or pharmaceutical compositions is at least 6 hours, 12hrs, 3 days and 4 days, 5 day, 6 day, 6 week, 2 week, 3 days, 4 week, 5 days, 6 week, 6 week, 2 week, 3 week, 4 week, 5 days, 6 week, 6 week, 2 week, 3 week, 4 week, or more. The art has many strategies to increase plasma half-life for peptides and protein drugs. (See Werle and colleagues,?Strategies for improving plasma half-life time of peptides and protein drugs. Amino Acids 2006, 30(4):351-67. The content of which is incorporated by reference herein in its entirety.

“Pharmaceutical Compositions”

“The compositions described herein could include pharmaceutical compositions containing the presently disclosed bacteria toxins, and formulated to be administered to a subject who requires them.” These compositions can be prepared and/or administered in doses and using techniques that are well-known to medical professionals. This includes factors such as age, gender, weight, and current condition.

“The compositions can include pharmaceutical solutions that contain carriers, diluents and excipients as well as surfactants. The compositions can also include preservatives, such as anti-microbial and anti-bacterial agents like benzalkonium salt. Buffering agents may also be included in the compositions to maintain the pH between 6.5-7.5.

The compositions can be used therapeutically. The compositions may be administered therapeutically to patients in an amount that is sufficient to produce a therapeutic effect.

“Novel Active Short-ACE2 Fragments”

“The inventors discovered novel fragments full-length ACE2?molecular mass about 110 kD. They have a shorter molecular weight (less that 70 kD) and have high enzymatic activities. These fragments of ACE2 could be used in pharmaceutical compositions and treatment methods. The disclosed methods can be used in some cases to lower AngII (1-8) levels in a subject who is in need. There are many other substrates that can be cleaved using the novel ACE2 segments. The subject could be a pharmaceutical composition that contains a fragment of angiotensin-converting enzyme 2 (ACE2, SEQ ID NO:1) in a suitable pharmaceutical vehicle. The disclosed treatment methods are suitable for subjects with or at risk of developing diabetes and non-diabetic kidney disease, acute kidney failure and its prevention and chronic kidney disease. The disclosed pharmaceutical compositions can be administered in any manner, including intravenous injection and subcutaneously. Patients could also inject the contents themselves at home. Inhalation is another method of administering the disclosed pharmaceutical compositions. This could be useful for treating idiopathic lung fibrosis or other conditions.

“The ACE2 activity of the polypeptide fragment of ACE2 is for converting AngII (1-8) to Ang(1-7).” The polypeptide fragment of ACE2 may be delivered more efficiently to the kidneys than full-length ACE2.

“In general, the polypeptide of ACE2 is of low molecular weight so that it can be filtered through glomerulus and delivered directly to the kidney. Some embodiments have a polypeptide fragment with a molecular mass of less than 70 kD.

“The disclosed polypeptide fragments may contain a deletion relative full-length ACE2 (SEQID NO:1). A deletion may be made from either an N-terminal or C-terminal portion of the disclosed polypeptide fragments. This deletion could be relative to full-length ACE2(SEQ ID NO. 1). In some cases, the disclosed polypeptide fragments might also include an internal deletion. The deletion could remove approximately 1, 2, 3, 4, 5, 15, 20, 25, 50. 100, 150, 200 or more full-length ACE amino acids. Some embodiments may result in the deletion of one or more glycosylation site. As such, polypeptides fragments from ACE2 may be less well glycosylated that full-length ACE2. This further reduces the molecular weights of polypeptides fragments of ACE2.

“ILLUSTRATIVE EMBODIMENTS”

“The following embodiments should not be taken to limit the scope and benefits of the claimed subject matter.

“Embodiment 1”

“A variant angiotensin converting enzyme 2, (ACE2, SEQID NO: 1), the variant ACE2 with ACE2 activity and a lower molecular weight than 70 kD.”

“Embodiment 2”

“The variant ACE2 of embodiment 1, in which the variant of ACE2 contains an N-terminal deletion or a C-terminal deletion or both relative to full-length ACE2 [SEQ ID No:1], for example, SEQ ID NOT:3 or SEQID NO:4.

“Embodiment 3”

“The embodiment 2 variant of ACE2 where the deletion removes a glycosylation spot present in full-length ACE2.”

“Embodiment 4”

“The variant of ACE2 in any of the above embodiments, wherein ACE2’s molecular weight is less than 60 kD.”

“Embodiment 5”

“The variant of ACE2 in any of the above embodiments, wherein ACE2 is more active than full-length ACE2 (SEQID NO:1) to convert AngII (1-8) to Ang(1-7).

“Embodiment 6”

“The variant of ACE2 in any of the above embodiments, wherein it is a truncated version of ACE2 with a plasma half-life of at least 6 hours or 12 hours, 1 week, 2 days 3 days, 4 weeks, 5 days and 6 days, or more.”

“Embodiment 7”

“A fusion protein that contains the variant of ACE2 from any of the above embodiments, such a truncated version, fused with a heterologous sequence of amino acids that increases the plasma half-life of this variant of ACE2.”

“Embodiment 8”

“The embodiment 7 fusion protein, where the fusion proteins has a half life in plasma of at minimum of 6 hours, 12hrs, 1 day 2 days 3 days 4 days 5 days 6 days 6 days 6 days one week two weeks three weeks four weeks or longer.

“Embodiment 9”

“The embodiment 7 or 8 fusion protein, wherein the heterologous amin acid sequence consists of an amino acids sequence chosen from the group consisting (i) of an amino Acid sequence of an antibody Fc or a fraction thereof; (ii), an amino Acid sequence of serum albumin (e.g. SEQID NO:6); (ii), an amino A sequence of streptococcal proteins G or a portion thereof such as the amino sequence of C-terminalalbumin binding domain 3 (ABD3) (ABD3) of streptococcalprotein G) of streptococcalprotein G’s (e.

“Embodiment 10”

“The fusion protein in any of the embodiments 7-9 also includes a linker amino sequence between the variant ACE2 or the heterologous sequence (e.g. a linker sequence comprising 5-15 amino acids selected glycine or serine).

“Embodiment 11”

“The fusion protein in any of the embodiments 7-10 further comprises an amino acid tag sequence, such as an amino sequence consisting of 5-10 histidine residues.”

“Embodiment 12”

“A conjugate containing the variant of ACE2 from any of the embodiments 1-6 (e.g. a truncated version of ACE2), or the fusion proteins of any one of embodiments 7-11. In which the variant or fusion protein is covalently linked to a polyethylene glycol-polymer,

“Embodiment 13”

“A conjugate containing the variant of ACE2 in any of the embodiments 1-6, or the fusion proteins of any embodiments 7-11. In which the variant or fusion protein is bound to a nanoparticle such as a biogel or a polymer-coated Nanobin or gold nanoparticles,

“Embodiment 14”

“The claim 12 or 13 conjugate, wherein the conjugate has at least a half-life in plasma of at minimum of 6 hours, 12hrs, 3 days and 4 days, 5 day, 6 day, one week, two weeks, three weeks, or four weeks or more.”

“Embodiment 15”

“A pharmaceutical composition that includes: (i) any one of the above embodiments reciting variants ACE2, fusion protein or conjugates thereof; (ii) a suitable drug carrier.”

“Embodiment 16”

“A method to reduce AngII (1-8) levels or increase Ang(1-77) levels in a subject who is in need thereof. The method includes administering the pharmaceutical composition of embodiment 15.

“Embodiment 17”

“The method according to embodiment 16 wherein the subject is suffering from a condition that includes diabetic or non-diabetic acute kidney disease, acute kidney failure and its prevention and chronic kidney disease, severe hypertension, chronic kidney disease and its prevention, acute kidney disease, chronic kidney disease, chronic renal failure and its treatment, chronic kidney disease, severe kidney disease, glomerulonephritis, scleroderma, scleroderma, pulmonary hypertension, malignant hypertension and renoesis, cardiac fibrosis and remodeling.

“Embodiment 18”

“The embodiment 16 or 17 where the pharmaceutical composition can be administered intravenously, subcutaneously, or pulmonarily (e.g. via inhalation with an inhaler, nebulizer, or inhaler).

“EXAMPLES”

“The following examples should not be taken to limit the scope and use of the claimed subject matter.

“Example 1?”Novel Active Ace2 Fragments

“Introduction & Aims”

“Activation and progression of diabetic kidney disease (DKD), is influenced in large part by the renin-angiotensin (RAS). The RAS can be overactive in several situations, including malignant hypertension, diabetic kidney disease systemic, systemic scleroderma and idiopathic lung fibrosis. Angiotensin Converting Enzyme 2 is a transmembrane monocarboxypeptidase which converts Angiotensin II (AngII), to Angiotensin (Ang (1-7), (Ang (1-7)). ACE2 can lower AngII levels and prevent or reduce the negative effects of excessive amounts of this peptide. Ang-1-7, which is formed by AngII cleavage, and works on its own receptor, has tissue protection functions that are generally different from those of AngII. It can also be used in conjunction with AngII to lower AngII. Our lab was able, during the previous funding period to produce mouse recombinant ACE2 (“mrACE2”) as a way of circumventing the immunogenicity human ACE2 when it was given to mice. We investigated the kidney effects of systemic administrations of mrACE2 via either daily injections mrACE2 and DNA minicircle delivery. The result was a significant and sustained increase in plasma ACE2 activity, which conferred an enhanced ability to metabolize acute AngII loads. Streptozotocin-induced DKD in mice was caused by Streptozotocin minicircle gene ACE2 administration or ACE2 delivery to the rACE2 receptor. There was no increase in urinary ACE2 activity, albuminuria and glomerular mesangial growth, glomerular cellularity, and glomerular hypertrophy. A profound augmentation of ACE2, confined only to the circulation, did not improve the glomerular lesion and hyperfiltration that were characteristic of STZ-induced DKD. Targeting the circulatory RAS with a marked ACE2 amplification fails to alleviate DKD. This is due to the fact that the systemic RA is not active and the blood pressure has not increased in the STZ model. Our preliminary data from a mouse renin transgenic mouse model of systemic AngII excess show that rACE2 can be used therapeutically to reduce RAS activity. We found that rACE2 was fused with an Fctag to prolong its duration of action, which lowered plasma AngII levels and markedly reduced hypertension and albuminuria.

“ACE2’s large molecular weight (?110 kDa), renders it unfilterable by a normal glomerulus, or early forms of DKD. This explains why there was no significant therapeutic benefit in the STZ model. These ACE inhibitors are effective in this model as well as other DKD models. This is due to the small molecules being easily filtered, which are capable of suppressing local kidney ACE. As seen in the col4A3/?? model, there was a marked increase in glomerularpermeability. We were able to demonstrate that infused rACE2 in a mouse model of Alport disease or CKD can be filtered, as it is easily extracted from the urine. It is not possible to reverse kidney alterations or reverse fibrosis at this stage. We are interested in developing and testing new forms recombinant ACE2 that have a reduced molecular structure to pass a normal kidney glomerular filtration filter barrier. The observation of a particular ACE2 species in human and mouse urine led to the design of a new ACE2 biologic. This shorter form of ACE2 is 75 kD and we found it to be more active than the intact 110 kDa ACE2 protein. To facilitate kidney delivery via glomerular filtration, we have decreased the molecular weight of ACE2 with the primary goal of treating DKD kidney RAS excess activity. We have created ACE2 truncates with a lower molecular weight (1-619 (71 kD) and 1-605 (69) kD), which can be used for kidney delivery via glomerular filtration. We also plan to attach a carrier tag on the shortest ACE2 proteins that maintain high enzymatic activities to prolong its biological half-life and facilitate its continued use. Enhancing the degradation AngII with rACE2 has the unique advantage of concurrently forming Ang 1-7, a renoprotective peptide. This is a natural physiologic approach to AngII blockage. We believe that ACE2, which continuously degrades AngII formation when used with ACE inhibitors will reduce the effectiveness of the traditional RAS blockers. These are the aims of this work: (1) To produce the shortest human and murine ACE2 protein fragments that are highly enzymatically active and deliverable to the kidney via glomerular filtration; (2) To evaluate the renoprotective effect of short rACE2 truncates on murine models early DKD. (3) To increase the duration of action using protein fusion technology and study their renoprotective effect in murine models of DKD with and without an ACE inhibitor Our ultimate goal is to create enzymatically active, shorter ACE2 proteins that have a longer half-life and are more effective in fighting DKD than existing RAS inhibitors. These shorter ACE2 proteins will also be tested in other conditions, such as systemic scleroderma and malignant hypertension, cardiac fibrosis, and cardiac hypertrophy.

“Summary Of Work To Date”

“Our research has been focused on ACE2 amplification to increase Ang II degradation to treat kidney disease. A podocyte-specific transgenic mice was created by Dr. Kevin Burns, our collaborator. It was used as a proof-of-concept to study the effects of glomerular ACE2 expression on STZ-induced DKD4. The podocyte-specific transgenic mouse showed a slight increase in ACE2 expression (2-5 fold) within the glomeruli. This relatively small increase in glomerulus-restricted ACE2 activity was nevertheless sufficient to confer significant renoprotection based on reduction of albuminuria and of mesangial expansion in the STZ model of DKD4. As a way to amplify endogenous ACE2 we performed studies using a small molecular compound (1-[(2-dimethylamino) ethylamine]-4-(hydroxyethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one) (XNT) that was initially described to be an ACE2 activator. XNT, however, exhibited its effects on AngII induced hypotension in ACE2KO mice to our surprise. This indicates that it acts by a mechanism other than ACE25. Moreover, LC-MS/MS results showed that XNT did no alter plasma Ang II or Ang (1-7), nor Ang (1)-5) levels. However, rACE2, which was used as a positive control, significantly increased Ang (1-7 and Ang (5-7) levels due to enhanced Ang II degrad5

“Because we couldn’t use XNT and DIZE (another presumed ACE2 activator), for the purpose robust and clear cut ACE2 amplification and because of the toxic nature these compounds, our mouse recombinant ACE26-7 was developed. Ex-vivo experiments were conducted to examine the effects of our mouse (mrACE2) upon angiotensin peptides dynamics within the physiological plasma environment. We used LC-MS/MS to simultaneously measure 10 angiotensin peptides7-8. Then, we administered mouse rACE2 chronically to diabetic and control mice via daily intramuscular injections. Or, we used mini-circles technology to deliver ACE2 directly to the mice9-10. Minicircle DNA delivery is more resistant to gene silencing than lentiviral delivery and so offers a promising platform for gene replacement strategies. The intact mouse ACE2 cDNA was cloned to a circular expression cassette. FVB mice were then injected with the resulting ACE2 miniaturecircle using an i.v. hydrodynamic approach. The minicircle injected ACE2 to mice was monitored for several weeks for blood pressure, serum ACE2 activity, and plasma Ang II levels. After several months of monitoring, Ang II was infused immediately. Compared to vehicle-treated mice, the plasma Ang II levels in mice that were treated with ACE2 had a significantly lower increase. Next, we infected mice with STZ with ACE2 pretreated via minicircle delivery. Despite an expected rise in serum ACE2 activity over 26 weeks, the development of albuminuria was not stopped.

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