Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides

Abstract

Described herein is the use of elastin-like polypeptides to generate hemoglobin-based oxygen carriers as a means of preventing and treating conditions caused by blood loss or anemia, for example, hemorrhagic shock. Elastin-like polypeptides are capable of creating therapeutically functional fusion proteins through genetic engineering with a therapeutic agent, for example, hemoglobin and biologic equivalent thereof. Specific forms of these fusion proteins have the ability to form into spherical nanoparticles possessing a therapeutically agent at their core. This provides a unique basis for employing elastin-like polypeptides as hemoglobin carriers in the manufacture of blood substitutes.

What is claimed is: 
     
         1 . A biocompatible pharmaceutical composition comprising a therapeutic amount of a complex comprising a polymer in association with a hemoglobin (Hb), a Hb subunit(s), a Hb fragment(s), a Hb derivative(s), or a functional equivalent thereof that stores and releases oxygen in accordance with an oxygen dissociation curve; wherein the therapeutic amount of the complex is effective to treat a condition caused by blood loss, anemia, or a hemoglobin disorder, and to improve subject survival relative to a control, wherein the polymer is a protein polymer, a polynucleotide polymer, a polysaccharide polymer, or a synthetic polymer. 
     
     
         2 . The biocompatible pharmaceutical composition of  claim 1 , wherein the condition caused by blood loss includes hemorrhagic shock. 
     
     
         3 . The biocompatible pharmaceutical composition of  claim 2 , the protein polymer is associated with the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof via a covalent bond, an ionic bond, a hydrogen bond, a hydrophobic force, encapsulation, or via fusion. 
     
     
         4 . The biocompatible pharmaceutical composition of  claim 1 , wherein the protein polymer is an elastin-like polypeptide (ELP). 
     
     
         5 . The biocompatible pharmaceutical composition of  claim 4 , wherein the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof are operatively linked to form a fusion protein, which is encoded by a polynucleotide comprising a nucleotide sequence that encodes the ELP and a nucleotide sequence that encodes the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof. 
     
     
         6 . The biocompatible pharmaceutical composition of  claim 4 , wherein the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof are operatively linked to form a fusion protein, which is obtained by chemically joining the ELP and the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof. 
     
     
         7 . The biocompatible pharmaceutical composition of  claim 4 , wherein the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof are operatively linked to form a complex, wherein the ELP is assembled into a spherical nanoparticle comprising a core into which the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is encapsulated. 
     
     
         8 . The biocompatible pharmaceutical composition of  claim 5 , wherein the fusion protein is assembled into a spherical nanoparticle comprising a core inside of which the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is enclosed. 
     
     
         9 . The biocompatible pharmaceutical composition of  claim 4 , wherein the ELP comprises a pentameric amino acid motif (Val-Pro-Gly-Xaa-Gly) n , wherein Xaa specifies any amino acid and n denotes a number of repetitive motifs. 
     
     
         10 . The biocompatible pharmaceutical composition of  claim 9 , wherein n=20-90, and Xaa is Serine or a conservative amino acid substitute thereof. 
     
     
         11 . The biocompatible pharmaceutical composition of  claim 10 , wherein the conservative amino acid substitute of Serine is Thr. 
     
     
         12 . The biocompatible pharmaceutical composition of  claim 9 , wherein n=20-90, and Xaa is Isoleucine or a conservative amino acid substitute thereof. 
     
     
         13 . The biocompatible pharmaceutical composition of  claim 11 , wherein the conservative amino acid substitute of Isoleucine is Leu or Met or Val. 
     
     
         14 . The biocompatible pharmaceutical composition of  claim 4 , wherein the ELP comprises a diblock copolymer comprising:
 a hydrophilic block comprising a pentameric amino acid motif (Val-Pro-Gly-Xaa-Gly) n , wherein n=20-90, and Xaa is a hydrophilic amino acid; and   a hydrophobic block comprising a pentameric amino acid motif (Val-Pro-Gly-Xaa-Gly) n , wherein n=20-90, and Xaa is a hydrophobic amino acid.   
     
     
         15 . The biocompatible pharmaceutical composition of  claim 14 , wherein for the hydrophilic block, the Xaa is selected from the group consisting of lysine (+), arginine (+), aspartate (−) and glutamate (−), serine, threonine, asparagine, glutamine, and histidine; and for the hydrophobic block, Xaa is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. 
     
     
         16 . The biocompatible pharmaceutical composition of  claim 15 , wherein for the hydrophilic block the Xaa is Serine or a conservative amino acid substitute thereof; and for the hydrophobic block the Xaa is Isoleucine or a conservative amino acid substitute thereof. 
     
     
         17 . The biocompatible pharmaceutical composition of  claim 16 , wherein the conservative amino acid substitute of Serine is Thr; and the conservative amino acid substitute of Isoleucine is Leu or Met or Val. 
     
     
         18 . The biocompatible pharmaceutical composition of  claim 14 , wherein n=48 for hydrophobic block and n=48 for hydrophilic block. 
     
     
         19 . The biocompatible pharmaceutical composition of  claim 14 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is operatively linked to the C-terminus of the ELP. 
     
     
         20 . The biocompatible pharmaceutical composition of  claim 14 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is operatively linked to the hydrophobic block of the ELP. 
     
     
         21 . The biocompatible pharmaceutical composition of  claim 1 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is of an amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6. 
     
     
         22 . The biocompatible pharmaceutical composition of  claim 14 , wherein the ELP is of amino acid sequence SEQ ID NO. 7. 
     
     
         23 . The biocompatible pharmaceutical composition of  claim 1 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3. 
     
     
         24 . The biocompatible pharmaceutical composition of  claim 1 , further comprising one or more pharmaceutically acceptable salts. 
     
     
         25 . A method of treating a condition due to blood loss and improving subject survival, the method comprising: (1) administering a biocompatible pharmaceutical composition comprising a therapeutic amount of a complex comprising a polymer associated with a Hb, subunit(s), a Hb fragment(s), a Hb derivative(s), or a functional equivalent thereof, wherein the polymer is a protein polymer, a polynucleotide polymer, a polysaccharide polymer, or a synthetic polymer; wherein the therapeutic amount is effective to store and release oxygen in accordance with an oxygen dissociation curve. 
     
     
         26 . The method of  claim 25 , wherein the condition caused by blood loss includes hemorrhagic shock. 
     
     
         27 . The method of  claim 26 , wherein the protein polymer is associated with the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof via a covalent bond, an ionic bond, a hydrogen bond, a hydrophobic force, encapsulation, or via fusion. 
     
     
         28 . The method of  claim 25 , wherein the protein polymer is an elastin-like polypeptide (ELP). 
     
     
         29 . The method of  claim 28 , wherein the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof are operatively linked to form a fusion protein, which is encoded by a polynucleotide comprising a nucleotide sequence that encodes the ELP and a nucleotide sequence that encodes the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof. 
     
     
         30 . The method of  claim 28 , wherein the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof are operatively linked to form a fusion protein, which is obtained by chemically joining the ELP and the ELP and the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof. 
     
     
         31 . The method of  claim 28 , wherein the ELP is assembled into a spherical nanoparticle comprising a core inside of which the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is encapsulated. 
     
     
         32 . The method of  claim 28 , wherein the fusion protein is assembled into a spherical nanoparticle comprising a core inside of which the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is enclosed. 
     
     
         33 . The method of  claim 28 , wherein the ELP comprises a pentameric amino acid motif (Val-Pro-Gly-Xaa-Gly) n , wherein Xaa specifies any amino acid and n denotes a number of repetitive motifs. 
     
     
         34 . The method of  claim 33 , wherein n=20-90, and Xaa is Serine or a conservative amino acid substitute thereof. 
     
     
         35 . The method of  claim 34 , wherein the conservative amino acid substitute of Serine is Thr. 
     
     
         36 . The method of  claim 33 , wherein n=20-90, and Xaa is Isoleucine or a conservative amino acid substitute thereof. 
     
     
         37 . The method of  claim 36 , wherein the conservative amino acid substitute of Isoleucine is Leu or Met or Val. 
     
     
         38 . The method of  claim 28 , wherein the ELP comprises a diblock copolymer comprising:
 a hydrophilic block comprising a pentameric amino acid motif (Val-Pro-Gly-Xaa-Gly) n , wherein n=20-80, and Xaa is a hydrophilic amino acid; and   a hydrophobic block comprising a pentameric amino acid motif (Val-Pro-Gly-Xaa-Gly) n , wherein n=20-80, and Xaa is a hydrophobic amino acid.   
     
     
         39 . The method of  claim 38 , wherein the Xaa is selected from the group consisting of lysine (+), arginine (+), aspartate (−) and glutamate (−), serine, threonine, asparagine, glutamine, and histidine in the hydrophilic block; and Xaa is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine in the hydrophobic block. 
     
     
         40 . The method of  claim 39 , wherein for the hydrophilic block the Xaa is Serine or a conservative amino acid substitute thereof; and for the hydrophobic block the Xaa is Isoleucine or a conservative amino acid substitute thereof. 
     
     
         41 . The method of  claim 40 , wherein the conservative amino acid substitute of Serine is Thr, and the conservative amino acid substitute of Isoleucine is Leu or Met or Val. 
     
     
         42 . The method of  claim 38 , wherein n=48 for hydrophobic block and n=48 for hydrophilic block. 
     
     
         43 . The method of  claim 38 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is operatively linked to the C-terminus of the ELP. 
     
     
         44 . The method of  claim 38 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is operatively linked to the hydrophobic block of the ELP. 
     
     
         45 . The method of  claim 25 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is of amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6. 
     
     
         46 . The method of  claim 38 , wherein the ELP is of amino acid sequence SEQ ID NO. 7. 
     
     
         47 . The method of  claim 25 , wherein the Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3. 
     
     
         48 . The method of  claim 25 , wherein the biocompatible pharmaceutical composition further comprises one or more pharmaceutically acceptable salts. 
     
     
         49 . The method of  claim 25 , further comprising constructing a vector and/or host cell comprising a fusion gene polynucleotide that comprises a polynucleotide sequence coding a fusion protein comprising ELP and Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof. 
     
     
         50 . The method of  claim 49 , further comprising preparing the fusion protein by expressing the fusion gene polynucleotide in an expression system. 
     
     
         51 . The method of  claim 50 , further comprising separating or purifying the fusion protein from the expression system. 
     
     
         52 . The method of  claim 51 , further comprising preparing the fusion protein by chemically operatively linking the ELP and Hb, the Hb subunit(s), the Hb fragment(s), the Hb derivative(s), or the functional equivalent thereof.