Recombinant host containing ferritin or a homolog thereof

Abstract

Disclosed herein is a recombinant host comprising at least a first set of chromosomes and a second set of chromosomes, wherein at least one of the first set of chromosomes and the second set of chromosomes comprises a recombinant gene encoding ferritin or a homolog thereof. Disclosed herein is also a recombinant host comprising a prototroph comprising a recombinant gene encoding ferritin or a homolog thereof. The recombinant host may be included in a composition, an ingestible article, a dietary supplement, or a pharmaceutical composition. Disclosed herein is also a method of administering the composition of the present invention to a subject.

Description

[Technical field]

[0001] The present disclosure relates to a recombinant host comprising ferritin or a homolog thereof. [Background technology]

[0002] Deficiency in iron absorption or excess iron loss leads to suboptimal blood and / or tissue iron levels with various symptoms. The current industry standard for iron replacement therapy is oral administration of ferrous sulfate supplements. However, such supplements have limited effectiveness because the iron is not fully absorbed or the supplements depend on environmental conditions to produce consistent levels of iron. The use of ferrous sulfate supplements can also lead to gastrointestinal discomfort. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein is a recombinant host comprising at least a first set of chromosomes and a second set of chromosomes, wherein at least one of the first set of chromosomes and the second set of chromosomes comprises a recombinant gene encoding ferritin or a homolog thereof.

[0004] Also disclosed herein are recombinant hosts, including prototrophs, that contain a recombinant gene encoding ferritin or a homolog thereof.

[0005] Also disclosed herein are compositions comprising the recombinant hosts of the invention.

[0006] Also disclosed herein are ingestible articles comprising the compositions of the present invention.

[0007] Also disclosed herein are dietary supplements comprising the compositions of the present invention.

[0008] Pharmaceutical compositions comprising the compositions of the invention are also disclosed herein.

[0009] Methods of administering the compositions of the invention to a subject are also disclosed herein. [Brief description of the drawings]

[0010] [Figure 1] The amino acid sequence of human H-ferritin (SEQ ID NO:1) is shown. [Diagram 2] The nucleic acid sequence of human H-ferritin (SEQ ID NO:2) is shown. [Diagram 3] Schematic diagram showing the structure of the ferritin expression cassette containing the human H-ferritin gene FTH1 and the selected URA3 gene. The ferritin expression cassette was amplified by PCR from plasmid RLK / pL5659 using oligonucleotide primers 0-730 and 0-731. The double horizontal line indicates the 3.2 kbp ferritin expression cassette, the open arrows indicate the positions of the primers with arrows showing the direction of DNA synthesis (5' to 3'), the hatched arrow indicates the open-reading frame (ORF) with the arrow tip showing the 3' end of the gene (carboxy-terminus of the encoded protein), the open arrow indicates the position of the constitutively highly expressed yeast TDH3 promoter with an arrow showing the direction of transcription (5' to 3'), and the open block indicates the yeast CYC1 terminator. [Figure 4A] The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Figure 4B] The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Figure 4C] The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Figure 4D] The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Figure 4E] The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Figure 4F] The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Figure 4G]The nucleic acid and amino acid sequences (SEQ ID NO:3) of the expression cassette shown in FIG. [Diagram 5] Schematic diagram showing chromosomal integration of a ferritin expression cassette containing the TDH3 promoter: double horizontal lines indicate the 3.2 kbp ferritin expression cassette, cross-hatched arrows indicate the location of primers with arrowheads indicating the direction of DNA synthesis (5' to 3'), hatched arrows indicate the open reading frame (ORF) with the arrow tip indicating the 3' end of the gene (carboxy-terminus of the encoded protein), open arrows indicate the location of the constitutively highly expressed yeast TDH3 promoter with arrowheads indicating the direction of transcription (5' to 3'), and open block indicates the yeast CYC1 terminator. [Figure 6A] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6B] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6C] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6D] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6E] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6F] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6G] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6H] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6I]The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 6J] The nucleic acid sequence (SEQ ID NO: 4) of the ferritin expression cassette integrated into the chromosome in TDH3 shown in FIG. 5 is shown. [Figure 7] Example 1 compares hemoglobin recovery in rats treated with diploid yeast-ferritin complex compared to rats treated with haploid yeast-ferritin complex. [Figure 8] 1 shows hematocrit recovery in rats treated with diploid yeast-ferritin complex compared to rats treated with haploid yeast-ferritin complex in Example 1. [Figure 9A] The iron content of haploid and diploid yeast samples based on weight percent of a 1 g sample of yeast is shown. [Figure 9B] The H-ferritin content of haploid and diploid yeast samples is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] For purposes of the following detailed description, it should be understood that the invention can assume various alternative variations and step sequences, unless expressly indicated to the contrary. Moreover, except in any operational examples or otherwise indicated, all numbers, such as numbers expressing values, amounts, percentages, ranges, subranges and proportions, can be read as if preceded by the word "about", even if the term is not expressly indicated. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired results obtained by the present invention. At the very least, no attempt is made to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should be construed by at least considering the number of reported significant digits and applying ordinary rounding techniques. When limiting or non-limiting numerical ranges are described herein, all numbers, values, amounts, percentages, subranges and proportions within or subsumed within the numerical ranges should be considered to be specifically included in and belong to the original disclosure of this application, as if all such numbers, values, amounts, percentages, subranges and proportions were expressly written out.

[0012] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0013] As used herein, unless otherwise indicated, plural terms can include their singular counterparts, and vice versa, unless otherwise indicated. For example, although reference may be made herein to "a" species of yeast or "a" set of chromosomes, combinations of these components (i.e., components) may be used. Additionally, in this application, the use of "or" means "and / or," unless otherwise specifically indicated, even though "and / or" may be expressly used in particular instances.

[0014] As used herein, the terms "including," "containing," and similar terms are understood to be synonymous with "comprising" in the context of the present application and are therefore open-ended and do not exclude the presence of additional, unrecited and / or unlisted elements, materials, components and / or method steps.

[0015] As used herein, "consisting of" is understood to exclude the presence of any unspecified elements, components and / or method steps within the context of the present application.

[0016] As used herein, "consisting essentially of" is understood in the context of this application to include the specified elements, materials, components and / or method steps "as well as those that do not materially affect the basic and novel characteristic(s) of what is being described."

[0017] As used herein, "recombinant host" refers to a host whose genome has been augmented or introduced with at least one recombinant gene. "Augmented" or "introduced" refers to an augmentation or introduction caused by human intervention. Exemplary recombinant hosts include, but are not limited to, plants, fungi, such as yeast, bacteria, or animals, such as dogs, pigs, or cows.

[0018] As used herein, a "recombinant gene" is a nucleic acid sequence into which one or more genes or segments of genes have been inserted to obtain a new genetic combination. A recombinant gene is generally a gene that is not found in nature in the context in which it is used. A recombinant gene may also be a gene that has been integrated into the chromosome of a recombinant host at an integration site where the sequence does not naturally occur. A recombinant gene may also be a DNA sequence from a species other than the recombinant host. A recombinant gene may be one or more additional copies of a gene that naturally occurs in a recombinant host that have been inserted into an additional location in the genome of the recombinant host to allow overexpression or modified expression of the gene product of that DNA sequence.

[0019] As used herein, "haploid" means a cell that contains one set of chromosomes.

[0020] As used herein, "a set of chromosomes" means a haploid genome.

[0021] As used herein, "diploid" means a cell that contains two homologous sets of chromosomes.

[0022] As used herein, "polyploid" means a cell that contains three or more homologous sets of chromosomes.

[0023] As used herein, "aneuploid" refers to a cell that contains an abnormal number of chromosomes. An "abnormal number of chromosomes" refers to a number of chromosomes that differs from the normal number of chromosomes for a given organism. For example, a S. cerevisiae cell contains 16 chromosomes. An aneuploid S. cerevisiae cell may, by way of example, contain 15 chromosomes or 17 chromosomes (i.e., any number other than 16).

[0024] As used herein, "homologous" refers to a pair of chromosomes of similar centromere position, genetic composition and length.

[0025] As used herein, "two sets of chromosomes" refers to a diploid genome.

[0026] As used herein, "homolog" means one chromosome of a homologous pair.

[0027] As used herein, "prototroph" or "prototrophic" refers to a microorganism capable of synthesizing nutrients from inorganic materials.

[0028] As used herein, "mate," "mated," or "mates" is the process by which two cells of opposite mating types, generally haploids, fuse to form a single cell, generally a diploid.

[0029] As used herein, "ferritin" is a globular protein that serves as the major intracellular iron storage protein in most microorganisms, including prokaryotes and eukaryotes. Ferritin is a large (nearly 480 kDa) multisubunit complex containing 24 polypeptide subunits, and contains as many as 4,500 atoms of iron ions (Fe ) within an iron hydroxide core. 2+ or Fe 3+ ).

[0030] As used herein, "ingestible" means capable of being taken into the body orally.

[0031] As used herein, "medical food" means a food designed to be ingested or enterally administered for the treatment of a disease that has specific nutritional requirements that cannot be met by the normal diet alone.

[0032] As used herein, "dietary supplement" or "nutritional supplement" means something taken in addition to meals to supplement a subject's diet.

[0033] As used herein, "composition" means a solution, dispersion, or a solid, e.g., a powder.

[0034] As used herein, "pharmaceutical composition" means any chemical or biological composition, material, agent, etc., capable of inducing a therapeutic or metabolic effect when properly administered to a subject, and includes the inactive form of the composition, material, agent, etc., as well as active metabolites thereof, which may be formed in vivo.

[0035] As used herein, a "subject" or "patient" refers to an animal, including a mammal, including a human, canine, feline, bovine, equine, porcine, primate, and / or rodent.

[0036] As used herein, "administering" an amount (e.g., a dose) of a composition may be performed by the subject himself or by another subject (e.g., a medical professional, a caregiver, a family member). The composition may be provided by the subject or by an administrator to the subject along with instructions for administering the composition (e.g., instructions on a label on a container that contains the composition).

[0037] As used herein, "treat", "treatment" or "treating" refers to a therapeutic, prophylactic or preventative measure administered to a patient or subject with the intent of preventing the onset or altering the pathology or symptoms experienced by the patient or subject, such as those resulting from a disorder, which may include, for example, an iron deficiency disorder or a gut microbiome disorder. A "treatment" administered to a patient or subject may achieve up to and including complete elimination and any clinically or quantitatively measurable reduction in the condition for which the patient or subject is being treated. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. "Treatment" may also be designated as a palliative treatment. "Treatment" may also include administration of the composition as a dietary iron source. Those in need of treatment include those already with one or more iron deficiency disorders, those in which a disorder is to be prevented, and those who may benefit from iron supplementation.

[0038] As used herein, "dietary management" means the treatment of a condition by the administration of a medical food, food ingredient, or dietary supplement.

[0039] As used herein, "salt" means an ionic compound consisting of a metal cation and a non-metallic anion and having a total charge of 0. Salts may be hydrated or anhydrous.

[0040] As used herein, "dry matter" in reference to the compositions of the present invention means that the composition contains no more than 10% water by weight, based on the total weight of the composition.

[0041] As used herein, "dry matter basis" refers to a method of expressing the concentration of a component in a composition by expressing the concentration of the component in terms of dry matter content.

[0042] As used herein, an "iron complex" or "organic iron complex" is a compound that contains iron in the (II) or (III) oxidation state complexed or otherwise bound (e.g., ionic) to an organic compound. Examples of suitable organic iron complexes include, but are not limited to, iron polymer complexes, iron carbohydrate complexes, and iron aminoglycosan complexes.

[0043] As used herein, "closely linked" means that a first gene is within 5 centimorgans of a second gene.

[0044] As used herein, "centimorgan" means a unit of measurement equal to a 1% chance that a first marker on a chromosome will become separated from a second marker on the same chromosome by a crossover in one generation.

[0045] As used herein, "iron" refers to elemental iron which may be in the form of salts such as, but not limited to, iron oxide, iron sulfate, and iron hydride.

[0046] As used herein, "iron-deficient" or "iron deficiency" refers to the condition in which a subject has a TSAT of ≦25% and a serum ferritin concentration of ≦100 ng / mL.

[0047] As used herein, "anemia" refers to a condition in which a subject has a hemoglobin concentration less than 13 g / dL, and the subject may also exhibit symptoms commonly associated with iron deficiency, iron uptake, and / or iron metabolism-related disorders or diseases based on responses to health-related quality of life questionnaires. As used herein, "iron deficiency anemia" refers to a condition in which a subject has a TSAT less than 20%, a serum ferritin concentration less than 50 ng / mL, and a hemoglobin concentration less than 13 g / dL. The subject may also exhibit symptoms commonly associated with iron deficiency, iron uptake, and / or iron metabolism-related disorders or diseases based on responses to health-related quality of life questionnaires. An example of an iron deficiency disorder includes iron deficiency caused by insufficient dietary intake or absorption of iron. Iron deficiency disorders can be associated, for example, with malnutrition, pregnancy (including the postpartum period), heavy uterine bleeding, chronic disease (including chronic kidney disease), cancer, renal dialysis, gastric bypass, multiple sclerosis, restless legs syndrome, diabetes (e.g., type I or type II diabetes), insulin resistance, and attention deficit disorder.

[0048] As used herein, "transferrin saturation (TSAT)" means the ratio of serum iron to total iron binding capacity (TIBC).

[0049] As used herein, "total iron binding capacity (TIBC)" means the total amount of iron that can be bound to serum proteins.

[0050] As used herein, a "functional selectable marker" or "functional selectable marker gene" means a genetic element that provides a growth advantage under particular conditions for growth of a recombinant host.

[0051] As used herein, "growth advantage" means that 90% or more of the progeny of the recombinant host contain the gene cassette.

[0052] As used herein, a "gene cassette" comprises a functional selectable marker gene and a gene encoding ferritin.

[0053] As used herein, "mutant" refers to a non-functional gene. As used herein, "non-mutant" refers to a functional gene.

[0054] Disclosed herein is a recombinant host comprising, consisting essentially of, or consisting of a recombinant gene encoding ferritin or a homolog thereof.

[0055] Also disclosed herein is a recombinant host comprising, consisting essentially of, or consisting of at least a first set of chromosomes and a second set of chromosomes, wherein at least one of the first set of chromosomes and the second set of chromosomes comprises a recombinant gene encoding ferritin or a homolog thereof.

[0056] Also disclosed herein are recombinant hosts, including prototrophs that contain, consist essentially of, or consist of a recombinant gene encoding ferritin or a homolog thereof.

[0057] As described above, the recombinant host may express ferritin (e.g., the recombinant host may include a gene encoding ferritin or a homolog thereof). The gene encoding ferritin or a homolog thereof allows the recombinant host to produce ferritin protein by expression of the ferritin gene. Suitable ferritins may include mammalian or plant H-ferritin subunits and / or L-ferritin subunits. Any H-ferritin subunit from any species may be used as long as it encodes H-ferritin. The H-ferritin subunit may be, for example, a mammalian H-ferritin subunit, such as human, canine, feline, bovine, equine, porcine, primate, and / or rodent. The H-ferritin subunit may be human H-ferritin (FTH1) (SEQ ID NO: 1, see FIG. 1, also see FIG. 2). The H-ferritin may also be a naturally occurring or synthetic homolog or variant of human H-ferritin. H-ferritin homologs may have 80% to 100% sequence identity with human H-ferritin, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with human H-ferritin, and retain the ability to bind iron and form a multi-subunit ferritin-iron complex (described below), but may be mutated to provide different binding and dissociation strengths between iron and ferritin. Optionally, the ferritin may be or include L-ferritin. For example, the ferritin subunits may comprise at least 20% H-ferritin compared to L-ferritin, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% H-ferritin compared to L-ferritin. Optionally, all of the ferritin subunits (i.e., 100% of the ferritin subunits) may be H-ferritin, or all of the ferritin subunits (i.e., 100% of the ferritin subunits) may be L-ferritin.

[0058] According to the present invention, the H-ferritin may be recombinant H-ferritin. For example, the H-ferritin may be human H-ferritin or a homologue thereof produced in a microbial strain containing a polynucleotide sequence encoding H-ferritin under the control of a suitable promoter. (See SEQ ID NO: 3, also see Figures 3 and 4). The recombinant gene encoding ferritin may be extrachromosomal (episomal) or chromosomally integrated. (See SEQ ID NO: 4, also see Figures 5 and 6).

[0059] The recombinant host may be prototrophic. Prototrophic recombinant hosts may be either diploid, polyploid, or aneuploid. Examples of suitable diploid recombinant hosts useful in the present invention include, but are not limited to, fungi, algae, protozoa, microscopic worms, microorganisms, or combinations thereof. Examples of suitable polyploid or aneuploid recombinant hosts useful in the present invention include, but are not limited to, fungi, plants, or combinations thereof. For example, recombinant microbial strains suitable for iron nutritional supplementation are capable of storing iron in a form that is highly bioavailable to mammals, including humans, and that meet, for example, the requirements of Generally Recognized As Safe (GRAS) for human consumption. Other microorganisms that can be used in processes to produce therapeutic compounds can also be used in the compositions of the present invention. The fungus may be, for example, a yeast. Non-limiting examples of yeast include various species of the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, or Pichia. Non-limiting examples of algae include various species of the genus Chlamydomonas. Recombinant hosts may contain impurities which may form part of the weight of the compositions herein, but whose weight is excluded from the total dry matter weight of the composition.

[0060] As mentioned above, the recombinant host may be diploid and prototrophic. When the recombinant host is diploid and prototrophic, a non-mutated copy of each gene is present in at least one of the two sets of chromosomes. This allows the diploid recombinant host to produce all the nutrients required for self-sustaining from inorganic materials, thus making it prototrophic.

[0061] As shown in Figures 3 and 5, in an example, the H-ferritin gene can be stably integrated into the chromosomes of a set of haploid recombinant hosts. In another example, the first set of chromosomes of a diploid, polyploid, or aneuploid recombinant host can contain the stably integrated H-ferritin gene. For the purposes of this application, the terms "first set of chromosomes" and "second set of chromosomes" are used for convenience, but do not indicate any limitation to a particular order or number of chromosomes (i.e., there may be two, three, or more complete or partial sets of chromosomes, referred to herein for convenience as "second set of chromosomes"). In yet another example, both sets of chromosomes of a diploid recombinant host or at least two chromosomes of a polyploid or aneuploid can contain the stably integrated H-ferritin gene. The H-ferritin gene can be closely linked to any functional selection marker gene to constitute a gene cassette. For example, suitable functional selection marker genes such as URA3, LEU2, TRP1, HIS3, or MET15 will be known to those skilled in the art. For example, the H-ferritin gene may be closely linked to the URA3 gene. The copy of the functional selection marker gene present in the chromosome of the recombinant host may be a mutant of the functional selection marker gene that renders it non-functional. As will be understood by those skilled in the art, this facilitates the selection of the haploids that contain the gene cassette of the first haploid, and the second haploid may be a mutant URA3 gene. For clarity, in addition to the H-ferritin gene and the functional selection marker gene, the gene cassette may further include other components known to those skilled in the art, including, for example, a promoter and a terminator.

[0062] The recombinant host containing the H-ferritin gene may contain at least one diploid, polyploid, or aneuploid cell. A haploid contains one complete set of chromosomes. When the recombinant host is diploid, two haploids of opposite mating type mate to form a diploid recombinant host. One chromosome of the first set of chromosomes of the recombinant diploid host may contain a recombinant H-ferritin gene. The recombinant H-ferritin gene may be part of a gene cassette integrated into the chromosome. The recombinant H-ferritin gene encodes an amino acid sequence for the production of H-ferritin. The H-ferritin coding sequence may be placed under the control of a suitable promoter in the gene cassette so that the recombinant host produces a high enough level of iron storage protein to serve as a suitable vehicle for iron supplementation. Suitable promoters are known in the art and include promoters that induce high levels of constitutive expression and promoters whose expression can be controlled by environmental conditions. For example, in a yeast recombinant host, a suitable constitutive promoter may be the yeast TDH3 transcription promoter. For example, in a yeast recombinant host, a suitable regulatable promoter may be the yeast GAL1 promoter. Additionally, the genetic makeup of the recombinant host may be further manipulated to achieve a variety of potentially advantageous outcomes. For example, proteolysis may be manipulated to improve the stability of iron storage proteins. Iron transport mechanisms may be manipulated to achieve desirable outcomes, such as altering iron concentrations in specific subcellular compartments, including but not limited to, cell surface, vacuole, or mitochondrial iron concentrations. The iron content of the recombinant host may be regulated by adding known amounts of iron compounds to the medium in which the recombinant host is grown. Using recombinant hosts, iron supplementation for humans and other animals can be achieved by any of a number of means, including but not limited to consumption or ingestion of the recombinant host. The recombinant host may be grown specifically for iron supplementation, or it may be a by-product of another process (e.g., fermentation). In an example, a diploid, polyploid, or aneuploid recombinant host may only have the typical nutritional requirements of a prototrophic yeast.That is, the nutritional requirement of one set of chromosomes encoding ferritin due to a genetic mutation is complemented by the presence of a non-mutated copy of the gene in a second set of chromosomes, and similarly, the nutritional requirement of the second set of chromosomes due to a genetic mutation is complemented by the presence of a non-mutated copy of the gene in the first set of chromosomes.

[0063] In an example, the second set of chromosomes may not express ferritin. The second set of chromosomes may not further express a functional selection marker. That is, the second set of chromosomes may not include a gene cassette. For example, the first set of chromosomes may include a mutated LEU2 gene involved in the synthesis of leucine, an amino acid essential for the production of proteins. The second set of chromosomes may include a non-mutated LEU2 gene, which allows a diploid, polyploid, or aneuploid comprising at least the first set of chromosomes and the second set of chromosomes to synthesize leucine. The ability of the second set of chromosomes to synthesize leucine compensates for the first set of chromosomes that cannot synthesize amino acids. As a result, the diploid, polyploid, or aneuploid recombinant host can produce all the normal nutritional materials that prototrophic yeast synthesizes (i.e., the recombinant host is prototrophic). That is, an unmutated copy of each gene is present on at least one of the two homologous chromosomes in a diploid recombinant host, on at least one of three or more homologous chromosomes in a polyploid recombinant host, or on at least one of at least two sets of chromosomes in an aneuploid or aneuploid recombinant host.

[0064] For example, a first set of chromosomes may contain a mutant URA3 gene involved in the synthesis of uracil, a nucleobase essential for the synthesis of ribonucleic acid (RNA). A second set of chromosomes may contain a non-mutant URA3 gene, which enables the second set of chromosomes to synthesize uracil. The ability of the second set of chromosomes to synthesize uracil compensates for the first set of chromosomes' inability to synthesize nucleobases. As a result, the diploid or polyploid recombinant host is capable of producing all the normal materials that prototrophic yeast synthesizes (i.e., the recombinant host is prototrophic).

[0065] In another example, the first set of chromosomes and the second set of chromosomes may code for ferritin. Both sets of chromosomes may further code for a functional selectable marker gene. That is, both sets of chromosomes contain a gene cassette. When both sets of chromosomes express the gene cassette, the gene cassette may be at a different locus, so that each set of chromosomes has a different mutation. For example, the first set of chromosomes may contain a mutated LEU2 gene and a non-mutated URA3 gene, while the second set of chromosomes contains a non-mutated LEU2 gene and a mutated URA3 gene.

[0066] The present invention also discloses a composition comprising one of the recombinant hosts disclosed herein. In an example, the composition may comprise, consist essentially of, or consist of a recombinant host comprising at least a first set of chromosomes and a second set of chromosomes, wherein at least one of the first set of chromosomes and the second set of chromosomes comprises a recombinant gene encoding ferritin or a homolog thereof. In another example, the composition may comprise, consist essentially of, or consist of a recombinant host comprising a prototroph comprising a recombinant gene encoding ferritin or a homolog thereof. The composition may further comprise iron. The ferritin and iron expressed by the recombinant host may form a ferritin-iron complex. The source of iron may be an iron salt, an organic iron complex, elemental iron nanoparticles, or a combination thereof. Examples of suitable iron salts include, but are not limited to, iron sulfate. Examples of suitable iron complexes include, but are not limited to, iron polymer complexes, iron carbohydrate complexes, and iron aminoglycosyl complexes. These organic iron complexes may be commercially available and / or may be synthesized by methods known in the art. Suitable non-limiting examples of iron carbohydrate complexes include iron sugar complexes, iron oligosaccharide complexes, and iron polysaccharide complexes, such as iron carboxymaltose, iron sucrose, iron polyisomaltose (iron dextran), iron polymaltose (iron dextrin), iron gluconate, iron sorbital, and iron hydrogenated dextran, which may be further complexed with other compounds such as sorbitol, citric acid, and gluconic acid, and mixtures thereof (e.g., iron dextrin-sorbitol-citric acid complex and iron sucrose-gluconic acid complex). Suitable non-limiting examples of iron aminoglycosan complexes include chondroitin sulfate, iron dermatin sulfate, and / or iron keratan sulfate, each of which may be further complexed with other compounds, and mixtures thereof. Examples of iron aminoglycosan complexes include, but are not limited to, iron hyaluronic acid, iron protein complexes, and mixtures thereof.

[0067] Iron may be present in the composition in an amount of at least 1% by weight, such as at least 3% by weight, such as at least 5% by weight, for example at least 5.5% by weight, based on the dry matter of the recombinant host expressing ferritin and the iron. Iron may be present in an amount of not more than 15% by weight, such as not more than 10% by weight, such as not more than 9.5% by weight, for example not more than 8% by weight, based on the dry matter of the recombinant host expressing ferritin and the iron. Iron may be present in the composition in an amount of 1% to 15% by weight, such as 3% to 12% by weight, such as 3% to 8% by weight, for example 5% to 10% by weight, for example 5.5% to 9.5% by weight, based on the dry matter of the recombinant host expressing ferritin and the iron.

[0068] The composition may include intracellular iron and / or extracellular iron that is not complexed with ferritin or a homologue thereof.

[0069] According to the present invention, in some cases, at least 5% of the iron may be complexed with ferritin, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, 100% of the iron may be complexed with ferritin. According to the present invention, in some cases, not more than 95%, for example not more than 90% of the iron may be complexed with ferritin. According to the present invention, 5% to 100%, for example, 10% to 100%, for example, 30% to 95%, for example, 50% to 90%, for example, 60% to 90%, for example, 75% to 90% of the iron may be complexed with ferritin.

[0070] The composition of the present invention may further include a second recombinant host. As used herein, the term "second" in relation to a recombinant host refers to a separate and different recombinant host, and does not necessarily mean that there are only two recombinant hosts. The second recombinant host may include any of the above recombinant hosts. The second recombinant host may include a recombinant host that expresses ferritin, a recombinant host that does not express ferritin, or a combination thereof. Additionally or alternatively, the second recombinant host may include a probiotic, a prebiotic, or a combination thereof.

[0071] Any of the compositions described herein may be included in an ingestible article. In an example, the recombinant host expressing ferritin may be included in an ingestible article. For example, the ingestible article may be a medical food, a food, a food ingredient, a dietary supplement, a pharmaceutical composition, or a combination thereof. In another example, any of the compositions described herein may be in the form of a suppository. In another example, any of the compositions described herein may be a dietary supplement or a nutritional supplement. In an example, the recombinant host expressing ferritin may be included in a dietary supplement or a nutritional supplement.

[0072] In another example, any of the compositions described herein may be included in a pharmaceutical composition. The pharmaceutical composition may be administered, for example, for the treatment of iron deficiency disorders, such as anemia. The pharmaceutical composition may be administered whenever a high dose of iron would be beneficial in treating iron deficiency.

[0073] The compositions described herein may be in the form of dry powder, dispersion of dry powder in liquid, suspension of dry powder in liquid, suppository, foam enema, liquid enema, etc., and may be formulated in a manner to be administered orally or rectally. The compositions of the present invention may include a pharma- ceutically acceptable carrier or diluent (as described herein) to form a solution, dispersion, emulsion, microemulsion, suspension, syrup, elixir, etc., so that the material can be swallowed. A pH adjuster (i.e., acid or base) may be included to adjust the pH to a suitable level, and / or a bacteriostatic and antifungal agent may be included to prevent microbial activity. The pharmaceutical composition may also include a formulation that controls or delays the release of the drug in the body. In some cases, the pharmaceutical composition may be contained in a dispenser, such as a syringe, a dosage vial, etc.

[0074] Examples of ingestible diluents or carriers, according to the judgment of the formulator, are sugars, such as monosaccharides, disaccharides, and the like; excipients, such as cocoa butter and waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; saline; Ringer's solution; ethyl alcohol; phosphate buffers; other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; coating agents; preservatives and antioxidants.

[0075] The compositions described herein may be released from a pressurized container or may be in the form of a powder, granule, or lozenge.The compositions may further comprise at least one binder and / or at least one filler.Examples of suitable binders and fillers include, but are not limited to, magnesium stearate, microcrystalline cellulose, cellulose gel, cellulose gum, carboxymethylcellulose, wood pulp, soy lecithin, glycine, sodium glutamate, vegetable protein, seaweed or extract, carrageenan, or combinations thereof.

[0076] As used herein, the term "pharmaceutical acceptable" means acceptable for use in pharmaceutical and veterinary technology, compatible with other ingredients of the formulation, commensurate with a reasonable benefit / risk ratio, and not toxic or otherwise unacceptable. As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening and emulsifying agents, stabilizers, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 19th Edition, edited by Gennaro, Mack Publishing, Easton, Pennsylvania, 1995, provides various carriers used in formulating pharmaceutical compositions and known techniques for their preparation.

[0077] Those skilled in the art will understand that various factors affect the dosage required to effectively treat a patient, and therefore dosage and administration can be selected by the attending physician in consideration of the patient being treated and adjusted for sufficient levels of active agent(s) or to maintain the desired effect. Additional factors that can be considered include the severity of the disease state, e.g., moderate or advanced stage of the disease; the age, weight, sex and general health of the patient; diet, time and frequency of administration; iron deficiency status; route of administration; drug combinations; reaction sensitivity; prior treatment; and tolerance / response to treatment. The pharmaceutical composition may be administered, for example, every 30 minutes, every hour or once a day, multiple times a day, once a week, multiple times a week, once every two weeks, once a month, etc.

[0078] The active agent of the present invention may be used to treat any disease, disorder, etc. disclosed herein, and may be administered as an effective dose suitable for the patient or subject to be treated. As mentioned above, the effective dose of the composition of the present invention may be determined by the attending physician within the scope of sound medical judgment and experience. The effective dose for the active agent can be estimated first in cell culture assays or animal models such as mice, rats, rabbits, dogs, or pigs. The animal cell model can be used to achieve or determine the desired concentration and total dosage range and route of administration, which can be used to determine the useful range and route of dosage for administration to humans. Furthermore, clinical trials and individual patient response may determine the recommended effective dose.

[0079] An effective dose of one of the compositions described herein may be administered at a dosage level of at least 13 mg per dose, for example at least 50 mg per dose, for example at least 75 mg per dose, for example at least 100 mg per dose or more. An effective dose of one of the compositions described herein may be administered at a dosage level not exceeding 1000 mg per dose, for example not exceeding 700 mg per dose, for example not exceeding 300 mg per dose. An effective dose of one of the compositions described herein may be administered at a dosage level of 13 mg to 1000 mg per dose, for example 50 mg to 700 mg per dose, for example 75 mg to 300 mg per dose, for example 100 mg to 300 mg per dose. As described herein, dosage levels may be administered as a single dose administered to a subject or patient, or by multiple administrations to achieve the dosage level during the day. The dosage levels may also be the total amount of iron administered for administration multiple times per week, weekly, biweekly, or monthly with days between administrations, and the dose administers iron at the above dosage levels per average day.

[0080] As stated above, the methods described herein generally include administering to a subject any of the compositions described herein. The methods may include, consist essentially of, or consist of administering a composition comprising, consisting essentially of, or consisting of (a) a recombinant host expressing ferritin and (b) iron in an amount of at least 1% by weight based on dry matter of the recombinant host expressing ferritin and iron, where at least 60% of the iron is complexed with ferritin. For example, methods are disclosed for treating a subject that include administering to the subject any of the compositions described herein. Administering may include administering to the subject an effective amount of at least one of the compositions described herein. As used herein, the term "effective amount" or "effective dose" refers to the amount of the composition indicated for treatment (i.e., to regulate, ameliorate, or prevent a symptom or condition of iron deficiency) while not exceeding any amount that may cause adverse effects. The effective dose may increase or decrease during the course of treatment. Methods for assessing the efficacy or toxicity of effective treatments are known to those skilled in the art, such as ED50 (the dose effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic effects to effective effects is the therapeutic index, expressed as the ratio LD50 / ED50. In particular, ED50 and LD50 may vary with the age or condition of the subject.

[0081] The composition may be administered as a single dose or as multiple doses (i.e., first, second, third, etc. doses) administered simultaneously or sequentially, such that a first dose of the composition is administered followed by a second dose of the composition, or vice versa. When the first and second doses are administered sequentially, the method may include a waiting period between the administration of the doses. The first, second, third, etc. doses may contain the same or different amounts of iron. As used herein, the term "sequentially" refers to a treatment protocol in which a first dose of the composition of the invention is administered followed by a second dose of the composition of the invention. As used herein, the term "concurrently" refers to the administration of a first dose of the composition of the invention and a second dose of the composition of the invention, where the first and second doses are administered separately and at substantially the same time.

[0082] According to the present invention, the iron deficiency of a subject can be treated by administering to the subject any of the compositions described herein, for example, by administering an effective amount of any of the compositions described herein. For example, a method of treating a subject can include, consist essentially of, or consist of administering to the subject a composition comprising (a) a recombinant host expressing ferritin and (b) iron, in an amount of at least 1% by weight based on the dry weight of the recombinant host expressing ferritin and iron. The administering can include oral administration or rectal administration. The subject can be determined to have at least one of the following prior to the administration: functional iron deficiency, iron deficiency, anemia, iron deficiency anemia, or gut microbiome disorder. The subject need not be iron deficient. A suitable subject would be any subject that would benefit from the incorporation of the composition. The treating can also include dietary management.

[0083] Although particular embodiments of the invention have been described in detail, those skilled in the art will recognize that various modifications and variations to those details may be developed in light of the overall teachings of the present disclosure. Accordingly, the particular arrangements disclosed are intended to be illustrative only and not limiting on the scope of the invention, which is set forth in the full scope of the appended claims and embodiments and any and all equivalents thereof.

[0084] Those skilled in the art will recognize that numerous modifications and variations are possible without departing from the broad inventive concept described in light of the above disclosure and exemplified herein. Accordingly, it should be understood that the foregoing disclosure is merely illustrative of various exemplary embodiments of the present application, and that numerous modifications and variations that are within the spirit and scope of the present application and the appended claims may be readily made by those skilled in the art. EXAMPLES

[0085] Twenty-day-old male Sprague-Dawley rats (Envigo) were housed one per cage in hanging wire cages in a temperature (23±2°C) and humidity (40%) controlled room maintained on a 12:12-h light / dark cycle (lights on from 06:00 to 18:00). Rats were fed ad libitum an iron-deficient diet ("ID diet") with 2 ppm iron or supplemented diets as indicated. ID diets were prepared without added iron according to the American Institute of Nutrition (AIN)-93G dietary recipes, with corn starch as the sole source of carbohydrate. Iron levels in all diets were verified using atomic absorption spectrometry after wet digestion with nitric acid (PerkinElmer). All experimental protocols followed the National Institutes of Health animal experimentation guidelines and were approved by the Institutional Animal Care and Use Committee of Pennsylvania State University.

[0086] Rats were fed the ID diet for 26 days (P21-P47), which resulted in anemia (mean hematocrit and hemoglobin levels were 5.3±0.2 and 16.2±0.1%, respectively). Then, on P47, rats were divided into two weight-balanced dietary groups (n=3-5 / group). Dietary group 1 was fed a diet containing the haploid yeast ferritin complex (53 μg iron / g diet, n=4). The haploid yeast ferritin complex contains a single chromosome containing the ferritin expression cassette integrated into the chromosome in FIG. 5. The haploid yeast ferritin complex is auxotrophic. Dietary group 2 was fed a diet containing the diploid yeast ferritin complex (49 μg iron / g diet, n=3) described in this application. The diploid yeast ferritin complex contains two chromosomes, the first containing the chromosomally integrated ferritin expression cassette of FIG. 5 and the second chromosome not containing the chromosomally integrated ferritin expression cassette. The diploid yeast ferritin complex is prototrophic. The rats were fed the assigned diet for 14 days and food intake was monitored throughout the study.

[0087] The results shown in Figures 7 and 8 indicate that the diploid yeast ferritin complex provided equivalent hemoglobin and hematocrit recovery, respectively, as the haploid yeast ferritin complex. EXAMPLES

[0088] Generation of haploid budding yeast Haploid Saccharomyces cerevisiae were grown for 2 days with shaking at 30°C in synthetic complete (SC) medium lacking uracil and containing 6 mM FeSO4. Cells were harvested by centrifugation and washed once with sterile water. After resuspension, cells were pasteurized by incubation at 65°C for 30 min. Cells were then washed two more times with sterile water to remove unincorporated iron and lyophilized.

[0089] Preparation of diploid budding yeast (S. cevervisiae) Diploid Saccharomyces cerevisiae was grown for 2 days with shaking at 30°C in synthetic complete (SC) medium lacking uracil and containing 6 mM FeSO4. Cells were harvested by centrifugation and washed once with sterile water. After resuspension, cells were pasteurized by incubation at 65°C for 30 min. Cells were then washed two more times with sterile water to remove unincorporated iron and lyophilized.

[0090] Analysis of haploid and diploid yeast samples To analyze haploid and diploid yeast samples for ferritin, aliquots of yeast preparations were examined by electron microscopy. To further demonstrate the presence of FHT1, Western blot analysis was performed on yeast lysates. Samples were run on a 4-20% Tri-glycine SDS gel. After this, it was transferred to a PVDF membrane and probed first with an antibody against H-ferritin (1:500, Cell signaling), followed by a secondary antibody (1:5000, GE healthcare). To determine whether ferritin in yeast contains iron, 25 μl of yeast lysate was run on a 4-20% Tris-glycine SDS gel (BioRad) and used to assess iron using the Perls strain. Briefly, the gel was rinsed once with deionized water and then incubated for 30 minutes with a solution consisting of 2 parts potassium ferricyanide (Sigma Aldrich, Cat #702587) and 1 part 10% HCl solution. The gel was then washed several times with deionized water to remove background and observed for the Blue Perls reaction.

[0091] To analyze the iron content of yeast samples, the yeast was digested in nitic acid and the iron content was measured using atomic absorption spectrometry.

[0092] The results shown in Figure 9A compared the iron content of the haploid and diploid yeast samples based on the weight percent of a 1 g sample of yeast. As shown, the diploid yeast samples had higher iron levels (9%) compared to the haploid yeast samples (5%). The results shown in Figure 9B compared the H-ferritin content of the haploid and diploid yeast samples. As shown, the haploid yeast samples had higher amounts of H-ferritin (4.12%) compared to the diploid yeast samples (2.76%).

[0093] Thus, despite the fact that there was less H-ferritin in the diploid yeast samples, there was more iron compared to the haploid yeast samples. Moreover, despite this difference, the haploid and diploid yeasts performed equally well in the in vivo tests, suggesting that the diploid yeast was more efficient at absorbing iron while still maintaining performance.

Claims

1. A recombinant host comprising at least a first set of chromosomes and a second set of chromosomes, wherein at least one of the first set of chromosomes and the second set of chromosomes comprises a recombinant gene encoding ferritin or a homolog thereof, said recombinant host.

2. The recombinant host comprises diploid cells, polyploid cells, and / or aneuploid cells, and the diploid cells, the polyploid cells, and the aneuploid cells comprise the first set of chromosomes and the second set of chromosomes. The recombinant host according to Claim 1.

3. The recombinant host according to Claim 1, which is prototrophic.

4. The recombinant host according to Claim 1, wherein the recombinant gene comprises SEQ ID NO: 2, or SEQ ID NO: 3, or a homolog thereof.

5. A recombinant host comprising a prototroph comprising a recombinant gene encoding ferritin or a homolog thereof.

6. The recombinant host comprises diploid cells, polyploid cells, and / or aneuploid cells, and the diploid cells, the polyploid cells, and the aneuploid cells comprise a first set of chromosomes and a second set of chromosomes. The recombinant host according to Claim 5.

7. The recombinant host according to Claim 5, wherein the recombinant gene encoding ferritin is integrated into a chromosome.

8. A composition comprising the recombinant host according to Claim 1.

9. The composition according to Claim 8, further comprising iron.

10. A dietary supplement comprising the composition according to Claim 8.

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