Patent Grant
11708561
Type 1 diabetes is caused by a malfunctioning immune system that targets and destroys healthy insulin-producing beta cells in the pancreas. Strategies to tame the immune attack on beta cells may ultimately help prevent type 1 diabetes before onset. However, individuals at risk of type 1 diabetes in the general population cannot yet be identified. Furthermore, immune-mediated destruction of beta cells often starts very early in life.
Consequently, a successful immune intervention would help prevent disease in some individuals but will not help patients with long-standing disease or in whom disease-onset could not be predicted in time for prevention. For these patients, a method to replace lost beta cells and restore insulin production is urgently needed and will remain essential even when successful immune therapies exist.
Two approaches have been proposed to replenish beta cell mass in patients with type 1 diabetes. Beta cells could be regenerated by stimulating the patient's own cells to cause beta cell replication or to cause other cell types (e.g. precursor cells, pancreatic ductal cells, exocrine cells, or alpha cells) to become beta cells. The advantages of this approach are that it is non-invasive and that new beta cells would be perfectly well tolerated by the patient compared to an allogenic islet transplant. The critical caveat is that newly generated beta cells will be as susceptible to autoimmune killing as the patient's original beta cells. Therefore, beta cell regeneration would only be effective in combination with a successful immune therapy that abrogates autoimmunity.
Alternatively, beta cells can be provided from external sources. Several groups have demonstrated that induced human pluripotent stem cells (hiPSCs) can be reprogrammed into beta-like cells that secrete insulin in response to glucose stimulation. hiPSCs can be derived from a patient's own blood or skin cells. Conceivably, one day clinicians may be able to use a patient's own cells to generate vast amounts of beta-like cells in the laboratory that could be transplanted back into the same individual. Alternatively, some researchers have postulated that cells could be generated that are ‘universal-donor’ cells and could be used to treat any patient. Regardless of the source of these newly generated beta-like cells, one key challenge will be to protect them against the autoimmune attack that underlies type 1 diabetes.
Protection against immune killing could be achieved by a physical barrier, and a number of laboratories have a long-standing interest in developing beta cell encapsulation whereby beta cells are packaged within a man-made device that keeps immune cells out. Encapsulation poses many challenges such as packaging a sufficiently large number of cells, allowing proper oxygenation of densely packed cells and preventing fibrosis around the artificial device. Furthermore, encapsulation of beta cell can cause a delay in insulin secretion and action.
Achieving protection without the need for a physical barrier would overcome all of these issues. The killing of beta cells by immune cells proceeds in two stages. First, immune cells must recognize beta cells by interacting with cell surface molecules including HLA molecules. In a second step, immune cells launch an attack to kill beta cells. This attack may come in the form of cytokines, death-receptor ligands (e.g. FasL) or cytolytic molecules (e.g. perforin and granzymes). With the advent of CRISPR/Cas technology and other highly-efficient genome editing methods, it has become possible to precisely engineer hiPSCs. This has led to the prospect of generating gene-modified beta cells with mutations that protect against immune killing.
One approach would then be to target known molecules involved in immune recognition. This strategy may seem promising, however the immune system has evolved many complementary and redundant mechanisms to identify and kill target cells. Driven by the pressures of constantly-evolving pathogens that try to evade host immunity, the immune system has become exceedingly flexible in its ability to find and destroy cells it believes to be diseased. Trying to subvert the immune system by mutating only those molecules we know to be involved in immune recognition may prove futile.
Described herein are unbiased experiments that assess a wide range of genetic modifications to find loss-of-function (LOF) mutations that protect beta cells from autoimmune destruction. These mutations provide therapeutic targets that, individually or in combination, allow genetic engineering to develop beta or beta-like cells resistant to the immune killing that underlies type 1 diabetes. Administration of these engineered beta or beta-like cells may be a novel means to promote survival of beta or beta-like cell transplants in patients with type 1 diabetes.
In accordance with the description, a composition is provided comprising a human beta-like cell, wherein the beta-like cell is capable of producing insulin in response to glucose, and wherein the beta-like cell is genetically or otherwise modified to inhibit expression of one or more of menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, the genetic modification inhibits expression of menin (SEQ ID No: 1).
In some embodiments, the genetic modification inhibits expression of transcription factor HIVEP2 (SEQ ID No: 2).
In some embodiments, the genetic modification inhibits expression of renalase (SEQ ID No: 3).
In some embodiments, the genetic modification inhibits expression of lengsin (SEQ ID No: 4).
In some embodiments, the genetic modification inhibits expression of eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5).
In some embodiments, the genetic modification inhibits expression of perilipin-4 (SEQ ID No: 6).
In some embodiments, the genetic modification inhibits expression of mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7).
In some embodiments, the genetic modification inhibits expression of protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8).
In some embodiments, the genetic modification inhibits expression of zinc finger BED domain-containing protein 3 (SEQ ID No: 9).
In some embodiments, the genetic modification inhibits expression of metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, the beta-like cell is a pancreatic beta cell isolated from a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent.
In some embodiments, the beta-like cell is generated from a stem cell. In some embodiments, the stem cell is an adult stem cell, pluripotent stem cell, or embryonic stem cell. In some embodiments, the stem cell is a hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell.
In some embodiments, the beta-like cell is a cell that is reprogrammed or transdifferentiated. In some embodiments, the reprogrammed or transdifferentiated cell is a pancreatic alpha cell. In some embodiments, the reprogrammed or transdifferentiated cell is a pancreatic exocrine cell. In some embodiments, the reprogrammed or transdifferentiated cell is a gut or stomach cell.
In some embodiments, the genetic modification is a substitution, insertion, deletion, or excision of one or more nucleotides.
In some embodiments, the genetic modification is performed using the CRISPR/Cas9 system. In some embodiments, the guide RNA is selected from the guide RNAs in Table 2 (SEQ ID Nos: 23-35).
In some embodiments, the genetic modification is performed using zinc-finger nucleases.
In some embodiments, the genetic modification is performed using transcription activator-like effector nucleases (TALENs).
In some embodiments, the genetic modification is performed using meganucleases.
In some embodiments, the genetic modification is performed using group one intron encoded endonucleases (GIIEE).
In some embodiments, the genetic modification is within the coding region of the gene, and no gene product is expressed, or a non-functional gene product is produced.
In some embodiments, the genetic modification is not within a coding region of the gene, and no gene product is expressed or a non-functional gene product is produced.
In some embodiments, survival of the beta-like cells over 1, 2, 3, 4, 5, 6, 12, 18, 24, or 36 months is improved compared to beta-like cells without the genetic modification.
In some embodiments, proliferation of beta-like cells over 1, 2, 3, 4, 5, 6, 12, 18, 24, or 36 months is improved compared to beta-like cells without the genetic modification.
In some embodiments, a method of lowering blood glucose in a subject comprises administering any one of the compositions described herein.
In some embodiments, a method of increasing insulin secretion in response to glucose in a subject comprises administering any one of the compositions described herein.
In some embodiments, a method of treating type 1 diabetes in a subject comprises administering any one of the compositions described herein.
In some embodiments, a method of treating type 1 diabetes in a subject comprises administering an agent that genetically modifies any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, a method of preventing the death of pancreatic islet cells comprises administering any one of the compositions described herein.
In some embodiments, a method of preventing the death of pancreatic islet cells comprises administering an agent that genetically modifies any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6) mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, a method of ameliorating type 1 diabetes in a subject comprises administering any one of the compositions described herein. In some embodiments, the subject has a blood sugar level higher than 11.1 mmol/liter or 200 mg/dl.
In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.
In some embodiments, a composition is administered in combination with an additional treatment.
In some embodiments, the additional treatment is insulin. In some embodiments, the insulin is a rapid-acting, intermediate-acting, or long-acting insulin.
In some embodiments, the additional treatment is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazolidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium/glucose transporter 2 inhibitor.
In some embodiments, the beta-like cells are administered by transplant into the pancreas, liver, or fat pads via surgery, injection, or infusion.
In some embodiments, a method of treating type 1 diabetes in a subject comprises administering a composition that inhibits the function of any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, the composition inhibits the function of metabotropic glutamate receptor 2 (SEQ ID No: 10). In some embodiments, the composition is LY341495, (25)-α-ethylglutamic acid (EGLU), or MGS-0039.
In some embodiments, a method of preventing the death of pancreatic islet cells comprises administering a composition that inhibits the function of any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, the composition inhibits the function of metabotropic glutamate receptor 2 (SEQ ID No: 10). In some embodiments, the composition is LY341495, (25)-α-ethylglutamic acid (EGLU), or MGS-0039.
Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.
Table 1 provides a listing of certain sequences referenced herein.
I. Definitions
In addition to definitions included in this sub-section, further definitions of terms are interspersed throughout the text.
In this invention, “a” or “an” means “at least one” or “one or more,” etc., unless clearly indicated otherwise by context. The term “or” means “and/or” unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term “or” refers back to more than one preceding claim in the alternative only.
“Autoimmune” or “autoimmune attack,” as used herein, refers to an attack by the subject's immune system against cells that are part of the subject. As such, an autoimmune disease is an abnormal immune response to a normal body part. In the case of type 1 diabetes, the autoimmune attack is predominantly against the beta cells of the pancreas that normally secrete insulin in a glucose-dependent manner.
As used herein, “beta-like cell” refers to any cell that secretes insulin in response to glucose. Thus, a pancreatic beta cell is a “beta-like cell.” Beta-like cells may be derived from cells that do not normally produce insulin in response to glucose. For example, a beta-like cell may be a stem cell that is induced to differentiate into a “beta-like cell” that produces insulin in a glucose-responsive manner. (see F W Pagliuca et al., Cell 159:428-439 (2014); E Kroon et al., Nature Biotech 26(4):443-452 (2008); and A Rezania et al., Nature Biotech 32(11): 1121-1133 (2014). Likewise, a “beta-like cell” may also be a pancreatic exocrine cell (see Q Zhou et al., Nature 455:627-633 (2008)), pancreatic alpha cell (see Li et al, Cell 168:86-100 (2017), or gut cell (see Ariyachet C et al., Cell Stem Cell 18(3):410-21 (2016)) that is induced to produce insulin in response to glucose. The term “beta-like cells” also includes cells that become glucose responsive insulin secretors after transplantation into a subject.
The term “genetically modified” or to “genetically modify,” as used herein, describes any method that reduces the expression or function of one or more protein in a cell from the baseline or unmodified state. Examples of means to genetically modify cells include decreasing expression of a protein, inhibiting expression of a protein, silencing expression of a protein, eliminating expression of a protein, reducing function of a protein, inhibiting proper confirmation of a protein, or any other means to change expression or function of a protein.
The term “inhibit expression of a gene” or “inhibiting expression of a gene,” as used herein refers to causing a decrease in expression of a protein product of the gene.
The term “silence a gene” or “silencing a gene,” as used herein refers to causing a lack of expression of the protein product of the gene.
The term “treatment,” as used herein, covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of diabetes type 1 subjects may comprise alleviating hyperglycemia as compared to a time point prior to administration or reducing the subject's need for exogenous insulin administration.
II. Compositions
In some embodiments, compositions are provided comprising modified beta-like cells. In general, the modifications allow the beta-like cell to survive when implanted into an animal model of type 1 diabetes, or when implanted into a human with type 1 diabetes. The modifications generally allow the beta-like cell to survive autoimmune attack.
In some embodiments, the genetic modification comprises any modification that results in a reduced expression of the following proteins: menin (SEQ ID No: 1), transcription factor HIVEP2 (SEQ ID No: 2), renalase (SEQ ID No: 3), lengsin (SEQ ID No: 4), eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5), perilipin-4 (SEQ ID No: 6), mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7), protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8), zinc finger BED domain-containing protein 3 (SEQ ID No: 9), and metabotropic glutamate receptor 2 (SEQ ID No: 10).
A. Types of Beta Cells to be Genetically Modified
1. Beta Cells Themselves
Beta cells of pancreas are the cells that normally can secrete insulin. These beta cells of the pancreas are located in pancreatic islets, also known as the islets of Langerhans.
In some embodiments, the genetically-modified beta-like cell is a beta cell of the pancreas. In some embodiments, the genetically-modified beta-like cell is a beta cell that has been genetically modified ex vivo, and reintroduced into the same or different individual from which it was isolated. When introduced into the same subject from which it was isolated it is an autologous genetically-modified beta-like cell. When introduced into a different subject from which it was isolated it is a heterologous genetically-modified beta-like cell.
2. Cells Induced to Have a Phenotype of a Beta-Like Cell
In some embodiments, the beta-like cell is a cell that does not normally produce insulin in response to glucose, but is induced or designed to have a phenotype of a beta-like cell, i.e., induced or designed to produce insulin in response to glucose. Beta-like cells include “designer beta cells,” which have been described as using synthetic pathways to produce insulin (see M Xie et al., Science 354(6317):1296-1301 (2016)).
a) Stem Cells
Any stem cell capable of differentiating into a beta-like cell may be genetically modified according to the invention. In some embodiments, the beta-like cell may be differentiated from a hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell.
Beta-like cells capable of secreting insulin in response to glucose can be generated from pluripotent stem cells (PSCs) (see FW Pagliuca et al., Cell 159:428-439 (2014)) or embryonic stem cells (ESCs) (see E Kroon et al., Nature Biotech 26(4):443-452 (2008) and A Rezania et al., Nature Biotech 32(11): 1121-1133 (2014)).
In some embodiments, the stem cell may be an embryonic stem cell. In some embodiments, the embryonic stem cell is taken from a blastocyst. In some embodiments, the embryonic stem cell may be derived from an embryo fertilized in vitro and donated. In some embodiments, the embryonic stem cell undergoes directed differentiation.
In some embodiments, the stem cell may be an adult stem cell. An adult stem cells may also be referred to as a “somatic” stem cell. In some embodiments, the adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ.
In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC).
In some embodiments, the stem cells may be from bone marrow, adipose tissue, or blood. In some embodiments, the cells may be from umbilical cord blood.
In some embodiments, stem cells undergo directed differentiation into beta-like cells. In some embodiments, the directed differentiation is based upon treatment of stem cells with modulators. In some embodiments, the directed differentiation is based on culture conditions.
In some embodiments, beta-like cells are generated from human PSCs (hPSCs) in vitro. In some embodiments, beta-like cells are generated from hPSCs using directed differentiation. In some embodiments, beta-like cells are generated from hPSCs using a multi-step protocol. In some embodiments, beta-like cells are generated from hPSCs using sequential modulation of multiple signaling pathways. In some embodiments, beta-like cells are generated from hPSCs using a three-dimensional cell culture system.
In some embodiments, beta-like cells are generated from human ESCs (hESCs) in vitro. In some embodiments, beta-like cells are generated from hESCs using directed differentiation. In some embodiments, beta-like cells are generated from hPSCs using a multi-step protocol. In some embodiments, beta-like cells are generated from hESCs using sequential modulation of multiple signaling pathways. In some embodiments, beta-like cells are generated from hESCs using a planar cell culture and air-liquid interface at different stages of differentiation.
b) Non-Stem Cells
In some embodiments, beta-like cells are produced from non-stem cells. In some embodiments, beta-like cells are produced from differentiated non-beta cells. In some embodiments, beta-like cells are produced from reprogramming or transdifferentiation of differentiated non-beta cells.
In some embodiments, the beta-like cell is a reprogrammed non-beta cell. In some embodiments, the beta-like cell is a transdifferentiated non-beta cell.
As all cells of the body contain the full genome, any type of cell could be induced into a beta-like cell based on principles of reprogramming and transdifferentiation. Thus, the invention is not limited by the original phenotype of the beta-like cell.
Pancreatic exocrine cells can be reprogrammed into beta-like cells that secrete insulin (see Q Zhou et al., Nature 455:627-633 (2008)).
In some embodiments, a pancreatic exocrine cell is reprogrammed into a beta-like cell. In some embodiments, the pancreatic exocrine cell is differentiated into a beta-like cell based on re-expression of transcription factors. In some embodiments, these transcription factors are Ngn3, Pdx1, and Mafa.
Pancreatic alpha cells can be transdifferentiated into beta-like cells. The anti-malarial drug, artemisin, inhibits the master regulatory transcription factor Arx (Aristaless related homeobox) and enhances gamma-amino butyric acid (GABA) receptor signaling, leading to impaired pancreatic alpha cell identity and transdifferentiation of alpha cells into a beta-like cell phenotype (see Li et al, Cell 168:86-100 (2017) and Ben-Othman N et al., Cell 168(1-2):73-85 (2017)).
In some embodiments, the beta-like cell is a transdifferentiated cell. In some embodiments, an alpha cell is transdifferentiated into a beta-like cell. In some embodiments, the transdifferentiation into a beta-like cell is due to inhibition of Arx. In some embodiments, the transdifferentiation into a beta-like cell is due to enhancement of GABA receptor signaling.
Stomach tissue can be reprogrammed into beta-like cells (see Ariyachet C et al., Cell Stem Cell 18(3):410-21 (2016)). In some embodiments, a gut or stomach cell is reprogrammed into a beta-like cell. In some embodiments, the reprogramming is based on expression of beta cell reprogramming factors. In some embodiments, cells of the antral stomach are reprogrammed into beta-like cells. In some embodiments, these cells of the antral stomach are antral endocrine cells. In some embodiments, reprogrammed antral endocrine cells can be assembled into a mini-organ of beta-like cells.
B. Types of Genetic Modification to the Beta-Like Cells
In some embodiments, genetic modification inhibits or reduces expression of a protein, thus leading to improved survival and/or proliferation of transplanted beta-like cells. In some embodiments, genetic modification silences expression of a gene, thus leading to improved survival and/or proliferation of transplanted beta-like cells.
In some embodiments, silencing of a gene or inhibiting expression of a protein is due to editing that removes all or a portion of the target gene, or all or a portion of a region of DNA that regulates the target gene. In some embodiments, editing that removes a portion of the target gene, or the DNA controlling its regulation, results in silencing the gene or inhibiting expression of the gene product.
A variety of methods of gene editing would be known to one skilled in the art, and this invention is not limited by the particular mechanism used for editing.
a) CRISPR/Cas9
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a prokaryotic immune system that confers resistance to foreign gene elements.
In some embodiments, the CRISPR/Cas9 system is used to genetically modify beta-like cells. In some embodiments, a synthetic guide RNA (gRNA) is used to direct the CRISPR/Cas9 system to a specific sequence within the genome of the beta-like cell to perform gene editing.
b) Zinc-Finger Nucleases
Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.
In some embodiments, a zinc-finger nuclease is used to genetically modify beta-like cells. In some embodiments, the zinc-finger nuclease targets to a specific sequence within the genome of the beta-like cell to perform gene editing.
c) Transcription Activator-Like Effector Nuclease (TALEN)
Transcription Activator-Like Effector Nucleases (TALEN) are Restriction enzymes engineered to cut specific sequences of DNA. TALEN are generated by fusion of a TAL effector DNA-binding domain to a nuclease.
In some embodiments, a TALEN is used to genetically modify beta-like cells. In some embodiments, the TALEN targets to a specific sequence within the genome of the beta-like cell to perform gene editing.
d) Meganuclease
Meganucleases are endodeoxyribonucleases with a large recognition site that often will only occur rarely within a genome. Modified meganucleases can have a targeted recognition site.
In some embodiments, a meganuclease is used to genetically modify beta-like cells. In some embodiments, the meganuclease targets to a specific sequence within the genome of the beta-like cell to perform gene editing.
e) Group One Intron Encoded Endonuclease (GIIEE)
In some embodiments, a GIIEE is used to genetically modify beta-like cells.
In some embodiments, the meganuclease or GIIEE is I-SceI, I-Cre, I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-DmoI, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, PI-SceI, I-SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, or I-VdI141I.
In some embodiments, gene modifications silence a gene or inhibit expression of a gene that promotes beta-like cell death. In some embodiments, gene modifications silence a gene or inhibit expression of a gene, thereby promoting beta-like cell survival or proliferation.
In some embodiments, the gene encoding human menin (SEQ ID No: 1), transcription factor HIVEP2 (SEQ ID No: 2), renalase (SEQ ID No: 3), lengsin (SEQ ID No: 4), eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5), perilipin-4 (SEQ ID No: 6), mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7), protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8), zinc finger BED domain-containing protein 3 (SEQ ID No: 9), or metabotropic glutamate receptor 2 (SEQ ID No: 10) is silenced or its expression is inhibited. In some embodiments, more than one gene encoding these proteins is inhibited or silenced. The term “gene encoding” includes any DNA encoding the amino acid or functional equivalents thereof. In some embodiments, the functional equivalent is a mutated or variated protein, wherein the protein has same or similar function. The term “gene encoding” further includes all isoforms, splice variants, and mature and immature forms of the protein. In some embodiments, inhibiting or silencing a protein includes inhibiting expression, function, structure, or any other property of a protein needed to perform its normal role in the body.
In some embodiments, a genetic modification is introduced into an exon, intron, promoter, or other region of the Men 1 (Gene ID No: 4221), HIVEP2 (Gene ID No: 3097), RNLS (Gene ID No: 55328), LGSN (Gene ID No: 51557), GCN1 (Gene ID No: 10985), PLIN4 (Gene ID No: 729359), MED11 (Gene ID No: 400569), TGM6 (Gene ID No: 343641), ZBED3 (Gene ID No: 84327), or GRM2 (Gene ID No: 2912), and the gene is silenced or its expression is inhibited by introduction of the genetic modification. In some embodiments, more than one of these genes is silenced or its expression is inhibited. In some embodiments, the genetic modification introduced is a deletion, substitution, or insertion of one or more nucleotides.
C. Non-Cellular Agents to Modify Function of a Protein Promoting Beta-Like Cell Death
The function of human menin (SEQ ID No: 1), transcription factor HIVEP2 (SEQ ID No: 2), renalase (SEQ ID No: 3), lengsin (SEQ ID No: 4), eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5), perilipin-4 (SEQ ID No: 6), mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7), protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8), zinc finger BED domain-containing protein 3 (SEQ ID No: 9), or metabotropic glutamate receptor 2 (SEQ ID No: 10) can also be inhibited by post-translational means. Any means of post-translational modulation may be used, including inhibiting binding of ligand, inhibiting function of an enzyme protein, allosteric modulation, or increasing degradation of the protein.
In some embodiments, small molecules can be used to inhibit the function of human menin (SEQ ID No: 1), transcription factor HIVEP2 (SEQ ID No: 2), renalase (SEQ ID No: 3), lengsin (SEQ ID No: 4), eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5), perilipin-4 (SEQ ID No: 6), mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7), protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8), zinc finger BED domain-containing protein 3 (SEQ ID No: 9), or metabotropic glutamate receptor 2 (SEQ ID No: 10). In some embodiments, the function of the target protein is inhibited without an effect on the expression level of the protein.
Renalase (SEQ ID No: 3) and protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8) are enzymes. In some embodiments, enzyme inhibitors of renalase or protein-glutamine gamma-glutamyltransferase 6 inhibit the function of the enzymes. In some embodiments, inhibition of renalase or protein-glutamine gamma-glutamyltransferase 6 promotes survival and/or proliferation of beta-like cells.
Inhibitors of menin have been described (see Grembecka J et al., Nature Chemical Biology 8:277-284 (2012)). In some embodiments, an inhibitor of menin promotes survival and/or proliferation of beta-like cells.
Metabotropic glutamate receptor 2 (mGluR2) inhibitors or negative allosteric modulators have been described (see Podkowa K et al., Psychopharmacology (Berl) 233(15-16):2901-14 (2016)). In some embodiments, an inhibitor or negative allosteric modulator of mGluR2 promotes survival and/or proliferation of beta-like cells. In some embodiments, LY341495 is the inhibitor or negative allosteric modulator of mGluR2. In some embodiments, (2S)-α-ethylglutamic acid (EGLU) is the inhibitor or negative allosteric modulator of mGluR2. In some embodiments, MGS-0039 is the inhibitor or negative allosteric modulator of mGluR2.
III. Methods of Treatment
In each embodiment of the invention, the subject treated is a mammal. In one embodiment, the mammal is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In embodiment, the subject is a human subject.
Glucose levels in the blood are normally tightly regulated to maintain an appropriate source of energy for cells of the body. Dysregulation of blood sugar must be ameliorated to maintain health and longevity, and therapies that are fast acting are especially desired. Such fast acting therapies allow subjects to monitor blood glucose in real time and immediately self-medicate themselves to bring glucose levels within normal limits. Dosing with exogenous insulin is one example of a fast-acting glucose modulator that has allowed subjects with diabetes to maintain relatively normal lifestyles. Described herein is a non-insulin fast-acting compound that regulates blood glucose levels in real-time.
Insulin and glucagon are principal hormones that regulate blood glucose levels. In response to an increase in blood glucose, such as after a meal, insulin is released from beta cells of the pancreas. Insulin regulates the metabolism of carbohydrates and fats by promoting uptake of glucose from the blood into fat and skeletal muscle. Insulin also promotes fat storage and inhibits the release of glucose by the liver. Regulation of insulin levels is a primary means for the body to regulate glucose in the blood.
When glucose levels in the blood are decreased, insulin is no longer released and instead glucagon is released from the alpha cells of the pancreas. Glucagon causes the liver to convert stored glycogen into glucose and to release this glucose into the bloodstream. Thus, insulin and glucagon work in concert to regulate blood glucose levels.
In one embodiment, treatment of diabetes mellitus is to administer a composition to a subject to lower blood glucose.
Hyperglycemia refers to an increased level of glucose in the blood. Hyperglycemia can be associated with high levels of sugar in the urine, frequent urination, and increased thirst. Diabetes mellitus refers to a medical state of hyperglycemia.
The American Diabetes Association (ADA) suggests that fasting plasma glucose (FPG) levels of 100 mg/dL to 125 mg/dL or HbA1c levels of 5.7% to 6.4% may be considered hyperglycemia and may indicate that a subject is at high risk of developing diabetes mellitus (i.e. prediabetes, see ADA Guidelines 2015).
The ADA states that a diagnosis of diabetes mellitus may be made in a number of ways. A diagnosis of diabetes mellitus can be made in a subject displaying an HbA1c level of ≥6.5%, an FPG levels of ≥126 mg/dL, a 2-hour plasma glucose of ≥200 mg/dL during an OGTT, or a random plasma glucose level ≥200 mg/dL in a subject with classic symptoms of hyperglycemia.
Diabetes mellitus can be broken into Type 1 and Type 2. Type 1 diabetes mellitus (previously known as insulin-dependent diabetes or juvenile diabetes) is an autoimmune disease characterized by destruction of the insulin-producing beta cells of the pancreas. Classic symptoms of Type 1 diabetes mellitus are frequent urination, increased thirst, increased hunger, and weight loss. Subjects with Type 1 diabetes mellitus are dependent on administration of insulin for survival.
Type 2 diabetes mellitus is a metabolic disease characterized by a relative decrease in insulin levels and/or a phenotype of insulin resistance. Insulin resistance refers to when cells of the body no longer respond appropriately to insulin. The risk of Type 2 diabetes mellitus is increased in individuals who are obese or who have a sedentary lifestyle.
In the absence of regulation of glucose levels in subjects with diabetes, a range of serious complications may be seen. These include atherosclerosis, kidney disease, stroke, nerve damage, and blindness.
A method of treating diabetes mellitus comprising administering a composition is encompassed. In one embodiment, the method comprises lowering blood glucose levels in the diabetic subject to below about 200 mg/dL, 150 mg/dL, 100 mg/dL, or about 125 mg/dL.
In some embodiments, treatment of diabetes is increasing insulin levels in the subject after administering a composition.
In some embodiments, administering a composition causes a decrease in blood glucose levels such that levels are less than 200 mg/dL.
In some embodiments, the subject treated with a composition has Type 1 diabetes mellitus. In some embodiments, the diabetic subject treated has a relative decrease in insulin levels. In some embodiments, the subject treated has decreased beta cell mass. In some embodiments, the decrease in beta cell mass in a subject is due to an autoimmune disease.
In some embodiments, the subject treated has diabetes mellitus based on diagnosis criteria of the American Diabetes Association. In some embodiments, the subject with diabetes mellitus has an HbA1c level of ≥6.5%. In some embodiments, the subject with diabetes mellitus has an FPG levels of ≥126 mg/dL. In some embodiments, the subject with diabetes mellitus has a 2-hour plasma glucose of >200 mg/dL during an OGTT. In some embodiments, the subject with diabetes mellitus has a random plasma glucose level ≥200 mg/dL or 11.1 mmol/L. In some embodiments, the subject with diabetes mellitus has a random plasma glucose level ≥200 mg/dL or 11.1 mmol/L with classic symptoms of hyperglycemia.
A. Treatment with Genetically Modified Beta-Like Cells
In some embodiments, a method of treating type 1 diabetes, improving glucose tolerance, lowering blood glucose, and increasing insulin secretion in response to glucose is encompassed comprising administering a composition comprising a human beta-like cell, wherein the beta-like cell is capable of producing insulin in response to glucose, and wherein the beta-like cell is genetically modified to inhibit expression of one or more of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, the method comprises administering an agent that genetically modifies any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10) to an individual with type 1 diabetes is encompassed.
In some embodiments, the administering prevents the death of pancreatic islet cells.
In some embodiments, the administering lowers blood glucose in a subject.
In some embodiments, the administering increases insulin secretion in a subject.
In some embodiments, the administering treats type 1 diabetes in a subject.
In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.
In some embodiments, the subject has a blood sugar level higher than 11.1 mmol/liter or 200 mg/dl.
In some embodiments, genetically modified beta-like cells are administered via subcutaneous or intraperitoneal injection. In some embodiments, genetically modified beta-like cells are administered by portal vein infusion.
In some embodiments, genetically modified beta-like cells are transplanted. The genetically modified beta-like cells may be transplanted into any tissue that can support their survival/growth. In some embodiments, genetically modified beta-like cells are administered by transplant into the pancreas, liver, or fat pads. In some embodiments, genetically modified beta-like cells are transplanted via surgery, injection, or infusion.
In some embodiments, transplanted genetically modified beta-like cells can survive for 1, 2, 3, 4, 5, 6, 12, 18, 24, 36 months or indefinitely. In some embodiments, transplanted beta-like cells can survive for a year. In some embodiments, transplanted beta-like cells can survive for two years. In some embodiments, transplanted beta-like cells can survive for three years.
In some embodiments, proliferation of genetically modified beta-like cells over 1, 2, 3, 4, 5, 6, 12, 18, 24, or 36 months is improved compared to beta-like cells without the genetic modification.
B. Treatment with Agents Modulating Function of Protein Promoting Beta-Like Cell Death
In some embodiments, a method of treating type 1 diabetes, improving glucose tolerance, lowering blood glucose, and increasing insulin secretion in response to glucose in a subject comprises administering a composition that inhibits the function of any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, a method of preventing the death of pancreatic islet cells comprises administering a composition that inhibits the function of any one of human menin (SEQ ID No: 1); transcription factor HIVEP2 (SEQ ID No: 2); renalase (SEQ ID No: 3); lengsin (SEQ ID No: 4); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 5); perilipin-4 (SEQ ID No: 6); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 7); protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 8); zinc finger BED domain-containing protein 3 (SEQ ID No: 9); and metabotropic glutamate receptor 2 (SEQ ID No: 10).
In some embodiments, composition inhibits the function of metabotropic glutamate receptor 2 (SEQ ID No: 10). In some embodiments, the composition is LY341495, (25)-α-ethylglutamic acid (EGLU), or MGS-0039.
C. Combination Treatment
In some embodiments, treatment further comprises an additional therapeutic agent.
In some embodiments, the further therapeutic agent is insulin. In some embodiments, the insulin is a rapid-acting, intermediate-acting, or long-acting insulin.
In some embodiments, the further therapeutic agent is an immunosuppressant or immunomodulatory agent. In some embodiments, the further therapeutic agent decreases the autoimmune response of the subject against beta-like cells.
In some embodiments, the further therapeutic agent is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazolidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium/glucose transporter 2 inhibitor.
Unbiased whole-genome screening is a powerful approach to discover novel genes and signaling pathways that underlie disease. An LOF screen was performed in a mouse model using CRISPR/Cas9 genome editing technology (see Komor A C et al., Cell 168(1-2):20-36 (2016)).
This screen took advantage of the NOD mouse model of type 1 diabetes (see Pearson J A et al., J. Autoimmun. 66:76-88 (2016)). NOD mice develop type 1 diabetes due to autoimmune attack on beta cells of the pancreas.
In addition, the model used NIT-1 cells that are an immortalized beta cell line derived from NOD mice (see Hamaguchi K et al., Diabetes 40:842-9 (1991)). NIT-1 cells can be implanted into NOD mice without triggering alloreactivity, because of the cells' NOD origin. However, NIT-1 cells express all the beta cell antigens that are targeted by the immune system during autoimmunity and so are subject to immune killing in a NOD mouse.
A mouse model of induced attack on beta cells was developed using NIT-1 and NOD.scid mice. NOD.scid mice are NOD mice lacking a normal immune system. The scid mutation prevents the development of mature T and B lymphocytes, so that NOD.scid mice are protected from autoimmunity. Therefore, transplanted NIT-1 cells will not be targeted for immune killing by the NOD.scid immune system, and NIT-1 cells can be used as surrogate beta cells.
To elicit an experimentally regulated autoimmune attack on NIT-1 cells transplanted into the NOD.scid mice model, lymphocytes were transferred from diabetic NOD mice into NOD.scid animals. Autoreactive NOD T cells from the donor lymphocytes can start killing endogenous NOD.scid beta cells, as well as experimentally implanted NIT-1 cells.
This model system was tested as shown in
A series of tools to run a CRISPR/Cas9 mediated whole-genome loss-of-function (LOF) screen were developed. The GeCKO V2 lentiviral pooled library was used that comprises guide RNAs (gRNAs) against every gene (Addgene, targeting >20,000 genes with 6 gRNAs/gene). This library was split into two sub-libraries (A and B), which each cover all targeted genes with 3 gRNAs/gene. These libraries cover the entire coding genome to potentially mutate every gene and also contain >1000 non-targeting gRNAs as internal negative controls. In the GeCKO library, Cas9 and gRNAs were incorporated into a single lentiviral vector to introduce these gene-targeting elements into beta cells by lentiviral infection. This CRISPR/Cas9 LOF system is superior to other previous LOF screening platforms because it is highly efficient and is likely to mutate both copies of a gene simultaneously.
A high-stringency LOF screen was done using a single mouse. In this model, 107 GeCKO library-A infected NIT-1 cells (multiplicity of infection (MOI)=0.3) were injected subcutaneously, and 107 diabetic NOD splenocytes were injected intravenously at the same time.
The workflow for the genome-wide LOF CRISPR-Cas9 screenings of beta cells is illustrated in
For sequencing, genomic DNA from explanted grafts was extracted with genome DNA midi prep kits, and the gRNA region was amplified from genomic DNA using established protocols. As illustrated in
Selection pressure (i.e., autoimmune attack) on transplanted NIT-1 cells was maximized by waiting until NOD.scid mice had become severely hyperglycemic (˜60 days, marked by the arrow) as shown in
Since the GeCKO library (A library) only contain approximately 60,000 unique gRNAs, 106-107 sequencing reads was sufficient to cover the whole library and to achieve statistical significance. Typically, one high throughput sequencing reaction using the second generation Illumina system (e.g. NextSeq 500) can yield ˜150 millions of sequence reads, so multiple samples from different grafts were barcoded, mixed and sequenced together, thus greatly reducing sequencing costs.
The sequencing data was de-barcoded and then analyzed by bioinformatics analysis. Established analysis tools such as HiTSelect or MAGeCK were used for gRNA enrichment analysis. In sum, the ability to amplify, sequence and sub-clone gRNAs after in vivo selection allowed identification of genes whose suppression improved beta cell survival.
Data from a single recipient of CRISPR-targeted beta cells yielded a surprisingly small number of target genes. As expected with the high stringency selection, only 13 unique gRNAs that target 12 genes out of 22 60,000 gRNAs present in pre-implantation cells were identified. The number of times the gRNA was present in sequence (count), the frequency of the gRNA in the total reads, and the gene targeted by the gRNA are shown in Table 2. Two different gRNAs targeting Men1 were found in the screen. The target genes in Table 2 encode for mouse SEQ ID Nos: 11-22 in Table 1, and the known human equivalents of these gene products are SEQ ID Nos: 1-10 in Table 1.
As shown in Table 3, ten of the mouse genes targeted by gRNAs in the LOF screen had a known human homolog.
Several of these selected gRNAs were enriched over 6000 times (>12% in the remaining transplanted cells) in the surviving graft. With the experimental set-up, any given gRNA is initially expected to infect only ˜150 cells. Because not every cell carries homozygous mutations, the selected gRNAs are likely to have conferred strong protection even when causing only partial loss of function.
Notably, two of the top gRNA hits target the same Men1 that had previously been implicated in beta cell biology, though not in the context of autoimmunity. This result provides strong evidence that our approach yields highly relevant targets.
Strikingly, the third most enriched gRNA targets the gene Rnls that is a lead candidate for a type 1 diabetes-associated region identified by genome wide association study (GWAS). The Rnls gene has been suggested to associate with the progression rate to overt type 1 diabetes, but how Rnls is involved in pathogenesis is unknown. The fact that the Rnls gene is associated with type 1 diabetes but with no other autoimmune disease indicates that it probably has a non-immune role, likely altering beta cell survival or function.
Interestingly, another one of the 12 targets identified in this preliminary screen is a candidate gene for a type 2 diabetes-associated region (Zbed3). This particular gene had been suggested to participate in insulin secretion. Again, this provides suggestive evidence that this gene's role in type 2 diabetes stems for a key function in beta cell biology.
The remaining candidate genes identified have no clear link with diabetes based on current knowledge. One of these genes has not even been annotated previously (Gm3604), let alone studied. The fact that this screen was able to discover genes that were already associated with type 1 and 2 diabetes or that are known to impact beta cell biology highlights the power of this unbiased yet stringent screening strategy.
In vitro experiments were performed to confirm the in vivo screen results that inhibition of the genes listed in Table 2 promoted survival of beta cells in a type 1 diabetes model.
NIT-1 stable cell lines were prepared for each candidate gene to investigate whether the gRNA used mediated editing of the target gene. To generate NIT-1 stable cell lines, the Cas9 gene and single gRNA targeting each candidate gene were stably incorporated into the genome of NIT-1 cells by lentivirus transduction. The T7E1 assay was used to detect mutations in the target genome locus, with activity based on the capability of T7 endonuclease I to cut mismatched double strand DNA. This strategy means that not all cells will have the homozygous null/missense mutation; therefore, a relative decrease (without total loss of native form) indicates successful editing of the target gene. Data shown in
As shown in
A cellular model of immune killing of stable NIT-1 cell lines was also developed, as shown in
These data confirm that suppression of expression of many of the target genes found in the LOF screen protects NIT-1 cells from NOD splenocyte attack in an in vitro system.
In some instances, beta cell death in Type 1 diabetes is induced by endoplasmic reticulum (ER) stress. Blocking ER stress and subsequent beta cell death has been shown to reverse early-onset Type 1 diabetes in mouse models (see Morita S. et al. Cell Metabolism 2017; 25(4):883-897). Therefore, one way to assess whether a therapeutic intervention is capable of protecting beta cells from autoimmune destruction is to assess whether that therapeutic can protect beta cells from death caused by ER stress.
NIT-1 beta cells were genetically modified at two genes—1) Rnls; and 2) Zbed3 with CRISPR/Cas9 and guide RNAs (gRNAs) targeting Rnls and Zbed3, respectively. The modified NIT-1 cells had reduced expression of renalase (Rnls mutants) or zinc finger BED domain-containing protein 3 (Zbed3 mutants). ER stress was induced by thapsigargin, which blocks the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump on the ER, thus depleting calcium from the ER and leading to misfolding of proteins in ER and eventually ER stress induced cell death.
All cells were treated with thapsigargin for 3 days, and cell viability was evaluated by a standard MTT assay (see Mosmann T, J. Immunol Methods 65(1-2):55-63 (1983)). Our studies showed that thapsigargin at different dosages induced beta cell death in control cells. However, in Rnls mutant and Zbed3 mutant cells were both resistant to thapsigargin-induced cell death in a dosage-dependent manner. Thus, inhibiting expression of the Rnls or Zbed3 protected beta cells from cell death induced by ER stress. See
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.
As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/−5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.
PCT/US2018/019862, which was filed on Feb. 27, 2018, which claims the benefit of priority to U.S. Provisional Application No. 62/464,532, which was filed on Feb. 28, 2017, and both of which are incorporated by reference in their entirety.
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| Number | Date | Country | |
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| 20190376042 A1 | Dec 2019 | US |
| Number | Date | Country | |
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| 62464532 | Feb 2017 | US |
| Number | Date | Country | |
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| Parent | PCT/US2018/019862 | Feb 2018 | US |
| Child | 16542721 | US |
