Patent Grant
8865160
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 21, 2010, is named SAMSF335.txt and is 12,940 bytes in size.
The present invention is directed to compositions and methods for enhancing viability of islets of Langerhans for purposes of transplantation, and for treating various diseases and other abnormal or pathological conditions, with DNA repair enzymes that are directed to the mitochondria.
Diabetes is a significant health problem worldwide. Present estimates indicate that diabetes is becoming an increasingly prevalent disease which is doubling every 10-15 years, and that by the year 2010 over 250 million people will be afflicted. P. Zimmet, et al., Diabetes global estimates and projections, ed. 1994-2010 (1994). Most individuals with idiopathic diabetes can be categorized as having insulin-dependent diabetes mellitus (type I diabetes) or noninsulin-dependent diabetes mellitus (type II diabetes). Although the etiologies and clinical manifestations of these two syndromes differ, it has become increasingly clear that a critical lesion in many of the subgroups under these large classifications is at the level of the β-cell. Mandrup-Poulsen, Biochem Pharmacol 66:1433-40 (2003); Halban, et al., Diabetologia 47:581-89 (2004).
Although new treatment protocols have had a significant impact on life expectancy and quality of life of individuals with either form of diabetes, there still remains no cure. Insulin treatment for absolute or relative insulin-deficiency cannot adequately compensate for the loss of physiological insulin secretion. Intra-hepatic islet transplantation is a promising strategy in connection with type I diabetes. Unfortunately, this approach has been plagued by the loss of function and viability of islet cells during isolation and in the early transplantation period. Weir, et al., Arch. Med. Res. 36:273-80 (2005). This has caused a dramatic increase in the number of islets required for transplantation, which has significantly impacted the amount of islets available for transplant. Additionally, this problem has contributed greatly to the graft failure that has been seen in most transplant recipients over time. See, Gaglia, et al., Arch. Med. Res. 36:273-80 (2005); Weir, et al., supra. Therefore, there is a belief that functional loss and death of β-cells play a predominant role in the pathogenesis of both types of diabetes and in the failure of islet transplants.
Inflammatory bowel disease (IBD), more common forms of which include Crohn's disease and ulcerative colitis, affects a staggering number of people in developed countries, estimated at around 0.1% of the population. As a group, these disorders are characterized by unrelenting inflammation of the lower gastrointestinal tract leading to periodic and sometimes chronic bouts of diarrhea, profound abdominal distress, malnutrition, and other symptoms. Pathologically, the colonic mucosa of afflicted individuals is overtly disrupted and shows signs if intense inflammation with invasion of commensal organism from the intestinal lumen into the mucosal wall. The cause of IBD remains unknown, although it is believed that both genetics and environmental factors make a significant contribution.
Ischemic heart disease (IHD) continues to be a leading cause of death and of health care expense in the United States. Although there are myriad risk factors for IHD (e.g., smoking, hypertension and obesity). In diabetic and obese prediabetic patients, there is a growing consensus about the growth of coronary collaterals being impaired. See, Abaci, et al., Circulation 99:2239-42 (1999); Kornowski, Coron. Artery Dis. 14:61-4 (2003); Turhan, et al., Coron. Artery Dis. 16:281-85 (2005); Waltenberger, Cardiovasc. Res. 49:554-60 (2001). This is critical because the presence of well-developed coronary collateral circulation makes an important impact on the morbidity and mortality associated with coronary disease. See, Hansen, Am. Heart J. 117:290-95 (1989). Because 30-40% of patients demonstrate an absence of a coronary collateral circulation and are at much higher risk of developing complications in the event of a coronary occlusion, new therapies are essential to protect these individuals.
Acute lung injury and the acute respiratory distress syndrome (ALI/ARDS) are syndromes of acute onset arising with non-cardiogenic pulmonary edema development, manifest as bilateral infiltrates on the chest radiograph coupled to acute hypoxic respiratory failure. Currently, it is estimated that 16%-18% of all ICU patients develop ALI/ARDS producing 6,368,000 hospital days per year in health care costs. The mortality estimates of patients suffering from this syndrome are still 30-50%. Recent estimates of incidence in the United States are around 190,000 cases of ALI and ARDS per year. These numbers are actually projected to rise with the aging of the population and the projected increase in severe sepsis cases, which is the most common cause of ALI/ARDS. See, Ware, et al., N. Eng. J. Med. 353:2788-96 (2005).
Bronchopulmonary dysplasia (BPD) is one such example of an acute lung disease that occurs primarily in preterm infants who require supplemental oxygen. Although the advent of surfactant therapy and use of high frequency low tidal volume ventilator approaches have improved the survival rates of extremely low birth weight infants, significant morbidity and mortality continues to be associated with BPD. In a recent review of clinical data, BPD was shown to be the most costly disease per child and second only to asthma in overall costs. See, Ireys, et al., Pediatrics 100:197-204 (1997). Due to the high rate of premature births and the recent increase in multiple births, BPD is rapidly becoming a common and costly healthcare challenge.
It was estimated that in 2005, approximately 18,500 new cases of primary central nervous system (CNS) tumors would be diagnosed and 12,760 deaths from primary CNS tumors would occur in the United States. See, Jemal, et al., Can. Cancer. J. Clin. 53:5-26 (2003). The majority of these deaths result from malignant gliomas. The optimum therapeutic modality presently consists of surgical tumor resection followed by radiotherapy and chemotherapy. The relationship between increased survival and increased radiotherapy dose was demonstrated by Walker, et al, Int. J. Radiat. Oncol. Biol. Phys. 5:1725-31 (1979). However, the total dose of radiation that can be administered safely is limited by the risk of normal brain morbidity. This can lead to devastating functional deficits several months to years after irradiation. See, Sheline, et al., Int. J. Radiat. Oncol. Biol. Phys. 6:1215-28 (1980); Leibel, et al., Int. J. Radiat. Oncol. Biol. Phys. 17:1129-39 (1989). Thus, in practice, the radiation dose applied to the brain must be limited. The need to both understand and minimize the side effects of brain irradiation is enhanced by the ever-increasing number of patients with secondary brain metastases that require treatment with large field or whole brain irradiation (WBI). Approximately 200,000-250,000 patients will be diagnosed with brain metastases in 2005, making this the second most common site of metastatic cancer as well as the most common neurological manifestation of cancer. Brain metastases are more common in incidence than newly diagnosed lung, breast, or prostate cancer. Currently, approximately 175,000 cancer patients per year receive large field or WBI. Yet, at the present time, there are no successful treatments for radiation-induced brain injury, nor are there any known effective preventive strategies.
A first aspect of the present invention is directed to method of enhancing viability of islets of Langerhans (β-cells) for purposes of transplantation, comprising contacting the β-cells with a composition comprising a multi-domain conjugate or fusion protein comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, in an amount effective to enhance viability of the β-cells.
In some embodiments, the contacting is achieved by administering the fusion protein to a recipient of a β-cell transplant, such as a type-I or type-II diabetic. In other embodiments, the contacting is achieved by administering the fusion protein to a donor of β-cells. In yet other embodiments, the contacting is achieved by storing the β-cells in a medium containing the fusion protein after they have been harvested from the donor. In yet other embodiments, the contacting is achieved by administering the fusion protein to both the donor and the recipient, optionally in combination with storage of the β-cells in fusion protein-containing medium. In some embodiments, the protein transduction domain contains a TAT protein or is related to a TAT protein, the mitochondrial targeting sequence comprises or is derived from an MNSOD sequence, and/or the DNA repair enzyme comprises or is related to human 8-oxoguanine DNA glycosylase (hOGG1).
A second aspect of the present invention is directed to a composition comprising a culture of β-cells and a composition comprising a multi-domain conjugate comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, in an amount effective to enhance viability of the β-cells. A related aspect is directed to the multi-domain conjugate, per se (e.g., which may be prepared recombinantly in the form of a continuous protein or fusion protein). Thus, this aspect is directed to a chimeric peptide, polypeptide or protein comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, linked together directly or indirectly (e.g., via a linking group) via covalent or peptide bonds.
A third aspect of the present invention is directed to a method of treating inflammatory bowel disease, comprising administering to a patient in need thereof, a composition comprising a multi-domain conjugate or fusion protein comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, in a therapeutically effective amount.
A fourth aspect of the present invention is directed to a method of treating ischemic heart disease, comprising administering to a patient in need thereof, a composition comprising a multi-domain conjugate or fusion protein comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, in a therapeutically effective amount.
A fifth aspect of the present invention is directed to a method of treating acute lung injury or acute respiratory distress syndrome, comprising administering to a patient in need thereof, a composition comprising a multi-domain conjugate or fusion protein comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, in a therapeutically effective amount. In one embodiment, the acute lung injury is bronchopulmonary dysplasia.
A sixth aspect of the present invention is directed to a method of treating radiation induced-brain injury, comprising administering to a patient in need thereof, a composition comprising a multi-domain conjugate or fusion protein comprising a protein transduction domain, a mitochondrial targeting sequence and a DNA repair enzyme, in a therapeutically effective amount.
Without intending to be bound by theory, Applicants hypothesize that an acceptable lesion equilibrium is normally maintained in mitochondrial DNA (mtDNA) in all cells through a balance between the damage to mtDNA caused by the normal endogenous production of reactive oxygen species (ROS) as a byproduct of oxidative phosphorylation and its subsequent repair. However, this equilibrium is altered during the pathogenesis of type I diabetes, the isolation of islets for transplantation, ischemic bowel and heart disease, acute lung injury, and radiation therapy due to increased exposure to elevated levels of ROS and reactive nitrogen species (RNS), resulting in increased damage to mtDNA. This causes alterations in mtDNA transcription to ensue, either through base mispairing which results in defective transcripts, or decreased transcription due to RNA polymerase blocking. Either process will change key electron transport complexes to cause decreased ATP production and alter the reduction of O2 to H2O, so that more one electron reductions of O2 will occur. This will cause enhanced amounts of reactive oxygen species (ROS) to be produced. This increased production of ROS and decreased ATP generation will exacerbate the ongoing stress in the cells and lead to defective normal cellular functions. As this process continues, mitochondrial genomes will become increasingly mutated, further decreasing the pool of viable genomes and intensifying the oxidative stress in the cell. When this process reaches a critical level, the cell will die, either through apoptotic, necrotic, or mixed necroapoptotic processes. The present invention aborts this vicious cycle in its initial stages, and normalizes or at least inhibits further damage to mtDNA, thus enhancing cell viability.
The Fusion Proteins
Mitochondrial Targeting Sequence
As used herein, the term Mitochondrial targeting Sequence (MTS) refers to a signal containing a polypeptide that directs a molecule to a specific organelle namely a mitochondrion.
The eukaryotic cell comprises a number of discrete membrane bound compartments, or organelles. The structure and function of each organelle is largely determined by its unique complement of constituent polypeptides. However, the vast majority of these polypeptides begin their synthesis in the cytoplasm. Thus organelle biogenesis and upkeep require that newly synthesized proteins can be accurately targeted to their appropriate compartment. This is often accomplished by amino-terminal signaling sequences, as well as post-translational modifications and secondary structure.
Mitochondrial localization/targeting sequences generally consist of a leader sequence containing positively charged amino acids. This allows the protein to be targeted to the highly negatively charged mitochondria. Unlike receptor:ligand approaches that rely upon stochastic Brownian motion for the ligand to approach the receptor, the mitochondrial localization signal is drawn to mitochondria because of charge. Thus, in some embodiments, the mitochondrial localization sequence contains at least two, and in some embodiments about 5-15, or about 11 charged groups.
In order to enter the mitochondria, a protein generally must interact with the mitochondrial import machinery, consisting of the Tim and Tom complexes (Translocase of the Inner/Outer Mitochondrial Membrane). With regard to the mitochondrial localization sequence, the positive charge draws the fusion protein to the complexes and continues to draw the fusion protein into the mitochondria. The Tim and Tom complexes allow the proteins to cross the membranes.
Representative examples of mitochondrial localization sequences that may be useful in the present invention include human manganese superoxide dismutase (hMnSOD, aa 1-24) MLSRAVCGTSRQLAPALGYLGSRQ (SEQ ID NO:1) (from Genbank CAA42066); mitochondrial isoform of human malate dehydrogenase (MDH2, aa 1-28) MLSALVRPVSAALRRSFSTSAQNNAKVA (SEQ ID NO.:2) (from Genbank CAG38785); human cytochrome c oxidase subunit VIII (COX, aa 1-29) MPLLRGRCPARRHYRRLALLGLQPAPRFA (SEQ ID NO.:3) (from Genbank Q7Z4L0); human ornithine transcarbamylase (OTC, aa 1-32) MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQ (SEQ ID NO.:4) (from Genbank AAI07154); human mitochondrial transcription factor A (TFAM, aa 1-42) MAFLRSMWGVLSALGRSGAELCTGCGSRLRSPFSFVYLPRWF (SEQ ID NO.:5) (from Genbank NP—003192) human DNA polymerase γ (PolG, aa 1-33) MSRLLWRKVAGATVGPGPVPAPGRWVSSSVPAS (SEQ ID NO.:6) (from Genbank NP 002684); and human uracil DNA glycosylase (UNG1, aa 1-35) MGVFCLGPWGLGRKLRTPGKGPLQLLSRLCGDHLQ (SEQ ID NO.:7) (from Genbank CAA61579). Yet other examples that may be useful in the present invention are set forth in Table 1 in U.S. Patent Application Publication 20070196334.
DNA Repair Protein or Enzyme
Cells have evolved the capacity to remove or tolerate lesions in their DNA. The most direct mechanisms for repairing DNA are those that simply reverse damage and restore DNA to its normal structure in a single step. A more complex mechanism, excision repair, involves incision of the DNA at the lesion site, removal of the damaged or inappropriate base(s), and resynthesis of DNA using the undamaged complementary strand as a template. This system of repair can further be categorized into base and nucleotide excision repair. These enzymes can be used in the practice of the present invention.
Base excision repair involves two major classes of repair enzymes, namely, N-glycosylases and AP endonucleases DNA N-glycosylases are enzymes that hydrolyze the N-glycosidic bond between the damaged base and the deoxyribose moiety, leaving behind an AP site on the DNA backbone. AP sites produced by the action of N-glycosylases are acted upon by AP endonucleases, which can make an incision either 3′ to the AP site (class I AP lyase) or 5′ to the AP site (class II AP endonuclease). All those enzymes shown to contain class I AP lyase activity possess an associated DNA glycosylase activity; however, not all glycosylases are AP lyases. Class II AP endonucleases are the major enzymes responsible for the repair of AP sites in DNA.
Several DNA glycosylases have been identified. They are classified into two major families: 1) enzymes that possess only DNA glycosylase activity; and 2) enzymes that contain both a DNA glycosylase activity and an associated class I AP lyase activity; that is, enzymes that catalyze a β-elimination cleavage of the phosphodiester bond 3′ to an AP site.
The major cellular enzymes initiating the repair process for AP sites, the so-called “class II” AP endonucleases, have been identified and characterized in bacteria, yeast and mammalian systems, including human cells. These repair proteins hydrolyze the phosphodiester backbone immediately 5′ to an AP site generating a normal 3′-hydroxyl nucleotide which can prime DNA repair synthesis. Moreover, these enzymes have also been shown to contain repair activity for 3′-terminal oxidative lesions. By hydrolyzing 3′-blocking fragments from oxidized DNA, these enzymes can produce normal 3′-hydroxyl nucleotide termini, permitting DNA repair synthesis.
In E. coli, the major AP endonuclease enzymes are exonuclease III and endonuclease IV. Exonuclease III comprises approximately 90% of the cellular AP endonuclease activity and greater than 95% of the total activity for removal of blocked 3′ ends. Endonuclease IV accounts for much of the residual activity.
Exonuclease III is the major class II AP endonuclease in E. coli and incises on the 5′ side of an AP site, leaving a 3′ hydroxyl and a 5′ phosphate. This 3′ hydroxyl group is a substrate for DNA polymerases. In addition to its AP endonuclease activity, exonuclease III demonstrates phosphodiesterase, exonuclease, phosphatase and RNAse H activities.
Endonuclease IV, encoded by the nfo gene, is the other main class II AP endonuclease of E. coli. Like exonuclease III, endonuclease IV exhibits many activities such as phosphatase, phosphodiesterase, as well as endonuclease activity against DNA containing urea residues.
S. cerevisiae contains a single major AP endonuclease/3′-repair diesterase encoded by the APN1 gene. Apn1 protein has been reported to have many biochemical properties in common with endonuclease IV.
AP endonucleases have been purified to apparent homogeneity from a variety of mammalian sources including mouse, drosophila, calf thymus, human placenta, and HeLa cells (Seki, et al., J. Biol. Chem. 266:20797-20802 (1991); Haukanes, et al., Nucleic Acids Res. 17(4):1493-1509 (1989); Ivanov, et al., Eur. J. Biochem. 172(1):155-9 (1988); Henner, et al., Nucleic Acids Res. 5(14):5529-44 (1987); Cesar, et al., J. Biochem. 129(3):509-517 (1983); Shaper, et al., J. Biol. Chem. 257(22):13455-8 (1982); and Kane, et al., J. Biol. Chem. 256(7):3405-14 (1981)). The activities have similar molecular weights around 37 kDa and require magnesium. Each of these enzymes appears to be a class II AP endonuclease.
Sequences of specific DNA repair enzymes proteins that may be useful in the present invention include those from the base excision repair (BER) pathway, e.g., AP endonucleases such as human APE (NAPE, Genbank Accession No. M80261) and related bacterial or yeast proteins such as APN-1 (e.g., Genbank Accession No. U33625 and M33667), exonuclease III (ExoIII, xth gene, Genbank Accession No. M22592) bacterial endonuclease III (EndoIII, nth gene, Genbank Accession No. J02857), huEndoIII (Genbank Accession No. U79718), and endonuclease IV (EndoIV nfo gene Genbank Accession No. M22591). Additional BER proteins suitable for use in the invention include, for example, DNA glycosylases/AP lyases such as, formamidopyrimidine-DNA glycosylase (FPG, Genbank Accession No. X06036), endonuclease VIII like 1 (NEIL1 Homo sapiens Genbank Accession No. AAH108761 endonuclease VIII-Like 2 (NEIL2 Homo sapiens Genbank Accession No. AAH13964), Glycosylases such as human 3-alkyladenine DNA glycosylase (HAAG, also known as human methylpurine-DNA glycosylase (hMPG, Genbank Accession No. M74905), NTG-1 (Genbank Accession No. P31378 or 171860), SCR-1 (YAL015C), SCR-2 (Genbank Accession No. YOL043C), DNA ligase (Genbank Accession No. M36067), β-polymerase (Genbank Accession No. M13140 (human)) and 8-oxoguanine DNA glycosylase (OGG1 Genbank Accession No. U44855 (yeast); Y13479 (mouse); Y11731 (human)). Proteins for use in the invention from the direct reversal pathway include human MGMT (Genbank Accession No. M29971) and other similar proteins.
Protein Transduction Domain
A Protein Transduction Domain or PTD refers to a polypeptide compounds that facilitates traversing a lipid bilayer, micelle, or cell membrane. The PTD allows the fusion protein, or at least the DNA repair enzyme and the mitochondrial targeting sequence to traverse the islet cell membranes, for example and move from extracellular space to intracellular space, or cytosol. Suitable PTDs for use in the present invention include the TAT PTD YGRKKRRQRRR (SEQ ID NO:8), the basic domain Tat(49-57) which is RKKRRQRRR (SEQ ID NO.:9); 11 arginine residues, and positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues. Several modifications to TAT, including substitutions of glutamine to alanine, have been reported to demonstrate an increase in cellular uptake anywhere from 90% to up to 33 fold in mammalian cells. Other suitable PTDs include penetratin, which is the PTD of the Drosophila homeotic transcription factor Antennapedia. Penetratin is an active domain of this protein and consists of the 16-amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO.:10).
The fusion proteins of the present invention may be prepared in accordance with standard techniques. In some embodiments, for example, the fusion proteins are expressed in E. coli and purified from bacterial lysate using metal affinity chromatography. The general procedure for the expression and purification of the proteins is as follows. Competent E. coli cells are transformed with plasmid DNA containing the protein of interest using an electroporation procedure. Transformed bacteria are grown in liquid culture media under optimized conditions to produce a sufficient bacterial mass. Bacterial pellets are harvested by centrifugation and bacterial cells are subsequently lysed by a sonication procedure. Clarified bacterial lysates contain the protein of interest in soluble form. Ni-NTA agarose is subsequently added to the lysates to specifically bind the recombinant histidine tag—containing the protein of interest. After washing off and the unbound material, the bound protein is eluted from Ni-NTA agarose with buffer containing a high concentration of imidazole. When necessary, gel-filtration chromatography is then used to remove the imidazole and/or exchange the salt composition in the eluted protein buffer.
DNAs encoding mitochondrial localization sequences, DNA repair proteins or enzymes and protein transduction domains are known in the art. To prepare a chimeric DNA molecule containing fragments encoding each of these domains, for purposes of expression in a host to produce a fusion protein wherein each domain is linked via a peptide bond, the respective DNA fragments may be individually assembled (e.g., using recursive PCR) and then ligated together in accordance with standard techniques. The chimeric DNA may further contain sequences e.g., to facilitate detection of other recombinant proteins (e.g., a HA tag) through the use of specific antibodies to the tag, or to facilitate purification of the expression product (e.g., a polyhistidine tag containing about 10 His residues (e.g., HHHHHHHHHH) (SEQ ID NO.:11)). The chimeric DNA molecules are preferably constructed so that the DNA repair protein is sandwiched between the mitochondrial targeting sequence and the protein transduction domain in order to protect the mitochondrial targeting sequence from cleavage. Exemplary chimeric DNA molecules encoding fusion proteins of the present invention, and the corresponding amino acid sequences are set forth below. Exemplary chimeric DNA molecules encoding fusion proteins of the present invention, and the corresponding amino acid sequences are set forth below.
The chimeric DNAs are typically introduced into hosts (cells or organisms) via a vector, which is generally regarded as a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into at least one cell where it can be replicated. The chimeric DNAs may be situated in the vector so as to be in operable association with one or more regulatory or other genetic elements, including promoters and enhancers that effectively directs the expression of the DNA in the host chosen for expression, initiation signals and internal ribosome binding sites, multiple cloning sites, splicing sites, termination signals, polyadenylation signals, origins of replication, and selectable markers.
A variety of vectors may be useful in connection with the present invention, depending upon the host. In the case of bacterial hosts, plasmids are appropriate. Vectors containing the chimeric DNAs of the present invention may be delivered to hosts using known techniques, including direct delivery of DNA such as by injection, electroporation, calcium phosphate precipitation, DEAE-dextran followed by polyethylene glycol, direct sonic loading, liposome mediated transfection, receptor-mediated transfection, microprojectile bombardment, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation of protoplasts and desiccation/inhibition-mediated DNA uptake. The targets may be stably or transiently transformed.
Representative hosts include prokaryotes and eucaryotes alike, including bacteria, yeast, animal cells and plant cells.
Following introduction of the chimeric DNAs into the host, the host is cultured under conditions suitable for expression of the chimeric DNA. The expression production is then extracted or harvested from the host, followed by purification. All such procedures, which vary from host to host, are known in the art.
Constructing TAT-PTD fusion proteins and producing them in relatively large amounts can be advantageously practiced in bacterial expression vectors. See, Schwarse, et al., Science 285:1569-72 (1998). In this system, in-frame polyhistidine-TAT fusion proteins are purified from a bacterial lysate under denaturing conditions through a series of affinity, ion exchange, and desalt columns.
In other embodiments, the fusion proteins are constructed non-recombinantly, in which case the polypeptide domains (e.g., the MLS, DNA repair enzyme/protein and the PTD) are linked together via covalent bonds, e.g., disulfide bonds. See, Wagstaff and Jans, Curr. Med. Chem. 13, 1371, 2006.
The fusion proteins of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
Methods of Using the Fusion Proteins
The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or concentration of the fusion proteins of the present invention utilized for period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome.
Enhancing Viability of Islets of Langerhans
For example, in the context of enhancing viability of islets of Langerhans, the term “effective amount” includes amounts that achieve the enhancement of the viability of islets. Effective amounts of the fusion proteins for use in the present invention include, for example, amounts that are effective for enhancing survival and/or improving function of islet cells in vivo and/or in vitro. Within the context of transplantation of individual islet cells or masses of islet cells, e.g., transplant donors and/or recipients, an effective amount of the fusion protein is that amount administered to the transplant donor and/or recipient sufficient to enhance survival of the cell or mass of cells, e.g. to reduce loss of the cell, or mass of cells, and/or to improve functional performance of a transplanted cell or a mass of cells. Within the context of treating cells outside a body, e.g., islet cells to be cultured and/or used for transplantation, an effective amount of the fusion protein is that amount with which the cells are incubated or stored in order to enhance preservation of the cells and/or to reduce cell loss, e.g., loss via apoptosis, and/or to enhance function. As used herein, the term “inhibiting” includes delaying the onset of, reducing, preventing, or alleviating a biological process, e.g., apoptosis.
The term “patient” is used throughout the specification to describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary applications are clearly anticipated by the present invention. The term “donor” or “donor patient” as used herein refers to an animal (human or non-human) from whom islet cells can be obtained for the purposes of storage and/or transplantation to a recipient patient. The term “recipient” or “recipient patient” refers to an animal (human or non-human) into which islet cells can be transferred.
The term “diabetes” is a general term to describe diabetic disorders as they are recognized in the art, e.g., Diabetes Mellitus. Diabetes Mellitus is characterized by an inability to regulate blood glucose levels. The two most prevalent types of diabetes are known as Type I and Type II diabetes. In Type I, or insulin-dependent diabetes (IDDM), the pancreas makes little or no insulin because the insulin-producing β-cells have been destroyed. In Type II, or noninsulin-dependent diabetes (NIDDM), the pancreas makes some insulin but the insulin is not effective. The term also encompasses the myriad secondary disorders caused by diabetes, both acute and chronic, e.g., diabetic complications, e.g., hypoglycemia and hyperglycemia, retinopathy, angiopathy, neuropathy, nephropathy, insulin-resistance, syndrome X, and the involvement of advanced glycation end products (AGE) in neuropathy and atherosclerosis.
The term “islet cell(s)” is used throughout the specification as a general term to describe the clumps of cells within the pancreas known as islets, e.g., islets of Langerhans. Islets of Langerhans contain several cell types that include, e.g., β-cells (which make insulin), α-cells (which produce glucagons), γ-cells (which make somatostatin), F cells (which produce pancreatic polypeptide), enterochromaffin cells (which produce serotonin), PP cells and D1 cells.
By “isolated cell” is meant that the cell is removed from the tissue or organ in which it (or its predecessor) naturally occurs. A cell can be just partially purified from its natural milieu and be deemed “isolated.” For example, an intact islet of Langerhans is considered to be made up of “isolated” cells, once the islet is removed from a pancreas and can be physically separated from other islets.
The term “transplantation” is used throughout the specification as a general term to describe the process of implanting mass of islet cells, or individual islet cells into a patient. The term “transplantation” is defined in the art as the transfer of living tissues or cells from a donor to a recipient, with the intention of maintaining the functional integrity of the transplanted tissue or cells in the recipient (see, e.g., The Merck Manual, Berkow, Fletcher, and Beers, Eds., Merck Research Laboratories, Rahway, N.J., 1992). The term “cell transplantation” is used throughout the specification as a general term to describe the process of transferring at least one islet cell to a patient. For example, such transplantation can be performed by removing the β-cells (or intact islets) from a donor's pancreas and putting them into a recipient patient whose pancreas cannot produce sufficient insulin. The terms include all categories of transplants known in the art, except blood transfusions. Transplants are categorized by site and genetic relationship between donor and recipient. The term includes, e.g., autotransplantation (removal and transfer of cells or tissue from one location on a patient to the same or another location on the same patient), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantations between members of different species).
The terms “transplant rejection” or “rejection” are art-recognized, and are used throughout the specification as general terms to describe the process of rejection of cells in a recipient. Included within the definition are, for example, three main patterns of rejection that are usually identified in clinical practice: hyperacute rejection, acute rejection, and chronic rejection (see, e.g., Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994).
The present invention contemplates the use of fusion protein compositions to treat donors, recipients, masses of islet cells, and/or individual islet cells at any step of the harvesting, storage and transplant process. A mass of cells or individual islet cells may be harvested from a donor, treated with a fusion protein composition ex vivo in accordance with the present invention, and transplanted into a recipient. Alternatively or in addition, islet cells can be treated in situ, while still in the donor. Optionally, a fusion protein composition can be administered to the recipient prior to, during, and/or after the surgery with the recipient's blood. The fusion protein may also be administered to the donor prior to or during the process of harvesting the islet cells.
Islet cells can be harvested from a donor and transplanted by any methods known to those of skill in the art (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press (1994)). The skilled practitioner will recognize that methods for harvesting and transplantation may vary depending upon many circumstances, such as the type of donor.
It is further contemplated by the present invention that the methods described herein can be used with islet cells ex vivo, such as a bioartificial pancreas (see, e.g., Sambanis et al., Cytotechnology 15:351-363 (1994)). The cells (or masses of cells) can be treated with the fusion protein either prior to putting them in the device, or while they are utilized in the device, or both. Alternatively or in addition, the donor animal can be administered fusion protein prior to removal of the islet cells for use in the device.
Alternatively or in addition, a cell can be cultured as described below and transplanted into a recipient.
In the context of the other diseases and conditions disclosed herein, inflammatory bowel disease includes for example, patients with Crohn's disease and ulcerative colitis. Other, less common forms of IBD include indeterminate colitis, infectious colitis (viral, bacterial or protozoan, e.g., amoebic colitis), pseudomembranous colitis (necrotizing colitis), and ischemic inflammatory bowel disease.
Crohn's disease is characterized by intestinal inflammation and the development of intestinal stenosis and fistulas. Neuropathy often accompanies these symptoms. Ulcerative colitis is a disease that causes inflammation and sores, called ulcers, in the lining of the large intestine. The inflammation usually occurs in the rectum and lower part of the colon, but it may affect the entire colon. Ulcerative colitis rarely affects the small intestine except for the end section, called the terminal ileum. Ulcerative colitis may also be called colitis or proctitis. The inflammation makes the colon empty frequently, causing diarrhea. Ulcers form in places where the inflammation has killed the cells lining the colon; the ulcers bleed and produce pus. Ulcerative colitis may occur in people of any age, but most often it starts between ages 15 and 30, or less frequently between ages 50 and 70. Children and adolescents sometimes develop the disease. Ulcerative colitis affects men and women equally and appears to run in some families. The most common symptoms of ulcerative colitis are abdominal pain and bloody diarrhea. Patients also may experience fatigue, weight loss, loss of appetite, rectal bleeding, and loss of body fluids and nutrients. About half of patients have mild symptoms. Others suffer frequent fever, bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitis is also known to cause problems such as arthritis, inflammation of the eye, liver disease (hepatitis, cirrhosis, and primary sclerosing cholangitis), osteoporosis, skin rashes, and anemia. A thorough physical exam and a series of tests may be required to diagnose ulcerative colitis. Blood tests may be done to check for anemia, which could indicate bleeding in the colon or rectum. Blood tests may also uncover a high white blood cell count, which is a sign of inflammation somewhere in the body. By testing a stool sample, the doctor can detect bleeding or infection in the colon or rectum.
Without intending to be bound by any particular theory of operation, Applicants hypothesize that the fusion proteins of the present invention suppresses IBD symptoms by ameliorating or even preventing persistent inflammatory injury (mediated by fragmentation of mitochondrial DNA) to the mucosal barrier.
Ischemic heart disease (IHD) includes coronary arteriosclerosis, angina pectoris, and acute and old myocardial infarction and refers to the serious disorder of the vital heart that mostly affects males and females in late middle age or older. Coronary arteriosclerosis is characterized by arteriosclerosis in the coronary artery that supplies nutrients to the heart. Angina pectoris is characterized by attacks of chest pain caused by impaired blood flow in the coronary artery. Myocardial infarction is characterized by myocardial necrosis caused by impaired blood flow in the coronary artery and by fatal complications coming there with such as arrhythmia, cardiac failure, cardiac rupture, and pump failure. Impaired blood flow to the heart, a vital organ, is an essential characteristic of these ischemic heart diseases. IHD patients who may be particularly suitable for treatment in accordance with the methods of the present invention include diabetics (e.g., type II diabetics) and obese pre-diabetics. Ischemic heart disease can be diagnosed using well-known techniques. See, e.g., U.S. Patent Application Publication 20080146498 A1. According to U.S. Patent Application Publication 20080081997 A1, conclusive diagnosis of ischemic heart disease is given by coronary angiography.
Without intending to be bound by any particular theory of operation, Applicants hypothesize that the fusion proteins of the present invention may exert a beneficial effect on coronary collateral growth, activation (e.g., phosphorylation) of key kinases in diabetics, maintain mitochondrial integrity, maintain mitochondrial energy production, and combinations thereof.
As used herein, “acute lung injury” refers to a critical illness syndrome consisting of acute hypoxemic respiratory failure with bilateral pulmonary infiltrates that are not attributed to left atrial hypertension. See, e.g., Rubenfeld, et al., N. Engl. J. Med. 353:1685-93 (2005). Acute lung injury also refers to a syndrome of life-threatening progressive pulmonary insufficiency or hypoxemic respiratory failure in the absence of known pulmonary disease (such as emphysema, bronchitis, or chronic obstructive pulmonary disease), usually following a systemic insult such as surgery or major trauma. In embodiments of the methods of the present invention, the acute lung injury is induced by diseases or disorders other than pulmonary diseases. In some embodiments of the methods of the present invention, the acute lung injury is pulmonary injury caused/induced by an extrapulmonary origin such as neurogenic pulmonary injury, secondary to severe CNS (central nervous system) trauma. In some embodiments of the methods of the present invention, the acute lung injury is acute lung injury induced by extrapulmonary diseases. In some embodiments of the methods of the present invention, the acute lung injury is indirect pulmonary injury from trauma, sepsis, and other disorders such as acute pancreatitis, drug overdose. In some embodiments of the methods of the present invention, the acute lung injury is acute lung injury induced by inhalation of noxious fumes, burn, or massive blood transfusion. In some embodiments of the methods of the present invention, the acute lung injury is acute lung injury induced by peritonitis during sepsis, acute lung injury induced by intravenous bacteremia during sepsis, acute lung injury caused by smoke inhalation, acute lung injury occurring in a premature infant with deficiency of surfactant, acute lung injury caused by oxygen toxicity or acute lung injury caused by barotrauma from mechanical ventilation. Thus, symptoms of acute lung injury may include lung hemorrhage, hyaline membrane formation, lung lesion, lung edema, lung inflammation, increased transvascular fluid flux, prevalent interstitial edema and alveolar collapse, and combinations thereof.
BPD affects 20-60% of all premature, very low birth weight infants. BPD is associated with substantial morbidity and mortality as well as extremely high health care costs. Although the widespread use of intra-tracheally administered exogenous surfactant and antenatal steroid therapy has reduced the overall severity of BPD, the prevalence of this condition has actually increased, due in part to improved survival of very low birth weight infants. More specifically, BPD is associated with alveolar simplification, capillary malformations, and interstitial cellularity. See, e.g., Coalson, Semin. Neonatol. 8:73-81 (2003). This impaired alveolar formation leads to a long-term reduction in alveolar number and a decrease in the gas-exchange surface area. While a variety of risk factors predispose infants to BPD, it has been reported that supplemental oxygen remains one of the prime sufficient conditions for its development. See, e.g., Chess, et al., Semin. Perinatol. 30:171-78 (2006). Thus, BPD is a multi-factorial disease process that is the end result of an immature, surfactant-deficient lung that has been exposed to hyperoxia, mechanical ventilation and infection.
To treat (and even prevent) BPD, the fusion proteins of the present invention may be administered during the first few days of life of a premature infant, thus providing improved lung function. The fusion proteins may be administered with or without, and before or after surfactant therapy. Thus, treatment with the fusion proteins may be initiated during the first day of an infant patient's life, e.g., within about 30 minutes of intubation and receipt of surfactant. Premature infants are typically intubated for the purposes of administering oxygen and inflating their lungs, which would collapse without intubation. Thus, premature or preterm infant patients are also suitable for treatment in accordance with the inventive methods. Intubation can then serve as a direct route for the intratracheal administration (also known as endotracheal administration) of medicines, such as surfactants, to the lungs. Thus the fusion proteins may be administered intra-tracheally as well as parenterally (e.g., injection into the muscle tissues or intravenous injection). Since problems related to BPD can persist, the present invention also contemplates administration not only to infants, but to adolescents and even adults if necessary.
ARDS is also known in the medical literature as stiff lung, shock lung, pump lung and congestive atelectasis, and its incidence has been estimated to be 1 out of 100,000 people. ARDS is accumulation within the lung which, in turn, causes the lung to stiffen. The condition is triggered by a variety of processes that injure the lungs. In general, ARDS occurs as a medical emergency. It may be caused by a variety of conditions that directly or indirectly cause the blood vessels to “leak” fluid into the lungs. In ARDS, the ability of the lungs to expand is severely decreased and damage to the alveoli and lining (endothelium) of the lung is extensive. The concentration of oxygen in the blood remains very low in spite of high concentrations of supplemental oxygen which are generally administered to a patient. Among the systemic causes of lung injury are trauma, head injury, shock, sepsis, multiple blood transfusions and medications. Pulmonary causes include pulmonary embolism, severe pneumonia, smoke inhalation, radiation, high altitude, near drowning, and more.
ARDS symptoms usually develop within 24 to 48 hours of the occurrence of an injury or illness. It is believed that cigarette smoking may be a risk factor. Among the most common symptoms of ARDS are laboured, rapid breathing, nasal flaring, cyanosis blue skin, lips and nails caused by lack of oxygen to the tissues, breathing difficulty, anxiety, stress and tension. Additional symptoms that may be associated with this disease are joint stiffness and pain and temporarily absent breathing. The diagnosis of ARDS is commonly done by testing for symptomatic signs. A simple chest auscultation or examination with a stethoscope, for example, will reveal abnormal breath sounds which are symptomatic of the condition. Confirmatory tests used in the diagnosis of ARDS include chest X-rays and the measurement of arterial blood gas. In some cases ARDS appears to be associated with other, diseases, such as patients with acute myelogenous leukemia, who developed acute tumour lysis syndrome (ATLS) after treatment with cytosine arabinoside. In general, however, ARDS appears to be associated with traumatic injury, severe blood infections such as sepsis, or other systemic illness, the administration of high dose radiation therapy and chemotherapy, and inflammatory responses which lead to multiple organ failure, and in many cases death.
Based on the time of clinical expression, radiation-induced brain injury has been described in terms of acute, early delayed and late delayed reactions Tofilon, et al., Radiat. Res. 153:357-70 (2000). Acute injury, expressed days to weeks after irradiation, is fairly rare under current radiotherapy regimens. Early delayed injury occurs from 1-6 months post-irradiation and can involve transient demyelination with somnolence. While both of these early injuries can result in severe reactions, they are normally reversible and resolve spontaneously. In marked contrast, late delayed effects, characterized by demyelination, vascular abnormalities and ultimate white matter necrosis (Schultheiss, et al., Br. J. Radiol. 65:737-53 (1992)), are observed for more than 6 months post-irradiation and are viewed as irreversible and progressive. In addition to these histopathologic endpoints, there is a growing awareness of intellectual deterioration in patients receiving brain irradiation. Crossen, et al., J. Clin. Oncol. 12:627-42 (1994). Thus, patients falling into any of these categories may be treated in accordance with the methods of the present invention. The fusion proteins can be administered before irradiation, or after irradiation.
Cognitive dysfunction, including dementia, induced by large field or WBI is reported to occur in 20-50% of brain tumor patients who are long-term survivors (greater than 15 months post-irradiation). Crossen, et al., supra; Imperato, et al., Ann. Neurol. 28:818-22 (1990); and Johannesen, et al., Radiother. Oncol. 69:169-76 (2003). Thus, these patients may be treated in accordance with the methods of the present invention.
For purposes of practicing any of the methods of the present invention, the fusion protein compositions can be combined in admixture with a pharmaceutically acceptable carrier or vehicle, optionally with other excipients (e.g., wetting agents, dispersing agents, surfactants, stabilizers, preservatives, etc.). The term “pharmaceutically acceptable carrier” refers to a carrier or adjuvant that may be administered to a patient, together with a fusion protein of this invention, and which does not destroy the pharmacological activity of the fusion protein and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the fusion protein. Therapeutic compositions are prepared by mixing the fusion protein with the carrier. They are typically formulated as reconstitutable lyophilized formulations or aqueous or oily solutions, dispersions, suspensions, or emulsions. Accordingly, acceptable carriers and other excipients include water, saline, fixed oils such as mono- and diglycerides, buffers such as phosphate, citrate and other organic acids (e.g., HEPES based buffer containing KCl, BSA and glycerol, pH 7.5); antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG ion exchangers, alumina, aluminum stearate, lecithin, and self-emulsifying therapeutic agent delivery systems (SEDDS) such as D-α-tocopherol polyethyleneglycol 1000 succinate.
The compositions of the present invention can be administered to patients in need thereof (e.g., an in the case of islet preservation, both donors and recipients), in accordance with well-known techniques and sound medical practice. One such mode is parenteral administration. As used herein, “parenteral administration” is characterized by administering a pharmaceutical composition through a physical breach of a subject's tissue. Parenteral administration includes administering by injection, through a surgical incision, or through a tissue-penetrating non-surgical wound, and the like. The compositions described herein are typically administered by injection, e.g., intravenously, intra-arterially, subdermally, intraperitoneally, intramuscularly, subcutaneously or intraductually. Intravenous administration is advantageous in terms of rapid delivery of the fusion protein throughout the body. In the case of islet preservation, intraductal administration to the common bile duct affords direct delivery of the fusion protein to the pancreas of donors or into the liver of recipients which is a site of transplantation. Intraperitoneal administration is relatively easy, but requires relatively more fusion protein. Other than modes described herein, further modes of administration e.g., intranasal and via suppository, might also be useful.
Thus, parenteral formulations containing the fusion protein may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. The formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers.
In terms of administration to human for any of the indications disclosed herein, dosage amounts of the fusion proteins generally ranges from about 1 to about 100 mg/kg of patient body weight. Some variation in the actual dosage amount will necessarily occur depending on physical and physiological factors such as the body weight and overall condition of the subject being treated including the type and severity of the disease or condition, as well as the route of administration and the molecular weight of a given fusion protein. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Experiments conducted with rats (and which are described in the working examples below) showed positive results with dosages of an embodiment of the present invention, as it pertains to the preservation of islets, ranging from 1.5 to 2.5 μg/g of body weight. Animal experiments provide reliable guidance for the determination of effective doses for human therapy, so interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The use of interspecies scaling in toxicokinetics,” in Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96. Lower or higher doses than those recited above may be required.
The timing and duration of treatment of the diseases and conditions disclosed herein are also variable, taking into account various factors including the half-life of the fusion proteins, which is approximately 72 hours. For example, in embodiments of the present invention pertaining to islet preservation, the fusion protein is administered to the donor prior to harvesting the islets, and to the recipient at the time of transplantation and at about 48-hour intervals thereafter as needed. Success of treatment (and the need for continuation of treatment) can be determined by assessing the number of islet equivalents required to maintain euglycemia in the recipient. Enhancement of the viability of the islets can also be evaluated via glucose tolerance tests and radioimmunoassay for insulin and/or C-peptide.
Cell Culture
In some embodiments of the present invention, the islets can be treated with the fusion proteins in vitro. The method can include the steps of providing a vessel containing the fusion protein, providing the islet cell in vitro and then culturing or simply maintaining in the presence of the fusion protein. In vitro treatment is advantageously performed immediately following harvesting of the islets from the donor and for the duration of storage prior to transplantation.
The method can be performed in any vessel suitable for culturing or holding islet cells, such as roller bottles, cell culture flasks, petri dishes, and test tubes.
The skilled practitioner will appreciate that culture conditions, e.g., temperature, can be selected and/or varied (see, for example, Cells: A Laboratory Manual, Spector and Leinwand, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997). For example, the murine insulinoma cell line βTC3 (DSMZ, Braunschweig, Germany) can be incubated in humidified 5% CO2/95% air at 37° C.
The islet cell may be disposed, e.g., suspended or bathed in, a liquid medium. The medium can be any medium known to those of skill in the art to be suitable for culturing, preserving, or washing the islet cells of interest (see, for example, Cells: A Laboratory Manual, Spector and Leinwand, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997). Such types of media include, but are not limited to, various buffers, Eagle's minimal essential medium (MEM), Dulbecco/Vogt modified Eagle's minimal essential medium (DMEM), or Roswell Park Memorial Institute (RPMI) Medium. Such media may also comprise appropriate supplements, e.g., fetal bovine serum (FBS), individual amino acids, antibiotics, and/or vitamins. For example, the medium can be RPMI medium 1640 (Life Technologies, Grand Island, N.Y.) supplemented with 2 mM L-glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin and 10% Fetal Calf Serum (FCS) (Life Technologies). The concentration of the fusion protein in the medium will generally range from about 1 μg/ml to about 100 μg/ml, and in some embodiments, about 25 μg/ml to 100 μg/ml.
In the context of transplantation, the present invention further contemplates that other procedures known in the art for enhancing graft survival/function can be used along with the methods described herein. Such procedures include, but are not limited to immunosuppressive therapies and donor specific transfusions (DSTs). For example, a DST can be administered to a recipient prior to, during and/or after the administration of CO, HO-1, other heme-associated products, and/or NO to a recipient. Such administration, e.g., administration of DST(s) along with a treatment described herein, can be carried out prior to, during, and/or after transplantation.
Embodiments of the present invention are now described in connection with the following non-limiting examples.
To show that the TAT protein can effectively target proteins to mitochondria in beta cells, the β cell line INS-1E was exposed to the fusion protein MTS-TAT-GFP. The experimental protocol was as follows: INS-1E cells were grown in RPMI 1640 medium containing 10 mmol/l HEPES, 10% fetal bovine serum, 50 μmol/l 2-mercaptoethanol, 50 μg/ml gentimicine sulfate, and 1 μmol/l pyruvate. The solution of MTS-GFP-TAT in the elution buffer was added directly to the medium in the culture dish to a final concentration of 80 μg/ml. After 12 h of incubation with the fusion protein, cells were trypsinized and replated onto glass cover slips. After attachment, cells were fixed in 4% formaldehyde, washed with PBS and mounted on a glass slide using a SlowFade Antifade Kit. To visualize mitochondria, live cells were incubated prior to fixation with 1 μM of Mito-Tracker Red CM-H2 XRos for 15 mins. Cells were observed by florescent microscopy. As shown in
We next replaced GFP in the construct with OGG1. Enzyme activity assays of the protein off the column showed that all four elution fractions had enzyme activity (
Finally, to explore whether the fusion proteins would work in vivo, we obtained IACUC approval to do a pilot study using mice. Two mice were injected i.v. with 100 μg of MTS-GFP-TAT diluted in glycerol buffer (phosphate buffered saline containing 0.25% glycerol). Another mouse received glycerol buffer only. One of the MTS-GFP-TAT mice was euthanized after 2 hr and its pancreas removed and frozen in liquid nitrogen for frozen sections. At 12 hr post injection, the other mouse receiving MTS-GFP-TAT and the control mouse were euthanized and their pancreata removed and frozen in liquid nitrogen for frozen sections. Pictures were made using identical exposure and contrast settings. See
To determine whether TAT proteins could be delivered to islets, a pilot study was conducted in which Lewis rats were injected intraperitoneally (i.p.) with recombinant protein containing the TAT sequence, the mitochondrial localization sequence from MnSOD and GFP. At 2 or 24 hours, the rats were euthanized, pancreata removed and islets isolated. It is clear from this study (
Studies were then performed using normal β-cells in monolayer culture. After establishment of cultures and removal of fibroblasts using published procedures [Nelson, et al., Diabetes 42:1187-94 (1993); LeDoux, et al., Diabetes 35:866-72 (1986); Wilson, et al., Diabetologia 27:587-91 (1984); Wilson, et al., Diabetes 46:1291-95 (1997); Wilson, et al., In Vitro 19:25-30 (1983); Wilson, Toxicol. Appl. Pharmacol. 68:375-9 (1983); Wilson, et al., Nature 285:112-3 (1980); Wilson, et al., Diabetologia 24:38-41 (1983); and Wilson, et al., Pancreatic islet cell cultures: Immunoperoxidase staining and autoradiography. Tissue Culture Association Manual, 1980. 5:p. 1193-97], cultures were given normal culture medium containing 300 mg/dl glucose. After 48 hrs the medium was removed and saved for radioimmunoassay of insulin (IRI). Two groups of cultures (4 cultures in each group) were replenished with culture medium containing MTS-OGG1-TAT (80 μg/ml) and two groups of controls were given only elution buffer (EB). The next day cultures were treated with 1 mM alloxan in HBSS or HBSS alone for 20 mins. The cultures were replenished with medium containing 300 mg/dl glucose. After 48 hrs the medium was removed, saved for IRI, and the cultures replenished with the same high glucose-containing medium. This process was repeated two more times. The saved culture medium was assayed for insulin and the results expressed as a percentage of the insulin released by the culture before the experimental treatment, which allowed correction for the differences in cell number in different cultures which occur when β-cells are cultured in monolayer. The results of these (
In summary, these studies showed that apoptosis can be prevented and cellular function enhanced by targeting specific repair proteins to mitochondria, and that these proteins can be delivered rapidly to mitochondria through protein transduction.
A fusion protein was constructed to contain the mitochondrial transport sequence from MnSOD, the bacterial glycosylase/AP lyase Endonuclease III and the TAT sequence from HIV. See paragraphs herein. In the first set of experiments, the fusion protein was given to rats i.p. in two different doses and islets isolated the next day. Control animals received buffer only. Both concentrations significantly enhanced the recovery of islets with the higher concentration enhancing recovery by almost 250% (
To determine the effect of the fusion protein on islet viability a double labeling technique was employed using ethidium bromide and acridine orange. Non-viable cells stain red while viable cells stain green. Representative islets are shown (
To evaluate whether the fusion protein from EXAMPLE 3 is able to restore functional viability to islets, ADP/ATP ratios were evaluated in brain-dead rats. ATP is generated from ADP in mitochondria and serves as the energy molecule in the cell. Normal ADP/ATP ratios are an absolute requirement for insulin secretion. Three rats obtained from Charles River Mouse Farms were either given 1.5 μg/g body weight of fusion protein (described in EXAMPLE 3) or glycerol-based dilution buffer and then made brain-dead. Two hours later, islets were isolated from the pancreata of these animals and evaluated for ATP and ADP levels. A second control group was comprised of animals which were not made brain-dead. The results which are illustrated in
This example demonstrates Use of a fusion protein construct ((described in EXAMPLE 3) to deliver DNA repair enzyme directly to mitochondria, and which protects against acute lung injury and pulmonary edema.
First, rat pulmonary microvascular endothelial cells were cultured to confluency on gold plated microelectrodes, and Transendothelial Electrical Resistance across cell monolayers was measured using an electrical cell-substrate sensor (ECIS) system (Applied Biophysics, Troy, N.Y.) as an index of pulmonary endothelial permeability. Current was applied across the electrodes by a 4 kHz AC voltage source with amplitude of 1 mV in series with a 1 WM resistance to approximate a constant current source. The small gold electrode and larger counter-electrode (1 cm2) were connected to a phase-sensitive lock-in amplifier (model 5301A, EG&G Instruments, Princeton, N.J.) with a built-in differential preamplifier (model 5316A EG&G Instruments). The phase-in and out-of-phase voltages between the electrodes were monitored in real time with the lock-in amplifier and converted to scalar measurements of transendothelial impedance of which resistances were the primary focus. In this system, addition of the hydrogen peroxide-generating enzyme, glucose oxidase, in the presence of glucose led to profound decreases in transendothelial electrical resistance. Importantly, and as shown in
In companion studies, intact, buffer-perfused rat lungs were challenged with intravascular glucose oxidase-glucose solutions to engender hydrogen peroxide-induced endothelial injury, while increases in weight lung weight were monitored as a function of time as an index of pulmonary edema formation. This isolated lung preparation extends observations from cultured cells because it more closely mimics the behavior of the in vivo lung. As shown in
Gestational day 15 rat fetuses were removed from timed-pregnant Sprague-dawley dams. Fetal hearts and lungs were removed enbloc, individual lung lobes were dissected from large airways and placed in culture on a transwell (Costar, USA). Lungs were cultured in a humidified chamber at the air-liquid interface for up to 72 hours at fetal oxygen tension (3% O2 or 27 mmHg) or hyperoxia (21% O2 or approx 140 mmHg). To test the capacity of the mtDNA repair fusion protein to repair fetal lung mtDNA and restore lung branching, the fusion protein construct (described in EXAMPLE 3) was added to lung cultures (10, 25, 50 and 100 μg/ml). Lung branching was assessed grossly by counting terminal lung bud branch points (15 lobes/experiment N=5). Tissues were also harvested for biochemical analysis. The fusion protein restored lung branching in hyperoxic fetal lungs. As measured by quantitative Southern analysis, hyperoxia damages mitochondrial DNA in the fetal lung, the fusion protein designed to facilitate repair of mitochondrial oxidative damage was found to restore mtDNA integrity as well as fetal lung branching in the hyperoxia exposed tissues.
All publications cited in the specification, including patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
The present application is a 35 U.S.C. §§371 national phase conversion of International Application No. PCT/US2009/000889, filed Feb. 9, 2009, which claims priority of U.S. Provisional Patent Application No. 61/065,044 filed Feb. 8, 2008, the disclosures of which are hereby incorporated herein by reference. The PCT International Application was published in the English language.
| Filing Document | Filing Date | Country | Kind | 371c Date |
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| PCT/US2009/000889 | 2/9/2009 | WO | 00 | 12/10/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/099679 | 8/13/2009 | WO | A |
| Number | Name | Date | Kind |
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| 20050255093 | Shone et al. | Nov 2005 | A1 |
| Number | Date | Country |
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| 2005042560 | May 2005 | WO |
| 2009097568 | Aug 2009 | WO |
| 2009098682 | Aug 2009 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20110110908 A1 | May 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61065044 | Feb 2008 | US |
