Patent Application
20060110359
Brain natriuretic peptide (BNP) is a peptide of 26 residues, with a remarkable homology to atrial natriuretic peptide (ANP). The peptide exerts natriuretic-diuretic activity as well as a potent smooth muscle relaxant activity (Sudoh et al., Biochem Biophys Res Commun, 1988, 155:726-732). ANP administered into the rat lateral ventricle, or administered intravenously, has been reported to inhibit brain water and sodium accumulation associated with ischemic infarcts and in sub-arachnoid hemorrage-induced brain edema (Nakao et al., Neurosurgery, 1990, 27:39-43; discussion 43-34; Naruse et al., Acta Neurochir Suppl (Wien), 1990, 51:118-121; Doczi et al., Acta Neurochir (Wien), 1995, 132:87-91). ANP given after a 4-hour delay significantly reduced brain water and sodium 24 hours after an experimental brain hemorrhage in rats. Neither mannitol nor 8-bromo-cGMP affected brain edema. The anti-edematous effect of ANP has been attributed to inhibition of sodium transport and the coupled water influx.
Administration of BMSC intravenously or intracerebrally has been shown to result in significant improvement in the rate of recovery from the neurological deficits produced in a rat model of stroke (Li et al., Journal of Cerebral Blood Flow & Metabolism, 2000, 20:1311-1319; Chen et al., Stroke, 2001, 32:1005-1011; Li et al., Neurology, 2002, 59:514-523). Similar treatment with BMSC has also enhanced recovery from traumatic brain injury (Mahmood et al., Neurosurgery, 2001, 49:1196-1204; Mahmood et al., Journal of Neurosurgery, 2001, 94:589-595; Mahmood et al., J Neurotrauma, 2002, 19:1609-1617). In all these reports, the structural repair of the brain lesion does not correlate with the recovery, leading to the hypothesis that secretion of growth factors and cytokines mediate, in part, the enhanced recovery process. Indeed, secretion of growth factors such as brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) have been demonstrated in vitro and in vivo after transplantation of BMSC (Chen et al., J Neurosci Res, 2002, 69:687-691; Chen et al., Neuropathology, 2002a, 22:275-279; Li et al., Neurology, 2002, 59:514-523). Moreover, an immune-mediated response elicited by grafted bone marrow cells may play a role in recovery (Li et al., Neurology, 2002, 59:514-523).
The present inventors have discovered a new way to deliver anti-edema agents utilizing a cellular vehicle that generates natural anti-edemic agents, the natriuretic peptides. In light of the beneficial effects of intravenous administration or intracerebral grafting of BMSC in animal models of stroke and brain trauma, it is strongly suggested that the enhanced recovery from neurologic deficits is mediated in part by BNP secreted by BMSC in situ.
The natriuretic peptides have as one of their physiological functions the ability to increase excretion of sodium and water by the kidney and to pump sodium and water from individual cells. ANP are produced in great amounts in the heart, especially during heart failure. BNP is found in the hypothalamus of the brain, but is also found at much higher concentrations in heart tissue. These peptides have been shown to be effective when delivered directly into brain tissue (through an indwelling probe), but until the present inventors' discovery, these peptides could not be delivered systemically because of rapid proteolysis in the system circulation.
The present invention pertains to a method for treating deficits caused by focal or generalized edema associated with injury to organs or organ systems, such as the central nervous system, heart, liver, and kidneys. According to the method of the present invention, edema associated deficits are treated by the administration of cells that produce natriuretic peptides, such as bone marrow stromal cells. Where bone marrow stromal cells are utilized, the cells are preferably conditioned with retinoic acid and nerve growth factor before, during, or after administration to the patient, in order to increase the cells' production of natriuretic peptide. The method of the present invention can be used to treat neurological deficits of the central nervous system that result from stroke, trauma, toxins, and other nervous system insults.
In another aspect, the present invention concerns pharmaceutical compositions comprising bone marrow stromal cells and effective amounts of retinoic acid and nerve growth factor to induce the bone marrow stromal cells to increase production of natriuretic peptides.
In another aspect, the present invention provides a method for producing natriuretic peptides comprising culturing bone marrow stromal cells and isolating the natriuretic peptides from the bone marrow stromal cells. Preferably, the bone marrow stromal cells are cultured in the presence of retinoic acid and nerve growth factor, thereby inducing the bone marrow stromal cells to increase production of the natriuretic peptides.
In another aspect, the present invention pertains to cells that have been genetically modified to produce a natriuretic peptide.
SEQ ID NO:1 is the gene encoding human brain natriuretic peptide (BNP) (NCBI ACCESSION # M31776; Seilhamer, J. J. et al., Biochem. Biophys. Res. Commun., 165(2):650-658, 1989):
SEQ ID NO:2 is the amino acid sequence of human BNP:
SEQ ID NO:3 is the gene encoding human atrial natriuretic peptide (ANP) (NCBI ACCESSION # NM—006172; Zivin, R. A. et al., Proc. Natl. Acad. Sci. USA, 81(20):6325-6329, 1984):
SEQ ID NO:4 is the amino acid sequence of human ANP:
SEQ ID NO:5 is the gene encoding dog (Canine) brain natriuretic peptide (BNP) (NCBI ACCESSION # M31777; Seilhamer, J. J. et al., Biochem. Biophys. Res. Commun. 165(2):650-658, 1989):
SEQ ID NO:6 is the amino acid sequence of dog (Canine) BNP:
SEQ ID NO:7 is the hBNP-exon M2 probe.
SEQ ID NO:8 is a forward primer for human BNP.
SEQ ID NO:9 is a reverse primer for human BNP.
SEQ ID NO:10 is the nucleotide sequence encoding human BNP (NCBI ACCESSION # M31776):
SEQ ID NO:11 is the nucleotide sequence encoding human ANP (NCBI ACCESSION # NM—006172):
SEQ ID NO:12 is the mouse type-B natriuretic peptide gene (NCBI ACCESSION # S58667; Steinhelper, M. E., Circ. Res. 72(5): 984-992, 1993; coding sequences (CDS)=join nucleotides 211-336, 531-753, 1188-1204):
SEQ ID NO:13 is the mouse BNP gene for natriuretic peptide (NCBI ACCESSION # D16497; Ogawa, Y. et al., J. Clin. Invest., 93(5):1911-1921, 1994; coding sequences (CDS): join nucleotides 210-335, 530-752, 1196-1212):
SEQ ID NO:14 is the rat BNP mRNA (NCBI ACCESSION # M25297; Kojima M. et al., Biochem. Biophys. Res. Commun., 159(3):1420-1426, 1989; coding sequence (CDS): join nucleotides 58-423):
SEQ ID NO:15 is the porcine BNP mRNA (NCBI ACCESSION # M25547; Porter et al., J. Biol. Chem., 264(12):6689-6692, 1989; coding sequence (CDS): join nucleotides 94-216, 463-718, 1273-1289):
SEQ ID NO:16 is the bovine ANP gene (NCBI ACCESSION # M13145; Vlasuk, G. P. et al., Biochem. Biophys. Res. Commun., 136(1):396-403, 1986; coding sequence (CDS): join nucleotides 313-432, 531-857, 1383-1394):
SEQ ID NO:17 is the rat iso-ANP gene (NCBI ACCESSION # M60731; Roy, R. N. and Flynn, T. G., Biochem. Biophys. Res. Commun., 171(1):416-423, 1990; coding sequence (CDS): join nucleotides 58-183, 405-627, 1080-1096):
SEQ ID NO:18 is the amino acid sequence of bovine natriuretic peptide:
1 mgssaitvsf llflafqlpg qtganpvygs vsnadlmdfk nlldrledkm pledeavpsq
61 vlseqneeag aplsplsemp pwmgevnpaq reggvlgrgp wessdrsall ksklrallta
121 prslrrsscf ggrmdrigaq sglgcnsfry rr.
SEQ ID NO:19 is the amino acid sequence of mouse BNP:
1 mdllkylsqm ilfflflyls plgghsyplg spsqspeqfk mqkllelire kseemaqrql
61 lkdqgltkeh pkrvlrsqgs tlrvqqrpqn skvthisscf ghkidrigsv srlgcnalkl
121 l
SEQ ID NO: 20 is the amino acid sequence of rat BNP:
1 mdlqkylpqm illllflnls plgghshplg spsqspeqst mqkllelire kseemaqrql
61 skdqgptkel lkrvlrsqds afriqerlrn skmahssscf gqkidrigav srlgcdglrl
121 f.
SEQ ID NO:21 is the amino acid sequence of porcine BNP:
1 mgprmalprv llllflhlll lgcrshplgg aglaselpgi qelldrlrdr vselqaertd
61 leplrqdrgl teaweareaa ptgvlgprss ifqvlrgirs pktmrdsgcf grrldrigsl
121 sglgcnylrr y.
The present invention is based at least partly on the discovery that brain natriuretic peptides (BNP) and atrial natriuretic peptides (ANP) are produced in high levels by human bone marrow stromal cells (BMSCs) in vitro.
The present invention pertains to a method for treating deficits caused by focal or generalized edema associated with injury to organs or organ systems, such as the central nervous system, heart, liver and kidney. According to the method of the present invention, edema associated deficits are treated by the administration of cells that produce natriuretic peptides, such as BMSCs. Where BMSCs are utilized, the cells are preferably conditioned with retinoic acid and nerve growth factor before, during, or after administration to the patient, in order to increase the cells' production of natriuretic peptide. However, conditioning of BMSCs with retinoic acid and nerve growth factor is not necessary since BMSCs produce BNP in vivo.
The natriuretic peptides delivered by the transplanted cells increase excretion of sodium and water within the patient, mimicking the effects of direct infusion of the peptides. For example, the method of the present invention can be used to treat neurological deficits of the central nervous system that result from stroke, trauma, toxins, and other nervous system insults. Administration of natriuretic peptide producing cells into the brain results in decreased brain edema at the penumbra zone of injury, facilitating a more rapid recovery. Thus, infusion of natriuretic peptide producing cells, such as BMSC, systemically or intracerebrally is a novel way to treat any acute injury to brain, spinal cord or peripheral organs that results in edema.
ANPs do not cross the blood-brain-barrier, but BMSCs infused systemically do penetrate preferentially at the zone of the infarct or injury. BMSCs have also been shown to localize at the ischemic heart following systemic intravenous administration.
In another aspect, the present invention concerns pharmaceutical compositions comprising bone marrow stromal cells and effective amounts of retinoic acid and nerve growth factor to induce the bone marrow stromal cells to increase production of natriuretic peptides. The pharmaceutical composition can be adapted for various forms of parenteral administration, such as intravenous or intracerebral routes. Administration can be continuous or at distinct intervals as can be determined by a person skilled in the art.
The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19th ed.) describes formulations which can be used in connection with the subject invention. Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question.
In another aspect, the present invention provides a method for producing natriuretic peptides comprising culturing bone marrow stromal cells and isolating the natriuretic peptides from the bone marrow stromal cells. Peptide isolation methods known in the art can be utilized. For example, antibodies specific for natriuretic peptide can be used to isolate the peptides from culture. Preferably, the bone marrow stromal cells are cultured in the presence of retinoic acid and nerve growth factor, thereby inducing the bone marrow stromal cells to increase production of the natriuretic peptides by the bone marrow stromal cells. According to one embodiment of the present invention, bone marrow stromal cells are conditioned for about two to about four days in vitro with retinoic acid (0.5 μM) and NGF (100 ng/μL). After the cultures reach confluency, the cells can be lifted by incubation with trypsin (0.25%) and 1 mM EDTA at 37° C. for 3-4 min. The BMSC cells are suspended in phosphate buffered saline (PBS) and infused into the systemic circulation (total of about 1-3 million cells).
A wide variety of media, salts, media supplements, and products for media formulation can be utilized to culture the BMSCs in the presence of retinoic acid and nerve growth factor, thereby conditioning the cells to increase production of natriuretic peptides according to the method of the present invention. Examples of these substances include, but are not limited to, carrier and transport proteins (e.g., albumin), biological detergents (e.g., to protect cells from shear forces and mechanical injury), biological buffers, growth factors, hormones, hydrosylates, lipids (e.g., cholesterol), lipid carriers, essential and non-essential amino acids, vitamins, sera (e.g., bovine, equine, human, chicken, goat, porcine, rabbit, sheep), serum replacements, antibiotics, antimycotics, and attachment factors. These substances can be present in various classic and/or commercially available media, which can also be utilized with the subject invention. Examples of such media include, but are not limited to, Ames' Medium, Basal Medium Eagle (BME), Click's Medium, Dulbecco's Modified Eagle's Medium (DMEM), DMEM/Nutrient Mixture F12 Ham, Fischer's Medium, Minimum Essential Medium Eagle (MEM), Nutrient Mixtures (Ham's), Waymouth Medium, and William's Medium E.
According to methods of the present invention, natriuretic peptide producing cells can be administered as cell therapy to alleviate the symptoms of a wide variety of disease states and pathological conditions associated with edema, in various stages of pathological development. Treatment with natriuretic peptide producing cells is intended to include prophylactic intervention to prevent onset of the symptoms associated with the particular edema-associated deficit, as well as intervention to alleviate or eliminate symptoms of an edema-associated deficit that are being presented by the patient at the time of administration of the cells. For example, natriuretic peptide producing cells can be used to treat acute disorders (e.g., stroke or myocardial infarction), and administered acutely, subacutely, or in the chronic state. Similarly, the natriuretic peptide producing cells can be used to treat chronic disorders (e.g., Parkinson's disease), and administered preventatively and/or prophylactically, early in the disease state, in moderate disease states, or in severe disease states.
The following pathological conditions are examples of diseases or conditions that cause edemic injury which may be treated in accordance with the present invention: cerebrovascular accidents (also referred to as stroke), brain trauma, and spinal cord injuries. The following are examples of deficits associated with or induced by edemic injury: paralysis, loss of sensation, inability to speak or understand language, loss of consciousness and coma.
Preferably, treatment will substantially correct an edema-induced deficit. However, that may not always be possible. Thus, treatment according to the present invention, and with the cells and pharmaceutical compositions of the invention, may lead to improvement in function without complete correction.
The number of cells to be used will vary depending on the nature and extent of the edemic tissue. Typically, the number of cells used in transplantation will be in the range of about one hundred thousand to several million. One or more transplants can be carried out to further improve function.
Methods for transplantation of cells into human and non-human mammals are known to those skilled in the art and described in the scientific literature. The terms “transplantation”, “administration”, and “implantation” are used herein interchangeably and include the transplantation of cells which have been grown in vitro, and may have been genetically modified to produce natriuretic peptides, as well as the transplantation of material extracted from another organism. Natriuretic peptide producing cells can be transplanted by means of microsyringe infusion of a known quantity of cells in the target area. The phrase “intracerebral transplantation” as used herein includes transplantation into any portion of the brain, and is not restricted to the front and/or larger portion of the brain.
The natriuretic peptide producing cells can be administered as autografts, syngeneic grafts, allografts, and xenografts, for example. As used herein, the term “graft” refers to one or more cells intended for implantation within a human or other animal. Hence, the graft can be a cellular or tissue graft, for example.
The natriuretic peptide producing cells can be administered in an open manner to the target anatomical site, as in the heart during open heart surgery, or in the brain during stereotactic surgery, or by systemic procedures such as intravascular interventional methods using catheters going to the blood supply of the specific organs, or by other interventional methods known in the art.
Mammalian species which benefit from the disclosed methods of treatment include, and are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions, elephant seals, otters, porpoises, dolphins, and whales. As used herein, the term “patient” is intended to include such human and non-human mammalian species. The cells used in the subject invention can be any human or non-human mammalian cells.
Combinations of cell types can be co-administered to enhance therapeutic potential. For example, a trophic factor-producing cell line can be co-administered with a neuronal cell line that has been genetically modified to produce a natriuretic peptide, such as BNP or ANP. Sertoli cells can be co-administered with natriuretic peptide producing cells of a species different from that of the recipient, such that the Sertoli cells provide local immunosuppression of the xenograft.
Methods for transplanting various nerve tissues as allografts and xenografts have been described previously (Freeman T. B. et al., Progress in Brain Research, 1988, Chapter 61, Elsevier Science Publishers, 78:473-477; Freeman T. B. et al, Parkinson's Disease: Advances in Neurology, 2001, Chapter 46, Lippincott Williams & Wilkins, 86:435-445; Freeman T. B. et al., Annals of Neurology, 1995, 38(3):379-387; Freeman T. B. et al., Progress in Brain Research, 2000, Chapter 18, Elsevier, Amsterdam, 127:405-411; Olanow C. W. et al. The Basal Ganglia and New Surgical Approaches for Parkinson's Disease, Advances in Neurology, 1997, 74:249-269; Bjorklund et al., Neural Grafting in the Mammalian CNS, 1985, p. 709, Elsevier, Amsterdam; Das G. D., “Neural Grafting in the Mammalian CNS, 1985, Chapter 3, p. 23-30, Elsevier, Amsterdam). These procedures include intraparenchymal transplantation, i.e., within the host brain tissue (as compared to outside the brain or extraparenchymal transplantation) achieved by injection or deposition of tissue within the host brain so as to be opposed to the brain parenchyma at the time of transplantation.
Methods for intraparenchymal transplantation include, for example: (i) injecting the donor cells within the host brain parenchyma (e.g., stereotactically, using image guidance, and/or with a catheter attached to a pump, such as a MEDTRONIC system); and (ii) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the graft into the cavity. Such methods provide parenchymal apposition between the graft and host brain tissue at the time of grafting, and both facilitate anatomical integration between the graft and host brain tissue.
Alternatively, the graft can be placed into the cerebral spinal fluid (CSF), either by open surgical injection, intraventricularly via a needle or ventricular reservoir, into the lumbar subarachnoid space using a lumbar puncture, or into any CSF site using a pump and a catheter (e.g., MEDTRONIC). These methods would lend themselves to repeated administration over time, to the CSF or to the brain. Grafting to the ventricle may be accomplished by injection of the donor cells or by growing the cells in a substrate such as 3% collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft. For subdural grafting, the cells may be injected around the surface of the brain after making a slit in the dura. Injections into selected regions of the host brain can be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted. The microsyringe can be mounted in a stereotactic frame and three-dimensional stereotactic coordinates are selected for placing the needle into the desired location of the brain or spinal cord. Image guidance methods can also be utilized. The cells of the subject invention can also be introduced into the putamen, caudate nucleus, pallidum, nucleus basalis, hippocampus, cortex, cerebellum, subcortical white matter, other regions of the brain, as well as the spinal cord.
The methods of the subject invention also contemplate the administration of cells that have been genetically modified to produce a natriuretic peptide, such as BNP or ANP. Such genetically modified cells can be administered alone or in combinations with different types of cells. Thus, genetically modified cells of the invention can be co-administered with other cells, which can include genetically modified cells or non-genetically modified cells. Genetically modified cells may serve to support the survival and function of the co-administered cells, for example.
SEQ ID NOs. 1 and 3 are genes encoding human BNP and human ANP, respectively. SEQ ID NOs. 10 and 11 are the coding sequences of human BNP and human ANP, respectively. In specific embodiments, the cells of the invention are genetically modified with nucleotide sequences encoding human BNP and/or human ANP (SEQ ID NOs. 2 and 4, respectively), such as the nucleotide sequences of SEQ ID NO: 1, 3, 10, or 11. In other embodiments, nucleotide sequences encoding non-human mammalian homologs of natriuretic peptides are used, such as nucleotide sequences encoding SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21. Exemplified nucleotide sequences encoding non-human mammalian homologs of natriuretic peptides are SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
The term “genetic modification” as used herein refers to the stable or transient alteration of the genotype of a cell of the subject invention by intentional introduction of exogenous nucleic acids by any means known in the art (including for example, direct transmission of a polynucleotide sequence from a cell or virus particle, transmission of infective virus particles, and transmission by any known polynucleotide-bearing substance) resulting in a permanent or temporary alteration of genotype. The nucleic acids may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful polynucleotides in addition to those encoding natriuretic peptides. A translation initiation codon can be inserted as necessary, making methionine the first amino acid in the sequence. The term “genetic modification” is not intended to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like. The genetic modification may confer the ability to produce natriuretic peptides wherein the cell did not previously have the capability, or the modification may increase the amount of natriuretic peptides produced by the cell, e.g., through increased expression.
Exogenous nucleic acids can be introduced into a cell by viral vectors (retrovirus, modified herpes virus, herpes virus, adenovirus, adeno-associated virus, and the like) or direct DNA transfection (lipofection, calcium phosphate transfection, DEAE-dextran, electroporation, and the like), for example.
Preferably, the exogenous nucleotide sequence encoding a natriuretic peptide is operably linked to a promoter sequence that permits expression of the nucleotide sequence in a desired tissue within the patient. The promoters can be inducible or tissue specific as necessary. Means for inducible regulation of human BNP are known (Quan H. et al., Am. J. Physiol. Heart Circ. Physiol., 2001, 280:H368-H376). Examples of other promoters that can be utilized to express nucleotides encoding natriuretic peptides within a mammalian cell, such as a human cell, include cytomegalovirus promoter (Boshart et al., Cell, 1985, 41:521-530) and SV40 promotor (Subramani et al., Mol. Cell. Biol., 1981, 1:854-864).
The term “operably-linked” is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably-linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably-linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered “operably-linked” to the coding sequence. Each nucleotide sequence coding for a natriuretic peptide will typically have its own operably-linked promoter sequence.
The nucleotide sequences encoding natriuretic peptides used in the subject invention include “homologous” or “modified” nucleotide sequences. Modified nucleic acid sequences will be understood to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to persons skilled in the art, and exhibiting modifications in relation to the normal sequences. For example, mutations in the regulatory and/or promoter sequences for the expression of a polypeptide that result in a modification of the level of expression of a polypeptide according to the invention provide for a “modified nucleotide sequence”. Likewise, substitutions, deletions, or additions of nucleic acid to the polynucleotides of the invention provide for “homologous” or “modified” nucleotide sequences. In various embodiments, “homologous” or “modified” nucleic acid sequences have substantially the same biological or serological activity as the native (naturally occurring) natriuretic peptide. A “homologous” or “modified” nucleotide sequence will also be understood to mean a splice variant of the polynucleotides of the instant invention or any nucleotide sequence encoding a “modified polypeptide” as defined below.
A homologous nucleotide sequence, for the purposes of the present invention, encompasses a nucleotide sequence having a percentage identity with the bases of the nucleotide sequences of between at least (or at least about) 20.00% to 99.99% (inclusive). The aforementioned range of percent identity is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 20.00% and 99.99%. These percentages are purely statistical and differences between two nucleic acid sequences can be distributed randomly and over the entire sequence length.
In various embodiments, homologous sequences exhibiting a percentage identity with the bases of the nucleotide sequences of the present invention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the polynucleotide sequences of the instant invention. Homologous nucleotide and amino acid sequences include, but are not limited to, mammalian homologues of the human natriuretic peptide sequences. Homologous nucleotide sequences, when expressed, exhibit substantially the same biological activity (e.g., anti-edema effects) as the native sequence, as can be determined in vitro or in vivo.
Both protein and nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman Proc. Natl. Acad. Sci. USA, 1988, 85(8):2444-2448; Altschul et al. J. Mol. Biol., 1990, 215(3):403-410; Thompson et al. Nucleic Acids Res., 1994, 22(2):4673-4680; Higgins et al. Methods Enzymol., 1996, 266:383-402; Altschul et al. J. Mol. Biol., 1990, 215(3):403-410; Altschul et al. Nature Genetics, 1993, 3:266-272).
In another embodiment, the natriuretic peptide producing cells are derived from transgenic animals, and thus are in a sense already genetically modified. There are several methods presently used for generating transgenic animals. A typical technique is direct microinjection of DNA into single-celled fertilized eggs. Other methods include retro-viral-mediated transfer, or gene transfer in embryonic stem cells. These techniques and others are detailed by Hogan et al. in Manipulating the Mouse Embryo, A Laboratory Manual (Cold Spring Harbor Laboratory Ed., 1986).
In addition to the objective of delivering natriuretic peptides to a patient in need thereof, the genetically modified cells of the subject invention can be administered to a patient for cell/gene therapy, e.g., for in vivo delivery of various biomolecules, such as the trophic factors described above. Alternatively, the genetically modified cells can be used as biological “factories” to provide the product of the exogenous DNA (e.g., natriuretic peptide) and/or the natural product of the modified cells in vitro, or in vivo within an animal.
Genetically modified cells can be stem cells or non-stem cells. Thus, non-stem cells (e.g., specialized or mature cells, and their precursor or progenitor cells) can be genetically modified to produce natriuretic peptides. Genetic modification with nucleotide sequences encoding natriuretic peptides, such as BNP and ANP, can be used to produce cells that have therapeutic potential for injuries related to multiple or specific organs. For example, cardiomyocytes or their progenitors can be genetically modified to target heart injury; hepatocytes or their progenitors can be genetically modified to target liver injury; and islet cells or their progenitors can be genetically modified to target diabetes. Any of the over 200 cells naturally occurring in mammals can be genetically modified to provide cellular delivery of one or more natriuretic peptides. Examples of cells that can be genetically modified include, but are not limited to, bone marrow cells, stem cells, neural cells, Sertoli cells, fat cells, placental cells, fibroblasts, and umbilical cord blood cells.
According to the methods of the present invention, whether genetically modified or non-genetically modified, the natriuretic peptide producing cells can be co-administered with other therapeutic agents useful in treating defects, trauma, or diseases, such as growth factors, antibiotics, or neurotransmitters.
Cells can be genetically modified to include regulators, inducible promoters, tissue-specific promoters, on-off genes, or suicide genes. Genetically modified cell lines can include more than one genetic construct.
Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, Calif. (1990). In-situ (In-cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al., Blood, 1996, 87:3822.)
Human Bone Marrow Stromal Cell Cultures. Human bone marrow cells are not easy to obtain for basic research since they are a scarce resource for treatment of certain leukemias. We used an easily accessible alternative source of bone marrow stromal cells (BMSC). The method has been described in detail (Song, S. and Sanchez-Ramos, J., “Preparation of Neural Progenitors from Bone Marrow and Umbilical Cord Blood” in Protocols for Neural Stem Cell Methods, Zigova, T. et al., Eds., 2002, pp. 79-88). Bone marrow from volunteer donors were routinely passed through a nylon filter to remove debris from the sample prior to preparation for bone marrow replacement. The nylon filter retains bony chips with adherent bone marrow cells, fatty tissue and other debris. The nylon filters were washed with sterile saline solution five times 4 of 19 and centrifuged to remove bone chips. The cells in the supernatant were then processed as described and plated in uncoated polyethylene culture flasks in Dulbecco's minimal essential medium (DMEM; Gibco/BRL, Inc) and fetal bovine serum 10% (FBS; Hyclone). When adherent cells reached 60-70% confluence, they were raised from the plates, resuspended and re-plated in new flasks.
Five human bone marrow donors provided the nylon filters that contain bone marrow debris from which BMSC were subsequently prepared.
1. Male 34 y/o BMSC; assays performed at passage 3 and passage 5
2. Male 50 y/o BMSC; assays performed at passage 1 and passage 3
3. Male 59 y/o BMSC; assays performed at passage 1 and passage 3
4. Female 44 y/o BMSC; assays performed at passage 11 and passage 13
5. Female 34 y/0 BMSC: immunocytochemical assay at passage 4.
The cultures were incubated under three distinct media conditions. Condition A: DMEM+FBS10% for 6 days. Condition B: DMEM+FBS10% for two days followed by Nerve Growth Factor (NGF 100 ng/mL)+all-trans retinoic acid (RA; 0.5 μg/mL) for four days. Condition C: N2+20 ng/mL of Epidermal Growth Factor (EGF; GIBCO)+10 ng/mL of basic Fibroblast Growth Factor (bFGF; GIBCO) for two days followed by NGF+RA for four days.
Immunocytochemistry and estimates of cell density. Cells from a 34 y/o female were plated in 35 mm multi-well plates at passage four with a density of approximately 7.5×105 cells per well. After a total of six days in vitro (under conditions described above), the cultures were fixed in 5% phosphate-buffered paraformaldehyde for 30 minutes, washed three times with phosphate buffered saline and incubated with rabbit antiserum to human BNP-32 (PENINSULA LABORATORIES, Inc.; Belmont, Calif.) at a dilution of 1:200 for 24 hrs at 4° C. FITC conjugated anti-rabbit antibodies (CHEMICON INTERNATIONAL Inc.) were used to visualize the BNP immunoreactive cells. Cultures were also incubated without primary antibody to control for non-specific staining by the anti-rabbit antibody, DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; MOLECULAR PROBES, Eugene Oreg.) staining of nuclei was used to visualize total number of cells per visual field. Estimates of cell density were based on counts of DAPI-stained nuclei in 10 visual fields per culture dish.
DNA Microarray Analysis. Total RNA was isolated from cell cultures using the RNA STAT-60 kit according to the protocol recommended by the manufacturer (TEL-TEST “B,” Inc. Friendswood, Tex. 77546). Following RNA isolation, its OD density was measured at 260 nm, and stored at 280° C. Integrity was tested on 1% nondenaturing Seakem LE agarose gel (FMC BIO-PRODUCTS, Rockland, Me.). As previously described the samples were probed on the human genome U95A array (HGU95A) from AFFYMETRIX, Inc (Sanchez-Ramos et al., 2001). The single array represents; 12,500 sequences. The experiment was done at the Functional Genomics Core, Microarray Facility, at the H. Lee Moffitt Cancer Center and Research Institute using GeneChip Fluidics station 400, a Gene-Chip Hybridization oven, and an HP GeneArray scanner. Analysis of the GeneChip microarray hybridization pattern was performed using GeneChip Analysis Suite 4.0 software.
RT-PCR. Both RT and PCR were performed with PERKIN ELMER's GENEAMP RNA PCR kit. RT was performed using total RNA (1 μg) and random hexamers as a primer. PCR was done using Human BNP (GeneBank accn.# M31776) primers (Forward: nt. 1320-1341 and Reverse: nt. 1597-1578) on a PE 9700 thermocycler (PERKIN ELMER, Foster City, Calif.).
Real Time Quantitative RT-PCR. Real-time quantitative RT-PCR analyses were performed using the ABI PRISM 7700 Sequence Detection System (APPLIED BIOSYSTEMS, Foster City, Calif.). Primers and probes for human BNP were designed and synthesized by APPLIED BIOSYSTEMS (Foster City, Calif.) from GenBank accession number M31776. The probe hBNP-exonM2 (5′-CGCTGCTCCTGTAAC-3′) (SEQ ID NO:7) was labeled with 6-carboxyfluorescein as the reporter and a minor groove binder moiety (MGB) on the 3′-end. The forward primer was 5′-GCCTCGGACTTGGAAACGT-3′ (SEQ ID NO:8) and the reverse primer was 5′-TGCAGCTCCGACAGTTTGC-3′ (SEQ ID NO:9).
Sample RNA (5 μl of 50 ng/μl, 5 μl of 400 ng/μl) was transcribed into cDNA using Superscript II-reverse transcriptase (LIFE TECHNOLOGIES, Inc.) and 250 ng of random hexamers (LIFE TECHNOLOGIES, Inc.) following the manufacturer's directions in a final volume of 20 μl. For the construction of standard curves RNA was diluted in DEPC-treated water (AMBION, Austin, Tex.) before reverse transcription.
PCR was carried out with the TAQMAN Universal PCR Master Mix (APPLIED BIOSYSTEMS) using 3 μl of diluted cDNA, 200 nM probe, and 900 nM in a 30-μl final reaction mixture. After a 2-min incubation at 50° C., AMPLITAQ GOLD was activated by a 10-min incubation at 95° C. Each of the 40 PCR cycles consisted of 15 s of denaturation at 95° C. and hybridization of probe and primers for 1 min at 60° C. Amounts of human BNP and 18s RNAs were calculated using linear regression analysis from an external standard curve.
Radioimmunoassay for Brain Natriuretic Peptide (BNP). The assay is based upon the competition of 125I-BNP and BNP of the sample binding to the limited quantity of specific antibodies for BNP. Cells and media were diluted with 100% ethanol (1:2 dilution), vortexed, and allowed to stand at 4° C. for 30 minutes. The samples were centrifuged and the supernatants were air-dried and stored at −80° C. until time of assay. The samples were incubated with rabbit anti-BNP antibody and normal rabbit serum for 24 hours at 4° C. Then, the samples were incubated with 125I-BNP (13,000 cpm) for 24 hours at 4° C. Goat antirabbit IgG serum were added to the samples and incubated for 2 hours at room temperature. The samples were centrifuged at 3000 g for 20 minutes at 4° C. The radioactivity was counted with a gamma-counter. A standard curve was constructed by measuring the amount of 125I-BNP bound to known concentrations of BNP.
Following is an example which illustrates materials, methods, and procedures for practicing the invention. The example is illustrative and should not be construed as limiting.
BMSC cultures were incubated with a) DMEM and FCS 10% for six days or with b) DMEM and FCS 10% for two days with a change of medium to retinoic acid and NGF for four days or with c) N2+EGF+bFGF for two days followed by NGF+RA for four days. Examination of the cultures under phase contrast microscopy revealed a change in morphology when growth factors were added (See
To validate the presence of BNP mRNA in the cell cultures, RT-PCR and quantitative RT-PCR was performed. In addition, a sensitive radioimmunoassay method was employed to quantify the levels of BNP protein produced within the cells and secreted into the medium.
This is the first report that bone marrow is capable of producing significant levels of BNP. The mean levels of BNP secreted by BMSC with and without addition of growth factors (mean of 75 pmol/L in DMEM and 317 pmol/L in growth factor-treated cultures) far exceeds the BNP found circulating in the serum and CSF of normal human subjects. The mean serum BNP level in 124 healthy subjects was 1.8+/−1.0 pmol/L (SD), with a range of 0.6-5.5 pmol/L (Jensen et al., Scand J Clin Lab Invest, 1997, 57:529-540). In eight patients with congestive heart failure, the median BNP level was 30.5 pmol/L, with a range of 3.9-65.3. The mean BNP concentration in human cerebrospinal fluid collected from fifteen patients without neurological disorders has been reported to be 0.27+/−0.10, pmol/L (mean+/−S.D (Kaneko et al., Brain Res, 1993, 612:104-109).
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
The present application claims the benefit of priority of U.S. Provisional Application Ser. No. 60/319,530, filed Sep. 6, 2002, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/28157 | 9/8/2003 | WO | 10/11/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60319530 | Sep 2002 | US |
