WO2003082187A2 - Purification et clonage de nmn adenylyltranserase et son utilisation therapeutique - Google Patents

Purification et clonage de nmn adenylyltranserase et son utilisation therapeutique Download PDF

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WO2003082187A2
WO2003082187A2 PCT/US2003/007563 US0307563W WO03082187A2 WO 2003082187 A2 WO2003082187 A2 WO 2003082187A2 US 0307563 W US0307563 W US 0307563W WO 03082187 A2 WO03082187 A2 WO 03082187A2
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cells
nmnat
nad
parp
sequence
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WO2003082187A3 (fr
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Hiremagalur N. Jayaram
Joel A. Yalowitz
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Indiana University Bloomington
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to methods and compounds for modifying NAD production for use in the therapeutics for treating for example, diabetes, cancer, myocardial infarction, stroke, neurodegenerative disorders, shock, and aging.
  • Nicotinamide adenine dinucleotide is a pyridine nucleotide involved in the intracellular energy transfer reactions. It is also required for repair of damaged DNA. NAD is synthesized in the nucleus of cells and is highly regulated. The rate- limiting and key enzyme in the synthetic pathway is NMNAT (nicotinamide mononucleotide adenylyltransf erase). NAD synthesis is controlled in cells by producing very low levels of NMNAT.
  • Pancreatic beta-cells that secrete insulin are especially affected by rapid utilization of NAD, as the nucleotide is required not only for the synthesis of ATP by glycolysis, but is also a substrate for DNA damage repair enzyme, PARP [poly(ADP-ribose)polymerase].
  • PARP poly(ADP-ribose)polymerase.
  • Activation of autoreactive T-cells (for example, by enteroviruses), releases cytokines (IL-1 beta) that upregulate nitric oxide synthesis in beta cells.
  • IL-1 beta cytokines
  • Nitric oxide is a reactive oxygen species that breaks DNA, giving rise to excessive synthesis of PARP to repair cellular DNA.
  • PARP requires NAD as a substrate to form poly(ADP-ribose) polymers to aid in the DNA repair process. However, since availability of NAD is inadequate for this process to continue, this results in beta cell death.
  • IMPDH potent IMP dehydrogenase
  • tiazofurin and benzamide riboside are anabolized to their respective 5'-monophosphates and then converted by NMN adenylyltransferase to their corresponding NAD analogues, in which nicotinamide moiety is replaced by thiazole-4-carboxamide (TAD) or benzamide (BAD) (1 -4).
  • TAD thiazole-4-carboxamide
  • BAD benzamide
  • tiazofurin in patients with advanced leukemia was studied (7-8). Thirty-two end-stage adult leukemia patients were accrued into this biochemically directed protocol that had no alternate therapy available, 25 patients could be evaluated under the stringent NCI protocol criteria. In this group of patients, five with acute myeloid leukemia (AML) and 4 with CML-BC (chronic myelogenous leukemia in blast crisis) attained complete remission. Three additional patients with CML-BC demonstrated hematological improvement. In another four patients (two with AML, one with CML-BC, and one with advanced stages of myeloid dysplastic syndrome), a marked antileukemic effect was seen.
  • AML acute myeloid leukemia
  • CML-BC chronic myelogenous leukemia in blast crisis
  • NMN adenylyltransferase plays a key role in the synthesis of pyridine nucleotide coenzymes, because NAD and NADP are required for the cellular energy transfer (electron transport and oxidative phosphorylation) reactions. Furthermore, NMN adenylyltransferase performs an important role not only as an anticancer drug- metabolizing enzyme but also in active cell death processes, however, the exact relationship is still unclear. Therefore in order to understand the molecular basis for the decreased activity of NMN adenylyltransferase in resistant cancer cells, it would be beneficial to purify and clone the enzyme.
  • this enzyme By purifying this enzyme, one can generate appropriate antibodies and find out whether the enzyme protein is present in the cells that acquire resistance to tiazofurin and benzamide riboside. Cloning of this enzyme allows understanding to development of the enzyme's role in pyridine nucleotide homeostasis and in apoptosis.
  • NMN adenylyltransferase which play central roles both in de novo biosynthetic and salvage pathways for nicotinamide nucleotides was studied.
  • This enzyme converts NMN (or NaMN) and ATP to NAD + (or NAD), and inorganic pyrophosphate (2) and its activity has been correlated with crucial cellular events, like mitosis and DNA synthesis (3,4).
  • NAD + or NAD
  • NMNAT NMN adenylyltransferase
  • This adenylyltransferase catalyzes the essential last step in the metabolic conversion of the potent antitumor agent tiazofurin from its pro-drug form to tiazofurin adenine dinucleotide. Low tumor levels of this enzymatic activity associated with the development of drug resistance (8,9). It would therefore be useful to develop methods of modifying NAD production.
  • a composition for modifying NAD production including an NMNAT modifying compound and a pharmaceutically acceptable carrier.
  • a cDNA sequence encoding NMNAT is also provided.
  • a method of treating either diabetes or cancer in a patient by administering to the patient an effective amount of a composition for modifying NAD production is also provided.
  • a gene therapy including a sequence encoding NMNAT included therein and the method for incorporating the sequence effectively into a host genome.
  • Figure 1 shows the sequence of events involved in ⁇ -cell death as a result of NO stress
  • Figure 2 shows the pathways of NAD synthesis and utilization
  • Figure 3 is a graph showing the influence of pH on NMNAT-2 activity
  • Figure 4 is a photograph showing the expression of hNMNAT-1 and hNMNAT-2 on K562 cells
  • Figures 5A through I are photographs showing infected cells wherein 5A shows mock-infect3ed HeLa cells, 5B shows HeLa cells infected with MOI 1 rAAV, 5C shows HeLa cells infected with MOI 10 rAAV, 5D shows mock-infected ⁇ HC9 cells, 5E shows ⁇ HC9 cells infected with MOI 1 rAAV, 5F shows ⁇ HC9 cells infected with MOI 10 rAAV, 5G shows mock-infected MIN6 cells, 5H shows infected MIN6 cells infected with MOI 1 rAAV, and 51 shows infected MIN6 cells infected with MOI 10 rAAV,
  • the present invention provides a composition for modifying NAD production, the composition including a NMNAT modifying compound and a pharmaceutically acceptable carrier.
  • the composition can be used for treating states or conditions.
  • state or condition it is intended to include diseases or states that are affected by NAD production. All states or conditions that are affected by NAD production can be treated using the methods and compounds of the present invention. Examples of such states or condition include, but are not limited to, diabetes, cancer, myocardial infarction, stroke, neurodegenerative disorders, shock, and aging. Other states, conditions, or diseases that are affected by NAD production can also be treated using the methods and compounds of the present invention.
  • NMNAT modifying compound as used herein, the phrase is intended to include, but is not limited to, any compound which is capable of modifying NMNAT production.
  • the compound can be a gene therapy composition that either increases or decreases NMNAT production dependent upon the treatment, such as cDNA, RNA, mRNA, cRNA, and tRNA.
  • the compound can be NMNAT, thus eliminating the need to include a compound that will increase the production of NMNAT.
  • NMNAT When NMNAT is being administered it can be administered as a cDNA sequence, for example the sequence set forth in SEQ ID No: 1 and analogs thereof or the protein encoded by the sequence.
  • Gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired.
  • the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide, or peptide of therapeutic value.
  • the genetic material of interest can encode a suicide gene.
  • ex vivo and (2) in vivo gene therapy Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy.
  • ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro.
  • a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
  • These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ that is within the recipient.
  • the host gene if the host gene is defective, the gene is repaired in situ [Culver, 1998]. These genetically altered cells have been shown to express the transfected genetic material in situ.
  • the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • the expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that may be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence that works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the expression vehicle can also include a selection gene as described herein below.
  • Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Ml (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Ml (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus-derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor that includes most cancers of epithelial origin as well as others.
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
  • Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.
  • Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus.
  • An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.
  • features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
  • recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if breast cancer were to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system were to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles that are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector will depend upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specif icity for infection.
  • An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid.
  • Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal.
  • non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Those of skill in the art know such transfection vehicles.
  • composition of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the composition of the present invention can be administered in various ways. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.
  • the composition can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the composition is also useful.
  • the patient being treated is a warm-blooded animal and, in particular, mammals including man.
  • the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses may be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the composition utilized in the present invention can be administered parenterally to the patient in the form of slow- release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, microspheres and nanospheres.
  • any compatible carrier such as various vehicle, adjuvants, additives, and diluents
  • the composition utilized in the present invention can be administered parenterally to the patient in the form of slow- release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, microspheres and nanospheres.
  • Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • a pharmacological formulation of the composition utilized in the present invention can be administered orally to the patient.
  • Conventional methods such as administering the composition in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques that deliver it orally or intravenously and retain the biological activity are preferred.
  • the composition of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level.
  • An oral dosage form then maintains the patient's composition levels.
  • other forms of administration dependent upon the patient's condition and as indicated above, can be used.
  • the quantity to be administered will vary for the patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably will be from 1 mg/kg to 10 mg/kg per day.
  • composition of the present invention can be used for treating cancer.
  • the composition includes a compound that down- regulates NMNAT thereby decreasing the production of NAD. This treatment induces cell death and therefore assists in eliminating cancerous cells.
  • composition of the present invention When the composition of the present invention is used for the treatment of diabetes or aging, the converse of the cancer treatment occurs.
  • the composition that is administered up-regulates NMNAT thereby increasing the production of NAD.
  • This treatment decreases the occurrence of ⁇ -cell death, thus eliminating one of the primary problems associates with diabetes.
  • NAD niacin or nicotinamide
  • NMN mononucleotide
  • NMNAT NMN adenylyltransferase
  • PARP inhibitors have multiple effects, including scavenging hydrogen peroxide, the contribution of PARP activity to ⁇ -cell death was not evident. Therefore, to understand the role of PARP in streptozotocin-induced ⁇ -cell death, PARP-deficient animal models were developed.
  • PARP-deficient mice were established by disrupting PARP exon 1 and comparing the sensitivity of these mice to streptozotocin-induced diabetes to that of wild type (PARP+/+) mice (19,20).
  • the results in PARP+/+ mice showed that streptozotocin injections elevated blood glucose levels, reaching a peak in 21 days and the levels remained elevated throughout the observation period.
  • glucose concentration remained at normal levels, except for a mild and transient elevation following streptozotocin administration.
  • PARP-1 knockout mice were resistant to diabetes induced by streptozotocin (19-22).
  • a transient activation in PARP activity has been shown to be required for inducing apoptosis in many cell lines (23,24), since the organism can selectively eliminate a particular cell via apoptosis without affecting others.
  • PARP Once PARP is activated, it is followed by cleavage by Caspase-3 (25).
  • Caspase-3 is a member of the caspase family and is one of 13 aspartate-specific cysteine proteases that play a central role in apoptosis and primarily responsible for cleavage of PARP during apoptosis (26,27). This inactivation is thought to prevent the depletion of NAD (substrates of PARP), and ATP, which is required for later steps in apoptosis.
  • NAD Pyridine nucleotide
  • NAD is required as a cofactor for many cellular processes. NAD is essential for oxidative catabolism, intermediary metabolism, and signaling of DNA damage through poly(ADP-ribose) polymer formation (27). NAD and its derivatives NADH, NADP and NADPH have regulatory functions in the formation of triose phosphates and pyruvate from glucose. In hyperglycemia, chronic glucose over-utilization causes an increase in sorbitol pathway activity resulting in a decrease in the NAD/NADH ratio (28,29). Preventing NAD depletion by selective continuous synthesis with NMNAT positively impact insulin-secreting cells (NIT-1 and TC-3 cells).
  • NIT-1 a pancreatic beta-cell line was established from a transgenic NOD/Lt mouse (35). NIT-1 cells secreted 638 ng insulin/million cells following stimulation with 16.5 mM glucose. DNA fragmentation (apoptosis) was shown to be an early event induced by cytokines [interluekin-1 , tumor necrosis factor-alpha (TNF- ⁇ ) or interferon- ⁇ ] in NIT-1 cells (36).
  • NIT-1 cells Minute oxidative stress in NIT-1 cells, such as exposure to 30 ⁇ M hydrogen peroxide for 15 minutes at 37°C, induced apoptosis (37). Since NIT-1 cells behave similar to human ⁇ -cells and are highly differentiated, these cells are valuable tools to study insulin-dependent (Type I) diabetes mellitus (IDDM). Moreover, TNF- ⁇ , a potential mediator of ⁇ -cell destruction in IDDM, activates apoptosis in NIT-1 cells mediated by caspase activation (38). Furthermore, by inhibiting NF-kappaB, NIT-1 cells can be protected from apoptosis induced by TNF- ⁇ (39).
  • IDDM insulin-dependent diabetes mellitus
  • mouse insulin secreting ⁇ -TC-3 cells (40, 41) were utilized to transfect NMNAT gene to examine the impact on NAD metabolism and protection from NO toxicity and oxidative stress. Studies have shown that in TC-3 cells, there was a progressive release of insulin following glucose (0.15 to 16.7 mM) stimulation similar to insulin release from normal islet cells (40, 41). This cell line expresses GLUT1 glucose transporter and has glucokinase and hexokinase levels similar to those of normal islets (42).
  • NMNAT prevents toxicity due to pathways involving NAD diminution. In essence, NMNAT prevents IDDM if given as a mode of gene therapy to high-risk patients.
  • NMN adenylyltransferase Cloning of NMN adenylyltransferase is helpful in examining whether the reduction in enzyme activity in cancer cells resistant to tiazofurin or the benzamide riboside class of drugs, is due to alteration in the enzyme protein (less enzyme protein synthesis, altered enzyme activity or presence of an inhibitory molecule), RNA (diminished or defective mRNA expression or transcription), or DNA (modification or alteration of gene coding for NMN adenylyltransferase).
  • RNA diminished or defective mRNA expression or transcription
  • DNA Modification or alteration of gene coding for NMN adenylyltransferase.
  • tiazofurin and benzamide riboside class of drugs In cancer cells resistant to tiazofurin and benzamide riboside class of drugs,
  • NAD is still present at a 40% concentration compared to the sensitive cells, and yet these cells exhibit undetectable levels of NMN adenylyltransferase.
  • tiazofurin and benzamide riboside resistant lines have provided an important tool to understand the pyridine nucleotide pathway and the influence of this pathway's perturbation in anticancer therapy, because of the defect observed in the function and/or synthesis of NAD pyrophosporylase activity in these cells. Therefore, cloning of NMN adenylyltransferase helps in understanding the role of this enzyme in the maintenance of pyridine nucleotide concentrations and the effect of its perturbation in cancer chemotherapy.
  • An Italian group has reported studies on NMNAT. All attempts at purifying this enzyme from human placenta using the techniques published by the Italian group were unsuccessful.
  • YGR010W was found, a close homolog of YLR328W with 82.3% homology. Applicants have expressed recombinant YGR010W and purified NMNAT enzyme. The substrate-kinetics, inhibitor-kinetics, and pH properties for NMNAT enzyme from YGR010W were similar to that from YLR328W.
  • human EST26487 was identified as homologous to yeast NMN adenylyltransferases YLR328W and YGR010W by BLAST search.
  • Human EST26487 was obtained from American Type Culture Collection (Manassas, VA) as a clone in pBluescriptl. The clone was characterized by sequencing from vector sites T3 and T7. The encoded cDNA was amplified using PCR and cloned into pTrcHis TA-TOPO (Invitrogen). The protein was expressed as a 6xHis tagged protein and purified by Ni-NTA chromatography (Qiagen).
  • SDS- PAGE gel electrophoresis revealed a single band at the predicted fusion mass of 42 kDa.
  • the protein exhibited NMNAT activity of 10 micromoles/min/mg protein.
  • the present human NMNAT gene can be expressed in human cells by utilizing standard gene transfer technology.
  • the cloned cDNA is different from the native cDNA.
  • ELISAs are the preferred immunoassays employed to assess a specimen.
  • ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature.
  • a radioimmunoassay can be developed on the basis of NMN adenylyltransferase protein that can be used to predict the sensitivity of a given patient, for example, with leukemia, to drugs, such as tiazofurin.
  • Patients who express adequate levels of NMNAT in circulating blood cells will be expected to convert the drug to its active form that will kill leukemic blast cells by their biochemical mode of action.
  • Patients who do not express adequate levels of NMNAT in their blood cells can be transfected with human NMNAT by the techniques described earlier, rendering those patients sensitive to drug therapy.
  • Diabetes mellitus is a group of debilitating disease with lethal complications. Diabetes exists primarily as Type I, insulin-dependent diabetes mellitus (IDDM) and Type II non-insulin mellitus (NIDDM). IDDM was formerly known as juvenile-onset diabetes, and has an early age onset than NIDDM (generally, 35 years). The Juvenile Diabetes Foundation estimates that more than 30,000 new cases of Type I diabetes are diagnosed annually (1). In other words, more than a million people in the U.S. suffer from this disease. Among the complications of diabetes are heart disease, peripheral, vascular and nerve disease, retinopathy, and neuropathy (2). Therefore, addressing treatment modalities are of prime importance for diabetes.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin mellitus
  • IDDM the pancreatic ⁇ -cells responsible for secreting insulin in response to blood glucose elevations, are ablated by the host immune system (3).
  • genetic susceptibility factors as particular genotypes of HLA-DQ are associated with increased risk of IDDM (4).
  • Viruses have been implicated in some instances, and molecular mimicry between the PC-2 protein of the coxsackievirus and glutamic acid decarboxylase (GAD) is a possible initiator of autoimmune destruction (5).
  • GID glutamic acid decarboxylase
  • anti-GAD autoantibodies are a sensitive predicator of IDDM (6). With the destruction of ⁇ -cells, the body loses its ability to produce insulin.
  • ⁇ -cells are recognized by cytotoxic T cells. Following recognition, T cells generate reactive oxygen intermediates (ROIs) and secrete interleukins - 1 ⁇ and -12, and TNF- ⁇ . IL-1 ⁇ and IL-12, as well as TNF- ⁇ , upregulate ⁇ -cell expression of nitric oxide synthase (NOS), leading to generation of NO, another ROI (12-14).
  • ROI toxicity to ⁇ -cells results in DNA damage and activates PARP [poly(ADP-ribose)polymerase] to enhance repair of damaged DNA (15).
  • PARP is a major utilizer of NAD to form poly(ADP-ribose) polymers. Since availability of NAD is already compromised due to low NAD/NADH ratios, excessive activation of PARP leads to greater cellular loss of NAD (16, 17). This results in ⁇ - cell death.
  • pseudohypoxia This decreased ratio of NAD/NADH, termed pseudohypoxia, mimics the effects of true hypoxia on vascular and neutral functions and plays an important role in the pathologies of diabetic complications (30,31 ). These effects are mediated in part by imbalances in lipid metabolism and increased production of superoxide anion.
  • IDDM is an autoimmune disease wherein pancreatic ⁇ -cells accountable for secreting insulin response to blood glucose stimulus are destroyed by the host immune system (3). Beta-cell death is a result of activation of auto-reactive T-cells. Although auto-reactive cells are found both in diabetic and non-diabetic cells, autoreactivity is specific in ⁇ -cells of IDDM patients wherein certain self-antigens (GAD 65 and IA-2) are found (2,3). When these auto-reactive T cells are activated, they attack ⁇ -cells. It has been shown that CD-4, a helper cell, locates the ⁇ -cells and secretes the cytokine IL-1 ⁇ .
  • cytokine TNF- ⁇
  • nitric oxide synthase transcription and NO production (12, 15).
  • NO is converted into peroxynitrite, a reactive oxygen species (ROS) and damages ⁇ -cell's DNA, causing fragmentation through free radical intermediates (13, 14).
  • ROS reactive oxygen species
  • ⁇ -cell-DNA fragmentation activates PARP to aid in DNA repair.
  • PARP utilizes NAD to synthesis poly(ADP-ribose)polymers to repair DNA. Excessive DNA damage leads to critical depletion of NAD pools, leads to homologous recombination and ⁇ -cell death. NAD is also utilized in the glycolysis pathway to synthesize ATP. Therefore, depletion of NAD results in lowered ATP and subsequent ⁇ -cell death.
  • NAD precursors such as nicotinamide or niacin to show that ⁇ -cell death induced by streptozotocin (a nitrosourea containing glucose analogue) can be prevented both in vitro and in vivo (32-34).
  • streptozotocin a nitrosourea containing glucose analogue
  • these precursors are known to influence ⁇ -cells by inhibiting PARP activity, and therefore, no direct demonstration of the effect on NAD pools and metabolism has been demonstrated until now.
  • the present application specifically directs attention to the influence of NAD concentration by transfecting the NMNAT (NMN adenylyltransferase) gene to upregulate the synthesis of NAD in the ⁇ -cells.
  • NMNAT NMN adenylyltransferase
  • NMNAT is a rate-limiting enzyme in the synthesis of NAD and thus, amplifying it maintains the ⁇ -cell's NAD pools, prevents NAD depletion and the cessation of ATP synthesis.
  • NMNAT genes, YLR328W and YGR010W were transduced into mouse insulin-secreting insulinoma NIT-1 and TC-3 cells. Transduction was confirmed by Northern blotting for transcript and increase in NMNAT enzyme activity. This permits the examination of the effects of increased NMNAT expression on intercellular NAD levels, and on the NAD-influenced substrates, ATP and GTP. Whether altered NAD levels influence insulin secretion in these cells was also examined.
  • NMNAT-transduced ⁇ -cells are less susceptible to streptozotocin toxicity.
  • the growth inhibition of cells and impact on NAD levels by streptozotocin was evaluated.
  • 3-aminobenzamide was incubated with a PARP inhibitor. T-Butyl peroxide, a known inducer of oxidative stress and PARP activator, to examine the influence of NAD depletion with and without 3-aminobenzamide.
  • NMNAT-transduced cells show decreased toxicity to destruction by immune cells. This is because damage of ⁇ -cells results in insulinitis and eventual destruction, and since ⁇ -cell destruction is correlated with toxicity and induction of nitric oxide synthase in the inflamed state.
  • transduced and control cells that are previously exposed to low-dose, nonablative streptozotocin were incubated with allogeneic mouse mononuclear cells. 51 Cr release and apoptosis (TUNEL) assays were used to quantitate ⁇ -cells lysis and immunotoxicity.
  • NAD depletion is the limiting step in IDDM. Therefore, one can either amplify NMNAT to produce more NAD or inhibit PARP that utilizes NAD. Upregulating NMNAT provides ⁇ -islet cells resistant to diabetogenic insulinitis and thus suitable for transplantation. Moreover, it has been suggested that maintaining overall optimal NAD levels with nicotinamide or its analogue, 6-aminonicotinamide, enhances glucose-induced release, in vitro (43). Nicotinamide 5'-mononucleotide adenylyltransferase (NMNAT) catalyzes the transfer of adenylyl group of ATP to NMN to synthesize NAD.
  • NMNAT Nicotinamide 5'-mononucleotide adenylyltransferase
  • NMNAT was purified from Saccharomyces cerevisiae and subjected to tryptic digestion. Through the database search a full match was found between the sequence of trypic fragments and the sequence of a hypothetical protein encoded by the yeast YLR328W open reading frame (GenBank number U20618) (44). The YLR328W gene was isolated, cloned into a T7-based vector and expressed in E. coli BL21 cells and yielded a high level of NMNAT activity (44). The purified recombinant NMNAT correlated with the predicted sequence.
  • EST 26487 GenBank Ace: AA323669, GenBank gi: 1975997
  • hNMNAT-2 is localized to chromosome 1. It is also known as C10RF15 and KIAA0479. It falls in 1q25.
  • YGR010W is closely related homolog of YLR328W, showing 82.3% homolog. Recombinant YGR010W was expressed and purified. The substrate-kinetics, inhibitor-kinetics, and pH properties of YGR010W were similar to that of YLR328W.
  • YGR010W was induced in yeast by mutagenesis using the alkylating agent, methymethanesulfonate (MMS) (45). In yeast and mammalian cells, MMS also promotes synthesis of poly(ADP-ribose)polymers (46). Cloning of veast YLR328W and YGR010W:
  • Oligonucleotide primers corresponding to the yeast ORFs were obtained from Gibco/Life Sciences (Bethesda, MD). The oligonucleotides were synthesized to incorporate Notl restriction sites at the 5' and 3' ends. The sequences are: 328- 5'Notl (TATAGCGGCCGCATGGATCCCACAAGSGCT), 328-rev (TGGTGCGGCCGCCCTGTAGGGGAAAATTGGAA), 010-5'Notl (TATAGCGGCCGCGATCCCACCAAAGCACC), 010-rev (TGTGGCGGCCGCGTGGTAAATTTGAACTGCTGC).
  • PRC was carried out using Taq polymerase (Perkin-Elmer Cetus, Madison, Wl) and purified high-molecular weight yeast DNA (Sigma-Aldrich Corporation). PCR product was gel purified and ligated into either pTrcHis-TA-TOPO-TA (prokaryotic vector) or pCDNA4/HisMax- TOPO-TA (eukaryotic vector) (Invitrogen Corporation, Carlsbad, CA). Ligase- independent ligation was carried out and transformed into TOP10 competent cells (Invitrogen). Final vectors were sequenced to ensure no errors were introduced by PCR and that insert was cloned in the proper orientation. Expression and purification of recombinant YLR328W and YGR010W
  • One-liter aliquots of TOP10 cells carrying pTrcHis+insert were grown to confluence and induced for five hours using 0.5 mM IPTG.
  • Bacteria were harvested, lysed with lysozyme in hypotonic buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, mM 2-ME, 0.5% Tween-20, 0.5 mg/mL lysozyme) and purified using Qiagen Ni-NTA resin. Protein was bound to resin in lysis buffer containing 500 mM NaCl and 5 mM imidazole, washed with buffer containing 40 mM imidazole and eluted with 200 mM imidazole.
  • hypotonic buffer 50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, mM 2-ME, 0.5% Tween-20, 0.5 mg/mL lysozyme
  • NMNAT activity was assayed as described (47) with some modifications.
  • reactions were performed using 900 ⁇ L of master mix and 50 ⁇ L of enzyme.
  • Master mix contained 240 ⁇ L 100 mM HEPES pH 7.5 containing 40 mM MgCI 2 ; 100 ⁇ L of 11 mM neutralized ATP; 20 ⁇ L of 60 mM NMN; 50 ⁇ L of yeast alcohol dehydrogenase (0.5 mg/mL); and 390 ⁇ L of ethanol reagent (35 mM semicarbazide, pH 7.5, with 0.1 M ethanol). Reaction was carried out at 37°C, and absorbance recorded in a 1 mL, 1 cm light path cuvet, at 340 nm.
  • NIT-1 cells were obtained from American Type Culture Collection (ATCC, Bethesda, MD) (49) and TC-3 cell line was obtained from Dr. Mark Deeg, Roudebush V.A. Medical Center, Indianapolis, IN. Media and supplements were obtained from Gibco/Life Technologies (Gaithersburg, MD). Heat-inactivated serum was obtained from Sigma Chemical Co. (St. Louis, MO). Cells were cultured in Hams F-12K medium supplemented with 10% heat- inactivated fetal bovine serum, 10,000 U/L penicillin and 50 mg/L streptomycin in an atmosphere of air with 5% Co 2 at 37°C. Doubling time for these cells was about 24 hours. Cells grew as monolayers and were disrupted with enzyme-free Hanks based cell-dissociation buffer (Gibco/Life Technologies, Gaithersburg, MD). Effect of various treatments on cell viability
  • non-adherent cells were collected by centrifugation and pooled with attached cells that were scrapped in lysis buffer (10 mM Tris-HCI, pH 7.5 containing 10.0 mM EDTA and 1 % Nonidet P-40, pH 7.5).
  • the lysates were centrifuged at 13,000 g for one minute.
  • the supernatant were first extracted with phenol and then with phenol-chloroform-isoamyl alcohol (25:24:1) to remove proteins and lipids.
  • DNA in the supernatants was precipitated in ethanol overnight at -20°C and pelleted at 13,000 g. After washing with 70% ethanol, the pellet was resuspended in Tris-EDTA buffer.
  • HIT-1 and TC-3 cells were treated with agents and harvested at indicated times to examine the time-course and dose-response kinetics.
  • Proteolytic cleavage of PARP and caspase-3 were detected by Western blot analyses as previously described (53,54).
  • PARP analysis cells were harvested after treatment at the indicated times. After one wash with PBS (phosphate buffered saline), cells were suspended at 5 x 10 6 cells/mL in sample buffer (62.5 mM Tris-HCI, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.00125% bromophenol blue, and 1 % 2- mercaptoethanol) and then sonicated for 15 seconds and incubated at 65°C for 15 minutes. Cell extract (15 ⁇ L each) was subjected to 7.5% SDS-PAGE determination.
  • sample buffer 125 mM Tris-HCI, pH 6.8, 5% glycerol, 2% SDS, 0.003% bromophenol blue, and 1% 2- mercaptoethanol
  • sample buffer 125 mM Tris-HCI, pH 6.8, 5% glycerol, 2% SDS, 0.003% bromophenol blue, and 1% 2- mercaptoethanol
  • caspase-3 The activity of caspase-3 was measured by the fluorometric technique described in the ApoAlert CPP32 fluorescence assay kit (Clontech, Palo Alto, CA). Briefly, fluorescence emission of the 7-amino-4-trifluoromethyl-coumarin (AFC) released on proteolytic cleavage of the fluorogenic substrate DEVD-AFC by caspase-3 activity was measured following excitation at 400 nm and emission at 505 nm (55). Poly (ADP-ribose) assays in whole permeabilized cells Poly (ADP-ribose) polymerase activity was assayed in logarithmically growing
  • NIT-1 and TC-3 cells (500,000-800,000 cells/mL) after appropriate treatment.
  • the cells were mutagenized with 10 ⁇ g MNNG for 30 minutes to induce double stranded DNA breaks.
  • cells were harvested at 4°C, and all subsequent steps, with the exception of the enzyme activity incubation, were performed on ice.
  • Cells were resuspended twice in PARP assay buffer (40 mM MgCb, 30 mM 2- mercaptoethanol, 10 mM Tris pH 7.80, 4 mM EDTA) at a concentration of 2-3 x 10 7 cells/mL.
  • Radioactive NAD reagent ⁇ [ 3 H] 2,8-(adenine)-nicotinamide adenine dinucleotide, 50 mCi/mmol, obtained from Moravek Radiologicals, Brea, CA ⁇ was added to prime the PARP reaction, cells were pelleted at 13,000 g for one minute, and immediately supernatant was aspirated. Then cells were extracted with 10% ice-cold TCA containing 30 mM sodium pyrophosphate. The pellet was vortexed to homogeneity, and centrifuged for two minutes at 13,000 g. Aqueous extract was aspirated, and acid insoluble radioactivity was left behind.
  • NMNAT activity was analyzed in logarithmically growing NIT-1 and TC-3 cells. 10 8 cells were harvested and lysed by vigorous vortexing in 1 mL of 100 mM Tris pH 8.0, 100 mM KCI, 5 mM 2-mercaptoethanol, 0.5% Tween-20, 1 mM PMSF, 0.01% leupeptin, and 0.01% aprotonin at 4°C. NMNAT activity was determined as described above and expressed as units per mg protein.
  • Ribonucleotide levels were analyzed as described (56). Briefly, 10 7 cells after appropriate treatment were harvested, extracted with ice-cold 10% trichloroacetic acid, and neutralized with 0.5 M tri-n-octylamine in Freon. The aqueous portion was analyzed by HPLC using ammonium phosphate buffer systems and a photodiode array detector. Drug sensitivity in NMNAT-transfected and control human cell lines
  • Quantitation of drug sensitivity was performed by dose-response to growth inhibition versus drug concentrations (streptozotocin and nicotinamide analogues). Growth inhibition assays were performed using tetrazolium reduction of MTS as described before with PMS (CellTiter Assay, Promega, Madison, Wl). Logarithmically growing cells were plated in 0.1 mL aliquots in 96-well plates. In the
  • control (sic) or NMNAT-transfected NIT-1 and TC-3 cell DNA was labeled by incubating 1 x 10 6 cells/mL with 1.62 ⁇ Ci/mL [ 125 l]UdR in complete medium for 16 hours.
  • cytoplasmic proteins was labeled in other cells by incubating 1 x 10 6 cells/mL with 10 ⁇ Ci/mL [ 51 Cr]sodium chromate in complete medium for 90 minutes.
  • the cells are then washed, seeded at a density of 1 x 10 6 cells/Petri dish (30 x 10 mm) and incubated at 37°C in 35% air and 5% C0 2 in 2.7 mL complete medium without and with cytokines (10-30 U each; IL-1 ⁇ [2-4 x 10 7 U/mg from Upjohn Co., Kalamazoo, Ml]; TNF- ⁇ [1-2 x 10 7 U/mg from Genentech, San Francisco, CA] and IFN- ⁇ [8 x 10 6 U/mg from San Francisco, CA] for 1-18 hour (36).
  • cytokines 10-30 U each; IL-1 ⁇ [2-4 x 10 7 U/mg from Upjohn Co., Kalamazoo, Ml]; TNF- ⁇ [1-2 x 10 7 U/mg from Genentech, San Francisco, CA] and IFN- ⁇ [8 x 10 6 U/mg from San Francisco, CA] for 1-18 hour (36).
  • the [ 125 l]UdR labeled cells were collected, DNA was counted separated into a detergent-soluble fraction (fragment DNA) and insoluble fraction, and these were counted in a gamma counter (57). DNA fragmentation is calculated as 100% x soluble [ 125 l]DNA/(soluble + insoluble [ 125 I]DNA).
  • Medium was collected from [ 51 Cr]-labeled cells and [ 51 Cr] was counted in a gamma counter.
  • Percent cytokine-induced cell lysis [ 51 Cr]-release was calculated as 100% x (cpm with cytokines and cpm without cytokines). Total cpm was determined by dissolving the cells in 4% Triton X-100.
  • cells were incubated in complete medium with and without cytokines, and with and without glucose, streptozotocin, nicotinamide or its analogues (0.1-10.0 mM) for periods up to 24 hours. DNA fragmentation and total DNA were analyzed as described above.
  • NMNAT The impact of NMNAT on the PARP-activation cycle is unknown.
  • a higher supply of NAD impacts positively on cell survival under NAD-depleting conditions of PARP activation.
  • Yeast knockouts of YLR328W and YGR010W show decreased survival following DNA damage either due to decreased DNA repair, depletion of NAD, or both.
  • a common mechanism of action of different PARP isoforms is NAD depletion with resulting decrease in ATP/ADP ratio that is due to inhibited glycolysis and oxidative phosphorylation.
  • a decreased ATP/ADP ratio is associated with loss of mitochondrial membrane potential, although the causality with respect to apoptosis is not known.
  • Increased expression of NMNAT prevents toxicity due to either pathway involving NAD depletion.
  • RIA radioimmunoassay
  • a sensitivity of 0.02 ng/mL of insulin measurement can be achieved.
  • a predetermined concentration of labeled tracer antigen is incubated with a constant dilution of antiserum such that the level of antigen binding sites on the antibody is limited (only 50% of the total tracer concentration is bound by the antibody). If unlabeled antigen is added to this system, there is competition between labeled tracer and unlabeled antigen for the limited and constant number of binding sites on the antibody. Thus, the amount of tracer bound to antibody decreases as the concentration of unlabeled antigen increases. This can be quantitated after separating antibody-bound from free tracer and counting one or the other, or both.
  • a calibration curve is set up with increasing levels of standard unlabeled antigen and from this curve the amount of antigen in unknown sample is calculated.
  • the four basic requirements for the RIA are: a specific antiserum to the antigen to be measured, the availability of a radioactive labeled form of antigen, a technique to separate unbound tracer, and an instrument to count radioactivity.
  • the LINCO sensitive rat insulin assay utilizes [ 125 l]- labeled insulin antiserum to determine the level of insulin in tissue culture media by the double antibody/PEG technique (58,59).
  • Human NMNAT-2 was first noticed during a BLAST search for homology to the recently cloned yeast NMNAT gene YLR328W.
  • the human cDNA of interest known as KIAA0479, encoded a 5,439 base transcript containing a 307-aa open reading frame (ORF) beginning from the first initiation codon.
  • ORF open reading frame
  • KIAA0479 was matched to another human cDNA, from the chromosome 1 ORF 15 (C10RF15) locus.
  • the C10RF15 cDNA was 5,563 bases, including a 5' extension of 1 12 bases.
  • This extended 5' region contained 3 stop codons in frame with the ORF, indicating that the ORF is complete and not a 3' fragment from a longer ORF.
  • the KIAA0479 cDNA was available as a TIGR EST (EST26487).
  • the ORF was cloned by PCR amplification using Pfu proofreading polymerase and the primers EST-ORF-FWD (5'-ATGACCGAGACCACCAAGAC), and EST-NCO-3' (5'- CGAACCATGGACTAGCCGGAGGCATTGATG).
  • a single, 979 bp fragment was amplified and cloned into pTrcHisTOPO-TA vector (Invitrogen, Carlsbad, CA).
  • NMNAT-2 was expressed using the plasmids pTrcHisTOPO-TA and pBADThiofusion in separate experiments. Proteins were expressed in E. coli TOP10 cells (Invitrogen) in LB broth containing 100 mg/L ampicillin and induced with 1 mM IPTG (pTrcHisTOPO-TA) or 0.2% arabinose (pBADThiofusion). Cells were grown to late log phase and induced, then harvested by centrifugation.
  • Cells were lysed using a French pressure cell in 100 mM KH 2 P0 , pH 7.6, containing 1 ⁇ M PMSF and 1 mg/mL benzamidine. Protein was purified using Qiagen Nickel-NTA IMAC chromatography. Protein was loaded in 1 mM histidine, washed with 5 mM histidine, and eluted with a linear gradient from 5- 100 mM histidine in 100 mM KH 2 P0 4 .
  • Protein was stored at 4 9 C during analysis and at -20 Q C in 50% glycerol for long-term storage. Protein stored at 4 Q C showed 90% of original activity after 2 days and 50% after 1 week. Purity was evaluated by SDS-PAGE and Coomassie Blue staining. Purity was estimated as greater than 95% as no other bands were visualized. Protein concentrations were determined by the method of Bradford and Lowry (Bio-Rad, Hercules, CA).
  • Enzyme activity Purified protein was analyzed for NMNAT activity by continuously coupled NMNAT and ADH reactions with product formation observed by absorbance at 340 nm. Reactions were performed using 900 ⁇ L of master mix and 50 ⁇ L of enzyme. Enzyme master mix contained 240 ⁇ L of 100 mM HEPES pH 7.5 containing 40 mM MgCI 2> 100 ⁇ L of 11 mM neutralized ATP, 20 ⁇ L of 60 mM NMN, 50 ⁇ L of yeast alcohol dehydrogenase (0.5 mg/mL), and 390 ⁇ L of ethanol reagent (35 mM semicarbazide, pH 7.5, with 0.1 M ethanol).

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Abstract

L'invention concerne une composition conçue pour modifier la production de NAD. Cette composition comprend un composant qui modifie les NMNAT ainsi qu'un excipient pharmaceutiquement acceptable. L'invention se rapporte également à une séquence d'ADN complémentaire codant les NMNAT. L'invention concerne par ailleurs un procédé permettant de traiter par exemple un diabète, un cancer, un infarctus du myocarde, un accident vasculaire cérébral, des troubles neurodégénératifs, un choc, et le vieillissement, par administration à un patient d'une dose efficace d'une composition conçue pour modifier la production de NAD. L'invention se rapporte en outre à une thérapie génique faisant appel à une séquence codant les NMNAT et un procédé permettant d'introduire cette séquence efficacement dans le génome d'un hôte.
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Cited By (8)

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WO2009108999A1 (fr) 2008-03-03 2009-09-11 Ross Stewart Grant Formulations pharmaceutiques de resvératrol et procédés d'utilisation de celles-ci pour traiter des troubles cellulaires
WO2011135332A3 (fr) * 2010-04-27 2011-12-22 Babraham Institute Modulateur de la nmn
EP2584041A1 (fr) * 2011-10-21 2013-04-24 Industrial Cooperation Foundation Chonbuk National University Composition pour prévenir ou traiter le diabète comprenant un inhibiteur nad glycohydrolase comme ingrédient actif
WO2013090732A3 (fr) * 2011-12-14 2013-09-26 The Board Of Regents Of The University Of Texas System Biomarqueurs d'inactivation de gène collatérale et cibles pour une thérapie anticancéreuse
WO2013130672A3 (fr) * 2012-02-27 2013-10-31 United States Of America As Represented By The Department Of Veterans Affairs Traitements et diagnostics améliorés du cancer
WO2014019108A1 (fr) * 2012-07-28 2014-02-06 深圳华大基因研究院 Gène mutant nmnat1, amorces, kit et son procédé de détection et son utilisation
CN103710321A (zh) * 2013-12-31 2014-04-09 邦泰生物工程(深圳)有限公司 烟酰胺单核苷酸腺苷转移酶突变体及其编码基因和应用
CN107475139A (zh) * 2017-09-30 2017-12-15 广州大学 一种pH稳定型发酵培养基及其应用

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009108999A1 (fr) 2008-03-03 2009-09-11 Ross Stewart Grant Formulations pharmaceutiques de resvératrol et procédés d'utilisation de celles-ci pour traiter des troubles cellulaires
WO2011135332A3 (fr) * 2010-04-27 2011-12-22 Babraham Institute Modulateur de la nmn
EP2584041A1 (fr) * 2011-10-21 2013-04-24 Industrial Cooperation Foundation Chonbuk National University Composition pour prévenir ou traiter le diabète comprenant un inhibiteur nad glycohydrolase comme ingrédient actif
WO2013090732A3 (fr) * 2011-12-14 2013-09-26 The Board Of Regents Of The University Of Texas System Biomarqueurs d'inactivation de gène collatérale et cibles pour une thérapie anticancéreuse
US9452182B2 (en) 2011-12-14 2016-09-27 Board Of Regents, The University Of Texas System Collateral gene inactivation biomarkers and targets for cancer therapy
WO2013130672A3 (fr) * 2012-02-27 2013-10-31 United States Of America As Represented By The Department Of Veterans Affairs Traitements et diagnostics améliorés du cancer
WO2014019108A1 (fr) * 2012-07-28 2014-02-06 深圳华大基因研究院 Gène mutant nmnat1, amorces, kit et son procédé de détection et son utilisation
CN103710321A (zh) * 2013-12-31 2014-04-09 邦泰生物工程(深圳)有限公司 烟酰胺单核苷酸腺苷转移酶突变体及其编码基因和应用
CN103710321B (zh) * 2013-12-31 2015-04-01 邦泰生物工程(深圳)有限公司 烟酰胺单核苷酸腺苷转移酶突变体及其编码基因和应用
CN107475139A (zh) * 2017-09-30 2017-12-15 广州大学 一种pH稳定型发酵培养基及其应用

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