WO2018152480A1 - Méthode d'identification, de ciblage et d'administration précis de thérapies dirigées pour la destruction de cellules cancéreuses - Google Patents

Méthode d'identification, de ciblage et d'administration précis de thérapies dirigées pour la destruction de cellules cancéreuses Download PDF

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WO2018152480A1
WO2018152480A1 PCT/US2018/018650 US2018018650W WO2018152480A1 WO 2018152480 A1 WO2018152480 A1 WO 2018152480A1 US 2018018650 W US2018018650 W US 2018018650W WO 2018152480 A1 WO2018152480 A1 WO 2018152480A1
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cell
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concentration
virus
cells
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Richard Postrel
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Richard Postrel
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • Cancer is not a single disease, but rather a class of diseases which are in a perpetual state of change and development. Each human organism is challenged by cancer millions of times in its lifetime. The difference between becoming a cancer patient and remaining a healthy individual is that in most cases the human immune system and the body's own defense mechanisms is sufficient to restore or rebalance its biochemistry to prevent undesirable and opportunistic contagions from dominating cellular growth and behavior.
  • Each cancerous cell presents an early onset bio-nanomarker in the form of one or more metabolic differentials that have shifted to support the massively enlarged number of chemical reactions/interactions necessary to support the enhanced replication, or simply
  • hypoproliferation that is characteristic of cells of the cancer group. Although different cancers may appear in disparate tissues and cancer cells may migrate from one tissue to another, at their root each cancer cell cohort involves a shift in normal metabolism from a lower to a higher metabolic rate, this shift being characteristic of hyperproliferating cancerous cells. As a cell transitions to become cancerous, it alters its metabolic pathways in various ways; down-regulating several, up- regulating others, possibly reinvigorating pathways used at an earlier time, for example during fetal development and turning off still others entirely.
  • hyperproliferation but with several modes of expression that are supra-dependent on the initial metabolic status of the cell and stresses or pressures that make or cause the metabolic changes to occur that are necessary to support hyperproliferation.
  • Cancer is an offshoot of mismatches in copying DNA (mutations) or opportunistic set of circumstances that supersedes cells' normal inhibition of growth of like neighboring cells. Evolution, survival of the fittest, requires differences between individuals of the species so that better suited members of the species survive to produce a next generation. Mismatched DNA are the means through which individual differences are possible. So it can be said that cancer is actually a result of evolution and that occasional mutations are advantageous to survival of the species. However, those that are cancerous, as with most mutations, are not.
  • cancer cells are living things and therefore follow chemical and physical laws and the principles of biology. Cancer itself is a complex disease. A cancer cell is not different in just a single respect from normal desirable cells. Many events are necessary to develop all the changes that make a cell cancerous.
  • Apoptosis is a process that has evolved to remove undesirable cells. For example, apoptosis is triggered to remove cells at the base of baby teeth to facilitate disposal when adult teeth are coming in. Apoptosis also often selectively removes cells at times of stress. For example, several cells may be sacrificed during lean times to preserve nutrition for remaining cells. Cells that misfunction for one reason or another, for example become leaky to Ca " " " , will present metabolic abnormalities.
  • Cancer cells have been altered or have altered themselves to follow a metabolic program to enhance necessary biosynthesis and support that cell's proliferation. The changes may not be in the best interests of the organism. But concomitant with these metabolic changes must be changes that evade the organism's control of inappropriately behaving cells and that evade the apoptotic cell death protocols that evolution has provided in each cell's genetic instruction set.
  • ATP adenosine triphosphate
  • This production process is carried out in the cytoplasm and produces less ATP per glucose molecule, and also ends with lactate, a three carbon molecule, instead of the single carbon molecule, C0 2 .
  • the metabolite, lactate is a chemically energetic molecule whose energy is lost to the cell when the lactate is excreted using a slow but effective transport protein, monocarboxylate transporter protein (usually MCT4 or MCT1). Lactate can be recycled by other organs in the body, e.g., the liver, to salvage the energy and carbon building capacities of the lactate molecule.
  • pyruvate kinase M2 plays a part in the altered glucose metabolism characteristic of cancer. Inhibiting one or more such enzymes using a virus, a small molecule or biological inhibitor and/or ligand starvation or product feedback negative feedback may be used with the systems and methods of this invention.
  • PEM2 pyruvate kinase M2
  • PLM2 interacts with phosphotyrosine-containing proteins, it inhibits their enzyme activities resulting in an increased availability of glycolytic metabolites the cell may then use to support and encourage cell proliferation.
  • PKM1 pyruvate kinase Ml
  • PKM1 pyruvate kinase Ml
  • a mutation in the pyruvate kinase gene itself may affect splicing
  • a mutation in another gene or even an extracellular signal turning on or accentuating another path within the cell may be part of this cell's path to cancer.
  • Cancer cells arise from diverse tissues and from many, many differentiated cell types, but at the root of all cancers is that cell's increased rate of making new cells, that is: hyperpro- liferation. Every time a cell proliferates it splits to create two cells - each of which requiring its own membrane, cytoskeleton, nucleus, mitochondria and other organelles. This duplication requires the cell to accelerate synthetic pathways and several additional pathways that support accelerated synthesis. The resulting two cells will require a doubling of DNA for duplicated nuclei, additional membrane lipids and proteins to cover the increased surface/volume ratio, extra endoplasmic reticulum, golgi, mitochondria, lysosomes, etc. to be split between two cells during mitosis.
  • Mitosis itself is a resource hungry process requiring a slew of catabolic and anabolic events.
  • a metabolic push is necessary to provide an additional set of all cellular components and the temporary resources and energy necessary to divide the cell into two. This accentuated metabolism can be employed to guide intercourse between i) an involved party, e.g., an anti-cancer compound, probe, or other therapy, and ii) the cancerous , i.e., metabolically modulated cell(s).
  • cancer cells will present an increased uptake of nutrient building blocks into the cell and increased use of the nutrients (reactants) in various chemical reactions to make increased products.
  • the products will include products useful for sustaining the cell and by-products such as waste chemicals and heat. While there are some common chemical waste products of metabolism, one ubiquitous product (since in general metabolism is exothermic) is an increased heat output.
  • cancer cells produce more heat than surrounding cells, increased temperature is a metabolism specific, local, let's say, "nanomarker”, that can be used to identify and target these cells for their destruction. While not an essential marker for all means of attacking cancer metabolism, heat can serve as a back-up confirmation or trigger signal for turning on natural innate and adaptive immunities and/or for making available one or more anti-cancer system(s) and method(s) in the identified cells.
  • Cancer cells are differentiated by their altered and increased metabolisms.
  • the altered metabolisms can serve as identifying markers, targeting markers and/or markers that signal or trigger a therapeutic intervention.
  • the unbalanced metabolism can be used as an important marker identifying the altered cells.
  • the identification, targeting and triggering can include mechanics that are very high tech.
  • nanoparticles can be configured with nanosensor capabilities. These nano- particles can be supplied in the vicinity of a tumor or may be applied more systemically, such as in blood or lymph vessels.
  • One species of particle we can make has a form of nano- motor, or means of moving itself. These can be random or can be configured to be thermotaxic (move towards or away from a heat source) or chemotaxic (move along a chemical gradient, such as a pH gradient).
  • Phototaxic (responsive to light - electromagnetic radiation, radio waves) sensors are another example, but these would be effective only close to the skin using ambient light or as secondary sensors responsive to a primary sensor that directs the secondary sensor to act at an identified location.
  • Nanoparticles can also be configured as receivers of electromagnetic radiation. Nanoparticles compartmentalized for example by physical and/or chemical means can be queried to confirm location and if desired about the particle's surroundings. For example, the particle may report back an indication of temperature, pH, and or other parameter programmed into the sensor.
  • electromagnetic energy can be transmitted and converted to heat energy at the target location.
  • nanomarkers While technology may be the source of many nanomarkers, naturally occurring events that produce a detectible signal when at the biologic or macromolecular scale in a sense these may also be termed as nanomarkers.
  • a sensor nanoparticle may also be a reporter nanoparticle, a courier nanoparticle and/or a signal nanoparticle able to deliver a preprogrammed substance or to recruit other couriers for delivery when a preprogrammed event is reported.
  • Nanoparticles can be mostly physical in their action, may include chemical elements to aid in sensing or for delivery and may even transport biologic cargo(es) depending on the whims of the nanoparticles creator(s).
  • Nanoparticle chemistry involves introducing seed particles with one portion having high affinity for a ligand of interest, for example, a membrane receptor, a metabolite, a specific nucleic acid. Nanoparticles can grow the seed to form a larger molecule, perhaps a stronger antenna, perhaps a stronger antigen for recruiting immuno-defenses of the organism, perhaps disabling nucleic acids and causing havoc in the vicinity. Necrotic or apoptotic death may be the desired response. Nanoparticles can self-direct movement along a chemical or biological gradient and when concentrated at a gradient maximum act as nano- identifiers.
  • a ligand of interest for example, a membrane receptor, a metabolite, a specific nucleic acid.
  • Nanoparticles can grow the seed to form a larger molecule, perhaps a stronger antenna, perhaps a stronger antigen for recruiting immuno-defenses of the organism, perhaps disabling nucleic acids and causing havoc in the vicinity. Necrotic or apoptotic death may be the
  • an enzyme may be activated by a chemo-attractant, e.g., H + , or a larger substrate, agonist, antagonist or cofactor, thereby providing a motive force in the direction of the higher concentration that is greater than the motive force where the concentration is lower.
  • chemo-attractant e.g., H +
  • Other examples include conscription of "walking" enzymes (picture a polymerase like DNA or RNA polymerase or ribosomal polymerases) that can transport a cargo as they move along a gradient.
  • a switch mechanism such as sensitivity to a physical or electromagnetic frequency can transform these nano-identifiers into targeting and delivering devices.
  • they may serve as primary identifiers and targeters serving as a nanomarker for a secondary triggered anticancer response.
  • Non-covalent binding e.g., hydrogen binding, reversible or equilibrium binding such as protonation, etc., or temporary or permanent modification such as hydroxylation, oxidation-reduction, phosphorylation, etc.
  • hydrogen binding reversible or equilibrium binding such as protonation, etc.
  • temporary or permanent modification such as hydroxylation, oxidation-reduction, phosphorylation, etc.
  • nano structures can be used to connect two distinct sites.
  • nano-tube structures can be made to conduct electricity or light between the site of interest a nd another device, perhaps outside the organism.
  • Many configurations using nano-tube structures are available including, but not limited to, for example: i.) The nano-tube may transmit information interacting between a sensor and receiver, ii.) The nano-tube may act as a courier for small molecules or biomolecules. iii.) A photo-activation signal can be transmitted through fiber-optic nano-tubes. iv.) Electrical pulses can be transmitted through conductive nano-tubes.
  • Salts and/or nutrients may be precisely delivered, vi.) Plasmids, phages, small bacteria, virus particles may be delivered to a precisely known site.
  • Nanotubes for biological applications have been synthesized as carbon nanotubes.
  • Membrane based (lipid bilayer) nanotubules projecting from one cell to another have been used for transporting cytoplasmic content, including structures as large as mitochondria, from one cell to another.
  • Synthetic nano-tubes can be nano-surgically manipulated using micro-robotic signaling to desired locations and effectors within or at the ends of such nano-tubes can react automatically to predetermined stimuli such as pH thresholds, enzymes or enzymatic substrates, and/or temperature. Reaction may involve turning on, e.g., an electronic, biochemical, physical or chemical signal to attract and/or induce biomarkers or events; and/or a signal effective at the site to modify the surrounding cells' behaviors.
  • predetermined stimuli such as pH thresholds, enzymes or enzymatic substrates, and/or temperature. Reaction may involve turning on, e.g., an electronic, biochemical, physical or chemical signal to attract and/or induce biomarkers or events; and/or a signal effective at the site to modify the surrounding cells' behaviors.
  • Chemicals especially lipid compositions, are heat responsive. Following the activation energy theories involved in completing a chemical reaction, including those facilitated by catalytic enzymes, chemical reactions are temperature dependent. Thermo-dependence is even more evident in enzymatic reactions where subtle temperature changes can induce profound changes in a protein's or RNA's folding and activity. According to these three-dimensional models, a complex molecule's binding site(s)require stability in the interactions of multiple hydrophobic and hydrophilic parts of a molecule.
  • the molecule's kinetic energies will be insufficient to dislodge hydrophobic and e.g., hydrogen bonds that maintain a three dimensional shape conducive to the catalyst presenting a ligand's reactive site(s) to another reactant.
  • Increased temperature can increase random kinesthesis in the molecule and disrupt the appropriate three-dimensional configuration.
  • interactions between lipids changes with temperature as the constituents in the bilayer present with a more solid or more melted form.
  • the melted state of the membrane or a portion thereof e.g., disordered or raft portions
  • Nucleic acids can be engineered to produce a protein of interest, including proteins whose range of temperatures where they are active is an engineering consideration, using available and improving software. Nucleic acids whose transcription, processing or translation is required to make the proteins can also be engineered for desired temperature dependence.
  • Another feature common to the metabolic shift of cancer cells is decreased reliance on the electron transport chain for making high energy phosphates, e.g., adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • cells switch metabolic paths to emphasize a glycosylation process that ends with lactate' " ' and hydrogen ion (H + ) as byproducts.
  • the additional H + ions depress the pH (a measurement indicative of H+ concentration).
  • Another common byproduct is an increased abundance of various reactive oxygen species (ROS) such as H 2 0 2 and -0 2 ⁇ .
  • ROS reactive oxygen species
  • the local pH can also be used as an activator or triggering mechanism extracellularly and/or intracellular ⁇ . Reactivity of molecules changes with protonation status which is dependent on pH. ROS species are very reactive and therefore will have greater applicability as an intracellular activator, but in specific circumstances these can be used as an activator signal or as a switch signal to be amplified in an extracellular application.
  • the increased metabolism results in a modified plasma membrane.
  • Some modifications are for stability, such as slightly longer fat chains in the membrane to raise the lipid melting point to coordinate with the increased heat of metabolism.
  • Most cells also have increased numbers of membrane transporters, e.g., to facilitate nutrient uptake and waste disposal; some cancer cells express binding or transport proteins not normally expressed in the neighboring more properly differentiated cells.
  • a transporter is found at elevated concentrations in the membrane to support the substantially increased needs to transport some raw nutrients, such as amino acids and/or glucose. While these may be available as secondary targeting or trigger mechanisms, the primary mechanism - increased need for certain chemical reactions within the hyperproliferating cell - is a fundamental mechanism underpinning the identifying, targeting mechanisms of this invention.
  • a virus e.g., a DNA or RNA virus can be engineered to deliver a therapy to the target cell's interior.
  • the activated ras oncogene renders the cell more prone to infection by a virus since the activated Ras system deactivates a cell's antiviral defenses.
  • Such an engineered retrovirus or other vector know in the art is therefore a viable courier for a variety of therapeutic strategies to modulate intracellular metabolism.
  • a phase l/ll study of intravenous reovirus in patients with melanoma MAYO-MC0672 (NCI trial)
  • patients received systemic administration of reovirus at a dose of 3xl0 10 TCID 50 per day on Days 1 - 5 of each 28-day cycle, for up to 12 cycles of treatment.
  • cancers of interest for reoviral therapy include: pericutaneous tumors, prostate cancer, glioma, metastatic ovarian tumors, head and neck tumors, metastatic sarcomas, non- small-cell lung cancer, squamous cell carcinoma lung cancer, pancreatic cancer, fallopian tube cancer, metastatic melanoma, colorectal cancer, etc.
  • Vesicles for example liposomes, are another alternative whose membranes can be engineered to be sensitive to heat, pH, ROS or other chemical attractant or binding agent.
  • Nanoparticles including nanosensor-particles can also be employed as couriers.
  • Viral particles may be allowed to interchange their lipid content with vesicles to change envelope fluidity and alter their selective merging with membranes they may encounter.
  • Non-biologic sensors e.g., nanochips, may be delivered to the cytoplasm by being inserted into a viral envelope to take advantage of the abilities viruses have developed to enter and infect cells.
  • the plasma membrane is a lipid bilayer and has a mosaic of proteins, glycolipids, lipoproteins, sterols, glycoproteins, etc.
  • the fluid mosaic membrane lipid bilayer model popularized in the 1970s has been updated to include a conceptual structure referred to as "lipid rafts".
  • Lipid rafts are believed to exist as constantly changing structural components floating in plasma membranes. Lipid rafts are believed to play an important role in many biological processes, especially signal transduction, apoptosis, cell adhesion and protein orientation and sorting.
  • Membrane proteins and lipidated peptides, carbohydrates or proteins either reside in, form the boundary of or be excluded from such rafts, depending on the molecule's physical/chemical properties.
  • lipid rafts are understood to play critical roles in many biological processes including viral infections.
  • the plasma membranes of eukaryotic cells comprise literally hundreds of different lipid species.
  • the bilayer has evolved the propensity to segregate constituents laterally. This segregation arises from dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization.
  • Eukaryotic membrane lipids are mostly glycerophospholipids, sphingolipids, and sterols.
  • Mammalian cell membranes predominately comprise but one sterol, namely cholesterol, but the membrane comprises several hundred of different lipid species of glycerophospholipids and sphingolipids.
  • glycerophospholipids the head group of varies, also the bonds linking the hydrocarbon chains to glycerol, and the length and location and degree of saturation fatty acids provide distinguishing molecular features including how they sort amongst each other.
  • sphingolipids have the combinatorial propensity to create diversity by different ceramide backbones and, above all, at least 500 different carbohydrate structures at the head groups of the glycosphingolipids.
  • Cholesterol interacts preferentially, although not exclusively, with sphingolipids due to their similar carbon chain structure and the saturation of the hydrocarbon chains. Although not all of the phospholipids within the raft are fully saturated, the hydrophobic chains of the lipids contained in the rafts are more saturated and tightly packed than the surrounding bilayer. Cholesterol then partitions preferentially into the lipid rafts where acyl chains of the lipids tend to be more rigid and in a less fluid state. Cholesterol is the dynamic "glue" that holds the raft together.
  • lipid is often close to half the cell membrane molecules. But, since it is smaller and weighs less than other molecules in the cell membrane, it makes up a lesser proportion of the cell membrane's mass, generally ⁇ 20 %. Cholesterol is also found in membranes of cell organelles, where it usually makes up a smaller but still significant proportion of the membrane. For example, the endoplasmic reticulum, which is involved in making and modifying proteins, is but 6% cholesterol by mass and the mitochondria, comprise about 3% cholesterol by mass.
  • Cancer cells exhibit an array of metabolic transformations induced by mutations leading to gain-of-function of oncogenes and loss-of- function of tumor suppressor genes that include increased glucose consumption, reduced mitochondrial respiration, increased reactive oxygen species generation and cell death resistance, all of which ensure cancer progression.
  • Cholesterol metabolism is disturbed in cancer cells and supports uncontrolled cell growth.
  • the accumulation of cholesterol in mitochondria emerges as a molecular component that orchestrates some of these metabolic alterations in cancer cells by impairing mitochondrial function.
  • mitochondrial cholesterol loading in cancer cells may contribute, in part, to the Warburg effect stimulating aerobic glycolysis to meet the energetic demand of proliferating cells, while protecting cancer cells against mitochondrial apoptosis due to changes in mitochondrial membrane dynamics.
  • the presence/absence of cholesterol regulates fluidity which is the reason why the contents of cholesterol and other lipids are critical cellular and organelle structural components.
  • Membrane dynamic processes involve biophysical concerns relating to fluidity which is controlled by lipid content and proteins in and on the membrane. Mitochondrial fusion/fission balance is critical to maintenance of proper cell functions. Altered fluidity can upset the balance and therefore the cell's energetic machinery.
  • the membrane is gel like.
  • Tm melting temperature
  • the membranes are in liquid disordered state, the rigidity of cholesterol ring reduces the freedom of motion of acyl chains (trans conformation tends to increase order and help define the rafts.
  • the decreased fluidity and higher order allows for a stronger resistance to disrupting influences such as polar molecules and thus decreases permeabilities to especially foreign substances such as water and nitrogen and oxygen containing compounds.
  • lipid rafts (straighter), they aggregate more, which cholesterol also helps. That ordered part of the membrane is also thicker, making it better suited for accommodating certain proteins. Since the fatty acids in lipid rafts are longer, raft phospholipids move in sync with the phospholipids on the opposite side of the membrane. In the disordered portions of the membrane, the phospholipids on one side of the membrane move independently of those on the other. By stabilizing certain proteins together in lipid rafts, cholesterol is important to helping these proteins maintain their function.
  • Lipids e.g., glycolipids such as a glycerolipid that has one fully saturated chain and one partially unsaturated chain could function as a surface-active component, a hybrid lipid or a linactant. These linactants would lower the line tension between domains by occupying the interface, with the saturated anchor preferring the ordered raft and the unsaturated fatty acid interacting with the less ordered lipid environment. Small finite-sized assemblies of disordered and ordered lipid domains separated and stabilized by these hybrid lipids could be expected to form as equilibrium structures. In the viral envelope especially but even in the plasma membrane with its generally lower protein content proteins, especially with multiple transmembrane domains such proteins should also act as linactants.
  • proteins that have both a GPI anchor and a trans-membrane domain have been identified, in which the GPI anchor could be raft- associated with the trans-membrane domain facing the non-raft bilayer.
  • Another such protein is the influenza virus M2 protein, which seems to occupy the perimeter of the raft domain that forms when the virus buds from the plasma membrane.
  • N-Ras has also been proposed to act as a linactant in the cytosolic leaflet of a raft.
  • membrane rafts must play an important role in the process of virus infection cycle and virus-associated diseases. Many viral components or virus receptors are exclusive to or concentrated in the lipid raft ordered microdomains.
  • Viruses have been divided into four main classes, non-enveloped RNA virus, enveloped RNA virus, nonenveloped DNA virus, and enveloped DNA virus.
  • General virus infection cycle is also classified into two sections, the early stage (entry) and the late stage (assembly and budding of virion).
  • RNA viruses classified into Picornaviridae, Caliciviridae, Astroviridae, Reoviridae, Flaviviridae, Togaviridae, Bunyaviridae, Coronaviridae, Rhabdoviridae, Arenaviridae, Filoviridae, Orthomyxoviridae, and Paramyxoviridae
  • DNA viruses classified into Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Hepadnaviridae, and Poxviridae).
  • RNA viruses initial viral infection arises via endocytosis or by injection of viral proteins and genes directly into the cytoplasm, by fusion of the viral envelope or by destruction of the viral capsids. Transcription and replication of DNA viruses except poxviruses generally happens inside the nucleus, whereas those of RNA viruses occur in the cytoplasm. However, influenza viruses are exceptional as RNA viruses with at least a major genome duplication occurring after transport to the target host cell nucleus. Before, after and during the transport and duplication processes, the innate immunity of the cell can act on the viral proteins and vRNA.
  • progeny viral components fragments are transferred to some organelles or to the plasma membrane, where formation of the progeny virus is processed by assembly and/or budding.
  • virus particles are classified into enveloped viruses (Herpesviridae, Hepadnaviridae, Poxviridae, Flaviviridae, Togaviridae, Retroviridae, Bunyaviridae, Coronaviridae, Rhabdoviridae,
  • Arenaviridae Filoviridae, Orthomyxoviridae, and Paramyxoviridae
  • nonenveloped viruses Parvoviridae, Papovaviridae, Adenoviridae, Picornaviridae, Caliciviridae, Astroviridae, and Reoviridae.
  • the envelope of virus particles is acquired from the plasma membrane of the cell surface, Golgi apparatus, and/or endoplasmic reticulum (ER) by budding off these membranes.
  • Influenza viruses which are highly transmittable pathogens of severe acute respiratory symptoms in various animals including human beings, internalize into host cells through multiple pathways including clathrin-independent and caveola-independent endocytosis after binding of the virus to a terminal sialic acid linked to glycoconjugates on the cell surface via viral surface glycoprotein, hemagglutinin. After transportation of the virus to late endosomes, low-pH-dependent conformation change of hemagglutinin induces membrane fusion of the viral envelope with the endosomal membrane. Then viral ribonucleoprotein complexes (RNP) including the viral genome are released to the cytoplasm of host cells by proton influx of viral ion channel M2 protein that requires binding with cholesterol.
  • RNP viral ribonucleoprotein complexes
  • Influenza virus particles consist of the viral RNP with an envelope that includes two spike glycoproteins, hemagglutinin and neuraminidase (NA), and ion channel M2 protein on the outer surface and internal Ml protein and nonstructural NS2 protein on the inner surface.
  • Membrane rafts are associated with the transmembrane domains and cytoplasmic tails of hemagglutinin and NA, with the short transmembrane domains of M2, and with NP but not with Ml. Domains of hemagglutinin and M2 contain palmitoylated cysteine residues that can associate with lipids and cholesterol in rafts. Although these domains of NA are essential for the association with rafts, there is no evidence that NA possesses palmitoylated residues.
  • the lipids are not randomly incorporated into the envelope, but virions seem to have a lipid composition different from the bulk host membrane.
  • the virion envelope appears to be determined both by the virion protein content helping to order and select to surrounding lipids AND the presence and availability of specific lipids from which to extract.
  • the cell and the culture conditions in which the progeny viruses are produced is a significant factor in determining lipid content and, to an extent, its Tm, the proteins present or deleted from the virus and mutations on these proteins as well as cell membrane lipid content and cell membrane proteins assisting in ordering the raft portions of the membrane are important factors.
  • Viruses may be engineered using molecular biology and/or mutated or adapted using for example serial culture to obtain viruses that recognize one or more selective feature.
  • Virus replication is a multi-stage process inside the respective host cell before viruses release to the environment to infect additional cells. Accordingly, to act as infectious agents viruses must cross the host cell boundary at least twice during a replication cycle, once when entering and once for exit and distribution.
  • Virus entry occurs by fusion of the incoming virus with, and cell lysis during budding of the nascent virus across a cellular membrane.
  • Virus entry is specific for susceptible host cells and depends on the viral surface proteins and receptors exposed on the target host cell membrane. Most cellular receptors are surface proteins of various functions, but sugars (i.e., one of the sialic acids for influenza virus) and lipids can function as receptors.
  • Virus entry is often enhanced by nonspecific binding, thus increasing viral residence times at the cell surface. This non-specific binding is often achieved by glycosaminoglycans (e.g., heparan sulfate), which promotes cell attachment of many different viruses by ionic or electric charge interactions.
  • glycosaminoglycans e.g., heparan sulfate
  • lipid rafts for entry. For example it has been shown that several non-enveloped viruses go through a raft-dependent entry pathway that requires cholesterol. Lipid rafts are also involved in enveloped virus entry as can be inferred from preferred binding to raft-associated viral receptors (e.g., GPI-anchored or raft-associated trans-membrane receptors). Enveloped viruses also present with a requirement for entry based on raft integrity and cholesterol.
  • the "endosomal sorting complex required for transport” (ESCRT) components found in cell and organelle membranes clearly play an important role in viral infection, especially in the release of many, but certainly not all enveloped viruses, important exceptions being the herpesvirus human cytomegalovirus, human influenza virus, and respiratory syncytial virus. These viruses recruit alternative cellular machinery or may employ viral proteins facilitating membrane scission.
  • the influenza virus M2 protein comprises an amphipathic helix that is both necessary and sufficient for vesiculation in vitro and generally for influenza virus budding in tissue culture.
  • Influenza M2 is a trans-membrane protein that self-associates to become a homotetramer providing proton-selective (H + ) ion channel activity through the virion membrane.
  • M2 binds to low cholesterol (lipid disordered) membrane regions to induce a positive curvature.
  • M2 preferentially sorts to the phase boundary of phase-separated vesicles causing extrusion of the lipid ordered (lo) domain, dependent on the presence of the amphipathic helix.
  • the M2 pore localizes to the neck of influenza virus buds in virus-producing cells. Mutation of its amphipathic helix causes late budding arrest similar to late domain mutations in other enveloped viruses.
  • M2 serves an analogous function as the ESCRT-lll/Vps4 complex in other viruses.
  • the influenza virus membrane is enriched in cholesterol and is more lo than the surrounding plasma membrane thereby creating line tension at the phase boundary demarcating the viral bud.
  • M2 preferentially sorts at this phase boundary and apparently modulates line tension through lipid membrane interaction of its amphipathic helices.
  • lipid rafts appear as subdomains of a cell's plasma membrane.
  • the rafts comprise elevated concentrations of cholesterol and glycosphingolipids. They exist as distinct liquid-ordered regions of the membrane that are resistant to extraction with nonionic detergents.
  • Lipid rafts generally contain 3 to 5-fold the amount of cholesterol found in the surrounding bilayer.
  • the lipid rafts are enriched in sphingolipids such as sphingomyelin, which is typically elevated by 50% in comparison to the disordered plasma membrane regions.
  • sphingolipids such as sphingomyelin
  • each raft is apparently small in size, but the many rafts constitute a relatively large area of each plasma membrane.
  • each raft must have a distinguishing protein and lipid composition different from the disordered lipid membrane through which it floats, but all rafts all rafts of a cell are not mandatorily identical in terms of either the proteins or the lipids that they contain.
  • vaccinia virus herpes virus, vesicular stomatitis virus, senaca virus, Semliki Forest virus, ECHO or REGVIR virus, and monstrously attenuated polio virus have been similarly tested and characterized in cancer cells or in animals or humans with cancers for their inherent cell killing effects primarily targeted at cancers.
  • the capsid is important throughout the life cycle. Quaternary arrangements in mature capsid cores are structurally conserved among retroviruses. Water and ions including H + and Ca " " " " control interactions at several interfaces in the mature capsids. Accordingly, successful viral infection is extremely dependent on the activity of water and the ions present in the environment surrounding and within the targeted cell.
  • blood will be in a slightly alkaline range of about 7.35 to 7.45.
  • Management of the pH is so important that the body's primary regulatory systems (especially breath, circulation, and renal controls) closely regulate the overall acid-alkaline balance and will counteract on a system wide or whole organism basis pH aberrations caused by local metabolic anomalies. The result is that the gross pH is generally maintained within a "normal" range irrespective of local stresses.
  • viruses including the rhinoviruses and coronaviruses that are most often responsible for the common cold and influenza viruses that produce flu
  • infect host cells by fusion with cellular membranes preferably modified by increased temperature and at low pH for optimal action in this invention.
  • pH-dependent viruses Viruses can exhibit similar temperature and/or pH sensitive selectivity through modification of the viral recognition proteins to bind, for example, an MCT4 or similar protein expressed on the surface of the metabolism-altered heat producing cells.
  • Influenza virus a member of the family Orthomyxoviridae that is an enveloped virus containing a genome comprising eight segments of negative-sense single- stranded RNA (ssRNA) has strains that are especially sensitive to pH for their target cell binding and thus can be used to preferentially target cells in low pH environs produced by cancer cells that skew metabolism towards lactic acid as a metabolic product.
  • Influenza is a lytic virus which rapidly kills the host cell when the offspring virus are released. Since flu is a lytic virus the host cell genome is immediately incapacitated and the cell can no longer divide to form offspring cancer cells.
  • retrovirus is a stronger candidate for genetic engineering of cells to correct genetic flaws, while non-lysogenic viral infection is better suited for targeting and destroying invading, diseased or otherwise unwanted cells.
  • Influenza A can swap one or more of its 8 RNA strands with co-infecting viral particles or may undergo a drift mutation, perhaps a single nucleotide base that changes a single amino acid or results in early truncation, with a robust change in transmissibility, infectivity ad/or mortality.
  • Hemagglutinin on the surface of the flu virus is instrumental for binding to and infecting target host cells.
  • the 2009 swine flu is particularly illustrative of this phenomenon. Hemagglutinin mutated to become more acid stable as this H1N1 virus shifted from swine to humans. This lowered the pH at which the flu hemagglutinin was activated. The activation process triggers an irreversible change in the hemagglutinin's shape that then fuses the virus and target cell.
  • the pH of activation is known to vary amongst various flu viruses. Avian and swine viruses are generally activated at about pH 5.5-6.0 compared to a two-fold higher [H + ] or pH about 5.0- 5.5 predominant for human flu viruses.
  • H1N1 swine viruses which were previously activated at pH 5.5-6.0 mutated to become activated at pH 5.5 at the pandemic inception and as the pandemic progressed, the activation pH of the H1N1 pandemic virus declined to 5.2-5.4.
  • This mutation process can occur naturally as pH of the target changes or for purposes of the present invention culturing susceptible cells at decreasing pH levels, where targets may be selectively cultured to decrease their pH ranges for survival and growth or by switching the target cell line if preferred. Lowering the activation pH of the hemagglutinin may be one means of selectively targeting cells that favor a more acidic metabolism.
  • Influenza viruses deliver their genomes into the nucleus as multiple single- stranded RNAs. Newly synthesized viral RNA will then exit the nucleus for assembly into virus particles on and with the plasma membrane.
  • the viral envelope can thus be engineered by choosing the host cell used to manufacture the virus particle.
  • the hemagglutinin is activated by a conformational change triggering its membrane fusion activity.
  • the viral membrane fuses with the limiting membrane of the endosome to release the nucleocapsid into the cytosol. Flu virus delivery of genetic material is rapid.
  • the total infection period - from docking onto the cell's surface to the RNA entering the cell nucleus - is two hours.
  • Influenza A because of its ability to mutate by both antigenic drift and shift is a preferred type of influenza virus for engineering select mutations in furthering this invention.
  • the pH stabilized viral particle may facilitate development or may take advantage of tunneling nanotubes to pass infectious RNA to neighbor cells without necessity for forming an envelope.
  • a class A influenza virus e.g., H3N2
  • H3N2 a class A influenza virus
  • the pH is gradually decreased with subsequent passaging. Attenuation is monitored to assure the virus remains infectious to human cells other than the cultured cell strain. In a preferred embodiment attenuation is observed at normal pH, but infectivity remains at elevated [H + ].
  • the temperature is also increased in culture to affect the content of the viral envelope to favor assimilation into membranes at increased temperatures.
  • the low pH stable virus is allowed to mix with liposomes with higher melting temperature to transfer the liposomic constituents to the viral envelope lipid coating.
  • infectious virus is again screened or tested for selective infection at depressed pH and elevated temperature.
  • Such virus may be delivered to a patient as a treatment for cancer, to target hyperproliferating cells and/or as a prophylactic event to seek out and eliminate cancerous cells that have not yet been outwardly observed, such as being palpated as a tumor mass.
  • H2 and H3, and Nl and N2 are the common human infecting hemagglutinins and neuraminidases respectively, others may mutate to be compatible with human cells as hosts and able to cause human disease and death.
  • the recent outbreak of bird flu was H7N9 killing several dozens of humans, but apparently was not able to replicate in a form transmissible from human to human.
  • virus with lytic potential but lacking transmission between untreated humans in contact with the recipient is prepared as a pH and heat targeting lytic vector.
  • Influenzas B and C may be cultured and applied in the invention for similar considerations.
  • An influenza A H5N1 virus, another drifted avian virus though weakly transmissible to humans, apparently requiring thousands of copies to infect a human can be extremely pathogenic as it may occasionally drift.
  • Influenza A viruses are especially capable of inducing the expression of cytokine and proapoptotic genes in infected cells. Pathogenicity, cell lethality, replication efficiency, and transmissibility of influenza viruses depend on both viral genetic and host factors.
  • Hemagglutinin protein binds receptors and mediates viral-cellular membrane fusion during viral entry is the primary antigenic target during infection.
  • Hemagglutinin protein is a trimeric class I membrane fusion protein that sports in its ectodomain a membrane-proximal, metastable stalk domain that is capped with a membrane-distal receptor-binding domain.
  • Hemagglutinin protein is readied for membrane fusion by cleavage of the hemagglutinin precursor into a fusion- capable hemagglutininl-hemagglutinin2 complex.
  • Some H5 and H7 hemagglutinin proteins can be cleaved by intracellular furin-like proteases to elicit systemic virus spread with enhanced virulence of such highly pathogenic avian influenza (HPAI) viruses.
  • HPAI highly pathogenic avian influenza
  • influenza virus' hemagglutinin surface glycoprotein binds sialic acid-containing receptors on the plasma membrane of a target host cell.
  • H5N1 influenza virus hemagglutinin proteins bind preferentially to (2,3)-linked sialosides.
  • human-adapted influenza viruses bind preferentially (2,6)-linked sialosides.
  • a switch from (2,3) receptor binding specificity to (2,6) receptor binding specificity may be preferred in adapting avian influenza viruses for mammalian hosts.
  • Hemagglutinin proteins from different strains and subtypes vary in activation pH values with a range from ⁇ 4.6 to ⁇ 6.0.
  • Hemagglutinin proteins from HPAI viruses normally exhibit an activation pH value at the higher end of the range ⁇ 6.0, while human seasonal viruses have lower pH activation values, ⁇ 5.0 or less.
  • H5N1 influenza virus isolates cluster in a range of ⁇ 5.3 to ⁇ 5.9. For individual viruses grown in sequential culture genetic drift is an effective tool for directed mutation towards a desired activation pH range to match that of a target host cell. For example, in HI, H3, and H7 influenza viruses, mutations that alter the hemagglutinin activation pH have been associated with changes in virulence in mice.
  • the hemagglutinin protein After receptor binding and internalization during influenza virus entry, the hemagglutinin protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion, and initiation of cell lethal infection either through apoptosis or other cell death or through lytic release of virus.
  • TLRs Toll-like receptors
  • TLRs 1-10 (appropriately numbered TLRs 1-10).
  • the various TLR proteins bind different type targets, for example, TLRs 1 and 2 are involved in bacterial infections through their recognition of lipopeptides (1) and lipopeptides, lipoproteins and glycolipids (2); TLR3 recognizes double stranded RNAs and thus is preferentially effective against viruses.
  • TLRs 7, 8 and 10 are activated in the presence of ssRNAs, especially of the types found in influenzas.
  • TLR7 and TLR8 especially recognize GU of AU rich sequences of ssRN A viruses such as the Orthomyxoviridae family that includes influenza virus.
  • TLR 10 expression Compared with seasonal influenza virus H1N1, highly pathogenic avian influenza virus H5N1 is a more potent inducer of TLR 10 expression. Influenza virus infection increases associated TLRs expressions which contribute to innate immunity through their sensing the viral infection. This leads to cytokine induction, especially proinflammatory cytokines and interferons. Since TLR 10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a less pathogenic H1N1 virus H5 influenzas are a preferred initiator of cell death.
  • H1N1 viruses may be effective for infecting human cells, previous exposures to similar H/N epitopes may compromise access to target cells. Accordingly, it is advised to be cognizant of recent flu outbreaks that may have produced antibodies and other humoral reservoirs that might neutralize specific cell lines.
  • influenza virus But as humans and other organisms have adapted to minimize and therefore better survive viral invasion, viruses also adapt to continue viral propagation.
  • the 11 proteins encoded by influenza virus the NS1 protein has been shown to block the production of IFN- ⁇ in infected cells. Such adaptations of an influenza virus allow it to evade host cell innate immunity.
  • influenza viral protein NS1 serves to bind viral RNA with its RNA binding domain to shield it from contacting ssRNA sensitive TLRs and retinoic acid inducible gene-l (RIG- I) a protein recognizing dsRNA including looped ssRNAs that complementarily bind.
  • TLRs, RIG1 and the like When stimulated by binding RNA, the TLRs, RIG1 and the like induce type I interferon production.
  • Some NS1 proteins also bind the tripartite motif-containing protein 25 (TRIM25) that works though the activation of RIG-I.
  • NSls apparently also can complex with RNA-dependent protein kinase (PKR) and inhibit it.
  • PTR RNA-dependent protein kinase
  • PKR is activated by binding double-stranded viral RNA and causes translation arrest in the cell nucleus including inhibition of viral protein synthesis.
  • influenza virus M2 protein can inhibit P58IPK also inhibiting protein synthesis, and arresting host cell apoptosis.
  • Influenza PB1-F2 with a serine at position 66 is especially adept at inhibiting type I interferon production.
  • This PB1-F2 binds to and inactivates mitochondrial antiviral signaling protein (MAVS).
  • MAVS mitochondrial antiviral signaling protein
  • PB1-F2 protein is also associated with the induction of apoptosis and has a synergistic effect on the function of influenza virus polymerases PA and PB2.
  • PB2 can also bind and inhibit the interferon promoter stimulator 1 (IPS-1) that normally promotes IFN- ⁇ production.
  • IPS-1 interferon promoter stimulator 1
  • defective viruses i.e., viruses lacking a full component of genetic material and associated proteins to reproduce more virus particles, and/or notably non-virulent viruses, e.g., viruses easily attacked by the host cell innate immunity, can be considered as viable or even preferred embodiments for use in the present invention.
  • flu viruses with one, two, three, four, five, six, seven, or even all eight RNA strands absent or modified to be incapable of expression or to result in a strong antiviral response and/or to lack viral defense against host cell antimicrobial defenses can still target the hyperproliferating cancer cell and by initiating cell apoptosis or other cell death, even non-productive lysis, serve the appropriate functions envisioned in this invention.
  • Ade no-associated virus in contrast, cannot gain cytoplasmic access by membrane fusion.
  • AAV comprises single stranded DNA (ssDNA).
  • the AAV genome is manipulatable by inserting chosen genes, including genes encoding interfering RNA molecules.
  • Various receptors can be selectively targeted.
  • secondary receptors including, but not limited to: fibroblast growth factor receptor (FGFR), 47/67kDa laminin receptor, 37/67kDa laminin receptor, hepatocyte growth factor receptor (HGFR), ⁇ / ⁇ 5 and 5 ⁇ 1 integrins, platelet-derived growth factor receptor (PDGFR), etc. have been observed as specific targets for binding by specific AAVs.
  • viruses of this sort may be selected to avoid undesired contagion of the treatments, generally gene therapy constructs in recent literature.
  • AAV has been mutated using site directed and random mutagenesis to alter its pH sensitive components.
  • PRRs Pathogen recognition receptors
  • RIG-I pathogen recognition receptors
  • Bcl-2, Bcl-xL, and Bcl-w Anti- apoptotic and pro-apoptotic (Bax, Bak, Bad, Bim, Bid, Puma, and Noxa) Bcl-2 proteins react to initiate the cascade of reactions leading to apoptosis with the early step involving mitochondria membrane permeabilization (MoMP) causing release of cytochrome c, apoptosome activation, ATP degradation, etc., ending with cell death.
  • MoMP mitochondria membrane permeabilization
  • the concentration of vRNA is a critical result-limiting factor so apoptosis becomes more likely as viral RNA is synthesized.
  • PB1- F2 protein of influenza A viruses depositing on mitochondria where it interacts with VDACl and ANT3 to decrease mitochondrial membrane potential (MMP), which induces the release and self-associations of proapoptotic proteins that cause cell death.
  • MMP mitochondrial membrane potential
  • PB1-F2 also forms non-selective protein channel pores that lead to the changed mitochondrial morphology, dissipation of MMP, and efficient cell death.
  • the M2 protein of influenza virus, another viroprotein that causes changed mitochondrial morphology and depletion of MMP, is another means whereby a viral infection can cause cell death.
  • the couriers may preferably transport a molecule whose effects are multiplied in the cell,
  • the courier may carry RNAi with downstream effects on one or more pathways, may carry transcription factors, methylation factors, demethylation factors, an engineering cassette such as used in CRISPR/cas, a plasmid that can infect mitochondria, a ligand that opens a pore in an organelle such as the nuclear membrane or mitochondrial membrane, packets that increase expression of a protein or group of proteins to favor or disfavor one or more metabolic pathways, such as the Electron transport pathway of mitochondria, mitochondrial fusion or fission, anti-apoptotic or pro-apoptotic compounds such as Bel or Bad, etc.
  • All cells are living entities and as living things they require raw materials to maintain function, to grow and to reproduce. Lone cells can obtain their nutrition from the immediate surroundings. But in complex organisms, where the cell may be distant from the outside environment a delivery service is necessary. In larger animals the circulatory system is responsible for delivering and clearing food and waste.
  • a blood supply transgressing through a system of tubes (blood vessels) is used. As the organism grows each part must be supplied with appropriate blood vessels for support. The formation of blood vessels requires migration and proliferation of endothelial cells. These endothelial cells must be fueled in order to form and maintain the circulatory system.
  • the circulatory system is also an information system. Blood can carry chemical messages to and from the cells it services. The message does not need a locational address. Since cells are in contact with the environment (interstitial space) they are constantly removing chemicals from the space and depositing chemicals into it. The tools on the cel l surface that help transport chemicals across the cytoplasmic membrane are exposed to the interstitial space. If a molecule has characteristic affinity for one of these "receptors" it will associate as a ligand with the receptor. A receptor may have one of many functional characteristics. It may serve to allow viral attachment to the cell membrane.
  • the receptor often will induce further changes inside the cell to manage (or metabolize) in some way the molecule being brought into the cell. While often signals are molecules manufactured by one cell and delivered to another to instruct that cell what it should do, simply classical food molecules can serve as signals to upregulate the pathways needed to metabolize that type of molecule.
  • the cell when the cell is behaving in a specialized manner, the cell often must alter its pathways to support the specialized needs. Or in the chicken-egg question, when the cell has activated surprising metabolic pathways, then the cell will by necessity be doing something distinct from "normal" cells.
  • a growth signaling receptor protein when activated will initiate a signal cascade through to the cell nucleus to build food receptors and carriers and to transport these receptors and carriers to the plasma membrane.
  • a sugar or amino acid then contacts the receptor and is carried inside.
  • the carrier/transporter will initiate or activate an appropriate pathway inside the cell to metabolize the cargo.
  • the cargo is aminated or otherwise modified to divert to a less common metabolic pathway or to serve as an intracellular signal.
  • Acetyl Co-A One popular branching point, i.e., a molecule that might be directed through several pathways is acetyl Co-A. Often acetyl co-A is produced from the degradation of carbohydrates and/or proteins. But, especially in circumstances where nucleic acid synthesis is required (e.g., rapidly proliferating cells or cells expanding mitochondria) mass) fatty acids may become a favored source of carbon. Acetyl-CoA is a lipogenic precursor for many lipid molecules including, but not limited to: isoprenoid, cholesterol and fatty acids.
  • oxaloacetic acid which may also be directly exported from the TCA cycle, supplies pools of non-essential amino acids.
  • Mitochondria are organelles in cells that are classically known for production of ATP from electron transfer (oxidation/reduction) reactions.
  • the size and shape of mitochondria can vary within a single cell and each mitochondrial package may contain plural copies of the mitochondrial genome which is a double stranded circular DNA molecule that encodes 37 genes.
  • Mitochondria are dynamic organelles that can migrate within a cell along cytoskeleton framework. Mitochondria can grow by fusing with other mitochondria and may dissociate in a process termed fission that allows split up smaller mitochondrial bodies to move more freely and to different locations within the cell. The smaller mitochondria produced through fission have reduced distance for diffusion of substances they make and use. Mitochondria can grow by adding additional membrane and protein materials and may be digested through a process termed mitophagy or autophagy. In general, smaller mitochondrial bodies will have better communication with the cytoplasm due to reduced volume to surface ratio.
  • One target of cancer treatment could theoretically involve hindering the ability of cancer cell mitochondria to participate in either of these fusion or fission processes and thereby impact general mitochondrial functioning.
  • accelerating the fission process in comparison to fusion may be one means through which neoplastic cells can diminish their death through apoptosis. Maintaining joined mitochondria as favored by fusion processes appears to make an apoptotic event more possible.
  • Several proposed rounds for use in practicing the present invention emphasize maintenance of fused mitochondria.
  • Mitochondria in cells are consistently changing. They are transported along the cytoskeleton to areas of need. They may change from more rodlike to more spherical shapes depending on location within a cell. During these processes, mitochondria may fuse together and may split apart under control of proteins within the cell.
  • Drpl mitochondrial membrane proteins essential for mitochondrial fusion
  • mitofusin 1 Mini
  • mitofusin 2 Mfn2
  • Drpl another essential protein for maintaining healthy mitochondria
  • Drpl a primarily cytosolic protein.
  • Drpl When bound to a mitochondrion, Drpl forms a constrictive ring around a mitochondrion to split it into two parts.
  • Drpl is one of the GTPase proteins in mammalian cells. Drpl interacts with several proteins including, but not limited to: Fisl, Mff, MiD49 and MiD51, that act on the mitochondrial surface to initiate and control mitochondrial fission.
  • Fission is important for maintaining a healthy mitochondrial population and appears to be necessary for cells to proliferate. Fission often precedes mitosis, perhaps to make equal division easier. Drpl activated mitochondrial fission is associated with inhibiting apoptosis, a property opposite that of eliminating the individual cell. Thus interfering with activity of any of these proteins may slow fission and maintain mitochondria in a fused state. Cancer cells are characterized by relatively fewer fused mitochondria with respect to more independent or smaller separate mitochondria than seen in non-malignant cells. Consistent with this observation is a finding that Drpl expression is elevated in cancer cells and that the fraction of Drpl phosphorylated at the serine residue at position 616 in Drpl, activated Drpl is elevated. Apparently, cancer cells increase phosphorylation at this spot with the effect of favoring fission activities.
  • Drpl Another inhibitor of Drpl is a compound known as P110.
  • the polypeptide P110, DLLPRGT appears more selective for blocking Drpl/Fist interaction than Drpl interaction with other ligands.
  • a novel Drpl inhibitor diminishes aberrant mitochondrial fission and neurotoxicity.
  • Xin Qi Nir Qvit, Yu-Chin Su, Daria Mochly-Rosen. J Cell Sci 2013 126: 789-802; doi: 10.1242/ jcs.1144391.
  • Delivering one or more Drpl inhibitors in a cocktail to the cancer cell targets can potentiate other pro-apoptotic interventions. Countering Cancer's Metabolic Changes
  • Cancer cells are distinguished from other cells usually based on their loss of controlled functions normally carried out by that organ or cell type and by their hyperproliferation. While the hyperproliferation can be understood from the viewpoint of the cell whose fittest life mission is to grow and continue its cell lineage, from the organism's point of view this group of rogue cells is not supportive of the survival life of the large organism: First, these cells are not performing activities for the good of the whole organism. Second, these cells are wasting nutrients. Third, the increased volume occupied by these cells interferes with communication and other functions of the non-cancer cells. Fourth, these cells are consuming (wasting) resources that could be more advantageously used. And fifth, these cells may be exporting toxic or problematic metabolites requiring surrounding tissues to expend resources and effort in clean-up operation.
  • the cells will also differ in the way they utilize intracellular and extracellular nutrients. Addressing these differences provides strategies for impeding tumor growth and tumor cell proliferation. For example, as the cells hyperproliferate, pathways for manufacturing purines and pyrimidines for nucleic acids must be accelerated.
  • Enhanced glucose uptake is a hallmark of several cancers and has been exploited in the clinic as a diagnostic tool through PET imaging of the glucose analogue 18F-deoxyglucose (18FDGPET).
  • FDGPET glucose analogue 18F-deoxyglucose
  • cancer cells preferentially convert glucose to lactate a three carbon molecule that retains and eventually removes energy unavailable for ATP synthesis.
  • the fate of glucose inside cells is influenced by the enzymatic properties of the specific glycolytic gene products expressed.
  • PKM2 pyruvate kinase
  • cancers are believed to present with a genetic abnormality.
  • genes have alleles that support or initiate development of cancer. For example, greater than 50 cancers are associated with genes that increase cancer risk in individuals inheriting one or more copies from a pa rent.
  • BRCA1 and BRCA2 associated with breast cancer
  • TP53 Li-Frau meni syndrome
  • PTEN Pin Syndrome
  • viruses e.g., human cytomegalovirus, Epstein-Barr virus, human papillomavirus, hepatitis B virus, and hepatitis C virus are also genetic factors that increase cancer risk, i.e., events contributing to su pporting a cancer cell's metabolic tra nsformations.
  • An external event switching a gene on or off may initiate or contribute to the ca ncer cascade. If the organism is inattentive to the changing cell, the cell may be allowed to continue development to a cancerous status. But to support the change the cell will have to adapt.
  • We have evolved means to halt the requisite adaptations. For example, either inherited or somatic mutations of TP53, a tumor suppressor gene for p53 protein, removes a brake on growth of abnormal cel ls and allows the metabolic transformations necessa ry for cancer cell proliferation to proceed.
  • Another gene with tumor suppressive activity a nd whose mutation removes restraints on uncontrolled growth is CHEK2.
  • Some adaptations will be built in, in accordance with feedback loops that evolution has given us; some may involve additional mutations in the nuclear or mitochondrial genomes; some may be more complex evolved responses, for example, an epigenetic modification like methylation.
  • One simple course of treatment will be to support "normal" metabolism. That is to provide raw material (nutrients) supporting normal metabolism, for example to favor electron transport chain activity. In concert with this can be a restriction on types of raw materials supporting the diverted or cancer enhanced or enhancing metabolic pathways. A more aggressive strategy may include inhibitors of one or more of these side pathways. When these cells are deprived of the environment in which they mutated and may have in fact contributed to, selective pressure will tilt against these cells in favor of the "normal" cells.
  • Nutrition can also be altered with a goal of supporting apoptotic activity and inhibiting cells that counter apoptosis.
  • Gene editing technologies Recent developments of technologies to permanently alter the hu man genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications in CNS disorders such as Parkinson's disease (PD) or Alzheimer disease (AD). These technologies are now commonly known as “genome editing.”
  • Current gene editing technologies comprise zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system or a combination of nucleases (e.g. mutated Cas9 with Fokl) (Tsai, S. Q., Wyvekens, N., Khayter, C, Foden, J.
  • Another approach to minimize off-target effects is to only introduce single strand breaks or nicks using Cas9 nickase (Chen et al., 2014; Fauser et al., 2014; Rong et al., 2014; Shen et al., 2014).
  • the CRISPR/Cas9 nuclease system can be targeted to specific genomic sites by complexing with a synthetic guide RNA (sgRNA) that hybridizes a 20-nucleotide DNA sequence (protospacer) immediately preceding an NGG motif (PAM, or protospaceradjacent motif) recognized by Cas9.
  • sgRNA synthetic guide RNA
  • PAM protospaceradjacent motif
  • PCSK9 For heart disease, permanent alteration of a gene called PCSK9 using CRIPR technology reduces blood cholesterol levels in mice (Ding et al., 2014). This approach was based on the observation that individuals with naturally occurring loss-of-function PCSK9 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels and protection against cardiovascular disease (Ding et al., 2014).
  • LDL-C blood low-density lipoprotein cholesterol
  • a second example for the feasibility of this approach is HIV. Individuals carrying the inherited Delta 32 mutation in the C-C chemokine receptor type 5, also known as CCR5 or CD195 are resistant to HIV-1 infection. Gene modification in CD4 T cells were tested in a safety trial of 12 patients and has shown a significant down- regulation of CCR5 in human (Tebas et al., 2014).
  • the present invention provides for the arrest and/or prevention of neurodegeneration associated with
  • arrest and/or prevention of neurodegeneration is accomplished using gene editing
  • the present invention provides a method of treating a neurological deficit associated with neuropathological disease comprising administering a genetically engineered vector comprising a gene for a nuclease and a promoter for the nuclease, as well as an appropriate molecular "guide” into a cell.
  • the vector facilitates an expression of a molecular component that alters a gene in the cell or expression of a targeted gene associated with the neuropathology in the cell.
  • the affected gene would be implicated in an etiology of the neurological deficit.
  • a medical composition for treating a neurological deficit in a patient includes a nuclease that introduces double strand break in a gene implicated a neurological deficit, a guide RNA that targets a gene implicated in neurological disease, and a delivery system that delivers the nuclease and guide RNA to a cell.
  • modification of gene and/or genomic region may be interpreted to include one or more of the following events (FIG. 1):
  • This modification in some embodiments provides a permanent mutation in a cell or population of cells having the modified gene.
  • other proteins e.g. transcription factors, polymerases or other proteins involved in transcription
  • CRISPR sgRNA introduces small insertions or deletions through non- homologous end joining (NHEJ), in general several nucleotides, rarely larger fragments (Swiech et al., 2014).
  • NHEJ non- homologous end joining
  • HDR Homology-directed repair
  • sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in promoter region.
  • sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in regulatory regions or intronically.
  • Gene editing or modification can be achieved by use of any variety of techniques, including zinc-finger nuclease (ZFN) or TAL effector nuclease (TALEN) technologies or by use of clustered, regularly interspaced, short palindromic repeat (CRIPSR)/Cas9 technologies or through the use of a catalytically inactive programmable RNA-dependent DNA binding protein (dCas9) fused to VP16 tetramer activation domain, or a Krueppel-associated box (KRAB) repressor domain, or any variety of related nucleases employed for gene editing.
  • ZFN zinc-finger nuclease
  • TALEN TAL effector nuclease
  • CIPSR short palindromic repeat
  • dCas9 catalytically inactive programmable RNA-dependent DNA binding protein
  • KRAB Krueppel-associated box
  • 20170015994 evidence the utility, feasibility and enablement of gene editing processes with high specificities a re well known and accepted in the art.
  • the weapon When a cell characteristic can be targeted, e.g., a Ras expressing cell targeted by a virus, the weapon might be factors to turn on, activate, augment, or duplicate activity of desired proteins. These can be proteins supporting and restoring more normal metabolism, but might also be proteins supporting cel l death, for example proteins supporting initiation or progression of apoptosis. On the flip side, anti-apoptotic protein activity or expression might be blocked.
  • Tra nscription factors or other manipulation of transcription may be used to increase expression of a protein or to throttle it down.
  • the ta rgeted gene need not be a gene mutated in the cancer process, so long as the cellular process is acceptably targetable.
  • These might be protein or nucleic acid based and could be directed against a modified gene, of course, or could be targeted against a more ubiquitously required or associated gene necessary to accomplish or prevent a proliferation event.
  • Genes involved in the cel l cycle, genes involved in cytoskeleton, genes required for mem brane integrity, genes required for a ny cellular or subcellula r process, etc., essentially any well used or essentially expressed gene might be selected for the ultimate target.
  • RNAi can be used to inhibit transcription and therefore protein activity.
  • DNA or modified DNAs may be incorporated into genomic material of the nucleus or mitochondrion.
  • RNAi molecules can be put to assorted applications including interruption of protein translation.
  • miRNA is encoded in nuclear DNA and several viruses as a means for turning off translation of messenger RN A (mRNA) molecules. Though shorter than common synthetic siRNA constructs their actions are similar to those of siRNA in ability to turn off protein production. But because of their shorter region of complementarity miRNAs often impact expression of several, possibly hundreds of mRNAs. Nuclear DNA includes portions that encode for miRNA. These portions may be found in either introns or exons and in many cases are constitutively expressed. Once in the cytoplasm miRNAs appear to have half-lives many fold longer (some estimate _ 10-fold longer) than their targeted mRNAs.
  • mRNA messenger RN A
  • the applicability exogenous miRNA for controlling cell function is evidenced by the ability of many viruses to use miRNAs to shut off a cell's anti-viral defenses.
  • Engineering a viral vector to include desired miRNA precursors can be useful for controlling expression of most proteins, including especially mitochondrial proteins.
  • Short interfering RNA are double stranded self-complementary RNA molecules popular for use in research and genetic therapeutic applications.
  • the siRNA is stabilized by proteins in a RSC complex within the cell to present a single stranded region available for complementary binding with mRNA. Once bound the mRNA is cleaved thereby preventing its translation to polypeptide and marking it for degradation.
  • Therapists have preferred siRNA over miRNA because of siRNA's shorter half-life and because its targets are more limited and therefore specific.
  • Synthetic siRNAs are deliverable to cells by methods known in the art, including, but not limited to: including cationic liposome transfection and electroporation.
  • exogenous siRNA have short term persistence of the silencing effect limiting risk of long term off-target effects.
  • siRNA or miRNA can be incorporated into a host genome through genetic engineering.
  • Genetic engineering can include miRNAs that down-regulate gene expression at the post transcriptional or translational level. Engineering may include substituting the native sequences of the imiRN A precursor with miRNA sequence complementary to a target mRNA encoding any carrier, enzyme, receptor, pore, etc. of choice.
  • the vectors delivering the novel or foreign miRNA can be used to produce siRNAs to initiate interference against specific mRNA targets.
  • siRNAs short interfering nucleic adds
  • siNAs short interfering nucleic adds
  • US Patents and Patent Applications such as: 20160244760, 20160053269 RNA Interference Mediated Inhibition Of Gene Expression Using Chemically Modified Short Interfering Nucleic Add (siNA), 20170022146 Novel Low Molecular Weight Cationic Lipids For Oligonucleotide Delivery (SIRNA Therapeutics (Merck), now owned by Anylam); 20160331828, 20160317647 Nucleic add Vaccines, 9464124, 20160271272
  • Pyruvate kinase catalyzes the last step of glycolysis, transferring the phosphate from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to yield adenosine triphosphate (ATP) and pyruvate.
  • PEP phosphoenolpyruvate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • pyruvate kinase In mammals, two genes encode a total of four pyruvate kinase isoforms.
  • the Pkrl gene encodes the PKL and PKR isoforms, expressed in the liver and red blood cells respectively. Either the PKM1 or PKM2 isoform encoded by the Pkm gene is found in cells.
  • PKM1 is found in many normal differentiated tissues whereas the PKM2 is expressed in most proliferating cells including all cancer cell lines and tumors tested.
  • PKM1 and PKM2 are derived from alternative splicing of a Pkm gene transcript by mutual exclusion of a single conserved exon that encodes 56 amino acids. Despite the similar primary sequences, PKM1 and PKM2 have different catalytic and regulatory properties. PKM1 appears always active, exhibiting high constitutive enzymatic activity. In contrast, PKM2 is less active, but is allosterically activated by the upstream glycolytic metabolite, fructose-l,6-bisphosphate (FBP).
  • FBP fructose-l,6-bisphosphate
  • PKM2 can interact with proteins harboring phosphorylated tyrosine residues thereby releasing FBP which, in a feedback mechanism, reduces the activity of the enzyme.
  • Low PKM2 activity in conjunction with increased glucose uptake, facilitates use of glucose carbons into anabolic pathways derived from glycolysis.
  • PKM2, but not PKM1 can be inhibited by direct oxidation of its cysteine 358 as an adaptive response to increased intracellular reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • PKM2 expression in cancer cells has been associated with enhanced phosphorylation of the Hu on phosphoglycerate mutase 1 (PGAMl) by PEP.
  • This pathway is an alternative route for pyruvate production but bypasses the generation of ATP via the pyruvate kinase step. This su pports high rates of glycolysis.
  • Replacement of PKM2 with the constitutively active isoform PKM1 results in reduced lactate production, enhanced oxygen consumption, and a decrease in PGAMl phosphorylation.
  • PKM2 expression may evidence selection against high pyruvate kinase activity and therefore against expression of PKM1. This rationale suggests that activation of PKM2 may impede cancer cell proliferation by interfering with regulatory mechanisms critical for proliferative metabolism.
  • PKM2 activators will mimic the regulatory properties of constitutively active PKM1, thereby promoting high PKM2 activity regardless of the known mechanisms cells use to decrease pyruvate kinase activity. Similar to results observed when PKM2 is replaced with PKM18, under standard tissue culture conditions, PKM2 activators had no significant effects on cell proliferation when tested across several lines. In contrast, when proliferation is assessed under hypoxic conditions ( 1% 0 2 ), PKM2 activator treatment results in decreased rate of cell proliferation in comparison to DMSO-treated cells. And expression of PKM1 in the presence of endogenous PKM2 has no effect on cell proliferation in standard tissue culture conditions, but inhibits proliferation under hypoxia to a similar degree as treatment with PKM2 activators.
  • PKM2 replacement of PKM2 with PKM1 also impairs cell proliferation under hypoxic conditions.
  • Cancer cells harbor genetic changes that allow them to increase nutrient uptake and alter metabolism to support anabolic processes, and interfering with this metabolic program is a viable strategy for cancer therapy.
  • Altered glucose metabolism in cancer cells is mediated in part by expression of PKM2, which has specialized regulatory properties.
  • PKM2 is allosterically activated by FBP and can interact with tyrosine-phosphorylated proteins to release FBP and decrease enzyme activity.
  • growth factor signaling promotes decreased PKM2 activity and availability of glycolytic metabolites for anabolic pathways that branch from glycolysis. This suggests that activation of PKM2 might oppose the effects of growth signaling and interfere with anabolic glucose metabolism.
  • Mitochondria have a limited set of genes in their genome. Most proteins in the mitochondrial mem branes and matrix are encoded in the nuclear genome before being translated on the cytoplasmic ribosomes. These nuclear encoded mitochondrial genes include, but are not limited to: mitochondrial enzymes, mitochondrial membrane pore and carrier proteins and chaperone or folding proteins.
  • the mitochondrial genome consists of one double stranded DNA polymer in a circular format, i.e., no apparent beginning or end. Mitochondrial genes can code for RNA or polypeptide polymers. The 37 mitochondrial genes are split between the two
  • a strand with higher guanine cytosine ration is called the H- strand and the com plement is dubbed the L-strand.
  • the H-strand is richer in genes with twenty-eight of the thirty-seven.
  • L-strand genes include TRNA, TRNC, TRN E, TRNY, TRN N, TRN P, TRNS1, N D6 AN D CR; while the H-stra nd genes encode TRNT, CYTB, N DS, TRN L2, TRN 2, TRN H, N D4, N D4L, TRN R, N D3, TRNG, COX3, ATP6, ATP8, TRN K, COX2, TRN D, COX1, TRNW, N D2, TRN M, TRN I, N D1, TRNL1, RRN L, TRNV, RRNS and TRN F.
  • a typical cell will contain between 10 2 and 10 4 DNA molecules (paired strands). However, sex cells vary with the egg carrying "2xl0 5 and sperm bringing 10 1 or fewer. Typically perhaps 15 mitochondria may harbor up to 500 genome molecules total. But numbers vary with cell type and with time in a given cell.
  • mitochondrial genes encode molecules that remain in the mitochondrion. Only humanin (an anti-apoptotic protein) is mtDNA encoded (by the larger ribosomal RNA encoding gene), but human is exported from the mitochondrion and exerts its effects after release into the cytoplasm.
  • the mitochondrion has its own ribosomal RNAs (2) and tRNAs (22). Leucine and serine each have two tRNAs.
  • Mitochondrial proteins encoded by mitochondrial DNA are involved in the electron transport chain which has five complexes: NADH:ubiquitone reductase, succinate dehydrogenase, cytochrome k J cytochrome c oxidase and ATP synthase. Each of these complexes resides in the inner mitochondrial membrane.
  • Ndufaf3 C3orf60
  • Ndufaf4 C6orf66
  • Ndufafl CIA30
  • C20orf7 C20orf7
  • Ecsit Indl
  • Ndufaf2 B17.2L
  • proteins encoded by nuclear DNA but transported into the mitochondria include but are not limited to: phosphoenolpyruvate carboxykinase, hinge protein (fragment), 14-3- 3 protein epsilon, tryptophanyl-tRNA synthetase, VDAC4 protein (Fragment), voltage-dependent anion-selective channel protein 3, voltage-dependent anion channel (fragment), voltage- dependent anion-selective channel protein 2 , voltage-dependent anion-selective channel protein 1, vesicle-associated membrane protein 1 (VAMP-1) (synaptobrevin 1), ubiquinol- cytochrome C reductase complex 11 kDa protein, ubiquinol-cytochrome C reductase iron-sulfur subunit, ubiquinol-cytochrome C reductase complex core protein 2, ubiquinol-cytochrome C reductase complex core protein I, ubiquinol-cytochrome C reductase complex 14 kD
  • dehydrogenase superoxide dismutase [Mn]
  • Smac protein sodium/hydrogen exchanger 6 (Na+/H+ exchanger 6) (NHE-6), ADP/ATP carrier protein, liver isoform T2 (ADP/ATP translocase 3), ADP/ATP carrier protein, fibroblast isoform (ADP/ATP translocase 2), ADP/ATP carrier protein, heart/skeletal muscle isoform Tl (ADP/ATP translocase 1), Phosphate carrier protein, mitochondrial precursor (PTP), mitochondrial 2-oxodicarboxylate carrier, mitochondrial carnitine/acylcarnitine carrier protein, mitochondrial deoxynucleotide carrier, solute carrier family 25, member 18, peroxisomal membrane protein PMP34 , mitochondrial ornithine transporter 1, brain mitochondrial carrier protein-1 (BMCP-1), calcium-binding mitochondrial carrier protein Aralar2, calcium-binding mitochondrial carrier protein Aralarl, mitochondrial 2- oxoglutarate/malate carrier
  • dehydrogenase ⁇ - ⁇ -subunit fragment
  • pyruvate dehydrogenase ⁇ - ⁇ -subunit fragment
  • peptide deformylase mitochondrial 28S ribosomal protein S30 (MRP-S30)
  • programmed cell death protein 8 phosphoenolpyruvate carboxykinase, mitochondrial precursor [GTP], phosphoenolpyruvate carboxykinase, cytosolic [GTP] , propionyl-CoA carboxylase ⁇ chain, propionyl-CoA carboxylase a chain, pyruvate carboxylase, transmembrane protein, succinyl- CoA:3-ketoacid-coenzyme A transferase, cytochrome oxidase biogenesis protein OXA1, ornithine carbamoyltransferase, mitochondrial ornithine transporter 2, optic atrophy 3 protein, dynamin- like 120 kDa protein, mitochondrial outer membrane protein
  • Gene editing involves excising, inserting or substituting one or more genes or epigenetic modification of a gene, i.e., modifying a gene sequence or modifying ability of a transcription factor to bind and initiate or halt a gene's transcription.
  • Excising a gene will require the DNA molecule to be cleaved at the beginning and end of the DNA strand being removed. Insertion requires but one cleavage point with each end of the opening being compatible (usually short complementary overlapping single stranded endpoints). Substitution events require both excision and insertion. Sometime the excision and insertion sites are identical, but this is not an absolute requirement.
  • DNA molecules are nucleotide acids and are cleaved by nuclease enzymes (nucleases).
  • nucleases nuclease enzymes
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR-Cas system CRISPR-Cas system
  • Gene editing systems can be made specific to mutated sequences, including epigenetic mutations. In cells then only undesired mutations could be made to serve as a check to prevent side effects on healthy cells.
  • the recognition site might be used simply to allow correcting a single mutation, but given that the cancer process involves many events in many of the cell's compartments in many instances the mutation recognition will serve as confirmation for the vector to effect a fatal cleavage or to insert a DNA sequence designed to be fatal to the cell.
  • nuclear DNA success rates are expected to be higher because of the limited number of targets in each cell as compared to the multiple copies in each mitochondrion and the multiple mitochondria per cell. To further improve efficacy and to take into account the continuing change as cancers develop and mature multiple targets and/or multiple fatal outcomes can be programmed into the editing processes.
  • One exploitable feature is that if a significant number of mitochondria are compromised, the mitophagy/autophagy process, Ca “ " " leakage, pore openings, cytochrome c release, etc. will induce or cause the targeted cell's death. Thus when the intent is to destroy rather than correct hyperproliferating cell only a portion of mitochondria need be compromised. Accordingly one preferred strategy for triggering death of cancer cells is delivery of a proliferating mitochondrial vector to a targeted cell wherein a sufficient number of the mitochondria are modified either in the mitochondrial genome, mitochondrial membrane, delivery of components to the mitochondria, etc., to cause the mitochondria to elicit cell death.
  • the invention may incorporate actions and/or compositions the impact transcription, translation, cytoskeleton control or other factors that modulate the propensity or ability of proteins which disfavor apoptotic events in the cell.
  • proteins include, but are not limited to: Bcl2, BclXI, BclxES, and Nip3.
  • the invention may incorporate actions and/or compositions the impact transcription, translation, cytoskeleton control or other factors that modulate the propensity or ability of proteins which favor apoptotic events in the cell.
  • proteins include, but are not limited to: Bax, Bak, Bad, Bid, Bim, NoxA, Puma, proline oxidase, p53, cytochrome C, HsplO,
  • SMAC/DIABLO apoptosis inducing Factor
  • AIF apoptosis inducing Factor
  • endonuclease G IAP inhibitor: omi/high tem perature requirement protein A2 (HtrA2), adenine nucleotide tra nslocator (ANT), cyclophilin D, peripheral benzodiazepine receptor, and procaspases.
  • HtrA2 omi/high tem perature requirement protein A2
  • ANT adenine nucleotide tra nslocator
  • cyclophilin D cyclophilin D
  • peripheral benzodiazepine receptor and procaspases.
  • the mitochondrial genome and the mitochondrion itself have evolved in parallel with the nuclear genome and the cells which mitochondria support. Metabolic processes (the bases of life are divided between these compartments.
  • the mitochondrion is best known for the Electron Transport Chain, the TCA or Krebs cycle for efficient production of ATP. Mitochondria also are responsible for producing acetyl CoA for use in the mitochondrion and cytoplasm. And fatty acid oxidation resides in the mitochondria) matrix.
  • Shorter fatty acids can diffuse into the mitochondrion. However, longer fatty acids are reacted with coenzyme A to become esterified as a fatty acyl-CoA. This complex is carried into the intermembrane space, but must be back-converted to acyl- CoA to cross the inner mitochondrial membrane and gain access to the enzymatically active matrix.
  • fatty acids B oxidation of fatty acids takes a long route. Free fatty acids are carried by a transporter protein e.g., FAT/CD36, SLC27, FATP, and FABP pm from the interstitial space to the cytoplasm. Or fatty acids can be made available internally by autophagy or other degradative processes. In the cytoplasmic compartment the fatty acid is adenylated consuming two active phosphates (ATP4 AMP) before a CoA group is added to the fatty acid by fatty acyl-CoA synthase (FACS) to make long-chain fatty acyl-CoA. But long chain fatty acyl-CoAs cannot cross the mitochondrion's outer or inner membranes. Carnitine pal mitoyltransferase 1 (CPT1) substitutes carnitine for CoA to form an carnitine-CoA which then crosses the outer membrane to the
  • the long-chain acyl-CoA enters the fatty acid B-oxidation pathway that produces one acetyl- CoA from each cycle of fatty acid B-oxidation.
  • each removal of acyl-CoA by acyl- CoA dehydrogenase yields a shortened fatty acid transenoyl-CoA and one FADH 2 .
  • the transenoyl-CoA Is hydrated by enoyl-CoA hydratase to hydroxyacyl-CoA. This is reduced by NAD + and B-hydroxyacyl-CoA dehydrogenase to B-ketoacyl-CoA.
  • Acyl-CoA acetyl-transferase then adds another CoA while cleaving one-acetyl CoA.
  • Acetyl-CoA can condense with oxaloacetate to enter the citric acid cycle as citrate.
  • NADH and FADH2 produced by both fatty acid B-oxidation and the TCA cycle are used by the electron transport chain to produce ATP.
  • a partial reverse of this process is used to produce ketone bodies especially essential to the central nervous system when glucose is unavailable.
  • Two acetyl CoAs can be converted by thiolase to acetoacylCoA which H NG-synthase catalyzes to form H MG-CoA.
  • H MG-CoA lyase forms one acetoacetate and regenerates a CoA.
  • B-hydroxybutyrate dehydrogenase converts the acetoacetate molecules to B-hydroxybutyrate available to maintain brain activity in the absence of available glucose.
  • [pentose phosphate pathway] phosphoglucose isomerase fructose-6-phosphate + ATP phosphofructokinase (PFK) (inhibitors: phosphoenolpyruvate (PEP), ADP fructose-l,6-bisphosphate + ADP aldolase glyceraldehyde-3-phosphate + dihydroxyacetone phosphate dihydroxyacetone phosphate triose phosphate isomerase glyceraldehyde-3-phosphate
  • acetyl-CoA The formation of blood vessels depends on the proliferation and migration of endothelial cells— processes that require production of the metabolite acetyl-CoA from mitochondria. Conversion of glucose, glutamine and other nutrients into acetyl-CoA is required for the production of energy and macromolecules, both of which promote endothelial-cell migration to the metabolizing site.
  • the interconnected metabolic pathways make the production of acetylCoA, from oxidation of fatty acids, essential for DNA synthesis and endothelial-cell and any other cell proliferation.
  • Vitamin B3, Niacin In addition to its well-known redox functions in energy metabolism, niacin, in the form of NAD, participates in a wide variety of ADP-ribosylation reactions.
  • Poly(ADP-ribose) is a negatively charged polymer synthesized, predominantly on nuclear proteins, by at least seven different enzymes.
  • Poly(ADP-ribose) polymerase-1 (PARP-1) is a major participant in polymer syntheses and is important in DNA damage responses, including repair, maintenance of genomic stability, and signaling events for stress responses such as apoptosis. PARP-1 is therefore a prime target when metabolic modulation is in play.
  • NAD is also used in the synthesis of mono(ADP-ribose), often on G proteins. Sequencing the human genome has made obvious the number and importance of G proteins for signal transduction, and as targets for therapeutic intervention.
  • cAM P cyclic AMP
  • PKA protein kinase A
  • PKA under different conditions phosphorylates many different downstream targets, including, but not limited to: anti-diuretic hormone (ADH, aka vasopressin), growth hormone releasing hormone (GHRH), growth hormone inhibiting hormone (GH IH, aka somatostatin), corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), thyrotropin releasing hormone (TRH), lutinizing hormone (LH), follicle stimulating hormone (FSH), parathyroid hormone (PTH), Calcitonin, glucagon, ( human) chorionic gonadotropin ((h)CG), and epinephrine.
  • ADH anti-diuretic hormone
  • GHRH growth hormone releasing hormone
  • GH IH growth hormone inhibiting hormone
  • ACTH corticotropin releasing hormone
  • TSH thyroid stimulating hormone
  • TRH thyrotropin releasing hormone
  • FSH parathyroid hormone
  • PTH par
  • NAD and NADP are required for the synthesis of cyclic ADP-ribose and nicotinic acid adenine dinucleotide (NAADP). These compounds control intracellular calcium signaling. Modulating any of these processes has the potential to impair genomic stability which might deregulate cell division and contribute to enhanced cancer activity.
  • Vitamin B6 Pyridoxine
  • Vitamin B6 is present in many foods so severe deficiency is uncommon. But even in the absence of a clinical deficiency availability of B6 may be sub-optimal, especially with respect to rapidly proliferating cancer cells. B6 is an important enzymatic cofactor. See, e.g., heme synthesis discussed later. Modulating B6 availability to the organism or to a cell or a group of cells in the organism can be one tool in modulating and balancing metabolism in favor of limited proliferation.
  • NADPH inhibits conversion of G6P to gluconolactone.
  • Ascorbate/Cu “ “ “ “ ” , ascorbate/Fe “ “ “ , Cu “ “ “ “ , diazotetrazole, and GSH are inhibitors of gluconolactonase that converts gluconolactone to 6- phospho-D-gluconate.
  • triphenylmethane and derivatives bromocresol purple, bromocresol purple - salt, bromochlorophenol blue - salt, bromophenol blue - salt, tetraiodophenol- sulfonephthalein - salt, ethylenesulfonic acid oligomer, 4-phospho-derythronate, 2-deoxy-6- phospho-D-gluconate, 5-phospho-D-ribonate, 6-aminonicotinamide, 6,7-dideoxy-7- phosphono-d-glucoheptonate, 6-deoxy-6-phosphono-d-gluconate, 5-phospho-dribonate, 4- Phospho-d-erythronate, each inhibit 6-phosphogluconate dehydrogenase thereby preventing additional NADPH and ribulose phosphate formation.
  • the inhibitory salts are salts most commonly using a monovalent cation, and very often sodium salt is most available in the open market. However, other salts may be selected when the benefits justify
  • Hh signaling pathway is a developmental pathway which plays a key role in directing growth and tissue patterning during embryonic development.
  • Dysregulation of Hh signaling contributes to the development of a variety of human tumors, including skin, brain, colon, pancreatic, and lung cancers. When constutively activated, this pathway results in the increased expression of Hh target genes, including several forms of the glioma-associated oncogene (Gli) family of signaling proteins. These events are associated with uncontrolled tumor proliferation.
  • Hh pathway inhibitors including, e.g., Cyc, GDC-0449, and VD3
  • VD3 cellular effects unrelated to Hh signaling likely result from activation of VDR signaling. Therefore, applying the anti-proliferative activity of the VD3 analogues could demonstrate ability to selectively inhibit the Hh pathway.
  • vitamin D metabolizing enzymes for example in U87MG cells suggests that the enhanced anti-proliferative effects may result from the cellular conversion of VD3 to 25-hydroxy-D3 and/or to la,25-hydroxy-D3 and to subsequent activation of VDR.
  • PI3K phosphatidylinositol 3-kinase
  • mTOR mammalian target of rapamycin
  • Growth factor-induced signaling is a common practice for organisms to coordinate these functions. Underlying this is a requirement for maintaining a bioenergetic state permissive for growth. For a cancer cell to proliferate it must have previously made the macromolecules necessary for both daughter cells and must have consumed and now stored sufficient energy to accomplish the task.
  • the PI3K/Akt/mTOR pathway is commonly activated in proliferating cell because it both stimulates a rapid increase in essential nutrient uptake and directs the allocation of these nutrients into catabolic and anabolic pathways needed to produce the energy and macromolecules. Interference with any of these downstream metabolic effects can render the growth factor initial stimulation ineffective.
  • AMP-activated protein kinase AMP-activated protein kinase
  • This serine- threonine kinase is a "fuel sensor” that becomes activated during a compromised bioenergetic state such as acute nutrient deprivation or hypoxia.
  • AMPK down-regulates energy-consuming, growth-promoting pathways like protein and lipid synthesis and up-regulates catabolism of fatty acids and other fuels. This enables the cell to rebalance energy supply with demand.
  • AMPK also regulates a p53-dependent, cel l-cycle checkpoint activated by glucose deprivation thereby limiting growth when glucose supply is weak.
  • AMPK also coordinates expression of stress response genes by migrating to chromatin and phosphorylating histone H2B on its S36. This modulating activity synergizes AMPK's effects on gene expression in the nucleus. As a result, AMPK executes and controls several activities that allow cells to respond emphatically and comprehensively to energy shortage. In mammals, cell growth and proliferation are controlled by extracellular factors that bind to receptors on the plasma membrane that include, but are not limited to: hormones, growth factors, cytokines, specific nutrients, etc.
  • ligands bind to cell surface receptors and initiate signal transduction cascades that stimulating numerous cellular activities to enable growth and replicative division. Appropriate control of metabolism is required for these effects to achieve valid results.
  • one of the proximal effects of growth factor signaling is to increase surface expression of transporters, for glucose and other nutrients, which when consumed provide energy and metabolic precursors to produce needed macromolecules. Catabolism of these nutrients generally ends with carbon dioxide and energy. If nutrients are present in excess so that flux through these foundational catabolic pathways is satisfied, other pathways branching from core metabolisms are induced to propagate growth signals internally and/or for export.
  • Hexosami ne biosynthesis reinforces growth signa ls by ena bl ing cel ls to maintain protein synthesis for exa m ple for cel l su rface expression of growth factor receptors a nd of n utrient tra nsporters.
  • Acetyl-CoA generated by acetyl-CoA synthetases (ACS) a nd ATP-citrate lyase (ACL) provides substrate needed to synthesize lipids a nd other macromolecu les a nd for acetylation reactions that regu late gene expression a nd resu lta nt enzyme fu nctions.
  • a favora ble energy state du ring growth factor signal ing a lso suppresses AM PK, thereby permitting cel ls to e ngage in energy-consu ming biosynthetic pathways a nd to progress th rough the cel l cycle.
  • the TCA cycle of the mitochond rion serves a biosynthetic role i n addition to its more fa mil ia r fu nction as energy deliver.
  • Requi reme nts of the ra pid ly proliferating cel ls for production of specific biosynthetic products would control the relative im porta nce of the TCA cycle i n tu mors.
  • Precursors for: protein, lipid, a nd nucleic acid synthesis are produced i n the TCA cycle.
  • Export of these precu rsors from the cycle to supply macromolecular synthesis is a prominent feature of proliferating cancer cells.
  • Pyruvate carboxylation is one of several mechanisms by which carbon can be resupplied to the TCA cycle to offset precursor export. Such processes, termed anaplerotic pathways, prevent TCA cycle intermediates from becoming detrimentally depleted during cell growth.
  • FDG-PET 18F-fluorodeoxyglucose— PET
  • Multiparametric MRI would also be particularly useful for this type of analysis, since it can assess regional heterogeneity of perfusion, oxygenation, cellularity, necrosis, temperature, and other characteristics relevant to cancer cell metabolism.
  • ATP adenosine triphosphate
  • glycolysis and oxidative phosphorylation In glycolysis, glucose is converted to pyruvate, while generating NADH from NAD + and ATP from ADP and inorganic phosphate. If the pyruvate is reduced to lactate, NAD + is regenerated and glycolysis continues. Although glycolysis is rapid, it is deemed inefficient because most of the energy that could be generated from glucose is lost when the cell secretes lactate, a three carbon molecule retaining significant energy in its bonding structure. In contrast, OXPHOS is highly efficient about 20-fold more efficient per ATP molecule generated.
  • Genes involved in the rebalancing relate to a large number of the cells pathways and their enzymes, including, but not limited to: STAT1, Akt, Jak/Tyk2, CUG2, HGPRT, SETDB1, LDH1, etc.
  • pathways leading to lactic acid formation thus sparing mitochondrial activity from having to metabolize pyruvate, pathways leading to puri ne a nd pyrimidi ne ma nufactu re to su pport n ucleic acid synthesis, pathways leading to a ngiogenesis, pathways spa ring the prolife rating cel l from cel l death or a poptosis, pathways that may be activated to d rive the proliferati ng cel l towa rds cel l death or a poptosis a nd pathways that control cel l division a nd cel l cycle.
  • a low-molecular-weight compound secreted appears responsible for enhanced CLL cell survival.
  • This compound is probably the amino acid cysteine, one of the three amino acids required to synthesize glutathione, and protector against oxidative damage and maintenance of volume in tissues such as the cornea.
  • hypoxic tumor cells appear to favor conversion of glucose to lactate, which is disposed of by secretion (or export) into the interstitial fluid (extracellular compartment) where it can be metabolized by cells in areas of more abundant oxygen - either because of better vascularization of because of lower metabolic demands.
  • tumor cells derived from the luminal epithelium synthesize glutamine de novo before secreting it. So while controlling glutamine availability may be an important support in methods of the present invention the same method(s) will not apply to all instances where the invention is practiced. Obviously these luminal epithelial derived cancer cells and other cancer cells with similar metabolic modifications can thrive under conditions of glutamine deprivation.
  • cells derived from the basal epithelium do not synthesize glutamine and therefore require an extracellular source. These cells can be rescued by co-culturing them with glutamine-secreting luminal cells, raising the possibility of regional heterogeneity in glutamine dependence in normal and tumor- tissue.
  • GAC glutaminase C
  • DON diazo-O- norleucine
  • allosteric GAC inhibitors have been identified and may present more promise as active ingredient compounds for cancer therapeutics.
  • One of these inhibitor groups consists of analogs of bis-2-(5-phenylacetamido-l,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a reversible GAC inhibitor.
  • BPTES bis-2-(5-phenylacetamido-l,2,4-thiadiazol-2-yl)ethyl sulfide
  • X-ray crystal structures of the GAC— BPTES complex show that BPTES effectively traps GAC as an inactive tetramer.
  • a second, more recently identified, class of allosteric GAC inhibitors a class that is highly specific for inhibiting cancer cell growth while having little effect on normal (nontransformed) cells is represented by the
  • Reductive carboxylation supports growth in tumor cells with defective mitochondria
  • Mitochondrial metabolism provides precursors for macromolecules in growing cancer cells.
  • the oxidative metabolism of glucose- derived and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, an important activity to support tumorigenesis.
  • some tumors bear mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function.
  • CAC citric acid cycle
  • ETC electron transport chain
  • Glutamine and cancer cell biology, physiology, clinical strategies
  • pyruvate dehydrogenase kinase (PDH), (PDK) blocks the activation of mitochondrial pyruvate dehydrogenase thereby limiting the pyruvate conversion into acetyl-CoA.
  • Hifla hyperoxia inducible factor la
  • LDH-A hyperactivity appears essential for scavenging pyruvate to maintain NAD + and/or to remove pyruvate stimulus of the mitochondrial pyruvate to acetyl-CoA Krebs mission.
  • Krebs is still able to partially cycle when glutamine is deaminated to glutamate in a reaction supporting synthesis of the pyrimidines and purines used for nucleic acids.
  • the glutamate enters the mitochondrion as a-ketoglutarate which progresses through maleate, exits the mitochondrion then is converted to pyruvate and lactate.
  • PKB/Akt Protein kinase B
  • P13K phosphatidylinositol 3 kinase
  • CREB cAMP response element binding protein
  • PER 1 and 2 time keeper genes - Periodl and Period2
  • the ta rget of CREB is the sequence TGACGTCA which wil l be left u n hindered when it benefits from C methylation.
  • CRE B a lso influences the plasma mem bra ne though its activation of P13K which controls positioning a nd pola rity of receptors i n plasma mem bra nes.
  • P13K activation is essentia l in foresta l ling differentiation in favor of prol iferation a nd thereby plays a key role in supporting ca ncer proliferation a nd slowing a poptosis.
  • HB9 phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase
  • PTEN phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase
  • GSK3B glycogen synthase kinase 3 ⁇
  • the NADPH is also a major anti-oxidant and participant in bio-synthesis.
  • the 3PG is a reactant for both amino acid and fatty acid and other synthesis.
  • 3PG is notable for its conversion to serine which serves as a carbon source for the folate cycle through its conversion of tetrahydrofolate (THF) to methyl-tetrahydrofolate (mTHF).
  • NADPH is oxidized as part of the folate cycle.
  • Monocarboxylate transporter 4 (MCT4) is necessary for removing the lactate from cell's cytoplasmic space. Since the lactate production is enhanced in proliferating cancer cells, especially growing, vascularization deprived, and/or hypoxic cancer cells, interference with formation of intact functioning transporter including, but not limited to stopping or altering: transcription, translation, expressing, processing, transport to or insertion in plasma membrane and maintenance within the membrane will seriously compromise cell survival.
  • a seemingly opposite strategy can augment or synergize this result.
  • neighboring cells especially neighboring well-oxygenated cancer cells, may remove lactate from interstitial space and cycle it though lactate dehydrogenase for metabolic use or may otherwise remove lactate, by blocking or slowing MCT1, the relevant lactate uptake transporter, a toxic buildup of lactate in the interstitial space which then would back up into cells to can promote necrosis or apoptosis of these cells.
  • blocking lactate uptake can severely increase the demand for glucose, which when unmet starves the cells into a necrotic or apoptotic, or extreme quiescent state.
  • Glycine also is involved in the folate cycle.
  • PSAT glycine C-transferase activity
  • SHMT serinehydroxymethlase
  • Akt phosphorylates and inactivates FOXO. This down regulates PGCla and inhibits mitochondrial biogenesis.
  • MYC glutaminolysis is induced - glutamine is converted to a-ketoglutarate (aKG).
  • NADPH- linked IDH2 results in isocitrate and more citrate available for export to the cytosol, where isocitrate is available for conversion back to aKG by NADr-linked IDH l.
  • citrate may be exported from the mitochondrial matrix to the cytosol where it is cleaved by ATP citrate lyase (ACL) to produce oxaloacetate (OAA) and acetyl-CoA.
  • Glutamine is an amino acid, one of the constituents of proteins. Glutamine, a carbon rich molecule, is also an acceptable substitute for glucose as the cell's fuel.
  • the ready alternatives available as substrate for various metabolic functions and alternative pathways available to achieve the necessary functions suggest two main approaches for external control of unwanted cell growth and proliferation.
  • a first approach would be to block metabolism at an initiation step critical to many downstream paths or to block a junction point critical to several alternative path.
  • Glutamine because it can participate in many functions, including, but not limited to: a carbon source for building biomolecules, an energy source for generating needed ATP, and a conduit of nitrogen between cells and parts of cells.
  • Glutamine with all its use is not surprisingly the most common amino acid (about 1/5 of the amino acids) free in circulating blood.
  • Glutamine although capable of being synthesized in mammalian cells, often is in short supply for all the metabolic demands it can satisfy.
  • Glutamine is exported to circulation as a non-toxic carrier of NH 4 + for example from breakdown of other amino acids.
  • Glutamine is a major source of urea, the chemical carrier of nitrogen out of the body in renal waste. As a nutrient for cancer cells glutamine is often, but not always, available from the circulatory system.
  • Another prime source of glutamine is proteins as they are recycled during normal metabolic processes.
  • the extraordinary consumption of glutamine in cancer cells is evident in the activity of oncogenic RAS to stimulate macropinocytosis, a process through which extracellular molecules, e.g., proteins are ingested by the cell in the form of macropinosome vesicles. These vesicles can merge to intracellular lysosomes for degradation of the engulfed proteins to useful building blocks.
  • Amplifying this macropinosomic lysosomic activity by internal or external signaling paths, like amplifying other lysosomic activities is one means of initiating apoptotic cell death. Mitochondria participate in glutamine recycling through several aminotransferases discussed below.
  • a glutamine transport protein e.g., SLC1A5 internalizes circulating glutamine. I n the cytoplasm, glutamine can be converted to nucleotides and uridine diphosphate N- acetylglucosamine (UDP-GlcNAc). Nucleotides are essential molecules for making genes in dividing cells.
  • N-glycosylation serves to stabilize proteins by maintaining appropriate 3D folded structure and to package for secretion to extracellular space.
  • glutamine can be converted to glutamate by glutaminase (GLS or GLS2).
  • the glutamate may be used to generate glutathione (an anti-oxidant protectant) or may be processed into other metabolic substrates, such as a- ketoglutarate (a-KG).
  • glutamate dehydrogenase GLUD which comes in two forms, GLUD1 and GLUD2, and ii.) aminotransferases.
  • GLUD is activated by ADP and inhibited by GTP, palmitoyl-CoA and SIRT4- dependent ADP ribosylation.
  • Leucine by itself allosterically activates GLUD and by acting through mTOR suppresses SIRT4 expression thereby accentuating GLUD activity even more.
  • ADP levels increase e.g., by consumption of ATP in excess of creation, this may operate as a signal for GLUD to increase its ATP output.
  • GLUD has NH 4 + as a product which might be detoxified by conversion to glutamine! Whereas the aminotransferase path used to make other amino acids, aminotransferase reactions can occur both in the mitochondria and in the cytoplasm.
  • Malate can leave the TCA cycle to produce pyruvate and NADPH. Remaining in the mitochondrion, malate cycles to oxaloacetate (OAA), which may leave the cycle as aspartate to support nucleotide synthesis, e.g., DNA or tRNA for a dividing or rapidly metabolizing cell.
  • OOA oxaloacetate
  • a-KG can reverse through the TCA cycle, in a process called reductive carboxylation (RC) to form citrate, to make acetyl-CoA and lipids.
  • RC reductive carboxylation
  • the requirements of tRNA (and probably to a lesser degree, mRNA) and DNA for the growing and proliferating cell are perhaps the most likely rational for a cancer cell's metabolic shift in favor of glutamine.
  • GLS glutaminase enzymes
  • GLS which has two alternative splice forms (GLC and KGA) is activated by phosphorylation, but receives feedback inhibition by its glutamate product.
  • GLS2 however increases activity as its NH 4 + increases abundance.
  • SIRT5 sirtuin 5
  • pH is an important modulator of GLS mRNA and its expression and activity can be controlled at the site of transcription (in the nucleus), and later in cytoplasmic environment by microRNAs and RNA binding proteins directing mRNA processing and alternative splicing.
  • GAC Splice variant GAC appears more prevalent in many cancers and is the more active variant. The cell's favoring of this variant would not be apparent in a nuclear genome sequencing, but might be seen in a complete sequence analysis that also monitors expression.
  • GLS2 can be turned off by methylation which has been observed in some cancers, especially hepatic forms. GLS2 methylation may also be important for cancer cell creation in that this enzyme may have another quality or side effect in its propensity to bind RAC1 cutting metastasis.
  • the aminotransferase family includes several forms. Better characterized family members include alanine aminotransferase (aka glutamate— pyruvate transaminase), aspartate aminotransferase and phosphoserine aminotransferase (PSAT1). Alanine aminotransferase comes in a mitochondrial isoform GPT2 and a cytoplasmic isoform, GPT. Similarly, aspartate aminotransferase has a cytoplasmic isoform, GOT1 and a mitochondrial isoform, GOT2. PAT1 appears to be preferentially expressed in tumor cells and thus controlling its activity can be one tool for stressing cancer cells. In cancer cells where hypoxia-inducible-factor-a (Hifa) is constitutively expressed or where mitochondrial participation in fatty acid synthesis is severely compromised, glutamine may see further use in reductive carboxylation to synthesize fats.
  • Hifa hypoxia-inducible-factor-a
  • Glutamine metabolism is crucial for cellular reactive oxygen species (ROS) homeostasis.
  • Glutathione GSG
  • Glutathione GSG
  • Glutathione GSG
  • Many studies have shown that glutamine availability is rate limiting in GSG synthesis.
  • ROS effects are complicated. U nder some conditions increased ROS (a sign of cell stress) initiates apoptosis. But some cancers as part of their development process have survived by downplaying the apoptotic input of increased ROS. In these cases ROS can cause internal oxidative damage within the cells.
  • Glutamine also is involved in the TOR pathway. TOR encourages growth and inhibits autophagy. Glutamine suppresses pro-apoptotic action of GCN2 and Integrated Stress Response (ISR).
  • ISR Integrated Stress Response
  • Oncogenic genes upregulate glutamine uptake and metabolism as observed in the Q (glutamine) metabolism stimulated by H IF2 and MYC.
  • glutamine When glutamine is metabolized, its carbon mass is preserved chiefly in amino acids and fats while the nitrogen is an integral component for nucleic acid synthesis.
  • glutamine Through aspartate transamination glutamine can also contribute carbon atoms to purines and pyrimidines of the nucleic acids.
  • Glutamine can serve an intracellular signal through mTOR to activate carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), responsible for using nitrogen from glutamine to synthesize pyrimidine.
  • CAD dihydroorotase
  • GLS phosphate-dependent glutaminase
  • GLS activity correlates with tumor growth rates in vivo, (Knox et al. 1969; Linder-Horowitz et al., 1969), and experimental models to limit GLS activity resulted in decreased growth rates of tumor cells and xenografts.
  • the rate-limiting step in the formation of hexosamine is catalyzed by glutamine:fructose-6- phosphate amidotransferase, which transfers glutamine's amido group to fructose-6- phosphate to form glucosamine-6-phosphate, a precursor for N-linked and 0-linked glycosylation reactions. These reactions are necessary to modify proteins and lipids for their participation in signal transduction, trafficking/secretion and other processes.
  • Impairment of glucosamine-6- phosphate production is thus expected to reduce cell growth a nd to interfere with cel l signaling.
  • glutamine:fructose-6-phosphate amidotransferase activity can be suppressed by expressing an antisense GLS complementary DNA in some breast cancer cells.
  • the disturbances of 0-linked glycosylation pathways alters glycosylation of the transcription factor Sp-1 and increases its transcriptional activity.
  • Glutathione (GSH) is the major thiol containing endogenous antioxidant and serves as a redox buffer against various sources of oxidative stress. In tumors, maintaining a supply of GSH is critical for cell survival because it allows cells to resist the oxidative stress associated with rapid metabolism.
  • GSH is a tripeptide of glutamate, cysteine and glycine and its formation is highly dependent on glutamine. Not only does glutamine metabolism produce glutamate, but the glutamate, pool is also necessary for cells to acquire cysteine, the frequent limiting reagent for GSH production. Glutaminase activity generates free ammonia, a potentially toxic metabolite. Without a mechanism to dispose of ammonia rapidly, intracellular ammonia concentrations would reach several hundred mmol/l within a few hours which would be expectedly toxic to most cells in the area. It is not understood how tumor cells dispose of ammonia during rapid glutamine catabolism.
  • glutamine was shown to be comparable to serum in preventing apoptosis, and it stimulated a sustained activation of ERK signaling. The importance of glutamine as a supporter of tumorigenic activity should not be downplayed.
  • Inhibitors of the ERK pathway have eliminated the protective effect of glutamine supplementation. It was not clear from these studies whether glutamine import alone was required for the effects, or whether the cells needed to metabolize glutamine to activate ERK signaling. Consistent with glutamine's effects on cell signaling, a number of reports have shown that it also influences gene expression. In cell lines, addition of glutamine increases expression of the pro-proliferation factors c-jun and c-myc within a few minutes and promotes cell survival through the negative effects on growth-inhibitory and pro-apoptotic factors such as CHOP, GADD45, Fas and ATF5. In Ehrich ascites tumor cells, GLS knockdown led to enhanced phosphorylation, DNA binding and transcriptional activity of Sot. In HepG2 hepatoma cells, glutamine was required for the induction of manganese superoxide dismutase expression that accompanied the depletion of essential amino acids.
  • Glutamine's involvement in manganese superoxide dismutase expression was blocked by inhibiting the TCA cycle, ERKl/2 or mTOR, suggesting that an integration between mitochondrial glutamine metabolism and signal transduction facilitates the effect.
  • Evidence shows that glutamine also modulates immune responses, though it is unclear exactly through which mechanistic paths these changes are achieved.
  • glutamine could exert its effects through redox homeostasis, bioenergetics, nitrogen balance or other functions.
  • pre-treatment of the animals with glutamine significantly decreased tissue inflammation and expression of nuclear factor-kB. So glutamine may be available to buffer the redox cell's capacity.
  • Nuclear factor-kB likely is a key mediator that links glutamine availability with stress responses, since there is an inverse correlation between glutamine abundance and nuclear factor- kB-mediated gene expression.
  • glutamine as an immunomodulator in cancer appears promising in that the avid consumption of glutamine by tumors reduces glutamine availability for neighboring cells, and can modulate local nuclear factor-kB signaling and expression of inflammatory mediators in the stroma. Because tumor cells are exposed to many nutrients simultaneously, achieving a comprehensive view of tumor metabolism requires an understanding of how cells relate these pathways into an over-arching metabolic phenotype. For different tumor cell types and for different tumors pathway emphases would most likely vary. It is expected that the skilled artisan in practicing this invention to its best advantages would investigate glutamine effects, either by assay or trial and error or a combination thereof.
  • this modified metabolism supports both bioenergetics and the production of biomacromolecule precursor pools while sparing other energy-rich substrates, such as fatty, acids and essential amino acids, for their direct incorporation into the biomacromolecules.
  • Increased glucose breakdown provides building blocks for the synthesis of nucleotides (via glucosamine and the pentose phosphate pathway) and amino and fatty acids (from intermediates formed in the glycolytic and tricarboxylic acid cycles).
  • local acidification of the tumor microenvironment may facilitate tumor invasion.
  • the enhanced activity of the pentose phosphate shunt may lead to an elevated production of NADPH and glutathione (GSH) (which would increase the resistance of tumor cells against oxidative insults and some chemotherapeutic agents). Modulating the pentose phosphate pathways is one process for compromising cancer metabolism.
  • the non-oxidative phase involving ribulose-5 phosphate as a substrate involves catalytic activities from enzymes including, but not limited to: ribose-5-phosphate isomerase, ribulose-5- phosphate-3-epimerase, transaldolase, transketolase, etc.
  • heme synthesis is an important component of cell's ROS defenses. And modulation of heme synthesis (several suggestions below) is a tool available for stimulating necrosis and/or apoptosis.
  • Heme synthesis is started in the mitochondrion where glycine, brought into the mitochondrial matrix by SLC25A and succinyl-CoA, react to form a-amino-B-ketoadipate in the presence of pyridoxal phosphate (vitamin B6) as a cofactor for the d-aminolevulinate synthase (ALAS) enzyme which then decarboxylates the complex to form d-aminolevulinic acid (ALA).
  • CLPX acts as a chaperone to coordinate association of B6 with ALAS thus stabilizing and activating the complex.
  • Nutritional deficiency of vitamin B6 can limit this reaction and thus heme synthesis.
  • the d- aminolevulinate synthase enzyme is not constitutively expressed and has a short half-life. Expression of the enzyme is induced in the presence of barbiturates and steroids such as testosterone and oral contraceptives that sport a 4,5 dou ble bond that is accessible to 5- ⁇ reductase which itself is ind uced du ri ng pu berty. Expression of d-a minolevu li nate synthase is in hibited by negative feed back from heme a nd by hemati n.
  • ALA the n is tra nsported to the cytoplasm where d-a m inolevu l inic acid hydratase (aka porphobili nogen synthase) condenses two ALA molecules to synthesize porphobilinogen.
  • Zn ++ is a cofactor for this enzyme.
  • Pb ⁇ has high affinity a nd ca n displace Zn ⁇ a nd inactivate this enzyme.
  • Lead poisoning effect on this enzyme results in increased ALA i n cel ls a nd blood. Since ALA ca n not progress to eventual heme synthesis there is no heme feedback to su ppress ALA synthesis.
  • ALA is a neu rotoxin possibly because of the ROS it creates and possibly because it mimics the
  • porphobilinogen molecules are condensed by uroporphyrinogen I synthase to form a linear tetrapyrrole which can isomerize non-enzymatically into
  • Uroporphyrinogen III is a substrate for vitamin B12 synthesis and chlorophyll synthesis as a branch off this heme synthesis pathway.
  • Uroporphyrinogen decarboxylase decarboxylates acetic groups of both
  • uroporphyrinogen I and uroporphyrinogen III changing these groups to methyl groups and forming coproporphyrinogen I and coproporphyrinogen III, respectively.
  • the fate of coproporphyrinogen I in the cell is unknown and may be a dead end synthetic product.
  • Coproporphyrinogen III migrates back into a mitochondrion through an ATP dependent carrier ABCB6 and is oxidized by coproporphyrinogen III oxidase to form protoporphyrinogen IX.
  • Protoporphyrinogen IX oxidase aromatizes the ring by converting methylene bridges of protoporphyrinogen IX to methenyl bridges in protoporyrin IX.
  • the resonance bonding improves stability of the molecule.
  • Ferrochelatase then adds Fe ++ to protoporphyrin IX while reducing ascorbic acid (vitamin C) and cysteine and releasing two H. Lead which inhibited ALA also inhibits ferrochelatase.
  • Iron is made available to FECH in the mitochondria though a transmembrane carrier, SLC25A37 stabilized with ABCB10 bound to FECH. Then finally the HEME is exported to the nucleus through FLVCRlb for cytosolic incorporation of heme into metaloproteins.
  • Cancer cells often upregulate the rate-limiting processes and enzymes of glycolysis, including glucose transporters, for instance as a result of the constitutive signaling through the Akt pathway or as a result of the expression of oncogenes including Ras, Src or Bcl-Abl. Failure to adapt these behaviors would be incompatible with the cell's survival. So, only cells effectively navigating these changes will survive to be observed. But since all living things in their creation have a built in drive to survive, when cell's begin to be stressed in a cancer leaning direction, the cell's evolved defense will kick in to preserve life of the cell but may not support survival strategies of the organism.
  • Cancer cells can accumulate defects in the mitochondrial genome, leading to deficient mitochondrial respiration and ATP generation.
  • mitochondrial germline mutations have been shown to provide a genetic predisposition to cancer development. This would be expected because all metabolic defects or changes can be expected to stimulate compensatory reactions which will induce further compensations, etc., within the cell.
  • a first category includes severe mutations that inhibit oxidative damage
  • ROS reactive oxygen species
  • Cancer cells may adapt to decreased oxygen tension (hypoxia) that is characteristic of most, if not all solid tumors as the pre-malignant lesion grows progressively further from the blood supply.
  • hypoxia decreased oxygen tension
  • the adaptation to hypoxia would be to durably shut down mitochondrial respiration and to switch on glycolytic metabolism.
  • mitochondrial enzymes can act as tumor-suppressor proteins whose mutation indirectly engenders aerobic glycolysis.
  • the inactivating mutation of mitochondrion-specific proteins such as succinate dehydrogenase (SDH subunits B, C or D) and fumarate dehydrogenase is an oncogenic event, causing phaeochromocytoma (in the case of SDH mutations) and leiomyoma, leiomyosarcoma or renal carcinoma (in the case of fumarate dehydrogenase mutations).
  • SDH subunits B, C or D succinate dehydrogenase
  • fumarate dehydrogenase is an oncogenic event, causing phaeochromocytoma (in the case of SDH mutations) and leiomyoma, leiomyosarcoma or renal carcinoma (in the case of fumarate dehydrogenase mutations).
  • the loss of function of succinate or fumarate deyhdrogenases
  • pseudohypoxic state accompanied by HIF-dependent reprogramming of the metabolism towards aerobic glycolysis.
  • Inhibitors of glycolytic enzymes that have been successfully used to slow down the growth in human tumors transplanted to mice include 3-bromopyruvate (an inhibitor of hexokinase) and oxythiamine (an inhibitor of the transketolase-like enzyme).
  • glycolytic inhibitors are already being evaluated in clinical trials. This applies to 2-deoxyglucose (an inhibitor of the initial steps of glycolysis) as well as to lonidamine (TH-070), an inhibitor of glycolysis that also has direct pro-apoptotic properties.
  • Nano devices underdevelopment might be used to administer these or other therapeutic compounds to relevant (diseased) locations.
  • These novel nano devices mentioned but not required to practice the present invention can in "smart" form be outfitted with sensors and brakes for attachment or movement stoppage to at that location deliver the ported therapeutic or they may remain as marker targets for a second porter to deliver one or more therapeutics to the relevant site.
  • these nanosensors are equipped with simple diagnostic tools and can be queried to report efficacy of any treatments in their vicinity.
  • a nanogel delivery system can be used for multiple therapeutics ortherapeutic combinations. Controlling Proliferation.
  • the cell cycle consists of a state of quiescence (Go), a first gap phase (Gl), the DNA synthesis (S phase) a second gap phase (G2), then mitosis (M), the actual cell division phase.
  • Retinablastoma protein phosphorylation by a CDK/cyclin complex allows release of transcription factor E2F that can activate several genes including, but not limited to: cyclins A, D and E.
  • CIP/KIP family members p21CIPl, p27KIPl and p57KIP2 assist CDK/cyclin association.
  • pl6INK4a and pl4ARF are tumor suppressors (encoded by the same gene in overlapping reading frames)!
  • pl6INK4a is inactivated in many cancers.
  • pl4ARF can maintain cycle arrest in Gl or G2. It complexes with MDM2 to prevent MDM2 from neutralizing p53 thereby transcriptionally activating cyclin-dependent kinase inhibitor 1A or may induce apoptosis. Hyperexpression of cyclins is one hallmark of cancer.

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Abstract

La présente invention concerne des systèmes et des méthodes d'identification, de ciblage et de destruction de cellules cancéreuses. Lorsque les cellules évoluent d'un état normal à un état cancéreux, leurs vitesses métaboliques accélérées et les voies adaptées génèrent une signature thermique supérieure qui sert de balise de ciblage pour un virus spécialement adapté. Ce virus adapté confirme ensuite l'état cancéreux de la cellule par sa sensibilité au pH. Une fois qu'un virus à nano-instruction selon la présente invention confirme ces deux critères, il est activé en vue d'infecter la cellule cancéreuse cible confirmée. Le cancer se développe du fait que les cellules ont été adaptées pour une division rapide, quasi-incontrôlée, pour former des cellules filles non nécessaires qui ont été adaptées afin d'échapper aux contrôles anticancéreux auto-immuns normaux du corps. La présente invention décrit comment utiliser le pH réduit et la température élevée de la cellule cancéreuse en tant que mécanisme pour attirer et activer le virus infectieux.
PCT/US2018/018650 2017-02-20 2018-02-20 Méthode d'identification, de ciblage et d'administration précis de thérapies dirigées pour la destruction de cellules cancéreuses WO2018152480A1 (fr)

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CN117153240A (zh) * 2023-08-18 2023-12-01 国家超级计算天津中心 基于氧自由基的关系确定方法、装置、设备及介质

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