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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

• RNAi Small interfering RNA
• C. elegans Fire, et al., (1998) Nature 391, 806-811
• Drosophila Kennerdell, J.R. and Carthew, R.W. (1998) Cell 95, 1017-1026.
• zebrafish Wargelius, et al., (1999) Biochem Biophys Res Commun 263, 156-161) and mice (McCaffrey, et al., (2002) Nature 418, 38-39).
• RNAi RNA induced silencing complex
• RISC RNA induced silencing complex
• RNAi produces sequence-specific reduction of gene expression in various mammalian in vitro systems. Recently, RNAi was shown to suppress the expression and activity of a luciferase transgene in adult mice (McCaffrey, et al., (2002)) suggesting that RNAi may become a valuable molecular and possibly clinical tool for use in vivo.
• RNAi over a similar protocol, using antisense oligonucleotides, is that the use of antisense oligonucleotides to produce chronic effects requires constant or repetitive delivery of a large molecule that is difficult to transport and maintain in appropriate conformation, is expensive, and is generally impractical for more than a few days.
• siRNA acts catalytically at sub-molar ratios to cleave up to 95% of the target mRNA in the cell.
• the RNA interference effect can be long-lasting and may be detectable after many cell divisions.
• non- viral methods have included the development of an intravascular delivery method that allows for the efficient delivery of siRNAs or other genetic material to liver cells (Herweijer, et al., (2001) Journal of Gene Medicine 3(3 :280-91: and Wolff et al., (1997) Hum Gene Ther 8:1763-72).
• non- viral DNA/polymer and/or DNA/lipid polyplexes have failed to deliver genes to cells in vivo as effectively as in vitro because the non-viral particles aggregate in physiologic solutions and the large size of the aggregates interferes with their ability to remain in the blood to access target cells.
• previously-developed non- viral particles required a net positive charge in order for the packaged DNA to be fully protected, preventing their contact with target cells in vivo.
• RNAi protocols can be used to decrease specific endogenous hypothalamic protein levels in the central nervous system leading to an increase in the metabolic rate and a decrease in body weight (Makimura, et al. (2002) BMC Neurosci 3, 18).
• body weight a measure of the degree of the central nervous system of mammals.
• siRNA and miRNA small regulatory RNA molecules, referred to as microRNAs
• microRNAs small regulatory RNA molecules
• the present invention is summarized as methods for affecting gene expression of a specific target gene in specific types of nerve cells in the central nervous system of a mammal.
• the methods include formulating an siRNA composition constructed to have a strand complementary to a portion of the target gene; and delivering the siRNA composition to a target site on the mammal to affect expression of the target gene in the nerve cell.
• the target injection site is a muscle tissue innervated by motor nerve cells or by localized injections within the cerebrospinal fluid, such that the expression of the target gene may be down-regulated.
• the target gene is brain derived neurotrophic factor
• the target gene is a receptor or a signaling molecule, such as
• the nerve cell is a motoneuron that sends processes from the cell body in the medulla or spinal cord to a target muscle, or sensory neurons that send processes to the muscle or skin.
• the muscle tissue is a tongue or a diaphragm muscle.
• the siRNA composition is delivered to the target site in the presence of a delivery reagent, preferably oligofectamineTM.
• the invention also provides a kit for use in affecting gene expression of a target gene in a nerve cell in the central nervous system of a mammal.
• the kit includes the siRNA composition; and instructions for practicing the method of affecting gene expression.
• the invention provides insight into the regulatory mechanisms of BDNF, MAP kinases and protein phosphatases in neuronal responses to intermittent hypoxia (and possibly a form of neuroplasticity in the control of breathing, known as respiratory long- term facilitation or LTF). Also provided is the utility of using siRNA technology directed to BDNF or protein phosphatases to regulate gene function in the medulla and spinal cord in vivo. It is also encompassed that the invention may be extended to other molecules and will be useful in the treatment of a variety of medical conditions. Accordingly, in another embodiment, genes in combination with BDNF or alone are targeted which could influence motoneuron function, such as for example, receptors and signaling molecules downstream of BDNF or that act in parallel with BDNF or that may regulate BDNF.
• motoneuron function such as for example, receptors and signaling molecules downstream of BDNF or that act in parallel with BDNF or that may regulate BDNF.
• the various modes of delivering the siRNA composition may prove highly useful in treating motoneuron related conditions, such as, for example, obstructive sleep apnea, spinal cord injury, degenerative motoneuron disease (ALS), polio.
• motoneuron related conditions such as, for example, obstructive sleep apnea, spinal cord injury, degenerative motoneuron disease (ALS), polio.
• ALS degenerative motoneuron disease
• siRNA administration to the skin or nerve(s) may be useful in treating chronic pain.
• Other applications where the methods of the invention may be useful are described in detail below.
• FIG. 1 A-B illustrates that intermittent hypoxia elicits a form of phrenic motoneuron plasticity known as pLTF and increased BDNF synthesis in ventral spinal segments associated with the phrenic motor nucleus;
• A shows a representative raw and integrated phrenic neurogram taken before and 60 min following three, 5 min episodes of hypoxia (11% O 2 ).
• phrenic amplitude is increased 80% above baseline levels 60 min post- hypoxia, indicating pLTF;
• FIG. 2 A-C graphically illustrates the regulation of ventral cervical BDNF following intermittent hypoxia;
• (A) shows the BDNF concentration expressed as a percentage increase from corresponding controls in rats pretreated with intrathecal artificial CSF, methysergide or emetine prior to hypoxia. Rats with intrathecal artificial CSF injections had a significant increase in ventral C 3 - C 5 BDNF concentration 60 min following intermittent hypoxia.
• B shows BDNF concentration expressed as a percentage increase from controls in rats exposed to intermittent hypoxia (CSNX-sham), intermittent hypoxia + carotid denervation (CSNX), or intermittent hypercapnia;
• C shows the correlation between average changes in BDNF (% control) in ventral cervical segments with average pLTF (% baseline) 60 min post-intermittent hypoxia or hypercapnia.
• Groups are: intermittent hypoxia with artificial CSF (•), intermittent hypoxia without intrathecal injections (•), intermittent hypoxia with intrathecal methysergide ( ⁇ ), intermittent hypoxia with intrathecal emetine (T), intermittent hypoxia in CSNX-sham (A), intermittent hypoxia with CSNX (A), intermittent hypercapnia ( ⁇ ) and intermittent hypoxia with siRNA ( ⁇ ).
• FIG. 3 A-C shows that intrathecal BDNF facilitates phrenic motor output;
• A shows a representative trace of integrated phrenic discharge before, during and 90 min following intrathecal BDNF (100 ng) injections;
• B shows the average data depicting the percentage change in phrenic burst amplitude 30, 60 and 90 min following intrathecal BDNF (•), vehicle (artificial CSF + 0.1% BSA; ⁇ ) or BDNF + K252a (A); and
• C shows that intrathecal BDNF did not affect XII activity, suggesting a spinal site of action. *significantly increased from baseline and significantly different from other groups (P ⁇ 0.05); data are mean + SEM.
• BDNF siRNA reduced BDNF mRNA in vitro and hypoxia-induced BDNF synthesis in vivo;
• A BDNF mRNA is reduced 75% from control 24 hours post-transfection in HT-22 cells;
• B shows no significant change in baseline BDNF protem levels in ventral gray matter (C 4 - C 5 ) 3 hours following intrathecal BDNF siRNA injections in adult rats.
• C 4 - C 5 ventral gray matter
• FIG. 5A-C illustrates that BDNF siRNA and Trk receptor inhibition with K252a block pLTF;
• A shows representative integrated phrenic neurograms illustrating the development of pLTF during and following three, 5 min hypoxic episodes;
• B shows average data illustrating that intrathecal BDNF (but not scrambled) siRNA pretreatment blocked pLTF 60 min post-intermittent hypoxia (% change from baseline activity);
• C shows average data illustrating that intrathecal K-252a pretreatment attenuated pLTF 60 min post-intermittent hypoxia (% change from baseline activity), when compared to rats receiving vehicle injections.
• FIG. 6 is a graph showing that an injection having a BDNF siRNA composition into the tongue muscle can inhibit LTF in hypoglossal nerve activity while leaving LTF in the phrenic nerve intact.
• This experiment shows that intramuscular injections can influence the function of discrete motoneuron populations 3 days after an intramuscular siRNA injection, expressed here as a block in hypoglossal (but not phrenic) LTF.
• FIG. 7 shows that diaphragm injections of BDNF siRNA abolish phrenic (but not
• FIG. 8A-B illustrates the underlying idea of the invention, where a differential balance of kinase and phosphatase activation accounts for differences between (A) intermittent and (B) sustained hypoxia in their capacity to elicit pLTF. During sustained hypoxia, phosphatase activation halts the mechanism leading to pLTF.
• a differential balance of kinase and phosphatase activation accounts for differences between (A) intermittent and (B) sustained hypoxia in their capacity to elicit pLTF.
• phosphatase activation halts the mechanism leading to pLTF.
• FIG. 10A-B shows intermittent (but not sustained) hypoxia elicits persistent activation of ERK1/2 MAP kinases.
• A immunoblot of phosphorylated (activated) ERK1/2 MAP kinase in ventral C4-C5 gray matter in rats exposed to normoxia (control), intermittent hypoxia (IH) or sustained hypoxia (SH). Tissues were harvested 60 min post-hypoxia. Also, shown is that (B) intermittent (but not sustained) hypoxia elicits persistent ERK1/2 MAP kinase activation in spinal regions associated with the phrenic motor nucleus.
• FIG. 11 shows ERK1/2 MAP kinase activation is BDNF-dependent.
• FIG. 12 shows sustained (but not intermittent) hypoxia increases protein phosphatase 2 (PP2) activity.
• FIG. 13 shows that sustained hypoxia elicits phrenic LTF following protein phosphatase inhibition. Intraspinal inhibition of protein phosphatases with okadaic acid reveals pLTF in a rat exposed to sustained hypoxia (SH+okadaic acid).
• the present invention generally relates to methods of affecting gene expression of a target gene in a nerve cell in the central nervous system of a mammal.
• the method includes formulating an siRNA composition constructed to have a strand complementary to a portion of the target gene; and delivering the siRNA composition to a target site in the mammal to affect gene expression of the target gene in the nerve cell.
• the target site may be a muscle tissue linked by nerve cell(s) or cerebrospinal space, such that the target gene or its ability to produce new protein is down-regulated in the nerve cell.
• one embodiment provides that direct delivery of the siRNA composition into the intrathecal space of a mammal, effectively interfered with BDNF mRNA and blocked increases in BDNF in the cervical spinal cord elicited by a reduced flow of oxygen called intermittent hypoxia. Intermittent hypoxia causes a form of serotonin- dependent spinal synaptic plasticity known as phrenic long-term facilitation (pLTF).
• pLTF phrenic long-term facilitation
• a target gene, preferably BDNF in a nerve cell in the central nervous system of a mammal was down-regulated.
• the method includes formulating an siRNA composition constructed to have a strand complementary to a portion of BDNF mRNA; and delivering the siRNA composition to a target site in the mammal to cause down-regulation of BDNF in the motor nerve cell.
• the target neurons are motoneurons in the medulla or spinal cord.
• the siRNA delivery site is the muscle linked to the motoneurons or the cerebrospinal space.
• an exemplary delivery reagent is a cationic lipid transfection reagent, such as oligofectamineTM used to deliver the siRNA composition to the target site. It is also noted that the nerve cells of the central nervous system are important for normal respiratory function and must provide an appropriate motor output, triggering respiration.
• another embodiment provides that indirect delivery of the siRNA composition intramuscularly into muscles innervated by motoneurons protected siRNA molecules from circulating RNAses in the blood and resulted in transport of intact siRNA molecules back to the nerve cells.
• This indirect transport of siRNA molecules from the muscles to the nerve cells resulted in blocking LTF following intermittent hypoxia in the hypoglossal nerve (the motor nerve of the tongue) but not phrenic nerve (associated with the diaphragm) motor output, when the siRNA was injected into the tongue muscle.
• the effect on motoneurons that attach to the tongue demonstrates specific delivery to the targeted motoneurons located in the medulla.
• the methods of the present invention are able to affect target gene expression and produce physiological affects by delivering siRNA molecules directly or indirectly into a target site.
• this embodiment discloses a method of down-regulating a target gene, in a nerve cell in the central nervous system of a mammal.
• an exemplary target gene is brain derived neurotrophic factor (BDNF); however, a receptor or a signaling molecule downstream of BDNF, or a molecule acting in concert with BDNF, or a molecule that regulates the expression of BDNF is also encompassed by the invention.
• the method includes formulating an siRNA composition constructed to have a strand complementary to a portion of BDNF; and delivering the siRNA composition to a target site on the mammal to cause down- regulation of BDNF in the nerve cell, wherein the target site is a muscle tissue linked to the nerve cell.
• the muscle tissue is a tongue or a diaphragm muscle.
• Other muscles and other motoneurons involved with motor functions other than breathing are encompassed by the invention.
• an exemplary delivery reagent is a cationic lipid transfection reagent, such as oligofectamineTM used to deliver the siRNA composition to the target site.
• oligofectamineTM used to deliver the siRNA composition to the target site.
• therapies include those known in the art, such as monoclonal antibody therapy, chemotherapy, radiation therapy, trophic factor supplementation, and analgesic therapy, or a combination thereof.
• kits that would include components to be used in practicing the methods of in vivo siRNA molecule delivery to a target cell, such as a nerve cell in the nervous system of a mammal to affect cellular gene expression.
• the subject kits may generally include siRNA molecules, as described herein, alone or complexed with a delivery reagent (siRNA composition) for delivery into the target cell.
• the subject kits may further include an aqueous delivery vehicle, e.g. a buffered saline solution, etc.
• the kits may include a competitor RNA, for competing with the target gene.
• kits the above components may be combined into a single aqueous composition for delivery into the host or separate as different or disparate compositions, e.g. in separate containers.
• the kit may further include an intrathecal or intramuscular delivery means for delivering the aqueous composition to the host, e.g. a syringe etc., where the delivery means may or may not be preloaded with the aqueous composition.
• the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
• One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g. a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
• Yet another means would be a computer readable medium, e.g. diskette, CD, etc., on which the information has been recorded.
• Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
• hypoxia refers to a state of oxygen deficiency in the body which is sufficient to cause an impairment of function.
• hypoxia is the reduction in partial pressure of oxygen within the blood caused by inadequate oxygen transport, such as a period without breathing or a reduction in the amount of oxygen in the air, such as at altitude.
• intermittent hypoxia is broadly defined as repeated episodes of hypoxia interspersed with episodes of normal oxygen.
• Intermittent hypoxia is caused by a frequent occurrence as exemplified by lung diseases and sleep-disordered breathing.
• Intermittent hypoxia triggers a cascade of physiologic and biologic intraneuronal events that are triggered by O 2 deprivation and that can lead to adaptation and survival or neuronal damage. Specifically, it causes a form of serotonin-dependent spinal synaptic plasticity known as respiratory long-term facilitation (LTF). Sustained hypoxia is triggered by long-term so journeyns at high altitude and chronic lung disease.
• LTF respiratory long-term facilitation
• LTF long-term facilitation
• Millhorn and colleagues more than two decades ago. They observed that integrated phrenic nerve activity remained elevated above pre-stimulation levels for at least 90 min following episodic stimulation of chemoafferent neurons in the carotid sinus nerve in anesthetized, paralyzed and ventilated cats. Even though the concept of LTF has been known for some time, researchers are just beginning to understand the detailed cellular and synaptic mechanisms giving rise to this form of neuroplasticity.
• anesthetized, paralyzed and ventilated rats as a model to study mechanisms of respiratory LTF in vivo.
• LTF is observed in several respiratory-related nerves, such as the phrenic (diaphragm innervation) and hypoglossal (tongue innervation), and is primarily revealed as an enhancement of nerve burst amplitude that develops 15-30 min post-episodic hypoxia, and lasts for more than 1 hour.
• phrenic LTF or "pLTF” arises from a central neural mechanism, largely within or near phrenic motoneurons.
• pLTF is generally due to a central mechanism since it can be elicited by electrical stimulation of carotid chemoafferent neurons in the absence of hypoxia and is not observed in carotid chemoafferent activity following intermittent hypoxia; is observed in short-latency spinally evoked responses in phrenic motor output and is blocked by spinal application of serotonin receptor antagonists, protein synthesis inhibitors and by small interfering RNAs (siRNAs) directed against BDNF mRNA.
• siRNAs small interfering RNAs
• pLTF requires spinal serotonin 5-HT2A receptor activation for its initiation, but not for its maintenance. Since pLTF requires spinal serotonin-dependent protein synthesis as early as 15 min post-intermittent hypoxia. It is believed that that intermittent 5-HT2A receptor activation on phrenic motoneuron dendrites initiates the synthesis of new proteins via translation of existing, dendritic mRNA (i.e., increased BDNF synthesis and pLTF are translation-dependent, but transcription-independent.) [00048] One protein that is critically involved in pLTF is brain derived neurotrophic factor (BDNF).
• BDNF brain derived neurotrophic factor
• RNA interference with small, interfering RNAs to block spinal BDNF synthesis following intermittent hypoxia, which abolished pLTF and demonstrated that new BDNF synthesis is necessary for its underlying mechanism.
• inhibition of the high affinity BDNF receptor abolishes pLTF, whereas spinal BDNF applications facilitate phrenic burst amplitude similarly to pLTF.
• siRNA refers to a (duplex) double stranded nucleic acid molecule capable of RNA interference "RNAi”, see for example Elbashir et al., (2001), Nature.411, 494-498.
• siRNA molecules need not be limited to those molecules containing only native or endogenous RNA nucleotides, but further encompass chemically modified nucleotides and non-nucleotides.
• siSTABLETM siRNA is a proprietary form of siRNA in which the complementary strands have been chemically modified to enhance duplex stability, silencing longevity and potency, without increasing cellular toxicity.
• siSTABLETM siRNA also includes modifications that also inactivate the sense strand of the duplex, eliminating its potential participation in off-target silencing.
• siRNA duplexes of the invention were designed and synthesized by Dharmacon. However, specific siRNA' s, which can be designed and manufactured, are available through Oligo Engine (Seattle, WA), Ambion (Austin, TX) or SiRNA Technologies among others.
• siRNA composition or "siRNA duplex pool” as used herein refers to naked siRNA molecules associated with a transfection, delivery reagent.
• the siRNA composition is delivered to a target site to down-regulate expression of a target gene or impair its ability to translate new protein through RNA interference.
• a "delivery reagent” as used herein is a compound or compounds used in the prior art that bind(s) to or complex(es) with polynucleotides, preferably siRNA molecules and mediates their entry into cells.
• the delivery reagent also mediates the binding and internalization of siRNA into cells.
• the siRNA molecules of the invention may be added directly, complexed with cationic lipids, packaged within a delivery reagent, or otherwise delivered to target cells or tissues under conditions suitable for administration.
• delivery- reagents include cationic liposomes and lipids, calcium phosphate precipitates, rechargeable particles and polylysine complexes.
• the delivery reagent has a net positive charge that binds to the siRNA's negative charge.
• the transfection reagent mediates binding of siRNA to a cell via its positive charge (that binds to the cell membrane's negative charge) or via ligands that bind to receptors in the cell.
• cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA.
• Other delivery reagents used to transfer genes into cells that are well known in the art are also encompassed within the scope of the invention. These include complexing the polynucleotides on particles that are then accelerated into the cell or elecroporation. The charge increases the permeability of the cell.
• a preferred transfection reagent of the invention is OligofectamineTM (Invitrogen;
• siRNA compositions can be achieved through a variety of different modes of administration. Exemplary methods of delivering siRNA compositions of the invention include intrathecal or intramuscular injections.
• the term "nerve cell” as used herein refers to a cell in the central nervous system which preferably includes the brain and spinal cord, but may also include peripheral nerves and ganglia. Multiple cell types may be included in this description, including neurons, astrocyte glial cells, microglial cells, oligodendrocytes, Schwann cells, and epithelial cells of the choroid plexus.
• target gene refers to a polynucleotide, preferably an mRNA which has a portion of its polynucleotide sequence complementary to an siRNA molecule of the invention. Both cytoplasmic and nuclear target genes are encompassed by the invention.
• a prefened target gene may include BDNF and any receptors or signaling molecules downstream of BDNF, or regulating BDNF, or acting in concert with BDNF.
• siRNA compositions targeted against the BDNF gene may be applicable to other mRNA targets that can influence motoneuron function.
• BDNF gene expression can be regulated by targeting receptors and signaling molecules downstream of BDNF, or molecules that regulate the expression of BDNF such as transcription factors (i.e., calcium response element (CRE), nuclear factor of activated T-cells isoform 4 (NFATc4), among others) or protein phosphatases (described below).
• transcription factors i.e., calcium response element (CRE), nuclear factor of activated T-cells isoform 4 (NFATc4), among others
• protein phosphatases protein phosphatases
• Major downstream targets of BDNF include the mitogen-activated protein (MAP) kinases ERK-1 and ERK-2, kinases that regulate ERK activation (MEKK), the high affinity tyrosine kinase receptor for BDNF (TrkB), protein kinase C, protein kinase A and CAMKII.
• protein phosphatases 1, 2A and 2B are the strongest candidates for a prominent role in pLTF and the possibility of enhancing respiratory motoneuron function.
• protein phosphatase 2 A (PP2) is preferred as detailed below in the examples, where sustained hypoxia was found to preferentially activate PP2, which acts as an endogenous "brake” and prevents the expression of pLTF by halting its fundamental mechanism at the level of kinase activation.
• PP2A substrates are serine-threonine protein kinases, and PP2A is sometimes regarded as a kinase phosphatase.
• PP2A inhibits protein kinase C (PKC) activation and there is a direct physical association between PP2A and PKC in mammalian cells. Indeed, prolonged activation of PKC causes its own dephosphorylation and the subsequent down-regulation of its own activity under the influence of PP2A.
• PKC protein kinase C
• PP2A also inactivates ERK 1/2 MAP kinases.
• NOGO an axon growth inhibitor in the adult CNS
• myelin basic protein serotonergic receptors
• GABA receptors GABA receptors
• glutamate receptors and their subunits
• specific potassium and chloride channels Specific targets can be chosen to promote plasticity and/or survival of motoneurons.
• BDNF brain-derived neurotrophic factor
• BDNF neurotrophins
• NT-3 neurotrophin-3
• BDNF is produced in the following relevant signaling cascade: 5-HT 2A receptors activate a G protein (G ⁇ q ), phospholipase C and then protein kinase C (PKC). PKC may then lead to new BDNF synthesis via (direct or indirect) phosphorylation of relevant translation initiation factors, such as the eukaryotic initiation factor 4E (eIF-4E).
• eIF-4E eukaryotic initiation factor 4E
• BDNF is released from the dendrites of phrenic motoneurons, and may act pre- and/or post-synaptically by activating the high affinity receptor tyrosine kinase (TrkB).
• TrkB high affinity receptor tyrosine kinase
• signal fransduction cascades are activated that establish pLTF, at least initially.
• ERKl/2 extracellular regulated kinases 1/2
• MAP kinase mitogen-activated protein kinase family
• BDNF BDNF synthesis is necessary for pLTF since interference with BDNF mRNA translation with small interfering RNAs (siRNA) can abolish pLTF. It is believed that BDNF likely induces pLTF via the high affinity TrkB receptor since inhibition of receptor tyrosine kinases abolished pLTF.
• the results presented herein also illustrate that BDNF is necessary and sufficient for pLTF following intermittent hypoxia.
• BDNF mRNA is the target of down regulation by siRNAs for purposes of blocking BDNF synthesis following intermittent hypoxia (loss of function).
• protein phosphatase 2 A would be a suitable target of down regulation to promote BDNF synthesis following sustained hypoxia (gain of function).
• BDNFor downstream molecules such as ERK or the activator s of ERKand MEKK are suitable gene targets.
• other molecules that regulate or work in concert with bBDNF to add function are suitable gene targets. Examples of such molecules would be the protein phosphatases.
• molecules such as purinergic receptors may be suitable targets, because intermittent hypoxia upregulates important ATP receptors such as the P2X7 receptor.
• plasticity refers to a change in system behavior based on experience. Such plasticity is suitably exhibited as a property of the neural network underlying respiratory control. Plasticity has many potential roles in guiding development and aging of the respiratory control system. Indeed, the neural elements that control breathing must adapt to a wide range of physiological and/or environmental changes throughout life, such as birth, pregnancy, obesity, respiratory infection, altitude exposure, neural injury, and even the normal deterioration of pulmonary mechanics and gas exchange with aging. Despite the critical importance of respiratory plasticity, particularly during disease, the detailed mechanisms giving rise to plasticity are not well understood.
• pLTF following intermittent and sustained hypoxia is a model of spinal, serotonin-dependent plasticity with great potential to advance the understanding of neurotrophins, their regulation, and their role in neuroplasticity.
• Such understanding of serotonin-dependent respiratory plasticity may have important implications in the development of therapeutic strategies to respiratory disorders including sudden infant death syndrome (SIDS), obstructive sleep apnea, respiratory insufficiency following spinal cord injury, respiratory insufficiency attendant to neurodegenerative motoneuron diseases (e.g., ALS), infectious motoneuron diseases (e.g., Polio) and other disorders that affect respiratory control (e.g., Rhett Syndrome).
• SIDS sudden infant death syndrome
• obstructive sleep apnea respiratory insufficiency following spinal cord injury
• respiratory insufficiency attendant to neurodegenerative motoneuron diseases e.g., ALS
• infectious motoneuron diseases e.g., Polio
• other disorders that affect respiratory control e
• the methods of the invention may be used in affecting gene expression of target genes to treat a variety of medical conditions.
• the methods of "the invention may be employed to use siRNA compositions directed against target molecules such as for example, BDNF and PP2A, to down regulate gene expression and facilitate treatment of motoneuron related conditions, such as, obstructive sleep apnea, spinal cord injury, degenerative motoneuron diseases (e.g., ALS and polio).
• motoneuron related conditions such as, obstructive sleep apnea, spinal cord injury, degenerative motoneuron diseases (e.g., ALS and polio).
• a similar approach may be used by applying siRNA to tissues such as skin and muscle, decreasing the expression of for example, BDNF and associated molecules to minimize chronic pain. This approach to minimize chronic pain will not be through effects on motoneurons, but via actions on sensory nerve cells.
• BDNF production in the dorsal horn of the spinal cord With respect to chronic pain it has been shown that there is increased BDNF production in the dorsal horn of the spinal cord. Thus, it is possible that siRNA directed to BDNF delivered specifically to this site could hold promise as a pain therapeutic.
• other types of pain such as discomfort caused by any one of carpal tunnel syndrome pain, back pain, neck pain, sciatica, intercostal neuralgia, opioid resistant pain, trigeminal neuralgia, arthritis, osteoarthritis and cancer-related pain are encompassed by the invention.
• the methods of the invention could be used to affect gene expression following intermittent hypoxic episodes caused by for example sleep apnea, central hypoventilation syndrome, and apnea of prematurity.
• Episodes of intermittent systemic or local hypoxia affect metabolic pathways, initiate neuroplasticity, induce angiogenesis, and affect inflammatory responses.
• the inability of cells to detect and adapt rapidly to changes in oxygen may underlie various vascular, pulmonary, coronary, cerebral, and sleep disorder states.
• hypoxia has also been shown to modulate the activity of gene regulators, growth factors, and reactive oxygen species that serve as intermediary signals in the cellular response to oxygen level changes.
• the methods of the invention can be used for indirectly treating and preventing cyclic reductions in blood oxygen saturation during sleep apnea which is associated with a loss of upper airway patency and causes increased risk of hypertension, myocardial infarction, cerebrovascular condition, and neurocognitive deficits.
• the following examples provide the materials and methods used to obtain and analyze the gene expression effects of the invention. These examples are intended to illustrate, but not limit, the present invention.
• the carotid sinus nerve was transsected bilaterally (CSNX) at the junction with the glossopharyngeal nerve (16).
• CSNX the carotid sinus nerves were identified, but not cut. Since CSNX rats did not receive drug treatments, laminectomy was not performed in these animals.
• BDNF protein concentration was measured in rat groups injected intrathecally with emetine (protein synthesis inhibitor; Sigma, St. Louis, MO) or methysergide maleate (serotonin receptor antagonist; Sandoz, Hanover, NJ) 30 min prior to intermittent hypoxia or isocapnic hyperoxia.
• emetine protein synthesis inhibitor
• methysergide maleate serotonin receptor antagonist
• Sandoz Hanover, NJ
• Emetine or methysergide were dissolved in artificial CSF and delivered intrathecally at concentrations of 1 ⁇ g /kg (70 ⁇ M) or 250 ⁇ g/kg (20 mM), respectively (10 - 15 ⁇ l injected volume).
• En bloc spinal cord segments C - C 5 were placed on a freezing microtome, and successive 50 ⁇ m sections of the dorsal horn were removed and discarded until the venfral aspect of the central canal was visible. BDNF and/or NT-3 analyses were performed on the remaining ventral spinal cord. Tissue samples were weighed and homogenized in cold extraction buffer (Tris-buffered saline, pH 8.0, with 1% NP-40, 10% glycerol, 5 mM sodium metavanadate, 10 mM PMSF, 100 ⁇ g ml aprotinin and 10 ⁇ g/ml leupeptin).
• cold extraction buffer Tris-buffered saline, pH 8.0, with 1% NP-40, 10% glycerol, 5 mM sodium metavanadate, 10 mM PMSF, 100 ⁇ g ml aprotinin and 10 ⁇ g/ml leupeptin).
• Homogenates were acidified with 1 N HCI (pH ⁇ 3.0), incubated at room temperature for 15 min, and neutralized with 1 N NaOH (pH ⁇ 7.6). Homogenates were then microfuged at 7000 g for 10 min, and the supernatants were assayed with antibody sandwich ELISAs (BDNF ELIS A, R & D Systems, Minneapolis, MN; NT-3 ELISA, Promega Corporation, Madison, WI). Neurotrophin concentrations were normalized per gram of tissue wet weight and per gram of total protein determined with the BCA (bicinchoninic acid) method (Pierce, Rockford, IL). Since both normalization methods produced qualitatively similar results neurotrophin concentrations are presented only per gram of tissue wet weight.
• Recombinant human BDNF (Promega Corporation, Madison, WI) was dissolved in artificial CSF + 0.1% bovine serum albumin (BSA).
• BSA bovine serum albumin
• K252a (Calbiochem; San Diego, CA) was initially dissolved in dimethylsulfoxide (DMSO; 100 ⁇ g K252a in 1.5 ml DMSO) and frozen in 200 ⁇ l aliquots.
• DMSO dimethylsulfoxide
• Vehicle delivery was as described above (DMSO + artificial CSF), except K252a was not added.
• arterial blood gases were analyzed to ensure that PaO 2 and PaCO 2 levels were similar to pre- injection values.
• BDNF siRNA small interfering RNAs directed against BDNF mRNA or a scrambled sequence
• the BDNF siRNA consisted of 4 pooled 21-nucleotide duplexes with syrnn etrical 3' overhangs (SMARTpool).
• sequences of the 4 duplexes were as follows: 1) TCGAAGAGCTGCTGGATGA (SEQ ID NO: 1); 2) TATGTACACTGACCATTAA (SEQ ID NO: 2); 3) GAGCGTGTGTGACAGTATT (SEQ ID NO: 3); and 4) GAACTACCCAATCGTATGT (SEQ ID NO: 4).
• BDNF siRNA and scrambled siRNA were suspended in siRNA Universal Buffer (Dharmacon; Layfayette, CO) to yield a concentration of 50 ⁇ M.
• the siRNA stocks were aliquotted and stored at 20°C.
• siRNAs directed against the mRNA of individual protein phosphatases could be developed, as described herein, for in vitro or in vivo applications or kinases (e.g., ERKl/2, CAMK ⁇ , PKC, PKA, MEKK) or translation factors.
• siRNAs of the invention may be chemically or enzymatically synthesized, as described in WO 99/32619 and WO 01/68836.
• Enzymatic synthesis of siRNA may use a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g. T3, T7, SP6) facilitated by expression constructs known in the art, such as for example described in U.S. Pat. No. 5,795,715.
• the contemplated constructs provide templates that produce RNAs which contain nucleotide sequences identical to and complementary to a portion of the target gene representing the sense and antisense strands, respectively.
• the length of identical sequences provided by these references is at least 25 base pairs in length.
• This method contemplates digesting longer dsRNAs to 19-25mer lengths with the endogenous nuclease complex that converts long dsRNAs to siRNAs in vivo. No distinction is made between the expected properties of chemical or enzymatically synthesized dsRNA for its use in RNA interference.
• RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
• the references described hereinabove also provide that in vitro synthesis may be chemical or enzymatic, for example using cloned RNA polymerase (e.g. T3, T7, SP6) for transcription of the endogenous DNA (or cDNA) template, or a mixture of both.
• cloned RNA polymerase e.g. T3, T7, SP6
• Murine HT-22 hippocampal cell line (Salk Institute, San Diego, CA) was grown to ⁇ 80% confluency and passaged in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Cells were plated overnight in Falcon 6 well plates at a density of 2 x 10 5 cells/well. The following day, cells were transfected with 200 nM scrambled or BDNF siRNA. Twenty minutes prior to transfection, siRNAs (BDNF or scrambled) or siRNA Universal Buffer was combined with OligofectamineTM (0.6 microliter from stock; Invifrogen, Carlsbad, CA) and added to the appropriate wells.
• OligofectamineTM OligofectamineTM (0.6 microliter from stock; Invifrogen, Carlsbad, CA)
• BDNF siRNA having siSTABLETM modification available through Dharmacon, Lafayette, Colorado.
• siSTABLETM is a proprietary form of siRNA with chemically modified strands that enhance stability and silencing longevity without compromising efficacy or increasing cellular toxicity.
• M-MLV Reverse Transcriptase Invitrogen; Carlsbad, CA. 18s rRNA was used to normalize BDNF mRNA values. Gene sequences were obtained from Genbank using the Unigene search engine (maintained by the National Center for Biotechnology Information).
• BDNF forward primer 5'-CTGACACTTTTGAGCACGTGATC-3' (SEQ ID NO: 5); reverse primer: 5'-AGGCTCCAAAGGCACTTGACT-3' (SEQ ID NO: 6); 18s Ribosomal Subunit forward primer: 5'-AACGAGACTCTCGGCATGCTAA-3' (SEQ ID NO: 7); reverse primer: 5'-CCGGACATCTAAGGGCATCA-3' (SEQ ID NO: 8).
• Genbank Accession numbers are as follows: BDNF, NM_012513; and 18s Ribosomal Subunit, X01117 K01593.
• Control rats or rats treated with scrambled sequence or BDNF siRNA were surgically prepared as described above.
• the siRNA 17. ⁇ l was combined with OligofectamineTM (2.5 ⁇ l) to make a siRNA composition.
• the siRNA composition was incubated at room temperature (22-24°C) for 15 min. Rats were anesthetized with isoflurane and the diaphragm exposed through a small ventral midline incision.
• the rats received 10, 4 ⁇ l bilateral diaphragm injections of siSTABLETM siRNA (BDNF or scrambled) or vehicle.
• the siRNA composition was injected over C 4 immediately following spinal cord exposure (two ⁇ 10 microliters injections spaced one minute apart).
• Awake rats were exposed to hypoxia in an environmental chamber designed in- house.
• the chambers were approximately 4 L in volume.
• Computer mixed gases were passed through the chamber at a flow rate of 4 L/min per chamber to assure levels of C O 2 accumulation below 0.5%, and to enable rapid dynamics in the on and off transients during hypoxic episodes (50 and 70 sec for down and up transients, respectively).
• Intermittent hypoxia consisted of 5, 5 min episodes of 9-10% O 2 , separated by 5 min intervals, since this pattern has been shown to elicit LTF in awake rats.
• Sustained hypoxia consisted of 25 min of 9-10% O 2 .
• pLTF magnitude was calculated at 60 min post-hypoxia or hypercapnia as a percentage change of the baseline value.
• Regression analysis was performed to determine the relationship between pLTF magnitude and the percentage change in BDNF from controls. For this analysis, pLTF was averaged and the change in BDNF from controls within each experimental condition is presented herein. [00094] A one-sample t-test was used to determine if rats pretreated with DMSO, K252a, scrambled siRNA or BDNF siRNA had significant pLTF.
• a Student's t-test was used to compare pLTF in the treatment groups with the controls (DMSO versus K252a, scrambled versus BDNF siRNA).
• a two-way ANOVA with a repeated measures design was used to compare phrenic responses before and after BDNF, vehicle and BDNF + K252a injections, and individual comparisons were made with the Student-Neuman-Kuels post hoc test. Differences were considered significant if P ⁇ 0.05. All values are expressed as mean ⁇ standard enor.
• an siRNA composition as described above directed to the BDNF target gene was injected into a rat tongue.
• the rat, Fisher (F344) was obtained from Harlan Sprague-Dawley, (Indianapolis, IN).
• the siRNA molecules were obtained from Dharmacon and consisted of four, 21-nucleotide duplexes as described herein.
• a stock of siRNA (50 ⁇ l) was combined with 7.5 ⁇ l OligofectamineTM (Invitrogen; Carlsbad, CA) forming an siRNA composition.
• the siRNA composition was maintained for 15 min at room temperature prior to injection into the rat tongue.
• Carprofen was administered sub ⁇ utaneously (5mg/kg) upon anesthesia induction to reduce pain.
• a series of eight, 7 ⁇ l injections of the BDNF siRNA compositions was made in the tongue, covering the top, bottom and base of the tongue bilaterally.
• the rat was allowed to recover for 2 days prior to being analyzed.
• the rat was then subjected to the LTF protocol as described above. The LTF was shown to be blocked 2 days later.
• FIG. 6 shows a graph of the functional result following tongue injection of BDNF siRNA molecules.
• the results suggest that the form of BDNF-dependent plasticity (LTF) was blocked in the targeted motoneuron pool, in the XII nucleus, but not blocked in a related (but separated) phrenic motor output. This indicates that the siRNAs were likely confined to the motoneuron pool that innervates the tongue XII nucleus and did not reach other, related targets.
• LTF BDNF-dependent plasticity
• BDNF siRNA can be transported to the motoneuron cell body from the tongue muscle, blocking BDNF functions.
• This approach allows delivery of siRNA to an accessible, peripheral site, the muscle, but affects nerve cells (motoneurons) located in the nervous system behind the blood brain barrier. It is envisioned that this approach will be applicable to different molecular targets of siRNA, allowing alterations of gene expression in a well defined cell, suitably motoneuron, which play s a role in neuro- muscular disorders such as sleep apnea, spinal cord injury, ALS and polio. Delivery of siRNA into diaphragm muscle
• siRNA compositions may be injected into the diaphragm muscle of a mammal, as well as in virtually any muscle in the body.
• an siRNA composition directed to the BDNF target gene is injected into a rat diaphragm muscle to reach phrenic motoneurons.
• the siRNA compositions Prior to the diaphragm injections, the siRNA compositions will be prepared by combining an siRNA stock (50 ⁇ l) with 7.5 microliters oligofectamineTM (RNase free conditions) and maintaining it at room temperature for 15 minutes. Carprofen was administered subcutaneously (5 mg/kg) immediately upon anesthesia induction.
• BDNF siRNA BDNF siRNA, scrambled siRNA or saline
• Rats were recovered for 2 - 5 days prior to the LTF protocol.
• the BDNF siRNA compositions injected into the rat diaphragm reached the phrenic motoneurons within three days, and affected spinal respiratory plasticity following intermittent hypoxia.
• tissue samples were homogenized in 1 ml phosphatase storage buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% /3-mercaptoethanol, 100 mM leupeptin, 75 mM pepstatin) and centrifuged for 1 hr at 100,000x g at 4°C. Phosphatase activity was immediately quantified in the supernatants as described hereinbelow.
• phosphatase storage buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% /3-mercaptoethanol, 100 mM leupeptin, 75 mM pepstatin
• Phosphatase activity was immediately quantified in the supernatants as described hereinbelow.
• the XII nucleus and ventral gray matter were homogenized in 250 ml RNA extraction reagent (Trizol Reagent, Invitrogen), respectively, according to manufacturers instruction.
• RNA samples will be stored at -80°C for later analysis.
• chloroform (1/5 Trizol volume) will be added to the samples to denature proteins.
• samples will be centrifuged (12,000g, 4°C, 15 min) to separate organic and inorganic layers. The organic layer will be collected and isopropanol (1/2 Trizol volume) will be added to precipitate RNA (10 min room temperature incubation).
• the RNA will be spun to a pellet (12,000g, 4°C, 10 min), washed with 500 ml of ice-cold 80% ethanol and centrifuged twice (12,000g, 4°C, 5 min) to remove excess ethanol. Pellets will be air-dried for 10 min and re-suspended in 30-50 ml DEPC-treated water. RNA samples will be stored at -80°C for later analysis.
• ELISA ELISA
• a BDNF ELISA (R&D Systems) was used to quantify BDNF changes following treatments.
• tissue samples and a standard curve will be added to a 96-well polystyrene plate pre-coated with a BDNF monoclonal antibody.
• the plate will then be incubated for 2 hrs at room temperature to allow BDNF in the experimental and standard samples to bind to the immobilized antibody.
• An enzyme-linked (horseradish peroxidase) BDNF monoclonal antibody will be added and incubated for 1 hr at room temperature, during which the conjugated antibody forms a "sandwich" with the immobilized antibody-BDNF protein complex.
• the plate will then be washed with buffer, and a substrate solution (hydrogen peroxide + chromogen) added. Color is allowed to develop for 30 min, and then an acidic solution (2 N sulfuric acid) is added to stop the peroxidase reaction. The color intensity is measured using a microplate reader (MRXI Absorbance Reader with MRX Revelation software; Dynex Technologies) set to 450 ran with wavelength coreection at 570 nm. The absorbances are directly proportional to the amount of bound BDNF. BDNF protein levels in the samples are normalized to both total protein levels and per gram of tissue, wet weight. Total protein is determined using a BCA assay kit (Pierce Biotechnology).
• Samples were diluted 1 :2 with 2x sample buffer (20 mM Tris, 2 mM EDTA, 1 mM Na3VO4, 2 mM dithiothreitol, 2% SDS, 20% glycerol) and boiled (105°C) for 5 minutes. Equal amounts of protein ( ⁇ 30 mg) from each sample were loaded per lane and separated by 10% SDS-PAGE gel. Proteins in the gels were transfened to hnmobilon polyvinylidene difluoride (PVDF) membrane (Millipore Corp.).
• PVDF polyvinylidene difluoride
• Membranes were blocked in 5% non-fat milk/TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) for 30 min at 37°C.
• An anti-phosphorylated ERKl/2 MAP kinase antibody (Cell Signaling Technology), which recognize enzymatically active ERKl/2, was used at a dilution of 1 :2500 overnight at 37 oC.
• the immunoreactive bands were visualized using secondary antibodies conjugated to horseradish peroxidase (goat anti-rabbit IgG-HRP; Santa Cruz Biotechnology) at a dilution of 1 :8000 and chemiluminescent detection with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Band density will be determined using the AC1 AutoChemi System (UVP) and quantified using Lab Works Image Acquisition and Analysis Software (UNP). All samples were run in duplicate.
• horseradish peroxidase goat anti-rabbit IgG-HRP; Santa Cruz Biotechnology
• membranes were stripped using Restore Western Blot Stripping Buffer (30 min at 37°C; Pierce Biotechnology), re-blocked in 5% milk/TBST, and probed using an anti-ERKl/2 MAP kinase antibody that recognizes both phosphorylated and non- phosphorylated forms of ERKl/2 (1:2500 dilution; Santa Cruz Biotechnology).
• the density of phosphorylated ERKl/2 MAP kinase was normalized to total ERKl/2 MAP kinase within the same lane; the percentage change in normalized phosphorylated ERKl/2 MAP kinase in treated versus control rats run on the same gel was then calculated.
• Assay System Promega Corporation according to manufacturers instructions.
• free phosphates in the samples will be removed using a Sephadex G-25 resin column and centrifuged at 600 g at 4°C for 5 min.
• a reaction buffer that preferentially targets protein phosphatase 2A (PPTase-2A 5x reaction buffer: 10 ml; 250mM imidazole, pH 7.2, 1 mM EGTA, 0.1% ⁇ - mercaptoethanol, 0.5 mg/ml BSA) and 1 mM phosphopeptide (5 ml) will be added to a 96 well plate and incubated at 30°C for 3 min.
• sample lysate (5 ml) diluted in phosphatase storage buffer (30 ml; see tissue preparation section) was added to the wells and incubated for 30 min at 30°C. Enzymatic activity of the protein phosphatases in the sample lysate was stopped with Molybdate Dye/ Additive mixture (50 ml), and the plate will be incubated at room temperature for 15 min. Optical density of the samples was read at 630 ran using a microplate reader (MRXI Absorbance Reader with MRX Revelation software; Dynex Technologies). The level of serine/threonine phosphatase activity in each sample was calculated using a standard curve generated by diluting the phosphate standard. To compare across samples, the level of phosphatase activity was divided by total protein in the sample lysates (determined using the Pierce BCA protein assay kit).
• each treatment group was paired with age-matched control rats with similar surgery and drug freatments, but did not receive intermittent hypoxia or hypercapnia.
• a Student's t-test was used to detect significant differences between matched controls and experimental groups exposed to intermittent hypoxia or hypercapnia.
• BDNF levels following intermittent hypoxia or hypercapnia were normalized as a percentage change from the appropriate controls prepared and analyzed on the same day (to control for batch differences).
• BDNF concenfration following hypoxia in rats prefreated with methysergide were expressed as percentage changes from the average BDNF concentration in methysergide confrols homogenized with the same lysis buffer and analyzed on the same ELISA plate.
• a one-way ANONA was used to test for statistical differences in the percent change in BD ⁇ F across experimental groups.
• a two-way A ⁇ ONA was used to make statistical inferences regarding baseline BD ⁇ F concentrations in control (no drug + scrambled siR ⁇ A) and BD ⁇ F siR A treated rats.
• BDNF protein concenfration in ventral C 3 - C 5 increased from 2476 ⁇ 321 pg/g tissue in control rats to 3940 ⁇ 308 pg/g tissue 60 min post- intermittent hypoxia (P ⁇ 0.05; FIG. IB).
• intermittent hypoxia increased BDNF protein concentration near the phrenic motor nucleus 56 ⁇ 12% (batch controlled; P ⁇ 0.05).
• This observation was confirmed in rats exposed to intermittent hypoxia, but without intrathecal aCSF; BDNF concenfration increased 49 ⁇ 9% 60 min post-intermittent hypoxia (P ⁇ 0.05; data not shown).
• ventral cervical NT-3 concentration was unaffected by intermittent hypoxia (control: 2397 ⁇ 457; 60 min post-hypoxia: 2291 ⁇ 389 pg/g tissue; P > 0.05). It is envisioned that the intermittent hypoxia also increases BDNF synthesis in other respiratory motor nuclei (for example, hypoglossal) as well as non-motor nuclei involved in other functions such as walking, posture, reaching and grasping or speech.
• intermittent hypoxia also increases BDNF synthesis in other respiratory motor nuclei (for example, hypoglossal) as well as non-motor nuclei involved in other functions such as walking, posture, reaching and grasping or speech.
• the primary oxygen-sensitive chemoreceptors in adult mammals are in the carotid body.
• carotid denervated rats CSNX
• intermittent hypoxia elicited a non-significant 19 ⁇ 11% increase in ventral C 3 - C 5 BDNF concenfration (P > 0.05; FIG. 2B).
• Althou h sham CSNX rats increased ventral cervical BDNF 38 ⁇ 12% post-intermittent hypoxia (P ⁇ 0.05; FIG. 2B), this value was not significantly different from changes in CSNX rats (P > 0.05).
• intact chemoreceptors appear necessary for the full effect of intermittent hypoxia on ventral cervical BDNF.
• Ventral C 3 - C 5 BDNF concentration was measured following intermittent hypercapnia to determine if increased BDNF concentration is a nonspecific response to increased respiratory (synaptic) activity. Although hypercapnia is a powerful respiratory stimulus, it does not elicit pLTF (3,13). The BDNF concentration was unchanged, 60 minutes post-intermittent hypercapnia, (change from control -10 ⁇ 13%, P > 0.05; FIG. 2B). In referring to FIG.2B, it suggests that increased BDNF in venfral C - C 5 following intermittent hypoxia at least partially requires carotid chemoreceptors, since intermittent hypoxia failed to significantly increase BDNF following CSNX.
• Intrathecal BDNF elicits long-lasting phrenic facilitation
• rats were injected with BDNF (0.1 ⁇ g) in the intrathecal space above the phrenic motor nucleus.
• vehicle (artificial CSF + 0.1% BSA) injections elicited no time-dependent change in phrenic activity (90 min post-injection: 36 ⁇ 24% above baseline, P > 0.05; FIG. 3B)
• intrathecal BDNF significantly increased integrated phrenic discharge (125 ⁇ 25%, 90 min post-injection, P ⁇ 0.05; FIG. 3A,B).
• Intrathecal BDNF elicited significant increases in phrenic burst amplitude 60 and
• BDNF-induced facilitation was blocked by pre-treatment with intrathecal K252a (9 ⁇ 10%, 90 min post-injection, P > 0.05; FIG. 3B), a Trk receptor inhibitor.
• Intrathecal BDNF effects were restricted to the spinal cord since there were no time-dependent changes in hypoglossal nerve activity, a reflection of brainstem respiratory motor output (30 ⁇ 7%, 90 min post-injection, P > 0.05; FIG. 3C).
• spinal BDNF facilitates phrenic motor output, likely via TrkB receptor activation.
• RNA interference is achieved with double-stranded RNA segments that elicit sequence specific inhibition or degradation of homologous mRNA via an endogenous pathway (14,15).
• siRNA small interfering RNA
• BDNF siRNA did not knock-down basal BDNF protein levels in this short time frame (gray bars). However, BDNF siRNA prevented hypoxia-induced increases in BDNF protein concentration (black bars).
• a typical rat exhibited a progressive increase in phrenic amplitude (pLTF) for at least one hour following intermittent hypoxia (upper trace).
• a rat pretreated with intrathecal BDNF siRNA or K252a showed no pLTF (bottom two traces).
• two individual BDNF siRNA duplexes were tested for their effect on pLTF.
• BDNF siRNAs inhibit hypoxia-induced BDNF mRNA translation and pLTF, providing compelling evidence that endogenous BDNF synthesis is necessary for pLTF following intermittent hypoxia. It is envisioned that BDNF is necessary for LTF or motor plasticity in other motor nuclei as well.
• K252a block pLTF. This finding was determined by testing the hypothesis that pLTF requires activation of a high affinity Trk receptor. Rats were pretreated with intrathecal K252a (0.13 - 0.2 ⁇ g), a non-specific Trk receptor inhibitor. In rats receiving intrathecal DMSO (vehicle), integrated phrenic burst amplitude was significantly increased from baseline 60-min post- intermittent hypoxia (109 ⁇ 19% above baseline, P ⁇ 0.05; FIG. 5A,C), indicating pLTF. In contrast, rats pretreated with K252a had no significant increase in phrenic burst amplitude 60 min post-intermittent hypoxia (25 ⁇ 6%, P > 0.05; FIG.
• Trk receptor activation is necessary for full expression of pLTF following intermittent hypoxia, which is consistent with the hypothesis that BDNF acts via the TrkB receptor to elicit pLTF.
• Example 5 Diaphragm injection of BDNF siRNA blocks phrenic LTF.
• experiments were performed to confirm the hypothesis that BDNF synthesis within phrenic motoneurons is necessary for pLTF and to show the feasibility of retrograde transport of siRNAs from a target muscle to the motoneurons that innervate that muscle.
• BDNF siRNAs were targeted to the phrenic motoneurons by siRNA injections into the diaphragm to prevent hypoxia-induced BDNF synthesis within phrenic motoneurons er se.
• applicants delivered BDNF siRNAs to the diaphragm for axonal uptake and retrograde transport.
• BDNF siRNA 10 depicts exciting preliminary data from one rat that received diaphragm injections of BDNF siRNA (10, 4 ml injections; 50 ml of a 50 mM solution of two siRNA duplexes added to 8 ml OligofectamineTM).
• BDNF siRNA was injected into the tongue, and applicants observed the reverse result (i.e. XII LTF was abolished, but pLTF was not; Fig. 6).
• BDNF siRNAs are transported to the target motoneurons where they degrade/inhibit BDNF mRNA and prevent LTF.
• Successful delivery of the siRNA should reduce BDNF protein levels due to normal protein turnover, and reduce hypoxia-induced translation of BDNF mRNA.
• this technique could be further developed by characterizing BDNF mRNA and protein changes following diaphragm injections of BDNF siRNA, as well as further experiments concerning the functional consequences on pLTF.
• this technique could be further developed by characterizing the effects of siRNAs that target other relevant molecules, such as the mRNA for serotonin receptors, kinases and phosphatases.
• Example 6 Intermittent (but not sustained) hypoxia increases BDNF in ventral cervical gray matter
• rats were returned to normoxia for 60 min.
• the C4-C5 ventral gray matter was harvested and assayed for BDNF protein concentration (ELISA; R&D Systems). Similar to anesthetized rats, intermittent hypoxia increased BDNF protein concenfration in the ventral gray matter of the cervical spinal cord of awake rats.
• ERK 1/2 MAP kinases have many of the requisite characteristics to play a prominent role in pLTF.
• ERK 1/2 MAP kinases are critically involved in important models of synaptic plasticity.
• ERK 1/2 is activated by 5-HT2A receptor activation, an effect associated with reactive oxygen species in some cell types.
• BDNF and TrkB receptor activation rapidly activate ERK 1/2 MAP kinases. Since ERK 1/2 MAP kinases are involved in glutamate receptor trafficking applicants believe that they are logical candidates to translate BDNF signaling into synaptic enhancement, thereby establishing pLTF.
• ERK 1/2 is activated in a manner consistent with a major role in pLTF.
• BDNF siRNA was injected over C4 in two anesthetized rats: one received normoxia (confrol), while the other received intermittent hypoxia. This technique effectively prevents hypoxia-induced BDNF synthesis near phrenic motoneurons. Tissues were harvested and ERK 1/2 activation was assessed via immunoblot.
• Example 9 Activation of ERKl/2 MAP kinase. but not CaMKII, is necessary for pLTF [000126] To determine if the activation of ERKl/2 MAP kinase alone is necessary for pLTF, one anesthetized rat was pre-freated with intrathecal UO126 (0.4 mg; 100 mM), a MAP kinase kinase inhibitor, 30 min prior to intermittent hypoxia. The inhibition of ERKl/2 MAP kinase activation blocked pLTF.
• Example 10 MAP kinase activation facilitates phrenic burst amplitude
• anisomycin 100 mg; 20 mM
• phrenic burst amplitude was increased by 60% above pre-injection levels.
• Example 11 Sustained (but not intermittent) hypoxia increases protein phosphatase (PP) activity
• Example 12 Sustained hypoxia elicits phrenic LTF following protein phosphatase inhibition
• protein phosphatase inhibition removes the inhibitory constraint and reveals pLTF following sustained hypoxia.
• 10 ml okadaic acid (0.15 mg; 20 mM)
• PP1 and PP2A protein phosphatase 1/2A
• genes such as BDNF and PP2A may be targeted by siRNA molecules through either direct (intrathecal) or indirect (inframuscular) in vivo delivery to affect gene expression and resulting in physiological change.
• the invention provides that direct delivery of the siRNA composition into the intrathecal space of a mammal, effectively interfered with BDNF mRNA.
• the interference with BDNF mRNA blocked increases in BDNF in the cervical spinal cord elicited by a reduced flow of oxygen called intermittent hypoxia, which causes a form of serotonin-dependent spinal synaptic plasticity known as phrenic long-term facilitation (pLTF).
• the invention provides that indirect delivery of the BDNF siRNA composition intramuscularly (into muscles innervated by nerve cells) protected siRNA molecules from circulating RNAses in the blood and resulted in transport of intact siRNA molecules back to the nerve cells.
• This indirect transport of siRNA molecules from the muscles to the nerve cells resulted in blocking LTF following intermittent hypoxia in the hypoglossal nerve (the motor nerve of the tongue) but not phrenic nerve (associated with the diaphragm) motor output, or in the converse depending on where the siRNA had been injected.
• Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia. J. Appl. Physiol. 90, 2001-2006 (2001).
• Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing N-methyl-D-aspartic acid receptor activity. Proc. Natl. Acad. Sci. USA 95, 10235-10239 (1998).

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• Virology (AREA)
• Pulmonology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Physical Education & Sports Medicine (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)

Priority Applications (3)

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Publications (2)

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ID=34699962

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Country Status (5)

Cited By (6)

Families Citing this family (9)

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• 2004
  • 2004-12-10 JP JP2006544003A patent/JP2007513968A/ja not_active Abandoned
  • 2004-12-10 CA CA002548972A patent/CA2548972A1/en not_active Abandoned
  • 2004-12-10 WO PCT/US2004/041339 patent/WO2005059135A2/en not_active Ceased
  • 2004-12-10 EP EP04813641A patent/EP1708756A2/en not_active Withdrawn
  • 2004-12-10 US US11/009,797 patent/US20050234000A1/en not_active Abandoned
• 2008
  • 2008-01-10 US US11/972,376 patent/US20090186844A1/en not_active Abandoned

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