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WO2003091387A2 - Administration non invasive de polypeptides a travers la barriere hemato-encephalique et selection in vivo de ligands endocytotiques - Google Patents

Administration non invasive de polypeptides a travers la barriere hemato-encephalique et selection in vivo de ligands endocytotiques Download PDF

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WO2003091387A2
WO2003091387A2 PCT/IB2003/002371 IB0302371W WO03091387A2 WO 2003091387 A2 WO2003091387 A2 WO 2003091387A2 IB 0302371 W IB0302371 W IB 0302371W WO 03091387 A2 WO03091387 A2 WO 03091387A2
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neurons
blood
polypeptide
ligand
brain barrier
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WO2003091387A3 (fr
WO2003091387A9 (fr
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Ian A. Ferguson
Hiroaki Tani
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Priority claimed from AUPS1935A external-priority patent/AUPS193502A0/en
Priority claimed from US10/188,184 external-priority patent/US20030083299A1/en
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Priority to AU2003233120A priority Critical patent/AU2003233120B2/en
Priority to JP2003587923A priority patent/JP2005535580A/ja
Priority to CA2483980A priority patent/CA2483980C/fr
Priority to EP03727872A priority patent/EP1503801A4/fr
Publication of WO2003091387A2 publication Critical patent/WO2003091387A2/fr
Publication of WO2003091387A3 publication Critical patent/WO2003091387A3/fr
Publication of WO2003091387A9 publication Critical patent/WO2003091387A9/fr
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Definitions

  • This invention relates to methods for delivering polypeptides into brain and spinal tissue in humans and other mammals, and in other higher animals that have a blood-brain barrier (BBB). It also relates to methods for targeting delivery of polypeptides to specific populations or types of neurons entirely within the brain or spinal cord. It further relates to treatments for neurological disorders in humans, and to other forms of treatment (such as to regulate fertility, maturation, growth rates, etc.) in livestock and other animals, using polypeptides that normally cannot cross the BBB.
  • BBB blood-brain barrier
  • the BBB protects the brain and spinal cord, it also excludes many therapeutic agents that could help treat diseases or injuries that affect the nervous system.
  • proteins and peptides cannot cross the BBB in any substantial quantities (e.g., Langer 1990).
  • a small number of exceptions involve limited transport systems and certain types of neuronal receptors, such as transferrin receptors (e.g., Kastin et al 1999 and Granholm et al 1998).
  • transferrin receptors e.g., Kastin et al 1999 and Granholm et al 1998.
  • those limited exceptions are not adequate for treating CNS disorders using various types of polypeptides.
  • polypeptide Any such molecule formed by expression of a gene sequence is referred to herein as a polypeptide, regardless of whether the polypeptide is later processed by "post-translation" steps such as cleavage, cell secretion, glycosylation, cysteine crosslinking, etc.
  • Neurotrophic comes from the Greek work for "nourishment” or "food”.
  • “Trophic” implies that a certain molecule is involved in the stimulation, growth, nourishment, sustenance, or similar support of a certain system.
  • Neurotrophic factors are molecules that promote neuronal growth, cause the formation of new synaptic connections between neurons, or carry out other stimulating or supporting neuronal activities. However, this term excludes: (i) nutrients (such as oxygen, glucose, amino acids, and nucleotides) that are required by all cells, and (ii) neurotransmitters which directly modulate nerve impulses between neurons (such as glutamate, acetylcholine, dopamine, serotonin, etc.).
  • Many neurotrophic factors are polypeptides, and these offer potential therapies for many CNS disorders, including: (1) treatments for brain damage caused by physical injuries, such as automobile accidents, concussions, etc.; (2) brain damage caused by a stroke, cardiac arrest, near-drowning or suffocation, loss of blood, or other problems involving ischemia (inadequate blood flow) or hypoxia (inadequate oxygen supply); and, (3) neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.
  • ischemia inadequate blood flow
  • hypoxia inadequate oxygen supply
  • neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.
  • disorders such as disorder, damage, and injury refer to any CNS disorder (whether from trauma, disease, etc.) that might be prevented or treated by one or more therapeutic polypeptides, if such polypeptides can be delivered to the proper regions of the brain or spinal cord.
  • peptide neurotransmitters act in a manner similar to classical neurotransmitters, such as glutamate or acetylcholine; they are released by a neuron at a synapse, where a binding reaction to a specific receptor on the other neuron at the synapse stimulates or inhibits the transmission of a nerve signal (also called a nerve impulse, firing, or depolarization).
  • a nerve signal also called a nerve impulse, firing, or depolarization
  • the purposes and goals of this invention are: (i) to provide methods for delivering foreign polypeptides into areas of brain and spinal tissue that are protected by the BBB, and (ii) to provide methods for delivering foreign polypeptides to targeted, specific, and limited regions of brain and spinal tissue, and/or to targeted, specific, and limited types or classes of cells inside the CNS.
  • CNS tissue is heterogeneous in the extreme, and nervous tissue from one region can not be substituted for nervous tissue from another region.
  • CNS neurons in adults are post-mitotic, and will not divide and repopulate if a vacancy is created.
  • nerve cell processes or axons, once broken, do not readily regrow and reestablish synaptic and other connections.
  • surgical or similar extraction of CNS tissue poses great risks to a patient, even if the surgery is done with the highest level of skill and care.
  • the viral vectors are derived from viruses, and make use of the lipid envelope or surface shell (also known as the capsid) of a virus. These vectors use a virus's ability to bind to one or more particular surface proteins on certain types of cells, and then inject the virus's DNA or RNA into the cell. These have become the dominant class of vectors that have been used in attempts at human gene therapy, and they are described in numerous published articles.
  • a non-viral genetic vector is created by adding, to a gene expression construct, agents that will enable and promote entry of the gene construct into target cells.
  • agents include cationic lipids, positively charged molecules such as polylysine or polyethylenimine, and/or ligands that bind to receptors expressed on the surface of the target cell (such as, in some cases, a viral ligand that was originally obtained from a virus).
  • ligands that bind to receptors expressed on the surface of the target cell (such as, in some cases, a viral ligand that was originally obtained from a virus).
  • Major categories of non- viral gene vectors include:
  • the Applicants herein were searching for ways to genetically transform neurons (and, in particular, neurons that straddle the blood-brain barrier and have fibers that extend outside the BBB). In addition, because they were aware of the problems and limitations that have plagued and thwarted the medical use of viral vectors, they were looking for other types of transport and delivery systems, which could pull strands of foreign DNA into cells (and, in particular, into neurons that straddle the BBB, and have fibers that extend outside the BBB).
  • Another object of this invention is to disclose a new method of transfecting sensory neurons or motor neurons that "straddle" the BBB, in a manner which causes the BBB- straddling neurons to deliver therapeutic polypeptides to neurons which are located entirely within the blood-brain barrier.
  • Another object of this invention is to disclose a new method for identifying and isolating ligand molecules that can be used to effectively transport therapeutic drugs, diagnostic or analytical compounds, or DNA sequences, into selected, targeted, and limited types and classes of animal cells.
  • a vector-borne gene encoding a CNS-active polypeptide will be transported by retrograde transport to the main cell body, where it will be expressed by the transfected neuron, to form therapeutic or otherwise useful polypeptide molecules.
  • These polypeptides will be of a type that normally are secreted by cells, or they can be provided with leader sequences that can promote secretion; accordingly, they will be transported within the transfected neuron to one or more secretion sites located within CNS tissue that is protected by the BBB.
  • the polypeptides that are secreted at such locations inside the BBB will then be able to contact targeted neurons that are located wholly within (and are therefore protected by) the blood-brain barrier.
  • this screening method uses the following steps: (1) emplacing multiple candidate ligands at a first location inside the body of a living animal, where the candidate ligands will directly contact nerve fibers (such as a sciatic nerve bundle, in the leg of a rat); (2) allowing enough time to pass for the nerve fibers to internalise those particular ligands which can activate and drive endocytosis; (3) harvesting segments of the nerve fibers, at a site (such as distal to a ligature that constricts the sciatic nerve, in a rat hip) that is sufficiently distant from the ligand emplacement site to avoid collecting ligand candidates that did not enter or were not transported by the nerves; and, (4) removing the internalised ligands from the harvested nerve segments.
  • nerve fibers such as a sciatic nerve bundle, in the leg of a rat
  • the DNA carried by the genetic vector 100 As shown on a different scale (at a microscopic, cellular level) in FIG. 3, once the DNA carried by the genetic vector 100 has entered a neuronal projection 212, it is carried to the main cell body 224, by retrograde transport (discussed below). Because the genetic vector in this example has been derived from a type of virus that is fully capable of infecting nasal receptor cells, the vector DNA (which carries passenger gene 160, as shown in FIG. 2, as merely one component inserted into an disabled but infective virus genome) is able to help promote and facilitate this process. Accordingly, the use of infective viruses to create the vector delivery system will help ensure that the transcribed portion of the passenger gene 160 will be expressed into messenger RNA strands 299 in at least some transfected neurons, as shown in FIG. 3.
  • neuroactive polypeptides are inherently secreted; as a general rule, their neuroactivity was discovered and recognized due to their ability to be secreted by one type of cell within the CNS, and to act upon other types of cells within the CNS. And second, if some particular candidate polypeptide that is of interest is not naturally and normally secreted by cells, that polypeptide sequence can be coupled to any of numerous known “leader” polypeptide sequences that cause cellular secretion.
  • basal forebrain cholinergic neurons, serotonergic raphe neurons, and noradrenergic locus coeruleus neurons do not have direct synaptic junctions with BBB-straddling olfactory receptor neurons; nevertheless, these classes of cholinergic, serotonergic, and noradrenergic neurons inside the BBB can be target neurons, if olfactory neurons are transfected by a genetic vector as disclosed herein, because they can take up polypeptides released by transfected olfactory receptor neurons.
  • Retroviruses including Lentiviruses
  • epi- refers to an exposed and accessible surface; as examples, the epidermis is the outermost dermal (skin) surface, and the epithelium is the exposed surface of a mucous membrane.
  • epitopic analysis is often used by researchers to help them determine the three-dimensional structure of a complex protein; as a general rule, the amino acid sequences which show up as epitopic sites in a protein are the amino acid sequences that are exposed, and accessible to antibodies, on the surface of the protein.
  • Examples 1-7 describe how NGF (or other neurotrophic or similar polypeptides) can be delivered to cholinergic neurons in the basal forebrain of a laboratory animal, such as a rat.
  • Examples 1-4 contain a complete embodiment, divided into various sequential steps.
  • Example 1 describes the assembly of the vectors;
  • Example 2 describes administration of those vectors to the nasal sinuses;
  • Example 3 describes methods of monitoring delivery of the polypeptide through the blood brain barrier; and
  • Example 4 describes methods of measuring the physiological and behavioral effects of such treatments on lab animals.
  • EXAMPLE 6 CONSTRUCTION AND USE OF LIPID-BASED VECTORS FOR NON- INVASIVE DELIVERY OF NGF
  • Various other known compounds also provide good candidates which can be evaluated for potential use as ligands which can bind to endocytotic receptors on one or more types of BBB-stiaddling neurons, and/or as peptide sequences which can promote retrograde tiansport or some other function after a genetic vector or some portion thereof has been taken inside a neuron.
  • Wiley and Lappi 1993 describes a conjugate formed by coupling (i) monoclonal antibody 192-IgG, which binds to the p75 neuronal receptor, to (ii) a plant-derived toxin called saporin, which is believed to promote intracellular tiansport to a neuronal cell body, where it inactivates ribosomes.
  • administración of these genetic vectors to olfactory epithelium, via nasal instillation of vectors suspended in an aqueous saline solution or other carrier liquid can use the same general procedures described in Example 2, adapted for such use by means known to those skilled in the art.
  • Such polypeptides can work by at least two known mechanisms: (i) by binding to NGF molecules in cerebrospinal fluid (CSF) or synaptic fluid, thereby inactivating the NGF molecules by rendering them unable to bind to NGF receptors; and/or, (ii) by binding to NGF receptor proteins on neurons, thereby occupying those receptors and rendering them inaccessible to NGF molecules, in a manner which does not trigger the cellular reactions that occur when free NGF molecules bind to the receptors.
  • CSF cerebrospinal fluid
  • synaptic fluid thereby inactivating the NGF molecules by rendering them unable to bind to NGF receptors
  • NGF receptor proteins on neurons thereby occupying those receptors and rendering them inaccessible to NGF molecules, in a manner which does not trigger the cellular reactions that occur when free NGF molecules bind to the receptors.
  • Anti-NGF polypeptides which bind to free NGF molecules in CSF or synaptic fluid are known, and are described in articles such as Ruberti et al 1993 and 1994. Gene sequences which encode these anti-NGF polypeptides are also known, and can be incorporated into complete gene constructs having suitable gene promoters which will drive gene expression in mammalian cells, as disclosed in Example 1 above.
  • CGRP promoter which drives expression of the CGRP gene (described in Watson et al 1995)
  • expression of a vector-carried anti-NGF gene can be restricted to tiansfected nocioceptive neurons which express the neuropeptide CGRP.
  • the CGRP promoter may be especially useful if one objective of a tieatment is to reduce the level of NGF in the spinal cord of a patient suffering from neuropathic or other chronic pain, because the activity of this promoter is enhanced in the presence of NGF.
  • this promoter may be able to increase synthesis and secretion of anti-NGF in response, thereby creating a self-limiting, self-regulating gene expression system.
  • Anti-NGF may be detected in the spinal cord after allowing sufficient time for gene expression in the dorsal root ganglion (anticipated to be about 24 to 72 hours) and anterograde transport and release of the anti-NGF into the dorsal horn regions of the spinal cord (anticipated to be another 8 to 24 hours).
  • anti-NGF polypeptides in the spinal cord can be monitored directly with appropriate methods, such as electron microscopy, immunological staining, and various methods of labelling the vector-derived anti-NGF, such as inclusion of an appropriate antigenic tag in the amino acid sequence of the polypeptides encoded by the vector gene. It also may be possible to monitor the concentration and/or effects of anti-NGF by measuring the concentration of free NGF. When anti-NGF is released, it will bind to and neutralize endogenous NGF in the spinal cord.
  • changes in the level of NGF in the spinal cord can be monitored by making use of various immunological procedures, such as those outlined in Example 3, and applying these procedures to examination of the spinal cord tissue receiving innervation from the tiansfected sensory neurons. Changes in the level of NGF in the spinal cord can also be inferred from characteristic neuroanatomical and behavioral changes, as outlined in more detail in Example 10.
  • GDNF glial cell line derived neurotrophic factor
  • the necessary tasks can be simplified and rendered more reliable by administering the gene vector to the muscles on one side of a test animal (such as injecting the vector solution into a hind leg), and tieating the other side of the same animal as a contiol, by using a control vector which carries, for example, an innocuous and/or non-functional gene, a nonsense DNA sequence which does not encode any polypeptide, or a marker gene which encodes a polypeptide that can be easily detected if expressed in mammalian cells, but which has no significant physiological effect.
  • a control vector which carries, for example, an innocuous and/or non-functional gene, a nonsense DNA sequence which does not encode any polypeptide, or a marker gene which encodes a polypeptide that can be easily detected if expressed in mammalian cells, but which has no significant physiological effect.
  • the left and right regions of the spinal cord can be compared against each other, to assess the movement, concentrations, and effects of the vector DNA and/or the polypeptide(s) encoded by the vector gene(s).
  • the effectiveness of gene vector delivery can be assessed by removing the lumbar spinal cord from some experimental animals and processing the tissue to monitor for expression of the neurotiophic factor gene within the spinal motor neurons.
  • analyses can use methods such as: (i) hybridization of cellular mRNA with DNA probes that are complementary to the gene vector mRNA, but not to endogenous mRNA sequences, using procedures as described in articles such as Xian and Zhou 2000; (ii) techniques which use "polymerase chain reaction” (PCR) reagents and methods to detect DNA or mRNA sequences from the viral vector, as described in articles such as Chie et al 2000; and, (iii) immunostaining, ELISA, or similar methods which use antibodies that selectively bind to the vector polypeptide but not to the endogenous polypeptide in the test species.
  • PCR polymerase chain reaction
  • a 1 mL aliquot of the glycerol- containing stock was thawed, and 100 ⁇ L of the thawed stock was added to 25 mL of 2TY broth containing 2% (w/v) filter-sterilized glucose and 100 mg/mL ampicillin.
  • the cells were grown at 37°C in a shaking incubator until they reached an OD 600 density of about 0.5 to 0.8.
  • M13KO7 helper phages were then added, to form a final concentration of 5 x 10 9 "colony forming units" (cfu) per mL.
  • the mixture was incubated for 30 minutes while stationary, then for 30 minutes in a shaker tray at 200 rpm.
  • the sciatic nerve can be clearly seen.
  • the sciatic nerve is only loosely attached to the surrounding tissues, by membranes that are easily separated.
  • the nerve itself is protected by a tough nerve sheath, and an estimated 50,000 axons may be contained within this nerve bundle, depending on the location. While the axons of some sympathetic or other nerve may not be myelinated, each motor axon (and most sensory nerve axons) is sunounded by a myelin sheath, contributed by Schwann cells, which make up the bulk of nerve tissue mass outside the blood brain barrier.
  • a fluorescent staining technique was used to generate photomicrographs that visually confirmed the accumulation of internalised and transported MC192-M13KO7 antibody-phage conjugates in the sciatic nerve bundle, just below the hip ligature.

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Abstract

La présente invention se rapporte à une méthode de traitement et à des vecteurs génétiques permettant l'administration non invasive de polypeptides à travers la barrière hémato-encéphalique (BBB), aux fins du traitement d'un tissu cérébral ou spinal. Un vecteur génétique est utilisé pour transfecter un ou plusieurs neurones qui 'chevauche(nt)' la barrière BBB, tels que des neurones sensitifs, des neurones nociceptifs ou des neurones moteurs inférieurs; pour ce faire, le vecteur est administré de manière à provoquer son contact avec des protubérances neuronales qui s'étendent à l'extérieur de la BBB. Une fois à l'intérieur d'une projection périphérique qui appartient à un neurone chevauchant la BBB, les vecteurs (ou une partie de ceux-ci) sont transportés vers le corps cellulaire principal du neurone, selon un processus appelé transport rétrograde. A l'intérieur du corps cellulaire principal, au moins un gène porté par le vecteur génétique sera exprimé, de manière à former des polypeptides. Certains de ces polypeptides (qui peuvent inclure des séquences de tête qui peuvent favoriser le transport antérograde et la sécrétion par des neurones chevauchant la BBB) sont transportés par les neurones vers des sites de sécrétion à l'intérieur de la BBB. Les polypeptides sont ensuite sécrétés par les neurones transfectés en des emplacements intérieurs à la BBB, et ils entrent ensuite en contact avec des neurones secondaires 'cible' et exercent leurs effets sur ces neurones situés entièrement à l'intérieur de la BBB. Grâce à ce système, les polypeptides qui stimulent la croissance ou l'activité nerveuse peuvent être utilisés pour traiter des maladies neurodégénératives, des membres endommagés chez des victimes d'accidents vasculaires cérébraux, etc., et les polypeptides qui suppriment l'activité neuronale peuvent être utilisés pour traiter une activité neuronale excessive non souhaitée, telle que la douleur neuropatique. L'invention se rapporte également à de nouvelles méthodes d'administration de polypeptides endocriniens et paracriniens au système nerveux central, ceci permettant l'amélioration de traitements médicaux et de traitements visant à favoriser la reproduction chez des sujets humains, ainsi que l'amélioration de la capacité à moduler la croissance, la maturation, la reproduction ou d'autres fonctions liées au système endocrinien sur du bétail, des espèces menacées et d'autres animaux.
PCT/IB2003/002371 2002-04-26 2003-04-28 Administration non invasive de polypeptides a travers la barriere hemato-encephalique et selection in vivo de ligands endocytotiques Ceased WO2003091387A2 (fr)

Priority Applications (4)

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AU2003233120A AU2003233120B2 (en) 2002-04-26 2003-04-28 Non-invasive delivery of polypeptides through the blood-brain barrier, and in vivo selection of endocytotic ligands
JP2003587923A JP2005535580A (ja) 2002-04-26 2003-04-28 血液脳関門を通したポリペプチドの非侵襲的な送達およびエンドサイトーシスリガンドの生体内選択
CA2483980A CA2483980C (fr) 2002-04-26 2003-04-28 Administration non invasive de polypeptides a travers la barriere hemato-encephalique et selection in vivo de ligands endocytotiques
EP03727872A EP1503801A4 (fr) 2002-04-26 2003-04-28 Administration non invasive de polypeptides a travers la barriere hemato-encephalique et selection in vivo de ligands endocytotiques

Applications Claiming Priority (4)

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AUPS1935A AUPS193502A0 (en) 2002-04-26 2002-04-26 Molecules that mediate internalisation in cells
AUPS1935 2002-04-26
US10/188,184 US20030083299A1 (en) 2000-11-04 2002-07-02 Non-invasive delivery of polypeptides through the blood-brain barrier
US10/188,184 2002-07-02

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WO2003091387A2 true WO2003091387A2 (fr) 2003-11-06
WO2003091387A3 WO2003091387A3 (fr) 2004-01-08
WO2003091387A9 WO2003091387A9 (fr) 2004-02-26

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EP1985311A4 (fr) * 2006-01-24 2011-04-13 Univ Kagoshima Agent pour cibler un médicament sur un neurone cérébral
CN113838541A (zh) * 2021-09-29 2021-12-24 脸萌有限公司 设计配体分子的方法和装置
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006070290A3 (fr) * 2004-06-23 2006-11-02 Ian A Ferguson Agents et procedes pour le diagnostic et le suivi precoces de la maladie d'alzheimer et d'autres troubles neurologiques
GB2431348A (en) * 2004-06-23 2007-04-25 Ian A Ferguson Agents and methods for early diagnosis and monitoring of alzheimer's disease and other neurological disorders
GB2431348B (en) * 2004-06-23 2009-12-30 Ian A Ferguson Agents and methods for early diagnosis and monitoring of alzheimer's disease and other neurological disorders
AU2005321021B2 (en) * 2004-06-23 2011-11-24 Ian A. Ferguson Agents and methods for early diagnosis and monitoring of Alzheimer's disease and other neurological disorders
EP1985311A4 (fr) * 2006-01-24 2011-04-13 Univ Kagoshima Agent pour cibler un médicament sur un neurone cérébral
US11259740B2 (en) 2011-05-03 2022-03-01 The Research Foundation for State University of New York Methods useful in optimizing the treatment of neuropathies and targeting tissues with cosmetic botulinum injections
CN113838541A (zh) * 2021-09-29 2021-12-24 脸萌有限公司 设计配体分子的方法和装置
CN113838541B (zh) * 2021-09-29 2023-10-10 脸萌有限公司 设计配体分子的方法和装置

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CA2483980A1 (fr) 2003-11-06
JP2013136586A (ja) 2013-07-11
JP2005535580A (ja) 2005-11-24
WO2003091387A3 (fr) 2004-01-08
WO2003091387A9 (fr) 2004-02-26
CA2483980C (fr) 2013-07-16
EP1503801A4 (fr) 2006-02-08
EP1503801A2 (fr) 2005-02-09

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