JP2005535580A - Non-invasive delivery of polypeptides across the blood brain barrier and in vivo selection of endocytic ligands - Google Patents

Abstract

Disclosed are treatment methods and gene vectors for non-invasive delivery of polypeptides through the blood brain barrier (BBB) to treat the brain or spinal cord. The gene vector is administered in contact with neural projections extending outside the BBB and transfected into neurons that span the BBB, such as sensory neurons, nociceptive neurons or lower motor neurons. When the vector enters a peripheral projection of a neuron that spans the BBB, it is carried by retrograde transport to the main cell body of the neuron where the polypeptide is expressed from at least one gene in the vector. The polypeptide (which may include a leader sequence that facilitates antegrade transport and secretion by neurons across the BBB) is transported to the secretory site in the BBB, secreted into the BBB, and entirely present in the BBB Acts in contact with secondary target neurons. By using a polypeptide that stimulates nerve growth or nerve activity by this method, it is possible to treat neurodegenerative diseases, limb disorders of patients with cerebral infarction, etc., and if a polypeptide that suppresses nerve activity is used, it is troublesome. Excessive neuronal activity, such as neuropathic pain, can be treated. Similarly, novel methods for delivering endocrine and paracrine polypeptides within the CNS are also disclosed. This can improve medical and reproductive treatment in humans and improve the ability to regulate growth, maturation, regeneration or other endocrine-related functions in livestock, endangered species, and other animals. it can.

Description

  The present invention relates to a method of delivering a polypeptide to the brain and spinal cord tissue to humans and other mammals or other animals in the presence of the blood brain barrier (BBB). Furthermore, it relates to a method of delivering a polypeptide to specific neurons and cell populations that are entirely present in the brain or spinal cord. Furthermore, the present invention relates to a method for treating human neuropathy and a method for treating livestock and other animals (control of fertilization rate, maturation rate, growth rate, etc.) using a polypeptide that cannot normally pass through the BBB.

  The higher-animal blood-brain barrier (BBB) ensures that brain and spinal cord neurons are not exposed to blood molecules that interfere with nerve function as they pass through the BBB. BBB is not a single membrane. Instead, capillaries in the brain and spinal cord are formed by endothelial cells that form tight junctions. In contrast, capillary walls outside the brain and spinal cord have “interstitial pores” between adjacent cells, greatly increasing the permeability of those capillaries. The BBB system has already been described in many textbooks and articles (eg Goldstein et al 1986; Pardridge 1998; Rubin et al 1999; Banks 1999; Kniesel et al 2000; Kniesel et al 2000). An animal model for investigating BBB passage is described in Bonate et al 1995, and an early study to develop a cell culture model is described in de Boer et al 1999.

  BBB protects the brain and spinal cord, but also eliminates many drugs to treat injuries and illnesses that affect the nervous system. In particular, proteins and peptides are unable to cross the BBB in substantial amounts with a few exceptions (eg Langer 1990). A few exceptions involve limited transport systems and certain neuroreceptors, such as transferrin receptors (eg, Kastin et al 1999, and Granholm et al 1998). However, those limited exceptions are not suitable for treating central nervous disease using various polypeptides. The need for better ways of passing therapeutic polypeptides (and other types of useful agents) through the BBB is well known and has been published in many reviews (Zlokovic 1995, Cloughesy et al 1995, Davis et al 1995). Abbott et al 1996, Begley 1996, Kroll et al 1998, Pardridge et al 1998, Rochat et al 1999 and Rapoport 2000).

  The present invention relates to a method of delivering a polypeptide into brain or spinal cord tissue protected by BBB. As used herein, “polypeptide” refers to any peptide molecule formed in a living cell by gene expression (formed by connecting amino acids). Intracellular gene expression is involved in: (i) transcription from a DNA gene sequence to form a messenger RNA (mRNA) chain, followed by (ii) translation of the mRNA chain with the correct sequence and consisting of amino acids A polypeptide chain is formed. Such molecules formed by expression of gene sequences are all referred to as polypeptides, regardless of whether they are post-translationally cleaved, cell secreted, glycosylated, cysteine cross-linked, or the like.

  Some scholars describe a completed functional polypeptide as a “protein” (as opposed to a polypeptide fragment or a polypeptide precursor that has not undergone the processing necessary to activate it). In the present invention, a polypeptide is not a subject unless it can perform its intended function, and therefore the terms “polypeptide” and “protein” are used interchangeably.

  The polypeptide of interest here (within the claims) requires three properties. First, the polypeptide must be expressed by an exogenous gene (also called an “exogenous” gene) inserted into one or more types of nerve cells, such as using the intervention methods described herein. Don't be. However, the exogenous (exogenous) gene carried by the vector may have the same sequence as the “natural” or “natural” gene normally expressed in the central nerve. This can occur, for example, in a procedure that increases the level of the polypeptide in a person suffering from a disease because the polypeptide is no longer being produced in the proper amount.

  Second, the polypeptide must be expressed by one or more types of neurons that release the polypeptide into brain or spinal cord tissue that is normally protected by the blood brain barrier. As used herein, the expression “delivering a polypeptide through the blood-brain barrier” or “releasing the polypeptide into (or into) brain or spinal cord tissue” means that the foreign polypeptide is completely present in the BBB Means and requires contact with at least one cell or cell type. This condition is not met when neurons across the BBB secrete the polypeptide only at the point of contact of the polypeptide with other neurons across the BBB. Instead, in most cases, this condition is met only when neurons secrete foreign polypeptides to cerebrospinal fluid (CSF) and / or synaptic fluid at locations within the BBB.

  Third, exogenous polypeptides are of interest only when there is desirable useful (rather than pathogenic) neural activity that is useful for therapeutic and / or other uses. This excludes viral infections and other non-useful infections, tests, and methods of invading or attacking brain or spinal cord tissue. Many tests have been performed using viruses that infect neurons or glial cells in laboratory animals. It analyzes brain tissue samples to test antiviral drug candidates, infect animals with viruses such as rabies virus, kill animals a few hours later, and see which neurons are infected by the virus Was done to do a “tracer” study to analyze neural networks and connections. In these experiments, viruses were used to introduce foreign polypeptides into brain or spinal cord tissue protected by BBB, but giving such infections to experimental animals is pathogenic rather than therapeutic. Obviously (most animals were sacrificed as part of the experiment). Thus, the present invention is significantly different from such tracers and other pathogen tests, which introduces therapeutically or otherwise useful polypeptides useful and beneficial to BBB-protected tissues, and human medical treatments, and It is designed to provide methods used in the field of breeding livestock and endangered animals.

  “Nerve activity” is a necessary part of the third element described above. The “neuroactive” polypeptides described herein are useful therapeutically or for other applications when properly delivered to the desired intended area in brain or spinal cord tissue protected by BBB, and / or Or a polypeptide that can produce the desired result or effect. Various examples are described below and in the table.

  It should be noted that useful neuroactive polypeptides may include polypeptides that exert only a direct effect on glial cells without a direct effect on nerve cells, in which case treated glial cells and liquids It is necessary to generate a response that produces a therapeutic effect or other beneficial outcome between the more closely communicating neurons.

  Furthermore, the term “polypeptide” includes naturally occurring polypeptides or genetically engineered variants, derivatives and fragments of the polypeptides. Examples of this include: (i) a neuroactive portion derived from one gene, and a `` leader '' from another gene (to enhance stability, transport, secretion, or other useful properties or activities) A chimeric or fusion polypeptide having the sequence; and (ii) a fragment or portion of a polypeptide having the desired neural activity (eg, a single chain-binding fragment isolated from the variable region of a monoclonal antibody).

  The next section lists the four categories of neuroactive polypeptides that can provide therapeutic benefits to people or animals suffering from neurological disorders.

The neurotrophic factor neurotrophic factor suffix “trough” comes from the Greek word “nutrition” or “food”. “Trophic” means that a molecule is involved in stimulating, growing, nourishing, maintaining, or supporting a system. A “neurotrophic factor” is a factor that promotes nerve growth, causes the formation of new synaptic connections between nerve cells, or stimulates or supports other nerve activity. However, this term excludes: (i) nutrients required for all cells (such as oxygen, glucose, amino acids, and nucleotides), and (ii) (such as glutamate, acetylcholine, dopamine, serotonin) A neurotransmitter that directly controls nerve impulses between neurons.

  Most neurotrophic factors act like hormones. It is first secreted by one type of cell and then interacts with other cells. However, they differ from hormones in that they typically interact only with neighboring cells after secretion. This works in some ways like a “paracrine” factor. Paracrine refers to hormone-like molecules that act only locally, while “endocrine” factors act systemically and can affect cells that are far from secretory cells.

  Neurotrophic factors are described in articles such as Lindsay 1994, Snider et al 1994, Bothwell et al 1995, Lewin et al 1996 and Skaper et al 1998 and numerous US patents. The first neurotrophic factor discovered by stimulating nerve cell growth was called nerve growth factor (NGF). Later, other neurotrophic factors were also discovered, elaborate names such as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor, and neurotrophin 3 Needed names using serial numbers like Neurotrophin 4.

  Many neurotrophic factors are polypeptides that provide promising treatments for many central nervous disorders, including: (1) brain disorders caused by physical injuries such as car accidents, concussion, etc .; (2) Brain damage caused by stroke, cardiac arrest, drowning, asphyxia, blood loss or other problems including ischemia (insufficient blood flow) or hypoxia (insufficient oxygen supply); and ( 3) Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These possibilities are described in articles such as Hefti 1994, Thoenen et al 1994, Ibanez et al 1995, McMahon and Priestley 1995 and Yuen and Mobley 1996.

  For convenience, terms such as disorders, injuries and injuries refer to any central nervous system disorder (trauma that is inhibited or treated by one or more therapeutic polypeptides when they are transported to the appropriate local area of the brain or spinal cord. Derived from diseases, etc.).

Hypothalamic release factor Hypothalamic release factor is a short polypeptide produced and secreted by neurons in the hypothalamus. These polypeptides stimulate the pituitary gland to control a number of important body functions involved in growth, metabolism, blood water and salt balance, reproduction, and secrete various hormones that affect it Is a subject of many studies. Articles describing hypothalamic release factors include Turnbull et al 1999, Phelps et al 1999 and Sawchenko et al 2000.

Peptide neurotransmitters Numerous peptide neurotransmitters such as substance P, enkephalin, endorphins, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), galanin, somatostatin have been described. In many cases, peptide neurotransmitters behave like classic neurotransmitters such as glutamate or acetylcholine. They are released by neurons at the synapse and bind to specific receptors of other neurons at the synapse to stimulate or suppress the transmission of nerve signals (these are also called nerve impulses, firing, or depolarization). In other cases, the binding of peptide neurotransmitters to synaptic receptors has a longer-lasting effect that increases or decreases the sensitivity of the target neuron to other neurotransmitters. In pain recognition, for example, substance P is released by the nociceptive (pain transmitting) nerve fibers of the spinal cord and is excitatory, while enkephalins and endorphins have opposite effects and suppress the transmission of pain information. Pain relief by morphine is due to binding to the endorphin receptor. However, the use of endorphins for pain treatment is currently not possible because it is difficult to transport such peptides safely into the brain.

Cytokines and other growth factors Certain cytokines and other growth factors act locally as neurotrophic factors to stimulate nerve growth. However, cytokines are distinguished from neurotrophic factors in terms of their ability to stimulate the growth and mitosis of various non-neuronal dividing cells with respect to the nervous system. Examples include acidic and basic fibroblast growth factors (aFGF and bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II) Tumor necrosis factor B (TGF-B), leukemia inhibitory factor (LIF), and various interleukins.

  These molecules are involved in normal physiological processes such as central nervous tissue growth, post-injury reconstruction and repair, and immune responses within the central nervous system. While they may be used to treat nerve center injury or infection, they are at risk of stimulating cancer cells, especially when administered systemically through the nerve center. Nonetheless, they are of great interest to neurologists, and the development of methods to transport cytokines and similar molecules to individual limited central nerve regions has attracted great interest and is potentially beneficial.

  Table 1 provides a partial list of commonly known neural central system active polypeptides. To explain in more detail one area of application, Table 2 lists some examples of neurological disorders and polypeptides that are currently very promising for treating these neurological disorders. Tables 1 and 2 contain only a partial list, and various other neuroactive polypeptides have been rapidly identified from numerous research attempts (including human, mouse, and rat genome sequencing projects). .

  The biggest and most difficult obstacles that hinder the evaluation of neuroactive polypeptides and the use of these therapies in patients in need of treatment are the great challenges of actually delivering neuroactive polypeptides to specific areas of the brain and spinal cord. In order to provide a therapeutic, prophylactic or other advantage in that area, the polypeptide needs to contact a specific group of target neurons or glial cells. Accordingly, the objects and goals of the present invention are as follows: (i) to provide a method for delivering an exogenous polypeptide to regions of the brain and spinal cord tissue protected by the blood brain barrier; and (ii) foreign Methods of delivering sex polypeptides to specific and limited regions of brain and spinal cord tissue targets and / or to specific and limited types or classifications of targets within the nervous system To provide.

Literature review of prior art drug delivery methods for central nervous system drug delivery (eg Langer et al 1989; Langer 1990; Madrid et al 1991; Thoenen et al 1994; Zlokovic 1995, Cloughesy et al 1995, Davis et al 1995, Abbott et al 1996, Begley 1996, Kroll et al 1998, Pardridge et al 1998, Rochat et al 1999, and Rapoport 2000), currently available, effective, and clinically useful for delivering polypeptides through the blood-brain barrier. Only a few alternatives that are relevant are identified. Attempts at various methods to deliver drugs across the blood brain barrier have not been very successful and are reviewed in articles such as Zlokovic 1995 and Rapoport 2000. These attempts can be classified and summarized as follows:

  1. Invasive methods that require physical invasion, such as surgery to puncture cells and membranes, or at least the use of hypodermic needles. An example is an injection or infusion into the ventricle, so that a neurosurgeon approaches one of the “ventric chambers” that function as the production site or accumulation nodule of the spinal fluid and puts the drug into the ventricle. It is necessary to pour into the liquid that accumulates. Most of these ventricles are deep inside the brain (the human fourth ventricle is somewhat accessible, but is not useful for delivering drugs to the majority of the brain because it is the outlet for fluid leaving the brain). Drug injection into the ventricle is difficult and dangerous, even if only a needle is used. Any penetration with a blade or needle will damage the capillary in the middle, and if blood leaks from the damaged capillary, it will come into contact with nerve cells that must be protected from the blood by the blood brain barrier. When cauterization is used to minimize blood leakage, cauterized capillaries, small arteries and veins no longer carry blood and nerve cells supplied by those vessels may die.

  Another invasive method involves the transplantation of genetically selected and / or engineered cells that can secrete neurotrophic factors at high rates into the target area of the brain (eg, US Pat. No. 5,082,670 Hefti et al 1992). The insertion of such cells into the target portion of the brain requires invasive access using a blade or needle, creating serious problems and dangers. Because of these problems, these invasive methods are not very limited clinical trials, patients suffering from end-stage illness, and patients with very severe and debilitating illness that cannot be helped by any treatment Cannot be used by humans.

  2. Non-invasive methods of delivering neurotrophic factors or similar molecules across the blood brain barrier by chemical, cellular, carrier transport, or other non-surgical methods. These methods usually require the injection of a fairly large amount of vesicles, complexes, or other macromolecules into the bloodstream, and even small amounts of injected “freight” molecules enter the brain and cross the blood-brain barrier. Expected to be delivered across to brain tissue. Examples of these methods are described in US Pat. No. 5,154,924 (Friden 1992), chapter by Thoenen et al in Racagni et al 1994, and Zlokovic 1995.

  One important issue with using these methods is that if a compound can cross the BBB in significant amounts, that same compound will also make the remaining permeable capillaries of the body faster and more abundant. Is to pass. This leads to two serious problems. The first is a side effect that occurs when large amounts of neuroactive compounds are delivered to non-central nervous system tissues, which can destroy the peripheral nervous system, or other types of cells and tissues. The second problem is the very high costs that occur when large quantities of complex drugs must be carefully synthesized, purified and quality tested before being injected into humans. In addition, such injections or infusions must usually be repeated many times frequently, and emergency measures should be taken immediately if there are side effects after injection or infusion of a large amount of neuroactive compound. Require patient monitoring at least one day, usually in a hospital or clinic rather than at home.

  In addition, the conventional method cannot administer a foreign polypeptide only to selected nerve cells, nerve cells, or brain regions. In contrast, the present invention provides several promising methods for administering foreign polypeptides to target neurons in a controllable and selective (or at least enriched) manner. By targeting only certain sites or regions within the central nervous system and / or only certain types or populations of neurons, the risk and severity of side effects can be minimized.

Gene therapy One method that may treat central nervous system disorders is gene therapy. Two main methods have been used in previous studies to genetically modify brain and spinal cord neurons. In one method, cells are genetically modified outside the body and then transplanted somewhere in the central nervous system, usually in a region within the BBB. In another method, a gene “vector” is injected into one or more regions of the central nervous system to genetically modify cells normally protected by the BBB. Since serious problems arise when applying methods that work well in other parts of the body to the treatment of brain or spinal cord tissue, these two methods are described in more detail below.

Transplanting genetically modified cells In traditional tissues, cell transplantation usually involves isolating a group of cells, treating them in cell culture (in vitro) conditions, and then transplanting the treated cells into an animal or patient It involves doing. In some cases, cells are obtained from the host to minimize potential rejection problems after transplanting the cells into the host. In other cases, cells are obtained from others (immortal cell lines, fetal or stem cells, etc.). Genetic manipulation may involve precise control techniques, or may include a “shot gun” method followed by selection testing.

  The use of this general method with central nervous system tissue is usually impractical for several reasons, including those briefly listed below. First, the central nervous system is very heterogeneous and neural tissue from one area cannot replace neural tissue from another area. Second, neurons in the adult central nervous system are post-mitotic and do not divide again when a deletion occurs. Third, neuronal processes or axons, once severed, cannot easily re-grow and reconstruct synapses and other connections. Fourth, surgical or similar extraction of central nervous system tissue poses a great risk to the patient even if surgery is performed using the highest level of technique and care.

  For these and other reasons, in most cases, removing a group of neurons from the central nervous system from a patient, genetically processing the neurons, and returning them to the patient's brain or spinal cord is not easily feasible. None or impractical. Thus, most trials of transplanting genetically modified cells into central nervous system tissue do not attempt to use cells taken from the same patient. Instead, early experiments use genetically modified cells (not necessarily nerve cells) that have been modified to secrete abnormally high amounts of the desired protein (for example, nerve growth factor when transplanted into the brain of Alzheimer's disease patients) It was done. Such trials and treatment attempts are described in articles such as Blech and Tuszynski 1996, Karpati et al 1996, Fick and Israel 1994, Fisher and Ray 1994 and Friedman 1994, and patents such as US Patent 5,082,670 (Hefti et al 1992). ing. Such studies are interesting and may be useful at some point, but great risks and dangers are unavoidable as long as it is necessary to transplant foreign cells into the central nervous system tissue. In such transplantation, the brain or spinal cord tissue must be punctured or cut, and the process of transplantation necessarily injures the brain or spinal cord tissue that must be invaded or replaced.

  In contrast, genetic manipulation of central nervous system cells (called in situ treatment) without moving the cells is a better hope to correct the problem without invasive damage to the brain or spinal cord tissue. I will provide a.

In Situ Gene Modification of Central Nervous System Cells This method aims to introduce a desired polypeptide source into the central nervous system by genetic modification without moving cells in the central nervous system. In the prior art, this has been achieved by injecting a gene vector carrying the foreign gene directly into the brain or spinal cord tissue several times. However, this method is invasive and the brain or spinal cord tissue must be penetrated or cut with a needle or blade. Instead, what is needed is to genetically transfect cells located within the BBB without puncturing, cutting, or otherwise invading or penetrating the tissue protected by the BBB, Or a non-invasive method of transformation.

  The terms “transfection” and “transformation” are used interchangeably. Both terms refer to a method of introducing a foreign (exogenous) gene into one or more existing cells so that the polypeptide is produced and expressed from the foreign gene. For some scientists, “transfection” means that the foreign gene is expressed only temporarily, similar to an infection that lasts for a while and eventually stops. In contrast, “transformation” means a permanent genetic change that is passed to any and all cell progeny, usually because a foreign gene is integrated into the chromosome of one or more cells. However, the boundaries between these terms are not clear, and the distinction between the use of these terms is not uniform by all scientists. Thus, “transfection” and “transformation” are used interchangeably herein, regardless of how long the foreign gene continues to be expressed after entering the target cell.

There are two major categories of gene vectors . Viral vectors are derived from viruses and utilize the lipid envelope or shell surface of the virus (known as the capsid). These vectors take advantage of the property that the virus binds to one or more specific surface proteins of certain cells and then injects the DNA or RNA of the virus into the cells. These have become a major class of vectors used in human gene therapy attempts and have been described in numerous published papers. Gene transfer to central nervous system neurons using vectors derived from herpes simplex viruses (eg European patent 453242, Breakfield et al 1996), adenovirus (La Salle et al 1993) and adeno-associated virus (Kaplitt et al 1997) It has been reported.

  All other gene vectors are generally divided into a wide variety of non-viral vectors. These vectors usually contain a “gene expression construct” as understood by those skilled in the art. For purposes of illustration, the construct may be a plasmid and usually comprises: (i) an origin of replication for immediate growth in a host cell such as E. coli; (ii) a host cell having the plasmid is At least one selectable marker gene to inactivate antibiotics, to grow on lactose without the need for any other carbon source, or to develop color in the presence of certain compounds, and (iii ) At least one transport gene comprising a coding sequence that expresses the desired polypeptide and a suitable promoter after the gene expression construct has been injected into the target mammalian cell.

  Non-viral gene vectors are made by adding to the gene expression construct an agent that allows and promotes the entry of the gene construct into the target cell. Some commonly used drugs are cationic lipids, positively charged molecules such as polylysine or polyethyleneimine, and / or ligands that bind to receptors expressed on the surface of target cells (sometimes from viruses) Virus ligand originally acquired). The main classifications of non-viral gene vectors are as follows.

  1. Cationic gene vector. Both the DNA strand and the cell surface are negatively charged. Thus, a positively charged drug eliminates the electrical repulsion of the DNA strand and assists in entering the target cell. Examples are polylysine or polyethyleneimine (PEI) and various cationic lipids. Such drugs are described in articles such as Sahenk et al 1993, Nabel et al 1997 and Li et al 2000.

  2. A receptor-targeting gene vector in which a DNA strand is bound to a molecule that normally enters a certain type of cell through a receptor molecule on the cell surface. This method was demonstrated in US Pat. No. 5,166,320 (Wu et al 1992) using liver cells. By using this method, DNA delivery can be directed to certain types of target cells by binding the DNA to molecules that are selectively taken up by specific types of cells. This method relies on cellular receptors, which are usually proteins that span the outer membrane, some of which are exposed to the outside of the cell, and some of which are immobilized in the cytoplasm. To be suitable for this type of uptake, the receptor must go through a process called “endocytosis”, which means that when the appropriate ligand binds to the receptor, both the receptor and the ligand bound to it It means that the process of being drawn into the cell is triggered.

  Endocytosis is more important in one aspect of the present invention (a novel in vivo sorting method that uses nerve fibers to identify ligands that activate and promote endocytosis) and will be described in more detail below.

  3. Other non-viral gene vectors. Because viral vectors have problems and limitations, and cationic compounds and receptor targeting ligand vectors are currently not as effective as viral vectors in inducing the expression of foreign genes by target cells, researchers Attempts are being made to develop methods to enhance gene delivery to cells. One such attempt uses viral capsid proteins that can rupture "endocytotic vesicles" that are formed when foreign molecules or particles are drawn through the extracellular membrane via endocytosis. Foreign DNA must escape from the endocytic vesicle before it can function as a gene, and certain viruses (eg, adenoviruses) have an evolved capsid molecule that ruptures the endocytic vesicle. This releases foreign DNA in the cytoplasm. An example of this type of study is described in Curiel 1997.

  Various other studies have been conducted in other fields, but none of these attempts has yet reached the mainstream of gene vector research.

Virus central nervous system infection and tracer studies Some viruses (eg, rabies and poliovirus) have the property of being transmitted from nerve cells to nerve cells within the central nervous system tissue, and researchers use virus transmission Using “inter-nerve tracking”, it was possible to track the nervous system and nerve pathways.

  There is an important similarity between the transmission pattern of motor neurons by poliovirus and the degeneration pattern of motor neurons in patients with amyotrophic lateral sclerosis. Similarly, cholinergic neuronal degeneration of the forebrain basal ganglia, an early feature of Alzheimer's disease, shows an important similarity to neuronal damage following administration of rabies virus to the olfactory epithelium. These previously unpublished observations are disclosed here. Because the virus transmission pattern of the central nervous system tissue is similar to the neurodegenerative pattern of a certain central nervous system disorder, the method disclosed here (ie, from one neuron to share contact with a carrier neuron) This is because the method of non-nociceptively delivering a therapeutic polypeptide to the brain or spinal cord in order to transport and deliver the polypeptide to other neurons that do so provides the following two possibilities: (i) Potent therapeutic treatments; and (ii) by researchers, for example, to selectively alter gene expression patterns and / or neuronal functions in brain or spinal cord tissues and / or various in central nervous system tissues Powerful experimental methods used to study various networks and principles, such as factors governing the response of central nervous system tissues to attacks ranging from viral infections to neurodegenerative diseases

Endocytosis Surface Molecules One aspect of the present invention is the in vivo of a “ligand” molecule that can be used to activate and promote endocytosis processes (ie, transport of foreign molecules into the cell interior). discovery and disclosure of novel methods for screening and identification in vivo. These types of ligand molecules are considered as vehicles, carriers or transport systems that can be used to draw or carry useful “transport” or “transport” molecules to specific targeted types of cells. be able to. If the goal is gene therapy or similar genetic engineering to deliver the polypeptide to brain or spinal cord tissue, or other target cells, the transport molecule may be part of a DNA with a foreign gene. However, this type of endocytotic delivery is not limited to gene therapy and can be used to deliver therapeutic agents, diagnostic compounds, or other useful agents to specific target cell types.

  This type of endocytotic delivery (also referred to interchangeably as uptake) involves mainly a class of proteins known as “receptors” on the cell surface. However, at least certain other surface molecules are also known to induce the endocytotic process (eg, cholera and tetanus toxins bind specifically to specific surface carbohydrate molecules and thus the resulting ligand and It is known to activate and promote the uptake of carbohydrate complexes). Thus, most of the references herein relate to endocytic receptor proteins as a typical classification used in the description of the present invention, but other types of endocytic surface molecules are also discussed herein. It can be used as a target for an endocytosis ligand.

  The structure of cell membranes and endocytosis receptor proteins, as well as the process and mechanism of endocytosis, are described in various reference articles such as Alberts et al, Molecular Biology of the Cell. In the third edition (1994) above, the relevant pages are 478-488, 618-625 and 731-744 (cell membrane and receptor proteins), 618-626 and 636-641 (proteins and processes involved in endocytosis) is there. Endocytosis also includes pinocytosis (when the ligand incorporated into the cell is relatively small) or phagocytosis (when the whole cell or a larger object such as a viral particle is incorporated into the cell).

  Not all candidate ligands that bind to the endocytosis receptor protein can be completed by promoting endocytosis. As an example, an important class of “low affinity” nerve growth factor receptors known as p75 receptors have been identified in mammals and a number of research teams have produced monoclonal antibodies that can bind to the receptors. . However, most of these monoclonal antibody lines cannot complete by promoting endocytosis after binding to the p75 receptor. Only a few specific monoclonal antibodies are taken up by the p75 receptor. This was first disclosed in Chandler and Shooter 1984 and was reported by Yan et al 1988 to include a specific antibody line known as the MC192 antibody and then taken up by cells bearing the p75 receptor.

  Considerable work has been done to identify ligands capable of endocytotic transport to mammalian cells. Many of the studies use genetic research tools called phage and phage display libraries. Very simply, a phage (which is a shorthand for bacteriophage) is a virus that can infect bacteria and reproduce. A long form of phage known as "filamentous phage" (abbreviated as "Ff") has been researched and developed by genetic researchers. This is because it can be designed to express accessible (displayed) foreign proteins on the surface of viable and infectious phage particles.

  A strain of filamentous phage was processed to contain a bacterial origin of replication and converted to a “phagemid”. The DNA is either in double-stranded form as a circular plasmid in E. coli cells, or in single-stranded form that is packed into phage coat proteins to be secreted as new infectious phage particles without killing host cells. Can proliferate. This phage strain is also given the kanamycin resistance gene as a selectable marker, so that E. coli harboring the phage can grow in a culture containing kanamycin, an antibiotic that kills non-resistant cells. This phage strain, called the M13 strain, is commercially available and is widely used for genetic studies.

  Various researchers and companies have created complete “phage display libraries” in which a large population of phage (eg, M13 phage) has been processed to display a variety of different foreign proteins. In order to create and test in vivo screening methods to identify endocytic ligands that can bind to target endocytic receptors on the surface of nerve fibers that extend outside the blood-brain barrier, Applicants A rally was used. One of the phage display libraries, known as scFv libraries, contains about 13 billion phagemids, each containing a foreign polypeptide sequence normally present in the antigen-binding “variable fragment” region of a human antibody. Including. Another phage display library, known as the PhDC7C library, contains small foreign polypeptide sequences each containing 7 amino acids randomly arranged using a method called “combinatorial chemistry”. Both phage display libraries are described in detail in the “Detailed Description” and “Examples” below.

  Phage display libraries provide a powerful and useful means for selecting a vast number of different “ligand candidates” or other polypeptide sequences. By using them, researchers have identified and isolated a small number of phage from a vast library that accidentally have a foreign polypeptide sequence of interest due to specific binding or cellular activity, and then Can be propagated.

  However, due to several cellular and physiological factors, screening a phage display library in vivo (ie in a living animal) is very difficult or completely (under prior art). It was impossible. Instead, almost all such screens were limited to affinity columns (with immobilized antigen or antibody, or similar binding reagent), or in vitro conditions (using cultured cells).

  As a result, in an attempt to identify and isolate ligand molecules that can activate and promote endocytosis, prior to the present invention, realistic and substantial in identifying ligands that can be delivered to cancer cells and cells involved in autoimmune diseases. There was a significant advance because these two types of cells could be easily grown and tested in in vitro cell culture. Examples of such studies include U.S. Patents 5,977,322 (Marks et al 1999), 6,113,898 (Anderson et al 2000), 6,376,170 (Burton et al 2002) and PCT applications WO 2000-29004 (Plaksin), 2000-38515 (Ferrone). And 2000-39580 (Christopherson et al). Similar successful advances were not observed when screening phage display libraries to identify ligands that facilitate endocytosis delivery to other types of tissue cells such as neurons.

  The present inventors have sought a method for genetically transforming nerve cells (particularly nerve cells having fibers that cross the blood-brain barrier and extend outside). In addition, the present inventors were aware of the problems and limitations that hindered and prevented the medical use of viral vectors, so that foreign DNA strands could be transferred to cells (especially nerve cells with fibers extending across and out of the BBB). We were looking for other types of transport and delivery systems that could be incorporated.

  All of these studies have been unified and have resulted in the present invention. Despite significant advances in molecular biology and neuroscience in recent years, prior to the present invention, useful polymorphs through the blood-brain barrier without invasive damage were provided to provide therapeutic effects to brain and spinal cord neurons. There was still a strong need for better ways to transport peptides.

  Accordingly, one object of the present invention is to disclose an improved non-invasive method for delivering polypeptides to target cells within the BBB.

  Another object of the present invention is to disclose a novel method for transfecting BBB "crossing" sensory or motor neurons, whereby the neurons crossing the BBB are completely within the blood brain barrier. The therapeutic polypeptide is delivered to the located nerve cell.

  Another object of the present invention is to use genetic engineering methods and compounds to selectively affect targeted portions, regions or classifications of neurons that are completely located within the blood brain barrier. It is to provide a method for transporting a polypeptide to nervous system tissue.

  Another object of the present invention is to effectively “transport” or “carry” within selected, targeted, limited types and categories of nerve fibers, and other selected targeted categories of animal cells. Disclose a new and practical in vivo method for identifying and isolating ligand molecules that can be used to deliver "molecule" molecules (including but not limited to DNA strands encoding foreign proteins) It is.

  Another object of the present invention can be used to efficiently deliver therapeutic agents, diagnostic or analytical compounds, or DNA sequences to selected, targeted, limited types and categories of animal cells. It is to disclose a new method for identifying and isolating ligand molecules.

  Another object of the invention is the identification of endocytotic transport into cells from libraries, repertoires, or other combinations containing multiple candidate polypeptides or other compounds made by combinatorial chemistry. A novel in vivo screening method for identifying and isolating these candidates is disclosed.

  The above and other objects of the present invention will become more apparent from the following summary, drawings, and description of preferred embodiments.

Summary of the Invention

  Non-invasive transport of the polypeptide through the blood-brain barrier (BBB) of mammals or other higher animals and into the central nervous system (CNS) so that the foreign polypeptide contacts cells completely located within the BBB And methods and compounds for delivery are disclosed. This method realizes a treatment method for humans and animals that has been impossible in the past, and can be used for commercial purposes in livestock and the like.

  This method is accomplished by transfecting a gene vector into a specific type of neuronal cell that “crosses” the blood brain barrier. Examples of such neurons that span the BBB are sensory neurons (eg olfactory receptor neurons and nociceptive neurons) and motor neurons. Autonomous neurons in the autonomic nervous system may also be useful for certain treatments. Multiple gene vectors are introduced and administered to a patient or animal in order to contact and transfect the vector with nerve "projections" that extend outside the BBB. After the vector has been introduced into the peripheral projection of neurons across the BBB, the vector carrying the gene encoding a polypeptide with central nervous activity is transported back to the main cell body where it is expressed by the transfected neurons Polypeptide molecules useful for therapeutic or other applications are produced. These polypeptides are of the kind normally secreted by cells, or they can be provided with a leader sequence that facilitates secretion. They are therefore transported in the transfected nerve cells and secreted at one or more sites in the central nervous system tissue protected by the BBB. Polypeptides secreted at such locations within the BBB can make contact with target neurons that are fully located (and thus protected) within the blood brain barrier.

  Using this method, polypeptides such as neurotrophic factors can be delivered non-invasively to targeted types and classifications of neurons that are located entirely within the BBB. This enables a new method of treating brain and spinal cord neurons that are degenerated or damaged due to aging, illness, or other disorders. Alternatively, by this novel method, a polypeptide capable of suppressing neural activity can be transported through the BBB to suppress damage due to unnecessary excessive neural activity such as neuropathic pain. This method also enables a new method of delivering polypeptides capable of modulating endocrine function within the central nervous system, thereby improving the treatment of various human health problems and also in domestic animals and endangered species. Can improve growth, maturation, reproduction, or other endocrine-related functions such as

  Further disclosed are novel in vivo screening methods for identifying and isolating endocytotic ligands that are taken up into and delivered within nerve fibers. This ligand can be used to prepare a gene vector that can deliver the vector to cells that span a particular BBB. Briefly, this screening method uses the following steps. (1) administering multiple candidate ligands to the first site in a living animal where the candidate ligands can directly contact nerve fibers (such as the rat leg sciatic nerve bundle); (2) endocytosis Provide sufficient time for nerve fibers to take up specific ligands that can be activated and promoted; (3) to avoid collecting ligand candidates that have not been taken up or delivered by nerves. Collecting a region of nerve fibers at a location sufficiently distant from the administration location (eg, downstream of the sciatic nerve ligation site in the rat buttock); and (4) removing the incorporated ligand from the collected nerve region.

  This method can be accomplished using a phage display library (of polypeptide ligands) or other ligand repertoires generated by combinatorial chemistry synthesis. Since this method is a preferred embodiment of the present invention, it is disclosed and claimed.

Detailed Description of the Invention

  As indicated schematically above, the present invention has four distinct elements or components. All four must work together in a cooperative and continuous manner in order to successfully carry out the present invention. These four elements are outlined below in numerical order.

  (1) A suitable gene vector is a DNA sequence containing at least one desirable “transport gene”, or a marker gene (to simplify detection and analysis in research) and / or a transport gene (other therapeutically desirable Possesses a DNA sequence comprising a useful polypeptide). This type of vector is used to (2) transfect one or more specific types of nerve cells (eg, olfactory receptor neurons, lower motor neurons) that cross the blood brain barrier. The transport gene enters a neuron across the BBB and is then transported by the neuron to the cell body of the neuron through a natural process called retrograde transport. After reaching the main cell body, the gene is expressed, thereby forming a “foreign” or “exogenous” polypeptide molecule within the transfected neuronal cell that spans the (3) BBB. Such exogenous molecules (which may be the same as “endogenous” or “natural” polypeptides that are not fully produced in the patient) may be contained within the BBB by neurons that span the transfected BBB. Is transported to the secretion site located in The polypeptide molecule is then secreted at a secretion site located within the BBB by neurons that span the transfected BBB. This secretion causes the exogenous polypeptide to contact (4) other “target” central nervous system neurons or other cells that are located entirely within the BBB (and treated therapeutically or in other useful ways, Adjustment or influence).

  Below, this therapeutic method is demonstrated using a virus-derived vector. However, non-viral vectors can also be used, as will be explained later.

  A first example illustrating the method of treatment is illustrated in FIGS. 1-3, using an instruction number common to all three drawings. FIG. 1 shows the treatment “macroscopically”. A fluid containing many gene vectors 100 is introduced into the nasal sinus of patient 80. The vector contacts the exposed “peripheral projection” 212 (see FIG. 3) of the olfactory receptor neuron 200. These neuroprojections are accessible on the surface in the nasal sinus and come into direct contact with various airborne compounds as one of the biochemical processes involved in odor sensation.

  As shown in another scale (microscopic, cell level) in FIG. 3, when DNA carried by the gene vector 100 enters the neuroprojection 212, it is carried to the main cell body 224 by retrograde transport (described later). Since the gene vector in this example is derived from a type of virus that sufficiently infects nasal receptor cells, the vector DNA (shown in FIG. 2 as just one component inserted into the inactive but infectious viral genome). Carrying the transport gene 160) facilitates this process. Therefore, creating a vector delivery system using infectious virus ensures that messenger RNA strand 299 is transcribed from transport gene 160 in at least some of the transfected neurons, as shown in FIG. Is done.

  In the transfected nerve cell 200, the mRNA chain transcribed from the transport gene 160 is translated into a plurality of foreign polypeptide molecules 300 by a normal cellular mechanism involving ribosome 226.

  For natural transport and secretion mechanisms (which can be realized or enhanced if desired by adding one or more special transport or secretion sequences to the end of the polypeptide chain by genetic engineering techniques). At least some of the molecules 300 are transported (by a process called antegrade transport) to the various synaptic terminals 242 (or other peptide secretion sites) of the neuron 200 across the BBB.

  The foreign polypeptide molecule 300 is released by the nerve cell 200 straddling the BBB at the site located completely within the central nervous system tissue protected by the BBB. This secretory process allows the secreted foreign polypeptide molecule 300 to directly contact (and provide a therapeutic or other regulatory effect) to another class of neurons, referred to as “target” neurons. These “target” neurons are located entirely within the BBB. In the description of the schematic diagrams of FIGS. 1 and 3, as described below, the polypeptide is transfected into an approximately spherical structure called “glomerular” 910 that contains various “target” neural structures, and is transfected into an olfactory receptor. Secreted by nerve cell 200.

  Briefly describing this process, the above overview and FIGS. 1-3 show how (1) a gene vector carrying a transport gene is used to (2) transfect neurons across the BBB, (3) Shows whether the foreign polypeptide is expressed and then secreted into the central nervous system tissue within the BBB, where it (4) contacts the “target” central nervous system nerve cells that are completely located within the BBB.

  Each of these four elements is described in more detail below. Since the optimal design of a gene vector depends on the neurons that cross the particular type of BBB being transfected, we first describe the neurons that cross the BBB.

Types of neurons that cross the BBB
Nerve cells that cross the BBB can be roughly divided into three types. Sensory neurons, motor neurons, and pre-autonomic ganglion cells. All of the neuronal cells that span these types of BBB discussed here have been extensively studied, and genetic vectors are known that can be used to transfect each of these types of neurons.

  The primary class of neurons that cross the BBB that are first described are sensory neurons, which are "front" cells that are directly involved in various sensations such as vision, olfaction, taste, and touch, pain, and limb position. It is.

  Sensory neurons have a special portion (referred to as “peripheral projection”) that extends to a surface or tissue region outside the BBB (or its immediate vicinity). A “peripheral projection” is the part of a neuronal cell that spans the BBB where the gene vector is most directly accessible. The gene vector does not need to cross the BBB to contact the peripheral projection.

  Conversely, the term “central projection” refers to a fibrous portion of a neuronal cell that extends away from the main cell body, which further includes (i) that the main cell body is BBB like many types of sensory neurons. If outside, into the BBB; or (ii) If the main cell body is in the BBB, such as motor and autonomic ganglion cells, it extends to the center of the brain or near the upper spinal cord.

  Note the term “axon” in relation to central or peripheral projections. In most cases, the “axon” refers to the single largest and longest projection that emerges from the main nerve cell body. However, many sensory neurons have two axons, one extending to the periphery and the other extending to the central part of the brain. Since one of the major roles of neurons across most of the BBB is to reciprocate nerve impulses between the peripheral and central nervous systems, that portion of the nerve cell that actually crosses the BBB across the BBB Can be appropriately classified as an axon in most cases.

  Four classes of neurons spanning the BBB deserve consideration in the methods of use disclosed herein. Two of them are sensory neurons (i) olfactory receptor neurons and (ii) nociceptive (pain signaling) neurons. The third classification is a motor neuron mainly involved in musculoskeletal control, and includes a sub-classification called a tongue motor neuron of special interest because the cell body is in the brainstem. The fourth category, called “autonomous preganglionic cells”, is promising for most human medical uses because the genetic changes in these neurons can affect the autonomic nervous system. Not a good candidate. However, it should be recognized as it may be useful for the treatment of certain medical conditions and for various studies. Each of these four categories will now be considered separately.

Olfactory receptor neurons One of the types of neurons that cross the BBB suitable for this use is olfactory receptor neurons. Because of the olfactory role, their peripheral projections are exposed and accessible on the inner surface of the nasal sinus and are directly contacted and activated by various air compounds sucked through the nose.

  These neurons are described in various physiology textbooks, eg, Guyton and Hall, Textbook of Medical Physiology 9th Edition (1996), pages 678-681, and cited references on pages 681-682. In brief, the human olfactory membrane has a surface area of about 2.5 square centimeters, and usually has about 100 million receptor neurons. At the exposed tip of each olfactory receptor neuron, there are usually about 6 to 12 tiny hairs called cilia, which extend into the mucus layer several microns below. Most molecules that trigger odor recognition are presumed to interact with receptor proteins that span the exposed surface membranes of cilia and / or receptor neurons. Most molecules that trigger the odor sensation do not penetrate the nerve cell body, but a few specific types of molecules (such as wheat germ agglutinin that binds non-specifically to most glycoproteins) are probably some types It is taken up and transported into olfactory receptor neurons by endocytosis. Also, according to several published reports, adenoviral vectors can be transfected into olfactory receptor neurons (inserting foreign genetic material) via contact with the olfactory membrane. This was shown by the expression of the marker gene by transfected neurons.

  As schematically illustrated in FIG. 3, the olfactory neuron cell 200 is composed of several cell parts, which interact with one or more gene vectors 100. The nasal sinus membrane 90 is shown schematically as a distinct layer and is composed of the exposed apical surface of the olfactory epithelial olfactory support cells. Nerve projection 210 passes through nasal sinus membrane 90 to form an exposed end 212 (also referred to as a mucosal tip, end, or knob). Since this exposed nerve tip 212 resides in the nasal sinus, the gene vector carried by the liquid nose spray can contact the nerve tip 212.

  The olfactory receptor neuron 200 is composed of a cytoplasm 220, a nucleus 222 located in the main cell body 224, a ribosome 226 that translates mRNA into a polypeptide, and an axon 240 that passes through the blood-brain barrier (BBB).

  As noted in the background art, the BBB is not a single membrane structure, but instead is a capillary wall network with very close adhesion between the endothelial cells that form the capillary wall. Some scholars consider the olfactory epithelium (the viscous membrane surface in the nasal sinuses with projections of olfactory receptor neurons) to be an extension of the brain, which is therefore a part of the central nervous system tissue where BBB is not present Although arguing to be a region, from physiological studies, the olfactory glomeruli are completely located within the BBB, and from unwanted molecules that may trigger false unwanted nerve impulses, It is clear that it is protected. In order to show the fact, FIG. 1 schematically shows BBB using a double broken line 900. The peripheral projection 210 and main cell body 224 of the olfactory receptor neuron 200 are located outside the BBB, but the axon 240 passes through it and a number of synaptic terminals located within the central nervous system tissue protected by the BBB Branch to 242. Therefore, the olfactory receptor nerve cell 200 straddles the BBB.

  The synaptic terminal 242 of the olfactory receptor neuron 200 is located in a substantially spherical structure, designated “glomerular” 910 in FIG. There are thousands of glomeruli in humans, and each glomerulus contains approximately 25,000 axon synaptic terminals from olfactory receptor neurons. Each glomerulus 910 is also the end of thousands of dendrites and projections 920 from large “mitral” cells and small “tufted” cells. The cell bodies and dendrites of mitral and tufted cells appear to be located in the glomeruli, but their main cell bodies are located in the bulbous structure above the glomeruli. In addition, each glomerulus (or surrounding area in close proximity to the glomerulus) is a neuron 930 (which is located in the basal ganglia and is mainly activated by the excitatory neurotransmitter acetylcholine. It also includes a terminal 934 located at the tip of a long fiber 932 (called a basal ganglia cholinergic neuron)) that extends down (called a process).

  The schematic diagram of FIG. 3 is a selective explanatory diagram and does not explain the entire target nerve cell structure. For example, in or near the glomerulus, ends are found from the following fibers: (i) fibers extending from “serotonergic” neurons in the brain region called raphe nuclei where the main cell body is in the midbrain; and (ii) A fiber extending from a "noradrenergic" nerve cell in which the main cell body is located in the brain part called the blue nuclei. Depending on the therapeutic polypeptide to be delivered, the above or other neuronal cells that are completely located within the BBB are targeted for the treatment disclosed herein.

  In general, polypeptides released from transfected olfactory receptor neurons 200 can be obtained from so-called “trans-synaptic tracer studies” (eg Lafay et al 1991; Barnett et al 1993). It is expected to exert a regulatory effect in contact with any (or almost any) type of neuronal cell found to be infected by virus particles released from the neuronal cell. Such neurons include, for example, mitral cells and tufted cells (which are many infected because they have many globules on the glomeruli), and at least some forebrain cholinergic neurons , Raphe nucleus serotonergic neurons, and locus coeruleus noradrenergic neurons, which have been shown to be infected by tracer studies.

  Various “glia cells” may also contact the polypeptide molecule 300 secreted from the olfactory receptor neuron 200. As detailed below, glial cells (also called neuroglia cells) are unable to receive or transmit neural signals, but instead support neurons located within the central nervous system tissue protected by the BBB And various cells to support.

  Thus, herein, the term “target cell” refers to the desired “target” of a foreign polypeptide molecule 300 that is located entirely within a central nervous system cell protected by BBB and that is encoded by the transport gene of gene vector 100. Is used to refer to cells that are Neurons that straddle the BBB actually contacted and transfected by the gene vector are not considered target cells here. Instead, neurons across such BBB should be considered as part of the delivery mechanism and are referred to as “delivery cells”, “transfection pathways”, or transfected “primary” neurons .

  When olfactory receptor neurons are used as delivery routes, another physiological factor can be important and should be recognized. Olfactory receptor neurons gradually die and are continuously replaced by newly generated neurons. In mice, the “half-life” of olfactory receptor neurons is about 3 months, which is estimated to be roughly equivalent to that of humans.

  Therefore, even if olfactory receptor neurons were stably transformed with a gene vector (ie, one or more foreign genes were inserted into the olfactory receptor cell chromosome by the gene vector), they were transformed regardless of it. Cells gradually die over the next weeks and months. As a result, additional vectors may need to be re-administered to the patient every few weeks, depending on the type of polypeptide and vector used and the effect desired for the particular patient.

Nociceptive neurons
Another classification of sensory neurons across the BBB sends pain signals from the body and skin to the spinal cord. These neurons generate nerve impulses in response to noxious substances, signals, or environmental events. Thus, these are nerve cells that begin the process of generating pain or discomfort when the skin is cut, rubbed, struck, or exposed to very high or low temperatures. The nerve cells that send these pain signals are usually called “nociceptors” or “nociceptive” neurons, and are sometimes called nociceptors or nociceptors. A nociceptive neuron is one of sensory neurons that span several BBBs with different functions that are part of the dorsal root ganglion.

  FIG. 4 very schematically displays the nociceptor nerve cell 400. Like most other sensory neurons, this neuron 400 has its main cell body 402 in a tissue region outside the BBB that is not protected by the BBB. In the case of nociceptor nerve cells 400, subject 402 is located in the dorsal root ganglion relatively close to spinal cord 480. The nerve cell has a peripheral projection 410 that extends out of the spinal cord 480 to the skin (other projections extend into deeper areas of the body). This peripheral projection 410 branches a number of “near-surface end” 412 into a shallow tissue layer just below the skin surface 405.

  In the opposite direction, nociceptor neurons 400 have axons 420 that pass through the blood brain barrier. As shown in FIG. 4B, the axon 420 branches into processes 422, 424 and 426 (shown in FIG. 4B) within the central nervous system protected by the BBB of the spinal cord 480. Each process ends with one or more synaptic connections so that nociceptor neurons send pain signals to the “secondary” neurons within the spinal cord 480.

  The surface of the spinal cord 480, shown as a cross-section, includes (i) a ventral midline fissure 482 (or anterior midline) located in front of the patient; and (ii) two smaller posterior lateral grooves (or posterior middles) There is a dorsal midline groove 484 (also referred to as a back midline groove) sandwiched between the grooves). The body of the spinal cord 480 consists of “white matter” 486 surrounding “ash matter” 490. Ash 490 consists of left and right front corners 492 and left and right rear corners 494. All four corners are connected to a central part called ash matter commissure. As shown in FIGS. 4 and 5, large bundles of nerve fibers that exit laterally from the spinal cord are commonly referred to as dorsal root (or dorsal root) and anterior root (or abdominal root). The term “ganglion” generally refers to a collection of neuronal cell bodies outside the central nervous system and is also used to refer to these nerve bundles.

  When skin surface 405 is cut or rubbed, a neural signal begins at terminal 412 near the surface of nociceptor neuron 400, passes through peripheral projection 410, passes through cell body 402, and passes through BBB. It passes through 420 and reaches the branched dendrites 422-426 inside the dorsal 494 of the spinal cord 480. A neurotransmitter is released from the apical synapse of dendrites 422-436, which induces nerve impulses in spinal nerve cells. The nerve impulse travels over the spinal cord and reaches a processing center in the brain, and the incoming neural signal is processed so that the head interprets it as pain.

  Various medical problems are classified as “neuropathic pain”. As the name suggests, neuropathic pain is associated with pathological conditions that affect nerve cells to generate unnecessary and excessive pain signals. This often involves anatomical reconstruction of the intra-BBB neural connection that produces chronic and inappropriate pain responses. The term “hyperalgesia” is also used as a synonym for “excess pain”, and “allodynia” is also used for this condition.

  Neuropathic pain is a collection of well-known medical problems, and this broad category includes diabetic neuropathy, “phantom limb pain” from amputated limbs or fingers, arachnoiditis, trigeminal neuralgia, This includes post-infectious pain (eg, the occurrence of “shingles” caused by herpes zostar virus) and chronic pain that does not regress even after a normal recovery period that occurs after traumatic injury and surgery. Burning pain (causalgia) is another type and involves burning sensations (the root word “Kauza” originates from the same root as “shochu” and has nothing to do with causation). Other types of neuropathic pain are also known, with neuropathic pain having a wide range of intensities, starting with a nuisance level and extending to debilitating, intolerable or very severe pain. In fact, neuropathic pain is an important factor in many suicides, chronic and incurable pain is very strong and heartless, and many patients commit suicide.

  The cellular mechanisms believed to be widely involved in many types of neuropathic pain are illustrated in FIG. 4B. In this schematic diagram, axon 420NP is involved in neuropathological problems, germinates too many dendrites 422, 424, 426, and spinal nerve cells 432, 434, And begin interacting with 436 (some of which may be inappropriate). Germination and chronic activation of too many dendrites from a single nociceptor axon causes transmission of too many pain signals into and through the spinal cord 480NP affected by pain, Or worsen. Too many neural connections involving pain-transmitting neurons are shown in FIG. 4B and are termed “excessive innervation”.

  4A-4C and as described below, the present invention provides a method for controlling and reducing neuropathic pain by administering a gene vector that transfects nociceptive neurons into or near an affected area. I will provide a. This type of vector, shown as vector 100NPC in FIG. 4A (“NPC” means “neuropathic pain control”), is designed to suppress neural activity (rather than increase), as described below. It also has a “NPC” shipment gene.

  Since nociceptive receptors are located in shallow areas under the skin, subcutaneous, intramuscular, or other relatively shallow infusions are preferred routes of administration. Alternatively, topical and other modes of administration can also be evaluated, for example (i) ointments, creams, etc. containing vectors, which may contain tissue penetration enhancers such as (i) dimethyl sulfoxide, mesyl salicylate Topical application of a solution or suspension; (ii) topical application of a formulation comprising a vector to a skin area that has been rubbed, worn or otherwise physically treated; and / or ( iii) The topical application of a vector-containing formulation to skin that has been chemically treated, such as with chemicals used in “peel peeling” treatment.

  For further discussion, a fluid containing the gene vector 100NPC for neuropathic pain control will have a clear “focal point” (position, loci) that senses neuropathic pain most strongly by the patient, in the shallow, subcutaneous area. Consider injecting into or near the skin (also called hot spots). As mentioned above, the vector 100NPC contains genes that are designed to suppress (rather than increase) neural activity. Such suppressor genes encode, for example: (i) monoclonal antibodies (or antibody fragments) that bind and inactivate one or more types of neurotrophic or other neurostimulatory peptides or compounds. Or (ii) a peptide fragment that competitively binds, occupies and blocks one or more types of neuronal receptors involved in neural stimulation. The NPC gene is delivered (via retrograde transport) to the cell body 402 of nociceptive neurons where the plurality of NPC polypeptides are expressed. The NPC polypeptide then passes through the BBB (via antegrade transport) by nerve axon 420 and is induced or exacerbated by excessive innervation (with components in the spinal cord) and is involved in pain Delivered within 480NP (see Figure 4B).

  NPC polypeptides are released into the spinal cord tissue protected by the BBB at or near the hyperinnervated site in the spinal cord. By exerting an inhibitory effect (by binding, blocking, competing or inhibiting cellular agents or processes that stimulate or maintain high neuronal activity), NPC polypeptides can be permanently or in number, depending on the type of treatment. Helps reduce and alleviate the pathology of neuropathic pain for days or weeks.

Motor neurons
Another class of neurons that cross the BBB is usually called motor neurons. They primarily send instructions from the central nervous system to the body muscles, "innervate" the skeletal muscle system, and put the muscle under control of the central nervous system. One major subclass of motor neurons, commonly referred to as “spinal motor neurons”, has a projection that places its cell body in the spinal cord and extends through the BBB to contact peripheral neurons and muscles.

  FIG. 5 (FIGS. 5A-5C) schematically illustrates the use of spinal neurons as a “transfection pathway”. Through this pathway, have therapeutic limbs weakened due to stroke attacks, traumatic injury, neurodegenerative disease or similar causes by delivering therapeutic polypeptides to upper motor neurons that are located completely within the BBB Stimulates and enhances nerve control of the patient's muscles. FIG. 5A shows spinal nerve cells 500 with cell bodies 502 in the BBB-protected tissue of spinal cord 520. FIG. The spinal cord 520 has the same structure as shown in FIG. 4A, and the cell body 502 of motor neurons is located in the cortex of the anterior 522. The axons of spinal motor neurons 500 form long “processes” 504 that pass through the BBB and extend to the muscle fiber bundle 550. Within the muscle fiber bundle 550, the nerve axon or process 504 branches to the dendrite and terminal 506, interacting with muscle tissue (in healthy people) to induce muscle contraction when needed, so arms, legs, etc. "Voluntary movement control" is provided.

  Figure 5B shows 3 of patients suffering from a stroke, brain or spinal cord injury, or a neurodegenerative disease (such as amyotrophic lateral sclerosis) or a similar disease that impairs voluntary motor control of the arm or foot The cell bodies of two separate spinal nerve cells (designated 512, 514, 516) are shown schematically. This dysfunctional state is at least partially due to the death or damage of upper motor neurons in the brain or brainstem located above the cell body of at least three motor neurons. Some of the killed or damaged upper motor neurons in the brain or brainstem located above the spinal cord cell bodies 512-516 provided spinal nerve cells 512 and 516 with nerve impulses. As illustrated in FIG. 5B, when various upper motor neurons are damaged or killed, processes 532 and 536 (dotted line in FIG. 5B) cease and begin to degenerate, resulting in three spinal motor neurons Only one (central process 534) remains as a cell that receives an active supply of neural signals coming from upper motor neurons. Since the two motor neurons 512 and 516 no longer receive nerve impulses, they are at risk of quiescence and immobilization, atrophy, alteration and death over time.

  FIG. 5C schematically shows the outcome of this symptom treatment after injecting a fluid containing multiple gene vectors 100 into a muscle bundle 550 that no longer works normally. Vector 100 contains a therapeutic gene encoding a neurotrophic factor or other polypeptide that stimulates neural activity or cell division. Genes contained in the vector are transported to cell bodies 512-516, from which neurostimulatory polypeptide molecules are expressed. These polypeptides are then secreted by three motor neurons and function as a signal to add nearby viable and active neurons, such as upper motor neurons with active processes 534 Stimulates and induces dendritic growth and / or activation. This allows the active protrusion 534 to germinate additional protrusions 534a and 534c and form synapses with other neurons. Additional newly generated synaptic connections then begin reactivating the processes from spinal motor neurons 512 and 516. Combined with physical therapy and exercise therapy, this leads to better spontaneous control of the patient's arms or legs.

Tongue Motor Neurons One subclass of motor neurons that may be very useful in the present invention has projections that extend through the BBB and govern the tongue muscles. These “tongue motor neurons” control the movement of the tongue for eating and talking. The cell body is in the brainstem. Thus, they provide a promising route for delivering foreign polypeptides to the brainstem portion of the central nervous system.

FIG. 6 shows the tongue motor nerve cell 600. It has a cell body 602 in the brainstem 950 and a long peripheral projection 604 that passes through the BBB of the patient 60 and ends in the tongue 62. The accessible tip of the neuroprojection 604 contacts the gene vector 100A via a carrier liquid injected into the tongue 62.
The vector DNA is retrogradely transported to cell body 602 through neuroprojection 604. The therapeutic polypeptide is expressed from the transport gene and secreted from the tongue motor neuron 600 into a region within the brainstem. These secreted polypeptides contact and exert effects on various other neurons 952 and 954 located in the brainstem 950.

Autonomic preganglionic cells
The last major class of neurons that cross the BBB is usually called autonomic ganglion cells. Like motor neurons, the cell body is located in the BBB. Axons extend through the BBB and connect with sympathetic and parasympathetic neurons. As part of the “autonomous” system, these neurons are involved in the control of various functions that are not under conscious control (eg blood pressure, digestion, excretion, sweating).

  The term “node” means a collection of nerve cells. Thus, “pre-nodal” neurons are cells that have cell bodies in the central nervous system as lumps (also called nuclei) whose axons are projected through the BBB and exist in ganglia outside the BBB. Includes cells that innervate and contact cells.

  It is believed that the methods and gene vectors of the present invention can be applied and used as appropriate to genetically transfect autonomic ganglion cells. In at least some cases, such transfected autonomic ganglion cells transport foreign polypeptides to the brainstem and / or spinal cord and possibly to other central nervous system neurons that are located entirely within the BBB. (See, for example, Pickard et al 2002 for general support data).

  However, it should be recognized that sensory and motor neurons are somewhat easier to manipulate and analyze, at least in the early stages of development of the present invention. This is due to several factors. On the one hand, projections of sensory and motor neurons are actually applied to exposed accessible surface sites (or in the vicinity) (in the case of olfactory neurons), and also to relatively shallow muscle and subcutaneous regions (or in the vicinity). Reach (for pain transmitting and motor neurons). In contrast, the pre-autonomic ganglion cell ends reach deep below the skin surface within the autonomic ganglion. Thus, when autonomic ganglion cells are used as a “transfection route” across the BBB, accurate vector delivery is somewhat more difficult (and somewhat dangerous) than sensory or motor neurons. is there).

  For this reason, most of the examples and text focus on the use of sensory or motor neurons as neurons across the BBB transfected by the gene vector to illustrate the present invention. Neurons that straddle this type of BBB are generally considered good candidates for initial development and testing of the present invention. However, autonomic ganglion cells are available as a “transfection route” across the BBB and may ultimately be very useful for various therapies or other treatments.

Gene cells carrying one or more useful “transport” genes (which may include marker genes and / or transporter genes) as summarized on gene vectors (eg, viral cells, liposomes, and neurons across the BBB) (Eg, a ligand vector that targets the endocytosis receptor above) promotes BBB to facilitate transfection of vector DNA (including useful transport genes) into neurons that span one or more BBBs. It contacts the peripheral projections of the neurons that straddle. Upon entering such a nerve cell, at least a plurality of transport genes are delivered to the main cell body through projection by a natural retrograde transport process. Upon entering the cell body, the polypeptide encoded by the gene is expressed from the transport gene by normal intracellular mechanisms.

  Most polypeptides of interest (such as nerve growth factors) function as hormones or growth factors, and such polypeptides must normally be secreted naturally from the cell to perform the required function. Would be suitable for secretion from the transfected neuronal cell when expressed by the neuronal cell across the transfected BBB.

  Thus, the present invention discloses gene vectors that are capable of transfecting certain types of specific central nervous system neurons that cross the BBB, whereby the transfected neurons express useful therapeutic polypeptides. , And normally secreted into the central nervous system tissues protected from foreign polypeptides by the blood brain barrier.

  FIG. 2 schematically shows the viral vector 100. This vector has three major components: outer shell portion 110, binding ligand protein 120, and genome 150 (shown as double-stranded DNA or dsDNA in FIG. 2).

  For viral vectors without lipidic “skins” (such as adenovirus-derived vectors), the outer shell portion 110 consists of capsid proteins 112 that are semi-linked with each other. The protein “shell” of such a vector is usually called a capsid, and the binding ligand protein 120 is usually just a domain of the capsid protein exposed on the outer surface of each virus particle.

  In other viral vectors (such as herpes virus-derived vectors), the outer shell (usually referred to as the “coat”) consists of lipids, which are usually comparable to the lipid bilayers that make up the outer membrane of most mammalian cells. Formed as a multilayer. In such vectors, the binding ligand protein 120 usually straddles the outer coat layer, and the external protrusion of each protein extends outward so that it can contact and bind to cell surface proteins.

  Regardless of whether there is a lipid coat, the viral binding ligand 120 attaches to a complementary protein on the surface of the cell that can be infected by that type of virus (a non-covalent binding mode equivalent to the binding reaction between antibody antigens). so). Certain viruses (and vectors derived from them) have very specific binding ligands. These types of viruses infect only those types of cells that have complementary surface proteins. Another type of virus (and the vector derived from it) has a less specific binding ligand and binds to and infects a wider variety of cells.

  The vector genome carried by the vector 100 shown in FIG. 2 consists of double-stranded DNA (dsDNA). Another type of viral vector can carry single-stranded DNA or double-stranded RNA. There are four classes of genomes known for the various gene vectors, and any such vector can be used as disclosed herein. The genome of a common viral vector carrying dsDNA is a good candidate for early evaluation of the present invention. For processing synthetic or isolated genes in the laboratory, dsDNA is easier to use than ssDNA (which is “sticky” because of its inherent base attraction) or RNA (which is somewhat more unstable than DNA). Thus, most of the viral vectors developed so far (such as those obtained from herpes viruses or adenoviruses) contain the dsDNA genome.

  Using conventional terminology developed for the use of viral vectors, the genome 150 carried by the viral vector (and other various gene vectors) consists of several “domains”. One or more “transport genes” 160 are generally inserted into the viral genome so that both ends of the viral genome can function properly after entering the virally transfected cell. . Thus, insertion of a transport gene 160 into the viral genome forms two “flanking sequences” 155 and 157, which contain natural or modified viral DNA sequences that sandwich the ends of the transport gene. It is out. The tips of the sandwich sequences 155 and 157 are often linked to viral polypeptides 158 and 159. In general, such polypeptide “cap” structures appear to be relatively stable and resistant to defense mechanisms in cells that normally attempt to destroy or degrade viral DNA after it has entered the cell. It has evolved into. If necessary, one or both of these polypeptide “caps” can be selected and / or modified to facilitate and accelerate various cellular processes (such as retrograde transport of viral DNA to the cell nucleus, active transport). .

  “Transport Gene” 160 has three distinct domains. Promoter region 162 contains a signal sequence that directs “transcriptase” to begin transcription of the messenger RNA strand from the double-stranded DNA strand immediately. This promoter region usually contains a “TATA” box or similar signal, which directs the enzyme to begin to transcribe mRNA from a DNA sequence usually located about 25 bases downstream from the TATA box. For ease of explanation, the approximately 25 base sequence between the TATA box and the first base transcribed into mRNA is considered part of the gene promoter. Alternatively, if preferred, it is referred to as part of the “non-transcribed leader sequence” and need not be considered part of the actual gene promoter.

  A gene promoter continues to a “coding” region 164 that defines the base sequence of the mRNA strand transcribed from the gene. This region is the DNA that defines the AUG “start” codon of the mRNA chain (which defines the methionine residue of the first amino acid at the N-terminus of the polypeptide) and the “end” codon (which cuts the translation of the mRNA chain by the ribosome). Contains an array. In some vectors, the transport gene coding region also includes an intron that is deleted from the final mRNA strand by an "editing" process in the host cell.

  The coding region continues to the “untranslated” sequence 166. This domain is transcribed in the host cell and becomes part of the mRNA strand. When these domains are on the mRNA strand, they stabilize the mRNA strand in the host cell and protect it against rapid enzymatic degradation. However, this untranslated sequence is not translated into a polypeptide sequence by the ribosome.

  Variations of this general genetic structure are possible and can also be used in the present invention. As an example, after an polypeptide coding region 164 and before the untranslated sequence 166, an “in-sequence ribosome re-entry site” (IRES) (eg, derived from an RNA virus such as encephalomyocarditis virus) 2 Inserted with the coding sequence to achieve co-expression of the second polypeptide. (Example: Wong et al 2000). Under the direction of the IRES, the ribosome binds to the second position of the mRNA and begins to express the second polypeptide in parallel with the first polypeptide.

  Viruses have evolved a variety of different ways to introduce their DNA (or RNA) into cells. Accordingly, genetically engineered viral vectors can utilize a similar infectious process to deliver DNA “carriers” to susceptible cells.

  As an example, in a process that is almost common among viruses without lipid envelopes, the viral capsid protein attaches to a protein molecule displayed on the host cell outer membrane. This attachment process initiates a process called “invagination”, so that the virus is drawn and packed into a lipid bilayer envelope or vesicle formed from cell membrane lipids, which are intracellularly Disperse in the cytoplasmic fluid. Subsequently, the bubble-like vesicles (often referred to as “endosomes”) are ruptured with the aid of viral capsid proteins or by digestive processes or digestive organs in the cytoplasm. Endosomal rupture releases viral DNA into the cytoplasm.

  In another example, in a process that is almost common between herpesviruses and other viruses that have a lipid bilayer envelope, the different lipid bilayers surrounding the virus and cells fuse and coalesce. As a result, the entire contents of the viral lipid envelope are transferred into the cytoplasm.

  Regardless of which mode of infection is used by a particular viral vector, as a result, an effective viral vector transfers some or all of its genetic material into the cytoplasm. Returning to the example of FIG. 1, the viral vector 100 inserts its DNA 150 into the exposed mucosal tip 212 of the olfactory receptor nerve cell 200.

  In some cases, after transfecting a gene vector containing a transport gene into a neuron that crosses the BBB, multiple results and effects are produced depending on the replication and migration characteristics of the vector system used. In most cases, the vector and transport gene remain in neurons transcending the transfected BBB, and the polypeptide expressed from the transport gene in the transfected neurons is the transfected gene. It is thought that it is only secreted from nerve cells. However, certain vectors (referred to as “trans-neuronal” vectors) can themselves be secreted into the BBB by neurons that straddle the BBB. This allows such a trans-neural vector (ie, its genetic material) to be transfected in contact with other neurons that are completely located within the BBB.

  When developing gene vectors and gene constructs for this method of use, various well-known techniques and DNA sequences for expressing the encoded polypeptide in large quantities can be used. Such techniques and sequences are discussed below under “Gene and vector constructs; expression, delivery, and secretagogues”.

A “marker gene” (also called a “reporter gene”) makes it easier, faster, cheaper, more certain, higher, to detect, quantify and identify transformed cells and their cell lineage and fate It is an important class of transport genes that can be carried by this gene vector. Such genes, well known in genetic engineering, can facilitate and improve their research, evaluation and development in a variety of ways that will be readily apparent to those skilled in the art in various fields such as business, commerce and medicine.
Additional comments on various gene vectors are given in the examples below.

Therapeutic polypeptide candidates The gene vector should basically be regarded as a carrier, carrier, or delivery system. Therefore, the goods and vehicles to be delivered and transported by the carrier are selected by the user intended for the specific use.

  In general, the present gene vectors are capable of essentially transporting and delivering any selected gene having any nucleic acid sequence such that the corresponding polypeptide is expressed by the transfected neuronal cell. As such, most polypeptides of interest to physicians and researchers can be secreted into the BBB by neurons that span the transfected BBB. However, in most cases where doctors and researchers are interested, the need for secretion is not a severe limitation for two reasons. First, almost all types of “neuroactive” polypeptides are secreted in nature. In general, such neural activity has been discovered and identified because the polypeptide is secreted from one central nervous cell and acts on other central nervous system cells. Second, if a particular candidate polypeptide of interest is not normally secreted naturally, a number of “leader” polypeptide sequences known to cause cell secretion can be linked to that polypeptide sequence.

  The endocytotic ligands disclosed herein do not deal with and are not directly involved in the secretion of polypeptides and other compounds by transfected neurons. Instead, these ligands are designed and intended to allow targeted delivery of gene vectors to neurons across specific BBBs. These endocytotic ligands can be used to transduce transport molecules such as therapeutic agents, diagnostic compounds, and other compounds into neurons that cross the BBB with other endocytic surface molecules to which the ligand specifically binds (other target cells). ) Can be used as a system for transportation and delivery.

  Thus, sometimes drugs or other non-DNA compounds that can be transported into neurons across the BBB enter via peripheral projections accessible outside the BBB and are secreted from the neurons by central projections within the BBB. (This was shown by a transsynaptic tracer molecule such as wheat germ agglutinin).

  If the transport component can dissociate from the ligand after the ligand transport complex has invaded the nerve cells that cross the BBB by using a linking component that links the transport component to the endocytic ligand, Such delivery routes for other compounds may be particularly promising. Such linking components are often referred to as “unstable” agents, meaning that they are not particularly adhesive or durable and can be released under a variety of conditions. A variety of labile linking components are well known (eg, acid sensitive spacer molecules are described in US Pat. Nos. 4,631,190 and 5,144,011 by Shen et al). If necessary, together with specific endocytic ligands (or ligands) and specific therapeutic agents, analytical compounds, and other transport components of interest to form molecular complexes such as: It can be determined what unstable connected components can be used. Such molecular complexes can (1) deliver transport components to neurons across the BBB via accessible peripheral projections; and (2) the complex invades neurons across the BBB After that, the transport component can dissociate from the endocytic ligand; and (3) the dissociated transport component can be secreted at a secretory location within the BBB by neurons across the BBB .

  Again, with respect to the range and types of polypeptides that can be introduced into central nervous tissue by introducing foreign genes into neurons across the BBB using gene vectors, Tables 1 and 2 list one application (human Some of the options available in the treatment of various neurodegenerative or neuropathic diseases are listed below. Since the table does not have a description, the following overview outlines the various polypeptides that can be used in human medical treatment and the specifics that can be regulated by such polypeptides to provide additional information on the listed items. List some of the disease groups. This description is not complete, and once the method of the present invention is known, the neurology or neuropharmacology specialist will recognize other potential uses and treatments.

  1. Various neurotrophic factors, growth factors, or neurite inhibitory factors, such as listed in Table 1, can cause central nervous system disorders such as neurodegenerative diseases, or ischemic or stroke attacks, cardiac arrest, asphyxia, blood loss, etc. Prevent or repair various forms of nerve damage resulting from hypoxic strokes or other physical or trauma.

  2. Various neurotrophic factor hormones, growth factors, or neurite inhibitory factors stimulate the formation of new synaptic connections between pre-existing neurons and / or induce neurite outgrowth while promoting synaptic connections However, it can be suppressed on the other side. Depending on the patient, this type of treatment promotes the recovery of neurological loss due to aging or various diseases. It also helps patients regain muscle, speech, and other functions after stroke attacks, head trauma, or other ischemic, hypoxic, excitotoxic, etc. attacks.

  3. A variety of endocrine, paracrine, and related analog polypeptides are useful in the treatment of various glandular, growth, maturity, sexual and other disorders.

  4). Polypeptides that can increase the amount of certain neurotransmitter molecules in the BBB can treat various neurodegenerative diseases. For example, polypeptides that can increase dopamine levels in the brain can be used to treat Parkinson's disease (by enzymes, hormones, secreted factors, or various other mechanisms). Polypeptides that can increase acetylcholine levels are also useful for the treatment of Alzheimer's disease.

  5). Cytotoxins or growth inhibitory polypeptides can be used within the BBB to treat certain cancers or other diseases.

  6). Various receptor antagonists, antibodies, or other polypeptides that can inhibit and inhibit one or more neural activities are used to control and reduce neuropathic pain, hyperalgesia, and similar symptoms be able to.

  7). Lysosomal storage diseases resulting from a deficiency of a specific polypeptide in the central nervous system can be treated by delivering the polypeptide into the central nervous system.

  8). Infection of the central nervous system with viruses, prions, or bacteria can be treated by delivering to the central nervous system something that controls or reduces the transmission of the infection. For example, delivery of polypeptides that bind to receptors and inhibit viral binding can reduce the spread of viruses such as HIV within the central nervous system.

  9. By delivering recombinant antibodies to antigens within the central nervous system, physiological processes can be controlled in a beneficial or useful manner. For example, delivery of recombinant antibodies to myelin conjugated to neurite inhibitor molecules such as No-Go allows for regrowth and regeneration of central nervous system nerves after spinal cord injury and other traumatic injuries.

The target central nervous system nerve cell or glial cell term “target cell” is (i) part of the central nervous system, located entirely within the BBB; and (ii) protected by the BBB by the method and the vector Refers to a neuronal or glial cell that contacts or is intended to contact an exogenous polypeptide delivered to the treated brain or spinal cord tissue.

  The term intentionally excludes neurons that cross the BBB. However, such neural cells are the “primary” or “starting” target of the gene vector used to contact and transfect, and are referred to by such terms. In the entire process of the present invention, transfection of an “initiating target” across the BBB is of value in a multi-step process, as long as it is followed by delivery of the vector-encoded polypeptide into central nervous system tissue protected by BBB. Is useful. Thus, the true “target” of the present invention is a neuron or glial cell that is completely located within the BBB and protected by the BBB, and a neuron that crosses the BBB used in the delivery mechanism is called a true target cell. Rather, it should be considered a route and a passage.

  Reference is made herein to “glia” cells. This is because the cells in the brain and spinal cord tissue protected by the BBB are divided into two divisions called nerve cells and glial cells (also called “neuroglia cells” in some medical texts). By definition, the term “neural cell” is limited to cells that can receive and transmit neural signals. The term “glia cells” has a broader meaning and includes all types of central nervous system cells that cannot receive and transmit neural signals. These glial cells perform various activities within the central nervous system that can be considered supportive, housekeeping, and “nurturing” functions. This helps nerve cells perform the necessary functions. “Glia” is derived from the same root word as “glue (adhesive)”. Glial cells were originally thought as “glue” to attach CNS cells together. Glial cells are divided into various categories and include oligodendroglial cells, astrocytes, ependymocytes, and microglial cells. These are an important part of the central nervous system and are described in almost every textbook in neurology.

  For several reasons, the initial work of the present invention focuses on the use of exogenous polypeptides that contact and regulate neurons rather than glial cells. One major reason for this tendency is to first confirm the reaction of the central nervous system, which is directly related to nerve cells, as compared to the reactions and effects that involve neurons and then only affect the neurons as a secondary effect, This is because it is much easier to quantify.

  Thus, although some researchers prefer to evaluate the present invention with a focus on “target” neurons that are completely located within the BBB, the following should be recognized: (1) Methods and vectors of the present invention Can also be useful to provide a method of treating central nervous system tissue by contacting and treating an exogenous polypeptide to glial cells; and (2) certain that glial cells are involved Symptoms include treatments for certain types of glial cell carcinoma (eg, glioblastoma and astrocytoma) and methods for controlling glial cell responses to insults such as traumatic injury, hypoxia, ischemia, etc. Be advantageous in relatively early research and evaluation.

  As the term “target” means, not all neurons or cells that are completely located within the BBB need necessarily contact the polypeptide delivered using the present invention. When transfecting a nerve cell (or nerve cell mass) that crosses the BBB with a gene vector, a neuron or glial cell that is completely located within the BBB is: (i) the transfected neuron located within the BBB Located near the synapse or other terminal; or (ii) a cell process or cell extension (e.g., a dendrite) of the transfected neuronal cell located near the synapse or other terminal located within the BBB. Axon, or terminal). When using “trans-neuronal” vectors (ie, the gene vector itself travels through the “primary” neurons across the BBB to the secondary or tertiary neurons located within the BBB, where they are translocated. When transfected and expressing the encoded polypeptide), the “target” neuron or glial cell is a transfected secondary or tertiary neuron that expresses and secretes the polypeptide encoded by the foreign gene contained in the vector. It may be located in the vicinity of the cell.

  Examples of how one or more neurons in the brain can become “target” neurons in the BBB are forebrain basal cholinergic, serotonergic raphe, and noradrenergic Sexual blue patch neurons do not synapse directly to olfactory receptor neurons that cross the BBB. Nevertheless, if olfactory neurons are transfected with this gene vector, these cholinergic, serotonergic and noradrenergic neurons in the BBB can become target neurons. This is because they can take up polypeptides secreted by transfected olfactory receptor neurons. This is evidenced by the fact that cholinergic, serotonergic, noradrenergic neurons in the BBB are infected by rabies or herpes simplex virus released from virus-infected olfactory receptor neurons ( Lafay et al 1991, Barnett et al 1993).

  If neurons or glial cells of the central nervous system are located in the BBB, next to, or in close proximity to, the central projection of cells that cross the BBB, they are the target of contact and treatment with exogenous polypeptides. Can be. This principle can be used most advantageously if supported by the latest information on the anatomical relationship between the transfected cells and the target cells. This type of anatomical knowledge can be found in the technical literature using viruses or other “trans neuronal labeling molecules”. For example, after trans-neuron virus is administered to a neuron across the BBB of an experimental animal, if a particular type of neuron or glial cell of the central nervous system is infected by that virus, then the infected central Neural neuronal cells or glial cell types can be used as target neurons or target glial cells for the purposes of the present invention. Such neural mapping has already been accomplished to some extent by various researchers and is described in literature such as Loewy 1998 and Norgren et al 1998. Furthermore, such research continues today and continues to provide more information on neural circuits. Those who implement the present invention can consider the new information.

  The preferred method of contacting the gene vector with the peripheral projection of sensory or motor neurons depends on the structure and location of the target peripheral projection. For example, administration to olfactory receptor sensory neurons can be performed by instillation into the nose, for example using a nasal spray or liquid filler that can be placed in the nasal sinus for a period of time (eg, 30-90 minutes), Direct contact with nasal surface including olfactory projection.

  If necessary, direct and sustained contact between the gene vector and olfactory neuron projection is further facilitated by the following processes: (i) Nasal surface using nasal decongestants (Ii) clean and prepare the contact surface using a cleaning agent or similar agent such as dilute isopropyl alcohol, acetone; and / or (iii) generally by an otolaryngologist Use mechanical friction methods used to treat recurrent sinus infections in patients. In general, surface neuroprojection that leads to moderate inflammation tends to be more sensitive to the uptake of foreign molecules into cells than quiescent or resting cells. Furthermore, it may be possible to promote the invasion of the gene vector by applying a charge or an electric field to the nasal cavity surface as in an in vitro cell transfection method called “electroporation”. If necessary, any or all of these methods can be performed under general or local anesthesia.

  If necessary, nerve contact and uptake can be further promoted and enhanced by compounds called “mucus deposits”. Such compounds have been developed to deliver larger amounts of various drugs into the bloodstream by membrane permeable methods such as nasal sprays. Examples of mucus deposits are chitosan (Janes et al 2001) and various polysaccharide colloids (Schipper et al 1999). See also Rillosi et al 1995 and Lim 2000 for details on mucus deposits.

  Administration to projections that are part of nociceptive neurons is also done via dermal and subcutaneous injection, using various controlled dermabrasion, and also by topical application if sufficient penetration can be achieved (Skin penetration can be accelerated if necessary by drugs such as dimethyl sulfoxide). When motor neuron projection is used as an access route, it is usually administered by subcutaneous or intramuscular injection. For example, projections that are part of the lower motor nerve cells of the hypoglossal nucleus can be accessed by injecting a gene vector into the muscles of the tongue. In some cases, the gene vector is administered intravenously (especially if it contains components directed to specific nerves). In general, however, gene vectors should be administered by intramuscular or similar injections that establish and maintain a high concentration of the vector at the desired target site.

  This points to an important difference between this method (targeted transfection of specific neurons) and delivery of genes, polypeptides or other compounds by injection into the cerebrospinal fluid. For physical delivery to the cerebrospinal fluid system (catheter injection into the subarachnoid space of the brain ventricle or spinal cord), the neuroactive agent is distributed to many and many types of cells in the central nervous system. In contrast, the present invention provides for targeted delivery of a desired therapeutic polypeptide to a limited number of cells or neurites that are in close proximity (or synaptic connection) to transfected neurons across the BBB. Or can be delivered preferentially under control.

  If necessary, the methods and gene vectors disclosed herein can be used to introduce polypeptides into the brain and spinal cord of mammals other than humans, and to other animals with blood-brain barriers such as reptiles and birds. It is thought that it can be used. As such, the present invention would be useful for controlling and regulating the growth rate and / or reproductive status of livestock, pets, and other animals. For this and other purposes, hypothalamic release factors can be delivered through olfactory receptor neurons to regulate the secretion of potent hormones from the pituitary gland. For example, GHRH is delivered to stimulate growth hormone secretion in order to accelerate the growth rate of animals. Also, to achieve or promote contraception, such as reversible contraception, an over-maximal dose of GnRH can be delivered continuously to suppress normal secretion of LH and / or FSH. When the methods disclosed herein are used on other mammals or other species of animals such as birds and reptiles, the following major modifications are required: (i) In selected species of animals, BBB Selection and use of gene vectors that are well-suited for transfecting neuronal projections, and (ii) selection and use of polypeptides that exhibit desirable activity in certain types of animals.

  While not neglecting these and other potential applications, the remaining description of gene vectors relates only to animal experiments to demonstrate the methods and procedures of the present invention, and human therapy for medical purposes.

Viral vectors Papers describing various mammalian viral vectors include Karpati et al 1996 and Kaplitt and Makimura 1997. At least four viral vectors were used for transfection of neurons. These viruses are as follows:

A. Adenoviruses These are double-stranded DNA viruses that are frequently found in various glandular tissues. Wild-type viruses cause respiratory infections, conjunctivitis, and various other disorders. Genetically modified adenoviruses that do not replicate (except for special cells and / or laboratory conditions) are the major types of viral vectors used in mammalian in vivo research, including human gene therapy. became. Adenoviral vectors have been used for transfection of various neuronal cells, as discussed in Smith and Romero 1999.

B. Adeno-associated virus (Depend virus, Adeno satellite virus)
These are single-stranded DNA viruses that require an adenovirus for replication. Methods for preparing adeno-associated virus vectors are described in the chapter by Bartlett and Samulski in Robbins (edited) 1997.

C. Herpes simplex virus (HSV)
These are double-stranded DNA viruses with a capsid surrounded by a lipid envelope. The use of genetically modified HSV vectors to transfect neurons is described by Staecher et al 1998.

D. Retroviruses, including lentiviruses These viruses contain RNA rather than DNA. The use of a lentiviral vector that delivers and expresses the GDNF gene in mouse lower motor neurons is described in Hottinger et al 2000.

  These four classes of viruses are non-pathogenic after transfecting cells but invading target cells (due to deletions or defects in one or more genes that normally encode proteins required for replication) It is currently the most studied and attracting interest in the production of viral vectors.

  However, other known classes of neuropathic viruses (eg, various viruses that cause viral encephalitis, such as RNA viruses) are promising candidates for vectors that can selectively (or at least preferentially) transfect neurons. provide. Any of the above classified viruses should be sufficient for this use if they can be made safe and non-pathogenic by the same genetic treatment as the methods used to make the vectors of other viruses non-pathogenic. Suitable.

  Alternatively, or additionally, using genetic engineering techniques to project neuronal projections across olfactory neurons, motor neurons, or other types of BBB into various viral vectors that do not have strong affinity for neurons Selected and / or modified surface proteins (including chimeric surface proteins) that bind preferentially to surface proteins can be provided. Increased speed, potency, and other benefits of viral vectors when used as disclosed herein, if modified viral surface proteins with increased neuronal cell binding affinity are properly developed and used be able to.

Several types of non-viral gene vectors are well known and evaluated for this use, as briefly summarized in the non-viral vector background art and described in detail in the various examples below and in many published papers. Is done. Major candidates for the classification of non-viral vectors capable of transfecting neuroprojections include: (1) cationic substances, such as cationic liposomes, and (2) polypeptide ligands that bind to endocytic receptors Protein / DNA complex.

  With regard to ligands that can bind to neuronal endocytic receptors, the Applicant is very useful and very useful for identifying and isolating ligands that can bind to endocytic receptors on the surface of nerve fibers outside the BBB. A powerful in vivo screening method has been developed. Briefly, this new in vivo screening method uses the following process:

(1) administering a number of candidate ligands in a living animal to a first site where the candidate ligands can directly contact nerve fibers (eg, the sciatic nerve bundle of the rat leg);
(2) allow sufficient time for specific ligands that activate and promote endocytosis uptake and retrograde transport to be taken up into nerve fibers;
(3) collecting a portion of the nerve fiber at a location sufficiently far from the site of ligand administration to avoid collecting candidate ligands that have not been taken up by nerve fibers or delivered by retrograde transport; And
(4) Remove the ligand from the collected portion of nerve fiber for replication and further processing.

  This novel in vivo screening method is for developing improved non-viral vectors (and target viral vectors) that can specifically bind to any specific endocytosis receptor or other endocytosis-promoting surface molecule. Provides a powerful means of Accordingly, it provides a preferred embodiment of the present invention and is described in detail in the following individual sections and in the various examples.

  Numerous methods and techniques are known to the expert to increase the likelihood of achieving the desired result using non-viral vectors. As an example, non-viral vectors that target neurons can use neurotensin to target plasmids to nigrostriatal and mesolimbic dopaminergic neurons. Reported (Martinez-Fong et al 1999). Various other examples are discussed in the next section.

  In general, plasmid vectors made for use in cationic or endocytotic delivery mechanisms include both: (i) sequences that allow replication of the plasmid in a host cell, such as E. coli, and (ii) A sequence that allows expression of the therapeutic polypeptide gene in the target cell. For replication, the vector usually contains a marker gene that allows easy selection of the origin of replication and transformed cells. For example, for replication in E. coli, the plasmid pBR322 (Bolivar et al 1977) contains ampicillin and tetracycline resistance genes, thus the use of their antibiotics allows for rapid and simple identification of transformed cells.

  Sequences that allow gene expression of the therapeutic polypeptide in the target cell usually include an origin of replication (if necessary), a promoter in front of the expressed gene, a necessary ribosome binding site and / or RNA splice site, a polyadenylation site, And a transcription termination sequence.

Gene and vector constructs; expression, transport and secretagogues Using various well-known genetic engineering techniques and DNA or polypeptide sequences, the following can be improved and increased: (i) The ability to successfully achieve the desired and intended results in a particular animal or patient at a recognizable level; (ii) the efficacy, effect, duration, or other desirable point of treatment in the treated animal or patient; and (iii) Researcher and physician activities that track and monitor the status, progression and outcome of treatment, and the location and concentration of exogenous genes and / or polypeptides.

  This section, along with various examples of such well-known techniques and sequences, briefly describes how they apply to the methods and vectors of the present invention. This list of illustrative examples is neither complete nor exclusive, and experts will recognize that various other genetic engineering techniques, reagents, genes and peptide sequences and fragments can be used for this use. .

  Moreover, it will be apparent to those skilled in the art that the techniques and arrangements disclosed herein may be combined with one another in various ways. As an example, to express a gene encoding mature neurotrophin in neurons across the BBB, a gene construct encoding a polypeptide comprising the following two can be developed: (i) the mature form An “epitope tag” placed at the N-terminal prepro BDNF leader sequence of the sequence encoding neurotrophin, and (ii) at other positions of the sequence encoding mature neurotrophin. Similarly, other additional genetic engineering techniques or sequences now known or discovered in the future can be adapted for this use.

  As an example of a technique for enhancing the expression of a transport polypeptide contained in a gene vector, the coding sequence of the polypeptide can be placed under the control of a strong “gene promoter”, whereby the mRNA containing the coding sequence Greatly facilitates transcription of the strands. Various promoter sequences are known that function as strong promoters in human cells (including human neurons), including, by way of example: cytomegalovirus (CMV) promoter, rhus sarcoma virus (RSV) terminal repeat ( LTR) promoter, and promoter sequences from various pathogenic viruses, such as the “early” promoter of Simian Virus 40 (SV-40).

  Alternatively, in some cases, one or more genes (e.g., a gene encoding a labeled polypeptide) may be placed under the control of a so-called "inducible" promoter that is only active under certain conditions or in the presence of certain compounds. desired.

  In addition, gene promoters usually referred to as “tissue-specific” promoters are also attracting attention. These types of promoters efficiently express polypeptide coding sequences in mRNA only in specific types of tissues or cells. Even if a gene construct containing a tissue-specific promoter is delivered to the wrong cell or tissue, those “unintentional” cells usually do not have a transcription factor or enzyme that can recognize the tissue-specific promoter. The gene constructs are not expressed (or only expressed at a low rate) in their “unintended” cells. Thus, tissue specific promoters are very useful candidates for the evaluation described herein.

  According to reports of genetically engineered animal studies, several tissue-specific promoters have already been identified and published, and other genetically engineered animal studies have been reported and the number is increasing. The following list includes multiple examples of tissue specific promoters that are not complete or exclusive and would be useful in the present invention.

  (i) As a tissue-specific promoter that is much more active in olfactory receptor neurons than other types of neurons, the olfactory marker protein promoter described in Servenius et al 1994, and described in Qasba and Reed 1998 There are M4 olfactory receptor protein promoters.

  (ii) A tissue-specific promoter described in Watson et al 1995 that is much more active in nociceptive sensory neurons than sympathetic neurons. This gene expressing the minimal “calcitonin gene-related peptide” (CGRP) is highly active in the presence of nerve growth factor (NGF) and may thus provide a compound-inducible gene promoter. Thus, the use of the CGRP gene promoter can minimize gene expression in sympathetic neurons and minimize undesirable side effects. This type of gene promoter can be particularly useful because nociceptive sensory and sympathetic neurons are known to take up NGF by receptor-mediated endocytosis. Because both nociceptive and sympathetic neuronal cell types are probably transfected, peripheral non-viral vectors that utilize NGF receptor-mediated endocytosis to deliver targeted genes to accessible neurons When administered, gene constructs that are not substantially expressed in sympathetic neurons transfected with a gene promoter such as the CGRP promoter can be very effective.

  (iii) Glycine is much more active in spinal motor neurons than nociceptive or other sensory neurons, as described in articles such as Bechade et al 1994, Rajendra et al 1997 and Zafra et al 1997 A tissue-specific promoter that promotes the expression of the alpha 1 subunit of the receptor.

  In general, in genetically engineered animal systems, when a transgene construct has been shown to restrict a given gene expression to a specific class of neurons, an appropriately prepared gene construct (same promoter and related gene expression elements) The same pattern of gene expression restriction can be expected when delivered in vivo by viral or non-viral vectors. Therefore, when designing gene expression constructs, reference should be made to transgenic animal technology (eg, Causing and Miller 2000).

  In related analogous methods, it may also be possible and preferred to use natural or synthetic promoters and / or promoters that stimulate, repress or control the expression of foreign genes in a variety of ways. For example, the use of gene promoters that are normally silent but activated after nerve injury (see Funakoshi et al 1998) can reduce foreign gene expression in damaged neurons without substantially affecting other neurons. It can be established or greatly enhanced. Alternatively, if the amount of glucocorticoid adrenal stress steroids is high as a result of the stress response (eg Ozawa et al 2000), a glucocorticoid responsive promoter can be used to promote gene expression after nerve injury. Alternatively, latency-related transcriptional promoters from herpes simplex virus (Lachmann et al 1997) can promote gene expression for a longer period than various other promoters.

  As another example of techniques for enhancing protein expression, one or more “non-preferred” codons are often deleted and replaced with “preferred” codons in order to create a transfection gene. The distinction between the non-preferred codon and the preferred codon is that most species (and many different cell types within a species) have (i) a preferred codon that passes quickly through the translation system within the ribosome, and (ii) It stems from the fact that it has evolved to include both unsuitable codons that slow down the translation process within the ribosome. This mode of preferred and non-preferred codons provides a very useful cellular mechanism for gene expression control so that the correct balance of many different proteins can all be present at a suitable concentration within a single cell. . However, in genetic engineering, gene vectors can bypass normal control mechanisms by excluding non-preferred codons and replacing them with preferred codons and promote higher production of foreign proteins than usual. This type of method is described in numerous research reports, such as US Pat. No. 5,795,737 (Seed et al 1998) and the various articles cited therein.

  Another well-known genetic engineering technique includes “cysteine depletion” polypeptide variants. As is well known, cysteine residues (amino acids with a highly reactive sulfhydryl group -SH) tend to react with other cysteine residues. When two cysteine residues react with each other, a disulfide bond is formed. The disulfide bond is very important to ensure that the polypeptide is synthesized intracellularly and “folded into the proper three-dimensional structure” during “post-translational processing”. However, when genetic engineering is used to make a protein, cysteine residues that are not involved in disulfide bonds can cause problems and interfere with the desired results. Such problems are often avoided and overcome by replacing one or more cysteine residues at certain positions in the polypeptide sequence with other residues. This can be done by replacing a codon specifying a cysteine residue at a specific position in a gene with a codon specifying an amino acid residue other than another cysteine. These types of cysteine-depleted “mutant proteins” are described in various patents such as US Pat. No. 4,737,462.

  In addition, a “leader sequence” and / or “signal sequence” can be added to a foreign polypeptide encoded by the present gene vector using genetic engineering techniques. The term “leader sequence” is not necessarily used consistently or correctly, but generally refers to a polypeptide sequence that has one or both of the following properties: (i) a leader sequence for a foreign polypeptide that does not normally undergo transport or secretion. Transport the leader + polypeptide molecule to a specific location within the cell, or promote the process, or cause the secretion of the leader + polypeptide molecule by the host cell, as indicated when (Ii) that part of the initial polypeptide that is cleaved from the shorter final (mature form) polypeptide by natural “post-translational processing”. In almost all cases, the leader sequence is located at the N-terminus of the polypeptide and is the first end created (often referred to as the “head”) and appears first from the ribosome at the same time the polypeptide is made.

  Similarly, the term “signal sequence” is not necessarily used consistently or accurately. In general, it refers to a polypeptide sequence that directs a particular result, effect or process, as indicated by the sequence that gives that result or process to other peptide sequences that do not normally result in that result or process. By way of example, the signal sequence induces: (i) “encapsulating” the polypeptide in a vesicle or other compartment, by storage, or other treatment; (ii) to a specific part or region of the cell Or (iii) secretion of the polypeptide by the host cell. Thus, “signal sequence” is more widely used than “leader sequence” and generally includes a leader sequence. Furthermore, the signal sequence need not be cleaved from the initial polypeptide to produce the mature final form of the polypeptide. The signal sequence is often kept in the mature polypeptide.

  Various neural leaders and / or signal sequences that are believed to enhance one or both of the two processes that are very useful in the present invention are well known. These two processes are as follows: (i) the polypeptide present in the synthesized cell passes through the “central projection” from the main body of the neuron, crosses the blood brain barrier, and / or Or antegrade transport of the polypeptide to be delivered in the vicinity of the desired target region of the brain; and / or (ii) secretion from the synaptic terminal or other terminal of the neuron by the neuron.

  One such type of leader sequence that can be very useful in various situations is the leader sequence present in the initial polypeptide called “preproBDNF”. BDNF is an abbreviation for brain-derived neurotrophic factor. BDNF is a homologue of nerve growth factor (NGF), but unlike NGF, it is known that prepro-BDNF is efficiently antegrade transported by nociceptive neurons. It has been confirmed by animal experiments and it has been shown that its BDNF expressed in nociceptive neurons is later detected in spinal cord tissue. The shorter final (or “mature”) BDNF is made by cleaving the leader sequence from a longer initial polypeptide. BDNF polypeptides and pre-pro BDNF leader sequences have been described in several published articles including Conner et al 1998, Altar et al 1999 and Tonra 1999. Anyone who analyzes BDNF polypeptides and pre-pro BDNF leader sequences will find this leader sequence, and any other peptide leader sequence known to perform or facilitate antegrade transport and / or secretion in neurons. It will be appreciated that it is very useful in increasing the success rate and efficiency of the present invention.

The epitope tag sequence prefix “epi” refers to an exposed accessible surface. As an example, the epidermis (epidermis) is the outermost dermis (skin) surface and the epithelium (epicerium) is the exposed surface of the mucosa.

  Similarly, the term “epitope” (antigenic determinant) is used to refer to a surface exposed region of a protein that is strongly bound by an antibody. The antibody does not cover the entire surface of the antigen protein; instead, the localized binding domain that is part of the antibody binds to the localized binding domain that is part of the antigen. This is similar to two jigsaw puzzle pieces that fit along one (only one) edge of each piece.

  It is not difficult to identify the epitope region of an antigen protein for a monoclonal antibody of a specific strain by passing a digested (cleaved) fragment of the antigen protein through a column in which the monoclonal antibody is fixed to extremely small beads. However, since different monoclonal antibodies bind to different surfaces of an antigenic protein, proteins generally have several different epitope regions, and the common factor that determines which region is an epitope is that region. Whether it is exposed to the protein surface and accessible to the antibody. For this reason, epitope analysis is frequently used by researchers to define the three-dimensional structure of complex proteins. In general, an amino acid sequence shown as an epitope site of a protein is an amino acid sequence that is easily accessible to an antibody exposed on the protein surface.

  The use of epitope “tag” sequences in genetic engineering is based on the following facts: it is relatively easy to generate and identify monoclonal antibodies that bind with very high selectivity to known epitope sequences. Several such monoclonal antibody strains (and the high affinity corresponding antigen amino acid sequences to which they bind) are well known. Examples include monoclonal antibody strains that bind to known amino acid sequences of polypeptides such as c-myc and influenza virus derived hemagglutinin proteins.

  Since cellular polypeptide expression begins at the N (amino) terminus and ends at the C (carboxy) terminus, generally there is a high probability that the amino acid region at or near the C terminus is exposed to the protein surface. This is based on the fact that the folding and formation processes that form the final three-dimensional polypeptide occur simultaneously with chain formation and elongation. Because, simultaneously with chain formation, various amino acid residues or domains at specific positions in the chain begin to attract or repel each other. This description is very concise and does not fully explain the details of protein folding, but the first part of the chain emerges from the ribosome as the formation of the “nucleus” of the polypeptide, and the rest of the chain is usually the first nucleus. You can imagine wrapping up or joining the first nucleus in another way.

  Because of this factor, the amino acid sequence at or near the polypeptide “tail” (carboxy terminus) is more likely to be exposed and accessible as an epitope region on the polypeptide surface. To reduce the risk that insertion of an epitope region into the central domain will destroy or reduce an important activity or property of the polypeptide, this factor should generally be used to modify it to include an epitope tag. The epitope tag sequence should be placed at or near the tail (carboxy terminus) of the polypeptide. In general, this is the best method for initial experiments using epitope tags and, if necessary, the experimental data obtained using this method can be used to optimize subsequent studies.

  Alternatively, or in addition, if the complete three-dimensional structure of the polypeptide molecule is known and the “active” site of the polypeptide essential for catalytic activity or receptor binding is known, the opposite surface of the polypeptide molecule It is possible to insert an easily accessible epitope region.

  A relatively simple in vitro screening test will test whether such modified “tagged” proteins still have suitable activity, and tagged proteins that have passed their first test will be tested in in vivo animal experiments. Inspected more extensively.

  Thus, the gene vector system disclosed herein adds (or inserts) a unique, or at least very unusual, detectable “epitope tag sequence” to almost any type of well-known protein (eg, nerve growth factor). It is possible to produce a gene construct. Using well-known genetic engineering techniques, it is easy to alter the coding sequence of the gene construct contained in the gene vector, thereby adding a relatively small number of additional codons to the “natural” coding sequence, or Replace a few codons in the native coding sequence with other codons not normally present in the native protein.

  In various studies and therapies disclosed herein, gene vectors containing gene constructs that express epitope-tagged polypeptides are very useful, and researchers and physicians can use the gene therapy disclosed herein. It can make it easier to monitor and survey the results and effects of (and in some cases, required to make it possible). As an example of a problem that arises during the analysis of natural polypeptides, in the current art, natural nerve growth factor (NGF) tends to be degraded by tissue fixation methods. Furthermore, NGF is difficult to measure accurately even by skilled researchers, and the related forms of NGF found in widely different animals such as rats and humans also have high homology and antibody cross-reactivity. Thus, such difficulties can be avoided or at least minimized by using epitope-tagged forms of NGF (and various other neuroactive polypeptides).

  Epitope-tagged polypeptides are widely used in research much more frequently than in human clinical therapy. In general, epitope-tagged polypeptides are used in cell culture and animal experiments to develop, optimize, and demonstrate the effectiveness and efficacy of certain therapeutic methods. In addition, they are sometimes used in small “Phase 1” or “Phase 2” human clinical trials approved under US “investigation drug” applications and under similar formal reviews and other approvals. The Once treatment can be validated in phase 3 clinical trials at multiple larger locations, all epitopes or other “exogenous” are usually used to reduce the risk of generating an immune response with antibodies that recognize and attack the epitope sequence. "Sex" sequences are excluded.

In Vivo Identification of Phage Libraries and Combinatorial Chemistry-derived Endocytosis Ligands As noted in the Summary, Applicants have identified a novel for identifying and isolating endocytosis ligands that are taken up and transported into nerve fibers. In vivo screening method was prepared. This novel method is a preferred embodiment for practicing the present invention and, as detailed herein, one skilled in the art will use this method to deliver polypeptides into CNS tissue protected by BBB. Improved gene vectors can be made that can be used for this purpose.

  In order to fully understand the in vivo screening method and the endocytosis ligand obtained from this screening method, background information on the endocytosis ligand receptor protein needs to be provided. Like almost all cell surface receptors, endocytic receptors span the outer membrane. They therefore have three distinct domains: (i) an extracellular part that is exposed and accessible to molecules carried by the aqueous fluid surrounding and in contact with the cell; (ii) the lipid bilayer surrounding the cell Embedded internal part; and (iii) an intracellular part exposed to the aqueous cytoplasm inside the cell.

  In general, ligand receptors have two properties that are different from various other non-ligand receptors. The first property is that each specific type of endocytic ligand receptor interacts with a single type of extracellular molecule (or possibly a limited number of structurally similar extracellular molecules) Induced and activated by. This coupling mechanism is thought to be comparable to the “key and lock” relationship. Suitable for locks with only a few keys of certain exact and specific size and shape. This property distinguishes ligand receptors from, for example, various transport proteins that capture nutrients floating outside the cell and carry them inside the cell.

  The second distinct property is that when the ligand and endocytic ligand receptor protein bind, the two molecules have bound to each other for a longer period of time. This distinguishes ligand receptors from neurotransmitter receptors that are stimulated by acetylcholine, glutamate, and other classical neurotransmitters. Instead of being measured in milliseconds (as seen in most neurotransmitter receptors), the ligand binding response is usually measured in minutes, hours, or days.

  In an endocytosis ligand binding reaction, the ligand binds to the ligand receptor to form a ligand-receptor complex that is taken up into the cell where typically the ligand and receptor are ultimately digested together. Those building blocks are recycled. To understand how this happens, polypeptide hormones that induce permanent changes in animal size, health, nerve or reproductive system, or other characteristics or organs, such as growth hormone, nerve growth factor It should be considered. If this type of hormone is released from the receptor, it floats back into the extracellular fluid and then contacts and activates another receptor on another cell, resulting in Uncontrolled and potentially unregulated effects will occur.

  As used herein, the terms “endocytosis” and “uptake” are used interchangeably, and are extracellular molecules (nutrients and oxygen) that specifically bind to molecules on the cell surface (usually referred to as “affinity binding”). Refers to the process by which cells are drawn into the cell. This process involves receptor-mediated endocytosis involving the binding of ligand and receptor protein. This also includes the process by which ligands specifically bind to other types of cell surface molecules (including surface carbohydrates) to form other types of ligand complexes that are taken up into cells. The terms “endocytosis” and “uptake” refer to the phagocytosis (if involved in the complexation of a ligand molecule and a cell surface molecule to form a complex that is then taken up by the cell ( It also includes processes that are involved in the ingestion of very small particles and soluble molecules) and phagocytosis (involved in the ingestion of large particles such as viral particles and bacterial cells).

  Processes related to endocytosis are well known and many documents such as Alberts et al, Molecular Biology of the Cell (3rd edition, 1994), pages 618-626 (Description of endocytosis), 636- 641 (Explanation of clathrin proteins and triskelions that assist in promoting endocytosis) and 731-734 (Explanation of internal structural changes when ligands bind to membrane proteins).

  Since most types of animal cells have a typical diameter in the range of about 10 microns or 1/100 millimeter, ligand-receptor complex endocytosis is the transport of lipid vesicles over very short distances. Involved in.

  However, neuronal ligand-receptor complexes can travel much longer distances. Many neurons in the brain and spinal cord have long fibers that extend multiple centimeters (eg, axons, dendrites, “processes”), and in humans they send neural signals from the limbs to the spinal cord (or vice versa). Some types of nerve cells that carry have more than a meter of fibers. Thus, due to the endocytosis that occurs within such long fibers, the ligand-receptor complex is from the forefront of the nerve fiber where several ligand-receptor complexes first enter the nerve cell, The entire body must be transported to the main cell body of the nerve cell no matter how long.

  As described above, “retrograde” transport occurs when a molecule is transported along the axon or dendrite from the tip (or end) of a neuronal cell to the main body of the cell where the nucleus is present. Transport in the opposite direction is called “forward” transport. In addition, the term “axon transport” should be recognized and understood. Many types of neurons have a single main fiber that is the longest fiber extending from its main cell body. This longest fiber is usually called an axon. The term “axon transport” refers to and includes any form of molecular transport within an axon regardless of the direction in which the molecule moves (ie, to or from the cell body). Thus, retrograde transport that occurs within axons is a type of axon transport. However, in this specification, the direction of movement is also pointed out, so the term “retrograde transport” is preferred.

  Not all candidate ligands that can bind to the endocytic ligand receptor are able to fully activate and induce the uptake process. As an example, monoclonal antibodies that actually bind to known receptors can be made fairly easily, but such antibody preparations successfully trigger and complete the entire process of receptor-mediated endocytosis. There are only a few reports that it can. An example of an antibody that was reported to be able to induce and complete the receptor-mediated endocytosis process in rat neurons was first a monoclonal antibody named IgG-192 and then called MC192 (Chandler and Shooter 1984). This antibody MC192 specifically known as “low affinity nerve growth factor receptor” in the mid-1980s and subsequently specifically binds to the rat neuronal receptor called p75 receptor. Several years after the monoclonal antibody MC192 was made, it was radiolabeled by other researchers and injected into rats, where it was taken up and transported back to the cell body of neurons that expressed the p75 receptor on the surface. (Yan et al 1988).

  This example shows that antibodies can be theoretically developed that can induce and complete ligand-receptor endocytosis. However, before such research results are used in human medicine, major obstacles still remain and two important problems arise immediately.

  The first problem relates to the difficulty of extending the above facts to human medicine. Obviously, it is very problematic and often unsuitable to inject humans with antigens or unproven antibody fragments. More importantly, screening tests that need to be performed to prove that certain antibodies that work well in animal tests work well in humans are not conducted in ways not currently accepted in human research. If so, it is very difficult and potentially impossible. If you begin to seriously consider the obstacles you face in such research trials (perhaps you need to remove and analyze a sample of spinal cord tissue to determine if the radiolabeled tracer molecule actually reaches the spinal cord ), Consider testing against murderers who have been sentenced to being executed in a few days, or who will die from cancer or other end-stage symptoms within a few days. However, tests that remove solid tissue from the human spinal cord to analyze tissue are not recognized in industrial countries using modern medicine.

  The second difficult problem is that if the antibody fragment itself functions as an endocytosis-inducing agent as desired, an "endocytosis receptor antibody fragment" Concerning the issue of whether to carry inside. Obviously, antibody fragments are of no therapeutic value if nothing is bound and nothing is drawn inside the nerve cell. Instead, such antibody fragments can only be considered as carriers (or locomotives that pull trains). They are not useful if certain “cargo” or “carrying” molecules cannot be carried or pulled into the nerve cells that are entering.

  However, it must be recognized from the outset that any additional molecular fragment attached to the endocytic antibody fragment will necessarily result in a larger complex. By increasing the complex, depending on its size, the property of being drawn into the cell with the same efficiency as the antibody fragment alone in the complex can be substantially reduced. In the research required for such antibody fragments, there is no good way to answer such problems at an early stage. Instead, the transport carrier must be evaluated and proved to function on its own before testing the nature of the carrier to carry (or pull) a “cargo” or “carry” component. Value is generated.

  Thus, reports published to date have shown that only a small set of antibodies made against endocytic receptors induce and complete the process of receptor-mediated endocytosis. Because the nature of the ligand cannot be mimicked, tens or hundreds of monoclonal antibody candidates are each tested to identify rarely occurring uptake antibodies, making the above problem even more difficult and expensive become.

  It should be further noted that there are two major problems with in vitro screening methods that evaluate candidate endocytotic ligands using cell culture conditions. These two problems have effectively made some progress in the field of research on cancer cells and blood cells involved in autoimmune diseases.

  To understand these two issues, the first thing to note is to identify and isolate a rare “needle in hay” ligand that can activate and induce endocytosis. Most evaluations of “libraries” or “repertoires” of candidate ligands usually begin with either (i) a phage display library or (ii) a preparation made by “combinatorial chemistry”. Phages (viruses that can infect bacterial cells) and phage display libraries are available such as Koivunen et al 1999, Cabilly 1999, Shusta et al 1999, Larocca et al 1999, 2001, 2002, Rader 2001 and Manoutcharian et al 2002 It is described in the literature. Combinatorial chemistry has been published in literature such as Lockhoff et al 2002, Flynn et al 2002, Edwards et al 2002, Ramstrom et al 2002, Ley et al 2002, Lepre et al 2002, Liu et al 2003, Edwards 2003 and Geysen et al 2003. Has been described.

  When researchers try to use in vitro cell culture preparations to screen phage display libraries to identify endocytic polypeptide ligands, there are two major problems: (i) incorporation into cells Multiple different phages usually bind to multiple different proteins and other molecules on the cell surface; and (ii) cells are killed or taken up without killing the cells Rinse, wash, or ensure removal of any and all phage that are attached to the cell surface and that have not been taken up into the cell without causing significant problems that interfere with other desired processing of the phage Very difficult to do. Due to these two factors, it is very difficult to suppress the selection of “false positives”. Thus, in vitro screening of endocytic ligands from phage libraries has been only successful to a significant extent in studies using cancer cells or blood cells that can be easily grown in cell culture. In contrast, in vitro screening of endocytic ligands from phage libraries has not succeeded to a substantial degree in cell culture involving anchorage-dependent cells that form aggregated tissues.

  The problems summarized above apply to the simplest tissue culture systems where all cells are clonal replicates and have exactly the same receptors. A normally surviving animal (where different tissues and cells, each having its own specific set of receptors and other surface molecules coexist closely with each other, such different tissue regions and cells The idea of easily performing a phage library screening test (with constant circulation of blood and lymph between them) is not only for technicians familiar with phage library screening techniques and even the simplest cell culture conditions. However, it is not seen by engineers who understand that it is quite difficult to do this well.

  Therefore, much research has been conducted when using phage display libraries to develop improved genetic engineering methods and vectors that can be used to genetically transform and treat cancer and autoimmune diseases. Many results were obtained. However, when using a phage display library to treat other diseases or to create genetic vectors that can transform neurons and other cells present in aggregated tissues in the body Little research has been done and only very limited results have been obtained. Under the prior art, in vivo testing eliminates false positives in normal animals when screening for phage uptake from phage display libraries into cells of neurons and other aggregating tissues via endocytosis Attempts and their difficulties are so hard and cumbersome that practical progress in this area of research has been virtually constrained. Prior to the present invention, no one knew how to use a phage display library for the results that can now be achieved by the in vivo screening methods of the present invention.

  Therefore, the newly developed in vivo screening method can be described below with reference to FIGS. More information is in Examples 22-31.

  In a preferred embodiment, this in vivo screening method uses rodent sciatic nerves such as rats and mice. Both species are inexpensive, easy to mate and grow, and are used as standard animal models in most genetic studies of small mammals. A vast amount of basic information, species-specific biomolecules (eg gene promoter sequences, gene coding sequences, monoclonal antibodies, etc.) and special animal families have been developed for genetic studies using mice. These information can be freely obtained from internet sites such as www.informatics.jax.org and www.ncbi.nlm.nih.gov/genome/seq/MmHome.html. Corresponding information, reagents and pedigrees for rat genetics are somewhat less, but still enormous and very useful, e.g. http://rgd.mcw.edu, http://ratmap.gen.gu. It can be obtained from Internet sites such as se and www.hgsc.bcm.tmc.edu/projects/rat.

  Rats are larger than mice, making it easier to study the sciatic nerve (on both sides of the animal, extending from the spinal cord through the waist and legs to the toes). This can be done by a known method as shown below.

  However, even in mice, the sciatic nerve is long enough and discriminable enough to allow the necessary surgical procedures by the methods disclosed herein (researchers who have made such studies still have Especially when operating). Furthermore, it is possible to perform a surgical operation on a mouse using a binocular microscope or a surgical tool generally used by ophthalmic surgeons and neurosurgeons. Additional explanation regarding the surgical procedure is in Example 25.

  If necessary, it can be evaluated whether other types of laboratory animals (primates, vertebrates other than mammals, or certain invertebrates) can be used in this in vivo screening method. In particular, some animals are known to have exceptionally large nerve axons. As an example, some squids have “giant axons” that control the muscles that produce the driving force. Similarly, Chinese hamster ovary (CHO) cells are widely used in laboratories because of their unusually large cellular components. Squids and other animals with abnormally large nerve fibers or bundles can be used as disclosed herein if desired.

  As shown schematically in FIG. 7, the candidate ligand can be placed in contact with the sciatic nerve bundle. This will be described in detail in Examples 25 and 26. Briefly, when targeting an inducible receptor (eg, a low affinity p75 nerve growth factor receptor), the first ligature 1102 is placed around the sciatic nerve bundle 1090 and tightened. This ligature 1102 is made by wrapping a suture around the nerve bundle and then tightening and tying it. The ligature made in this way functions as a tourniquet and interferes with the normal flow of fluids and molecules within nerve fibers.

  As shown in FIG. 7, the ligature 1102 is positioned so that the sciatic nerve bundle 1090 is adjacent to the “tibial bifurcation” 1092 where it splits into two major branches that act on different parts of the leg and foot. be able to. The ligature 1102 should preferably be placed upstream of and very close to the tibial bifurcation 1092. As stated in the background, the terms “upstream” and “proximal” refer to a position closer to the spinal cord of the animal (rightward direction in FIG. 7). Conversely, the terms “downstream” and “distal” refer to a position away from the spinal cord and closer to the legs and feet (leftward direction in FIG. 7).

  The purpose of ligation 1102 is to increase the number of p75 receptors expressed on the surface of the sciatic nerve bundle. The p75 receptor interacts with neurotrophic factors (also called nerve growth factors), which are polypeptides that exhibit hormone-like effects on nerve cells. The most known such molecule discovered fairly early was called nerve growth factor. Furthermore, such a molecule was discovered and named, for example, brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4 / 5, and the like. Neurotrophic factors effectively stimulate nerve cells, usually leading to increased metabolic activity, additional formation of synaptic connections with other nerve cells, and the like. If the nerve fiber of a motor nerve cell is damaged or damaged, one of the ways that the motor nerve cell responds is to increase the number of p75 receptors on the nerve fiber and any that may be present in the surrounding extracellular fluid By increasing the opportunity to capture and bind the nerve growth factor molecules. The process of “upregulating” certain neuroreceptors on the surface of nerve fibers has been found, and this development is associated with certain neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS, In Lou Gehrig's disease and motor neuropathy). It also occurs after various traumas.

  Accordingly, the ligature 1102 is designed to take advantage of such neural responses. Increasing p75 receptors along the entire length of the nerve fiber between the spinal cord and the ligation site by wrapping and tightening a suture around the nerve fiber and making a ligation to stop fluid flow in the fiber Can do. Based on various tests, such as staining tests using monoclonal antibodies that bind to the p75 receptor, the increase in p75 receptor is estimated to be about 10-15 fold.

  The receptor expression response to ligation by such nerve fibers occurs for about a week. Thus, after placing and tightening the ligature 1102 around the sciatic nerve bundle, the wound is closed and sutured, and the animal is allowed to recover for at least several days, preferably about one week, before performing the next surgery. Should.

  In the second step, two different sites are surgically opened approximately 2-3 cm apart from each other. One site is close to the site where the ligature 1102 is placed, and in fact, the same site as before may be surgically opened. At this site, the sciatic nerve bundle is cut using a scalpel or scissors to produce two ends (which may be right angle, diagonal, etc.). These cuts are performed immediately upstream and downstream of the ligature 1102, and a small portion of the nerve bundle containing the ligature can be cut out and discarded. These cuts produce two ends in the nerve bundle, a distal end 1094 (which is no longer active) and a proximal end 1130 (the spinal side).

  A lump of material 1150 is placed at the site where the sciatic nerve bundle 1090 is cut. The mass 1150 is composed of a porous and permeable material (eg, collagen gel foam) containing a large number of phage particles (preferably millions or billions of “colony forming units (cfu)”). This lump 1150 should be fixed at this position so that (i) the phage particles contained in the lump 1150 and (ii) the cut end 1130 of the sciatic nerve bundle 1090 are in close contact with each other.

Such arrangement and fixing can be performed as follows, for example.
(a) tie the two cut ends 1094 (distal cut end) and 1130 (proximal cut end) of the sciatic nerve bundle 1090 with a suture 1132; and
(b) Wrap and secure a small sleeve or cuff 1136 made of a waterproof material such as silicone rubber around the lump 1150 and around the two cut ends 1094 and 1130 of the sciatic nerve 1090; Two nerve edges are contained in a small waterproof cylinder. If necessary, secure the sleeve 1136 by tying one or more sutures around it, placing a drop or bead of adhesive on the outer surface of the sleeve, or by other suitable means can do.

  Therefore, the part where the substance mass containing the phage particles is arranged is called a phage arrangement part or a phage contact part. This is the site where the phage particle library or repertoire contacts the cut end of the sciatic nerve. Phage particles that have surface manifested polypeptide sequences that induce endocytosis (eg, via p75 receptors on the surface of nerve fibers) are taken up by nerve fibers at this site.

  At a separate site, preferably about 1 cm or more away from the phage placement site, the second site is surgically opened and a second ligature 1202 (made by a loop of suture) is placed around the sciatic nerve bundle And tighten and tie. Since the rat buttock is a convenient location sufficiently away from the phage placement site to eliminate the great risk of false positives caused by phage attached to the outside of the sciatic nerve fiber, the site is preferably located in the buttock area. It should be, and is referred to herein as a buttock ligation site. The buttock ligature 1202 acts as a stenosis or tourniquet around the sciatic nerve bundle 1090 and must be tight enough to prevent substantial flow of fluid or molecules within the nerve fiber beyond its occlusion point. Therefore, this ligation forms a phage accumulation region inside the nerve bundle and downstream of the buttock ligation 1202.

  A phage particle carrying a ligand that can effectively activate and induce the endocytosis process by closing and suturing the rat wound and allowing it to stand for an appropriate period of time (eg about 18 hours). Allow enough time to enter the nerve fiber and subsequently be transported back to the spinal cord through a length of nerve fiber.

  After this standing time, the rats are killed painlessly, the buttock ligation site is opened, and the adjacent sciatic nerve bundle region immediately downstream of the buttock ligation is removed (collected). The short nerve fiber bundle is then divided into small pieces and treated with chemicals that partially digest the cell membrane (consisting of a lipid bilayer) without damaging the phage particles. By this treatment, live phage particles taken up into nerve fibers can be recovered and isolated.

  Phage particles selected by performing the series of in vivo screens described above can be propagated and / or manipulated in a desired manner. As an example, any or all of the following methods can be performed using a phage group selected by the in vivo screening method disclosed in the present specification.

  (1) If the phage is a “phagemid” (the phage simply needs to have a bacterial origin of replication), it is amplified using E. coli without a helper phage to produce double-stranded DNA in the form of a plasmid. Can be produced. Almost all modern phage display libraries use phagemids because various useful techniques are possible, such as the synthesis of a desired amount of circular plasmid DNA.

  (2) The selected phage can be amplified using E. coli with helper phage to produce new fully infectious phage particles containing ssDNA. These phage particles can be used as starting materials in another in vivo screening cycle, which (in the initial cycle) can be used to further enhance the “enriched population” of endocytosis phage and increase the efficiency of inducing endocytosis. It can be purified to "elite" groups that will contain higher ligands.

  (3) After performing a sufficient number of screening cycles until suggested to be suitable for phage analysis (often after at least 1 to about 3 screening cycles, in some cases perhaps more After many times), one or both of the following can be made using the phage selected by the final round of in vivo screening (actually by any number of in vivo screenings). (i) a desired amount of double-stranded or single-stranded DNA to perform nucleotide sequencing to determine the exact sequence of a gene encoding a particular endocytotic ligand; and / or (ii) specific A desired amount of soluble form of the coat protein with an endocytotic ligand for processing and sequencing to determine the amino acid sequence of the ligand domain in the coat protein.

Photographic confirmation of in vivo screening results The effectiveness and success of the in vivo screening methods disclosed herein are visually illustrated by the photographs in FIG. This photograph was taken using a fluorescent reagent in an actual test of the in vivo selection method and shows the location and concentration of the phage incorporated into the sciatic nerve bundle (preparation of the phage used in this test) And staining reagents are described in Example 27 below). The left side of the photograph in FIG. 8 shows a considerably high concentration of fluorescently labeled phage in the nerve portion corresponding to the phage accumulation region 1204 shown in FIG. A narrow zone in the middle of the photo was created by a ligature 1202 at the waist. The right side of the picture shows the sciatic nerve (ie, in the spinal direction) on the side close to the lumbar ligation. Because there is little or no phage that could pass through the lumbar ligature 1202 and reach that part of the sciatic nerve, there will be little fluorescent labeling on that part.

Combined in vivo screening and combinatorial chemistry The general methods disclosed herein for in vivo screening and identification of endocytic ligands can be used with candidate ligand molecules made by “combinatorial” chemical synthesis. it can. Over the past 20 years, chemical synthesis and screening in this area has become extremely active. A database search of the National Library of Medicine in April 2003 revealed over 900 review articles in this research area. Recent reviews include Lockhoff et al 2002, Flynn et al 2002, Edwards et al 2002, Ramstrom et al 2002, Ley et al 2002, Lepre et al 2002, Liu et al 2003, Edwards 2003 and Geysen et al 2003 . A wide variety of combinatorial libraries can be provided using the synthetic methods and techniques described in these reviews.

  One essential feature of such a combinatorial library is that it needs to be applied to at least one screening test. Otherwise, a mix of hundreds of thousands or millions of different candidates would be completely worthless. This is because no one can predict which compounds out of tens of millions and millions of candidate mixtures are useful for some purpose.

  Thus, any type of combinatorial library can be used to identify or manipulate candidate compounds in one or more useful ways (ie, specifics that are modified, isolated, or identified by reactions or screening methods). To identify candidate compounds having a kind of “handle”.

  A wide variety of such “handles” are known, and a combinatorial library using at least one, usually a plurality of such “handles”, using the manipulation of nerve fibers disclosed herein by various well-known methods. It can be applied to in vivo screening. For example, well-known classes of “handle” methods that can be used to process and adjust all items of combinatorial chemistry include:

  1. Minute beads, tubes or other solid surfaces, usually made from plastic, starch or similar compounds. These beads and other solid surfaces usually have a reactive group (on their surface) that acts as a substrate or “anchor” and serves as a binding site for the chemical chain. Added. If necessary, the microbeads can be made with a diameter that is well suited for phagocytosis by mammalian cells.

  2. A special reactive moiety that occurs only once for each candidate compound in a combinatorial library. Such unique reactive moieties can be used for binding, chemical reactions, or other manipulations that can be used to transport to the target location at any stage of the screening method or after its execution. Precipitation, enrichment, or other accumulation of specific compounds that participated in a particular chemical or cellular reaction of interest, or that acted differently from unsuccessful candidates during a screening test , Isolation, binding, labeling, or manipulation.

  3. A non-toxic fluorescent “label” or “tag” that emits light of another wavelength when excited by light of one wavelength. Thus, cells or particles having fluorescent activity can be separated using a device called “flow cytometer” (also called a cell sorter). In a typical flow cytometer with a sorting function, millions of cells or particles can pass through a thin tube at a known or controlled rate at a time. At one point in this path, each cell or particle passes through the excitation wavelength light. Each cell or particle that contains or is bound to a fluorescent label or tag responds by emitting light of a different wavelength. This fluorescence generated in nanoseconds is detected by an optical sensor matched to the fluorescence wavelength. When the fluorescence sensor detects fluorescence emitted by a cell or particle, it triggers a small amount of gas or liquid injection at a position slightly downstream of the cell or particle flow path. This jet of gas or liquid is aligned with the point in time when the fluorescent cells or particles pass through a junction in the path. Fluorescent cells or particles are pushed to one side in the flow path by jetting gas or liquid, enter the separation collection tube, and are carried to the collection container. In this way, the flow cytometer can process millions of cells or particles for hours or minutes and even isolate single fluorescent cells or particles from among millions of populations. .

  4). Other types of signs or tags. For example, radioisotopes or compounds containing special molecular structures that can be localized and tracked by modern analytical methods such as magnetic resonance imaging, Raman scattering.

  5). When a class of candidate compounds includes or is associated with a polypeptide, a phage display library is a very powerful, flexible, and compatible “handle” for processing such polypeptides. Provide the system. Once a single phage particle with a highly effective and potentially useful ligand polypeptide is isolated, the single phage particle can be rapidly propagated into a complete colony strain. Using the methods described herein and methods well known to those skilled in the art, an unlimited number of polypeptides and genes encoding the polypeptides can be supplied.

  In fact, the PhD-C7C phage display library provides an example of a combinatorial method adapted for use with polypeptides. In this library, an essentially random 7 amino acid short polypeptide was made by combinatorial chemistry. These randomly generated short polypeptide sequences were incorporated into phage particles. These phage particles provide a “handle” that can be used to manipulate, propagate and sort combinatorial taxon polypeptides in the PhD-C7C library.

  As described above, a combinatorial library must be made to accept at least one system or mechanism that allows researchers to process and manipulate candidate compounds in the library. Otherwise, it would be meaningless to make such a library if it cannot be sorted out by an effective and logical method. Therefore, the extensive and diverse methods developed in the last 20 years for screening combinatorial libraries are very sophisticated and powerful. Accordingly, the in vivo screening methods disclosed herein are merely considered to provide one newer (and potentially powerful and useful) method for screening candidate compounds made by combinatorial chemistry. be able to.

Extending in vivo screening to other receptors, other species, and other neuronal cells The invention developed and tested with can be extended and expanded in several directions that are of interest to researchers, doctors and others.

  As an example, the present invention can be expanded to other than sciatic motor neurons by methods apparent to researchers in the field of neuroanatomy and neurotracking studies, and (i) after the phage library is injected into the anterior chamber, Along with sympathetic neurites extending from the cervical ganglion and sensory neurites extending from sensory nerves of the trigeminal ganglion; (ii) olfactory receptor sensory neurons (collecting olfactory bulb tissue), trigeminal nerve after intranasal administration of the test library With ganglion sensory nerves and sympathetic nerves of the superior cervical ganglion; (iii) with posterior chamber injection followed by sensory neurons in the retinal ganglion; and (iv) injection into the lateral ventricle and other ventricles Can then be used with various neurons of the central nervous system. By such a method, the endocytic receptor naturally expressed by the specific neuron group described above is targeted for use in diagnosis and treatment of a disease in which the neuron of the specific taxonomic group is involved. Ligands can be identified and isolated.

  Similarly, the present invention, originally developed and tested with rat p75 receptor, was expanded and expanded by methods apparent to cell engineering and molecular biologists to: (i) With cytosis receptors; (ii) with endocytosis surface molecules other than receptors; (iii) with endocytosis receptors present in species other than rats, such as human receptors; and (iv) other than neurons Can be used with endocytosis receptors present in certain types of cells and tissues (eg, receptors that are usually present in significant numbers only on the surface of cells such as kidney, liver, lung, heart, etc.).

  This type of research has been done in many cases. This is because once a human gene sequence encoding a specific type of human receptor protein is known, the human gene sequence (or part thereof) can be used to transform different types of animals such as mice and rats. Because. The genetically transformed animal expresses a human receptor protein having the exact same sequence as the human amino acid sequence. By way of example only, mice were transformed with human receptor proteins that can infect certain motor neurons in humans and other primates with poliovirus. Therefore, this transformed mouse could be used as a cheap animal model for polio and poliovirus studies.

  Similarly, it is well known and available in the art as an example showing how this type of genetic engineering can be adapted to perform the in vivo screening disclosed herein. Using the appropriate DNA sequences, reagents and methods, one of ordinary skill in the art can perform the following series of steps.

  1. The fully sequenced human homologue of the p75 gene (Johnson et al 1986) can be incorporated as a chimeric gene under the control of the p75 gene promoter normally found in mice or rats;

2. This p75 gene chimera (mouse or rat promoter / human coding sequence) is used to genetically characterize a selected type of mouse or rat, for example, a mouse strain having a knockout mutation that appropriately suppresses expression of the p75 receptor. Such a family can be obtained from Jackson Laboratories (www.jax.org or www.jaxmice.jax.org) as stock number 002213 (family name B6.12954-Ngfr ^tm1Jae );

  3. This transformed mouse or rat expresses the human p75 receptor protein, which is the same as the rat p75 receptor protein normally exists, such as the sciatic nerve fibers that extend outside the blood brain barrier. Appear on the surface;

  4). A ligand library (eg, scFv or PhD-C7C phage display library) is then screened by the same method disclosed herein, via binding of the phage particle ligand domain to the human p75 receptor. Identifying candidate phage that are taken up by endocytosis and transported retrogradely;

  5). Alternatively or additionally, a ligand found to enter the cell via the rat p75 receptor protein was tested in vitro to determine if the ligand had a human p75 cell receptor (eg, suspension culture). It can be determined whether it can easily enter into a human neuroblastoma cell line that grows easily and expresses native human p75;

  6). Alternatively or additionally, the efficiency of endocytosis of a ligand that easily enters the rat nerve fiber via the rat p75 receptor is not very high, but quite high, and the ligand interacts with the human p75 receptor Starting with the ligand, (i) making various analogs of rat p75 binding ligand using site-directed or random mutagenesis, and (ii) using human in vivo screening Promising analogs that can easily enter cells via receptors can be identified and evaluated.

  These are the ways in which the in vivo screening methods disclosed herein can be used within human cells for use in human cells for the treatment, diagnosis, analysis and research. Only a few examples show whether ligands that can efficiently transport molecules can be adapted for use in discovering, isolating and analyzing.

Molecular Complexes and Methods Realized by the Present Invention The real value of the screening methods disclosed herein is not the act of identifying specific ligands that can activate and perform endocytosis uptake, , Derived from the ability to use such selected ligands incorporated into "molecular complexes" that can be used for therapeutic, diagnostic and similar purposes.

  As used herein, the term “molecular complex” refers to a molecular assembly comprising at least two different components, namely at least one ligand component and at least one transport or transport component.

  In order to be included within the claims referring to the ligand or a molecular complex comprising the ligand, the ligand component must meet the following two criteria:

  First, the ligand component needs to have been identified by the in vivo sorting method disclosed herein (ie, to be protected by claims such as claim 1, the ligand component is In order to make a proper selection, it must be identified by an in vivo selection method that requires at least endocytosis uptake into nerve fibers). This type of identification is an essential step in the screening method of the present invention and in the formation of molecular complexes with the ligands actually identified by this method. To illustrate this fact, phage libraries containing billions of candidate ligand polypeptides (such as the scFv library screened using Examples 28 and 29) are expressed in cells with p75 receptors on the surface. Hundreds, thousands of actually having candidate ligand sequences that can fully function as effective, specific, and effective endocytotic ligands that can be used to transport shipment components Or it can be estimated to actually contain millions of phage particles. However, in such a large library, hundreds, thousands, or millions of theoretically competent phage particles are completely useless for that purpose and are bound to the transport molecules. Surrounded by billions of other phage particles that would cause all undesirable reactions when injected into an animal or human being in need of treatment.

  Clearly, the screening and processing steps required to identify and isolate the phage with a ligand sequence having a known useful endocytotic activity will result in the desired useful therapeutic, analytical or similar results. Is an absolutely important step in the creation of molecular complexes that can actually be achieved.

  The second condition applied to the ligand component used to transport the transport or transport component in the molecular complex as described herein or in the claims is the life conditions disclosed herein. The molecular complex containing the ligand component first identified by the in vivo screening method must actually be able to enter the target type or taxonomic cell with the endocytic surface molecule to which the ligand binds. is there. If such a molecular complex is unable to enter the cells of at least one of the above taxonomic groups, the molecular complex is not protected by the claims and is not intended to be protected.

  Clearly, however, once a specific ligand has been identified that can enter the cell via a specific and targeted type of endocytotic surface molecule (and if that specific ligand is a polypeptide, then Once the amino acid sequence is known, the specific ligand can be synthesized in the desired amount and used as an endocytotic transport system to target a wide range of useful “cargo” or “cargo” molecules Can be carried into cells. Thus, after such a ligand component has been identified by the novel and powerful in vivo screening methods disclosed herein, when such a ligand is used as an endocytosis transport component, it is referred to as “transport” or “transport”. In molecular complexes that also include “product” molecules, they are not limited to a particular type or taxon of transport or transport molecules.

  As used herein, the terms “passenger molecule” and “payload molecule” are interchangeable and the transport molecule has a target having an endocytotic surface molecule to which a ligand component binds. A part in a complex molecule (including the above-mentioned ligand) that exerts one or more useful functions or effects after entering the cell. The term is to be construed broadly, and in general, a shipment or package molecule should be construed in recognition of how the term is used in other modes of transport. I must. Cars, buses, trains, aircraft, and bicycles are all useful because they can carry passengers at speeds and distances that are not easily achieved by other means of transportation. Similarly, freight trains, trailer cars, tank cars and ships are useful because they can carry freight (which can be considered paid to be transported to a destination). Cars, buses, trains, aircraft, bicycles and ships are very useful and useful modes of transportation, not just because one particular person can be transported to one particular place, but many different This is because people or cargo can be transported to a number of selected destinations.

Accordingly, the carriage or carriage described and considered herein should be construed broadly and includes, but is not limited to, the following major classifications:
(1) A DNA segment that is part of a gene vector for genetically transforming an animal or medically treating a human in need of gene therapy. In fact, one of the primary goals of this study is to specifically target only certain types of cells without destroying the state or activity of other types of cells so as to greatly increase the risk and extent of undesirable side effects. To identify and create cell targeting components that can be used to create new types of genetic vectors that can be transformed as targets;
(2) therapeutic and / or diagnostic compounds (eg pharmaceuticals, imaging compounds, etc.) for use in human or veterinary medicine;
(3) Analytical compounds, reagents, and other substances that are more useful in industrial research and development if they can be efficiently transported to target cell types.

  Finally, it should also be noted that a molecular complex comprising both a ligand component and a transport or transport component has some effective means of binding and uniting the two components, and at least the transport or It is necessary to form a united complex until the transport component is indeed taken up into the target cell. In some cases, depending on the transport or transport component, either by direct covalent bonding or by “coordinating” bonding (this term is a class of species having intermediate strength and / or stability between covalent and ionic bonds. Refers to a molecular bond) that allows the transport or transport component to be linked directly to the ligand component. However, this type of molecular complex would not be suitable for at least some end uses if the transport component was directly linked to the ligand component. Because, after the molecular complex has entered the target cell, the transport component often needs to be released from the ligand component, so the transport component cannot perform its intended function if the ligand component remains linked. is there. Similarly, this compares to the fact that cars, trucks or buses are substantially more useful because they have doors that can take passengers down once they arrive at their destination.

  Thus, most types of molecular complexes having a ligand component and a transport component disclosed herein should further include an appropriate “linking component”, which has two different requirements: Linking the ligand component and the transport component by a suitable means to adjust: (i) The link means, the ligand component performs its function and allows the transport component to enter the cell, while the molecular complex And (ii) in many cases, the desired effect is present while the transport component is linked to the ligand component. If unsuccessful, the linking means must have a type of structure or mechanism that allows the transport component to eventually be released or separated from the molecular complex after the molecular complex has indeed entered the cell. A.

  This patent application is not suitable to comprehensively describe candidate linking components that can achieve and / or tune the above two antagonistic requirements. A wide variety of linking component candidates are well known to those skilled in the art of drug delivery, and therefore, after the ligand component, the transport component and the target cell type are all known in full detail, Any candidate linking component can be evaluated for use in a molecular complex.

  To outline and provide a practical introduction to the range of options available when designing specific types of molecular complexes to optimally combine known ligands, known transport components, and known target cell types The following briefly describes some of the wide variety of connected component types.

  1. A cross-linking agent that forms a covalent bond with a relatively non-specific reactive group, such as glutaraldehyde, and a compound having two aldehydes or other non-specific reactive groups at both ends of a spacer chain of adjustable length.

  2. A cross-linking agent that forms a covalent bond only through specific molecular groups. For example, “sulfo-SMCC” described in Example 24, which is used to crosslink the ends of DNA strands to lysine residues of polypeptides for uses such as gene vectors and affinity purification. .

  3. Affinity binders with very high levels of adhesion and affinity (such as occurs between two polypeptides, biotin and avidin as described in Example 27), and lower but substantially desired Affinity binders that actually have a certain level of adhesion and affinity (which can be adjusted by various means such as adjusting the elution conditions in the affinity purification method).

  4. A binder that uses ionic attraction and / or hydrogen bonding to bind the two components. Since ionic attraction and hydrogen bonding are not particularly strong, typically this type of binder is a compound with multiple ionic charges, all of the same polarity, gathered together in a fairly narrow range. For example, polylysine, polyethyleneimine, and other positively charged compounds that attract and bind to negatively charged phosphate groups in the backbone of DNA and RNA strands.

  5). A special type of connecting molecule designed to weaken and break when a molecular complex is exposed to acid (for example, it occurs in lysosomes, the acidic digestive tract in cells). Such types of connecting molecules are described in US Pat. Nos. 4,631,190 (Shen et al 1986) and 5,144,011 (Shen et al 1992).

  The above is only a brief overview and other types of connecting molecules are also known to those skilled in the art. However, it is clear that a variety of well-known options with a wide variety of strengths, stability and other properties can be used to link the package component to the ligand component.

Summary of Examples 22-32 (in vivo screening method)
The methods described in Examples 22-31 are complex, and those tests use specific types of phage and cells as reagents, and some test methods are based on previous methods that Applicants had not succeeded. It is selected after analysis and finally solved. Therefore, this section gives an overview and brief description of these examples and the structure of the information obtained.

  Example 22 describes the three bacteriophages used. These are (1) M13KO7 helper phages that do not possess endocytic ligands; (2) phage display libraries called scFv libraries with about 13 billion phagemids, where each phagemid usually appears in human antibodies A candidate phage; and (3) a phage display library referred to as a PhD-C7C library carrying a small foreign polypeptide sequence with 7 amino acid residues that are randomly sequenced by “combinatorial chemistry” .

  Example 23 describes the host cells used (mainly E. coli TG1 strain) and some techniques used to amplify their specific phage group in various phage groups and determine their titer.

M13KO7 helper phage does not carry a ligand polypeptide but is first described in Example 22 because it is used to make antibody-phage conjugates. These conjugates are prepared to display multiple MC192 monoclonal antibodies known to be taken up by the rat p75 receptor, crosslinked to the surface of this helper phage.
These antibody-phage conjugates were first tried and shown to be taken up by sciatic nerve fibers. Thus, using a set of tools established from these (comparable to probe drugs), Applicants can screen endocytosis in vivo by manipulating sciatic nerve fibers in rats. I was able to come up with the concept and details of an effective method that would be possible. Example 24 describes a method for cross-linking antibodies to helper phage. Examples 25 and 26 describe methods that ultimately achieved endocytosis and retrograde transport of antibody-phage conjugates in the sciatic nerve. Example 27 describes methods and reagents for taking photographs demonstrating endocytosis and retrograde transport of antibody-phage conjugates, as shown in FIG.

  After developing a set of consistent methods for reliable uptake of phage particles via p75 using antibody-phage conjugates, Applicants have tested a phage display library called the scFv library. Started. Example 22 describes the main properties of this library. Briefly stated, this library has a vast number (approximately 13 billion) of foreign gene inserts originally obtained from human B cells. These gene inserts encode a “variable fragment” of a wide range of human antibodies from humans of various ancestry. Consistent results were not obtained from the initial in vivo screening tests performed with this library. Accordingly, the Applicant has addressed these issues and finally solved it by a method of pre-screening the scFv library in vitro by a technique referred to as “Biopanning” described in Example 28. In short, this prescreen uses a p75 polypeptide immobilized on a hard plastic surface. By selecting phage particles that bind to these immobilized p75 polypeptides by this step and using them for subsequent in vivo screening, more consistent results were obtained that could be interpreted. Example 29 describes these methods and the results obtained.

  Subsequently, Applicants tested in vivo screening methods on another phage display library called the PhD-C7C library. As summarized in Example 22, this library has a short polypeptide fragment (7 amino acid residues), created by combinatorial chemistry, randomly inserted into the phage pIII coat protein. Example 30 describes these tests and results.

  As a result of all these screening tests, in-vivo screening of phage libraries to select specific phages that are taken up and transported by nerve fibers can be performed from a large display library into the nerve fibers. It is confirmed that this is a practical and efficient method for identifying and isolating specific phages that accidentally carry a ligand component that can activate and carry out the cytosis process.

  Together, these results also confirm that a stochastic process is involved that depends on the probability and the size of the population tested by a highly specialized set of tests. The claim and claim of the present invention is that this type of selection method is not successful in every screening test. Rather, it is claimed and claimed in the present invention that this selection method is used to perform in vivo testing on live animals in ways that were previously unknown or impossible. Identify, select, from potentially large and / or random starting libraries or repertoires, a small number of display phage clones that certainly penetrate into nerve fibers and are retrogradely transported (if used repeatedly) And the fact that it can be isolated.

  Ligand molecules and ligands that can also perform in vivo screening methods according to the method from titration measurements and photographic data and activate and execute endocytosis steps even when bound to macromolecular complexes It clearly proves that fragments can be identified efficiently. Based on these results, this type of in vivo screening method allows researchers to identify such molecules (referred to as endocytotic ligands) and to transfer them as “transport” or “transport” components to cells. It can be used as part of a molecular transport system (also referred to as a carrier) for transporting into the interior. As such a transport component, if it can be efficiently transported into a cell, for example, a DNA fragment that is part of a gene vector, therapeutic, diagnostic, or other medical effects can be provided. Possible drugs or diagnostic molecules, as well as analytical compounds that are more useful in industrial research and similar efforts.

  In addition, as described and submitted in this patent application, neither the nucleotide gene sequence encoding a polypeptide ligand that functions well in in vivo screening tests nor the amino acid sequence of such a polypeptide ligand is known. Applicant's laboratory (Australia) does not have equipment to determine nucleotide or amino acid sequence information. Therefore, we sent several selected phages that function well in in vivo screening tests to an external contract laboratory where such data can be determined. However, the result data has not yet been received, and such data is not known as of the filing date of this patent.

Use of genetic vectors for drug administration / delivery into the BBB Transfection of one or more types of neurons across the BBB into or on tissues that are not protected by the BBB using the present invention One or more copies of the gene vector can be administered. Clearly, the preferred and possible method of administration depends on the type of neuronal cell that spans the BBB that is transfected with the vector, and the type and location of the peripheral projection that the vector contacts. As described above and in the examples, administration to olfactory receptor neurons can be by intranasal instillation; administration to nociceptive neurons can be by intradermal, subcutaneous, or intramuscular, and local (This may involve one or more agents or techniques that enhance epidermal passage and tissue penetration, as described above); administration to lower motor nerves innervating skeletal muscle Administration can be performed by intramuscular injection; and administration to the lower motor nerve of the hypoglossal nucleus can be performed by injection into the muscle of the tongue.

  It will be apparent to those skilled in the medical or neuroscience field that alternative methods exist for administering gene vectors to peripherally projecting neurons. It is also apparent to the expert that (i) other types of neurons spanning the BBB, including various types of sensory and motor neurons, and presyndromic neurons of the sympathetic and parasympathetic nervous system ) Is a potentially useful transfection target; and (ii) from the well-known anatomical and structural characteristics and properties of such types of neurons, neurologists or researchers Various methods for administration to the nerve cells can be developed.

Regulation of endocrine and paracrine hormone systems It is also clear that the present invention is a novel method that has not previously been used, in which a polypeptide encoded by a specific gene is not invasive to cells, systems and regions in the brain. Delivery, for example, protected brain tissue, such as the CNS (such as the pituitary gland and pineal gland), or other parts of the body (such as the thyroid, thymus, adrenal glands, other secretory glands, or pancreas, Provide methods to modulate various “downstream” effects or activities by increasing or inhibiting the release of various endocrine and / or paracrine hormones from various glands or organs, such as in any reproductive organ can do.

  This patent application is not a good place to analyze endocrine or paracrine in detail, and the following description is merely a very brief introduction and overview. More information on the endocrine and paracrine systems is provided as a suitable summary in most good physiology textbooks, and a number of complete textbooks such as Wilson & Foster 1992, Barrow & Sleman 1992, Brown 1993 , DeGroot 1994, etc. Recent review articles are not particularly useful for satisfying working knowledge of the endocrine or paracrine system. Because their focus is mainly on problems (eg glandular tumors, hormone destroyers such as insecticides), treatment (surgery or drugs), or hormonal systems and other systems such as immune response systems This is because it is an action. However, review articles are good if you go beyond working knowledge of the endocrine or paracrine system and aim for potential treatment for purposes such as human disease or congenital treatment, or livestock breeding Provide basic information.

One important component of the endocrine system is the pituitary gland, which lies at the base of the brain and hangs from a region of brain tissue called the hypothalamus. The anterior pituitary gland is known to release at least six hormones, and the release of each of these hormones is stimulated or suppressed by “upstream” hormones called hypothalamic hormones. These hormonal systems (ie pairing, association) are as follows:
1. Hypothalamic hormone, called thyrotropin releasing hormone (abbreviated as TRH; formally thyroid stimulating hormone releasing hormone), releases a hormone called thyrotropin (formally called thyrotropin, TSH) from the pituitary gland;
2. Hypothalamic hormone, called corticotropin releasing hormone (CRH), releases adrenocorticotropin from the pituitary gland;
3. Hypothalamic hormone, called growth hormone releasing hormone (GHRH), releases growth hormone (also called somatotropin, often referred to as hGH or HGH in the case of human growth hormone) from the pituitary gland;
4). Hypothalamic hormone called growth hormone inhibitory hormone (GHIH, also somatostatin) suppresses the release of growth hormone (somatotropin) from the pituitary gland;
5. Hypothalamic hormones called gonadotropin-releasing hormone (GRH or GnRH) release two “gonadotropic” hormones called luteinizing hormone and follicle-stimulating hormone from the pituitary gland;
6). Hypothalamic hormone, called prolactin-inhibiting hormone (PIH), suppresses the release of a hormone called prolactin from the pituitary gland.

  In brain tissue protected by the BBB, by administering a gene vector that increases the concentration of one of the above-mentioned hypothalamic hormones (transient rather than permanent by the methods disclosed herein) And transient increases are usually considered favorable for the treatment of human medical or developmental diseases), “downstream” or “dependent” in a regulatable manner “It is likely to be able to stimulate or inhibit the release of pituitary hormones. Thus, the stimulation or suppression of the pituitary gland in this way by the gene vector and / or the targeted result stimulates, suppresses or suppresses the same type of physiological effect caused by the release or suppression of pituitary hormones, or It is possible to adjust.

  Alternatively, stimulating or suppressing the targeted endocrine or paracrine system by administering a gene vector that directly encodes a pituitary hormone, rather than an “upstream” hypothalamic hormone, may be at least some It would be possible in the system. Along this direction, studies of radiolabeled tracers have shown that at least some types of proteins delivered into the CNS by direct injection into the ventricles are eliminated fairly rapidly from the CNS into the blood circulation. Indicated (eg Ferguson 1991). Thus, if the rate of delivering hormone-type polypeptides into CNS tissues protected by BBB is high enough, some of these hormone polypeptide molecules diffuse into the circulating blood and systemically Distributed.

  With respect to delivering hormones and other polypeptides into the brain to contact specific regions, cells or structures in the brain, the present invention is at least in some cases in a manner not previously available. A method of delivering to a target can be provided. Most prior art methods of delivering neuroactive molecules (eg, neurotrophic factor) into the CNS assume that an endocrine model of drug delivery is suitable as a delivery method for the molecule. Evidence for this premise is widespread in numerous animal experiments and limited human clinical studies in which recombinant neurotrophic factor is injected or infused into the cerebrospinal fluid (typically in the lateral ventricle). These drug administration methods are based on the premise that the flow of cerebrospinal fluid in the brain and spinal cord is the circulation inside the CNS as well as the blood circulation in the periphery.

  However, this assumption is not guaranteed. This is because the flow of CSF within the CNS is more similar to the peripheral lymphatic system than the blood circulation. Like lymph flow, CSF flow is closer to unidirectional drainage rather than fluid recirculation.

  Thus, if a neuroactive molecule (eg, neurotrophic factor) exerts a physiological effect rather than endocrine (systemic) rather than paracrine (local), a suitable drug delivery method is preferably, for example, It should not be by systemic administration such as intravenous infusion or injection. Because (i) a lot of very expensive drug compounds are wasted; and (ii) when systemically injected molecules react with various cells and organs other than the desired target, undesired adverse effects can occur Because there is sex. Instead, if available, systems that focus on a narrower range and systems that target the target should be used.

  The present invention provides a substantially improved drug delivery method that mimics paracrine delivery from several perspectives. This method involves transfecting only a selected population of neurons across the BBB and subsequently releasing the polypeptide into the desired limited “secretory zone”, thereby allowing the target cell Alternatively, limited and focused delivery of the drug to the target area can be performed or at least facilitated (especially when compared to other methods such as intravenous injection).

  To achieve continued drug delivery in the art, as demonstrated by many studies of transplanting cells that have been genetically engineered to secrete specific recombinant molecules, such as neurotrophic factors, into the CNS It is known that gene therapy can be used. However, most such studies explain or suggest that the goal is that the transplanted cells secrete the drug so that it is distributed systemically and achieve endocrine-like drug delivery. ing. In contrast, the present invention discloses a completely different form of gene therapy, and by transfecting a limited number of adjacent cells across the BBB, a specific population of neurons within the BBB and In certain types, therapeutic polypeptides can be delivered in a limited manner, such as paracrine.

Selective Modulation of the Nervous System The present invention allows the development of completely new methods for treating nervous system diseases. The disclosed paracrine-like drug delivery methods allow the development of new methods for selectively modulating specific nervous system functions within the CNS.

  Physiologically, the function of neuronal cell systems within the central nervous system can change in response to changes in the function of neurons that penetrate the BBB. For example, changes in electrical activity of nociceptive neurons that penetrate the BBB result in changes in electrical activity of secondary sensory neurons and related CNS neurons related to pain perception and response to pain . The anatomical connection between the neurons that project through the BBB and the neurons that are entirely within the BBB is not fixed and is not static. Instead, the nature, number, and distribution of these connections can change over time and in response to various events (which often results in downstream changes in other neuronal cell systems within the CNS) ). That is, neuronal cell systems within the CNS are plastic, changeable, and respond to changes in the function of neurons that penetrate the blood brain barrier.

  Based on such physiological facts, in at least some cases, the present invention alters innervation patterns due to synaptic connections between transfectable peripheral projecting neuronal cell lines and target neuronal cell lines in the CNS. Thus, the structure and function of at least some types of neural cells in the CNS can be selectively regulated and altered.

  As an example, in the art, neurotrophic factors are synaptic density and innervation patterns in the CNS system that respond to changes in electrical activity from other neurons located near the periphery, such as the number and frequency of nerve pulses. It is well known that it is involved in plastic changes. Physiologically, these neurotrophic factors act paracrine like in the above. Accordingly, the paracrine drug delivery method disclosed herein is used to selectively select the nature and extent of the connection between one or more peripherally projecting neurons and the CNS neurons or neural cell systems with which they interact. Can be adjusted to. In other words, by transfecting a neuron cell that spans the BBB with a gene vector, it is possible to modify the nature and degree of connection between the neuron that spans the BBB and the CNS neuron that is entirely present in the BBB. (At least in some cases).

For example, this principle can be illustrated by the following two preferred embodiments:
(i) being able to modulate a neuronal cell line protected by the BBB involved in pain perception by administering recombinant anti-NGF via transfection of nociceptive neurons across the BBB; and
(ii) By administering recombinant NT-3 or GDNF via transfection of spinal motor neurons across the BBB, the CNS neural cell system involved in voluntary control of motor function can be regulated .

Treatment of Neurological Diseases The present invention would be useful in the treatment of CNS neurodegenerative diseases (eg, Alzheimer's disease) by a method of delivering neurotrophic or neuroprotective factors to nerve cells at risk of degeneration. For example, Alzheimer's disease can probably be effectively treated by administering a neurotrophic factor (eg, nerve growth factor) into the CNS. In line with this objective, the present invention provides a relatively intensive and targeted delivery of neurotrophic factors to cholinergic neurons in the basal forebrain that are at risk of degeneration in Alzheimer's disease patients. it can. In at least some patients, this type of treatment could slow and potentially stop this neurodegeneration.

  The invention will also be useful in the treatment of CNS trauma or injury (eg head injury) by delivering neuronal growth factors (eg neurotrophic factor) to damaged and surviving neurons. Let's go. By way of example only, GDNF acting on cortical motor nerves (often damaged in cerebral infarctions and sometimes in head trauma) can be delivered to those neurons by the methods disclosed herein. Such potential effectiveness is described in many articles, such as Schacht et al. 1996.

  The present invention also relates to the treatment of learning or memory impairment cases that occur after various major operations, especially after surgery using a cardiopulmonary bypass device, in cases of learning or memory impairment, such as aging or dementia, after head trauma or injury. Will be useful in. Such trauma and damage often result in loss of function within one or more regions of the CNS. If possible, restoration of function includes compensatory changes in the composition of the CNS, for example, the sprouting and growth of affected neurons, and the formation of functional synapses with other neurons. Necessary and involved. This “neuroplastic” process has been shown in animal studies and human cases where the fingers and limbs have been severed. There, the victims of cerebral infarction were able to use the paralyzed limbs again after receiving treatment. In this treatment, the patient fixes one or two healthy limbs to the body with a bandage and repositions the non-functional limbs to widen it depending on the extent of movement and control remaining in the limbs after cerebral infarction. Moved and repeated exercises that actively worked to begin using.

  Neurotrophic factors can exert important functions in the process as described above (eg Lo 1995), and administration of NGF into the brain leads to various CNS activities and functions such as memory and learning. Have been shown to increase (eg Fischer et al. 1987 and 1991).

  Therefore, the present invention uses this improved delivery system to deliver this type of very useful therapy by delivering NGF and other neurotrophic and neurostimulatory factors into the CNS and into the brain. The law can be explained in more detail.

  Furthermore, the present invention relates to a disorder caused by excitotoxic damage to nerve cells, or ischemia (lowering blood flow as occurs during cerebral infarction or cardiac arrest), hypoxia (dripping, carbon monoxide poisoning). It may be useful in the treatment of disorders caused by diseases or injuries involving reduced oxygen supply) or head trauma. In animal experiments, NGF injection suppresses retrograde atrophy of cholinergic nerve cell bodies and fiber networks, as well as other changes in the cholinergic system induced by infarct and detected by infarct size and severity. Or could be recovered (eg Cuello et al 1992). Administration of NGF (or in vivo induction of NGF synthesis by Clenbuterol) has been shown to reduce infarct size in a rat model of permanent middle cerebral artery occlusion (Semkova et al 1999). Other animal data also suggest that NGF acts after brain injury to reduce the progression of nerve damage (eg Guegan et al 1998). Since the present invention would be useful for administering NGF after cerebral infarction or other brain injury, it would be possible to reduce the extent and severity of subsequent neurological deficits.

  The invention further relates to a method of treating sensory dysfunction by modulating the function of sensory neurons and / or neurons in the CNS that synapse with the cells. For example, persistent major pain has been linked to changes in nociceptive nerves and CNS (spinal cord or brain), and intrathecal administration of anti-NGF has been reported to recover and relieve pain. (Eg Christensen et al 1996 and 1997). Delivering polypeptides that act on one or more components of the sensory system (eg, antibodies that bind and inactivate NGF, antibodies that occupy and interfere with NGF receptors) and alter their physiological function according to the present invention The function of the sensory system can be adjusted.

  The present invention also expresses a neuropeptide gene, for example, a gene that expresses a polypeptide capable of blocking and suppressing pain (such as so-called “endorphin”); a gene that expresses a growth factor; a polypeptide that regenerates or prolongs the life of a cell A gene that expresses; and a gene that expresses a toxic polypeptide, eg, a toxic polypeptide that kills tumor cells, can be used to modulate neurophysiology.

  In addition, the present invention provides methods that can be used to treat various CNS-related neurological diseases or deficiencies that correlate with either a deficiency or an excess of some specific polypeptides. To accomplish this, the method can be used to deliver any of the following polypeptides into CNS tissue protected by BBB: (i) additional amounts to alleviate the deficiency Or (ii) inhibit or antagonize a particular molecule, receptor or response to help regulate a disease of the CNS that is induced, ie characterized by an excess of the particular molecule Or other inhibitory polypeptides.

The following embodiments are configured as follows.
Examples 1 to 8 transcribe olfactory receptor neurons using a neuron-stimulating polypeptide in a neuron completely located within the blood brain barrier and a vector having a gene encoding such a polypeptide. It is related to delivery by For illustration purposes, human NGF is used as an exemplary polypeptide. As recognized by those skilled in the art, other forms of NGF (mouse, other rodent or monkey NGF, or any mutated NGF, epitope-tagged NGF, which may be of pharmaceutical or research interest) Alternatively, a gene encoding a fragmented NGF or other form of NGF) can be used as an alternative.

  Examples 1-7 show how NFG (or other neurotrophic polypeptide or similar polypeptide) can be delivered to cholinergic neurons in the basal ganglia of experimental animals such as rats. Is described. Examples 1-4 include complete embodiments divided into various continuous processes. Example 1 describes the assembly of vectors; Example 2 describes the administration of these vectors into the sinuses; Example 3 describes a method for monitoring delivery of polypeptides through the blood brain barrier. Example 4 describes a method for measuring the physiological and behavioral effects of such treatment on experimental animals.

  Following its "from beginning to end" description, Examples 5-8 describe the delivery of genes encoding NGF or similar genes to olfactory receptor neurons using different types of vectors. Yes. Example 5 describes a herpesvirus-derived vector; Example 6 describes a liposomal vector; Example 7 describes a vector having a ligand that binds to an endocytic receptor on the surface of a neuronal cell. Example 8 describes a vector using a “transneuronal” polypeptide that can facilitate transport of the complete vector from one neuron to another.

  Examples 9-13 involve a different approach using gene vectors encoding neurosuppressive (not stimulating) factors, using as an example a polypeptide called “anti-NGF” that binds to and inactivates NGF. . These vectors are injected into skin or muscle sites to transfect pain sensation (pain signaling) neurons in areas that suffer from excessive pain signals that are not desired (often called neuropathic pain or allodynia) Is done. By suppressing excessively active pain signaling circuits, this approach can help reduce and control neuropathic pain.

  Examples 14-20 describe a third major approach strategy in which a gene vector carrying a gene encoding a neurostimulator is injected into muscle tissue that has been damaged due to stroke, spinal cord injury, and the like. Muscles with these types of disabilities often fail to properly function contact between upper motor neurons (completely located within the blood-brain barrier) and lower motor neurons (cross the blood-brain barrier) Suffers from a lack of spontaneous control (or disability) caused or exacerbated by Transfecting lower motor neurons in such impaired muscles using a gene encoding a neurostimulatory factor forms new additional connections between lower and upper motor neurons and / or Expand and repair damaged motor control networks by means such as increasing the level of synaptic activity between different neuronal classes, thereby reconstructing the correct innervation and central nervous system control for such muscle systems Can help you reconnect.

  Example 21 describes transfection of certain types of motor neurons located inside the tongue. This route of administration deserves special attention as it provides a route for delivering the polypeptide to some portion of the brainstem.

To deliver NGF to cholinergic neurons in the forebrain basal ganglia, construct an adenoviral vector to transfect olfactory receptors with the NGF gene. Replicate except in transgenic host cells that exist only in the laboratory. Methods for preparing non-pathogenic vectors derived from adenovirus that cannot be released have been published in articles such as Graham and Prevec 1995. NGF (or some other central nervous system such as GDNF, NT-3, CNTF, or BDNF) at a significant level inside the transfected human neurons, small enough to be carried and delivered by an adenoviral vector Methods for making genetic constructs that express active polypeptides (or any of a number of other polypeptides as listed in Table 1) are described by Romero et al 2000, Baumgartner and Shine 1998, Dijkhuizen et al 1997 And in articles such as Gravel et al 1997. Methods for propagating, purifying, concentrating and titrating adenoviral vectors carrying such gene constructs are described in the chapter by Engelhardt of “Methods in Molecular Medicine: Gene Therapy Protocols” (P. Robbins Publication 1997). (Pages 169-184).

  Genetic constructs that perform expression of NGF polypeptides (or other selected central nervous system active polypeptides) require precise selection of all gene-related sites. As used herein, terms such as “gene” or “gene construct” can usually be manipulated under laboratory conditions using known methods to transfect nerve cells across the blood brain barrier. (I) cause normal transcription and translation mechanisms within the neuronal cell body to synthesize the polypeptide encoded by the gene, and (ii) dendrites and processes located within the blood-brain barrier. ), A synapse, or other term that refers to a complete and functional transcription unit that can direct the cell to properly process and secrete the mature polypeptide from the end.

  Many of the mere “simple basic” types of genetic construct variants and enhancements are known to those skilled in the art, including the numerous variants and enhancements described in the “Detailed Description of the Invention”. is there. Any such variants or enhancements are included within the term “gene” or “gene construct” as used herein.

  The complete amino acids encoding the sequences of human NGF, mouse NGF, and other types of NGF (without introns) are publicly known and obtained from various researchers in the form of a simple engineered plasmid. can do. Alternatively, they can be made using known techniques or published information.

  Furthermore, if a polypeptide with a specific “epitope tag” sequence (such as c-myc) is desired, the coding portion of the gene construct must include a DNA sequence that encodes the “epitope tag” sequence. Inclusion of an epitope tag allows an investigator to distinguish between an exogenous polypeptide molecule encoded by a gene vector and an endogenous (natural) polypeptide native to the blood brain barrier of the test animal or patient. In situations where sequencing can help, the invention will be illustrated, refined, and otherwise enhanced.

  Another very important part of the gene construct is the gene promoter, and many known gene promoters that can carry out gene expression in olfactory receptor neurons are known and available. Specific examples of “selective” gene promoters known to perform specific gene expression in olfactory receptor neurons include the olfactory marker protein gene (Servenius et al 1994) and the M4 olfactory receptor gene Promoters identified in (Qasba and Reed 1998) are included. The use of such a selective promoter helps to minimize the chance that unwanted or uncontrollable expression of external genes occurs in the surrounding cells or tissues.

  Alternatively, if desired, various types of viruses and other promoters that are known to be unusually strong promoters in mammalian cells may contain NGF (or other central nervous system) in transfected cells. Active) polypeptides can be used for such purposes as involving expression at the most practical level. Examples of such strong promoters include the early gene promoter from cytomegalovirus and the late gene promoter from simian virus 40. If desired, an inducible gene promoter, so long as it can be administered in a manner that ensures that the inducer that activates the selected promoter is transported into a sufficient amount of transfected neurons. Can also be used.

  As noted above, to simplify the discussion herein, the term “gene promoter” as used herein includes the so-called “TATA box” and about between the TATA box and the actual transcription start point. Contains a 25 base sequence.

  Once the promoter and gene construct coding sequences are selected, various other gene construct portions may be used to enhance expression, packaging, transport, secretion, and / or performance of the vector encoded polypeptide. Can be selected. These gene parts include, for example, (i) a nerve that has been transfected to properly process and secrete the vector-encoded polypeptide (or “mature” form that can remove the leader or signal sequence). A DNA sequence that encodes a "leader" or "signal" peptide sequence that directs the cell; (ii) serves as a "tail" untranslated region that follows the "stop" codon in the mRNA to facilitate translation and release by the ribosome A DNA sequence that is transcribed into an mRNA sequence, and (iii) a transcription terminator sequence that directs RNA polymerase to cleave the mRNA strand when the DNA sequence is transcribed into mRNA. These types of functional sequences are known and can be obtained or adapted from almost any mammalian gene cloning vector that allows the cloned gene to be expressed at high levels by transfected mammalian cells. By replacing the prepro NGF sequence preceding the mature NGF sequence with the prepro BDNF sequence preceding the mature BDNF polypeptide, at least in some types of neurons, from sensory nerve cell terminals within the blood brain barrier Can facilitate antegrade transport and release.

Administration of adenoviral NGF vector to the olfactory epithelium to deliver NGF to cholinergic neurons in the forebrain basal ganglia . NGF (or Adenovirus-derived (or other virus-derived) vectors carrying gene constructs encoding several other central nervous system active polypeptides), human patients (for medical purposes) or test animals (test or other) Can be administered to any olfactory epithelium (for research purposes).

  The aqueous carrier solution must be compatible with adenovirus and active olfactory epithelium. Saline (buffer, if desired) can be used, and solutions containing hypotonic or hypertonic solutions or any other component capable of inducing higher levels of viral transfection are routine. Can be tested using experiments. Articles such as Holtmaat et al 1996 provide information on dosages and techniques for efficient administration of adenoviral vectors via the nasal nose in mice, and such methods are known to those skilled in the art. Can be adapted for use in larger rodents such as rats or rabbits or other mammals.

  If desired, (i) to increase the degree and / or retention period of contact between the fluid containing the gene vector and the olfactory neurons, and (ii) to increase the acceptability of the neurons to incorporate such gene vectors A process can be taken. This can be done, for example, by administering a nasal decongestant to a test animal (or human patient) several hours prior to gene vector administration, and immediately rinsing and / or washing the sinuses prior to vector administration. Swabing step with a dilute solution such as isopropyl alcohol or acetone that can help remove any aqueous solution (and possibly mucous, oily or other viscous coatings) ) Can be used. Olfactory neuronal receptivity can also be increased by using gentle mechanical friction using small wire loops, round spatula tips, or similar tools. In general, neuronal projections within the surface area stimulated to the point of mild inflammation tend to be more receptive to cellular uptake of external molecules than cells that are found to be dormant or resting.

  After sufficient time for gene expression (usually about 24 to 72 hours), sacrifice some test animals, remove the olfactory epithelium, olfactory bulb, and forebrain basal ganglia, and treat each tissue by type. By measuring the location and concentration of the polypeptide encoded by the vector in that type of tissue, the delivery efficiency of the gene vector can be assessed.

  If the polypeptide encoded in the vector is significantly different from the endogenous (probably rodent) polypeptide, the measurement and monitoring effort can be relatively simple. These types of analyzes (i) hybridize cellular mRNA with DNA probes complementary to vector mRNA sequences rather than endogenous mRNA sequences, using methods such as those described in articles such as Xian and Zhou 2000 (Ii) techniques using "polymerase chain reaction" (PCR) reagents and methods for detecting DNA or mRNA sequences from vectors, as described in articles such as Chie et al 2000, and ( iii) Methods such as immunostaining or similar methods using a monoclonal antibody that selectively binds to the polypeptide or epitope tag encoded by the vector used and does not bind to the endogenous polypeptide of the test species can be used. (These antibodies are commercially available and, if desired, can be prepared using methods disclosed in articles such as Conner 2000, Rush et al 2000, and Zhang et al 2000. Can also be made).

  If desired, the time-dependent level of exogenous mRNA and / or polypeptide expression by the transfected olfactory receptor can be measured by repeating one or more tests over a time range after gene vector administration. it can.

  If desired, a gene vector carrying a human (or epitope-tagged animal) polypeptide gene construct can be used to provide a control population of cells and animals for the purpose of data analysis. A control vector that can be administered to the olfactory epithelium on one side and carries several other genes (such as a marker gene encoding a polypeptide that is easily detected) is on the other side of the animal's sinus Can be administered to the olfactory epithelium. After sacrificing the animal, using histological and immunological examinations of the left and right olfactory receptors, olfactory bulb, and forebrain basal ganglia region, (i) polypeptide expression by transfected olfactory receptors And (ii) polypeptide transport and delivery by receptor cells across the blood brain barrier to other classes of neurons located within the blood brain barrier can be evaluated.

Monitoring NGF polypeptide delivery to cholinergic neurons in the forebrain basal ganglia Transfusing olfactory receptor neurons (or across the blood-brain barrier) completely transfected into a central nervous system region within the blood-brain barrier, and A test that sacrifices the amount of NGF polypeptide (or other central nervous system active polypeptide encoded in the vector) delivered via other types of sensory neurons transfected with the gene vector Histological and immunological analysis of cells and tissues from animals can be used to monitor specific types or areas of neurons of interest.

  After administration of a gene vector to olfactory receptor neurons or other types of sensory neurons, human NGF at any target location within the central nervous system (or other genes encoded by gene vectors administered to animals) Use well-established immunization methods such as immunohistochemistry and “enzyme-linked immunosorbent assay” (ELISA) tests to monitor and quantify the production, release and distribution of any central nervous system active polypeptide) Can do. When human NGF is a vector-encoded polypeptide, preparations of monoclonal and polyclonal antibodies that recognize and bind to human NGF rather than mouse, rat, or other rodent NGF are commercially available, Alternatively, it can be generated using methods well described in the literature (eg Conner 2000; Rush and Zhou 2000). Methods for measuring NGF levels using immunohistochemistry are described in articles such as Conner et al 1992, and methods for measuring NGF levels using ELISA are purchased from articles such as Zhang et al 2000 and commercial sources. It can be found in the manufacturing instructions included in the possible NGF-ELISA detection kit.

  As described briefly above, when using a monoclonal antibody that recognizes and binds to the (possibly human) polypeptide expressed by the vector, but not to the homologous mouse or rat polypeptide, the mouse or Monitoring of the vector-encoded human polypeptide in laboratory animals such as rats can be made simpler and more reliable. Hybridoma cell lines expressing monoclonal antibodies that selectively bind to human NGF rather than mouse or rat NGF have been created and such monoclonal antibody preparations are available. Similar hybridoma cell lines expressing monoclonal antibodies that selectively bind to other central nervous system active polypeptides, rather than rodents, can also be generated or constructed using methods known to those skilled in the art.

  Alternatively, or in addition, as described above, an epitope tag (such as c-myc) is incorporated into the sequence of a neurotrophic gene construct using methods described in articles such as Moller et al 1998 Immunological detection of the polypeptide expressed by the gene construct in the cell can be facilitated. Using such an approach, the polypeptide expressed by the vector construct can perform all the steps necessary to accurately deliver it to the neuronal cell targeted for treatment with the polypeptide. It is important to make sure that you can.

  For example, the purpose of certain methods is to deliver therapeutic polypeptides to the main cell body of forebrain basal ganglia cholinergic neurons, using “epitope tag” sequences to facilitate polypeptide monitoring In some cases, target forebrain basal ganglia cholinergic neurons take up a polypeptide encoded and epitope-tagged polypeptide (receptor-mediated endocytosis) secreted by neurons transcending the transfected blood-brain barrier. The target forebrain basal ganglia cholinergic neurons can be assessed to ensure that they have undergone retrograde transport. These types of confirmation tests have been performed on the epitope tag sequence (and / or natively together with the tag sequence) to demonstrate that the polypeptide encoded by the vector is taken up by the target forebrain cholinergic neuron. In articles such as Altar & Bakhit 1991, Ferguson et al 1991, DiStephano et al 1992, and von Bartheld 2000, using reagents such as monoclonal antibodies that specifically bind to a domain in a fusion polypeptide containing a portion of the amino acid sequence This can be done using the methods described.

  If the vector-derived NGF carries an antigen or epitope tag, monoclonal antibodies that selectively bind to the tagged polypeptide can be generated using known methods, followed by immunohistochemical or ELISA measurements. Any of these can be used. If desired, delivery of NGF or some other central nervous system active polypeptide (and subsequent neurological and physiological effects on tissues inside the blood-brain barrier) administration of gene vectors to olfactory receptor neurons Can be confirmed by removing these neurons in control animals by treatment with a compound such as a zinc sulfate solution as described by Horowitz et al 1991. .

  Based on transsynaptic tracking studies (eg Lafay et al 1991, Barnett et al 1993), polypeptides expressed by vectors such as NGF have sufficient time for gene expression in olfactory receptor neurons (approximately 24 to 72 hours). It is generally expected that the forebrain basal ganglia cholinergic receptor neurons can be detected after giving a time range of time). During this period, retrograde transport of the polypeptide (after expression of the polypeptide) to the synaptic terminals of receptor neurons in the olfactory glomerulus, secretion of the polypeptide in the olfactory glomerulus (from about 8 hours) 24 hours), and transport of the polypeptide to the forebrain basal cholinergic neurons (approximately 8 to 24 hours) is expected to be included.

  As discussed in Example 8, certain types of “transneuronal” vectors are also disclosed herein. The class of vectors transfects the recipient neuron with an external gene to provide a therapeutic external polypeptide to one or more classes of “downstream” neurons located entirely within the blood brain barrier. Would be able to be transferred by sensory neurons to other types of neurons inside the blood brain barrier.

  However, unless specific steps are taken to confer “transneuronal” transport between neurons (i) early or “primary” neurons (blood-brain barrier) with a gene vector as disclosed herein. Transfusion of neurons) does not result in the transport of the vector to other non-primary neurons within the blood-brain barrier, and (ii) a transient gene rather than a permanent gene transformation Expression may occur as a result. When sensory neurons, such as olfactory receptor neurons, are used as transfection targets, vector-mediated gene expression in such cells can be from days to weeks after expression of the highest peak or plateau level of the polypeptide. It is expected that it will decrease substantially and will stop completely. If desired, an additional supply of NGF can be delivered into the central nervous system tissue protected by the blood brain barrier by re-applying the same or similar gene vector to olfactory receptor neurons.

Monitoring the physiological effects of NGF delivery to cholinergic neurons in the forebrain basal ganglia Central nervous system in central nervous system tissues protected by the blood-brain barrier using the gene vector method described herein In order to allow and improve the analysis of the actual physiological, behavioral and other effects of delivering an active polypeptide, laboratory animals (or humans volunteering for clinical trials of this type of treatment) (i ) A test group that accepts a gene vector encoding NGF or some other selected neurotrophic or central nervous system active polypeptide; (ii) can be divided into two groups: a control group receiving placebo treatment . Placebo treatment is followed by the same treatment at the injection site (same treatment with decongestation, rinsing, swabbing, scratching, and / or other stimulation of the inner nasal wall) followed by (a) pure saline without the gene vector Nasal, or (b) for example, a harmless and / or non-functional gene, a nonsense DNA sequence that does not encode any polypeptide, or can be easily detected if expressed in mammalian cells, but significant Either the nasal solution of the solution containing the gene vector that may carry a marker gene encoding a polypeptide that has no physiological effect must be used.

  For animal studies, at least two categories of observable differences by introducing central nervous system active polypeptides (such as NGF or some other neurotrophic factor) through the blood brain barrier into protected brain tissue (I) Brain nerves that can be assessed by histological, immunological, or other biological analysis of brain tissue sites removed from sacrificed animals Effects on anatomy, and (ii) observable and measurable effects on animal behavior.

  Based on observable and measurable behavioral forms (such as the ability of a treated mouse or rat that remembers faced in previous attempts including mazes, water mazes, etc.) to occur within the central nervous system of laboratory animals A number of trials have been developed to evaluate what seems to be. In addition, other such tests are under development, and any such tests currently known or later discovered are those that are physiologically associated with polypeptides against target neurons that are located entirely within the blood brain barrier. It can be used under the condition that the evaluation of the effect is appropriate.

  Many of these trials involve surgical or drug interventions that cause some type of damage to the animal's central nervous system to model injury, seizures, neurodegenerative diseases, or other central nervous system disorders. It should be noted that this is accompanied. Subsequent trials will then seek to assess whether a treatment will help recover from the damage or disease that such animals suffered.

  An example of such a test is the “hippocampal forebrain arch injury model”, which is a commonly used model that “axonally cuts” the forebrain basal cholinergic neuron axons projected onto the hippocampus (ie, neuronal arch injury model). The major axons of the cells are surgically cut, which if not treated, typically causes atrophy and death of neurons in a period of about 2 weeks or less in most species). This induces atrophy and degeneration of forebrain basal ganglia cholinergic neurons (eg Hefti 1994). However, this is not a preferred injury model to evaluate the invention disclosed herein, because the forebrain cholinergic neurons that project to the hippocampal arch do not project to the olfactory bulb. Let ’s go.

  Instead, a preferred injury model for use herein must directly participate in the type of treatment disclosed herein or disrupt one of the affected neural pathways. For example, while surgically cutting the cortical projections of the broker's hindlimb neurons (projecting to both the olfactory bulb and cortex), to the olfactory bulb (so that the NGF retrograde axonal flow from the olfactory bulb is not blocked) Leaving this projection in place would provide a better form of trial that would enable researchers to evaluate the effectiveness of the treatments disclosed herein.

  An alternative approach to animal models that cut axonal pathways is to remove endogenous NGF from forebrain basal cholinergic neurons and thereby induce atrophy and degeneration of forebrain basal cholinergic neurons To remove certain appropriate target tissues. This approach can generally model the effects of excitotoxic injury associated with stroke. Published examples include Sofroniew et al 1993, which describes the removal of hippocampal tissue. This method can be modified to remove the olfactory cortical tissue that receives innervation from the broker hind limb (Wenk et al 1980) to induce atrophy of the forebrain cholinergic neurons in the broker hind limb. .

  Using the general method described in Sofroniew et al 1993, NGF delivery to forebrain basal ganglia cholinergic neuron body size (such as transfection of olfactory receptor neurons was disclosed herein). The effect of using a gene vector can be monitored. And / or can be monitored by assessing the level of one or more enzymes or other polypeptides that are directly affected by the presence or amount of NGF. Such an enzyme or polypeptide can thus be used as an indicator polypeptide (an example of a candidate enzyme that would be useful for such a measurement is choline acetyltransferase that is required for acetylcholine synthesis, ie ChAT). To test animals with selected areas of cortical central nervous system tissue that has been removed, axotomized, or otherwise attacked, the central nervous system (such as by intraventricular injection or infusion) Another neuro-anatomical effect that can be observed and measured when administered to is demonstrated by the ability of NGF administration to prevent or reduce the density reduction of ChAT immunoreactive fibers in the living cortex induced by injury (Eg Garofalo et al 1992). Thus, the effects of NGF delivery on the density of ChAT immunoreactive fibers in the living cortex after surgical or other interventions that remove or damage selected areas in one or more cortex (herein Methods described in articles such as Garofalo et al 1992 can be used.

  In addition to neuroanatomical changes, removal of the cortex reduces certain measurable behaviors, distinguishing normal and worsened behaviors for various tests that require learning and memory in test animals be able to. Examples include the “Morris Water Maze”, passive avoidance test, response test, and various other animal models as described in articles such as Garofalo and Cuello 1994. It has been shown that administration of exogenous NGF to central nervous system tissues (such as intraventricular injection or infusion) in such test animals can significantly reduce animal-induced behavioral deficits . Thus, such behavioral, memory and / or learning tests can be applied and used to monitor the effects of NGF delivery to the forebrain region via gene vector processing of neurons across the blood brain barrier.

  As measured by tests such as the Morris water maze (Fischer et al 1987 and 1991), aging also results in decreased memory and learning performance, but aging-related declines in memory and learning performance are, for example, It has been used to measure and quantify memory and learning losses that occur in Alzheimer's disease (both human patients with Alzheimer's disease and various animal models). It has been shown that administration of exogenous NGF to central nervous system tissues (such as intraventricular injection or infusion) can significantly reduce at least some types of aging-related declines in memory and learning Yes. Thus, the types of learning and memory tests used in such animal models can be used to monitor the effects of NGF delivery to the central nervous system by the methods and gene vectors disclosed herein (Fischer et al 1987 and 1991).

  The monitoring and measurement methods referred to in Examples 3 and 4 herein are not exhaustive or exclusive and may be supplemented by other monitoring and measurement methods known to those skilled in the art or later discovered. it can.

Construction and Use of Herpes Simplex Virus-Derived Vectors for Non-invasive Delivery of NGF The above examples illustrate the blood brain as a route and method for non-invasive delivery of NGF to central nervous system tissues protected by the blood brain barrier. The use and evaluation of adenovirus-derived vectors to transfect genes for olfactory receptor neurons across the barrier are described. However, it should be appreciated that adenoviral vectors are not the only type of gene vector that can be used for non-invasive delivery of polypeptides to central nervous system tissues protected by the blood brain barrier. Thus, Examples 5-8 describe the use of several other classes of gene vectors.

  To construct a genetic vector disclosed herein carrying one or more “transport” genes obtained from a herpes virus and encoding a central nervous system active polypeptide, Goins et al 1999 or Methods described in articles such as Federoff et al 1992 can be used. HSV-derived vectors are capable of transfecting a wide variety of human cells, including olfactory receptor neurons, expressing transport genes in neurons across the transfected blood-brain barrier, and such Secretion of the polypeptide to a significant level can be induced.

  Thus, HSV-derived vectors can be used in a format that can be directly compared to the use of the adenovirus-derived vectors described in Examples 1-4, if desired. The methods described in Examples 2-4 (supplemented where appropriate by other or alternative methods known to those skilled in the art) may be used for nasal and post-transcriptional monitoring and evaluation of HSV-derived vectors. it can.

Construction and use of lipid-based vectors for non-invasive delivery of NGF Methods for preparing DNA plasmid-lipid complexes designed for transfection of mammalian cells are described in Methods in Molecular Medicine: Gene Therapy Protocols. (P. Robbins Publication 1997) and is described in numerous publications such as the chapter by Nabel (pages 127-133). Various liposome preparation kits designed for use in gene transfer are commercially available or can be designed and made by skilled technicians using published methods. Preferred selection of lipid formulations and preparation parameters (including lipid and DNA concentrations and ratios in the preparation mixture, temperature, etc.) to be used with a specific size, type or concentration of plasmid DNA or other DNA preparation Determine the various prepared mixtures by routine testing, using the parameter ranges and confirmatory tests discussed in numerous articles and instructions on most commercially available kits. Can do.

  Administration of the lipid-based gene vector to the olfactory epithelium by the nasal nose of a lipid-gene complex suspended in saline solution or other carrier solution uses the same general method as described in Example 2. And can be adapted for such use by means known to those skilled in the art. The methods described in Examples 3 and 4 (as appropriate, supplemented by other or other methods known to those skilled in the art) can be used for post-transfection monitoring and evaluation.

Construction and Use of DNA Vectors Targeting Endocytosis Receptors; General Principles and Methods that can be Used to Prepare the Cyclic Ligand Selection Method “Receptor Targeted Gene Vectors” are described in “Methods in Molecular Medicine: Gene Therapy Protocols "(P. Robbins Publication 1997), in various publications such as the Findeis chapter (pages 135-152). As mentioned above, these vectors contain ligands that bind to certain receptors that are exposed on the surface of nerve cells.

  As mentioned in “Background”, many known types of neuronal surface receptors undergo a process called “endocytosis” after the ligand molecule binds to the receptor. As the name suggests, the endocytosis receptor is taken up inside the cell after the ligand molecule binds to it. Such activity can be demonstrated by testing with radiolabeled ligand.

  An example of an endocytic receptor is the “p75” receptor accessible on the surface of various sensory neurons, including olfactory receptor neurons. The receptor has been shown to be present in both humans and rats and is very useful in various studies. This is also known as the p75-NGFR receptor (where NGFR stands for "Neural Growth Factor Receptor") and is "up-regulated" in various neurons under crisis or stress conditions (Ie, increased expression of the mRNA encoding the receptor and an increased number of receptors appearing on the surface of the neuronal cell) has shown particular interest in the uses described herein. . Specifically, p75 expression was increased in rat motor neurons after peripheral nerve injury (Yan et al 1998), and p75 expression was also increased in motor neurons of human patients with amyotrophic lateral sclerosis (Seeburger et al. al 1993).

  Selection and / or production of an appropriate ligand that binds to an endocytic receptor on the target type of neuronal cell (ideally in the species to be tested or treated) is such effective receptor targeting. Gene vector construction is key. A variety of such ligands are already known, and other ligands can be made using methods such as those briefly summarized above.

  Since the endocytosis receptor is a protein, a complementary polypeptide that binds to any such receptor is usually produced by using the receptor-derived polypeptide sequence as an antigen during the production of monoclonal antibodies. Is possible. Using known techniques, the receptor-derived antigen sequence is injected into an animal such as a mouse, rat or rabbit, then the resulting antibody-producing cells are fused with an immortal cell line, and the resulting “hybridoma” cells are screened Thus, a clonal cell line secreting a monoclonal antibody that binds with high affinity to the receptor of interest is identified and isolated. Once the desired monoclonal antibody strain has been generated and identified, it is usually referred to by the variable binding portion of the monoclonal antibody (often referred to by the initial “scFv” fragment, etc., where “sc” refers to “single chain” “Fv” refers to a variable fragment comprising a binding domain), from which smaller domains or fragments can be isolated. The gene sequence encoding the binding fragment can then be incorporated into a plasmid or other vector and a limited amount of the receptor ligand fragment can be synthesized by fermentation of the host cell.

  That general approach can generate monoclonal antibodies that bind essentially to any known endocytic receptor on any sensory neuron that has one or more peripheral projections.

  Continuing with a specific example of the p75 receptor, a monoclonal antibody known as 192-IgG (described in Chandler et al 1984) has been shown to bind with high affinity to the rat p75 receptor. The 192-IgG monoclonal antibody (and various fragments derived from it) has been shown to undergo endocytosis and retrograde transport in peripherally projecting neurons expressing the p75 receptor (Yan et al. al 1988).

  Once a polypeptide sequence capable of functioning as a receptor binding ligand is identified, various reagents can be used to temporarily link the DNA moiety to the ligand polypeptide, thereby creating a polypeptide-DNA complex. it can. In one preferred method, a polypeptide having a “polylysine” domain consisting of a series of lysine residues (or other amino acid residues that are positively charged at physiological pH) can be linked to a ligand polypeptide. it can. Adding a series of lysine codons to a polypeptide-encoding gene used to express a polypeptide in a host cell, or chemically linking polylysine to a polypeptide using a bifunctional cross-linking reagent This can be done by some method. Because of chemical affinity (the polylysine strand has a positive charge and the DNA strand has a negative charge), the DNA portion is attached in a non-covalent manner to a polypeptide having a polylysine “tail”. When the ligand portion of the polypeptide binds to the endocytosis receptor, a process is initiated in which the entire polypeptide-polylysine-DNA complex (and also the receptor protein) is taken up into the cell. After the complex is taken up into the cell, the DNA molecule can be released and transported retrogradely within the cell.

  Alternatively, normal ligands for neurotrophin receptors (such as NGF binding to NGF receptors) can be used. Radiolabeled NGF has been shown to be receptor-mediated internalization and retrogradely transported in sensory neurons expressing a receptor called “trkA” (eg, Barbacid 1995). Thus, the NGF polypeptide itself can be used as a ligand for cells having that receptor. Note that the NGF polypeptide itself (and various other neuronal receptor ligands) has a net positive charge similar to that found in histones, a class of DNA binding proteins that bind to chromosomes. I must. Thus, it is not necessary, or minimal, to first add polylysine or other similar positively charged domains or conjugates to NGF or other positively charged polypeptides, and the polypeptide itself is a receptor target It could function as both a ligand and a DNA carrier.

  Various other known compounds can also be used as ligands that can bind to endocytic receptors on nerve cells that cross one or more blood brain barriers and / or gene vectors or certain portions thereof in nerve cells. It is a good candidate that can be evaluated for potential use as a peptide sequence that can promote retrograde transport or some other function after being incorporated. By way of example only, Wiley and Lappi 1993 (i) used a monoclonal antibody 192-IgG that binds to the p75 neuronal receptor, and (ii) a plant-derived toxin called saporin (which facilitates intracellular transport to the neuronal cell body. The conjugates formed by linking to ribosomes believed to inactivate them). According to Wiley and Lappi, this conjugate was reported to be useful in causing specific targeted nerve damage for research purposes. Therefore, it is considered herein a “probe” compound. Using methods known to those skilled in the art, a portion of DNA is (i) non-covalently linked to a `` probe '' compound, (ii) transported to the neuronal cell body with the aid of a probe compound, and (iii) Modify and adapt the above or similar probe compounds in a manner that allows them to be released from the probe compound to the cell body after the uptake and transport steps are complete, making them non-lethal to neurons It is possible to make it.

  A wide variety of neurotoxins are derived from spiders, bees, snakes, marine snails, and other poisonous organisms. One of the more common features of neurotoxin is that it binds (often with a very high level of binding affinity) to one or more types of receptors and / or ion channels on the surface of the neuroprojection. It is to be. This is one of the important mechanisms that wildlife uses to paralyze or inactivate prey, or to defend against attackers. In search of therapeutic agents that can be medically useful without causing the level of pain normally caused by biting or stinging a highly toxic animal, a significant number of such neurotoxins are present in neuronal receptors or ion channels. Modified by biochemical researchers to form analogs, conjugates, and other variants and derivatives that exhibit lower non-toxic levels of binding affinity.

  In addition, various toxins from pathogens (including tetanus toxin, cholera toxin, etc.) and various compounds from plants (including compounds belonging to the category known as lectins or agglutinins) are used in the uses described herein. To evaluate whether a gene vector becomes a selective ligand that binds to and helps transfect neurons that cross one or more types of blood brain barriers Can do.

  Thus, any such known or later discovered neurotoxins, microbial toxins, plant lectins, or one or more receptors, ion channels, proteins found on the surface of nerve cells that straddle the blood brain barrier , Sugar conjugates, or other functionally similar compounds that can selectively bind to other molecules, and analogs, conjugates, or other variants or derivatives of such neuronal cell binding compounds As a ligand capable of helping gene vectors to selectively bind and transfect neurons that cross the blood brain barrier of one or more types, the potential use disclosed herein Can be evaluated.

  As a third alternative approach, a “combinatorial chemistry” approach using a phage display library as described in “Preferred Embodiments” and illustrated in FIG. 7 can be used. Briefly, this approach uses a phage display library in which each phage expresses a single polypeptide fragment to produce an array of ligand polypeptide candidates. All arrays of phage are screened using a receptor-mediated endocytosis process performed by in vivo neurons such as rats with occlusion by ligatures located on nerve bundles such as the sciatic nerve. Subsequently, viable phage particles (which can infect host bacteria) are collected from the “distal” excised neuronal segment immediately adjacent to the location of the ligature. The collected phage particles are then inoculated into E. coli cells to grow the next generation of phage. This new generation of phage contains the specific phage that was present in the neuronal cell at a position next to the ligature. The specific phage selected by this screening method in terms of the method of inoculating the laboratory animal with the phage and subsequently recovering from the treated laboratory animal, as described in more detail in “Background” A ligand polypeptide sequence that (i) incorporates the specific phage into the peripheral projection of the neuronal cell by an endocytic process, and (ii) retrogradely transports within the neuronal axon to the site next to the ligature. Probably expressed.

  By repeating such screening and selection methods several times (and by causing random or targeted mutagenesis in high performance phage strains, if desired), binding to the endocytosis receptor Clonal phage strains can be identified that have an inserted gene that encodes a polypeptide sequence that is highly effective as a ligand causing endocytosis. Those phages are analyzed to determine the gene and amino acid sequences of the ligands, which are then used in creating highly effective receptor-targeted transfection vectors.

  If desired, such cyclic screening methods can be performed by the use of various tests, such as a "fluorescence activated cell sorter" (FACS), which can be performed using a known machine commonly referred to as "flow cytometry". Can be further improved. As an example of how this method can be used, plasmid, phage, or “phagemid” DNA can be fluorescently labeled with a compound such as rhodamine. Cells grown in tissue culture known to have the p75 receptor on the surface are incubated with various polypeptide-DNA complexes and then the cells are passed through a cell sorter equipped with a fluorescence detector. . If the fluorescence emitted from a particular cell is relatively strong, this indicates that the cell has acquired a large amount of labeled polypeptide-DNA complex, which is caused by a very short synchronized gas or liquid jet. The liquid stream containing the specific cells is divided into separate collection devices. In this way, cells containing large amounts of “incorporated” DNA can be isolated quickly and easily using a high-speed automated device, followed by high levels of polypeptide-DNA complex intracellular uptake. The resulting polypeptide can be reproduced, analyzed, or processed to evaluate or replicate. This is just one example of how automated analysis and manipulation can be used to simplify and improve the invention, and other methods will be apparent to those skilled in the art.

  At present, another alternative DNA-protein complex can be prepared by ligating a DNA segment to a known capsid protein from adenovirus (or from various other viruses that act similarly). . This adenovirus capsid protein is known to help facilitate efficient release of vector-bearing DNA from endocytic vesicles after the complex enters the target cell. This approach is described in the chapter by Curiel (pages 25-40) of “Methods in Molecular Medicine: Gene Therapy Protocols” (P. Robbins Publication 1997). If desired, the adenovirus capsid protein sequence and the receptor binding ligand sequence can be combined and integrated in the fusion protein.

  The same general method as described in Examples 2 and 6 can be used for the nasal nose of such receptor-targeted transfection vectors. These general methods can be adapted by means known to those skilled in the art, particularly for use with receptor-targeting ligand vectors. The methods described in Examples 3 and 4 and other methods known to those skilled in the art can be used for post-transfection monitoring and evaluation.

Construction and use of transneuronal vectors to deliver NGF genes to secondary neurons in the brain In this example, foreign genes (expressed by foreign genes) in neurons that are completely located within the blood brain barrier. A method for constructing a “transneuronal” gene vector capable of transporting (not just the polypeptide) is described. In other words, the goal of such transneuronal gene vectors is not just neurons that cross the blood-brain barrier, but other “two” that share synaptic junctions with “primary” neurons that cross the transfected blood-brain barrier. Transfecting and genetically transforming "secondary" or "secondary" neurons (and possibly tertiary neurons that share synaptic junctions with secondary neurons). Transneuronal vectors can greatly increase both the amount and distribution of new and / or supplemented polypeptides that can be secreted by neurons located entirely within the blood brain barrier. This treatment can be an effective method for treating certain brain degenerative diseases characterized by widespread and disseminated brain injury, such as Parkinson's disease and Alzheimer's disease.

  It should be recognized that these transneuronal vectors are not expected to replicate and produce their own multicopy after invading secondary (or tertiary or later) neurons. Instead, the goal of such transneuronal vectors is to simply place these vectors in “downstream” neurons that are entirely within the blood brain barrier, and span the blood brain barrier. It is not to localize the vector to “primary” neurons. However, when taking additional measures to promote the integration of the gene sequence into the chromosome in the downstream neuron, such as by interposing a plasmid transcription unit with adeno-associated virus-derived terminal repeats In some cases (prone and more likely to occur), this approach involves non-invasive gene therapy for central nervous system neurons that are completely within and protected by the blood brain barrier. It is recognized that this can be done.

  Currently anticipated approaches for constructing transneuronal vectors include (i) “non-toxin fragment C” of tetanus toxin (eg, Knight et al 1999); (ii) barley lectin (Horowitz et al 1999); and (iii) Based on various facts known for transneuronal transport of certain pathogens and polypeptides, such as wheat germ agglutinin (Yoshihara et al. 1999). Barley lectin and wheat germ agglutinin are actively passed from olfactory receptor neurons to forebrain basal ganglia cholinergic neurons, as well as other types of neurons, such as neurons of the locus coeruleus and raphe nucleus. It has been shown to be neuronally transported. Other polypeptide sequences with transneuronal transport capability can also be discovered and generated using the same type of combinatorial selection method described in Example 7. This is done by performing a cyclic test of the phage display library to identify phage having a gene sequence that encodes a polypeptide that translocates the phage to secondary (or tertiary or later) neurons. be able to.

  Known methods can be used to construct gene vectors using polypeptides known to have transneuronal transport ability. For example, a polylysine can be covalently linked to a transport polypeptide using chemical reactions or using a recombinant gene encoding multiple lysine (or other positively charged) residues at the end of the polypeptide. Since polylysine is positively charged, mixing the polypeptide with the DNA segment in solution attracts the (negatively charged) DNA segment. This results in moderately strong but non-covalent binding between the polypeptide and DNA. This type of preparation is described in Knight et al 1999.

  Administration of the gene vector to the olfactory epithelium by the nasal nose of a vector suspended in saline solution or other carrier solution is the same as described in Example 2 adapted for this use by means well known to those skilled in the art. The method can be used.

  The methods described in Examples 3 and 4 (supplementarily supplemented with other methods or additional methods known to those skilled in the art) can be used for post-transfection monitoring and evaluation. Demonstrating the expression of transneuronally transfected NGF gene constructs in neurons within the blood-brain barrier (eg, forebrain basal cholinergic neurons) in situ hybridization and / or Alternatively, it can be performed using a sensitive method such as polymerase chain reaction (PCR). As described in Example 3, detection of expression can be facilitated by utilizing an NGF gene sequence that expresses a polypeptide distinctly different from the corresponding polypeptide in the host.

  Additional monitoring of the physiological and behavioral effects produced by such transneuronal vectors can be performed in the manner described in Example 4.

Construction and use of viral vectors to deliver anti-NGF polypeptides into the dorsal horn region of the spinal cord Examples 1-8 include nerve growth factor (NGF) etc. to help stimulate nerve cell growth or repair While describing the delivery of a neurotrophic polypeptide of Example 9, Example 9 and several subsequent examples include completely different types of polypeptides referred to herein as “anti-NGF” polypeptides. Is described. Such polypeptides help treat and reduce a variety of symptoms such as neuropathic pain or autonomic dysfunction when transported through the blood brain barrier and delivered to the correct targeted location. be able to. Such polypeptides (i) bind to NGF molecules in cerebrospinal fluid (CSF) or synaptic fluid and inactivate NGF molecules by preventing them from binding to NGF receptors And / or (ii) binds to NGF receptor proteins on neurons and occupies those receptors in a manner that does not cause the cellular response that occurs when free NGF molecules bind to the receptor. Can work by at least two known mechanisms by making the NGF molecule inaccessible.

  Anti-NGF polypeptides that bind to free NGF molecules in CSF or synaptic fluid are known and described in articles such as Ruberti et al (1993 and 1994). The gene sequences encoding these anti-NGF polypeptides are also known and are incorporated into complete gene constructs with appropriate gene promoters that carry out gene expression in mammalian cells as described in Example 1. Can do.

  If such a gene construct is intended to be expressed in nociceptive neurons rather than olfactory receptor neurons or other types of sensory neurons, it can be a gene promoter used to carry out gene expression. Care must be taken. The range of potential gene promoters that function in nociceptive neurons is known, and from these candidate promoters, appropriate promoters capable of carrying out desired gene expression levels in nociceptive neurons are selected by routine experimentation. be able to. Preferred promoter choices can also be influenced by the level of expression naturally occurring in various neural cells.

  For example, by using a gene promoter (described in Watson et al 1995) that carries out the expression of the CGRP gene, the expression of the anti-NGF gene carried by the vector is transformed into a nociceptive neuronal cell that expresses the neuropeptide CGRP. Can be limited to. If one purpose of treatment is to reduce NGF levels in the spinal cord of patients suffering from neuropathic pain or other chronic pain, the CGRP promoter is enhanced by the presence of NGF, so It will be particularly useful. Thus, when NGF levels in the spinal cord are increased, this promoter can increase the synthesis and secretion of anti-NGF in response, thereby creating a gene expression system that is self-limiting and self-regulating.

  A gene construct having a sequence encoding anti-NGF and a selected promoter is transformed into a gene derived from a replication-deficient adenovirus using methods such as those described and cited in Example 1 and Ruberti et al (1993 and 1994). It can be placed in a vector or can be placed in a gene vector derived from a replication-deficient herpesvirus using methods such as those described in Example 5 and the articles cited herein.

  Since this example relates to a method for reducing and controlling neuropathic pain exacerbated by NGF, the anti-NGF vector disclosed herein is inserted into sensory receptor neurons such as olfactory neurons. There is no. Instead, these vectors are most effective using methods such as those described in Sahenk et al 1993, or as being most effective in clinical trials or therapeutic procedures involving specific patients. High concentrations of nociceptive nerves, such as subcutaneous tissue or muscle tissue in areas afflicted by neuropathic pain such as allodynia, and / or areas that are innervated by nociceptive nerves that cross the blood-brain barrier Injected into the region containing

  After injection into a region having a projection of nociceptive neurons, at least some anti-NGF gene vectors contact the projection of nociceptive neurons outside the blood brain barrier. Using the same mechanism that viruses normally use to inject DNA into cells during infection, viral vectors (or similar mechanisms used by non-viral vectors as described in Examples 6-8) are The genetically modified vector DNA is injected into a projection outside the nociceptive neurons that straddle the blood-brain barrier that innervates the tissue. By retrograde transport, vector DNA is transported through the cytoplasm into a nerve cell region called the dorsal root ganglion located on the side of the spinal cord. The gene that encodes the anti-NGF polypeptide is expressed in nociceptive neurons within the ganglia (also spelled “ganglia”), thereby causing anti-NGF polypeptides in neurons across the blood brain barrier. A peptide molecule is generated.

  At least some anti-NGF polypeptides are located in “back horn” regions (and possibly other regions) of the spinal cord in tissues that are enclosed within and protected by the blood brain barrier It is transported through nerve cell axons across the blood-brain barrier by normal anterograde transport until the end of the synapse or similar exit point is reached. A leader or signal peptide that directs the transfected cell so that the polypeptide is packaged in a vesicle containing a neurotransmitter peptide that is antegradely transported and released to synapses located within the blood brain barrier. By adding to the starting point of the secreted or mature polypeptide, the efficiency of this antegrade transport process can be enhanced. A specific example of a leader sequence suitable for use in nociceptive neurons is the prepro BDNF sequence, which directs nociceptive neurons to anterograde transport of mature BDNF into the dorsal horn region in the spinal cord (These neurons normally synthesize).

  When released by nociceptive nerve cell terminals at these locations, the anti-NGF molecule can bind to the NGF molecule, thereby reducing the amount of free NGF in the spinal cord tissue at that location. Reduced NGF binding does not kill mature nociceptive neurons, but as demonstrated in papers such as Christensen et al 1996, nociceptive function has been demonstrated by direct administration of anti-NGF into experimental animals And reversible suppression of activity (often referred to as “down-regulation”).

  Gene expression in dorsal root ganglia (expected to be approximately 24 to 72 hours) and anterograde transport and release of anti-NGF into the dorsal horn region of the spinal cord (expected to be approximately 8 to 24 hours) After giving enough time for anti-NGF can be detected in the spinal cord.

  Various methods for labeling vector-derived anti-NGF in sacrificed laboratory animals, such as electron microscopy, immunological staining, and appropriate antigenic tags in the amino acid sequence of the polypeptide encoded by the vector gene The release of anti-NGF polypeptide in the spinal cord can be monitored directly by any suitable method, such as adding. It is also possible to monitor the concentration and / or effect of anti-NGF by measuring the concentration of free NGF. When released, anti-NGF binds and neutralizes endogenous NGF in the spinal cord. Thus, changes in the levels of NGF in the spinal cord were subject to innervation from transfected sensory neurons using various immunological methods such as those outlined in Example 3 and these methods. It can be monitored by applying it to the examination of spinal cord tissue. Changes in the level of NGF in the spinal cord can also be inferred from intrinsic neuroanatomical and behavioral changes, as outlined in more detail in Example 10.

The experimental animals are divided into two groups to allow statistical comparison of the physiological and behavioral monitoring results of delivery of anti-NGF to the dorsal horn region in the spinal cord . One group (test animal) enjoys anti-NGF gene vector as described above, and the other group (control group) is non-coding DNA that has no known neuroactive effect on central nervous system tissue, harmless Receiving a gene or a vector containing a marker gene. Physiological effects of administration of anti-NGF to the spinal cord include effects on brain neuroanatomy and animal behavior as discussed in articles such as Christensen et al 1996 and 1997.

  Appropriate selection of animal models is important to assess the physiological effects of anti-NGF delivery to the spinal cord. In addition to animal models of inflammatory pain, hyperalgesia and “allodynia” (chronic and / or neuropathic pain where the central nervous system interprets mild irritation as severe pain) by damaging or attacking peripheral nerves Various models can be made.

  To model neuropathic pain and to test the therapeutic potential of neuropathic pain, as just one example of a general method, a ring of suture ("ligature") is placed on the hind limb of an animal such as a rat. It can be surgically placed around one half to two thirds of one sciatic nerve and tightened. Within days to a week, the limb becomes hypersensitive to mild stimuli and suffers severe pain. The treated rat is then placed in a special box with an automatic sensor to determine how many seconds have passed since the rat began to warm its bottom plate until the rat lifted the sensitive leg off its warmed surface. Can be measured and recorded. If the candidate treatment can extend the average number of seconds taken to raise the nerve-ligated leg to the comparison time of untreated control animals, that means that the treatment reduces neuropathic pain It is effective to do so and will prove to be more detailed in large animals and / or clinical trials in human patients.

  However, peripheral nerve ligation or other attacks or injuries interfere with retrograde transport of vector DNA from the injection site to the dorsal root ganglion and the test used to evaluate the treatments described herein. It should be recognized that it would complicate the design and interpretation of. Thus, an attack that directly affects and participates in the spinal cord may be preferable in at least some situations. Chronic or neuropathic pain resulting from spinal cord injury is often seen clinically, and NGF overproduction in the spinal cord is a condition known as “primary afferent sprouting” often associated with spinal cord injury (Eg Krenz et al 2000). Thus, a spinal cord injury model that reproducibly causes hyperalgesia or chronic pain can be used to monitor the effects of anti-NGF delivery to the spinal cord on hyperalgesia or chronic pain. A specific example is the spinal cord injury model described in Krenz et al 1999, which is suitable for assessing the physiological effects of anti-NGF delivery to the spinal cord using the methods described herein. I believe it.

  Neuroanatomical changes in dorsal horn sensory innervation patterns associated with hyperalgesia or chronic pain include abnormal germination of “A” fibers and nociceptive CGRP-containing fibers into dorsal horn layer II (Eg Bennet et al 1996). Labeling A fibers transganglionically with agents such as the cholera toxin B subunit while using the methods described in Bennet et al 1996 or Christensen et al 1997, and CGRP immunohistochemistry Can be used to visualize the effects of administration of anti-NGF gene vectors on such neuroanatomical changes by visualizing nociceptive (NGF responsive) fibers in the dorsal horn.

  Alternatively, the effect of anti-NGF delivery on the number and extent of synaptic connections between peripherally projecting sensory neurons and secondary neurons in the spinal cord can be achieved using methods such as those described in Blessing et al 1994. It can be evaluated by a neuronal tracking method.

  Established functional monitoring methods for pain responses in animal models include: (i) Legs in response to temperature stimuli as briefly outlined above and described in detail in articles such as Romero et al 2000. Measuring the withdrawal retention time, and (ii) measuring the response to application of the von Frey hair stimulant as described in articles such as Deng et al 2000.

  Other symptoms that can be treated with anti-NGF or equivalent polypeptides are often referred to as "autonomic dysfunction". This symptom most often occurs in patients with spinal cord injury in the upper chest or cervical region, and the normal connection with prenodal sympathetic neurons in the upper spinal cord is broken. As a result, normal feedback control mechanisms in the autonomic nervous system are disrupted, and normal stimuli (eg, swelling of the large intestine that indicates fullness of the intestine) can cause other reflex results that may be related to life and death ( Such as increased heart rate and blood pressure to a level that greatly increases the risk of serious cardiovascular disease such as heart attack or heart attack).

  As shown in animal model studies (eg, Krenz et al 1999), direct injection of anti-NGF into the spinal cord of animal models in this state can cause dorsal horn abnormalities due to nociceptive neurons that can occur after such spinal cord injury. In addition, by suppressing unnecessary innervation, this unnecessary reflex response can be prevented or controlled.

  Thus, in the spinal cord region protected by the blood brain barrier, the gene vector is transfected into a projection of exposed accessible nociceptive neurons (and possibly other spinal neurons such as motor neurons) The method of use of the present invention for introducing an anti-NGF polypeptide into a drug is quite promising for the treatment of autonomic dysfunction.

Construction and use of lipid-based gene vectors for delivery of anti-NGF polypeptides into the dorsal horn region in the spinal cord. Anti-NGF gene constructs can be made that are expressed in multiple targeted classes of nociceptive neurons. Using known methods, the resulting gene construct can be placed in a DNA vector such as a plasmid or other stable form. The DNA vector is then transformed into lipid vesicles using methods such as those described in Nabel's chapter (pages 127-133) of "Methods in Molecular Medicine: Gene Therapy Protocols" (P. Robbins Publication 1997) and Example 6. (Liposome) can be placed inside.

  Suffering from neuropathic pain or other chronic pain using methods such as those described by Sahenk et al 1993 or as being most effective in clinical trials or therapeutic procedures involving specific patients In the region, the resulting liposome vector can be used for peripheral projection of nociceptive neurons by means such as injecting a liposome suspension in saline into regions containing high concentrations of nociceptive nerves such as subcutaneous tissue or muscle tissue. Can be administered. At least some liposome vectors attach and fuse to projections of nociceptive neurons located outside the blood brain barrier in muscle tissue, so that the anti-NGF-encoding DNA carried by the liposomes is supplied into the nerve projection. Retrograde transport of DNA segments transports at least some anti-NGF DNA to the neuronal cell body where the anti-NGF polypeptide is expressed from the anti-NGF gene.

  Subsequent delivery of the anti-NGF polypeptide into the spinal cord and the physiological and behavioral effects of the anti-NGF polypeptide in the spinal cord tissue were measured using methods as described in Examples 9 and 10, Can be monitored.

Construction and use of DNA vectors targeting endocytic receptors on nociceptive neurons for delivery of anti-NGF polypeptides into the dorsal horn region of the spinal cord Various known methods such as the method described in Example 9 This method can be used to produce an anti-NGF gene construct that is expressed in nociceptive neurons. Using other known methods, such as the method described in Example 11, in plasmid bodies and other stable bodies that can be transported into nociceptive neurons by non-viral vectors such as liposomes, anti-NGF A genetic construct can be placed.

  These stable DNA bodies can then be used to form protein-DNA complexes using methods such as those described in Example 7. This complex incorporates a polypeptide segment as a ligand that specifically binds to the receptors of nociceptive neurons that induce endocytosis. Various such receptor-specific polypeptide segments are already known and others can be identified and developed using the phage library approach described in Example 7.

  Thus, when these steps are organized in the correct order, the end on the peripheral projection of nociceptive neurons is an area that is not surrounded by the blood brain barrier and thus provides relatively easy access to the peripheral projection. A gene vector is created that can specifically target the cytosis receptor. Using such a receptor-specific endocytosis gene vector, the neuronal cell can be transfected with the anti-NGF gene expressed in the neuronal cell. The nerve cells themselves then deliver and secrete anti-NGF polypeptides in the dorsal horn region of the spinal cord so as to control and reduce neuropathic pain and other pain disorders.

  Delivery of such anti-NGF polypeptides into spinal cord tissue can be monitored using methods such as those described in Example 9, and the physiological of anti-NGF polypeptides within such spinal cord tissue. Effects and behavioral effects can be evaluated using methods such as those described in Example 10.

Examples of gene constructs (including appropriate gene promoters) capable of expressing anti-NGF polypeptides in transneuronal anti-NGF vector nociceptive neurons that transport anti-NGF genes to secondary neurons in the spinal cord 9 can be made. These gene constructs can be placed in plasmids or other stable bodies using known methods.

  Such anti-NGF plasmids or other DNA vectors are then ligated with “transneuronal polypeptides” that can realize and facilitate the transport of protein-DNA complexes from one neuron to another. can do. A variety of such “transneuronal polypeptides” are known, eg, “non-toxin fragment C” of tetanus toxin (eg Knight et al 1999), barley lectin (Horowitz et al 1999), wheat germ agglutinin (Yoshihara et al 1999) (also listed in Example 8).

  Such a `` reversible binding to a protein that is exposed at the peripheral synaptic end (i) and (ii) internalized to the synapse within the blood brain barrier by nerve cells that straddle the blood brain barrier. Transneuronal polypeptides pass through the cytoplasm of “primary” nociceptive neurons across the transfected blood-brain barrier, through the blood-brain barrier, and into nerve cell terminals and other spinal cord tissues located in the dorsal horn. The protein-DNA complex can be transported to With the help of transneuronal proteins, the protein-DNA complex then exits the synaptic terminals of nociceptive neurons that straddle the blood-brain barrier and enters the nearby spinal nerve cells located entirely within the blood-brain barrier. , Thereby transfecting “secondary” spinal neurons protected by the blood brain barrier.

  Delivery of such anti-NGF gene vectors and their coding sequences into “secondary” spinal nerve cells protected by the blood brain barrier is performed in situ using spinal cells and tissues obtained from sacrificed laboratory animals. It can be monitored by methods such as DNA probe hybridization and PCR analysis. Expression of anti-NGF polypeptides by transfected “secondary” spinal neurons can be monitored using methods such as those described in Example 9, and the physiology of anti-NGF genes and polypeptides in spinal cells. Effects and behavioral effects can be evaluated using methods such as those described in Example 10.

Use of adenoviral vectors to transfect spinal motor neurons that transport neurotrophic polypeptides into upper motor neurons. A method for preparing non-pathogenic adenoviral vectors that cannot replicate in normal cells is described in Graham and Published in papers such as Prevec 1995. Articles describing specific examples of such vectors containing genes encoding various neurotrophic factors include Dijkhuizen et al 1997, Gravel et al 1997, Baumgartner et al 1998 and Romero et al 2000.

  The gene sequence encoding “glial cell line-derived neurotrophic factor” (GDNF) has been isolated, and is made into a form that is easy to handle such as plasmids and adenovirus vectors. Lindsay et al 1996, Choi-Lundberg et al 1997, Baumgartner described in papers such as et al 1997 and various other papers cited herein.

  A gene sequence encoding “neurotrophin-3” (NT-3) has been isolated and made into an easy-to-handle form such as a plasmid or an adenoviral vector in articles such as Snider 1994 and Bothwell 1995 and in this specification. It is described in various other papers cited.

  GDNF and NT-3 have a relatively strong activity and effect (as compared to other known neurotrophic factors), especially when administered to motor neurons, as demonstrated by tests performed in the prior art As such, it is a preferred candidate for initial testing for use in the treatment of motor neurons disclosed herein.

  Many gene promoters that perform gene expression in motor neurons are known and available and can be used in the tests described herein. One class of promoters that are particularly noteworthy for use in transfection of spinal motor neurons include those that carry out the expression of the so-called α-1 subunit of the glycine receptor in spinal motor neurons. Since glycine receptors are not present in high concentrations in nociceptive neurons or other sensory neurons, glycine receptors (or other genes that are more active in spinal motor neurons than in sensory neurons) Use of one or more types of promoters from genes that express one or more subunits helps increase the selective expression of neurotrophic genes in the vector in the desired target neuron, while non-targeting It is expected that potential adverse side effects that may be caused by unwanted expression in the cells can be minimized.

  Another class of promoters that may be useful in some situations involved in transfection of spinal motor neurons performs the expression of a protein called the poliovirus receptor. This receptor protein is not present at a substantial level in sensory neurons and therefore the promoter is presumed to be silent in sensory neurons. However, various experimental animals (including mice and rats) do not have the poliovirus receptor gene or its promoter, and therefore using a poliovirus promoter makes it somewhat difficult to conduct various studies in animal models. It should be noted that it would be fun.

  Alternatively, if desired, it is very potent in mammalian cells, such as to induce the highest possible level of expression of NGF (or other central nervous system active polypeptide) in transfected cells. Various viral promoters known as promoters and other promoters can be used. Examples of such strong promoters include the early gene promoter from cytomegalovirus and the late gene promoter from simian virus 40. If desired, use an inducible gene promoter so long as it can be administered so that an appropriate amount of transport in the transfected neuronal cell can be ensured that induces activation of the selected promoter. You can also

  Thus, adenoviral vectors carrying neurotrophic genes that function in motor neurons such as GDNF or NT-3 can be assembled using various elements and methods as disclosed in the above cited papers. .

  If desired, an “epitope tag” sequence can be incorporated into the coding region of the vector gene to facilitate detection and monitoring of the polypeptide encoded by the vector gene in various test animal tissues. This approach is described in detail in Example 1 and in articles such as Moller et al 1998. Using such an approach, polypeptides expressed by the vector construct can be expressed using methods described in articles such as Altar & Bakhit 1991, Ferguson et al 1991, DiStephano et al 1992, and von Bartheld 2000. It is important to ensure that all steps necessary to be accurately delivered to the neurons targeted for treatment with the polypeptide can be performed.

  Adenovirus carrying the desired gene construct by intramuscular injection of adenovirus vector suspended in a certain amount of solution (such as saline solution) compatible with adenovirus and active tissue (tissue vigor) Vectors can be administered to spinal motor neurons. The method of growing, purifying, concentrating, and titrating a solution containing an adenoviral vector is described in the Engelhardt chapter (pages 169-184) of “Methods in Molecular Medicine: Gene Therapy Protocols” (P. Robbins Publication 1997). Haase et al 1998 found that doses and administration techniques in the effective administration of adenoviral vectors by intramuscular injection for gene transfer into spinal motor neurons of experimental rats. Provide information about. If desired, electromyography can be used to help ensure that the fluid is injected at the exact desired location.

Testing of transfected motor neurons, superior motor neurons, and vector animals and polypeptides in brain tissue monitoring laboratory animals (i) various cells and spinal cords that can be affected by contact with the polypeptide Measure and monitor the movement, location, and concentration of the gene vector and polypeptide encoded by the vector in the region, and (ii) obtain reliable and useful statistical data that accurately reflects such results It should be designed to simplify the work involved. For example, a harmless and / or non-functional gene, a nonsense DNA sequence that does not encode a polypeptide, or a polypeptide that can be readily detected when expressed in mammalian cells but has no significant physiological effects By using a control vector carrying a marker gene that encodes a gene vector administered to one side muscle of the test animal (eg, by injecting a vector solution into the hind limb) and treating the other side of the animal as a control , Can simplify the necessary work and make it more reliable. During subsequent histological processing, the left and right regions of the spinal cord (and brain, if desired) are compared with each other to assess the movement, concentration and effect of the vector DNA and / or polypeptide encoded by the vector gene. can do.

  After allowing sufficient time for gene expression (24 to 72 hours), the lumbar spinal cord is removed from the experimental animal and the tissue is processed to monitor neurotrophic factor gene expression in spinal motor neurons The effect of gene vector delivery can be evaluated. For these analyses, (i) DNA probes that are complementary to the mRNA of the gene vector but not to the endogenous mRNA sequence, using methods such as those described in articles such as Xian and Zhou 2000 (Ii) DNA from viral vectors or polymerase chain reaction (PCR) reagents and methods, as described in articles such as Chie et al 2000, using methods for detecting mRNA sequences, and (iii) immunostaining, ELISA, or similar methods using antibodies that selectively bind to the polypeptide of the vector but not to the endogenous polypeptide in the test animal. Can be used. Many such antibody preparations are commercially available or, if desired (such as to detect polypeptides with specific “epitope tags”), Conner 2000, Rush et al 2000 and Zhang et al. It can be prepared using methods disclosed in articles such as 2000.

  The goal of the method outlined in Examples 14 and 15 is to use spinal motor neurons that transfect the transfected blood brain barrier to treat upper motor neurons that are completely within the blood brain barrier and protected. Delivering the polypeptide. This means that (i) expression of the vector-encoded neurotrophic factor gene creates a vector-encoded neurotrophic factor polypeptide, and then (ii) is protected from external polypeptides by the blood brain barrier. Can be achieved by transporting and delivering the polypeptide to a location in the spinal cord that is accessible to the axons of upper motor neurons.

  To facilitate the method of identifying the exact region in the spinal cord where the polypeptide is most likely to be delivered by transfected spinal motor neurons, using appropriate tracking methods and reagents, “transneuronal labeling” Can do research. Such studies using pseudorabies virus are described in articles such as Card et al 1990.

  Using transneuronal labeling, polypeptides such as NT-3 and GDNF are delivered to the upper motor neurons, resulting in germination of the upper motor neurons and lower motor movement across the transfected blood-brain barrier. Evidence of synaptic connections with neurons can also be obtained. The number of transneuronally labeled upper motor neurons in the brain and brain stem is increased by increasing the strength and number of synaptic connections between the upper and lower motor neurons.

  When control and test vectors are administered to both sides of the experimental animals, reasonable differences between the left and right sides of the spinal cord can be observed. To confirm that NT-3 or GDNF detected in the brains of experimental animals is derived from retrograde transport from transfected lower motor neurons, using incision of descending axons from other animals Can do.

Monitoring of physiological and muscular effects resulting from delivery of neurotrophic factors to upper motor neurons Neuronal factors such as NT-3 or GDNF into the spinal cord or brain using the gene vectors disclosed herein Physiological effects of administration include effects on spinal cord and brain neuroanatomy and effects on animal physiology and behavior. In particular, the improvement and benefit of muscle strength, muscle control, and muscle tone provided by using the gene vectors disclosed herein can be assessed using a variety of methods.

  These methods require a basic understanding of what has been seen so far in various conventional tests using animal models. Most of such tests first cause neuronal injury or damage to the animal's spinal cord or motor neuron system, then wait for a period of time for the damage to manifest more fully, and then receive test treatment (subcutaneous needles) (E.g., direct injection of neurotrophic factor into the cerebrospinal fluid of the animal), and finally, whether the test treatment was able to reduce the extent of injury caused by similar injury in the control animal or limb It should also be recognized that this is always done by conducting animal tests to determine. As outlined in Example 4, comparative studies usually involve one or both of the following: (i) treating different control animals, or (ii) treating two different sides of the same animal in different formats.

  It should be appreciated that many such tests use “axonectomy” referred to as surgical axotomy. As mentioned in Example 4, axons are extremely important for the functioning of nerve cells, and when a nerve cell axon is cut at a position relatively close to the nerve cell body, it spans a measurement period of several days. Even if the rest of the nerve cells are not damaged, the nerve cells begin to atrophy and eventually die. The reason for this is complex and is generally believed to involve cellular factors (and especially neurotrophic factors) that are involved in the development of the nervous system. According to the so-called “neurotrophic theory”, the developing brain of the fetus is initially oversupplied with neurons and then the pruning process begins. During the pruning phase, neurons that do not continue to actively receive incoming neural signals (and / or do not come into contact with one or more neurotrophic factors) die out as programmed cell death called “apoptosis”.

  Many animal studies have shown that the application of various neurotrophic factors (usually injections into the cerebrospinal fluid or spinal cord tissue) prevents atrophy, degeneration, and death induced in higher motor neurons by axotomy damage. I knew it was possible. Examples of such animal studies and the results that occur when neurotrophic factors are applied to neurons are reported in articles such as Novikova et al 2000, Giehl and Tetzlaff 1996 and Giehl et al 1997.

  However, if the axotomy damage is remote from the lower motor neurons transfected with the foreign gene (eg, when the gene vector disclosed herein is used as in Example 14), the axon Since the cut injury interferes with normal retrograde axonal transport of neurotrophic factors by transfected peripheral motor neurons, the vectors and genes of the present invention against atrophy and degeneration induced by axotomy. It is not easy to prove that it has an effect. If the transfected lower motor neuron is within a few millimeters of the cut edge of the upper motor neuron, the upper motor neuron is expressed by a neurotrophic polypeptide expressed and secreted by the transfected motor neuron. Easy access. Thus, in an appropriate experimental design using axotomy injury, the axotomy location may be within a reasonably close distance to the nearest tip of the motor neuron that can be reasonably transfected by intramuscular injection. preferable.

  It has been publicly shown that administration of neurotrophic factors stimulates the germination and regeneration of damaged axons (eg Schnell et al 1994). Therefore, using the gene vectors disclosed herein, factors such as NT-3 or GDNF are passed through the lower motor neurons that are within about 1-5 mm of the cut end of the damaged upper motor neurons. When delivered, various effects (such as stimulation of germination and regeneration) were studied using an antegrade pathway tracking study (Ferguson et al 2001) involving the injection of tracking compounds such as biotinylated dextran into the motor cortex. Can be demonstrated.

  In the use of the present invention to deliver NT-3 or GDNF via lower motor neurons, the highest concentration of attracting compound is concentrated near the transfected lower motor neurons (the molecule is other It is also expected that a chemoattractant gradient will be established (unless rapidly removed or dispersed, such as by active uptake into the cell). The regeneration of damaged motor neurons typically grows in the direction of increasing chemoattractant concentration in response to a chemoattractant gradient. By using an antegrade pathway tracking method as described in Ferguson et al 2001, the effects of chemoattractant gradients on directional growth in regeneration of damaged upper motor neurons can be assessed and demonstrated .

  The chemoattractant gradient that emerges from the transfected lower motor neurons reveals a new synapse between the regenerating damaged upper motor neurons that release chemoattractant compounds such as GDNF or NT-3. Accelerate the connection speed. This increase in synaptic connection speed has been studied over time in animal models, and the appearance of transneuronal tracking compounds in the upper motor neurons over time when tracers are injected into areas outside the blood brain barrier. This can be verified by monitoring. Suitable transneuronal tracking methods are described in articles such as Ugolini 1995, Travers et al 1995 and Card et al 1990.

  The establishment of a functional synaptic connection between the upper and lower motor neurons also changes the electrical properties of the lower motor neurons. The initial effect of upper motor neuron activity tends to be inhibitory, so if this inhibition is lost, lower motor neurons tend to enter an abnormally active state. Thus, recovery or regenerated connections with upper motor neurons are expected to restore lower motor neuron activity to a more normal level, which appears as a more normal sensory reflex.

  Electrophysiological methods can be used to monitor such changes over time and provide an indication of whether functional synaptic reconnection is occurring between upper and lower motor neurons Can do.

  The establishment of functional reconnection between upper and lower motor neurons also results in observable changes in motor function such as muscle strength, muscle tone, and coordination. Changes in muscle strength over time and the ability of animals to perform fine and coordinated exercise tasks (such as overcoming the load to obtain food) are tested using methods described in the literature or methods known to those skilled in the art. Can be monitored.

Use of lentiviral-derived vectors to transfect spinal motor neurons that transport neurotrophic polypeptides to upper motor neurons
Using methods described in articles such as Hottinger et al 2000, (i) a sequence encoding GDNF, NT-3 or other neurotrophic polypeptide, and (ii) a transfer sequence as described in Example 14. It is possible to construct a lentiviral vector capable of carrying a gene construct having a gene promoter suitable for gene expression in the discharged spinal motor neurons. This lentiviral-derived gene vector can be administered to the accessible projection of spinal motor neurons by intramuscular injection using the method described in Example 14.

  Following transfection of spinal motor neurons, expression of the encoded polypeptide in the transfected nerve cells and delivery of the polypeptide into the spinal cord or brain tissue protected by the blood brain barrier is an example. It can be measured and monitored by the method described in 15. The neurotrophic polypeptide's muscle and other physiological effects, as well as the ability of the neurotrophic polypeptide to prolong neuronal survival after axotomy or similar attack, is a method as described in Example 16. Can be measured and monitored.

Preparation and use of liposome vectors for transfection of spinal motor neurons that transport neurotrophic polypeptides to higher motor neurons As mentioned in Example 6, DNA plasmid-lipid complex for transfection of mammalian cells Methods for preparing the body are described in publications such as the chapter by Nabel (pages 127-133) in “Methods in Molecular Medicine: Gene Therapy Protocols” (P. Robbins publication 1997), and routine tests of various preparation mixtures are carried out. Can be adapted to cell types or specific uses.

  Using methods such as those described in Example 14, a liposomal vector carrying a neurotrophic factor gene can be administered to an accessible projection of spinal motor neurons by intramuscular injection. Following transfection of spinal motor neurons, expression of the encoded polypeptide in the transfected nerve cells and delivery of the polypeptide into the spinal cord or brain tissue protected by the blood brain barrier is an example. It can be measured and monitored by the method described in 15. The neurotrophic polypeptide's muscle and other physiological effects, as well as the ability of the neurotrophic polypeptide to prolong neuronal survival after axotomy or similar attack, is a method as described in Example 16. Can be measured and monitored.

Construction and use of DNA vectors targeting endocytic receptors on spinal motor neurons that transport neurotrophic polypeptides to higher motor neurons Genes using ligands that bind to endocytic receptors on neurons Reagents and methods for preparing vectors are described in Example 7. These include methods using monoclonal antibodies or iterative selection of phage display libraries to identify and generate high affinity ligands that actively bind to endocytic receptors on spinal motor neurons. The method used is included.

  As introduced in Example 7, a ligand for the p75 receptor can be used to target gene delivery to spinal motor neurons. Spinal motor neurons normally express only low levels of p75, but increase p75 expression in response to injury or neurotrophic factor deficiency and in various diseases such as amyotrophic lateral sclerosis. Thus, gene vectors that target p75 make it possible to enhance or target the delivery of genes encoding therapeutic proteins to spinal motor neurons that require therapeutic support.

  The efficiency of gene vectors targeting spinal motor neurons depends on the compound that accumulates the gene vector on the “motor end plate” in the injected muscle, either in the vector construct itself or in an injectable solution containing the gene construct. Inclusion can be enhanced. Botulinum toxin may be useful for this purpose. Alternatively, a monoclonal antibody can be produced by using a peptide sequence of 17 amino acids derived from the α1 subunit of the acetylcholine receptor as an antigen (Yoshikawa et al 1997). In a disease called myasthenia gravis, patients produce antibodies against acetylcholine receptor epitopes that are localized on the muscle endplate in the synaptic cleft.

  Thus, using “receptor targeted” gene vectors with such receptor binding ligands and other enhancers, as described in Example 14, GDNF, NT-3 or other neurotrophic A gene construct having a sequence encoding the polypeptide and a gene promoter suitable for gene expression in transfected spinal motor neurons can be carried. Administration to the spinal motor neurons by intramuscular injection of the ligand-DNA complex can use the method as described in Example 14. After transfection of spinal motor neurons, expression of the encoded polypeptide in the transfected neurons and delivery of the polypeptide into the spinal cord or brain tissue protected by the blood brain barrier is an example. It can be measured and monitored by the method described in 15. The neurotrophic polypeptide's muscle and other physiological effects, as well as the neurotrophic polypeptide's ability to prolong neuronal survival after axotomy or similar attack, is a method as described in Example 16. Can be measured and monitored.

Example 8 is a gene vector capable of transneuronal transport of spinal motor neurons using a transneuronal vector for delivering a neurotrophic factor gene to central nervous system neurons in contact with spinal motor neurons It is described in. Such a vector designed for use in transfection of spinal motor neurons can be assembled using the method described in Example 8 and possesses the gene construct described in Example 14. Can do. Administration to spinal motor neurons by intramuscular injection can use the method as described in Example 14, and monitoring after transfection uses the method as described in Examples 15 and 16. be able to.

Delivery of neurotrophic factors into the brainstem by injecting the vector into the tongue to transfect lower motor neurons of the hypoglossal nucleus, gene vectors derived from adenovirus, herpesvirus or lentivirus, or cationic liposomes , Ligands that bind to endocytic receptors, and / or transneuronal polypeptides can be used as described in the examples above. This type of vector is a neurotrophic gene construct (or one such as IN1 or No-Go, if desired) expressed in transfected motor neurons, as described in the Examples above. Or a recombinant antibody or a genetic construct that expresses a similar polypeptide that inhibits a plurality of neurite inhibitor molecules.

  As shown in FIG. 6, such gene vectors can be used to transfect a class of lower motor neurons with accessible projections outside the blood-brain barrier of the tongue. These neurons, known as “motor neurons in the hypoglossal nucleus”, are synapse connected to other neurons in the brainstem area. Thus, motor nerve cell terminals within the tongue deliver neurotrophic polypeptides to brainstem neurons that have, or are synapse with, projections that are close to the transfected lower motor neurons To provide a relatively direct route to

  Administration to these motor neurons can be performed by injection into the tongue, using a general method as described in Example 14, but adapted for injection into the muscle of the tongue. Post-transfection monitoring in laboratory animals is suitably adapted to monitor the delivery of exogenous peptides (such as epitope-tagged NGF, NT-3 or GDNF) to brainstem neurons, and General methods such as those described in 16 and other methods known to those skilled in the art can be used. As an example for animal use, transfected submotion in the sublingual nucleus using various transneuronal tracking methods such as those described in Ugolini 1995, Travers et al 1995, or Card et al 1990 Neurons in the brainstem that come into contact with neurons can be labeled. By counting the number of secondary neurons in contact with the hypoglossal nucleus in animals treated using the present invention, and comparing such data with the numbers found in control animals, the secondary neurons in the brainstem Evidence for the delivery of neurotrophic factors can be obtained.

  As another example of a monitoring method useful for human patients, abnormal excitatory reflex blinking is a symptom of Parkinson's disease and is clinically associated with disease severity. Persistently active serotonergic "rabbit neurons" usually suppress the spinal trigeminal nerve cells involved in the reflex blink circuit, and the deterioration of these serotonergic raphe neurons Produces abnormal excitatory reflex blinks (eg Basso and Evinger 1996). Thus, evidence of the clinical use of the present invention to reduce the rate of brainstem serotonergic neuronal degeneration can be obtained by monitoring aberrant excitatory reflex blinks in Parkinson's disease patients.

  Another way to monitor brainstem neuron maintenance in Parkinson's disease human patients is to measure the patient's swallowing reflex. The swallowing reflex involves the coordinated movements of the tongue and other mouth and respiratory muscles, and the time from stimulation to reflex swallowing is abnormally prolonged in Parkinson's disease. The swallowing reflex test involves the delivery of a small amount of water to the throat via an intranasal catheter as a swallowing reflex stimulus. The onset of swallowing reflex can be accurately measured using surface electrode electromyogram recordings of throat muscles and the time from stimulation to reflex swallowing can be calculated (Iwasaki et al 2000). Thus, evidence of the clinical use of the present invention to reduce the rate of brain stem nerve cell degeneration can be obtained by monitoring the swallowing reflex in Parkinson's disease patients.

In vivo screening: phage types and libraries
M13KO7 helper phage can be purchased from various commercial vendors such as New England Biolabs (www.neb.com) and Amersham Biosciences (www4.amershambiosciences.com). This helper phage strain contains a fully functional gene encoding both pIII and pVIII coat proteins. It also contains an origin of replication (from plasmid p15a) that is well controlled to give low copy numbers in bacterial cells. It also contains a mutation in the phage M13 pII gene that is essential for phage replication (Met-40-Ile). This mutation allows secretion as phage particles at low copy numbers by E. coli cells. It also carries a kanamycin resistance gene inserted at the AvaI site within the M13 replication origin. This kanamycin gene functions as a selectable marker in E. coli host cells that are not resistant to kanamycin. Additional information regarding the use and culture of these helper phages is available from commercial sources and from various published articles describing their use.

The scFv phage library was supplied by Cambridge Antibody Technology (Cambridge, UK www.cambridgeantibody.com). The scFv phage library has been described in various patents (such as US Pat. No. 6,172,197) and published articles. By inserting a gene sequence derived from B lymphocyte cells (making an antibody) into a gene sequence encoding a pIII coat protein (located at a relatively small number at one end of a linear M13 phage particle), the scFv A phage library was prepared. Each foreign gene sequence in the scFv library contains both the “heavy variable” (V _H ) and “light variable” (V _L ) domains of a single antibody in a single gene sequence, with an average molecular weight of about 35 It expresses the V _H and V _L domains as a “single chain (sc)” polypeptide that is a kilodalton. The scFv library has an estimated 13 × 10 ⁹ (ie 13 billion) different recombinants. To ensure maximum diversity, this includes “variable fragment” (Fv) antibody domains obtained from a large number of people with different ancestry. The library is evaluated to encode a wide variety of different Fv antibody domains that could be generated by the immune system of 10 different groups of people from different races and races.

  It should also be noted that the scFv phage is a phagemid. The phage has a bacterial origin of replication that can be grown in E. coli cells as a double-stranded plasmid with high copy number. The phage also contains a phage origin of replication that can cause synthesis of single-stranded DNA for assembly into phage particles. However, the ssDNA synthesis requires the presence of a phage ssDNA transcription protein, and the scFv phage does not encode that protein. The phage ssDNA transcription protein must be supplied by a helper phage such as the M13KO7 helper phage described above.

  Therefore, when co-infecting E. coli cells already infected with scFv phagemid using M13KO7 helper phage, cells already containing a large number of dsDNA plasmids (derived from scFv phagemid) will be treated with the above ssDNA (derived from helper phage). Addition of the transcription protein causes the formation of multiple ssDNA strands from the scFv phagemid plasmid. These newly formed ssDNA strands are then packaged inside a coat protein (mainly a pVIII coat protein from a particular scFv strain that has infected the host cell). The newly packaged ssDNA and its coat protein are secreted from host cells as filamentous phage particles. Since only low copy number helper phage DNA sequences are present in the host cell (due to the low copy number plasmid p15a origin of replication in helper phage DNA), most of these secreted phage particles are M13KO7 helper phage DNA Rather, it contains scFv phagemid DNA.

  The pVIII coat protein in phage particles secreted by a particular E. coli host cell contains a cloned copy of a specific antibody “variable fragment” polypeptide sequence encoded by a DNA sequence obtained from human B lymphocytes. This human antibody DNA sequence was inserted into the scFv phagemid DNA at a predetermined and target site near the center of the phagemid gene encoding the pVIII coat protein.

The PhD-C7C phage display library was obtained from New England BioLabs ( www.neb.com , catalog number 8120). This library contains an estimated 2 × 10 ⁹ different recombinants, and the foreign DNA insert encoding a random sequence of 7 amino acids contains a DNA sequence encoding the N-terminus of the pIII coat protein of M13 phage. It is inserted nearby. This library provides a repertoire of essentially random peptide sequences that are tested to determine whether a phage is taken up and transported by neurons of the sciatic nerve bundle.

Except where noted by cell type, characteristics, and method , all phage amplification and titrations were performed using the Cambridge Antibody Technology E. coli strain TG1. This strain, specially designed and developed to work with M13 phage, is also sold by companies such as Stratagene (La Jolla, California; www.stratagene.com). Additional information on the culture and transformation methods of this strain can be downloaded free of charge from the commercial website.

  Among other features, the TG1 strain has a “lacIq” repressor gene. This negatively regulates the “lac” promoter placed under control of the expression of the M13 gene encoding the pIII phage coat protein, along with the suppression of degradation products by glucose. Most types of M13 phage used with TG1 cells contain an “amber” stop codon inserted at the start of the pIII gene. As described below, this allows expression of soluble pIII polypeptides (including chimeric pIII polypeptides containing foreign amino acid sequences) in non-suppressed E. coli strains such as HB2151 without the need to re-clone the gene. .

  Transferring TG1 cells into a medium that does not contain glucose and instead contains lactose (a specific sugar molecule) as the only carbon source for bacteria can effectively inactivate the “amber” stop codon . Also added is a compound called isopropylthiogalactopyranoside (IPTG). This strongly induces expression of the “lac” operon that allows host cells to metabolize lactose molecules as nutrients. This allows the transformed cells to grow in a medium with lactose as the only carbon source, in addition to allowing the transformed cells to light a chemical called X-Gal. Conversion is also possible, so that transformed colonies are easily identified and isolated on agar plates.

Grown on agar plates in a typical method used to “amplify” (reproduce) a particular phage, such as after in vitro panning or in vivo selection methods described herein TG1 cell colonies were inoculated into a liquid medium containing 16 g of tryptone per liter, 10 g of yeast extract, and 5 g of NaCl (this liquid medium containing tryptone and yeast extract is called 2TY medium). Cells were grown in a shaker incubator to an optical density of approximately 0.5-0.8 units (OD ₆₀₀ measured at a wavelength of 600 nanometers) (all cultures were performed at 37 ° C. unless otherwise indicated). The phage preparation was added to E. coli medium and the mixture was incubated. In order to facilitate the binding of phage to bacterial cells, the initial culture was performed at rest for 30 minutes. This was followed by incubation for 30 minutes in a shaking incubator operating at 200 rpm in order to maximize the exposure of the cells to new nutrients.

  The cells were then centrifuged at 3500 rpm for 10 minutes and the supernatant containing the old broth and metabolites was discarded. The cell pellet is resuspended in 500 microliters (μL) of fresh 2TY culture broth and the mixture is transferred to four very wide square plates (24.3cm x 24.3cm) containing 2TY agar medium supplemented with ampicillin and glucose. ) Spread on the surface. Plates were incubated overnight at 30 ° C. Due to the presence of ampicillin, only E. coli cells containing scFv phagemid or PhDC7C phage formed colonies on the plate.

  The next day, to prepare a standardized phage solution that could be frozen until needed, colonies were scraped from each agar plate into 10 mL of 2TY broth and placed in 50 mL tubes. Half of the sterile 100% glycerol was added, and the solution was then mixed by placing the tube in an inverted stirrer at room temperature for 10 minutes. The 1 mL volume was frozen at -70 ° C. for storage.

When testing requires a batch of phage, thaw a 1 mL volume of glycerol-containing stock and add 100 μL of the thawed stock to 2TY broth containing 2% (w / v) glucose sterilized and 100 mg / ml ampicillin. Added to mL. Cells were grown at 37 ° C. in a shaker incubator until the OD ₆₀₀ density was approximately 0.5-0.8. Then, M13KO7 helper phage was added so that the final concentration was 5 × 10 ⁹ cfu (colony forming unit) per mL. The mixture was incubated at rest for 30 minutes and then in a 200 rpm shaking tray for 30 minutes. The cells were then centrifuged for about 10 minutes at 3500 rpm and the cell pellet was resuspended in 25 mL of pre-warmed (without glucose) 2TY medium containing kanamycin (50 μg / mL) and ampicillin (100 μg / mL). These were incubated overnight at 25 ° C. with high speed shaking to produce phage particles.

  Phage particles were purified from the supernatant by precipitation with 20% polyethylene glycol (PEG) and 2.5 M NaCl. These particles were then resuspended at 4 ° C. in a final volume of 1.5 mL sterile phosphate buffered saline (PBS).

To “titrate” the solution containing the phage particles (ie, to assess how many infectious phage particles were present in each 1 mL of solution), the OD ₆₀₀ density was about 0.5-0.8, TG1 cells are grown in 2TY medium for about 4 hours in a 300 rpm shaking incubator. The phage supernatant sample was diluted 10-fold sequentially in 2TY medium. That is, 50 μL of each diluted solution was sequentially added to 450 μL of the TG1 cell suspension in the Eppendorf tube. The tube was gently incubated for 30 minutes followed by shaking at 300 rpm for 30 minutes. 2TY agar plate pre-warmed with 100 μL of each dilution of infected TG1 cells (2TY medium containing ampicillin 100 μg / mL, filter sterilized glucose 2% (w / v), and agar 1.5% (w / v)) Stretched upwards. Plates were incubated overnight and the number of colonies counted the next day. TG1 cells were able to grow on an ampicillin-containing medium only if they possessed a phage-derived ampicillin resistance gene.

Cross-linking of p75 receptor-binding antibody (MC192) to M13KO7 helper phage
Monoclonal antibody preparations known as MC192 (also clone 192 first described in Chandler et al 1984) are commercially available from companies such as Cell Sciences (www.cellsciences.com) and Chemicon (www.chemicon.com). ing. These monoclonal antibodies bind to the “low affinity” (p75) nerve growth factor receptor on rat neurons. Monoclonal antibodies that bind to the human p75 receptor are also available from companies such as United States Biological (www.usbio.net).

  Unlike various other monoclonal antibodies that bind to the rat p75 receptor, the MC192 antibody can cause endocytosis of the antibody-receptor complex so that the MC192 antibody is taken up into neurons. This was shown by studies with radiolabeled antibodies (Johnson et al 1987, Yan et al 1988).

  To evaluate the ability of the MC192 antibody to perform phage endocytosis on rat neurons, a multi-step method was used to create a preparation containing the MC192 antibody chemically cross-linked to M13KO7 helper phage. . First, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (abbreviated as sulfo-SMCC, purchased from Pierce, Australia) with the primary amino group on the MC192 antibody ( (Via active sulfo-NHS ester end). This forms an amide bond between the antibody and each bridging group, and sulfo-NHS is released as a byproduct. To remove unreacted sulfo-SMCC, the activated antibody was purified using a Microcon YM-100 centrifugal filter (100 kilodalton cutoff, catalog number 42412, Millipore, USA).

  In the next step, M13KO7 phage was incubated with 2-iminothiolane (2-IT; also known as trout reagent) to form free sulfhydryl groups. These groups were located at the short ends attached to lysine residues in the phage pVIII coat protein. These phages were filtered using a YM-100 kDa filter to remove excess reagents.

  The antibody preparation was then mixed with the phage preparation in a 1:10 ratio to form thioester crosslinks from the maleimide group of the activated antibody and the sulfhydryl groups on the activated phage. The reaction product was filtered to remove excess antibody and iodoacetamide (Sigma Chemical) was added to block remaining free active sulfhydryl groups. The reaction product was precipitated twice in PEG / NaCl (20% (w / v) polyethylene glycol (average molecular weight 8000) in 2.5 M NaCl aqueous solution) to remove free antibody.

  The resulting phage mixture contained various numbers of MC192 antibodies randomly located along the entire length of the phage. Since the ratio of antibody to phage in the reaction mixture was 10: 1, it was estimated and estimated that on average about 2 to about 20 antibodies were bound to most phage particles.

Surgical treatment of rats and phage placement
Female Sprague-Dawley rats were used and surgery was performed under halothane anesthetic (2% concentration in oxygen, administered by a nose cone tube-connected to an anesthetic device). Alternatively, long-acting injection anesthetics such as sodium pentobarbiturate can be used if desired.

  Some comments on the preferred method of treatment to make this surgery a rat are given below because the chances of success are greatly increased by using the right method of treatment. Many people who are very familiar with cells and phage will not be familiar with the small animal surgery necessary to perform the in vivo treatment of the present invention.

  Note that whenever you do this type of surgery for the first time, you will almost always use a binocular microscope that can give you a 20x magnification for training to help you focus your field of view and attention to the critical areas and aspects of the procedure. I want to be. After a few minutes of treatment, the microscope is used for subsequent treatments (or when delicate and difficult parts of the treatment, such as placing the phage-containing gel foam between the nerve ends and then suturing the two ends of the sciatic nerve together) Whether or not to use can be selected. When using mice, the smaller size will use a binocular microscope for all procedures until the technician has a very high level of experience and familiarity with the procedure.

  When the rats were 6 weeks old, the first surgery was performed to “upregulate” (increase) the expression of the p75 cell receptor. When the sciatic nerve is damaged in this way, motor neurons (which have cell bodies in the spinal cord and axon fibers that project through the sciatic nerve) are stimulated and on the cell and axon surface Increased expression of p75 receptor. A test comparing the difference between the uptake of phage by pre-ligated neurons and the uptake of phage by non-ligated neurons showed that the pre-ligation step increased p75 receptor concentration and phage uptake by approximately 13-fold. Indicated. These tests included a phage uptake test and staining of tissue fragments obtained from the lumbar region of the rat spinal cord (with Mc192 antibody).

  Two other factors should also be noted for the rat p75 receptor. First, p75 receptors are present on the surface of motor neurons that send axons or other nerve fibers to muscle tissue that originates from the spinal cord and is not surrounded by the blood-brain barrier. This includes the sciatic nerve. However, there are a significant number of p75 receptors on the surface of the sciatic nerve only about two weeks after the rat's rapid growth. After about 1 to 2 weeks, the number of receptors on the sciatic nerve decreases, and until about 6 weeks of age, it is present in very small, often undetectable amounts on the sciatic nerve surface.

  The second factor of attention is as follows. Since there are not many p75 receptors on the surface of the sciatic nerve in rats older than 3 weeks, the p75 receptor endocytosis system saturates fairly easily even after controlled injury to the sciatic nerve. During this in vivo screening test, it was often clearly saturated. However, none of the results disclosed herein will be invalidated and this factor should be regarded as a “ceiling” value that cannot be exceeded. Therefore, these saturation limits can be studied and utilized in a way that confirms and validates the mechanisms and effects believed by the applicant to be active in the in vivo screening test using the p75 receptor.

  During the initial surgery, the use of any restraining device is not necessary. The animal is set on its side with its hind limbs raised, shaved, and the skin wiped. Using a surgical scissor or scalpel, an incision was made in the skin at the center of the thigh about 1-3 cm in parallel with the femur. Using the tip of a closed surgical scissors (blade length 2-4 cm), locate the femur by palpation and place the tip of the scissors through the muscle layer to the tail of the femur, depending on the size of the animal. Pressed to a depth of 2 cm. The scissors were then opened, the muscle was dissected with minimal bleeding, and a 1-2 cm window was formed exposing the sciatic nerve under the muscle. A retractor or suture can be used to keep the muscle open and maximize the surgical window, but this is not necessary for a skilled technician.

  With this invasion procedure, the sciatic nerve can be clearly seen. The sciatic nerve is only loosely attached to the surrounding tissue by an easily separated membrane. The nerve itself is protected by a strong nerve myelin sheath, and there are an estimated 50,000 axons in the nerve bundle depending on the location. Although sympathetic nerves and other nerve axons are not myelinated, motor nerve axons (and most sensory nerve axons) are derived from Schwann cells and are nerve tissue masses outside the blood-brain barrier. Surrounded by the majority of the myelin sheath.

  Place the ligature by inserting a bent forceps under the sciatic nerve. Then, the nerve is gently lifted using forceps, and if present, the loosely attached membrane is removed to a length of 1 to 2 cm. The forceps are opened, a 6/0 silk suture of a length is grasped using the forceps, and the silk suture is pulled under the nerve by pulling the forceps. The suture is then placed at a site slightly above the location where the sciatic nerve bundle is divided into two smaller bundles by tibial branch bifurcation, tied tightly around the nerve and ligated. It is important to use a non-absorbable suture such as silk or nylon. Black silk is generally preferred. This is because the elasticity is lower (to facilitate tight tightening of the ligature) and the black ligature is more easily located during subsequent surgery.

  The instrument is collected, the separated muscles are rejoined, and the skin incision is closed with 1-3 sutures depending on the length of the incision. The animal is then allowed to recover from anesthesia.

  After 7 days, the sciatic nerve was again exposed using surgical scissors and a 2-3 mm sciatic nerve fragment containing the ligature was cut.

An approximately 9 cubic millimeter collagen matrix gel foam (having a titer of about 3.3 × 10 ⁶ to about 2.1 × 10 ⁹ cfu / mL) containing 10 μL of MC192-M13KO7 antibody-phage conjugate is applied to the two cut ends of the nerve bundle. Inserted between.

  A 10-0 nylon surgical suture was used to suture together the incised ends of the sciatic nerve located on both sides of the gel foam containing the phage. A small flexible sleeve made of silicone rubber was placed around the gel foam containing the nerve ends and antibody-phage conjugate so that the antibody-phage conjugate in the gel foam remained in direct contact with both ends of the nerve fiber.

  During the same surgery with cutting and phage placement, the sciatic nerve bundle was ligated with a 6-0 silk suture at a position approximately 2 cm above the cutting site near the rat buttock. This ligation is referred to herein as a buttock ligation and is distinct from the initial ligation used to increase p75 receptor expression.

  The buttock ligation created an occlusion site that prevented antibody-phage conjugates incorporated into the sciatic nerve cells from being retrogradely transported all the way to the spinal nerve cells. Thus, antibody-phage conjugates accumulated in nerve fibers just downstream of the buttock ligation (distal).

  Rats were sacrificed by chloroform inhalation 18 hours later to allow sufficient time for endocytosis uptake and retrograde transport, and as described in the next example, a nerve segment distal to the buttock ligation Were collected.

Collecting nerves and collecting collected incorporated phage As mentioned in the previous examples, (i) a collagen gel containing phage particles was placed, and (ii) rats were sacrificed 18 hours after placing the buttock ligature. It was.

  Nerve segments were collected, including the buttock ligation and the nerve fiber segment just distal to the ligation. To that end, an additional ligature was placed around the sciatic nerve and tightened approximately 0.5 cm downstream (distal) of the buttock ligature to prevent loss of phage particles from the cut end of the nerve bundle. The segment of the sciatic nerve bundle was then excised and removed, including both two ligature rings tightly tied around the ends of the nerve segment.

  The resected nerve bundle was scrubbed 3 times with sterile PBS using forceps with sterile tissue paper until the outer membrane was removed. The nerve cells were then transferred onto a dry glass plate and the ligature was removed.

  Then 450 μL of lysis buffer (digesting cell membrane but not bacteriophage particles) was applied. The lysis buffer contains 1% Triton X-100 at pH 8, 10 mM Tris and 2 mM EDTA, and a 1/100 volume protease inhibitor mixture (Sigma Chemical) (4- (2-amino in dimethyl sulfoxide). Ethyl) -benzenesulfonyl fluoride, pepstatin A, E64, bestatin, leupeptin, and aprotinin). Nerve fibers were cut into small pieces using a scalpel in lysis buffer and the resulting suspension was transferred to an Eppendorf tube and incubated on a vortex for 1 hour at room temperature. The tube was then centrifuged at 10000 rpm for 10 minutes at 4 ° C. to pellet the sciatic nerve debris.

The supernatant (including phage particles) was collected and stored on ice. On the other hand, the debris pellet was further incubated with 300 μL of lysis buffer and vortexed for an additional hour at room temperature. The lysed debris was then incubated at room temperature for 1 hour and the sample was transferred to another eppendle tube and centrifuged at 10000 rpm for 10 minutes at 4 ° C. to pellet the remaining debris. The supernatant was collected and added to the previous supernatant, and 20% by volume CaCl ₂ was added to inactivate EDTA in the lysis buffer.

  As described in Example 25, a certain amount of the resulting mixed supernatant was titrated to determine the phage particle concentration. For another aliquot, 50% by volume of glycerol was added and the mixture was stored frozen at −20 ° C. for subsequent use or analysis. At all times except the final round of in vivo selection, an additional constant amount was used to infect E. coli cells and the same procedure as above was used to transfer the amplified phage preparation to the reagents for the next in vivo selection cycle. Used as.

Histological photo of nerve segment Using a fluorescent staining technique, a micrograph was taken to visually confirm the MC192-M13KO7 antibody-phage conjugate taken up and transported into the sciatic nerve bundle just below the buttock ligation .

  To confirm these photographs, animals were euthanized with an excessive amount of anesthesia (80 mg / kg sodium pentobarbital injected intraperitoneally). The animals were then perfused through the heart for 30 minutes with 400 mL of ice-cold 0.1 M sodium phosphate buffer containing 2% paraformaldehyde and 0.2% parabenzoquinoline. The sciatic nerve segment containing the buttock ligature was then cut and placed in the same fixative for an additional hour before being transferred to 30% sodium sucrose phosphate buffer.

  The intact nerve bundle was then embedded in OCT compound (TissueTek, Sakura Finetechnical, Tokyo, Japan) and frozen. Using a microtome, cryostat longitudinal sections (thickness 50 microns) were prepared from embedded frozen nerve bundles.

  A specific longitudinal tissue section prepared from near the center of the nerve bundle was then treated with an immunoreagent to reveal the presence and concentration of phage particles. One reagent was a rabbit-derived antibody preparation (Sigma Chemicals, catalog number B2661) with a biotin sequence that binds to the pIII capsid protein on bacteriophage particles. These antibodies were incubated with tissue sections for 1 hour at room temperature. Alexa Fluor 488 (Molecular Probes, Catalog No. S-11223) containing a streptavidin sequence that binds very tightly to the biotin sequence on the rabbit antibody that binds to the phage was then added and incubated at room temperature for 1 hour.

  Fluorescence pictures of nerve bundles covering the portion of nerve fibers containing nerve fiber segments on both sides of the buttock ligation were taken. One of those photographs copied in black and white is provided as FIG. 2 in the figure.

  The picture clearly shows that the antibody-phage conjugate has indeed accumulated at the distal side of the buttock ligation (other pictures showed very similar results). From these photographs and similar photographs from other studies, it is clear that the binding, endocytosis, and transport mechanisms described herein functioned effectively as disclosed herein. It is confirmed.

  Similar photographs from a control treatment injected with M13KO7 phage not cross-linked with a receptor-specific uptake antibody and other analytical tests showed no control phage at the same position distal to the ligation. It was.

  The foregoing tests and results using monoclonal antibodies with attached phage particles confirmed several important aspects of the invention disclosed herein. In particular, these results indicate that (i) intact linear phage particles that can bind to the p75 receptor of rat neurons can be taken up by sciatic nerve fibers and then retrograde transported, and (ii) Using the above ligation, placement, and collection process, complete and viable infectious phage particles can be incorporated into neuronal fibers based on the contact between phage and fibers that occur outside the blood brain barrier. It has been confirmed that certain ligands can be screened and selected in vivo that allow them to be taken up into and then transported within the fiber.

  Confirming a general approach to in vivo screening using nerve fibers, and devising, developing, and optimizing combinations of methods and reagents that can perform such in vivo screening tests reliably and effectively, And after confirmation, the Applicant has expanded the application of these approaches by actually testing in vivo screening of phage display libraries containing a large number of candidate ligands.

In vitro biopanning of scFv phage library
As described above, rat neuronal p75 receptors are known to have endocytic activity. It is also known that the number of receptors on the surface of nerve cells increases approximately 10 to 15 times in response to various nerve cell injuries. In rats, such damage can be controlled and reproduced in a controlled and reproducible manner by placing a rigid ligature ring around the sciatic nerve bundle just above the tibial bifurcation where the sciatic nerve divides into two main branches. Can be produced. Ligation at that site causes a substantial increase in the number of p75 receptors along the sciatic nerve fiber between the spinal cord and the ligation site. In addition, since the nerve fiber segment of the affected area with the increased number of p75 receptors is sufficiently long, it is possible to place a phage-containing gel at a certain position and subsequently collect the phage that has been taken up and transported at a remote position Become.

  All these factors make the p75 receptor useful for initial testing and validation of the in vivo selection methods disclosed herein using phage display libraries with a large number of candidate ligands. It became a possible target.

  Accordingly, Applicants choose to limit initial testing of scFv phage display libraries to candidate ligands that can specifically bind to the p75 receptor. Applicants have shown a display library (as described in Example 22 with an estimated 13 billion variable fragment antibody sequences designed to be comparable to 10 different human immune systems derived from multiple ancestors. Understand that other ligands in (containing) will be able to bind to other neuronal surface receptors that cause endocytosis and retrograde transport, but this using the scFv library The test decision and goal was to limit the test to ligands that bind to the p75 receptor.

  After confirming the general principles by the first series of studies, but also obtaining various results data (these tests and results are described in Example 30 and the selected phage in one cycle Used to evaluate the cycle iterations of in vivo screening methods to be used as starting materials for), the Applicant uses scFv phage display using an in vitro method called “biopanning” It was decided to focus on p75 receptor endocytosis by processing the library.

This in vitro method used a recombinant human p75 receptor polypeptide preparation. This polypeptide is commercially available and encodes 1 to 250 amino acid residues of the extracellular domain of the human nerve growth factor (NGF) receptor, which is linked to the Fc region of the human IgG1 protein via a peptide linker (
The carboxy terminus is fused to 6 histidines). To obtain glycosylated proteins, the chimeric protein is expressed in eukaryotic cells rather than bacterial cells using an insect cell line known as Sf21 (derived from Spodoptera frugiperda) and a baculovirus expression vector. The recombinant mature chimeric protein exists as a disulfide-linked homodimer. Each monomer contains 466 amino acids and has a calculated weight of 51 kDa. However, since glycosylation increases protein size and weight, each monomer migrates as a 90-100 kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

  These polypeptides were coated on the surface of immunotubes. For this, a 7 mL MAXISORP ™ tube (catalog number 444474, Nalge Nunc International, Denmark) with a polystyrene hydrophilic surface was used. Recombinant human p75 receptor protein at a concentration of 5 μg / mL suspended in 0.5 ml phosphate buffered saline (PBS) was incubated overnight at 4 ° C. in an immunotube.

  The next day, the tube was drained and then rinsed with PBS, filled to the edge with PBS containing 3% (w / v) skim milk and blocked for 2 hours at room temperature. On the other hand, 50 μL of scFv phage display library was pre-blocked with 450 μL of PBS containing 3% skim milk for 1 hour at room temperature in an Eppendorf tube. The immunization tube was rinsed with PBS, and 500 μL of a previously blocked phage solution was added to the tube and incubated at room temperature for 2 hours.

  After washing 15 times with PBS containing 0.1% Tween 20, the remaining bound phage was eluted by incubation with 500 μL of fresh 100 mM triethylamine (TEA) solution (pH 11) for 15 minutes at room temperature. The eluted phage was transferred to an Eppendorf tube and neutralized by adding 250 μL of 1 M Tris-HCl buffer (pH 7.4).

Half of the eluate is used to infect TG1 E. coli host cells and cultured to an OD ₆₀₀ optical density of 0.5-0.8. The other half of the eluate was saved as a reserve. The biopanned phage preparation was incubated with TG1 cells for 30 minutes at rest, followed by 30 minutes at 300 rpm shaking for 1 hour at 37 ° C. The serially diluted infected cell solution was then spread on 2TY agar plates containing ampicillin to determine the phage titer.

The remaining cells were spread on 2TY agar plates (243 mm x 243 mm) containing 4 ampicillins for growth. These plates were incubated overnight at 30 ° C. Colonies were scraped and placed in 2TY broth and grown to an OD ₆₀₀ level of 0.5-0.8. Subsequently, M13KO7 helper phage was added to a final concentration of 5 × 10 ⁹ cfu (colony forming units) per mL. This mixture was incubated at rest for 30 minutes and then in a 200 rpm shaking tray for 30 minutes. The cells were then centrifuged at 3500 rpm for 10 minutes and the cell pellet was resuspended in 25 mL of pre-warmed (without glucose) 2TY medium containing kanamycin (50 μg / mL) and ampicillin (100 μg / mL). These were incubated overnight at 25 ° C. with high speed shaking to produce phage particles. Precipitation was performed using 20% polyethylene glycol (PEG) and 2.5 M NaCl. These particles were then mixed with a collagen gel and a gel mass containing approximately 50 billion (5 × 10 ¹⁰ ) cfu of phage was placed in the rat leg during the in vivo screening process.

  Applicants wanted to preserve the diversity of p75 binding ligands to be tested in vivo as much as possible, so they performed in vitro biopanning only once before in vivo screening. Since many types of p75 binding antibodies are known not to be taken up by cells bearing the p75 receptor, this diversity would be compromised by continued in vitro screening. The concern is that tightly binding ligands appear to block and / or deform the p75 receptor in vivo, thereby preventing the ability of the p75 receptor to perform the normal endocytosis process. Additional implementations of in vitro biopanning tend to select tightly bound ligands, regardless of their ability to induce endocytosis, and are therefore significantly effective in achieving endocytotic transport into cells in vivo. The candidate ligand that is the target will be excluded.

  Nevertheless, three consecutive biopannings were performed using the scFv phage library to evaluate how this library responds to in vitro screening iterations. In order to establish comparable results, each phage solution used in the second and third rounds was diluted with PBS to match the titer of the solution used in the first round.

  The first biopanning resulted in an scFv titer of approximately 3000 cfu (as opposed to a control value of approximately 1200 cfu when PBS was tested). On the other hand, the second biopanning resulted in a very large increase to about 84 million cfu. The third biopanning resulted in a titer of about 85 million cfu, and there was no significant increase over the second biopanning.

In vivo (intrasciatic) selection of phage incorporated from scFv library Single biopanning (using human p75 receptor polypeptide) as described in Example 29, as mentioned in previous examples The “enrichment” of the scFv phage display library selected by was used as the starting reagent for a series of in vivo screening in rats. In these in vivo screens, treatments and methods developed, tested and optimized using MC192 / M12KO7 antibody-phage conjugates as described in Examples 25 and 26 were used.

  Briefly, in the first manipulation, the first ligature was placed just above the tibial nerve bifurcation of the sciatic nerve and induced an increase in p75 receptor expression on the sciatic nerve fiber above the ligation. One week later, in the second operation, the sciatic nerve bundle was cut, and a collagen gel containing about 50 billion cfu of scFv phage obtained by one p75 biopanning was packed inside the silicone rubber sleeve at the cut end. It was. During the second operation, the ligature is placed around the sciatic nerve in the buttocks area and tightened to create a barrier, and the captured and retrogradely transported phage particles are placed inside the nerve fiber immediately distal to the ligature. Accumulated. After 18 hours, in a third manipulation, the rats were sacrificed and nerve fiber segments containing nerve fibers approximately 0.5 cm distal to the buttock and ligated were collected. The collected nerve fibers were washed, cut into small pieces, and phage particles were taken out and isolated. The phage particles were amplified using E. coli cells and helper phage, and the titer was measured.

  Although the absolute number of phage recovered from excised nerve segments varied from experiment to experiment, a standardized uptake and transport measurement was obtained by always testing the control phage group and comparing the results with the test phage group data. Produced.

To illustrate, the absolute numbers from representative experiments (n = 3 for both control and test selections) are used. 5 × 10 ¹⁰ (ie, 50 billion) cfu (titer estimate) control phage (unmodified M13KO7 helper phage) was placed at the phage contact site. The value of the control phage recovered from the nerve segment excised 18 hours later was 14650 (± 2975, mean standard error (SEM), n = 3) by titration. The same amount (5 × 10 ¹⁰ cfu) of scFv phage (one biopanned for p75 recognition as described in Example 28) was placed at the phage contact site and recovered from the excised nerve segment 18 hours later The value of scFv phage was 190400 (± 14415 SEM, n = 3) by titration. Comparing these two results, it was calculated that 13 times more scFv phage (one biopanned for p75) was recovered from the excised nerve segment than the control phage. This experiment was repeated three times with similar results each time.

In order to test whether this significant increase in scFv phage uptake and transport is indeed the result of scFv binding to p75, the sciatic nerve is not pre-ligated (thus, motor neurons are more than about 2 weeks old). The experiment was repeated with another set of animals that did not increase the expression of p75 above the very low and often undetectable levels that appear in rats. In these tests, the amount of control phage (M13KO7) and test phage (scFv) applied was 5 × 10 ¹⁰ cfu as before. The value of the control phage recovered from the nerve segment excised 18 hours later was 14815 ± 4481 (n = 3). The value of scFv phage (biopanned once for p75 recognition) recovered from nerve segments excised 18 hours later was 16413 ± 4541 (n = 3). From these data, when rats with very low levels of p75 receptor originally found in rats over 6 weeks of age were tested, cell uptake of scFv phage (one biopanned for p75 recognition) and It was clearly shown that the efficiency of transport is not essentially different from that of control phage. This confirmed that there was a clear relationship between the expression level of p75 and the efficiency of uptake and transport of scFv phage biopanned for p75 receptor recognition. This test was repeated three times with similar results.

Cycle test of scFv library without panning In a series of tests conducted prior to determining and using in vitro biopanning treatment (described in Example 28), sciatic nerve fibers were analyzed as described in Examples 25 and 26. In use, scFv libraries were tested by a series of cycling in vivo screening methods. These screens are referred to as “cycled” because the screen-selected phage population obtained from one test is used as the starting reagent in the next test cycle.

Although these tests provided clear evidence that the phage library contains specific phage-mediated ligands that activate and perform endocytotic uptake and retrograde transport, the results of these cyclic tests are highly variable did. Due to data variability, Applicants have concluded that the data is consistent with the following interpretation:
(i) Due to the limited number of p75 receptors on the nerve fiber surface, the ligand-receptor binding and uptake process was saturated;
(ii) The administered phage population is very diverse, with only one or a small number of any particular phage present at the first time, and hundreds or even thousands of different phage candidates in subsequent rounds. Seemed to be;
(iii) Therefore, it was very unlikely that any one particular phage was selected in two different experiments in different animals (this possibility is that any particular phage in the test group Can be considered to be approximately equal to the number of copies divided by the number of phage instead of efficiently harvesting from the nerve bundle).

  These factors can clearly explain the very high variability seen between different experiments with different animals. Thus, after confronting and contemplating such high variability, Applicants reduced the variability within the dosing group and determined the copy number of p75 binding phage candidates that could be used to extract from the nerve bundle. It was decided to perform the experiment with a pre-screening step (ie in vitro biopanning) to increase substantially.

  From the data obtained from the scFv library test performed prior to the pre-screening step, the first, second and possibly third consecutive screening cycles all seem to provide better efficiency in cellular uptake and retrograde transport. It was shown that there is. However, under the specific conditions used, their efficiency tended to decrease when more in vivo screening was used. The cause of the resulting decline was not clear. On the other hand, it is reasonable that a significant proportion of phage selected from the second or third round of selection display ligands that bind to neuronal receptors and stimulate uptake and retrograde transport. Can be predicted (because they were selected repeatedly, they are unlikely to be false positives).

  Cycle in vivo screening has not yet been thoroughly evaluated, and has not yet been tested, particularly with phage groups that have been prescreened by biopanning or similar approaches. Nevertheless, I believe that the work done so far has clearly demonstrated and confirmed that:

  (1) With at least some types of phage groups, a series of cyclic in vivo screens using a candidate group selected in one screening as the starting reagent in the next screening is actually possible, and at least some In some cases, it appears to help identify and isolate candidate ligands that are highly effective in inducing and performing endocytotic transport into the cell.

  (2) It is possible to develop a numerical index that gives a useful index for the point in time at which the cycle method should be stopped, and thus candidate ligands that seem to have given the best performance identified up to that point in the screening method Can be carefully analyzed and sequenced.

  (3) It appears that at least some phage ligands stimulated uptake and retrograde transport after binding to surface molecules not previously known to mediate endocytosis.

  Finally, when considering the implications of these analyses, the fundamental and critical goal of this type of in vivo screening is highly enriched, which can provide thousands of phage candidates that are taken up by nerve fibers. Note that it is not to create “good” phage populations. Instead, this goal is to identify and isolate only one or a few specific ligands (and in the case of polypeptide ligands) that are very effective in activating and executing the endocytotic uptake process. Determining its nucleotide gene sequence and / or amino acid sequence). These types of ligands, once identified and isolated, can be replicated in large quantities and are useful transports into specific classes of cells that have specific target endocytosis receptors or similar molecules on their surface. Alternatively, it can be incorporated into a molecular complex that transports a carrier molecule.

  The biopanning process described above using p75 polypeptide sequences to pre-screen scFv phage libraries can be performed on any known or suspected endocytosis receptor or other surface molecule suspected of having endocytosis activity. Note that known polypeptide sequences or fragments derived from them can be used (as “antigen” molecules that adhere to the surface of immune tubes). Also, any glycosylated cell surface molecule suspected of having endocytotic activity can be used.

In Vivo Selection Using PhD-C7C Phage Library As briefly mentioned in Example 22, the PhD-C7C phage display library was inserted near the DNA sequence encoding the N-terminus of the pIII capsid protein of M13 phage 7 Contains an estimated 2 billion phage with foreign DNA inserts encoding a random sequence of amino acids. This library provided a repertoire of essentially random peptide sequences that were tested to determine whether a phage was taken up and transported by neurons in the sciatic nerve bundle.

  In vivo screening of the PhD-C7C library using the sciatic nerve processing described above showed that this library was implemented exactly as expected. A considerable number of phages were taken up by the sciatic nerve fibers and transported to the phage accumulation region just distal to the buttock ligation. Specific phages are removed from the collected nerve segments in a viable form, and these p75-selected viable phages can be replicated and manipulated by any of the methods described above.

  If desired, using biopanning techniques as indicated above (as described above for p75 biopanning of scFv libraries), using any known and available receptor polypeptide sequence as a biopanning antigen, The PhD-C7C library can be screened in advance.

  Thus, (i) to identify ligands that efficiently activate and carry out the endocytosis process of cells through specific endocytosis molecules present on the surface of only a limited number and type of cells. In addition, new and useful methods for using in vivo screening, and (ii) efficient transport of useful transport or transport molecules into cells with targeted endocytosis receptors or other surface molecules New and useful methods for incorporating their ligands into molecular complexes that can be used to do so are described. Although the present invention has been described with reference to particular embodiments for purposes of illustration and description, it will be apparent to those skilled in the art that various modifications, variations, and equivalent exchanges of the illustrated examples are possible. it is obvious. Any modification obtained directly from the teachings herein and without departing from the spirit and scope of the invention is deemed to be within the scope of the invention.

  Thus, novel for the non-invasive transport of therapeutic or other useful polypeptides through the blood brain barrier into brain and spinal cord tissue (especially into nerve cells located entirely within the blood brain barrier) And useful means, and in vivo screening to identify ligands that efficiently activate and execute cellular endocytosis processes through specific endocytotic molecules present on the surface of only a limited number and type of cells A new and useful means for using is shown and described. Although the present invention has been described with reference to certain specific embodiments for purposes of illustration and description, it will be appreciated that various modifications, changes, and equivalent exchanges of the illustrated examples are possible. It will be apparent to those skilled in the art. Any modification obtained directly from the teachings herein and without departing from the spirit and scope of the invention is deemed to be within the scope of the invention.

Cited references
Abbott, NJ, et al, "Transporting therapeutics across the bloodbrain barrier," Mol. Med. Today 2 (3): 10613 (1996)
Altar, CA, et al, "Receptormediated transport of human recombinant nerve growth factor from olfactory bulb to forebrain cholinergic nuclei," Brain Res. 541: 828 (1991)
Banks, WA, et al, "Passage of peptides across the bloodbrain barrier: pathophysiological perspectives," Life Sci. 59 (23): 192343 (1996)
Banks, WA, "Physiology and pathology of the bloodbrain barrier: implications for microbial pathogenesis, drug delivery and neurodegenerative disorders," J. Neurovirol. 5 (6): 53855 (1999)
Barbacid, M., "Neurotrophic factors and their receptors," Curr. Opin. Cell Biol. 7: 148-155 (1995)
Barde, YA "The nerve growth factor family," Prog. Growth Factor Res. 2: 237-248 (1991)
Bartlett, JS, et al, "Methods for the construction and propagation of recombinant adeno-associated virus vectors," pp. 25-40 in Robbins 1997
Barnett, EM, et al, "Two neurotropic viruses, herpes simplex virus Type 1 and mouse hepatitis virus, spread along different neural pathways from the main olfactory bulb," Neurosci. 57: 1007-1025 (1993)
Baumgartner, BJ, et al, "Neuroprotection of spinal motor neurons following targeted transduction with an adenovirus vector carrying the gene for glial cell-derived neurotrophic factor," Exp. Neurol. 153: 102-112 (1998)
Becerril, B., et al, "Towards selection of internalizing antibodies from phage libraries," Biochem. Biophys. Res. Comm. 255: 386-393 (1999)
Begley, DJ, "The bloodbrain barrier: principles for targeting peptides and drugs to the central nervous system,"
J. Pharm. Pharmacol. 48 (2): 13646 (1996)
Bennet, DL, et al, "NGF but not NT-3 or BDNF prevents the A fiber sprouting into lamina II of the spinal cord that occurs following axotomy," Mol. Cell. Neurosci. 8: 211-220 (1996)
Blesch, A., et al, "Ex vivo gene therapy for Alzheimer's disease and spinal cord injury," Clin. Neurosci. 3: 268-274 (1996)
Blessing, WW, et al, "Transneuronal labeling of CNS neurons with herpes simplex virus," Prog. Neurobiol. 44: 37-53 (1994)
Bolivar, F., et al, "Construction and characterization of new cloning vehicles: A multipurpose cloning system," Gene 2: 95113 (1977)
Bonate, PL, "Animal models for studying transport across the bloodbrain barrier," J. Neurosci. Methods 56 (1): 115 (1995)
Bothwell, M., "Functional interactions of neuurotrophins and neuurotrophin receptors," Ann. Rev. Neurosci. 18: 223-253 (1995)
Card, JP, et al, "Neurotropic properties of pseudorabies virus: Uptake and transneuronal passage in the rat central nervous system," J. Neurosci. 10: 1974-1994 (1990)
Causing, CG, et al, "Construction of transgenic animals overproducing neurotrophins and their receptors," Methods in Molecular Biology 169: 167-191 (2000)
Chie, E., et al, "Quantification of neurotrophin mRNA by RT-PCR," Methods in Molecular Biology 169: 81-90 (2000)
Choi-Lundberg, DL, et al, "Dopaminergic neurons protected from degeneration by GDNF gene therapy," Science 275: 838-841 (1997)
Christensen, MD, et al, "Anti-NGF prevents thermal allodynia in chronic central pain following spinal cord injury," Soc. Neurosci. Abstr. # 208.4 (1996)
Christensen, MD, et al, "Spinal cord injury and anti-NGF treatment results in changes in CGRP density and distribution in the dorsal horn of the rat," Exp. Neurol. 147: 463-475 (1997)
Cloughesy, TF, et al, "Pharmacological bloodbrain barrier modification for selective drug delivery," J. Neuroncol. 26 (2): 12532 (1995)
Conner, JM, et al, "The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation," J. Comp. Neurol. 319: 454-462 (1992)
Conner, JM, "Localization of neurotrophin proteins within the central nervous system by immunohistochemistry," Methods in Molecular Biology, Vol 169: 3-20 (2000)
Cuello, AC, et al, "Trophic effects on cholinergic innervation in the cerebral cortex of the adult rat brain," Mol. Neurobiol. 6: 451-461 (1992)
Cunningham, AM, et al, "Olfactory receptor neurons exist as distinct subclasses of immature and mature cells in primary culture," Neurosci. 93: 1301-1312 (1999)
Curiel, DT, "Methods for gene transfer using DNA-Adenovirus conjugates," Methods in Molecular Medicine: 25-40 (1997)
Davis, TP, et al, "Conformationally constrained peptide drugs targeted at the bloodbrain barrier," NIDA Res. Monogr. 154 (1): 4760 (1995)
de Boer, AG, et al, "The transference of results between bloodbrain barrier cell culture systems," Eur. J. Pharm. Sci. 8 (1): 14 (1999)
Deng, YS, et al, "Effects of inherent neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury," Exp. Neurol. 164: 344-350 (2000)
Dijkhuizen, PA, et al, "Adenoviral vector-directed expression of neurotrophin-3 in rat dorsal root ganglion explants results in a robust neurite outgrowth response," J Neurobiol. 33: 172-184 (1997)
DiStephano, PS, et al, "The neurotrophins BDNF, NT-3 and NGF display distinct patterns of retrograde transport in peripheral and central neurons," Neuron 8: 983-993 (1992)
Engelhardt, JF, "Methods for adenovirus-mediated gene transfer to airway epithelium," pp. 169-184 in Robbins 1997
Federoff, HJ, et al, "Expression of nerve growth factor in vivo from a defective herpes simplex virus 1 vector prevents effects of axotomy on sympathetic ganglia," Proc. Natl. Acad. Sci. USA 89: 1636-1640 (1992)
Ferguson, IA, "Basic fibroblast growth factor: Receptor mediated internalization, metabolism, and anterograde axonal transport in retinal ganglion cells," J. Neurosci. 10: 21762189 (1990)
Ferguson, IA, "Receptormediated retrograde transport in CNS neurons following intraventricular administration of NGF and growth factors," J. Comp. Neurol. 313: 680692 (1991)
Fiers, W., et al, "Complete nucleotide sequence of SV40 DNA," Nature 273: 11320 (1978)
Findeis, MA, et al, "Methods for targeted gene transfer to liver using DNA-protein complexes," pp. 135-152 in Robbins 1997
Fischer, W., et al, "Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor," Nature 329: 65-68 (1987)
Fischer, W., et al, "NGF improves spatial memory in aged rats as a function of age," J. Neurosci. 11: 1889-1906 (1991)
Fisher, LJ, "In vivo and ex vivo gene transfer to the brain," Curr. Opin. Neurobiol. 4: 735-41 (1994)
Funakoshi, H., et al, "Targeted expression of a multifunctional chimeric neurotrophin in the lesioned sciatic nerve accelerates regeneration of sensory and motor axons," Proc. Natl. Acad. Sci. USA 95: 5269-5274 (1998)
Garofalo, L., et al, "Nerve growth factor-induced synaptogenesis and hypertrophy of cortical cholinergic terminals," Proc. Natl. Acad. Sci. USA 89: 2639-2643 (1992)
Garofalo, L., et al, "Nerve growth factor and the monsialoganglioside GM1," Exp. Neurol. 125: 195-217 (1994)
Gavel, C., et al, "Adenoviral gene transfer of ciliary neurotrophic factor and brainderived neurotrophic factor leads to longterm survival of axotomized motor neurons," Nat. Med. Jul; 3 (7): 76570 (1997)
Giehl, KM, et al, "BDNF and NT-3, but not NGF, prevent axotomy induced death of rat corticospinal neurons in vivo," Eur. J. Neurosci. 8: 1167-1175 (1996)
Giehl, KM, et al, "GDNF is a trophic factor for adult rat corticospinal neurons and promotes their long-term survival after axotomy in vivo," Eur. J. Neurosci. 9: 2479-2499 (1997)
Goins, WF, et al, "Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxidase toxicity," J. Virol. 73: 519-532 (1999)
Goldstein, GW, et al, "The bloodbrain barrier," Sci. Am. 255: 7483 (1986)
Graham, FL, et al, "Methods for construction of adenoviral vectors," Mol. Biotechnol. 3: 207-220 (1995)
Granholm, AC, et al, "A noninvasive system for delivering neural growth factors across the bloodbrain barrier: a review,"
Rev. Neurosci. 9 (1): 3155 (1998)
Guegan, C., et al, "Reduction in cortical infarction and impairment of apoptosis in NGF-transgenic mice subjected to permanent focal ischemia," Brain Res. Mol. Brain Res. 55: 133-140 (1998)
Hart, SL, et al, "Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide," J. Biol. Chem. 269: 12468-12474 (1994)
Hefti, F., "Neurotrophic factor therapy for nervous system degenerative diseases," J. Neurobiol. 25: 1418-1435 (1994)
Holtmaat, AJGD, et al, "Efficient adenoviral vector-directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium," Mol. Brain Res. 41: 148-156 (1996)
Horowitz, LF, "A genetic approach to trace neural circuits," Proc Natl. Acad. Sci. 96: 3194-3149 (1999)
Hottinger, AF, et al, "Complete and long-term rescue of lesioned adult motoneurons by lentiviral-mediated expression of glial cell line-derived neurotrophic factor in the facial nucleus," J. Neurosci. 20: 5587-5593 (2000)
Iwasaki, K., et al, "The effects of the traditional Chinese medicine" Banxia Houpe Tang (Hange-Koboku To) "on the swallowing reflex in Parkinson's disease," Phytomedicine 7: 259-263 (2000)
Janes, KA, et al, "Polysaccharide colloidal particles as delivery systems for macromolecules," Adv. Drug Deliv. Rev. 23: 8397 (2001)
Kaplitt, MG, et al, "Defective viral vectors as agents for gene transfer in the nervous system," J. Neurosci. Methods 71: 125-132 (1997)
Karpati, G., et al, "The principles of gene therapy for the nervous system," Trends Neurosci. 19: 49-54 (1996)
Kastin, AJ, et al, "Peptides crossing the bloodbrain barrier: some unusual observations," Brain Res. 27 848 (12): 96100 (1999)
Kniesel, U., et al, "Tight junctions of the bloodbrain barrier," Cell. Mol. Neurobiol. 20 (1): 5776 (2000)
Knight, A., et al, "Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin," Eur. J. Biochem. 259: 762-769 (1999)
Krenz, NR, et al, "Neutralizing intraspinal nerve growth factor blocks autonomic dysreflexia caused by spinal cord injury," J. Neurosci. 19: 7405-7414 (1999)
Krenz, NR, et al, "Nerve growth factor in glia and inflammatory cells of the injured rat spinal cord," J. Neurochem. 74: 730-739 (2000)
Kroll, RA, et al, "Outwitting the bloodbrain barrier for therapeutic purposes: osmotic opening and other means," Neurosurgery 42 (5): 108399; discussion 1099100 (1998)
Lachmann, RH, et al, "Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system," J. Virol. 71: 3197-3207 (1997)
Lafay, F., et al, "Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation," Virol. 183: 320-330 (1991).
La Salle, G., et al, "An adenovirus vector for gene transfer into neurons and glia in the brain," Science 259: 988-990 (1993)
Langer R., "New methods of drug delivery," Science 249: 1527-33 (1990)
Langer, R., et al, "New directions in CNS drug delivery," Neurobiol. Aging 10: 642-4 (1989)
Lewin, GR, et al, "Physiology of the neurotrophins," Ann. Rev. Neurosci. 19: 289-317 (1996)
Li, S., et al, "Nonviral gene therapy: Promises and challenges," Gene Therapy 7: 31-34 (2000)
Lim, ST, et al, "Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan," J. Control Release 66: 28192 (2000)
Lindsay, RM, "Neurotrophins and receptors," Prog. Brain Res. 103: 3-14 (1994)
Lindsay, RM, et al, "GDNF in a bind with known orphan: Accessory implicated in a new twist," Neuron 17: 571-574 (1996)
Lo, DC, "Neurotrophic factors and synaptic plasticity," Neuron 15: 979-981 (1995)
Madrid, Y., et al, "New directions in the delivery of drugs and other substances to the central nervous system," Adv. Pharmacol. 22: 299324 (1991)
Martinez-Fong, D., et al, "Neurotensin-SPDP-poly-L-lysine conjugate: a non-viral vector for targeted gene delivery to neural cells," Brain Res. Mol. Brain Res. 69: 249-262 ( 1999)
McMahon, SB, et al, "Peripheral neuropathies and neurotrophic factors: Animal models and clinical perspectives," Curr. Opin. Neurobiol. 5: 616-624 (1995)
Moller, JC, et al, "Subcellular localization of epitope-tagged neurotrophins in neuroendocrine cells," J. Neurosci. Res. 51: 463-472 (1998)
Nabel, EG, "Methods for liposome-mediated gene transfer to the arterial wall," pp. 127-133 in Robbins 1997
Novikova, L., et al, "Survival effects of BDNF and NT-3 on axotomised rubrospinal neurons depend on the temporal pattern of neurotrophin administration," Eur. J. Neurosci. 12: 776-780 (2000)
Ozawa, CR, et al, "Neuroprotective potential of a viral vector system induced by a neurological insult," Proc. Natl. Acad. Sci. USA 97: 9270-9275 (2000)
Pardridge, WM, "Bloodbrain barrier biology and methodology," J. Neurovirol. 5 (6): 55669 (1999)
Pardridge, WM, "CNS drug design based on principles of bloodbrain barrier transport," J. Neurochem. 70 (5): 178192 (1998)
Phelps, CJ, et al, "Pituitary hormones as neurotrophic signals," Proc. Soc. Exp. Biol. Med. 222: 3958 (1999)
Pickard, GE, et al, "Intravitreal injection of the attenuated pseudorabies virus PRV Bartha results in infection of the hamster suprachiasmatic nucleus only by retrograde transsynaptic transport via autonomic circuits," J Neurosci 22: 2701-2710 (2002)
Price, DL, et al, "Tetanus toxin: direct evidence for retrograde intraaxonal transport," Science 188: 945-947 (1975)
Qasba, P., et al, "Tissue and zonal-specific expression of an olfactory receptor transgene," J. Neurosci. 18: 227-236 (1998)
Rapoport, SI, "Osmotic opening of the bloodbrain barrier: principles, mechanism, and therapeutic applications," Cell. Mol. Neurobiol. 20: 21730 (2000)
Rillosi, M., et al, "Modeling mucoadhesion by use of surface energy terms obtained from the Lewis acidLewis base approach.II. Studies on anionic, reactive, and unionisable polymers," Pharm. Res. 12: 66975 (1995)
Robbins, P., ed., Methods in Molecular Medicine: Gene Therapy Protocols (Humana Press, Totowa, NJ, 1997)
Rochat, B., et al, "Drug disposition and targeting. Transport across the bloodbrain barrier," Pharm. Biotechnol. 12: 181200 (1999)
Romero, MI, et al, "Adenoviral gene transfer into the normal and injured spinal cord," Gene Ther. 5: 1612-161 (1997)
Romero, M., et al, "Extensive sprouting of sensory afferents and hyperalgesia induced by conditional expression of nerve growth factor in the adult spinal cord," J. Neurosci. 20: 4435-4445 (2000)
Ruberti, F., et al, "Cloning and expression of an anti-nerve growth factor (NGF) antibody for studies using the neuroantibody approach," Cell. Mol. Neurobiol. 13: 559-568 (1993)
Ruberti, F., et al, "The use of the RACE method to clone hybridoma cDNA when V region primers fail," J. Immunol. Methods 173: 33-39 (1994)
Ruberti, F., et al, "Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe defects in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy," J. Neurosci. 20: 2589-2601 ( 2000)
Rubin, LL, et al, "The cell biology of the bloodbrain barrier," Annu. Rev. Neurosci. 22: 1128 (1999)
Rush, RA, et al, "Neurotrophin immunohistochemistry in peripheral tissues," Methods in Molecular Biology, Vol 169: 21-29 (2000)
Sahenk, Z., et al, "Gene delivery to spinal motor neurons," Brain Res. 606: 126-129 (1993)
Sawchenko, PE, et al, "Circuits and mechanisms governing hypothalamic responses to stress," Prog. Brain Res. 122: 6178 (2000)
Schacht, CM, et al, "GDNF application into cerebrospinal fluid prevents axotomy-induced death of adult rat corticospinal neurons in vivo," Soc. Neurosci. Abstr. # 767.1 (1996)
Schatz, PJ, "Construction and screening of biological peptide libraries," Curr. Opin. Biotech. 5: 487-494 (1994)
Schipper, NG, et al, "Chitosans as absorption enhancers of poorly absorbable drugs. 3: Influence of mucus on absorption enhancement," Eur. J. Pharm. Sci. 8 (4): 33543 (1999)
Schwab, ME, et al, "Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport," J. Cell Bio. 82: 789-810 (1979)
Schnell, L., et al, "Neurotrophin 3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion," Nature 367: 170-173 (1994)
Semkova, I., et al, "Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors," Brain Res. Rev. 30: 176-188 (1999)
Servenius, B., et al, "Metastasizing neuroblastomas in mice transgenic for SV40T under the olfactory marker protein gene promoter," Cancer Res. 54: 5198-5205 (1994)
Skaper, SD, et al, "Neurotrophic molecules: Strategies for Designing Effective Therapeutic Molecules in Neurodegeneration," Mol. And Cell. Neuroscience 12: 179-193 (1998)
Smith, GM, et al, "Adenoviralmediated gene transfer to enhance neuronal survival, growth, and regeneration," J. Neurosci. Res. 55: 14757 (1999)
Snider, WD, "Functions of the neurotrophins during nervous system development," Cell 77: 627-638 (1994)
Sofroniew, MW, et al, "Atrophy but not death of adult septal cholinergic neurons after ablation of target capacity to produce mRNAs for NGF, BDNF, and NT-3," J. Neurosci. 13: 5263-5276 (1993)
Staecher, H., et al, "Brain derived neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss," Otolaryngol Head Neck Surg. 119: 7-13 (1998)
Thoenen, H., et al, "Neurotrophic factors: Possibilities and limitations in the treatment of neurodegenerative disorders," Int. Acad. Biomed. Drug Res. 7: 197-203 (1994)
Travers, JB, et al, "Transneuronal labeling in hamster brainstem following lingual injections with herpes simplex virus-1," Neurosci. 68: 1277-1293 (1995)
Turnbull, AV, et al, "Regulation of the hypothalamic-pituitary-adrenal axis by cytokines," Physiol. Rev. 79: 171 (1999)
Ugolini, G., "Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups," J. Comp. Neurol. 356: 457-480 (1995 )
von Bartheld, CS "Tracing with radiolabeled neurotrophins," Methods in Molecular Biology 169: 193-214 (2000)
Watson, A., et al, "A minimal CGRP gene promoter is inducible by nerve growth factor in adult dorsal root ganglion neurons but not PC12 phaeochromocytoma cells," Eur. J. Neurosci. 7: 394-400 (1995)
Wenk, H., et al, "Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats," Brain Res. 2: 295316 (1980)
Wiley, RG, et al, "Preparation of anti-neuronal immunotoxins for selective neuronal immunolesioning," Neurosci. Protocols 93-020: 1-11 (1993)
Winter, G., et al, "Making antibodies by phage display technology," Ann. Rev. Immunol. 12: 433-455 (1994)
Wong, ET, et al, "Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes," Gene Therapy 9: 337-344 (2002)
Xian, CJ, et al, "Sensitive and non-radioactive in situ detection of neurotrophin mRNAs in the nervous system," Meth. Molec. Biol. 169: 91-96 (2000
Yan, Q., et al, "Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: Assessment of potential neurotrophic effects," Neuron 1: 335-343 (1998)
Yoshihara, Y., et al, "A genetic approach to visualization of multi-synaptic neuronal pathways using plant lectin transgene," Neuron 22: 33-41 (1999)
Yuen, EC, et al, "Therapeutic potential of neurotrophic factors for neurological disorders," Ann. Neurol. 40: 346-354 (1996)
Zhang, S.-H., et al, "Extraction and quantification of neurotrophins," Meth. Molec. Biol. 169: 31-41 (2000)
Zlokovic, BV, "Cerebrovascular permeability to peptides: manipulations of transport systems at the bloodbrain barrier,"
Pharm. Res. 12 (10): 1395406 (1995)
Barry, MA, et al, "Toward celltargeting gene therapy vectors: selection of cellbinding peptides from random peptidepresenting phage libraries," Nature Medicine 2: 299305 (1996).
Becerril, B., et al, "Toward selection of internalizing antibodies from phage libraries," Biochemical & Biophysical Research Communications 255: 38693 (1999).
Chandler, CE, et al, "A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells," Journal of Biological Chemistry 259: 68829 (1984)
Edwards, PJ, "Purification strategies for combinatorial and parallel chemistry," Comb Chem High Throughput Screen 6: 1127 (2003)
Edwards, PJ, et al, "Solidphase compound library synthesis in drug design and development," Curr Opin Drug Discov Devel 5: 594605 (2002)
Ernfors, P., et al, "Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons," Neuron 2: 160513 (1989).
Ferguson, IA, et al, "Comparison of wheat germ agglutininhorseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury," Journal of Neuroscience Methods 109: 819 (2001)
Flynn, DL, et al, "Recent advances in chemical library synthesis methodology," Curr Opin Drug Discov Devel 5: 58093 (2002)
Geysen, HM et al, "Combinatorial compound libraries for drug discovery: an ongoing challenge," Nat Rev Drug Discov 2: 22230 (2003)
Hart, SL, et al, "Cell binding and internalization by filamentous phage displaying a cyclic ArgGlyAspcontaining peptide," Journal of Biological Chemistry 269: 1246874 (1994)
Johnson, D., et al, "Expression and structure of the human NGF receptor," Cell 47: 54554 (1986)
Johnson, EM, Jr. et al, "Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system," Journal of Neuroscience 7: 9239 (1987)
Griffin, JW, et al, "The pathogenesis of reactive axonal swellings: role of axonal transport," Journal of Neuropathology & Experimental Neurology 36: 21427 (1977)
Griffiths, AD & Duncan, AR, "Strategies for selection of antibodies by phage display," Current Opinion in Biotechnology 9: 1028 (1998)
Ivanenkov, V., et al, "Uptake and intracellular fate of phage display vectors in mammalian cells," Biochimica et Biophysica Acta 1448: 45062 (1999)
Ivanenkov, VV & Menon, AG, "Peptidemediated transcytosis of phage display vectors in MDCK cells," Biochemical & Biophysical Research Communications 276: 2517 (2000)
Johnson, EM, et al, "Expression and possible function of nerve growth factor receptors on Schwann cells," Trends in Neurosciences 11: 299304 (1988)
Larocca, D., et al, "Targeting bacteriophage to mammalian cell surface receptors for gene delivery," Human Gene Therapy 9: 23939 (1998)
Larocca, D., et al, "Receptortargeted gene delivery using multivalent phagemid particles," Molecular Therapy: the Journal of the American Society of Gene Therapy 3: 47684 (2001)
Lepre, CA, "Applications of SHAPES screening in drug discovery," Comb Chem High Throughput Screen 5: 58390 (2002)
Ley, SV, et al, "New tools and concepts for modern organic synthesis," Nat Rev Drug Discov 1: 57386 (2002)
Liu, R., et al, "Combinatorial peptide library methods for immunobiology research," Exp Hematol 31: 1130 (2003)
Lockhoff, O., et al, "Glycoconjugate libraries accessed by multicomponent reactions," Comb Chem High Throughput Screen 5: 36172 (2002)
Poul, MA, et al, "Selection of tumorspecific internalizing human antibodies from phage libraries," Journal of Molecular Biology 301: 114961 (2000)
Poul, MA & Marks, JD, "Targeted gene delivery to mammalian cells by filamentous bacteriophage," Journal of Molecular Biology 288, 20311 (1999)
Ramstrom, O., et al, "Chemical biology of dynamic combinatorial libraries," Biochim Biophys Acta 1572: 17886 (2002)
Yan, Q., et al, "Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects," Neuron 1: 33543 (1988)

FIG. 1 shows that a genetic vector is transfected into an exposed projection (“mucus tip”) of olfactory receptor neurons to deliver therapeutic polypeptides through the BBB to neurons completely located within the BBB. . In transfected olfactory receptor neurons, polypeptides are expressed from genes in the vector and transported through nerve axons that penetrate the BBB. The polypeptide can then be secreted into the BBB by neurons across the BBB and come into contact with central nervous system neurons that are completely located within the BBB, such as cholinergic neurons in the basal forebrain. FIG. 2 shows a viral vector for transfecting neurons. This vector includes a capsid shell, a surface-bound ligand, and a genetically modified genome containing a useful “cargo” or “cargo” gene. FIG. 3 shows transfection of olfactory receptor neurons. In addition, the polypeptide encoded in the vector moves and reaches and contacts the terminal end located at the tip of the neurite that is part of the cholinergic neuron in which the main cell body exists in the basal ganglia For the path through the olfactory bulb. FIG. 4 (FIGS. 4A, 4B, and 4C) shows the complete location within the BBB using genetic vectors to suppress unnecessary and excessive neural connections or activity seen in conditions such as neuropathic pain. Shows delivery of an anti-neurotrophic polypeptide to a neurotrophic factor producing cell. In this method, an “NPC (Neuropathic Pain Control)” vector is transfected into nociceptive neurons that innervate tissues such as skin. Nociceptive neurons express polypeptides with anti-neurotrophic activity encoded in the vector and release them to the spinal cord tissue. As shown in FIG. 4B (showing neural connections that send a large number of unwanted pain signals abnormally before treatment) and FIG. 4C (showing neural connections that send reduced unwanted pain signals after treatment), Blocking or inhibiting neurotrophic receptors or factors results in suppression or atrophy of excessive neural connections that induce or exacerbate neuropathic pain symptoms. FIG. 5 (FIGS. 5A, 5B, and 5C) shows intramuscular “spinal motoneurons” to deliver therapeutic polypeptides to a type of neuron that is completely located within the BBB (referred to as upper motor neurons). Shows the transfection of the gene vector. This type of treatment is performed in patients with limbs that are paralyzed or damaged due to, for example, stroke, injury or illness. As shown in FIG. 5A, gene vectors are injected into muscle tissue and transfected into spinal motor neurons that cross the BBB, and those neurons express a polypeptide with neurotrophic activity encoded in the vector. Then release those polypeptides into the spinal cord tissue, thereby delivering the neurotrophic polypeptides to the upper motor neurons that are completely located within the BBB. FIG. 5B shows the stroke status or similar patient condition prior to treatment. There, some (but not all) of the neural “processes” of the upper motor neurons degenerate and / or atrophy, and can no longer fully interact with spinal motor neurons, leading to limb damage or paralysis. FIG. 5C shows the improved condition after treatment with nerve growth factor. In that state, still functional neurites belonging to one of the upper motor neurons form new and additional synaptic connections ("sprouting"), and spinal motor neurons that were not well innervated before treatment Can interact with cells. FIG. 6 shows transfecting a gene vector to the projection of lower motor neurons of the hypoglossal nucleus (part of the brainstem) to deliver therapeutic polypeptides through the BBB to neurons located entirely within the BBB. Indicates. The exposed end of these neuroprojections is on the tongue. Thus, this type of nerve cell provides a relatively short route for introducing a foreign polypeptide into the brainstem portion of the central nervous system. FIG. 7 shows that two ligatures were placed around the rat sciatic nerve bundle, and that the collagen gel containing the phage was incorporated into the sciatic nerve bundle so that the phage was incorporated into the sciatic nerve fiber via endocytosis. It schematically shows that it is arranged in contact with the cut end. FIG. 8 is a photograph of a fluorescently labeled antibody-phage conjugate collected in the sciatic nerve bundle next to the buttock ligation. Ligation (rings clamped with sutures) prevents antibody-phage conjugates taken up by sciatic nerve fibers from being retrogradely transported across the ligation site. FIG. 9 schematically shows a cycle-type in vivo ligand selection method. In this method, according to the in vivo methods disclosed herein, a ligand-displayed phage is selected from a phage display library for endocytosis uptake by neurons and the group of phage selected by one cycle is selected for the next cycle. Used as starting material for screening.

Claims (93)

A method for delivering a neuroactive polypeptide molecule through the blood-brain barrier of a higher animal comprising the following steps:
(A) Generating a gene vector having at least one gene encoding a neuroactive polypeptide and configured to be able to transfect at least one accessible neuron across the blood brain barrier The process of
Wherein each accessible neuron has at least one peripheral projection capable of accessing a compound that has not crossed the animal's blood-brain barrier;
When a gene vector is administered to a higher animal, it is administered in such a way that at least one gene vector and at least one peripheral projection are in direct contact;
After transfection of the gene vector into accessible neurons, the gene encoding the neuroactive polypeptide can express a plurality of neuroactive polypeptides in the accessible neurons; and (b) (i) at least One gene vector is transfected in contact with at least one accessible neuron; and (ii) at least one accessible neuron expresses a neuroactive polypeptide encoded by the gene; Subsequently administering the gene vector to the higher animal in a position and manner that ensures that the neuroactive polypeptide is secreted into the central nervous system tissue protected by the blood brain barrier.   The method of claim 1, wherein the accessible neuronal cell that spans the blood brain barrier is a sensory neuron.   The method of claim 2, wherein the sensory neuron is an olfactory receptor neuron.   The method of claim 2, wherein the sensory neuron is a nociceptive neuron.   The method of claim 1, wherein the accessible neurons that straddle the blood brain barrier are motor neurons.   6. The method of claim 5, wherein the motor neuron is a spinal motor neuron.   6. The method of claim 5, wherein the motor nerve cell is a motor nerve of the hypoglossal nucleus.   2. The method of claim 1, wherein the accessible neuronal cell that spans the blood brain barrier is a parasympathetic preganglionic neuron.   The method of claim 1, wherein the accessible nerve cells that cross the blood brain barrier are sympathetic preganglionic neurons.   Neuroactive polypeptide is neurotrophic factor, endocrine factor, growth factor, paracrine factor, hypothalamic release factor, neurotransmitter polypeptide, antibody that binds to neurotrophic factor, antibody that binds to neurotrophic factor receptor, central 2. The method of claim 1 selected from the group consisting of polypeptide antagonists, agonists, and polypeptides involved in lysosomal storage diseases of receptors expressed by neural cells.   2. The method of claim 1, wherein the genetic vector comprises a cell transfection component comprising at least a portion of a viral capsid that is active on mammalian cells.   2. The method of claim 1, wherein the gene vector comprises cationic liposomes or other gene transfection lipids.   2. The method of claim 1, wherein the gene vector comprises a ligand that specifically binds to at least one endocytic receptor on accessible neuronal cells. 14. The method of claim 13, wherein the ligand has been identified through a selection step comprising the following steps:
(A) contacting a plurality of selected types of neurons with a phagemid or a phage display library;
Here, each phage in the library has a candidate DNA sequence and has a polypeptide encoded by the candidate DNA on the exposed phage surface,
And the phage display library comprises a plurality of phage particles having a plurality of candidate DNA sequences and corresponding polypeptides exposed on the phage surface; and (b) from the phage display library, at least one clonal phage Selecting a stock,
Here, the selected clonal phage strain is one that has been transported into the selected type of nerve cell.   A gene vector transfers related genetic material from a selected type of accessible neuron across the cranial nerve barrier to at least one type of neuron completely located in the central nervous system tissue protected by the animal's blood-brain barrier. The method of claim 1, comprising a macromolecule that has been shown to facilitate transport through nerve cells. 2. The method of claim 1, wherein the gene vector encodes a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or facilitates antegrade transport of the chimeric polypeptide within the nerve cell expressing the chimeric polypeptide; and (b) within the blood brain barrier by an accessible nerve cell that crosses the blood brain barrier. A neuroactive sequence that, after being secreted, can produce a therapeutic effect by reacting with cells located entirely within the central nervous system tissue protected by the cranial nerve barrier. 2. The method of claim 1, wherein the gene vector encodes a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or facilitates secretion of the chimeric polypeptide at the nerve cell terminus located within the central nervous system tissue protected by the animal's blood brain barrier; and (b) accessible across the blood brain barrier. A neuroactive sequence that is secreted by the nerve cell into the blood-brain barrier and can then produce a therapeutic effect by reacting with cells that are completely located in the central nervous system tissue protected by the cranial nerve barrier. A method of delivering a neuroactive polypeptide molecule through the blood brain barrier of a higher animal comprising the step of administering to the higher animal a gene vector having at least one gene encoding a neuroactive polypeptide:
Here, the gene vector is configured to be able to transfect at least one accessible neuronal cell across the blood brain barrier,
When a gene vector is administered to a higher animal, it is administered in a location and manner such that the gene vector and accessible nerve cells are in direct contact; and
(i) at least one gene vector is transfected in contact with at least one accessible neuron; (ii) at least one transfected accessible neuron is carried by the gene vector. Expressing a plurality of at least one neuroactive polypeptide encoded by at least one gene; and (iii) at least one transfected accessible neuronal cell expressing at least one neuroactive polypeptide, blood brain barrier The gene vector is administered to higher animals in a location and manner that ensures multiple secretion to a location within the central nervous system tissue that is protected by.   19. The method of claim 18, wherein the gene vector is designed to be transfected into sensory neurons.   19. The method of claim 18, wherein the gene vector is designed to be transfected into motor neurons.   19. The method of claim 18, wherein the gene vector is designed to be transfected into presympathetic neurons of the parasympathetic nervous system.   19. The method of claim 18, wherein the gene vector is designed to be transfected into sympathetic preganglionic neurons.   Neuroactive polypeptide is neurotrophic factor, endocrine factor, growth factor, paracrine factor, hypothalamic release factor, neurotransmitter polypeptide, antibody that binds to neurotrophic factor, antibody that binds to neurotrophic factor receptor, central 19. The method of claim 18, wherein the method is selected from the group consisting of polypeptide antagonists, agonists of receptors expressed by neural cells, and polypeptides involved in lysosomal storage diseases.   19. The method of claim 18, wherein the genetic vector comprises a cell transfection component comprising at least a portion of a viral capsid that is active on mammalian cells.   19. The method of claim 18, wherein the gene vector comprises cationic liposomes or other gene transfection lipids.   19. The method of claim 18, wherein the genetic vector comprises a ligand that specifically binds to at least one endocytic receptor on accessible neuronal cells.   A gene vector transfers related genetic material from a selected type of accessible neuron across the cranial nerve barrier to at least one type of neuron completely located in the central nervous system tissue protected by the animal's blood-brain barrier. 19. The method of claim 18, comprising a macromolecule that has been shown to facilitate transport through nerve cells. 19. The method of claim 18, wherein the gene vector encodes a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or facilitates antegrade transport of the chimeric polypeptide within the nerve cell expressing the chimeric polypeptide; and (b) within the blood brain barrier by an accessible nerve cell that crosses the blood brain barrier. A neuroactive sequence that, after being secreted, can produce a therapeutic effect by reacting with cells located entirely within the central nervous system tissue protected by the cranial nerve barrier. 19. The method of claim 18, wherein the gene vector encodes a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or facilitates secretion of the chimeric polypeptide at the nerve cell terminus located within the central nervous system tissue protected by the animal's blood brain barrier; and (b) accessible across the blood brain barrier. A neuroactive sequence that is secreted by the nerve cell into the blood-brain barrier and can then produce a therapeutic effect by reacting with cells that are completely located in the central nervous system tissue protected by the cranial nerve barrier. In higher animals, designed to initiate and be able to initiate neuronal processes that deliver neuroactive polypeptide molecules non-invasively into central nervous system tissues protected by the blood brain barrier. Gene vectors:
here,
The genetic vector comprises a cell transfection component and associated genetic material assembled therein,
The gene vector transfects at least one accessible neuronal cell with at least one peripheral projection that spans the animal's blood brain barrier and has access to the gene vector that did not cross the blood brain barrier. Are suitable and can be transfected,
(A) The cell transfection component is such that the gene vector binds to (i) the selected type of accessible nerve cell, and (ii) the related genetic material possessed by the gene vector is inserted into the accessible nerve cell. And (b) the related genetic material comprises at least one gene encoding a neuroactive polypeptide;
The gene has a gene promoter that causes a neuroactive polypeptide or precursor thereof to be expressed from the gene in a transfected accessible neuronal cell;
A plurality of neuroactive polypeptides or precursors thereof,
(i) within the transfected accessible neuronal cell, transported to a neuronal secretory location within the central nervous system tissue protected by the animal's blood brain barrier; and
(ii) It has been shown that the transfected accessible neuronal cells are secreted at the secretory location of the neuronal cells within the central nervous system tissue protected by the animal's blood brain barrier.   In vivo mammalian studies can sufficiently initiate a neuronal process that delivers a neuroactive polypeptide molecule non-invasively, and the neuroactive polypeptide molecule is encoded by a gene vector 31. The gene vector of claim 30, which has been shown to be expressed in transfected accessible neurons in central nervous system tissues protected by the blood brain barrier in at least one mammal.   In human clinical trials, the neuronal process of non-invasively delivering a neuroactive polypeptide molecule can be fully initiated, and the neuroactive polypeptide molecule is encoded by a gene vector and treated human 31. The gene vector of claim 30, which has been shown to be expressed in a central nervous system tissue protected by a blood brain barrier in a transfected accessible neuronal cell in a patient.   32. The gene vector of claim 30, wherein the gene vector is designed to be transfected into at least one sensory neuron.   34. The gene vector of claim 33, designed to be transfected into olfactory receptor neurons.   34. The gene vector of claim 33, designed to be transfected into nociceptive neurons.   32. The gene vector of claim 30, wherein the gene vector is designed to be transfected into at least one motor neuron.   31. The gene vector of claim 30, wherein the gene vector is designed to be transfected into at least one prenodal nerve cell belonging to a higher animal sympathetic or parasympathetic nervous system.   Gene vector is neurotrophic factor, endocrine factor, growth factor, paracrine factor, hypothalamic release factor, neurotransmitter polypeptide, antibody that binds to neurotrophic factor, antibody that binds to neurotrophic factor receptor, central nervous system 31. The gene vector of claim 30, which encodes a neuroactive polypeptide selected from the group consisting of polypeptide antagonists, agonists of receptors expressed by cells, and polypeptides involved in lysosomal storage diseases.   32. The gene vector of claim 30, wherein the cell transfection component comprises at least a portion of a mammalian viral capsid.   32. The gene vector of claim 30, wherein the cell transfection component comprises a cationic liposome.   32. The gene vector of claim 30, wherein the cell transfection component comprises a ligand that specifically binds to at least one endocytic receptor on accessible neuronal cells. 42. The gene vector of claim 41, wherein the ligand has been identified through a selection process comprising the following steps:
(A) contacting a plurality of selected types of neurons with a phagemid or a phage display library;
Here, each phage in the library has a candidate DNA sequence and has a polypeptide encoded by the candidate DNA on the exposed phage surface,
And the phage display library comprises a plurality of phage particles having a plurality of candidate DNA sequences and corresponding polypeptides exposed on the phage surface; and (b) from the phage display library, at least one clonal phage Selecting a stock,
Here, the selected clonal phage strain has been efficiently transported into the selected type of nerve cell.   A cell transfection component from a selected type of accessible neuronal cell that spans the cranial nerve barrier to at least one neuronal cell located entirely within the central nervous system tissue protected by the blood brain barrier of the animal 32. The gene vector of claim 30, comprising a polypeptide that has been shown to facilitate transport of substances through nerve cells. The gene vector of claim 30, which encodes a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or facilitates antegrade transport of the chimeric polypeptide within the nerve cell expressing the chimeric polypeptide; and (b) within the blood brain barrier by an accessible nerve cell that crosses the blood brain barrier. A neuroactive sequence that, after being secreted, can produce a therapeutic effect by reacting with cells located entirely within the central nervous system tissue protected by the cranial nerve barrier. The gene vector of claim 30, which encodes a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or facilitates secretion of the chimeric polypeptide at the nerve cell terminus located within the central nervous system tissue protected by the animal's blood brain barrier; and (b) accessible across the blood brain barrier. A neuroactive sequence that is secreted by the nerve cell into the blood-brain barrier and can then produce a therapeutic effect by reacting with cells that are completely located in the central nervous system tissue protected by the cranial nerve barrier. In higher animals, designed to initiate and be able to initiate neuronal processes that deliver neuroactive polypeptide molecules non-invasively into central nervous system tissues protected by the blood brain barrier. A gene vector encoding a chimeric polypeptide comprising the following sequence:
(A) a leader sequence that performs or promotes at least one activity selected from the group:
(i) antegrade transport of the chimeric polypeptide within a nerve cell expressing the chimeric polypeptide, and
(ii) secretion of chimeric polypeptides at nerve cell terminals located within central nervous system tissue protected by the animal's blood brain barrier;
and,
(B) Producing therapeutic effects by reacting with cells located entirely within the central nervous system tissue protected by the cranial nerve barrier after being secreted into the blood brain barrier by neurons straddling the blood brain barrier A neuroactive sequence that can.   The neuroactive sequence of the polypeptide is neurotrophic factor, endocrine factor, growth factor, paracrine factor, hypothalamic release factor, neurotransmitter polypeptide, antibody that binds to neurotrophic factor, antibody that binds to neurotrophic factor receptor 49. The gene vector of claim 46, selected from the group consisting of: a polypeptide antagonist of a receptor expressed by a central nervous system cell, an agonist, and a polypeptide involved in lysosomal storage diseases. Molecular complex containing the following components:
(A) at least one ligand component identified by an in vivo selection method that requires uptake by endocytosis into neurons to effect selection; and (b) the ligand component is specific At least one passenger component capable of producing a desired effect after being transported into at least one target animal cell having an endocytotic surface molecule that binds to.   When the molecular complex is administered to an animal having the following target animal cells, the ligand-mediated endocytosis causes at least one target animal cell having an endocytotic surface molecule to which the ligand specifically binds. 49. The molecular complex of claim 48, capable of transporting a shipment component.   49. The molecular complex of claim 48, wherein the ligand component is derived from a molecule or molecular complex collected from nerve cells in vivo.   When the molecular complex is transported into the following cells by ligand-mediated endocytosis, the transport component is ligated to the ligand so that the transport component can enter at least one target mammalian cell. 49. The molecular complex of claim 48, further comprising at least one linking component linked to the component.   49. The molecular complex of claim 48, wherein in the in vivo selection method of the ligand component, it is further necessary that the ligand component is taken into the nerve cell by endocytosis and then transported into the nerve cell fiber. 49. The molecular complex of claim 48, wherein the in vivo selection method comprises the following steps:
(1) placing a plurality of candidate ligand components within an emplacement site of a living animal so that the candidate ligand components and the neuronal cell surface are in contact; and (2) being taken up by the neuronal cells; Collecting a region of nerve cell fibers comprising a ligand component transported by nerve cell fibers at a collection site remote from the placement site.   54. The molecular complex of claim 53, wherein the candidate ligand component is exposed on the surface of phage particles in a phage display library during the in vivo selection method.   54. The molecular complex of claim 53, wherein the candidate ligand component is produced by a combinatorial chemical synthesis method.   54. The molecular complex of claim 53, wherein the candidate ligand component has been processed by an affinity binding step prior to the in vivo selection method.   49. The molecular complex of claim 48, wherein the ligand component specifically binds to a low affinity nerve growth factor receptor. Molecular complex containing the following components:
(A) at least one endocytic ligand component selected by an in vivo selection method that requires uptake by endocytosis through a target type of endocytotic surface molecule to perform in vivo selection And (b) at least one transport component capable of producing a desired effect after being transported into at least one mammalian cell having a target type of endocytotic surface molecule.   When the molecular complex is transported into the following cells by ligand-mediated endocytosis, the transport component is ligated to the ligand so that the transport component can enter at least one target mammalian cell. 59. The molecular complex of claim 58, further comprising at least one linking component linked to the component.   59. The molecular complex of claim 58, wherein the ligand component in vivo selection method requires that the ligand component enter the nerve cell fiber by endocytosis. 59. The molecular complex of claim 58, wherein the in vivo selection method comprises the following steps:
(1) placing a plurality of candidate ligand components within a placement site of a living animal so that the candidate ligand components and nerve cell surface are in contact; and (2) being taken up by nerve cells and by nerve cell fibers Collecting a region of nerve cell fibers containing the transported ligand component at a collection site remote from the placement site.   62. The molecular complex of claim 61, wherein candidate ligand components are exposed on the surface of phage particles in a phage display library during the in vivo selection method.   62. The molecular complex of claim 61, wherein the candidate ligand component is produced by a combinatorial chemical synthesis method.   62. The molecular complex of claim 61, wherein the candidate ligand component has been processed by an affinity binding step prior to the in vivo selection method.   62. The molecular complex of claim 61, wherein the ligand component specifically binds to a low affinity nerve growth factor receptor. Comprising a plurality of endocytotic ligands having a molecular structure identified by an in vivo selection method that requires endocytotic uptake into neurons to perform in vivo selection;
The ligand comprises a transport component such that the ligand produces a molecular complex capable of transporting the transport component into at least one target cell having an endocytotic surface molecule to which the ligand specifically binds. Suitable to be linked to the
Purified preparation of endocytosis ligand.   68. The purified preparation of claim 66, substantially free of another ligand candidate that cannot bind to an endocytotic surface molecule to which the endocytotic ligand specifically binds.   68. The purified preparation of claim 66, further comprising in the in vivo selection method, the candidate ligand component is taken up into the nerve cell fiber by endocytosis and then transported intracellularly within the nerve cell fiber. 68. The purified preparation of claim 66, wherein the in vivo selection method comprises the following steps:
(1) placing a plurality of candidate ligand components within a placement site of a living animal so that the candidate ligand components and nerve cell surface are in contact; and (2) being taken up by nerve cells and by nerve cell fibers Collecting a region of nerve cell fibers containing the transported ligand component at a collection site remote from the placement site.   68. The purified preparation of claim 66, wherein the candidate ligand component is exposed on the surface of the phage particles in the phage display library during the in vivo selection method.   68. The purified preparation of claim 66, wherein the candidate ligand component selected by the in vivo selection method is produced by a combinatorial chemical synthesis method.   68. The purified preparation of claim 66, wherein the candidate ligand component selected by the in vivo selection method has been processed by an affinity binding step prior to the in vivo selection method.   68. The purified preparation of claim 66, wherein the ligand component specifically binds to a low affinity nerve growth factor receptor. Endocytosis can incorporate a molecular complex comprising an endocytosis ligand linked to a transport component into a target cell having an endocytosis surface molecule to which the endocytosis ligand specifically binds. In vivo selection method comprising the following steps for isolating a cytotic ligand:
(1) a step of arranging a plurality of candidate ligand components in an arrangement site of a living animal so that the candidate ligand components and a nerve cell surface come into contact with each other in a living body; Collecting a region of nerve cell fibers comprising a ligand component transported by the cell fibers at a collection site remote from the placement site.   75. The in vivo selection method of claim 74, wherein during the in vivo selection method, candidate ligand components are exposed on the surface of phage particles in a phage display library.   75. The in vivo selection method of claim 74, wherein the candidate ligand component is produced by a combinatorial chemical synthesis method.   75. The in vivo selection method of claim 74, wherein the candidate ligand component has been processed by an affinity binding step prior to the in vivo selection method.   75. The in vivo selection method of claim 74, wherein the nerve cell fibers are treated such that low affinity nerve growth factor receptor expression is increased prior to the in vivo selection method. Target animal cells are composed of the following components:
(A) at least one ligand component identified by an in vivo selection method that requires uptake by endocytosis into neurons to effect selection; and (b) an end to which the ligand component specifically binds. Contacting with at least one endocytotic molecule complex comprising at least one transport component capable of producing a desired effect after being transported into a target mammalian cell having a cytotic surface molecule A method of introducing molecules into selected types of target cells.   When the endocytosis molecular complex is transported into the following cells by ligand-mediated endocytosis, the transport component is made into a ligand component so that the transport component can enter the target mammalian cell. 80. The method of claim 79, further comprising at least one linking component to be linked. 80. The method of claim 79, wherein the in vivo selection method comprises the following steps:
(1) placing a plurality of candidate ligand components within a placement site of a living animal so that the candidate ligand components and nerve cell surface are in contact; and (2) being taken up by nerve cells and by nerve cell fibers Collecting a region of nerve cell fibers containing the transported ligand component at a collection site remote from the placement site.   32. The method of claim 31, wherein during the in vivo selection method, candidate ligand components are exposed on the surface of phage particles in a phage display library.   35. The method of claim 32, wherein the candidate ligand component is produced by a combinatorial chemical synthesis method.   35. The method of claim 32, wherein the candidate ligand component has been processed by an affinity binding step prior to the in vivo selection method.   80. The method of claim 79, wherein the nerve cell fibers have been treated to increase low affinity nerve growth factor receptor expression prior to the in vivo selection method.   80. The method of claim 79, wherein the ligand component specifically binds to a low affinity nerve growth factor receptor. A plurality of phage displaying at least one candidate ligand sequence in at least one coat protein;
In order to select phage particles contained in the library, they were pre-screened by an affinity binding step that utilizes the affinity to bind to polypeptides known to have endocytotic activity on animal cells. ,
Phage display library.   A purified gene vector encoding a polypeptide sequence comprising an endocytic ligand sequence identified by an in vivo selection method that requires uptake by endocytosis into a neuronal cell to effect selection. 90. The purified gene vector of claim 88, wherein the in vivo selection method comprises the following steps:
(1) placing a plurality of candidate ligand components within a placement site of a living animal so that the candidate ligand components and nerve cell surface are in contact; and (2) being taken up by nerve cells and by nerve cell fibers Collecting a region of nerve cell fibers containing the transported ligand component at a collection site remote from the placement site.   90. The purified gene vector of claim 88, wherein the candidate ligand component is exposed on the surface of phage particles in a phage display library during the in vivo selection method.   90. The purified gene vector of claim 88, wherein the candidate ligand component is produced by a combinatorial chemical synthesis method.   90. The purified gene vector of claim 88, wherein the candidate ligand component has been processed by an affinity binding step prior to the in vivo selection method. 90. The purified gene vector of claim 88, wherein the ligand component specifically binds to a low affinity nerve growth factor receptor.

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