WO2000023083A1

WO2000023083A1 - Neuroprotective truncated bax polypeptides

Info

Publication number: WO2000023083A1
Application number: PCT/US1999/024747
Authority: WO
Inventors: Eugene M. Johnson, Jr.; Rachael Easton
Original assignee: Washington University
Priority date: 1998-10-22
Filing date: 1999-10-22
Publication date: 2000-04-27
Also published as: AU1130600A

Abstract

Truncated BAX (tBAX) polypeptides which are anti-apoptotic in neurons are disclosed. The tBAX polypeptides are defined as lacking the C-terminal transmembrane domain of BAXα. Methods of inhibiting apoptosis of neurons using tBAX polypeptides and their encoding polynucleotides are described. Transgenic, nonhuman animals expressing tBAX polypeptides are also provided.

Description

NEUROPROTECTIVE TRUNCATED BAX POLYPEPTIDES

Reference to Government Grant

This invention was made with government support under NIH Grant Number R37-AG 12947. The government has certain rights in this invention.

Background of the Invention (1) Field of the Invention

This invention relates generally to the regulation of apoptosis and to compounds which regulate apoptosis, and more particularly, to a novel inhibitor of neuronal apoptosis. (2) Description of the Related Art

Programmed cell death (PCD) is a genetically controlled and evolutionarily conserved mechanism by which cells undergo a cell-autonomous death. (For review, see Hale et al., Ewr. J. Biochem. 236:1-26, 1996). During development, PCD is responsible for the elimination of surplus cells during the sculpting of tissues and organs as well as cells that are no longer needed as the organism proceeds from one developmental stage to another. PCD can also result from pathological states such as trauma, infection, neoplasm and degenerative disease. Although the sequence of biochemical and molecular events in different forms of PCD vary, at least to some degree, depending on the cell type and death-inducing stimulus, the dying cells in all forms appear to undergo the common terminal events of apoptosis as defined by its distinct morphological features of cytoplasmic shrinking, plasma membrane blebbing, condensation of nuclear chromatin, and fragmentation of genomic DNA. The apoptotic cells are eventually engulfed by neighboring cells or phagocytes, thereby avoiding an inflammatory response. Deshmukh et al., Molec. Pharmac. 51:897-906, 1997. In contrast, necrosis is a pathological type of cell death observed following physical or chemical injury, exposure to toxins, or ischemia and characterized by swelling, rupture of the plasma membrane and cellular organelles, and in vivo inflammation due to release of the cellular content into the surrounding tissue. Hengartner, M., in Molecular Biology and Biotechnology, Robert A. Myers, ed., NCH Publishers, Inc., 1995, p. 158.

In the nervous system, PCD occurs during both developmental and pathological processes. For example, depending on the neuronal population examined, 20-80% of all neurons produced during embryogenesis die prior to reaching adulthood (for review, see Oppenheim, Annu. Rev. Neurosci. 14:453-501, 1991). PCD is also believed to play a role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (Stefanis et al., Curr. Opin. Neurol. 0:299-305, 1997) as well as in acute neurological injuries such as ischemic and spinal cord injuries (Martinou et al., Neuron 13:1017-1030, 1994; Choi, Curr. Opin. Neurol. 6:667-672, 1996; Crowe et al, Nat. Med. 3:73-76, 1997; Freidlander et al., J. Exp. Med. 185:933-940, 1997; Hara et al., Proc. Natl. Acad. Sci. USA 94:2007-2012, 1997; Liu et al., J. Neurosci. 17:5395-5406, 1997; Portera-Cailliau et al., J. Comp. Neurol. 378:70-87, 1997; Kato et al, J. Thorac. Cardiovasc. Surg. 114:609-618, 1997; Cheng et al., J Clin. Invest. 101:1992-1999, 1998).

Our understanding of neuronal PCD derives largely from the study of neurotrophic factor dependence in the developing nervous system during the period when neurons innervate their targets. Deshmukh et al., supra. Immature cells that obtain trophic factors survive, whereas those that fail to obtain sufficient trophic factors die, thus conforming the number of innervating neurons to the size of target cell populations. As neurons mature, they become progressively less dependent upon neurotrophic factors for survival, a phenomenon which prevents irreversible loss of postmitotic neurons due to temporary changes in trophic factor levels (Easton et al., J. Neurosci. 17:9656-9666, 1997).

The most extensively studied in vitro model of neuronal PCD is nerve growth factor (ΝGF) deprivation of rat sympathetic neurons (Deshmukh et al., supra; Edwards et al., J. Cell Biol. 124:537-546, 1994; Deckwerth et al, J. Cell Biol. 123:1207-1222, 1993). Sympathetic neurons obtained from embryonic-day-21 rats and grown in the presence of NGF for five to seven days undergo PCD within 24-48 hr after the rapid removal of NGF by addition of anti-NGF antibodies (Martin et al, J.

Cell Biol. 106:829-844, 1988). However, as these neurons "mature" in culture, they become progressively less dependent on NGF for survival such that, after 25 days in culture, NGF deprivation does not result in cell death even by two weeks after removal of NGF (Easton et al., supra). A similar phenomenon is also seen in sensory neurons (Eichler et al., Brain Res. 482:340-346, 1989).

Neuronal PCD is believed to be regulated, at least in part, by members of the BCL-2 family of proteins (Deshmukh et al., supra). This family, which includes both anti-apoptotic (BCL-2, BCL-X_L, BCL-W, MCL-1, Al) and pro-apoptotic (BAX, BCL-Xs, BAK, BAD, BID, BIK, HRK) members, is defined by four regions of homology called BCL-2 Homology (BH) domains 1-4 (See (Reed, J.C., Advances in Pharmac. 41:501-532, 1997; Reed et aX., Adv. Exp. Med. Biol. 406:99-112, 1996). The BH1 and BH2 domains are believed to be required for the anti-apoptotic members to heterodimerize with the pro-apoptotic members and block cell death (Reed et al., 1996, supra; Hunter et al, 1996; Sedlak et al, 1995; Yin et al, 1995; Zhang et al., 1995; Borner et al, 1994). Although the BH3 domain is present in both the anti- and pro-apoptotic family members, this domain is reportedly required for homo- and heterodimerization and in promoting apoptosis by the pro-apoptotic family members (Ink et al., 1997; Simonen et al, 1997; Hunter et al., 1996; Zha et al., 1996; Chittenden et al., 1995). In addition, several pro-apoptotic members of the BCL-2 family contain only the BH3 domain (BIK, BIM, HRK, BID) (Inohara et al., 1997; Han et al., 1996; Wang et al, 1996; Kelekar et al., 1997). Recently, one group investigated the role played by various BCL-2 family members in regulating neuronal PCD by performing trophic factor deprivation studies of neurons from mice in which the gene encoding one of these proteins is inactivated by homologous recombination, i.e., knockout mice. Neurons from bcl-2 knockout mice die slightly faster than neurons from wild-type animals, as would be predicted for loss of an anti-apoptotic protein (Greenlund et al., 1995). Sympathetic neurons from knock-out mice deficient in BAD or BAK die normally after NGF deprivation, thus these proteins are not critically involved in the PCD pathway of sympathetic neurons (unpublished data). However, box knockout mice exhibit a much different neuronal phenotype.

Sympathetic neurons from these BAX-deficient mice do not die when deprived of trophic factor and axotomy of the facial nerve in BAX-deficient neonates does not result in motoneuron death. (Deckwerth et al., Neuron 17:401-411, 1996). Cerebellar granule cells from these animals are similarly protected from K⁺ deprivation-induced apoptosis but not from NMDA-induced excitotoxicity (Miller et al., J. Cell Biol.

139:205-217, 1997). Naturally occurring neuronal PCD is also prevented, or at least greatly reduced, in these animals as they are born with increased numbers of sympathetic and motor neurons (Deckwerth et al, 1996). These BAX-deficient mice also exhibit greatly reduced PCD in sensory, motor, and retinal ganglion cells during nerve system development (White et al., J. Neurosci 18:1428-1439, 1998). Thus, it is believed that BAX is required for naturally occurring or axotomy-induced apoptosis in many, and perhaps all, neurons.

However, BAX-deficient mice do not have decreased numbers of TUNEL- positive glial cells in the developing optic nerve, indicating that BAX is not required for glial cell apoptosis (White et al., supra). Similarly, lymphocytes from these mice die normally in response to several apoptotic stimuli and other systems examined, except for testicular and ovarian development, appear normal (Knudson et al., Science 270:96-99, 1995). Thus, BAX does not appear to be required for PCD in most nonneuronal cells.

The specificity of BAX in promoting neuronal PCD suggests that an inhibitor of BAX function is likely to be a highly neuronal selective anti-apoptotic agent, which would be largely free of the adverse effects of less selective anti-apoptotic proteins such as BCL-2, BCL-X, and caspase inhibitors, whose inappropriate expression in many cell types can lead to cancer and autoimmune disorders. Thus, there is a need for the identification of BAX inhibitors capable of selectively blocking neuronal apoptosis for use in treating neurodegenerative diseases, peripheral nerve injury, spinal cord injury, head trauma, and stroke.

Summary of the Invention

In accordance with the present invention, it has been discovered that certain truncated polypeptides derived from BAXα, referenced herein as tBAX, prevent apoptosis of neurons, in particular sympathetic neurons, after trophic factor deprivation. This discovery was unexpected in that BAXα is pro-apoptotic. BAXα, also commonly referred to as wild-type BAX, is the major species of several splice variants of BAX described in the literature (Apte et al, Genomics 26:592-594 (1995), Oltvai et al, Cell 74:609-619 (1993) (Figure 1). Anti-apoptotic tBAX polypeptides particularly exemplified herein contain the

N-terminal region and most or all of the BH3 domain, but lack the BH1, BH2 and C- terminal transmembrane domains present in full-length BAXα (see Fig. 1). However, it is believed that tBAX polypeptides lacking only the transmembrane domain will have anti-apoptotic activity in neurons. This belief is supported by the following evidence: (1) as reported below, NGF-deprivation of neurons leads to a macromolecular synthesis-dependent translocation of BAX to the mitochondria; and (2) BAX does not translocate to the mitochondria if it lacks its transmembrane domain (Wolter et al., 1997). Thus, a tBAX polypeptide lacking the transmembrane domain is predicted to bind to BAXα, or to whatever BAXα binds to, to prevent translocation of BAXα to the mitochondria, thereby inhibiting apoptosis in neuronal cells, which require normal BAX function to die. It is further believed that the tBAX polypeptides described herein can selectively inhibit neuronal apoptosis by acting as a dominant-negative inhibitor of BAX function, which is not required for apoptosis of nonneuronal cells. Accordingly, an aspect of the invention provides an isolated and purified polypeptide comprising a truncated BAX (tBAX) polypeptide that inhibits apoptosis of neuronal cells. Anti-apoptotic tBAX polypeptides identified herein include tBAX70 (SEQ ID NO: 1-2) and tBAX78 (SEQ ID NO:3-4), which are fragments of human and mouse BAXα, and mutants of these fragments, fBAX70jyι (SEQ ID NOS:5-6) and tBAX78_M (SEQ ID NOS:7-8), which contain mutations of the naturally occurring sequence in or around the BH3 domain (see Fig. 2). A particularly preferred tBAX polypeptide is human tBAX78_M (SEQ ID NO:7).

In another embodiment, the invention provides isolated polynucleotides encoding a tBAX polypeptide. These polynucleotides may be used to transfect a target neuron in which expression of the encoded tBAX polypeptide inhibits apoptosis of the target neuron. A recombinant cell stably transformed with a polynucleotide encoding for expression a tBAX polypeptide is also provided by the invention. The recombinant cell may be used in a method for producing the tBAX polypeptide.

In another embodiment, the present invention provides a composition comprising a tBAX polypeptide which has anti-apoptotic activity in neurons and a pharmaceutically acceptable carrier.

The tBAX polypeptides of the invention are also useful in a method for inhibiting apoptosis in a target neuron which comprises treating the neuron with an effective amount of a tBAX polypeptide. The target neuron may be treated in vitro or in vivo in a patient. In one embodiment, the neuron is treated with a tBAX polypeptide by administering to the neuron a polynucleotide encoding the tBAX polypeptide, through which the tBAX polypeptide is expressed in the neuron. Alternatively, the treating step comprises administering the tBAX polypeptide to the neuron, preferably with a carrier that facilitates delivery of the polypeptide into the neuron. Where the target neuron is in a patient, the method is useful for promoting survival of neurons under attack in a patient suffering from a neurodegenerative disease, trauma to the nervous system, or stroke.

Another aspect of the invention provides a transgenic nonhuman animal expressing a tBAX polypeptide which inhibits apoptosis of neurons. Such tBAX- expressing animals may be used to examine the role of naturally-occurring cell death during development of the nervous system and in evaluating the importance of apoptosis in various neuronal injury paradigms and in animal models of neurodegenerative diseases.

Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of new tBAX polypeptides which inhibit apoptosis of neurons; the provision of polynucleotides encoding these polypeptides; the provision of tBAX polypeptide compositions which can be readily delivered intracellularly to produce a death antagonist activity; the provision of a method for inhibiting neuronal apoptosis with these compositions, and the provision of tBAX- expressing transgenic animals as experimental tools. Brief Description of the Drawings

Figure 1 illustrates the predicted protein products of four alternatively spliced transcripts of the bax gene, with the numerals above BAXα indicating the amino acid residues defining the conserved BH3, BH1, BH2 and transmembrane (TM) domains; Figure 2 shows the C-terminal amino acid sequences (SEQ ID NOS:9-l 1) of three tBAX polypeptides derived from murine BAX that inhibit neuronal apoptosis, with the wild-type murine BH3 domain underlined in tBAX78;

Figure 3 shows aligned BH3 amino acid sequences of BCL-2 family members;

Figure 4 shows photographs of in vitro maintained primary rat sympathetic neurons stained with an anti-BAX antibody (FIGS. 4A-4C) or bisbenzimide (FIGS.

4D-4F) following a 36 hr treatment of: (FIG. 4A, 4D) continued growth in the presence of NGF; ( FIG. 4B, 4E) deprivation with NGF in the presence of cycloheximide; and (FIG. 4C, 4F) deprivation with NGF in the presence of the caspase inhibitor BAF; Figure 5 shows photographs of in vitro maintained primary rat sympathetic neurons stained with an anticytochrome c antibody (FIGS. 5A-C) or bisbenzimide

(FIGS. 5E-F) following a 3 day treatment of: (FIG. 5A, 4D) continued growth in the presence of NGF; ( FIG. 4B, 4E) deprivation with NGF in the presence of cycloheximide; and (FIG. 4C, 4F) deprivation with NGF in the presence of the caspase inhibitor BAF; and

Figure 6 illustrates the death suppressing activity of IBAX78M in NGF- deprived primary rat sympathetic neurons, showing the percentage of NGF-deprived

(-NGF) or NGF-maintained neurons (+NGF) surviving 16 to 24 hr after microinjection with a polynucleotide encoding tBAX78_M or the vector alone as a control;

Detailed Description of the Invention

The present invention is based on the surprising discovery that certain truncated BAX polypeptides lacking the C-terminal transmembrane domain inhibit neuronal apoptosis induced by trophic factor deprivation. This discovery arose out of research into the role played by BAX in neuronal PCD which demonstrated that BAX is required for naturally occurring or axotomy-induced apoptosis of many, and possibly all, neurons but is not required for apoptotic death of any nonneuronal cell type examined to date. Thus, it is believed that the antiapoptotic truncated BAX polypeptides described herein represent the first relatively selective anti-apoptotic agents for neurons and as such may be used for treating neurodegenerative diseases, stroke, or nervous system trauma without interfering with physiological PCD in nonneuronal cell types.

Accordingly, one embodiment of the invention provides an isolated and purified polypeptide comprising a truncated BAX (tBAX) polypeptide which inhibits apoptosis of neurons. As used herein, a tBAX polypeptide lacks the transmembrane domain of a BAXα protein. By BAXα protein is meant a full-length, naturally- occurring BAX protein containing N-terminal, BH3, BHl , BH2, and transmembrane domains that is isolated from any species and identified by alignment to human BAX (SEQ ID NO: 12) or murine BAX (SEQ ID NO: 13).

In preferred embodiments, the tBAX polypeptides of the invention are further defined as comprising an N-terminal region and at least part of a BH3 domain. The N-terminal region corresponds to that portion of a BAXα protein located N-terminal to the BH3 domain, e.g., amino acid 1-58 of human or mouse BAXα. The BH3 domain in human and mouse BAX consists of amino acids 59 to 73 of SEQ ID NOS:12 and 13, respectively. While the shortest anti-apoptotic tBAX polypeptide exemplified herein truncates at amino acid 70 of the murine BAX BH3 domain (Fig. 2), it is believed that tBAX polypeptides truncating at amino acid 68 of the BH3 domain of a BAXα protein will similarly have anti-apoptotic activity because aspartic acid at position 68 has been shown to be important for Bax activity. In addition this residue is conserved at the corresponding position in all known BCL-2 family members from mammalian species. (See Figure 3). It is also contemplated that in some embodiments, anti-apoptotic tBAX polypeptides will also contain a BAX BHl and/or a BAX BH2 domain, which are defined by amino acid 98-118 and amino acid 150-165 of human and mouse BAXα (Fig. 1).

The amino acid sequence of the tBAX polypeptide may contain one or more amino acid substitutions as long as such substitutions do not destroy anti-apoptotic activity. The skilled artisan can readily prepare tBax polypeptides with amino acid substitutions and test them for anti-apoptotic activity. Preferably, amino acid positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains. For example, one grouping of amino acids includes those amino acids that have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those amino acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those amino acids having basic side chains (K, R, and H); another grouping is those amino acids having acidic side chains (D and E); another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those amino acids having aliphatic-hydroxyl side chains (S and T); another grouping is those amino acids having amine-containing side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those amino acids having sulfur-containing side chains (C and M). Preferred conservative amino acid substitutions groups are: R-K; E-D; Y-F; L-M; N-I; and Q-H.

It is also contemplated that a tBAX polypeptide can comprise a modified amino acid or unusual amino acid at one or more positions in its amino acid sequence, and can also comprise amino acids that are glycosylated or phosphorylated so long as the tBAX polypeptide is anti-apoptotic in neurons. Some embodiments of the tBAX polypeptide terminate with one of the amino acid sequences shown in Fig. 3 (SEQ ID ΝOS:9-l 1). Preferably, the tBAX polypeptide consists of SEQ ID NOS:l, 3, 5 or 7.

The tBAX polypeptides of the present invention can be made by recombinant DNA technology by expressing a nucleotide sequence encoding the desired amino acid sequence in a suitable transformed host cell. Using methods well known in the art, a polynucleotide encoding a tBAX polypeptide may be operably linked to an expression vector, transformed into a host cell and culture conditions established that are suitable for expression of the tBAX polypeptide by the transformed cell.

Any suitable expression vector may be employed to produce recombinant tBAX polypeptides such as, for example, the mammalian expression vector pCB6 (Brewer, Meth Cell Biol 43:233-245, 1994) or the E. coli pET expression vectors, specifically, pET-30a (Studier et al, Methods Enzymol 155:60-89, 1990). Other suitable expression vectors for expression in mammalian and bacterial cells are known in the art as are expression vectors for use in yeast or insect cells. Baculovirus expression systems can also be employed.

A number of cell types may be suitable as host cells for expression of recombinant tBAX polypeptides. Mammalian host cells include, but are not limited to, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo 205 cells, 3T3 cells, CN-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells. Yeast strains that may act as suitable host cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,

Candida, and any other yeast strain capable of expressing heterologous proteins. Host bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium and any other bacteria strain capable of expressing heterologous proteins. If the tBAX polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide, for example, by phosphorylation or glycosylation of the appropriate sites using known chemical or enzymatic methods, to obtain a biologically active tBAX polypeptide.

The polypeptide of the invention may also be expressed in transgenic animals, e.g., cows, goats, pigs, or sheep whose somatic or germ cells contain a recombinant nucleotide sequence encoding tBAX.

The expressed tBAX polypeptide can be isolated and purified using known purification procedures, such as gel filtration and ion exchange chromatography. As used herein, "isolated and purified" means that a designated polypeptide constitutes at least about 50 percent of a composition on a molar basis compared to total proteins or other macromolecular species present in the composition. Preferably, the polypeptide of the invention will constitute at least about 75 to about 80 mole percent of the total protein or other macromolecular species present. More preferably, an isolated and purified polypeptide will constitute about 85 to about 90 mole percent of a composition and still more preferably, at least about 95 mole percent or greater. Purification may also include affinity chromatography using an agent that will specifically bind the tBAX polypeptide, such as a polyclonal or monoclonal antibody raised against the tBAX polypeptide or fragment thereof. Other affinity resins typically used in protein purification may also be used such as concanavalin A- agarose, heparin-toyopearl^® or Cibacrom blue 3GA Sepharose^®. Purification of tBAX polypeptides can also include one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether. It is also contemplated that a tBAX polypeptide may be expressed as a fusion protein to facilitate purification. Such fusion proteins, for example, include a tBAX polypeptide fused to a histidine tag such as when expressed in the pET bacterial expression system as well as a tBAX polypeptide fused to maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Similarly, the polypeptide of the invention can be tagged with a heterologous epitope and subsequently purified by immunoaffmity chromatography using an antibody that specifically binds such epitope. Kits for expression and purification of such fusion proteins and tagged proteins are commercially available.

Alternatively, the tBAX polypeptides of the invention may be produced by chemical synthesis using methods known to those skilled in the art.

Once prepared, the tBAX polypeptide can be tested for neuroprotective activity using one or more well-characterized models of neuronal death. For example, anti-apoptotic activity in sympathetic neurons deprived of NGF can be assessed using the superior cervical ganglion survival assay described in Example 2 below. Similarly, a tBAX polypeptide can be tested for the ability to prevent apoptosis of sensory neurons following NGF withdrawal using dissociated cell cultures of sensory ganglion neurons prepared as described by Eichler and Rich, Brain Res. 482, 340-346, 1989.

In another embodiment, the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence that encodes a tBAX polypeptide. As used herein, a polynucleotide includes DNA and/or RNA and thus the nucleotide sequences recited in the Sequence Listing as DNA sequences also include the identical RNA sequences with uracil substituted for thymine residues. Nucleotide sequences included in the invention are those encoding the tBAX polypeptides set forth in SEQ ID NOS:l-8. It is understood by the skilled artisan that degenerate nucleotide sequences can encode the tBAX polypeptides described herein and these are also intended to be included within the present invention. Such nucleotide sequences include modifications of naturally-occurring sequences in which at least one codon is substituted with a corresponding redundant codon preferred by a given host cell, such as E. coli or insect cells, so as to improve expression of recombinant tBAX therein.

Preferred polynucleotides of the invention encode the human and mouse versions of tBAX70, tBAX78, tBAX70_M and tBAX78_M as set forth in SEQ ID

NOS: 1-8. Particularly preferred polynucleotides encode the mouse or human version of tBAX78_M and comprise SEQ ID NO: 16 or SEQ ID NO:17, respectively.

The present invention also encompasses vectors comprising an expression regulatory element operably linked to any of the tBAX-encoding nucleotide sequences included within the scope of the invention. This invention also includes host cells, of any variety, that have been transformed with such vectors.

Also included in by the present invention are therapeutic or pharmaceutical compositions comprising a tBAX polypeptide which has antiapoptotic activity in neurons and a method for inhibiting apoptosis of a target neuron which comprises treating the neuron with an effective amount of the tBAX polypeptide.

In one embodiment, the neuron is treated with the tBAX polypeptide by administering to the neuron a polynucleotide encoding the tBAX polypeptide. The polynucleotide comprises a nucleotide sequence encoding a tBAX polypeptide operably linked to a promoter that produces expression of the tBAX polypeptide in the neuron. The polynucleotide can comprise an expression plasmid, a retrovirus vector, an adenovirus vector, an adenovirus associated vector (AAV) or other viral or nonviral vector used in the art to deliver genes into cells. Alternatively, the polynucleotide can be administered to the neuron by microinjection.

In embodiments where the neuron being treated is in a patient, such as neurons comprising a tissue involved in a neurodegenerative disease, the polynucleotide encoding tBAX is administered to the patient. Any of the vectors discussed above, as well as nonviral methods such as lipid-based systems and polycation-based systems, can be used. It is also contemplated that the neuron be treated with the tBAX polypeptide by coinfection with a replication-defective adenovirus expressing the tBAX polypeptide and another replication competent adenovirus that complements the replication defective virus to increase the expression of in the infected cells.

Preferably, the polynucleotide is selectively delivered to target neurons within the patient so as not to affect apoptosis in other tissues. Targeted delivery of the polynucleotide can be done for example by using delivery vehicles such as polycations, liposomes or viral vectors containing a targeting moiety that recognizes and binds to a specific marker on the target neuron. Such methods are known in the art, see, e.g., U.S. Patent No. 5,635,383. Another targeted delivery approach uses viral vectors that can only replicate in specific cell types which is accomplished by placing the viral genes necessary for replication under the transcriptional control of a response element for a transcription factor that is only active in the target cell. See, e.g., U.S. Patent No. 5,698,443. Neuron specific promoters such as neuron specific enolase can also be used to limit expression of the tBAX polypeptide to neurons. In other embodiments of the invention, the neuron is treated by administering to the neuron a composition comprising a tBAX polypeptide which inhibits apoptosis of the neuron. Preferably, the tBAX polypeptide is administered with a carrier that facilitates delivery of the polypeptide into the cell, such as liposomes. Where the tBAX polypeptide is being administered to a patient, the liposomes can have targeting moieties exposed on the surface such as antibodies, ligands or receptors to specific cell surface molecules to limit delivery of tBAX to targeted cells. Liposome drug delivery is known in the art (see, e.g., Amselem et al., Chem. Phys. Lipid 64:2X9-231, 1993). Alternatively, the tBAX polypeptide can be modified to include a specific transit peptide that is capable of delivering the peptide into the cytoplasm of neuronal cell. Examples of such transit peptides include but are not limited to the TAT protein from HIV-1 (Frankel et al, Cell 55:1189-1193, 1988; Fawell et al., Proc. Natl. Acad. Sci. USA 91:664-668, 1994; Ezhevsky Proc. Natl. Acad. Sci. USA 94:10699-10704, 1997), the third helix of the Antennapedia homeodomain (Derossi et al., J. Biol. Chem. 271:18188-18193, 1996), and penetratins, which are 16 mer peptides derived from the Antennapedia homeodomain (Derossi et al., Trends Cell Biol. 8:84-87, 1998). Alternatively, the polypeptide can be delivered directly into a neuron by microinjection.

Compositions comprising a tBAX polypeptide can be administered by any suitable route known in the art including, for example, intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). The tBAX polypeptide can also be administered with one or more agents capable of promoting penetration of the polypeptide across the blood-brain barrier.

The tBAX polypeptide can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties, including for example, substances known in the art to promote penetration or transport across the blood-brain barrier such as an antibody to the transferrin receptor (Friden et al., Science 259:313-311, 1993), or a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties (Davis et al. Enzyme Eng 4:169-73, 1978; Burnham, Am J Hosp Pharm 57:210-218, 1994).

For nonparenteral administration, the compositions can also include absorption enhancers which increase the pore size of the mucosal membrane. Such absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl-β- cyclodextrin, lauroyl-1 -lysophosphatidylcholine and other substances having structural similarities to the phospholipid domains of the mucosal membrane. The compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection. The primary solvent can be aqueous or alternatively non-aqueous.

The carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.

It is also contemplated that certain formulations comprising a tBAX polypeptide are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that diminish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents.

The tBAX polypeptide is administered to patients in an amount effective to inhibit apoptosis of target neurons within the patient. The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in cell death assays. Exact dosages are determined in conjunction with standard dose- response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used. The invention also provides a transgenic, nonhuman animal expressing a tBAX polypeptide in its neurons. Neuron-specific expression of the tBAX polypeptide is achieved by placing expression of a polynucleotide encoding the tBAX under the control of a neuron-specific promoter, such as the Neuron-Specific-Enolase (NSE) promoter. The NSE promoter has been used extensively in transgenic studies to drive the neuron-specific expression of various genes, including bcl-2. The tBAX- encoding polynucleotide can also be operably linked to a polyadenylation site. Animals that are homozygous or hemizygous for the tbax transgene are included in the scope of the invention. In one embodiment, a transgenic mouse expressing an N-terminal FLAG- tagged tBAX polypeptide is contemplated. To prepare this transgenic mouse, standard molecular techniques are used to generate a recombinant gene construct encoding a N-terminal FLAG-tagged tBAX operably linked to an upstream NSE promoter and a downstream 3 ' untranslated region, including the polyadenylation sites, of the human growth hormone gene. This recombinant gene construct is then injected into mouse oocytes which are then implanted into pseudopregnant females. Genomic DNA from founder animals is subjected to PCR and Southern blot analysis to screen for transmission and to identify transgenics in subsequent generations. The expression of transgenic RNA can be confirmed by RT-PCR using a 5 ' primer that contains a sequence corresponding to the FLAG epitope to distinguish the expression of tBAX from endogenous BAX.

Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples below.

Example 1 This example illustrates that NGF-deprivation of immature neurons leads to translocation of BAX from the cytoplasm to the mitochondria and translocation of cytochrome c from the mitochondria to the cytoplasm. Primary cultures of sympathetic neurons from superior cervical ganglion (SCG) were prepared by dissecting tissue from Day 20-21 rat embryo (E20-E21). The SCG's were placed in Leibovitz's L15 with L-glutamine medium (Cat. #11415- 053, Gibco-BRL, Gaithersburg, MD), digested for 30 minutes with 1 mg/ml collagenase (Cat #4188 Worthington Biochemical, Freehold, NJ) in Leibovitz's L15 medium at 37° C, followed by a 30 minute digestion in trypsin-lyophilized & irradiated (Type TRLVMF Cat. #4454 Worthington Biochemical, Freehold, NJ) which was resuspended in modified Hanks' Balanced Salt Solution (Cat #H-8389 Sigma Chemical Co., St. Louis, MO). The digestion was stopped using AM50 which contains Minimum Essential Medium with Earle's salts and without L-glutamine (Cat #11090-016 Gibco-BRL), 10% fetal calf serum (Cat #1115 Hyclone Laboratories, Logan, UT), 5 mM L-glutamine (Cat #G5763 Sigma Chemical Co.), 20 μM fluorodeoxyuridine (FUdR) (Cat #F-0503, Sigma), 20 μM Uridine (Cat #3003, Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 ng/ml 2.5 S NGF. The cells were dissociated into a suspension of single cells using a silanized and flame- polished Pasteur pipet. After filtration of the suspension through a nitex filter (size 3- 20/14, Tetko Inc., Elmsford, NY), the cells were placed in AM50 medium as above and preplated on a 100 mm Falcon or Primaria culture dish (Becton Dickinson Labware, Lincoln Park, NJ) to reduce the number of non-neuronal cells. After 2 hours, the medium containing the unattached neuronal cells was removed from these dishes and triturated again through a silanized and flame-polished Pasteur pipet. The single cell suspension was plated on 24-well tissue culture plates (Costar, Wilmington, MA) that have been previously coated with a double layer of collagen, one layer of collagen that had been ammoniated and a second layer of collagen that had been air dried. They were allowed to attach for 30 minutes to 2 hours prior to filling the wells with AM50. A specific number of viable cells, usually about 1200 to about 3000 total cells per well, or a specific percentage of the ganglion, usually 25% of the cells obtained per ganglion were plated into each well. When cell counts were to be performed they were placed in the 24-well dishes as stated above or, alternatively, on 2-well chamber slides (Nunc, Naperville, IL). Cultures were then incubated for 5 days at 37° C in AM50 medium in a 5% CO₂/95% air atmosphere.

To investigate the intracellular location of BAX in NGF -maintained and NGF- deprived neurons, the in vitro maintained primary neurons were stained with an anti- BAX antibody or bisbenzimide 36 hr after continued maintenance in the presence of NGF or removal of NGF in the presence of either the protein synthesis inhibitor cycloheximide or the caspase inhibitor BAF. NGF removal was accomplished by exchanging the medium with medium lacking NGF and containing 0.05% goat anti- NGF (final titer in the wells is 1 : 10). This NGF-deprivation results in death of unprotected neurons over a period of 24-72 hours. The results are shown in Figure 4.

In the immunostained neurons maintained in NGF, the pattern of BAX staining is diffuse indicating a cytosolic location (Fig. 4A). The BAX staining pattern is also diffuse in neurons deprived of NGF in the presence of cycloheximide (Fig. 4B), but is observed as a punctate (mitochondrial) pattern in NGF-deprived neurons incubated in BAF. The bisbenzimide staining shows the corresponding nuclear morphology. These results indicate that NGF-deprivation of immature neurons leads to a macromolecular synthesis-dependent, caspase independent translocation of BAX to the mitochondria. Furthermore, this translocation occurs at approximately the time the neurons become committed to die (Deckwerth and Johnson, supra).

To investigate the intracellular location of cytochrome C, primary sympathetic neurons from embryos of wild-type and BAX knock-out mice were prepared and maintained in vitro for 5 days in the presence of NGF as described above. The wild- type neurons were either maintained in NGF for 3 additional days or deprived of NGF for 3 days in the presence of BAF and then immunostained with an anti-cytochrome C antibody. The box-deficient neurons were also immunostained for cytochrome C three days after NGF-deprivation. The results are shown in Figure 5.

In NGF-maintained wild-type neurons, the cytochrome c staining pattern is punctate (mitochondrial) (FIG. 5A). However, in wild-type neurons deprived of NGF, the staining pattern is diffuse (FIG. 5C), indicating translocation of cytochrome c to the cytoplasm. This translocation is not observed in NGF-deprived neurons treated with cycloheximide (data not shown) and is also dependent upon BAX, as demonstrated by the cytochrome c punctate staining pattern observed in x-deficient neurons deprived of NGF (FIG. 5B). In addition, cytochrome c translocation is not prevented when apoptotic death is prevented by treatment with the caspase inhibitor BAF (FIG. 5C). In double-labeling experiments, BAX and cytochrome c were rarely found in the cytoplasm at the same time (data not shown), indicating that cytochrome c is released shortly after BAX translocates to the mitochondria. The collective results of these experiments suggest that NGF deprivation of immature neurons leads to a macromolecular synthesis-dependent, BAX-dependent, caspase-independent translocation of cytochrome c from the mitochondria to the cytoplasm.

Since mature neurons do not die after NGF-deprivation, the localization of BAX and cytochrome c in these neurons was also examined. BAX remained in the cytoplasm of mature neurons following removal of NGF and immunostaining for cytochrome c indicated there was no release of cytochrome c from the mitochondria. These results are consistent with a model that BAX translocation to the mitochondria is necessary for cytochrome c release from the mitochondria and the apoptotic death of neurons.

Example 2 This example illustrates the neuroprotective activity of three tBAX polypeptides derived from murine BAXα. The three tBAX polypeptides used in this study were tBAX78_M (SEQ ID

NO:8), tBAX70_M (SEQ ID NO:6) and tBAX78 (SEQ ID NO:4). The tBAX78 polypeptide consists of the first 78 amino acids of murine BAXα and is encoded by SEQ ID NO: 14. Each of tBAX70_M (SEQ ID NO:6) and tBAX78_M (SEQ ID NO:8) consist of the first 54 amino acids of murine BAXα but terminate with the sequences shown in Figure 2, which contain serine to alanine substitutions at positions 55 and 60. The tB AX78_M polypeptide also has a novel C-terminal sequence of 8 amino acids not present in BAXα. The polynucleotides encoding the murine versions of tBAX70_M and tBAX78M comprise the nucleotide sequences set forth in SEQ ID NO: 15 and 16, respectively. The anti-apoptotic activity of these polypeptides was assessed using a modification of the superior cervical ganglion survival assay previously reported (Martin et al., J of Cell Biol. 106:829-844, 1988; Deckwerth and Johnson, J. Cell Biol. 123:1207-1225, 1993).

Cultures of primary SCG neurons were prepared and maintained in vitro in NGF for 5-7 days as described above. The neurons were then co-injected with a plasmid encoding green fluorescent protein (GFP) and either expression vector alone as a control or the expression vector encoding a tBAX polypeptide. Sixteen to 24 hrs. after injection, microinjected cells were counted on the fluorescent microscope, and this number was used as a baseline. After the baseline count, the primary SCG neurons were maintained in NGF (+NGF) or deprived of NGF (-NGF) by exchanging the medium with medium lacking NGF and containing 0.05% goat anti-NGF (final titer in the wells is 1:10). This NGF-deprivation results in death of unprotected neurons over a period of 24-72 hours. At 24 and 48 hr after NGF deprivation, the number of neurons remaining GFP-positive and phase-bright were counted by a naive observer. The numbers of neurons scored at 24 and 48 hr are expressed as a percentage of the baseline count and the mean and s.d. of 5 experiments for IBAX78_M are shown in Figure 6.

Expression of tBAX78-vi for one day prior to NGF deprivation prevented apoptosis of the majority of the neurons in that over 80% of tBAX-expressing neurons were viable at 24 hr following NGF deprivation as compared to less than 20% of the noninjected neurons. Surprisingly, the neuroprotection provided by tBAX78_M is consistently superior to that produced by BCL-2 expression via the same promoter (data not shown). The tBAX78 and tBAX70_M polypeptides were also active as death suppressors, and were comparable to BCL-2 in their anti-apoptotic activities (data not shown).

These data indicate that the serine to alanine substitutions at positions 55 and 60 do not significantly affect the anti-apoptotic activity of a tBAX polypeptide. The greater effectiveness of tBAX78_M suggests that the novel 8 amino acid C-terminus either makes the molecule more stable or more potent than the other tBAX polypeptides tested.

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. All references cited in this specification are hereby incorporated by reference.

The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

Claims

What is Claimed is:

1. An isolated and purified polypeptide comprising a truncated BAX (tBAX) polypeptide which inhibits apoptosis of neuronal cells.

2. The isolated and purified polypeptide of claim 1 wherein, the tBAX polypeptide comprises an N-terminal region and at least part of a BH3 domain.

3. The isolated and purified polypeptide of claim 2, wherein the tBAX polypeptide further comprises a BHl and a BH2 domain.

4. The isolated and purified polypeptide of claim 2, wherein the tBAX polypeptide consists of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

5. The isolated and purified polypeptide of claim 4, wherein the tBAX polypeptide consists of SEQ ID NO:7.

6. An isolated and purified polynucleotide comprising a nucleotide sequence encoding a tBAX polypeptide.

7. The isolated and purified polynucleotide of claim 6, wherein the tBAX polypeptide consists of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

8. The isolated and purified polynucleotide of claim 7 which comprises SEQ ID NO:16 or SEQ ID NO:17.

9. A vector comprising a recombinant DNA molecule comprising an expression regulatory element operably linked to a nucleotide sequence encoding the tBAX polypeptide of claim 2.

10. A host cell transformed with the vector of claim 8.

11. A method for inhibiting apoptosis of a target neuron comprising treating the neuron with an effective amount of a truncated BAX (tBAX) polypeptide which inhibits apoptosis of neuronal cells.

12. The method of claim 11, wherein the target neuron is in a patient.

13. The method of claim 12, wherein the treating step comprises administering to the patient a polynucleotide encoding the tBAX polypeptide, wherein the polynucleotide is internalized in the target neuron and the tBAX polypeptide is expressed.

14. The method of claim 12, wherein the treating step comprises administering the tBAX polypeptide to the patient.

15. The method of claim 14 wherein the tBAX polypeptide is administered with a carrier which facilitates delivery of the tBAX polypeptide into the neuron.

16. The method of claim 12 wherein the patient suffers from a neurodegenerative disease, peripheral nerve injury, spinal cord injury, head trauma or stroke.

17. A composition comprising a truncated BAX (tBAX) polypeptide and a carrier suitable for facilitating delivery of the tBAX polypeptide into a neuron.

18. The composition of claim 17, wherein the tBAX polypeptide consists of SEQ ID NO:7.

19. A transgenic, nonhuman animal expressing a tBAX polypeptide which inhibits apoptosis of neurons.

20. The transgenic animal of claim 19, wherein the tBAX polypeptide consists of SEQ ID NO:8.