HK1069322B - Tryptophanyl-trna synthetase derived polypeptides useful for the regulation of angiogenesis - Google Patents

Description

tryptophanyl-tRNA synthetase derived polypeptides for modulating angiogenesis

Priority requirement

This application claims priority from U.S. provisional application serial No. 60/270,951 filed on 23/2/2001.

Government rights

The invention was made with government support from the U.S. government national ministry of health, GM23562 fund, and therefore the U.S. government has certain rights in the invention.

Technical Field

The invention relates to compositions comprising truncated tRNA synthetase polypeptides, and nucleic acids encoding the truncated tRNA synthetase polypeptides. Methods of making and using the compositions are also disclosed.

Background

aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA molecules, which are ancient proteases that decode genetic information during translation. In higher eukaryotes, 9 aminoacyl-tRNA synthetases associate with at least 3 other polypeptides to form supramolecular multienzyme complexes (Mirande et al, Eur. J. biochem. 147: 281-891985)). Each eukaryotic tRNA synthetase consists of a core enzyme, which is closely related to the prokaryotic counterpart of the tRNA synthetase, and additional domains attached to the amino-or carboxy-terminus of the core enzyme (Mirandde, prog. nucleic Acid Res. mol. biol. 40: 95-1421991)).

In most cases, the additional domain appears to be for assembly of a multi-enzyme complex. However, the presence of additional domains is not strictly associated with the association of synthetases into a multi-enzyme complex.

Mammalian TrpRS molecules have an amino-terminal additional domain. In normal human cells, two forms of TrpRS can be detected: major forms consisting of the full-length molecule (amino acid residues 1-471 of SEQ ID NO: 1) and truncated minor forms ("Mini-TrpRS"; amino acid residues 1-424 of SEQ ID NO: 3). The minor form is formed by the removal of the amino-terminal domain by alternative splicing of the pre-mRNA (Tolst rup et al, J.biol. chem.270: 397-403 (1995)). The amino terminus of Mini-TrpRS has been determined to be the methionine amino group (id.) at position 48 of the full-length TrpRS molecule. Alternatively, truncated TrpRS can be produced by proteolysis (Lemaire et al, Eur.J.biochem.51: 237-52 (1975)). For example, bovine TrpRS is highly expressed in the pancreas and secreted into pancreatic juice (Kisselev, Biochimie 75: 1027-39(1993)), resulting in the production of truncated TrpRS. These results indicate that truncated TrpRS can have functions other than aminoacylated tRNA (id.).

Angiogenesis, or the proliferation of new capillaries from existing blood vessels, is the fundamental process of embryonic development, subsequent growth and tissue repair. Angiogenesis is a prerequisite for vascular tree development and differentiation, as well as a variety of basic physiological processes, including embryogenesis, body growth, tissue and organ repair and regeneration, periodic growth of the corpus luteum and endometrium, and development and differentiation of the nervous system. In the female reproductive system, angiogenesis is present in the developing corpus luteum, in the post-ovulatory corpus luteum, and in the placenta to establish and maintain pregnancy. Angiogenesis also occurs as part of the process of body repair, such as wound and fracture healing. Angiogenesis is also a factor in tumor growth because tumors must continually stimulate the growth of new capillaries in order to grow. Angiogenesis is an integral part of the growth of human solid cancers, and abnormal angiogenesis is associated with other diseases such as rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J.and Klagsbrunn, M., Science 235: 442-447 (1987)).

Some factors are involved in angiogenesis. Both acidic and basic fibroblast growth factor molecules are mitogens for endothelial cells and other cell types. Although not functionally known, Angiotropin and angiogenin can induce angiogenesis (Folkman, J., cancer medicine, pp.153-170, Lea and Febiger Press (1993)). A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor or VEGF (Ferrara, N., et al., Endocr. Rev.13: 19-32, (1992)).

Most diseases that result in high loss of vision are those that result from ocular neovascularization; age-related macular degeneration (ARMD) affects 1200-1500 ten thousand Americans over the age of 65 and causes 10-15% of them to die due to the direct effects of choroidal (subretinal) neovascularization. Among americans over age 65, the leading cause of vision loss is diabetes; in the united states, 1600 million people suffer from diabetes, with 40,000 people per year developing ocular complications, usually due to retinal neovascularization. Although photocoagulation is effective in preventing severe vision loss in a subset of high-risk diabetic patients, the overall 10-year incidence of retinopathy remains largely unchanged. Photocoagulation is ineffective in preventing vision loss in patients with choroidal neovascularization due to ARMD or inflammatory eye diseases such as ocular histoplasmosis, with few exceptions. Although a non-destructive photodynamic therapy was recently developed with the hope of temporarily reducing vision loss in patients with previously treatment-ineffective choroidal neovascularization, only 61.4% of patients treated every 3-4 months improved or stabilized vision, while the placebo-treated group was 45.9%.

In normal adults, angiogenesis is tightly regulated and limited to wound healing, pregnancy and the uterine cycle. Angiogenesis is activated by specific angiogenic molecules such as basic and acidic Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF), angiogenin, Transforming Growth Factor (TGF), tumor necrosis factor alpha (TNF-alpha), and platelet-derived growth factor (PDGF). Angiogenesis can be inhibited by inhibitory molecules such as interferon alpha, thrombospondin-1, angiostatin, and endostatin. The balance of these naturally occurring stimulants and inhibitors controls the normally quiescent capillary system. When this balance is disturbed, for example in certain disease states, capillary endothelial cells are induced to proliferate, migrate and eventually differentiate.

Angiogenesis plays a central role in a variety of diseases, including cancer and ocular neovascularization. It has also been demonstrated that the continued growth and metastasis of a variety of tumors depends on the growth of new host blood vessels into the tumor in response to tumor-derived angiogenic factors. Proliferation of new blood vessels in response to a variety of stimuli is a major finding in most eye diseases and blindness, including Proliferative Diabetic Retinopathy (PDR), ARMD, iridocytic glaucoma, interstitial keratitis, and early onset retinopathy. In these diseases, tissue destruction can stimulate the release of angiogenic factors, leading to capillary proliferation. VEGF plays a major role in iris neoangiogenesis and neovascular retinopathy. Although reports clearly show the relationship between intraocular VEGF levels and ischemic retinopathy of the eye neovascularization, FGF is likely to play a role. Basic and acidic FGFs are known to be present in the normal adult retina, although detectable levels are not always associated with neovascularization. This is likely due to the fact that FGF binds very tightly to charged components of the extracellular matrix and does not readily exist in a freely diffusing form that can be detected by standard intraocular fluid assays.

The most common pathway for angiogenic responses involves the formation of integrin-mediated information exchange between proliferating vascular endothelial cells and the extracellular matrix. This class of adhesion receptors, called integrins, is expressed as heterodimers with alpha and beta subunits on all cells. An integrin, alpha_vβ₃Are the most promiscuous members of this family and allow endothelial cells to interact with a variety of extracellular matrix components. The integrin peptides and antibody antagonists inhibit angiogenesis by selectively inducing apoptosis of proliferating vascular endothelial cells. There are two cytokine-dependent angiogenesis pathways and integrin α can be targeted by them to different vascular cells_vβ₃And alpha_vβ₅Is determined. Specifically, basic FGF and VEGF induced angiogenesis are alpha dependent, respectively_vβ₃And alpha_vβ₅Since antibody antagonists of each integrin selectively block one of these angiogenic pathways in the rabbit cornea and chick chorioallantoic membrane (CAM) models. Block all alpha_vPeptide antagonists of integrins inhibit FGF and VEGF stimulated angiogenesis. Although normal ocular vessels are not apparentShowing any one of the integrins, alpha_vβ₃And alpha_vβ₅Selectively displayed on blood vessels in the tissue of a patient with active neovascular ocular disease. Although only alpha_vβ₃Alpha can be observed continuously in the tissues of ARMD patients_vβ₃And alpha_vβ₅Are present in the tissues of PDR patients. Systemic administration of peptide antagonists of integrins can block neovascularization in a mouse model of retinal angiogenesis.

Anti-angiogenic agents therefore play a role in treating retinal degeneration to prevent the damaging effects of these tropism and growth factors. Angiogenic agents have the effect of promoting desirable vascularization to delay retinal degeneration by increasing blood flow to the cells.

Summary of The Invention

tryptophanyl-tRNA synthetase derived polypeptides that are shorter than the naturally occurring form have chemokine activity and are useful for research; diagnosing; prognostic and therapeutic uses. In one embodiment, these tRNA synthetase derived polypeptides are useful for modulating vascular endothelial cell function, in particular for inhibiting angiogenesis, particularly ocular neovascularization.

These truncated tryptophanyl-tRNA synthetase derived polypeptides have an amino-terminal truncation, but may also comprise a Rossmann fold nucleotide binding domain. These polypeptides may modulate vascular endothelial cell function.

A preferred truncated tryptophanyl-tRNA synthetase derived polypeptide comprises a sequence consisting essentially of SEQ ID NO: 1 and angiogenesis inhibiting fragments thereof, in particular amino acid residues 94-471 of SEQ ID NO: 10 and SEQ ID NO: 11 or a fragment comprising at least one of these signal sequences. In a preferred embodiment, the truncated tRNA synthetase polypeptide is mammalian, more preferably human.

In another embodiment, the invention includes an isolated polynucleotide having a nucleotide sequence at least 95% identical to a sequence of a polynucleotide selected from the group consisting of: SEQ ID NO: 6; can be compared with SEQ ID NO: 6; encodes the amino acid sequence of SEQ ID NO: 7; encoding the amino acid sequence of SEQ ID NO: 12; encoding the amino acid sequence of SEQ ID NO: 7; and may be identical to a nucleic acid encoding SEQ ID NO: 7, or a polynucleotide that hybridizes to a polynucleotide of the polypeptide epitope of 7. The invention also includes a recombinant expression vector comprising an isolated nucleic acid molecule encoding any of the tryptophanyl-tRNA synthetase derived polypeptides described above. Another aspect is a host cell comprising the recombinant expression vector.

The invention also provides compositions and dosage forms comprising a truncated tryptophanyl-tRNA synthetase derived polypeptide and a pharmaceutically acceptable excipient. The compositions are suitable for intraocular, e.g., intravitreal, subretinal, etc., as well as systemic, e.g., transdermal, transmucosal, enteral or parenteral administration.

In another embodiment, the invention provides a method of treating an ocular neovascular disease, such as age-related macular degeneration, diabetic ocular complications, rubeosis iridis, early onset retinopathy, keratitis, ischemia, retinopathy (e.g., sickle cells), pathological myopia, ocular histoplasmosis, pterygium, punitate endochoroidopathy, and the like, by administering an angiogenesis inhibiting amount of a polypeptide and an appropriate physiologically compatible excipient or carrier.

Brief Description of Drawings

In the drawings, there is shown in the drawings,

FIG. 1 shows a tryptophanyl-tRNA synthetase polypeptide (SEQ ID NO: 1) comprising a signal sequence (SEQ ID NO: 10 and SEQ ID NO: 11) shown in boxes, also comprised in a truncated form (amino acid residues 94-471 of SEQ ID NO: 1).

Fig. 2 is a micrograph illustrating retinal vascular development in a mouse model.

Figure 3 is a graphical representation of the data reported in example 3 below.

Figure 4 is a graphical representation of the data reported in example 4 below.

Fig. 5 is a micrograph illustrating the binding site of a fragment of TrpRS (T2) in the retina of a mouse model.

Detailed Description

Definition of

By "truncated tRNA synthetase polypeptide" is meant a polypeptide that is shorter than the corresponding full tRNA synthetase.

"TrpRS" refers to tryptophanyl-tRNA synthetase.

"cell culture" includes both culture media and cultured cells.

The phrase "isolating a polypeptide from a cell culture" includes isolating a soluble or secreted polypeptide from the culture medium, as well as isolating an integral membrane protein from the cultured cells.

"cell extract" includes medium from which cells have been removed, particularly spent medium. It is understood that a cell extract comprising a DNA or protein of interest means a homogeneous preparation or a cell-free preparation obtained from cells expressing the DNA or protein containing the protein of interest.

A "plasmid" is an autonomous, self-replicating extra-chromosomal DNA molecule, previously indicated by the lower case letter "p" and/or subsequently indicated by the size letter and/or number. The starting plasmids herein are commercially available, publicly available on an unlimited basis, or may be constructed from potential plasmids according to the disclosed procedures. In addition, plasmids equivalent to those known in the art will be apparent to those skilled in the art.

"digesting" DNA refers to the catalytic cleavage of DNA with restriction enzymes that act only on certain sequences in the DNA. Various restriction enzymes useful herein are commercially available, and their reaction conditions, cofactors and other requirements used are well known to those skilled in the art. For analytical purposes, 1. mu.g of plasmid or DNA fragment and about 2 units of enzyme are generally used in about 20. mu.l of buffer. For the purpose of isolating DNA fragments from plasmid constructs, 5-50. mu.g of DNA fragments are generally digested with 2-250 units of enzyme in a larger volume. The appropriate buffer and substrate amounts for a particular restriction enzyme are specified by the manufacturer. Incubation times of about 1 hour at 37 ℃ are common, but can vary according to the supplier's instructions. After digestion, the desired fragment was isolated by direct electrophoresis on a polyacrylamide gel. The standard one-letter symbols (A, T, C, G, U) used in the art are used herein to denote the nucleotides in the various DNA and RNA fragments.

"Polynucleotide" in the present invention may be in the form of RNA or in the form of DNA, including cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (anti-sense) strand. The coding sequence encoding the mature polypeptide may be identical to SEQ ID NO: 6, which coding sequence encodes a polypeptide which is identical to or may be a different coding sequence than the coding sequence set forth in SEQ ID NO: 7 identical mature polypeptide sequence.

The term "polynucleotide encoding a polypeptide" includes polynucleotides that contain only the coding sequence for the polypeptide and polynucleotides that contain additional coding and/or non-coding sequences.

"oligonucleotide" refers to a single-stranded polynucleotide or two complementary polynucleotide strands that can be chemically synthesized. The synthetic oligonucleotide has no 5' phosphate and therefore will not be linked to another oligonucleotide as long as the phosphate is not added by ATP in the presence of the kinase. The synthetic oligonucleotide will be ligated to the non-dephosphorylated fragment.

"amino acid residue" refers to an amino acid that is part of a polypeptide. The amino acid residues described herein are preferablyThe "L" configuration. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue, as long as the desired functional properties of the polypeptide are retained. NH (NH)₂Refers to the free amino group at the amino terminus of the polypeptide, and COOH refers to the free carboxyl group at the carboxyl terminus of the polypeptide. And standard polypeptide nomenclature (described inJ.Biol.Chem.,243: 3552-59(1969) and adopted under 37c.f.r. § 1.822) and the abbreviations for the amino acid residues are given in the following table.

TABLE 1

Correspondence table

Symbol

All amino acid residue sequences represented by the formulae herein are in the conventional amino-terminal to carboxy-terminal left-to-right orientation. Furthermore, the phrase "amino acid residue" has a broad definition, including the amino acids and modifications listed in table 1, as well as unusual amino acids, such as those mentioned in 37c.f.r. § 1.822, which are incorporated herein by reference. The dashes at the beginning or end of the amino acid residue sequence indicate a relationship with other sequences or one or more amino acid residues, or amino end groups such as NH₂Or a peptide bond between the carboxyl end groups, such as COOH.

Suitable conservative substitutions of amino acids in a peptide or protein are well known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in The art understand that, in general, single amino acid substitutions made in non-critical regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al molecular Biology of The Gene, 4th Edition, 1987, The Benjamin/Cummings pub. Co., p.224).

The substitution is preferably performed according to table 2:

TABLE 2

Other substitutions are also permissible and can be determined empirically or made by conservative substitutions well known in the art.

A "complementing" plasmid describes a plasmid vector that delivers nucleic acid into a packaging cell line for stable integration into a chromosome in the genome of a cell.

By "delivery plasmid" is meant a plasmid vector that carries or delivers a nucleic acid encoding a therapeutic gene, or encoding a therapeutic product or precursor thereof, or a regulatory gene or other factor, which results in a therapeutic effect when delivered or delivered in vivo to a cell line, such as, but not limited to, a packaging cell line to propagate a therapeutic viral vector.

Various vectors are described herein. For example, a vector is used to deliver a particular nucleic acid molecule into a packaging cell line for stable integration into a chromosome. These types of vectors are generally identified herein as complementing plasmids. Another vector described herein carries or delivers nucleic acid molecules in or to a cell line (e.g., a packaging cell line) for the propagation of therapeutic viral vectors, and is generally referred to herein as a delivery vector. A third "type" of vector described herein is for carrying a nucleic acid molecule that encodes a therapeutic protein or polypeptide or a regulatory protein or that is a regulatory sequence of a particular cell or cell type in a subject in need thereof; these vectors are generally identified herein as therapeutic viral vectors or recombinant adenoviral vectors or viral Ad derived vectors and are in the form of viral particles encapsulating viral nucleic acid containing expression cassettes for expression of the therapeutic genes.

"DNA or nucleic acid homolog" refers to a nucleic acid comprising a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide. The term "substantially homologous" means having at least 80%, preferably at least 90%, most preferably at least 95% homology, or having less homology or identity than above but conserved biological activity or function.

The terms "homology" or "identity" are generally used interchangeably. In this regard, the degree of homology or identity can be determined by comparing sequence information, for example, using the GAP computer program. The GAP program utilizes Needleman and Wunsch, j.mol.biol.48: 443(1970) by Smith and Waterman, adv.appl.math.2: 482(1981) modifications. Briefly, the GAP program determines similarity as the number of similar symbols in the aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Preferred default parameters for the GAP program may include: (1) unary comparison matrices (identical value 1, different value) and Gribskov and Burgess, nuclear. 6745(1986) weighted comparison matrices, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National biomedical research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 per gap and a penalty of 0.10 per symbol in each gap; and (3) no penalty for end gaps. For example Pearson and Lipman, proc.nati.acad.sci.usa85: 2444(1988) determining whether two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical using well-known computer algorithms such as the "FASTA" program. Alternatively, identity can be determined using the BLAST function of the national bioinformatics database. "identity" itself has a meaning well known in the art and can be calculated using well known techniques (see, e.g., Computer Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, (1988); Smith, D.W., ed., Biocomputing: information and Genome Projects, Academic Press, New York, (1993); Griffin, A.M., and Griffin, H.G., eds., Computer Analysis of Sequence, Data, Parti, HumanaPress, New Jerses, (1994); yon health, G., Sequence Analysis Biology, Academic Press, (1987); and Griffinov, M.M., device, J., priority, software, New York, N.S.J., New York, and New York, N.S.. Although there are many ways to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those skilled in the art (Carillo, H. & Lipton, D., SIAMJ. applied Math.48: 1073 (1988)). Methods commonly used to determine identity or similarity between two sequences include, but are not limited to, those disclosed in martin j. bishop, ed., Guide to huge computers, Academic Press, San Diego, (1994), and carllo, H. & Lipton, d., siamj. applied math.48: 1073 (1988). Methods for determining identity and similarity are compiled in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research12 (I): 387(1984)), BLASTP, BLASTN, PASTA (Atschul, S.F., et al., J.Molec.biol.215: 403 (1990)).

The term "identity" refers to a comparison between a test and reference polypeptide or polynucleotide. For example, a test polypeptide can be identified as any polypeptide that is 90% or more identical to a reference polypeptide. The term "90% identical" as used herein refers to a percent identity of 90-99.99% relative to a reference polypeptide. An identity of 90% or higher indicates that the test and reference polypeptides are assumed to be compared in length of 100 amino acids. No more than 10% (i.e. 10 out of 100) of the amino acids in the test polypeptide differ from the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences may be expressed as point mutations randomly distributed over the full length of the amino acid sequence, or they may be clustered at one or more positions of different length up to a maximum number allowed, e.g., 10/100 amino acids different (about 90% identity). Different is defined as a nucleic acid or amino acid substitution or deletion.

The term "gene therapy" refers to the transfer of heterologous DNA to certain cells, target cells, of a mammal, particularly a human, suffering from a disease or condition targeted by the therapy. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and the therapeutic product encoded thereby is produced. Alternatively, the heterologous DNA may in some way mediate the expression of the DNA encoding the therapeutic product, it may encode a product, such as a peptide or RNA, which in some way directly or indirectly mediates the expression of the therapeutic product. Gene therapy may also be used to encode nucleic acids for gene products, to replace defective genes or to supplement the production of gene products in introduced mammals or cells. The introduced nucleic acid may encode a therapeutic compound, such as a growth factor inhibitor, or a tumor necrosis factor or inhibitor thereof, such as a receptor thereof, which is not normally produced in the mammalian host or produced in a therapeutically effective amount or produced at a therapeutically useful time. Heterologous DNA encoding a therapeutic product may be modified prior to introduction into cells of a diseased host to enhance or alter the product or its expression.

"heterologous DNA" is DNA that encodes RNA and proteins that are not normally produced in vivo in the cell in which it is expressed, or that mediates endogenous DNA expression or encodes a mediator by affecting transcription, translation, or other regulated biochemical processes. Heterologous DNA may also be referred to as exogenous DNA. Any DNA known or believed to be heterologous or foreign to the cell expressing the DNA by those skilled in the art is included herein in the scope of heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA encoding traceable marker proteins, such as proteins conferring drug resistance, DNA encoding therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA encoding other types of proteins, such as antibodies. The antibody encoded by the heterologous DNA can be secreted or expressed on the cell surface into which the heterologous DNA is introduced. As used herein, "heterologous DNA" or "foreign DNA" refers to DNA molecules that are not precisely oriented or positioned relative to the corresponding DNA molecule present in the corresponding wild-type adenovirus. It may also be referred to as a DNA molecule from another organism or species (i.e. foreign) or from another Ad serotype.

By "therapeutically effective DNA product" is meant a product encoded by DNA which, when introduced into a host, expresses a product effective to ameliorate or eliminate symptoms, i.e., manifestation or cure of an innate or acquired disease. Typically, the DNA encoding the desired heterologous DNA is cloned into a plasmid vector and introduced into a producer cell, such as a packaging cell, by conventional methods, such as calcium phosphate-mediated DNA uptake or microinjection. After expansion in the producer cells, the vector containing the heterologous DNA is introduced into the selected target cells.

By "expression or delivery vector" is meant any plasmid or virus into which foreign or heterologous DNA may be inserted for expression in an appropriate host cell, i.e., the protein or polypeptide encoded by the DNA is synthesized in the host cell system. A vector capable of directing the expression of a DNA segment (gene) encoding one or more proteins is referred to herein as an "expression vector". It also includes vectors that allow the cloning of cDNA (complementary DNA) from mRNA using reverse transcriptase.

A "gene" is a nucleic acid molecule whose nucleotide sequence encodes an RNA or polypeptide. The gene may be RNA or DNA. Genes may include intervening sequences (introns) between regions preceding and following the coding region (leader and trailer) and the respective coding segments (exons).

"isolated" for a nucleic acid molecule, polypeptide, or other biological molecule means that the nucleic acid or polypeptide has been separated from the genetic environment in which the polypeptide or nucleic acid was obtained. It may also indicate that a change has occurred from the natural state. For example, as the term is used herein, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but the same polynucleotide or polypeptide separated from its naturally occurring coexisting materials is "isolated". Thus, a polypeptide or polynucleotide produced in and/or comprised by a recombinant host cell is considered isolated. An "isolated polypeptide" or "isolated polynucleotide" also includes polypeptides or polynucleotides that are partially or substantially purified from a recombinant host cell or natural source. For example, recombinantly produced forms of the compounds may be produced by Smith and Johnson, Gene 67: 31-40(1988) in the one-step process. The terms "isolated" and "purified" are sometimes used interchangeably. The polynucleotide may be part of a vector, and/or the polynucleotide or polypeptide may be part of a composition but still be isolated in that the vector or composition is not part of its natural environment.

By "isolated polynucleotide" is meant that the nucleic acid does not contain the coding sequences of the gene that flanks the gene encoding the nucleic acid of interest (if any) in the genome of the naturally occurring organism. The isolated DNA may be single-stranded or double-stranded, and may be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It may be identical to the native DNA sequence or may differ therefrom by deletion, addition or substitution of one or more nucleotides.

When referring to a preparation prepared from a biological cell or host, "isolated" or "purified" means any cell extract containing the DNA or protein in question, including crude extracts of the DNA or protein of interest. For example, for proteins, purified preparations may be obtained according to various techniques or a series of preparative or biochemical techniques, and the DNA or protein of interest may be present in these preparations in various purities. These procedures may include, for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, density gradient centrifugation, and electrophoresis.

A "substantially pure" or "isolated" preparation of DNA or protein refers to a preparation that is free of naturally occurring substances with which the DNA or protein is normally associated in its natural state. "substantially pure" is understood to mean a "highly" purified preparation which contains at least 95% DNA or protein of interest.

A "packaging cell line" is a cell line that provides a deleted gene product or its equivalent.

An "adenovirus particle" is the smallest structural or functional unit of a virus. A virus may refer to a single particle, a collection of particles, or a viral genome. Adenovirus (Ad) particles are relatively complex and can be split into multiple substructures.

A "post-transcriptional regulatory element (PRE)" is a regulatory element present in the unspliced viral or cellular messenger RNA, i.e., the intron-free message. Examples include, but are not limited to, human hepatitis virus, woodchuck hepatitis virus, TK gene, and mouse histone gene. The PRE may be placed before the polyadenylation sequence or after the heterologous DNA sequence.

"pseudotyped" describes the production of adenoviral vectors with modified capsid proteins or capsid proteins from a serotype different from the serotype of the vector itself. One example is the generation of adenovirus vector particles containing Ad38 fiber protein. This can be accomplished by generating the adenoviral vector in a packaging cell line expressing different fiber proteins.

The "promoter of interest" may be inducible or constitutive. An inducible promoter will initiate transcription only in the presence of other molecules; constitutive promoters do not require any other molecule to regulate gene expression. A regulatable or inducible promoter can also be described as a promoter in which the rate or extent of RNA polymerase binding and initiation is regulated by an exogenous stimulus. Such stimuli include, but are not limited to, a variety of compounds or compositions, light, heat, pressure, and chemically energy-regulatable promoters. Inducible, repressible and repressible promoters are considered regulatable promoters. Preferred promoters herein are those that are selectively expressed in ocular cells, particularly photoreceptor cells.

"receptor" refers to a biologically active molecule that specifically binds to other molecules. The term "receptor protein" may be used more specifically to denote the protein nature of a specific receptor.

"recombinant" refers to any progeny that result from genetic engineering. It can be used to describe viruses formed by recombination of plasmids in packaging cells.

"transgenic" or "therapeutic nucleic acid molecules" include DNA and RNA molecules that encode RNA or polypeptides. These molecules may be "natural" or sequences of natural origin; they may also be "non-natural" or "foreign", i.e. of natural or recombinant origin. The term "transgene" as used interchangeably herein with the term "therapeutic nucleic acid molecule" is generally used to describe a heterologous or foreign (foreign) gene carried by a viral vector and transduced into a host cell. Therapeutic nucleic acid molecules include antisense sequences or nucleotide sequences that can be transcribed into antisense sequences. All therapeutic nucleotide sequences (or transgenes) include nucleic acid molecules that produce the desired effect in the cell or nucleus into which the therapeutic sequence is delivered. For example, a therapeutic nucleic acid molecule can include a nucleotide sequence that encodes a functional protein for delivery into a cell that is incapable of producing the functional protein.

By "vitreous of the eye" is meant a substance (i.e., aqueous humor or vitreous) that fills the posterior chamber of the ocular lens.

"promoter region" refers to a portion of the DNA of a gene that controls transcription of the DNA to which it is operably linked. The promoter region includes specific sequences of DNA sufficient for RNA polymerase recognition, binding, and initiation of transcription. This part of the promoter region is called the promoter. In addition, the promoter region includes a region that regulates this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis-acting or may be reactive to trans-acting factors. Depending on the nature of the regulation, the promoter may be constitutive or regulated.

"operably linked" means that the sequences or fragments have been covalently linked to a single or double stranded DNA segment such that the control sequences on one segment can control expression or replication, or exert such control over the other segment. However, the two fragments need not be contiguous.

"packaging" refers to a solid substrate or material, such as glass, plastic (e.g., polyethylene, polypropylene, or polycarbonate), paper, foil, etc., into which a polypeptide, polyclonal antibody, or monoclonal antibody of the invention can be immobilized. Thus, for example, the package may be a glass vial for containing milligram quantities of the polypeptide of interest or a microtiter plate well to which microgram quantities of the polypeptide or antibody of interest may be operatively attached (i.e., linked) so as to be capable of immunobinding to the antibody or antigen, respectively.

"Instructions for use" generally include tangible expressions that describe the concentration of a reagent or at least one parameter of the assay, such as the relative amounts of reagent and sample to be mixed, the maintenance time for mixing of the reagent and sample, temperature, buffer conditions, and the like.

The "diagnostic system" in the context of the present invention also comprises a marker or indicator means capable of signaling the formation of an immune complex comprising a polypeptide or antibody molecule of the invention.

As used herein, "complex" refers to the product of a specific binding reaction, such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immune reaction products.

"labels" and "indicating means" used in their various grammatical forms refer to individual atoms and molecules that directly or indirectly participate in the generation of a detectable signal to indicate the presence of a complex. Any labeling or indicator means may be attached to or incorporated into the expressed protein, polypeptide or antibody molecule as part of the antibody or monoclonal antibody composition of the invention, or used independently, and the atom or molecule may be used alone or in combination with other agents. These markers are known per se in clinical diagnostic chemistry and form part of the present invention to the extent that they are used by other novel protein methods and/or systems.

Discussion of the related Art

The polypeptide shown in figure 1 is SEQ ID NO: 1 (as shown in SEQ ID NO: 12), and amino acid residues 94-471 of SEQ ID NO: 7, or a polypeptide consisting of the amino acid sequence of SEQ id no: 6, which form part of the invention. In addition to variants of the above polypeptides, the present invention also includes variants of the polynucleotides. Included polynucleotide variants may be naturally occurring allelic variants of polynucleotides or non-naturally occurring variants of polynucleotides. Accordingly, the invention includes a polypeptide encoding the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 12, and a polynucleotide consisting of the polypeptide set forth in SEQ ID NO: 6, and variants of said polynucleotides encoding the polypeptide encoded by the cDNA of SEQ ID NQ: 7 and SEQ ID NO: 12, an angiogenesis inhibiting fragment, derivative or analogue thereof. The nucleotide variants include deletion variants, substitution variants, and addition or insertion variants.

As described above, the polynucleotide may have a sequence as SEQ ID NO: 6 or an allelic variant of the coding sequence shown in seq id no. As known in the art, an allelic variant is a polynucleotide sequence having a substitution, deletion, or addition of one or more nucleotides that does not substantially alter the function of the encoded polypeptide.

The term T1 as used herein refers to a polypeptide having the sequence of SEQ ID NO: 13 and a polypeptide having the amino acid sequence of SEQ ID NO: 5 containing His of the amino acid sequence₆A labeled polypeptide. The term T2 as used herein refers to a polypeptide having the sequence of SEQ ID NO: 12 and a polypeptide having the amino acid sequence of SEQ id no: 7 containing His of the amino acid sequence₆A labeled polypeptide. The term TrpRS as used herein refers to a polypeptide having the sequence of SEQ ID NO: 1 and an amino acid sequence having residues 1-471 of SEQ ID NO: 1, comprising His of the amino acid sequence of₆A labeled polypeptide.

The invention also includes polynucleotides in which the coding sequence for the mature polypeptide may be fused in the same reading frame with a polynucleotide sequence that aids in the expression and secretion of the polypeptide by a host cell, e.g., a leader sequence that is a secretory sequence that controls the transport of the polypeptide from the cell. The polypeptide having a leader sequence is a proprotein and may have a leader sequence which is cleaved by the host cell to form the mature form of the polypeptide. The polynucleotide may also encode a preprotein, i.e., a mature protein plus additional 5' amino acid residues. The mature protein with the pro sequence (prosequence) is a proprotein and is the inactive form of the protein. Once the pro sequence is cleaved, the active mature protein is retained.

Thus, for example, a polynucleotide of the invention may encode a mature protein, or encode a protein having a pre-sequence and a pre-sequence (leader sequence).

The polynucleotides of the invention may also have coding sequences fused in frame to a marker sequence that allows for purification of the polypeptides of the invention. In the case of bacterial hosts, the tag sequence may be a hexa-histidine tag provided by the pQE-9 vector, thereby providing for purification of the mature polynucleotide fused to the tag, or, when a mammalian host such as COS-7 cells is used, the tag sequence may be, for example, a Hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37: 767 (1984)).

The invention further relates to polynucleotides which hybridize to the sequences described above if there is at least 50%, preferably 70% identity between the sequences. The present invention relates specifically to polynucleotides which hybridize under stringent conditions to the polynucleotides described above. The term "stringent conditions" as used herein means that hybridization will only occur if there is at least 95%, preferably at least 97% identity between the sequences. Polynucleotides that hybridize to the polynucleotides described above encode, in preferred embodiments, polynucleotides that retain the sequence of SEQ ID NO: 6 encodes a mature polypeptide having substantially the same biological function or activity as the mature polypeptide.

When referring to SEQ ID NO: 7, SEQ ID NO: 12 or a polypeptide consisting of SEQ ID NO: 6, the terms "fragment," "derivative," and "analog" refer to a portion of a polypeptide that retains substantially the same vascular inhibitory (i.e., angiogenesis inhibitory) function or activity as the polypeptide. Thus, an "analog" includes a preprotein that can be activated by cleavage of the preprotein moiety, resulting in an mature polypeptide that has angiostatic activity.

The polypeptide of the invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

SEQ ID NO: 7, SEQ ID NO: 12, or an angiogenesis inhibiting fragment, derivative or analogue of SEQ ID NO: 6 may be (i) wherein one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue, preferably a conserved amino acid residue, and the substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code, or (ii) wherein one or more amino acid residues comprise a substituent, or (iii) wherein the polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) wherein additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence used for purification of the polypeptide or proprotein sequence. Such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art in view of the teachings herein.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, preferably purified to homogeneity.

The invention also includes vectors comprising the polynucleotides of the invention, host cells genetically engineered with the vectors of the invention and production of the polypeptides of the invention by recombinant techniques.

The host cell is genetically engineered (transduced or transformed or transfected) with the vector of the present invention, which may be a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, or the like. The engineered host cell can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the tRNA synthetase polypeptide gene. Culture conditions, such as temperature, pH, and the like, previously used to select host cells for expression, will be apparent to those skilled in the art.

The polynucleotides of the invention may be used to produce the corresponding polypeptides by recombinant techniques. Thus, for example, the polynucleotide sequence may be comprised in any expression vector, particularly a vector or plasmid for expressing a polypeptide. The vectors include chromosomal, nonchromosomal and synthetic DNA sequences, such as derivatives of SV 40; a bacterial plasmid; phage DNA; a yeast plasmid; vectors derived from a combination of plasmid and phage DNA, viruses such as vaccinia virus, adenovirus, fowlpox virus and pseudorabies virus DNA. A preferred vector is pET20 b. However, any other plasmid or vector may be used as long as it can replicate and survive in the host.

As described above, the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into the appropriate restriction enzyme site by procedures well known in the art. Such procedures and others are considered to be within the knowledge of those skilled in the art.

The DNA sequence in the expression vector is operably linked to appropriate expression control sequences (promoters) to direct mRNA synthesis. Representative examples of the promoters shown include the LTR or SV40 promoter, the E.coli 1ac or trp promoter, the bacteriophage lambda PL promoter, and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also contain appropriate sequences for amplifying expression.

Furthermore, the expression vector preferably comprises a gene which provides a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or E.coli tetracycline or ampicillin resistance.

Vectors containing the appropriate DNA sequences described above and appropriate promoter or control sequences may be used to transform an appropriate host to allow the host to express the protein. Representative examples of suitable hosts include bacterial cells, such as E.coli, Salmonella typhi, Streptomyces; fungal cells such as yeast; insect cells such as Drosophila and Sf 9; animal cells such as CHO, COS or Bowes melanoma; plant cells, and the like. The selection of an appropriate host is considered to be within the knowledge of one skilled in the art in light of the teachings herein.

More specifically, the invention also includes recombinant constructs comprising one or more of the sequences broadly defined above. The constructs include vectors, such as plasmids or viral vectors, into which the sequences of the invention are inserted in either a forward or reverse orientation. In a preferred aspect of this embodiment, the construct comprises regulatory sequences, including, for example, a promoter operably linked to the sequence. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are provided by way of example: bacteria: pQE70, pQE-9(Qiagen), pBs, phagescript, PsiX174, pBluescriptSK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, p0G44, pXT1, pSG (Stratagene) pSVK3, pBPV, PMSG, pSVL (Pharmacia) and pET 20B. In a preferred embodiment, the vector is pET 20B. However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two suitable vectors are pKK232-8 and pCM 7. Specifically named bacterial promoters include lacI, lacZ, T3, T7, gpt,. lamda.PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-I. Selection of appropriate vectors and promoters is within the level of ordinary skill in the art.

In another embodiment, the invention relates to a host cell comprising the above construct. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a eukaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be achieved by calcium phosphate transfection, DEAE-mediated transfection or electroporation (Davis, l., Dibner, m., Battey, i., Basic methods in molecular biology, 1986).

The construct in the host cell may be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be produced synthetically by conventional peptide synthesizers.

The protein may be expressed in mammalian cells, yeast, bacteria or other cells under the control of a suitable promoter. The proteins may also be produced using cell-free translation systems using RNA derived from the DNA constructs of the invention. Suitable Cloning and expression vectors for prokaryotic and eukaryotic hosts are described in sambrook, el, Molecular Cloning: a. the

Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is incorporated herein by reference.

Transcription of a DNA encoding a polypeptide of the present invention can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to about 300 base pairs (bp), that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the origin of replication (bp100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers.

In general, the recombinant expression vector will include an origin of replication and a selectable marker that permits transformation of the host cell, such as the E.coli ampicillin resistance gene and the s.cerevisiae TRP1 gene, as well as a promoter derived from a highly expressed gene for directing transcription of downstream structural sequences. The promoter may be derived from an operon encoding a glycolytic enzyme such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock protein, etc. The heterologous structural sequence is assembled with transcription start and termination sequences in a suitable bacteriophage, preferably with a leader sequence capable of directing secretion of the post-translational protein into the periplasmic space and the extracellular matrix. Optionally, the heterologous sequence may encode an N-terminal identification peptide comprising stabilization or simple purification of the recombinant product conferring the desired characteristics, such as expression.

After transformation of an appropriate host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by an appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

The cells are typically harvested by centrifugation, physical or chemical disruption, and the resulting crude extract is retained for further purification.

Microbial cells used in protein expression can be disrupted by conventional methods including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

Recombinant proteins can also be expressed using a variety of mammalian cell culture systems. Examples of mammalian expression systems include, for example, Gluzman, Cell, 23: 175(1981), COS-7 lines describing mouse kidney fibroblasts, other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors contain an origin of replication, an appropriate promoter and enhancer, as well as any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, e.g., SV40 origin, early promoter, enhancer, splice and polyadenylation sites, may also be used to provide the required non-transcribed genetic elements.

The polypeptide is recovered and purified from recombinant cell cultures by previously used methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Low concentrations (about 0.1-5mM) of calcium ions are preferably used in the purification (Price, et al.J.biol.chem., 244: 917 (1969)). If necessary, a protein refolding step can be used to complete the conformation of the mature protein. Finally, a final purification step can be performed using High Performance Liquid Chromatography (HPLC).

The polypeptides of the invention may be naturally purified products, or products of chemical synthetic procedures, or produced by recombinant techniques from prokaryotic or eukaryotic hosts, such as cultured bacterial, yeast, higher plant, insect and mammalian cells. According to recombinant production procedures, the polypeptides of the invention may be glycosylated with mammalian or other eukaryotic carbohydrates, or may be non-glycosylated.

The polypeptides of the invention may be modified to improve stability and increase potency by methods well known in the art. For example, the L-amino acid may be substituted with a D-amino acid, the amino terminus may be acetylated, or the carboxy terminus may be modified, such as by capping with ethylamine (Dawson, D.W., et al., mol. Pharmacol., 55: 332-338 (1999)).

According to the present invention, the polypeptide of the present invention can also be used as a gene therapy by expressing the polypeptide in vivo.

Various viral vectors taught herein that may be used for gene therapy include adenovirus, hepatitis virus, vaccinia virus, adeno-associated virus (AAV), or preferably RNA viruses such as retrovirus. Preferably, the retrovirus is a murine or avian retrovirus derivative, or a lentiviral vector. Preferred retroviral vectors are lentiviral vectors. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to: moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). Many additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a selectable marker gene, allowing the identification and generation of transduced cells. The vector is made target-specific, for example, by incorporating the zinc finger-derived DNA-binding polypeptide sequence of interest into a viral vector along with another gene encoding a ligand for a receptor on a particular target cell. Retroviral vectors can be made target-specific by inserting, for example, a polynucleotide encoding a protein. Preferably targeting is achieved by using antibodies directed to the retroviral vector. Those skilled in the art will know or be able to readily determine, without undue experimentation, the specific polynucleotide sequences that may be inserted into a retroviral genome to allow target-specific delivery of a retroviral vector containing a zinc finger-nucleotide binding protein polynucleotide.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. For example, the helper can be provided by using a helper cell line containing plasmids encoding all retroviral structural genes under the control of regulatory sequences within the LTRs. These plasmids lack the ability to identify the packaging machinery for encapsidationNucleotide sequence of the RNA transcript in effect. Helper cell lines lacking a packaging signal include, but are not limited to, for example2, PA317 and PA 12. These cell lines produce empty virions because no genome is packaged. If a retroviral vector is introduced into a cell in which the packaging signal is intact, but the structural gene is replaced by another gene of interest, the vector can be packaged and a vector virion can be produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as the NIH 3T3 cell line, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for polynucleotides encoding zinc finger-derived DNA-binding polypeptides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vacuoles used for delivery of vectors in vivo and in vitro. It has been found that Large Unilamellar Vacuoles (LUVs) of size 0.2-0.4 μm can encapsulate a large proportion of an aqueous buffer containing macromolecules. RNA, DNA, and intact virions can be encapsulated inside water and delivered to cells in a biologically active form (Fraley, et., Trends biochem. sci., 6: 77, (1981)). In addition to mammalian cells, liposomes have been used to deliver polynucleotides in plant, yeast and bacterial cells. In order for liposomes to be effective gene transfer vectors, the following characteristics should exist: (1) the gene is encapsulated with high efficiency without damaging the biological function; (2) preferentially and abundantly bind to target cells compared to non-target cells; (3) delivering aqueous components in the vacuole to the cytoplasm of the target cell with high efficiency; and (4) accurate and efficient expression of genetic information (Mannino, et al, Biotechniques, 6: 682, (1988)).

The composition of liposomes is usually a combination of phospholipids, especially high phase transition temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

Examples of lipids for liposome preparation include phosphatidyl compounds such as phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, sphingomyelin, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, wherein the lipid moiety contains 14-18 carbon atoms, particularly 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

Liposome-directed classification is based on anatomical and mechanical factors. Anatomical classification is based on the level of selectivity, such as organ-specific, cell-specific and organelle-specific. Mechanical guidance can be differentiated according to whether it is passive or active. Passive targeting uses the natural tendency of liposomes to distribute cells of the reticuloendothelial system (RES) to organs containing the capillary sinuses. Active targeting, on the other hand, involves altering the liposomes by coupling them to specific ligands such as monoclonal antibodies, sugars, glycolipids or proteins, or by altering their composition or size, to accomplish targeting to organs and cell types other than the naturally occurring localization sites.

The surface of the targeted delivery system can be modified in a variety of ways. For liposome-directed delivery systems, lipid groups can be incorporated into the lipid-bilateral sides of the liposomes to maintain stable association of the targeting ligand to the lipid-bilateral sides. A variety of linking groups may be used to link the lipid chain to the targeting ligand.

In general, compounds that bind to the surface of a targeted delivery system will be ligands and receptors that allow the targeted delivery system to look for and "home" on the desired cells. The ligand may be any compound of interest that binds to another compound, such as a receptor.

In general, surface membrane proteins that bind to specific effector molecules are called receptors. In the present invention, antibodies are preferred receptors. Antibodies can be used to target liposomes to specificityA cell surface ligand. For example, certain antigens expressed specifically on tumor cells, called tumor associated antigens (TTAs), can be used to direct antibody-zinc finger-nucleotide binding protein-containing liposomes directly to malignant tumors. Since zinc finger-nucleotide binding protein gene products can be distinguished according to the cell type they act upon, the targeted delivery system provides a significant improvement over randomly injected non-specific liposomes. A number of procedures can be used to attach polyclonal or monoclonal antibodies to the liposome bilayer. Antibody-directed liposomes can include monoclonal or polyclonal antibodies or fragments thereof, such as Fab, or F (ab')₂So long as they effectively bind to antigenic epitopes on the target cells. Liposomes can also be targeted to cells expressing receptors for hormones or other serum factors.

A variety of viral and non-viral methods are available to those of skill in the art that are suitable for introducing nucleic acids into target cells. Genetic manipulation of primary tumor cells is well known in the art. Genetic modification of cells can be accomplished using one or more techniques well known in the art of Gene Therapy (Mulligan, R.C. human Gene Therapy, 5 (4): 543-. The viral transduction method may comprise infecting a target cell with a recombinant DNA or RNA virus comprising a nucleic acid sequence that drives or inhibits expression of a protein having sialyltransferase activity. Suitable DNA viruses for use in the present invention include, but are not limited to, adenovirus (Ad), adeno-associated virus (AAV), hepatitis virus, vaccinia virus, or poliovirus. Suitable RNA viruses for use in the present invention include, but are not limited to, retroviruses or Sindbis viruses. It will also be appreciated by those skilled in the art that some of the DNA and RNA viruses described may be present and suitable for use in the present invention.

Adenoviral vectors can be used to transfer genes to eukaryotic cells, to study eukaryotic gene expression, for vaccine development, and in animal models. Ad-mediated gene therapy has also been used in humans, such as for the transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the lung. Routes of administering recombinant Ad to different tissues in vivo include, for example, intratracheal instillation, intramuscular injection, peripheral intravenous injection, and stereotactic vaccination into the brain. Then, the skilled person can widely obtain adenovirus vectors and apply to the present invention.

Adeno-associated virus (AAV) has recently been introduced as a gene transfer system with potential applications in gene therapy. Wild-type AAV has been reported to exhibit high levels of infectivity, broad host range, and specificity for integration into the host cell genome. Herpes simplex virus type 1 (HSV-1) is attractive as a vector system, particularly for use in the nervous system, because of its neurotropic properties. Vaccinia viruses of the poxvirus family have also been developed for use as expression vectors. Each of the above vectors is widely available to those skilled in the art and is suitable for use in the present invention.

Retroviruses are capable of infecting a large proportion of target cells and integrating into the cell genome. Retroviruses were earlier developed as gene transfer vectors relative to other viruses and were first successfully used for gene tagging and transduction of Adenosine Deaminase (ADA) cDNA into human lymphocytes. Preferred retroviruses include lentiviruses. In a preferred embodiment, the retrovirus is selected from the group consisting of HIV, BIV and SIV.

"non-viral" delivery techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, calcium phosphate precipitation, gene gun techniques, electroporation, liposomes, and lipofection. Any of the above methods are widely available to those skilled in the art and are suitable for use in the present invention. Other suitable methods will be available to those skilled in the art, and it will be appreciated that the invention may be carried out using any available transfection method. Several such methods have been used with varying degrees of success by those skilled in the art. Lipofection can be accomplished by encapsulating the isolated DNA molecule in a liposome particle and contacting the liposome particle with a target cell membrane. Liposomes are self-assembled colloidal particles in which a lipid bilayer composed of amphiphilic molecules such as phosphatidylserine or phosphatidylcholine encapsulates a portion of the surrounding medium, leaving the lipid bilaterally surrounding the hydrophilic contents. Unilamellar or multilamellar liposomes can be constructed such that the contents contain the desired chemical, drug, or, as in the present invention, isolated DNA molecules.

Cells may be transfected in vivo, ex vivo or in vitro. The cells may be transfected as primary cells isolated from the patient or cell lines derived from primary cells, and need not be autologous to the patient to whom the cells are ultimately administered. After ex vivo or in vitro transfection, the cells may be implanted into a host. To obtain transcription of the nucleic acids of the invention in target cells, transcriptional regulatory regions are used which are capable of driving gene expression in the target cells. Transcriptional regulatory regions may comprise promoter, enhancer, silencer or repressor elements and are functionally associated with the nucleic acids of the invention. Preferably, transcriptional regulatory regions suitable for use in the present invention include, but are not limited to, the human Cytomegalovirus (CMV) mediated immediate early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyoma virus promoter, the albumin promoter, the PGK, and the alpha-actin promoter coupled to the CMV enhancer.

The vectors of the invention can be constructed using standard recombinant techniques widely available to those skilled in the art. Such techniques can be found in common Molecular biology references such as Sambrook, et al, Molecular Cloning: a laboratory Manual, Cold Spring harbor laboratory Press (1989), D.Goeddel, ed., Gene expression technology, Methods in Enzymology series, Vol.185, academic Press, San Diego, CA (1991), and Innis, et al PCR Protocols: a guidelines Methods and Applications Academic Press, San Diego, Calif. (1990).

In vivo administration of a polypeptide or nucleic acid of the invention to a target cell can be accomplished using a variety of techniques well known to those skilled in the art.

The carriers of the present invention may be administered orally, parenterally, by inhalation spray, rectally or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneal administration. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature, so that the cocoa butter and polyethylene glycol melt in the rectum and release the drug.

The dosage regimen for treating a disorder or disease with a vector of the invention and/or a composition of the invention is based on a variety of factors, including the type of disease, age, weight, sex, medical condition of the patient, severity of the condition, route of administration and the particular compound used. Thus, the dosage regimen may vary widely, but can be routinely determined using standard methods.

The pharmaceutically active compounds of the present invention can be processed according to conventional methods of pharmacy to produce pharmaceutical compositions for administration to patients, including humans and mammals. For oral administration, the pharmaceutical composition may be, for example, a liquid, an ocular insert, a capsule, a tablet, a suspension. The pharmaceutical compositions are preferably prepared in dosage unit form containing an amount of the active agent. For example, they may contain about 10³-10¹⁵Viral particles, preferably 10⁶-10¹²And (c) viral particles. The appropriate daily dosage for a human or other mammal may vary widely depending on the condition of the patient and other factors, but may also be determined using conventional methods. Administration may be by injection of the active agent as a composition with a suitable pharmaceutically acceptable carrier, such as saline, dextrose or water.

Although the nucleic acids and/or vectors of the invention may be administered as the sole active pharmaceutical agent, they may also be used in combination with one or more carriers or other agents of the invention. When administered in combination, the therapeutic agents may be formulated as separate compositions for simultaneous or non-simultaneous administration, or the therapeutic agents may be administered as a single composition.

The polypeptides of the invention may also be used in combination with a suitable pharmaceutical carrier. The compositions comprise a therapeutically effective amount of the protein and a pharmaceutically acceptable carrier or excipient. The carrier includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should be suitable for the mode of administration.

The invention also provides a pharmaceutical package or kit comprising one or more containers of one or more ingredients of the pharmaceutical composition of the invention. Associated with the container may be instructions in the form of government regulations governing the manufacture, use or sale of a pharmaceutical or biological product, which instructions reflect approval by the manufacture, use or sale department for human administration. In addition, the polypeptides of the invention may be used in combination with other therapeutic compounds.

The pharmaceutical compositions may be administered by convenient means such as intraocular, ophthalmic and systemic routes. The amount of tRNA synthetase-derived polypeptide administered to the patient and the dosage regimen will depend on a number of factors, such as the mode of administration, the nature of the condition being treated, the weight of the subject being treated and the judgment of the prescribing physician. Generally, the polypeptide is administered at a therapeutically effective dose of at least about 10 μ g/kg body weight per day. Preferably, the dose is about 10 μ g/kg body weight to 1mg/kg body weight per day, taking into account the frequency of administration, route of administration, symptoms, etc.

Angiogenesis activity against endogenous and exogenous angiogenic factors can be treated with angiostatic trpRS and prevented from further growth or even inhibited solid tumors, as angiogenesis and neovascularization are essential steps in the growth of solid tumors. The treatment may also be used to treat rheumatoid arthritis, psoriasis and diabetic retinopathy, all of which are characterized by aberrant angiogenesis.

Compositions comprising therapeutically effective amounts and concentrations of recombinant adenoviral delivery vectors are provided for delivering therapeutic gene products to cells expressing specific receptors. These cells include the eye. Of particular interest are photoreceptor cells of the eye. Administration can be accomplished by any method that achieves contact with the photoreceptor. Preferred modes of administration for reaching the photoreceptor cells include, but are not limited to, subretinal injection or intravitreal injection.

The recombinant viral compositions may also be formulated for implantation into the anterior or posterior chamber of the eye, preferably the vitreous chamber, in a sustained release formulation, such as adsorbed to a biodegradable support, including collagen sponges, or in liposomes. Sustained release formulations may be formulated for multiple dose administration such that several doses are administered over a selected period of time, such as a month or to about a year. Thus, for example, liposomes can be prepared such that a total of about 2-fold to about 5-fold or more of a single dose is administered in a single injection.

The carrier may be formulated in an ophthalmically acceptable carrier for intraocular, preferably intravitreal, administration in a volume of about 0.05ml to 0.15ml, preferably about 0.05 to 0.1 ml.

The composition may be provided in a sealed sterile vial containing an amount of the active agent to deliver a sufficient amount of the viral particles to the photoreceptors upon intraocular administration in a volume of about 50-150 μ l, the volume containing at least about 10⁷More preferably at least about 10⁸Individual spots form a unit. Thus, typically, the vial contains about 0.15ml of the composition.

To prepare the composition, the viral particles are dialyzed against an appropriate ophthalmically acceptable carrier or the virus can be concentrated and/or mixed therewith. The resulting mixture may be a solution, suspension or emulsion. In addition, the viral particles may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other agents useful for treating a particular disease.

For administration by intraocular injection or by eye drop, suitable carriers include, but are not limited to, physiological saline, Phosphate Buffered Saline (PBS), Balanced Salt Solution (BSS), ringer's lactate solution, and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. They can be prepared according to methods known to the person skilled in the art. Suitable ophthalmologically acceptable carriers are well known. Solutions or mixtures for ophthalmic use can be formulated with suitable salts as 0.01% to 10% isotonic solutions, pH about 5-7 (see, e.g., U.S. patent 5,116,868, which describes typical compositions for topically applied ophthalmic solutions). The solution adjusted to pH7.4 contains, for example, 90-100mM sodium chloride, 4-6mM dipotassium hydrogenphosphate, 4-6mM disodium hydrogenphosphate, 8-12mM sodium citrate, 0.5-1.5mM magnesium chloride, 1.5-2.5mM calcium chloride, 15-25mM sodium acetate, 10-20mM sodium D, L-beta hydroxybutyrate and 5-5.5mM glucose).

The compositions may be prepared with carriers that protect the active agent against rapid bodily clearance, such as time release formulations or coatings. Such carriers include controlled release formulations such as, but not limited to, microencapsulated delivery systems, and biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other types of implants that can be placed directly into the anterior or posterior chamber of the eye or into the vitreous cavity. The composition may also be administered in granules, e.g.Pellets (ethylene-vinyl acetate copolymer resin, Dupont).

Liposomal suspensions, including tissue-targeted liposomes, may also be suitable for use in pharmaceutically acceptable carriers. For example, liposomal formulations can be prepared by methods well known to those 60 skilled in the art (see, e.g., Kimm et al Bioch. Bioph. Acta728: 339-398 (1983); asil et al Arch Ophthalmol.105: 400 (1987); and U.S. Pat. No. 4,522,811). Viral particles may also be encapsulated into the aqueous phase of the liposomal system.

The active substance or agent may also be mixed with other active substances which do not impair the desired action, or with substances which supplement the desired action or have other effects, including viscoelastic substances, e.g. toHyaluronic acid sold under the trademark Pharmacia, Inc, which is a high Molecular Weight (MW) molecule of about 300 million sodium hyaluronate fractions (see, e.g., U.S. Pat. Nos. 5,292,362, 5,282,851, 5,273,056, 5,229,127, 4,517,295 and 4,328,803) to name a fewResins sold under the trademark Alcon (available from Alcon Surgical, inc.), which are fluorine-containing (meth) acrylates such as 1H, 2H-heptadecafluorodecyl methacrylate (see, e.g., U.S. Pat. nos. 5,278,126, 5,273,751 and 5,214,080); to be provided withResins sold under the trademark Optical Radiation Corporation (see, e.g., U.S. Pat. No.5,273,056), and methylcellulose, hyaluronic acid methyl ester, polyacrylamide, and polymethacrylamide (see, e.g., U.S. Pat. No.5,273,751). The amount of viscoelastic material is generally about 0.5-5.0%, preferably 1-3% by weight of the conjugate and is used to coat and protect the treated tissue. The composition may contain a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye. Other active agents may also be included.

The composition may be contained in ampoules, disposable syringes or multi-or single dose vials made of glass, plastic or other suitable material. The contained composition may be provided in a kit. Specifically, provided herein are vials, ampoules, or other containers and kits, preferably having a disposable vial for delivering a sufficient amount of about 0.100ml of the composition, and a disposable needle, preferably a self-sealing 25-33 gauge or smaller needle.

Finally, the compositions may be packaged as an article of manufacture comprising packaging material, typically a vial, an ophthalmically acceptable composition comprising a polypeptide of the invention, and a label indicating therapeutic use of the composition.

Kits for practicing the invention are provided herein. The kit contains one or more containers, such as a sealed vial, sufficient composition for single dose administration, and one or more needles, such as a self-sealing 25-33 gauge or smaller needle, preferably a 33 gauge or smaller needle, and a precisely calibrated syringe or other precisely calibrated delivery device suitable for intravitreal injection.

The compositions are preferably administered by intraocular injection, but other modes of administration may be effective if a sufficient amount of the compound is available for contact with the vitreous cavity. Intraocular injection can be achieved by intravitreal injection, aqueous humor injection, or injection into the outer layers of the eye, such as subconjunctival injection or sub-tenon's injection, or by topical application to the cornea if a penetrating agent is used.

For a particular patient, the particular dosage regimen may be adjusted over time according to the individual needs and the discretion of the practitioner administering and monitoring the administration of the recombinant virus. The concentration and amount ranges set forth herein are exemplary only and do not limit the scope of the claimed methods.

The following examples are presented to illustrate specific embodiments of the present invention and its various uses. They are used for purposes of illustration and explanation only, and not by way of limitation.

Example 1 preparation of endotoxin-free recombinant TrpRS

Recombinant human TrpRS without endotoxin was prepared as follows. Preparation of a recombinant plasmid encoding full-length TrpRS (amino acid residues 1-471 of SEQ ID NO: 1) or a recombinant plasmid consisting essentially of SEQ ID NO: 1 (i.e., residues 94-471 of full-length TrpRS), a truncated TrpRS designated T2(SEQ ID NO: 12), and a polypeptide consisting essentially of SEQ ID NO: 1, hereinafter referred to as T1 (SEQ ID NO: 13). Each plasmid also encoded a C-terminal tag containing 6 histidine residues (e.g., amino acid residues 472-484 of SEQ ID NO: 1) and an initial methionine residue. Containing His₆Labeled T1 has seq id NO: 5, and His₆Labeled T2 has the amino acid sequence of SEQ ID NO: 7.

The above plasmid was introduced into E.coli strain BL21(DE3) (Novagen, Madison, Wis.). Human mature EMAPII also encoding a C-terminal 6 histidine residue tag was prepared in a similar manner for use. By treating the cells with isopropyl beta-D-thiogalactopyranosideOver-expression of recombinant TrpRS was induced for 4 hours. The cells were then lysed, according to the procedure recommended by the manufacturer, at HISThe protein was purified from the supernatant on a nickel affinity column (Novagen). After purification, the mixture was purified by a column containing 1. mu.M ZnSO₄Incubation of TrpRS protein with Phosphate Buffered Saline (PBS) followed by removal of free Zn²⁺(Kisselev et al.，Eur.J.Biochem.120：511-17(1981))。

Endotoxin was removed from the protein sample by phase separation using Triton X-114 (Liu et al, Clin. biochem.30: 455-63 (1997)). By using E-TOXATEGel-coagulation assay (Sigma, st. louis, MO) determined that protein samples contained less than 0.01 units of endotoxin per mL. Protein concentration was determined by standard Bradford assay (Bio-Rad, Hercules, Calif.) using Bovine Serum Albumin (BSA) as a standard.

Example 2 cleavage of human TrpRS with PMN Elastase

Cleavage of human TrpRS with PMN elastase was examined. In the case of protease: TrpRS0, 15, 30 or 60 min was treated with PMN in PBS (pH7.4) at a protein ratio of 1: 3000. After cleavage, the samples were analyzed on a 12.5% SDS-polyacrylamide gel. PMN elastase cleaves peptides consisting of DNA SEQ ID NO: 2 nucleotide 3428 and 4738 encodes a full length TrpRS of about 53kDa yielding a major fragment of about 46kDa (SEQ ID NO: 5, T1 with a C-terminal histidine tag) and a minor fragment of about 43kDa (SEQ ID NO: 7, T2 with a C-terminal histidine tag).

Carboxy-terminal His with recombinant TrpRS protein₆Western blot analysis of the labeled antibodies revealed that both fragments had a carboxyl-terminal His₆And (4) marking. Thus, only the amino-terminal ends of the two TrpRS fragments were truncated. The amino-terminal sequence of the TrpRS fragment was determined by Edman degradation using an ABI494 type sequencer. Sequencing of these fragments indicated that the amino-terminal sequenceShown as S-N-H-G-P (SEQ ID NO: 8) and S-A-K-G-I (SEQ ID NO: 9), indicate that the amino terminal residues of the major and minor TrpRS fragments are located at positions 71 and 94, respectively, of the full-length TrpRS. These human TrpRS constructs are summarized in figure 1. The signal sequences-HVGH- (SEQ ID NO: 10) and-KMSAS- (SEQ ID NO: 11) are shown in boxes.

The angiostatic activity of the major and minor TrpRS fragments was analyzed in an angiogenesis assay. Both major and minor TrpRS fragments having a C-terminal histidine tag (amino acid residues 472-484 of SEQ ID NO: 1) SEQ ID NO: 5 and SEQ ID NO: 7 in recombinant form. Both TrpRS fragments were able to inhibit angiogenesis.

Example 3 truncated fragments of Trp-RS exhibit potent angiostatic effects on retinal angiogenesis

The angiostatic activity of truncated forms derived from tryptophanyl rRNA synthetase (TrpRs, 53 kd; SEQ ID NO: 1) was examined in a postnatal mouse retinal angiogenesis model. Friedlander et al abstracts709-B84and714-B89, IOVS41 (4): 138-139(March15, 2000) reported postnatal retinal angiogenesis in mice that was staged. The present invention provides a method for measuring angiogenesis inhibition by using the staged retinal vascularization.

Endotoxin-free recombinant Mini-TrpRS (48 kDa splice variant of histidine-tagged TrpRS; SEQ ID NO: 3) and T2 (43 kDa cleavage product of histidine-tagged TrpRS; SEQ ID NO: 7) were prepared as recombinant proteins. These proteins were injected intravitreally into neonatal Balb/C mice at postnatal (P) days 7 or 8, and retinas were harvested at P12 or P13. Blood vessels in intact preparations of the retina were visualized with collagen type IV antibody and fluorescein conjugated secondary antibody. Anti-angiogenic activity was assessed by co-aggregation microscopy based on the effect of injected protein on the formation of deep, external vascular plexus. Intravitreal injection and retinal detachment were performed with a dissecting microscope (SMZ645, Nikon, Japan). An eyelid fissure was created with a thin blade in postnatal day 7 (P7) mice, exposing the eyeball for injection of T2(5pmol) or TrpRS (5 pmol). The sample (0.5. mu.l) was injected using a syringe (Hamilton Company, Reno, NV) fitted with a 32-gauge needle. The injection is made between the equator and the limbus, and the positioning of the needle tip is monitored by direct visualization during the injection to confirm that it is in the vitreous cavity. Eyes with needle-induced lens or retinal damage were excluded from the study. After injection, the eyelids were repositioned and the fissure closed.

At 12 days postnatal (P12), the animals were anesthetized and the eyeballs removed. After 10 minutes in 4% Paraformaldehyde (PFA), the glass was cut through an edge cut. Isolated retinas were prepared for staining by immersion in methanol on ice for 10 minutes, then blocked with 20% normal goat serum (The jackson laboratory, Bar Harbor, ME) in PBS on ice for 1 hour in 50% fetal bovine serum (Gibco, Grand Island, NY). By using in blocking buffer 1: the retina was stained with 200-diluted rabbit anti-mouse collagen IV antibody (Chemicon, Temecula, Calif.) at 4 ℃ for 18 hours to specifically visualize the blood vessels. Mixing ALEXA594 conjugated goat anti-rabbit IgG antibodies (Molecular Probes, Eugene, OR) (1: 200 dilution in blocking buffer) were incubated with the retina at 4 ℃ for 2 hours. The retinas were mounted with slow-fading mounting medium M (Molecular Probes, Eugene, OR).

The vascular inhibitory activity was evaluated based on the deep outer retinal vascular layer (secondary layer) formed between P8-P12. Normal development and toxicity symptoms were assessed by the appearance of the inner vascular network (primary layer). None of the protein constructs used in this example produced any side effects on the primary layer.

Figure 2 provides a graphical representation of T2 inhibition of vascularization of the deep vascular network of mouse retinal secondary. In FIG. 2, row A represents the retinal vascular network exposed to TrpRS, row B represents the retinal vascular network exposed to Mini-TrpRS, and row C represents the vascular network of the retina exposed to the polypeptide T2 of the present invention. Column 1 (left) represents the primary superficial vascular network and the second represents the secondary deep vascular network. As demonstrated in fig. 2, none of the polypeptides affected the primary vascular network, whereas only T2 significantly inhibited vascularization of the secondary, deep vascular network.

Most of the eyes treated with PBS showed normal retinal vascular development, but complete inhibition of the outer vascular layer was observed in about 8.2% (n-73) treated eyes. Complete inhibition of the external vascular network was observed in 28% (n-75) mini-TrpRS (0.5mg/ml) treated eyes. The smaller, truncated form (T2) is a much stronger inhibitor of angiogenesis, acting in a dose-dependent manner, with 14.3% (n-14) being completely inhibited after treatment with 0.1mg/mlT2, 40% (n-20) after treatment with 0.25mg/ml and 69.8% (n-53) after treatment with 0.5 mg/ml. The data for the 0.5mg/ml treatment is illustrated in FIG. 3. Mouse retina extracts contain proteins with the same apparent molecular weight and immunoreactivity as human mini-TrpRS, which was obtained by SDS-PAGE and Western blot analysis. Full-length mouse and human TrpRS have about 88% amino acid identity and contain 475 and 471 amino acids, respectively. Truncated forms of TrpRS, particularly T2, have potent angiostatic effects on retinal vascular development.

Example 4 Matrigel angiogenesis assay

According to Brooks et al methods mol.biol., 129: 257-269(1999) and Eliceiri et al. mol. cell, 4: the method described in 915-924(1999) examined the vascular inhibitory activity of T2(SEQ ID NO: 7) using the mouse matrigel angiogenesis assay. The following modifications were made. Athymic wehi mice were subcutaneously implanted with 400 μ l of growth factor-depleted matrigel (Becton Dickinson, Franklin Lakes, NJ) containing 20nM VEGF. The angiostatic activity of T2 was initially tested by adding 2.5 μ l of T2 to matrigel plug. Potency was determined by adding various concentrations of T2 to the plug. On day 5, mice were injected intravenously with fluorescein-labeled endothelial-binding lectin griffonia (bandeiraea) Simplicifolia I, isolectin B4(vector laboratories, Burlingame, CA), and matrigel plugs were excised. The plugs were ground in RIPA buffer (10mM sodium phosphate, pH7.4, 150mM sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and the fluorescein content of each plug was quantified by spectrophotometric analysis.

Example 5 positioning of T2 incorporation in the retina

To assess uptake and localization of T2 injected into the retina, fluorescein-labeled on postnatal day 7 (P7) (II)488, Molecular Probes, Inc., Eugene OR) T2 was injected into the vitreous of the eye. The eyeballs were harvested at P8 and P12 and fixed in 4% PFA for 15 min. The retina and adherent non-retinal TISSUE were further separated and placed in 4% PFA overnight at 4 ℃ and then embedded in media on dry ice (TISSUE-O.c.t., Sakura FineTechnical co, Japan). The lyophilized sections (10 μm) were rehydrated with PBS and blocked with PBS containing 5% BSA, 2% normal goat serum. Blood vessels were visualized with anti-mouse collagen IV antibodies as described above. By containingDAPI nuclear dye (Vector Laboratories, Burlingame, Calif.) tissue was mounted in coverslips.

Alternatively, unstained retinas were incubated overnight at 4 ℃ with 200nM fluorescein-labeled full-length TrpRS or fluorescein-labeled T2 in buffer. Sections were washed 6 times in PBS for 5 minutes each, and then incubated with 1. mu.g/ml DAPI for 5 minutes for visualization of nuclei. Pre-blocking with unlabeled T2 was performed by incubating 1. mu.M unlabeled T28 hours at 4 ℃ prior to incubation with fluorescein-labeled T2. The retinas were examined with a multiphoton BioRad MRC1024 confocal microscope. Confocal assist software (BioRad, Hercules, CA) was used to generate 3-dimensional vessel images from a set of Z-series images.

Vascular inhibitory potency in a mouse Matrigel plug assay.We examined T2(SEQ ID NO: 7) to determine whether it has vascular inhibitionActivity, although it has lost aminoacylation activity. The in vivo angiostatic activity of T2 was tested using the mouse matrigel assay. VEGF₁₆₅Vascular development was induced into the mouse matrigel plug. When it reacts with VEGF₁₆₅When T2 was added to matrigel together, angiogenesis, IC, was blocked in a dose-dependent manner₅₀It was 1.7nM, as shown in FIG. 4.

Fluorescein-labeled T2 localized to retinal blood vessels.To visualize the intraocular localization of T2(SEQ ID NO: 7), we examined the distribution after intravitreal injection of fluorescein-labeled T2 at day 7 after birth. The retinas were isolated the next day, sectioned, and viewed with a confocal microscope. By labeling with fluorescein: (594) The labeled T2 treated eyes were co-stained with anti-collagen IV antibody to confirm localization (data not shown). The green fluorescence of labeled T2 was still visible 5 days after injection of fluorescein labeled T2 (at P12) (fig. 5A). In these retinas, no secondary vascular layer was observed at P12, indicating that fluorescein-labeled T2 retained vasosuppressive activity similar to that of unlabeled T2. Retinas injected with fluorescein-labeled full-length TrpRS at P7 developed secondary vascular layers within 12 days, but no vascular staining was observed (fig. 5B). In fig. 5, the fluorescein-labeled protein is green, the collagen-labeled blood vessels are red, and the nucleus is blue.

To further assess the inhibitory properties of labeled T2, transverse sections of normal neonatal retina were stained with fluorescein-labeled T2. Under these conditions, fluorescein-labeled T2 bound only to blood vessels (fig. 5C). Binding was specific as it could be blocked by pre-incubation with unlabeled T2 (data not shown). When fluorescein-labeled full-length TrpRS was administered to the retina, no retinal vascular staining was observed (fig. 5D), consistent with the lack of angiostatic activity of the full-length enzyme.

As shown in fig. 5, fluorescein-labeled T2 was angiostatic and localized to retinal blood vessels. At 7 days postnatal (P7), fluorescein-labeled T2 (FIG. 5A) or full-length TrpRS (FIG. 5B) was injected (0.5. mu.l, intravitreally). Retinas were harvested at P8, stained with anti-collagen IV antibody and DAPI nuclear stain, and labeled T2 (up arrow pointing to blood vessels in fig. 5) was located in blood vessels (1 °) in the primary surface vascular network. Note the complete absence of the secondary deep vascular network (2 °). No labeling was observed, although both primary (1 °) and secondary (2 °) vascular layers were present in fluorescein-labeled full-length TrpRS-injected eyes (arrows in fig. 5B).

In a separate set of experiments, frozen P15 retinal sections were stained with fluorescein-labeled T2 (fig. 5C) or fluorescein-labeled full-length TrpRS (fig. 5D) and imaged in a confocal scanning laser microscope. Labeled T2 selectively localizes to blood vessels and is shown as bright green blood vessels under the label "2" of fig. 5C across primary and secondary retinal vascular layers. No staining was observed for full-length TrpRS (fig. 5D).

Full-length TrpRS contains a unique NH₂A terminal domain and lack vasoinhibitory activity. Removal of part or all of the entire domain results in a protein with angiostatic activity. The structure responsible for the angiostatic activity of T2 appears to be contained within the core of the Rossman folded nucleotide binding domain. NH removable by alternative splicing or proteolysis₂The terminal domain may modulate the angiostatic activity of TrpRS, possibly by exposing the binding sites necessary for angiostatic, which are not available in full-length TrpRS.

VEGF-induced angiogenesis is completely inhibited by T2 in the mouse matrigel model, which is physiological angiogenesis in neonatal retina. Interestingly, the most potent anti-angiogenic effects of the TrpRS fragment in vitro, in CAM and in matrigel models were observed in VEGP-stimulated angiogenesis. The association between retinal angiogenesis results in neonatal mice and VEGF-stimulated angiogenesis and angiostatic effects of TrpRS fragments is consistent; retinal angiogenesis in this system can be driven by VEGF. Furthermore, the inhibition observed in the retinal model is specific to newly developing blood vessels; this treatment did not alter the pre-existing (upon injection) vessels of the primary vascular layer. Although the mechanism of the angiostatic activity of T2 is unknown, the specific localization of T2 in the retinal endothelial vasculature and the selective effect of T2 on newly developing blood vessels suggest that T2 may act through endothelial cell receptors expressed on proliferating or migrating cells. Further understanding of the angiostatic activity of T2 requires the identification of relevant cellular receptors.

A variety of cell types that produce angiostatic mini TrpRS upon interferon-gamma stimulation also produce angiostatic factors such as IP-10. These results therefore suggest the possibility of a role for TrpRS in the normal, physiologically relevant pathways of angiogenesis. Another ubiquitous cellular protein, pro-EMAPII (p43), had two apparently unrelated effects similar to that reported for TrpRS. Pro-EMAPII aids in protein translation by associating with a multiple synthetase complex of a mammalian aminoacyl tRNA synthetase. It is processed and secreted as EMAPII, which has been proposed to act as a vasoinhibitory mediator in lung development.

Thus, T2 may be useful for physiologically relevant angiogenic remodeling observed under normal or pathological conditions. In normal angiogenesis, T2 may assist in the establishment of a physiologically important avascular zone, which is present in some organs, such as the avascular zone of the fovea. If cleavage of full-length TrpRS is inhibited, pathological angiogenesis can occur, leading to vessel overgrowth.

In eye diseases, neovascularization can lead to severe loss of vision. These patients potentially could benefit from inhibition of angiogenesis. Vascular endothelial growth factor is associated with neovascularization in the retina and macular edema, but other angiogenic stimulators are thought to play a role in retinal angiogenesis. We have observed VEGF-stimulated angiogenesis and potent angiostatic activity of the TrpRS fragment, making these molecules useful in the treatment of vision loss and other proliferative retinopathies. There is no report in the literature of anti-angiogenic agents that completely inhibit angiogenesis (FIG. 5) 70% of the time as in T2 of the present invention. Another advantage of TrpRS fragments is that they are naturally occurring and, therefore, potentially non-immunogenic anti-angiogenic agents. Thus, these molecules can be delivered by target cell-or viral vector-based therapies. Because many patients with ocular neovascular diseases have associated systemic ischemic disease, anti-angiogenic therapy with genetically engineered cells or viral vectors placed directly into the eye is required.

In addition to the treatment of angiogenic retinopathy, TrpRS fragments of the present invention, particularly T2 and its angiogenesis-inhibiting fragments, may also inhibit solid tumor growth by preventing tumor angiogenesis. The TrpRS fragments of the invention block VEGF-induced endothelial cell proliferation and chemotaxis in vitro and are therefore useful in the treatment of any pathology involving undesired endothelial vascular proliferation and angiogenesis.

Sequence listing

<110> P. Schmeier

K. Waka Suji

M, Friedel

Stickrips research college

<120> tryptophanyl-tRNA synthetase derived polypeptides for modulating angiogenesis

<130>TSRI-813.1PC

<150>60/270,951

<151>2001-02-23

<160>13

<170>FastSEQ for Windows Version4.0

<210>1

<211>484

<212>PRT

<213> Artificial sequence

<220>

<223> recombinant human trpRS

<400>1

<210>2

<211>4877

<212>DNA

<213> Artificial sequence

<220>

<223> recombinant human mini-TrpRS in pET20B

<221>CDS

<222>(3428)...(4738)

<400>2

<210>3

<211>437

<212>PRT

<213> Artificial sequence

<220>

<223> human TrpRS in pET20B

<400>3

<210>4

<211>4811

<212>DNA

<213> Artificial sequence

<220>

<223> cleavage product T1 of recombinant human TrpRS

<221>CDS

<222>(3428)...(4672)

<400>4

<210>5

<211>415

<212>PRT

<213> Artificial sequence

<220>

<223> cleavage product T1 of recombinant human TrpRS

<400>5

<210>6

<211>4742

<212>DNA

<213> Artificial sequence

<220>

<223> cleavage product T2 of recombinant human TrpRS

<221>CDS

<222>(3428)...(4603)

<400>6

<210>7

<211>392

<212>PRT

<213> Artificial sequence

<220>

<223> cleavage product T2 of recombinant human TrpRS

<400>7

<210>8

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>8

<210>9

<211>5

<212>PRT

<213> human (Homosapiens)

<400>9

<210>10

<211>4

<212>PRT

<213> human (Homo sapiens)

<400>10

<210>11

<211>5

<212>PRT

<213> human (Homo sapiens)

<400>11

<210>12

<211>378

<212>PRT

<213> human (Homo sapiens)

<400>12

<210>13

<211>401

<212>PRT

<213> human (Homo sapiens)

<400>13

Claims (15)

1. An isolated, water-soluble polypeptide consisting essentially of a sequence of amino acid residues

SAKGIDYDKL IVRFGSSKID KELINRIERA TGQRPHHFLR RGIFFSHRDM

NQVLDAYENK KPFYLYTGRG PSSEAMHVGH LIPFIFTKWL QDVFNVPLVI

QMTDDEKYLW KDLTLDQAYG DAVENAKDII ACGFDINKTF IFSDLDYMGM

SSGFYKNVVK IQKHVTFNQV KGIFGFTDSD CIGKISFPAI QAAPSFSNSF

PQIFRDRTDI QCLIPCAIDQ DPYFRMTRDV APRIGYPKPA LLHSTFFPAL

QGAQTKMSAS DPNSSIFLTD TAKQIKTKVN KHAFSGGRDT IEEHRQFGGN

CDVDVSFMYL TFFLEDDDKL EQIRKDYTSG AMLTGELKKA LIEVLQPLIA

EHQARRKEVT DEIVKEFMTP RKLSFDFQ(SEQ ID NO：12)

Or an angiogenesis inhibiting fragment thereof; the isolated polypeptide is no more than about 45 kilodaltons in size.

2. An isolated polypeptide according to claim 1, which is an angiogenesis-inhibiting fragment capable of inhibiting ocular neovascularization.

3. The isolated polypeptide according to claim 1, which is an angiogenesis inhibiting fragment capable of inhibiting ocular neovascularization and comprises at least one of the amino acid residue signal sequences HVGH (SEQ ID NO: 10) and KMSAS (SEQ ID NO: 11).

4. An isolated polypeptide according to claim 1, which is an angiogenesis-inhibiting fragment capable of inhibiting ocular neovascularization and comprises the amino acid residue signal sequence HVGH (SEQ ID NO: 10).

5. An isolated polypeptide according to claim 1, which is an angiogenesis-inhibiting fragment capable of inhibiting ocular neovascularization and is less than about 43 kilodaltons in size.

6. A polypeptide having the sequence of SEQ ID NO: 7.

7. An isolated polynucleotide having a nucleotide sequence at least 95% identical to a sequence of a polynucleotide selected from the group consisting of: SEQ ID NO: 6; can be compared with SEQ ID NO: 6; encoding the amino acid sequence of SEQ ID NO: 7; encoding the amino acid sequence of SEQ ID NO: 12; encoding the amino acid sequence of SEQ ID NO: 7; and may be identical to a nucleic acid encoding SEQ ID NO: 7, or a polynucleotide that hybridizes to a polynucleotide of the polypeptide epitope of 7.

8. A recombinant vector comprising the isolated polynucleotide of claim 7.

9. A recombinant host cell comprising the vector of claim 8.

10. A recombinant host cell expressing the polypeptide of claim 1.

11. Use of a water-soluble polypeptide consisting essentially of a sequence of amino acid residues in the manufacture of a medicament for inhibiting ocular neovascularization in a patient

SAKGIDYDKL IVRFGSSKID KELINRIERA TGQRPHHFLR RGIFFSHRDM

NQVLDAYENK KPFYLYTGRG PSSEAMHVGH LIPFIFTKWL QDVFNVPLVI

QMTDDEKYLW KDLTLDQAYG DAVENAKDII ACGFDINKTF IFSDLDYMGM

SSGFYKNVVK IQKHVTFNQV KGIFGFTDSD CIGKISFPAI QAAPSFSNSF

PQIFRDRTDI QCLIPCAIDQ DPYFRMTRDV APRIGYPKPA LLHSTFFPAL

QGAQTKMSAS DPNSSI FLTD TAKQIKTKVN KHAFSGGRDT IEEHRQFGGN

CDVDVSFMYL TFFLEDDDKL EQIRKDYTSG AMLTGELKKA LIEVLQPLIA

EHQARRKEVT DEIVKEFMTP RKLSFDFQ(SEQ ID NO：12)

Or an ocular neovascularization inhibitory fragment thereof.

12. An injectable vasculostatic composition comprising a sequence of substantially amino acid residues

SAKGIDYDKL IVRFGSSKID KELINRIERA TGQRPHHFLR RGIFFSHRDM

NQVLDAYENK KPFYLYTGRG PSSEAMHVGH LIPFIFTKWL QDVFNVPLVI

QMTDDEKYLW KDLTLDQAYG DAVENAKDII ACGFDINKTF IFSDLDYMGM

SSGFYKNVVK IQKHVTFNQV KGIFGFTDSD CIGKISFPAI QAAPSFSNSF

PQIFRDRTDI QCLIPCAIDQ DPYFRMTRDV APRIGYPKPA LLHSTFFPAL

QGAQTKMSAS DPNSSIFLTD TAKQIKTKVN KHAFSGGRDT IEEHRQFGGN

CDVDVSFMYL TFFLEDDDKL EQIRKDYTSGAMLTGELKKA LIEVLQPLIA

EHQARRKEVT DE IVKEFMTP RKLSFDFQ(SEQ ID NO：12)

Or an angiogenesis inhibiting fragment thereof, and a pharmacologically acceptable aqueous vehicle, the composition comprising the polypeptide at a concentration of at least 0.1mg per ml of aqueous vehicle.

13. The angiostatic composition according to claim 12, wherein the polypeptide has the amino acid sequence of SEQ id no: 7 or SEQ ID NO: 12 and is present at a concentration of about 0.1-0.5mg per ml of aqueous vehicle.

14. A kit for inhibiting ocular neovascularization comprising, packaged in a suitable sealed container, an amount of the polypeptide of claim 1 sufficient for at least single dose administration; at least one self-sealing, less than about 33 gauge syringe needle suitable for intravitreal injection; and at least one precisely calibrated syringe.

15. The kit of claim 14, further comprising printed informational material describing the composition, method of administration thereof, and safety and efficacy information as may be required by government regulations.