Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1077—Pentosyltransferases (2.4.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1258—Polyribonucleotide nucleotidyltransferase (2.7.7.8), i.e. polynucleotide phosphorylase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/02—Pentosyltransferases (2.4.2)
  • C12Y204/02007—Adenine phosphoribosyltransferase (2.4.2.7)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• chemotherapeutic drugs derive anti-tumor specificity from the ability to kill dividing, as opposed to non-dividing, cells.
• Many chemotherapies are suitable for systemic administration specifically because they are most toxic to cells that are dividing. This leads to an acceptable level of damage in other, rapidly proliferating, tissues and cells such as the bone marrow, intestinal tract and hair follicles, among others.
• many refractory tumors are refractory precisely because they have a very low growth fraction; i.e. a relatively small percentage of tumor cells are dividing at any particular point in time.
• the encoded adenine phosphoribosyltransferase is preferably a mammalian adenine phosphoribosyltransferase, such as a human adenine phosphoribosyltransferase.
• a nucleic acid sequence encoding human adenine phosphoribosyltransferase encodes amino acids 1-180 of Seq ID No 1.
• An expression vector including a nucleic acid encoding an adenine phosphoribosyltransferase may be any of various types of expression vector. General expression vector types include plasmids and viruses.
• the prokaryotic purine nucleoside phosphorylase is an E. coli purine nucleoside phosphorylase.
• the expression vectors including the nucleotide sequence encoding a prokaryotic purine nucleoside phosphorylase and the nucleotide sequence encoding an adenine phosphoribosyltransferase may be the same type of vector or different types of vector. Each vector may independently be a plasmid or a virus, for instance. In a preferred embodiment, the expression vectors encoding the purine nucleoside phosphorylase and the adenine phosphoribosyltransferase are both plasmids or both viruses.
• a single expression vector may contain a nucleic acid encoding both the purine nucleoside phosphorylase and the adenine phosphoribosyltransferase.
• the substrate for the purine nucleoside phosphorylase includes a purine nucleoside analog which is non-toxic to cells and which is capable of being cleaved by a purine nucleoside phosphorylase to yield a substrate for adenine phosphoribosyltransferase.
• a composition which includes a bicistronic expression construct including a first nucleic acid encoding a prokaryotic purine nucleoside phosphorylase and a second nucleic acid encoding a mammalian adenine phosphoribosyltransferase, the first and second nucleic acids both operably linked to a promoter.
• Such a composition is useful in methods to express the encoded proteins and particularly in methods for inhibiting a cell according to the present invention.
• the bicistronic expression construct further includes an internal ribosome entry site disposed between the first nucleic acid encoding a prokaryotic purine nucleoside phosphorylase and the second nucleic acid encoding a mammalian adenine phosphoribosyltransferase.
• a first nucleic acid encoding a prokaryotic purine nucleoside phosphorylase encodes an E. coli purine nucleoside phosphorylase.
• This first nucleic acid encoding an E. coli purine nucleoside phosphorylase preferably encodes a protein which is at least 90% identical to a E. coli purine nucleoside phosphorylase of SEQ ID No. 3.
• the first nucleic acid encoding a prokaryotic purine nucleoside phosphorylase is generally at least 80% identical to a E. coli purine nucleoside phosphorylase encoding portion of a nucleic acid of SEQ ID No. 4.
• the second nucleic acid encoding a mammalian adenine phosphoribosyltransferase optionally and preferably encodes a human adenine phosphoribosyltransferase.
• the second nucleic acid encoding a human adenine phosphoribosyltransferase preferably encodes a protein which is at least 90% identical to a human adenine phosphoribosyltransferase of SEQ ID No. 1.
• the second nucleic acid encoding a human adenine phosphoribosyltransferase is at least 80% identical to a human adenine phosphoribosyltransferase encoding portion of a nucleic acid of SEQ ID No. 2.
• the nucleic acid encoding a human adenine phosphoribosyltransferase encodes a protein which is at least 90% identical to a human adenine phosphoribosyltransferase of SEQ ID No. 1.
• Figure 1 is a schematic illustration of the role of APRTase in tumor cell killing in the context of recombinant E. coli PNP and APRT delivery and administration of particular prodrugs.
• Figure 2 illustrates glycosidic cleavage of nucleoside prodrugs to purine bases.
• Figure 3 is an image of a fluorescently labeled nucleic acid on a gel illustrating a 543 bp band which is a PCR product encoding full length human APRT.
• Figure 4 is a graphic representation of an expression vector according to the present invention containing a DNA sequence encoding human APRT.
• Figure 5 is an image of immunoprecipitated E. coli PNP detected using an antibody according to the present invention.
• Figure 6 is an image of E. coli PNP detected by Western blot using an antibody according to the present invention.
• Figure 7 is a graph illustrating generation of F-Ade from the active PNP substrate, F-dAdo.
• Figure 8 is an image showing in vitro bystander activity of MeP-dR in D54 human glioma cells expressing E. coli PNP.
• Figure 9 is a graph illustrating bystander killing by MeP-dR when E. coli PNP is expressed using a MuLV expression vector.
• Figure 10 is a graph illustrating effects of recombinant lentivirus expression of E. coli PNP on D54 glioma tumors in vivo with and without a prodrug.
• Figure 11 is a graph illustrating effects of recombinant lentivirus expression of E. coli PNP on D54 glioma tumors in vivo in which only 1% of the tumor cells are PNP expressing cells.
• Figure 12 is a graph illustrating a study in which 100% of cells express a lentivirus encoded transgene.
• Figure 13 is a graph illustrating dose dependence upon the amount of prodrug added.
• Figure 14 is a graph illustrating that tumors with lower proportions of PNP- expressing cells (10%, 5%, 2.5%), exhibit dose dependence upon intratumoral PNP activity.
• Figure 15 is a graph illustrating effects of control treatments to be compared with graphs in Figures 13 and 14.
• Figure 16 is a graph illustrating some effects of different schedules of prodrug dosing.
• Figure 17 is an image illustrating cell killing effects using an EIa deleted adenoviral vector encoding a transgene according to the present invention.
• Figure 18 is a graph illustrating anti-tumor effects of F-araAMP following delivery of an adenovirus expression vector according to the present invention.
• Inventive methods and compositions are active to inhibit cells expressing the exogenous enzymes as well as bystander cells.
• Bystander cells are cells other than those in which the exogenous enzymes are expressed.
• the term "inhibiting a mammalian cell" in the context of a process according to the present invention refers to disruption of cellular processes, such as transcription, translation and ATP-dependent processes. Death of the mammalian cell results from inhibition.
• compositions and methods active against both dividing and non-dividing cells designed to inhibit tumors with a low growth fraction are provided according to the present invention. Specific strategies to kill low growth fraction tumors are required to eliminate the common cancers most refractory to conventional treatment.
• a method of inhibiting a mammalian cell which includes introducing an expression vector including a nucleotide sequence encoding an adenine phosphoribosyltransferase (APRT or APRTase) into the mammalian cell and contacting the mammalian cell with an effective amount of a substrate for the adenine phosphoribosyltransferase. Activation of this substrate by the adenine phosphoribosyltransferase yields a compound which is toxic to the mammalian cell.
• APRT is an enzyme which belongs to the purine/pyrimidine phosphoribosyltransferase family.
• the enzyme catalyzes the formation of AMP and inorganic pyrophosphate from adenine and 5-phosphoribosyl-l- pyrophosphate.
• Purine bases are substrates for cellular APRTases, which convert the compounds to phosphorylated metabolites. Because the APRTase carries out the first step in intracellular adenine activation, this enzyme is expressed in most cells, as well as in malignant cell types and tissues.
• APRT is active to convert a purine analog to a cytotoxic compound, particularly a cytotoxic nucleotide analog. Such cytotoxic nucleotide analogs are incorporated into cellular RNA, disrupting both RNA and protein synthesis.
• a method of inhibiting a mammalian cell includes introducing a first expression vector including a nucleotide sequence encoding a prokaryotic purine nucleoside phosphorylase (PNP) and a second expression vector comprising a nucleotide sequence encoding an adenine phosphoribosyltransferase into the mammalian cell.
• PNP prokaryotic purine nucleoside phosphorylase
• the cell is contacted with an effective amount of a prodrug which is a substrate for the purine nucleoside phosphorylase.
• Cleavage of the prodrug by the purine nucleoside phosphorylase yields a substrate for the adenine phosphoribosyltransferase.
• Subsequent activation of the adenine phosphoribosyltransferase substrate by APRT enzymatic action yields a compound toxic to the mammalian cell, thereby inhibiting the cell.
• Figure 1 illustrates the role of APRTase in tumor cell killing in the context of recombinant E. coli PNP delivery and administration of particular prodrugs.
• APRTase is rate limiting for conversion of the purine base analogs, MeP and F-Ade, produced by PNP cleavage of the prodrugs, to a phosphorylated form.
• increased APRTase may shift the equilibrium of the reaction shown at (1) towards intratumoral accumulation of the toxic bases.
• APRTase represents the rate limiting step for PNP toxin activation in vitro as described in Parker, W. B., et al., 1998, Biochem. Pharmacol. 55:1673-1681.
• APRTase governs the pathway by which MeP and F-Ade become phosphorylated, trapped in tumor cells, and mediate anti-tumor effects, illustrated in Figure 1. After tumor cells die, MeP and F-Ade are regenerated, released, and recycled to neighboring tumor cells to elicit further rounds of bystander killing.
• a prokaryotic PNP and APRT allow for improved cell inhibitory effects compared to administration of either agent alone.
• APRT activity represents a rate limiting step in producing cytotoxins from administered prodrug substrates.
• Overexpression of APRT in conjunction with delivery of a prokaryotic PNP provides for increased toxin production, increased cell inhibition and increased bystander cell inhibition.
• increased tumor regressions are provided with administration of both a prokaryotic PNP and APRT. Such methods allow shorter treatment times and better effects with large tumor masses.
• E. coli PNP cleavage of prodrugs are substrates for APRT.
• the toxins liberated by E. coli PNP are activated by APRT to cytotoxic metabolites which are incorporated into cellular RNA, disrupting both RNA and protein synthesis. Cell death results over a period of days, causing RNA degradation and release of the toxins from nucleic acid pools into the extra-cellular space.
• An expression vector including a nucleic acid encoding an adenine phosphoribosyltransferase and/or prokaryotic PNP may be any of various types of expression vector.
• a suitable vector is adapted to express APRT and/or PNP in a mammalian cell.
• Such vectors include vectors derived from bacterial plasmids and from viruses such as adenoviruses; adeno-associated viruses; papovaviruses such as SV40; poxviruses; pseudorabies viruses; retroviruses such as lentiviruses; herpesviruses; and vaccinia viruses.
• An expression vector which is a virus may be replication-competent, conditionally replication-competent or replication defective.
• Various cloning and expression vectors are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1989).
• General expression vector types include plasmids and viruses containing one or more regulatory elements sufficient or desirable for expressing an encoded transgene.
• expression vector refers to a recombinant DNA molecule containing a desired nucleic acid coding sequence encoding APRT and/or a prokaryotic PNP, and containing appropriate regulatory elements necessary or desirable for the expression of the operably linked coding sequence in a particular cell.
• regulatory element refers to a nucleotide sequence which controls some aspect of the expression of nucleic acid sequences.
• exemplary regulatory elements illustratively include an enhancer, an internal ribosome entry site ("IRES"), an origin of replication, a polyadenylation signal, a promoter, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing and/or translation of a coding sequence and/or encoded polypeptide in a cell.
• IRES internal ribosome entry site
• operably linked refers to connection of two or more nucleic acid molecules, including a nucleic acid sequence to be transcribed to produce an mRNA encoding a desired peptide or protein and a regulatory element such as a promoter or an enhancer element, which allows transcription of the nucleic acid sequence to be transcribed.
• promoter refers to a DNA sequence operably linked to a desired nucleic acid sequence encoding APRT and/or a prokaryotic PNP which is capable of controlling the transcription of the nucleic acid sequence.
• a promoter is generally positioned upstream of a desired nucleic acid sequence encoding APRT and/or a prokaryotic PNP to direct transcription, although a promoter may be positioned alternatively.
• a promoter may provide a site for specific binding of various factors involved in transcription, such as an RNA polymerase and/or other transcription factors.
• a promoter included in an inventive expression vector may be a promoter naturally associated with APRT or PNP.
• a heterologous promoter may be used. Promoters drive constitutive expression, cell or tissue specific expression and/or regulated or inducible expression.
• Exemplary constitutive promoters include viral promoters such as CMV, SV40, and RSV promoters.
• Exemplary constitutive mammalian promoters include the beta-actin promoter, the ubiquitin- 1 promoter and the glyceraldehyde dehydrogenase promoter. Additional promoters are known in the art and some specific promoters are described in examples herein.
• Further suitable regulatory elements include, the egr-1 promoter, the EF-I alpha promoter, the WPRE regulatory element and hypoxia responsive elements.
• a substrate for adenine phosphoribosyltransferase administered according to the present invention is a purine analog which is converted to a cytotoxic nucleotide analog by adenine phosphoribosyltransferase.
• purine analogs illustratively include 6- methylpurine and 2-fluoroadenine.
• a mammalian cell inhibited according to a method of the present invention may be a tumor cell.
• it is desirable to inhibit a mammalian cell which is inhibited is abnormal or which is contributing to a disease or other pathological process.
• a cell infected with a microbe may be inhibited according to an inventive process in order to eliminate the cell and microbe.
• microbes include bacteria, viruses and protozoa.
• cells contributing to inflammatory processes causing pain or degeneration, as in rheumatoid arthritis may be inhibited.
• a vector administered according to methods of the present invention may be targeted to particular cells. Targeting may be achieved by association of the vector with a targeting moiety.
• a targeting moiety may be a receptor ligand, an antibody, a lectin or other binding partner specific for a complementary receptor on a target cell, such as a tumor cell,
• a vector may be administered in conjunction with a transfection or transduction enhancer in embodiments of the invention.
• a gene delivery compound may be used in conjunction with virus vectors.
• Gene delivery compounds are active to stimulate uptake of a virus into a cell. Such compounds are described in U.S. Patent Publication 20040204375 and U.S. Patent Application 10/520,377.
• adjunctive compounds for stimulating uptake and/or transgene expression of vectors encoding PNP and/or APRT may be used.
• Such adjunctive compounds illustratively include liposomal formulations, alginate formulations, or poloxamer installation such as described in Toyoda K, et al., 2001, J Cereb Blood Flow
• the expression vectors including the nucleotide sequence encoding a prokaryotic purine nucleoside phosphorylase and the nucleotide sequence encoding an adenine phosphoribosyltransferase may be the same type of vector or different types of vector. Each vector may independently be a plasmid or a virus, for instance. In a preferred embodiment, the expression vectors encoding the purine nucleoside phosphorylase and the adenine phosphoribosyltransferase are both plasmids or both viruses.
• a composition provided according to one embodiment of the present invention includes a single expression vector containing a nucleic acid encoding both a prokaryotic purine nucleoside phosphorylase and an adenine phosphoribosyltransferase.
• a bicistronic nucleic acid is included in an expression vector.
• a bicistronic nucleic acid for expression of two proteins may include an internal ribosome entry site
• IRES permitting translation of two open reading frames from one mRNA.
• IRES are exemplified by the encephaomyocarditis virus IRES described in Jang et al., J. Virol., 1988, 62, 2636-2643.
• a nucleic acid sequence encoding an APRT is preferably a mammalian APRT.
• Exemplary nucleic acid sequences encoding mammalian APRTases include those detailed in Sikela JM, et al., Gene, 1983, 22(2-3):219-28; Stambrook PJ, et al., Somat CeIl MoI Genet., 1984, 10(4):359-67; LowyI, etal.,Cell, 1980, 22(3):817-23; Wilson, J. M., et al, J. Biol. Chem., 1986, 261:13677-13683; and Murray AM, et al., Gene, 1984, 31(l-3):233-40.
• a human APRT cDNA is isolated as described in further detail in examples included herein.
• Cloning and expression vectors are provided according to embodiments of the present invention which contain a nucleic acid sequence encoding a human APRT of SEQ ID No. 1.
• a nucleic acid sequence encoding a prokaryotic PNP encodes an E. coli PNP in a preferred embodiment.
• Nucleic acid sequences encoding E. coli PNP as well as cloning and expression vectors containing such sequences are described in detail in examples included herein, in U.S. Patent Nos. 5,552,311; 6,017,896; 6,491,905; 6,958,318 and
• mutant E. coli PNPs are detailed in U.S. Patent No. 7,037,718 which are suitable for use inmethods and compositions according to the present invention.
• a wild-type E. coli protein is detailed in the present specification in SEQ ID No. 3.
• An isolated nucleic acid sequence encoding human APRT or E. coli PNP may be identical to the coding portion of sequences shown in SEQ ID No. 2 or SEQ ID No. 4, respectively.
• a different isolated nucleic acid encoding a protein having activity substantially similar to human APRT or E. coli PNP, as shown in SEQ ID No. 1 or SEQ ID No. 3, respectively, may be used owing to the redundancy or degeneracy of the genetic code.
• an isolated nucleic acid sequence encoding human APRT or E. coli PNP is at least 80%, 85% or 90% identical to the nucleic acid sequences of SEQ ID No. 2 or SEQ ID No. 4.
• an isolated nucleic acid sequence encoding human APRT or E. coli PNP is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences of SEQ ID No. 2 or SEQ ID No. 4.
• nucleic acids encoding APRT and/or PNPs include coding sequences for conservative amino acid substitutions which have little or no effect on enzyme activity compared to the wild-type proteins.
• the enzyme activity of mutant APRT and PNPs may be assessed by functional assays, such as those described herein.
• a conservatively modified APRT or prokaryotic PNP is one which includes a substitution of an amino acid present in the wild-type protein with a chemically similar amino acid.
• each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic.
• a conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic.
• Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, threonine, valine, tyrosine and tryptophan; and hydrophilic amino acids include , Amino acids may also be described in terms of relative size, alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine, all
• an isolated nucleic acid sequence encoding human APRT or E. coli PNP encodes a protein which is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences of SEQ ID No. 1 or SEQ ID No. 3.
• mutant APRT and PNPs may be generated which have substantially similar or better enzyme activity compared to the wild-type proteins.
• the enzyme activity of mutant APRT and PNPs may be assessed by functional assays, such as those described herein.
• an isolated nucleic acid sequence encoding mutant human APRT or E. coli PNP encodes a protein which is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences of SEQ ID No. 1 or SEQ ID No. 3.
• Certain specific mutants of E. coli PNP which may be used include M65V and A157V, among others, as described in detail in U.S. Patent No. 7,037,718.
• isolated as used herein to refer to a nucleic acid or amino acid sequence is intended to indicate that the nucleic acid or amino acid sequence has been removed from the original environment in which it naturally occurs and separated from other nucleic acids present in the natural source of the molecule.
• Prokaryotic PNP mediates the glycosidic cleavage of nucleoside prodrugs, to highly toxic purine bases.
• Figure 2 illustrates specific examples of such cleavage, showing conversion of MeP-dR to MeP and 2-F-dAdo to F-Ade. Also shown is cleavage of F-araA, the bioavailable fo ⁇ n of the clinically approved chemotherapeutic fludarabine monophosphate, to F-Ade .
• Table 1 describes the kinetic constants underlying these enzymatic reactions with E. coli PNP.
• the substrate for the purine nucleoside phosphorylase includes a purine nucleoside analog which is non-toxic to cells and which is capable of being cleaved by a purine nucleoside phosphorylase to yield a substrate for adenine phosphoribosyltransferase.
• purine nucleoside analogs include 9-(2- deoxy-beta-D-ribofuranosyl]-6-methylpurine; 9-(beta-D-ribofuranosyl)-2-amino-6- chloro-1-deazapurine; 7-(beta-D-ribofuranosyl)-3-deazaguanine; 9-(beta-D- arabinofuranosyl)-2-fluoroadenine; 2-fluoro-2'-deoxyadenosine; 9-(5-deoxy-beta-D- ribofuranosyl)-6-methylpurine; 2-fluoro-5'-deoxyadenosine2-chloro-2'-deoxyadenosine; 5'-amino-5'-deoxy-2-fluoroadenosine; 9-(5-amino-5-deoxy-beta-D-ribofuranosyl)-6- methylpurine; 9-(alpha-D-ribofura
• Methods and compositions are provided for multi-modality approaches to inhibition of cells, and particularly for inhibition of tumors.
• administration of radiation or conventional chemotherapy is a contemplated embodiment for enhancement of anti-tumor methods and compositions including APRT and/or PNP according to the present invention.
• a method according to the present invention further includes administration of a therapeutic agent.
• a therapeutic agent is illustratively an anti-tumoral agent.
• Anti-tumoral agents are described, for example, in Goodman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
• Such anti-tumoral agents illustratively include acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calusterone, capecitabine, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, ce
• a second type of anti-tumoral treatment may also be administered in conjunction with PNP and APRT.
• radiotherapy may be administered to a tumor before and/or after administration of PNP and APRT.
• Parameters for radiation therapy are known as exemplified in Washington, C. M. and Leaver, D. (Eds.), Principles and Practice of Radiation Therapy, CV. Mosby; 2nd ed., 2003.
• adjunctive therapeutic agents may be administered according to methods and in compositions of the present invention including analgesics, anesthetics, antibiotics, anti-inflammatory agents, nutritive supplements, vitamins, and other such agents beneficial to the subject.
• compositions of the present invention including analgesics, anesthetics, antibiotics, anti-inflammatory agents, nutritive supplements, vitamins, and other such agents beneficial to the subject.
• a pharmaceutical composition for inhibiting a cell which includes an expression vector including a nucleotide sequence encoding an adenine phosphoribosyltransferase.
• a pharmaceutically acceptable carrier is also included in such a pharmaceutical composition.
• compositions according to the present invention include an expression vector which includes a nucleotide sequence encoding a prokaryotic purine nucleoside phosphorylase.
• a further embodiment is provided in which the expression vector including a nucleotide sequence encoding an adenine phosphoribosyltransferase and the expression vector including a nucleotide sequence encoding a prokaryotic purine nucleoside phosphorylase are the same vector, the nucleotide sequence encoding an adenine phosphoribosyltransferase and the nucleotide sequence encoding a prokaryotic purine nucleoside phosphorylase operably connected to a regulatory element in a bicistronic nucleic acid.
• pharmaceutically acceptable as used herein is intended to mean a material that is not biologically or otherwise undesirable, which can be administered to an individual without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
• the composition can be a pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
• Time release preparations are specifically contemplated as effective dosage formulations.
• compositions will include an effective amount of the selected expression construct in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents.
• nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose and magnesium carbonate.
• Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving or dispersing an active compound with optimal pharmaceutical adjuvants in an excipient, such as water, saline, aqueous dextrose, glycerol, or ethanol, to thereby form a solution or suspension.
• the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, for example, sodium acetate or triethanolamine oleate.
• fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents maybe included. Tablets and granules are preferred oral administration forms, and these may be coated. Parenteral administration is generally by inj ection. Inj ectables can be prepared in conventional forms, either liquid solutions or suspensions, solid forms suitable for solution or prior to injection, or as suspension in liquid prior to injection or as emulsions.
• compositions and Substrates A pharmaceutical composition according to the present invention and/or substrate is administered by a route determined to be appropriate for a particular subject by one skilled in the art.
• a composition and/or substrate is administered orally, parenterally (for example, intravenously), by intramuscular injection, by intraperitoneal injection, intratumorally, or transdermally.
• Intratumoral injections may be a single injection or, preferably, multiple passes in multiple locations within the tumor. Intratumoral instillation or infusion methods may also be used.
• composition and/or substrate required will vary from subject to subject, depending on the age, weight and general condition of the subject, the severity of the disease that is being treated, the location and size of the tumor, the particular compound used, its mode of administration, and the like. An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Generally, dosage will preferably be in the range of about 0.5-500 mg/m 2 , when considering 5'-methyl(talo)MeP-R as a substrate for example, or a functional equivalent. For viral vectors, dosage is generally in the range of 5 x 10 3 - 5 x
• a subject having a tumor cell to be inhibited according to the present invention is a human or other mammal, illustratively including rodents, cats, dogs, rabbits, horses, cows, pigs, sheep and non-human primates.
• compositions and methods according to the present invention are described primarily herein with reference to prokaryotic PNPs and particularly with reference to E. coli PNPs.
• prokaryotic enzymes which are capable of cleaving purine containing nucleoside analog substrates to generate a substrate for APRT enzymatic activity and generation of a cytotoxic compound are considered within the scope of the present invention.
• prokaryotic enzymes include various prokaryotic hydrolases and phosphorylases as disclosed in U. S. Patent No.6,491,905.
• a cDNA encoding full length human APRT is isolated, the cDNA encoding the APRT protein of SEQ ID NO 1.
• cDNAs are synthesized from HeLa total RNA using an RNeasy kit from Qiagen and used in a PCR reaction to amplify APRT cDNA.
• Figure 3 shows a 543 bp band on a gel which is a PCR product encoding full length human APRT.
• the PCR products are cloned into a pCR4-Blunt Topo vector, available commercially from Invitrogen. The resulting clone is verified by DNA sequencing.
• This 543 bp APRT coding sequence may be amplified with primers to introduce restriction sites, such as Ncol and Xhol, into the product suitable for cloning into an expression vector.
• Figure 4 shows an example of an expression vector according to the present invention containing a DNA sequence encoding human APRT.
• E. coli PNP-encoding sequence is inserted into an expression vector.
• E. coli (strain, JMlOl) chromosomal DNA template is obtained using the method described in NJ. Gay, J. Bacterid., 158:820-825 (1984).
• GTACGCGGCCGCTTACTCTTTATCGCCCAGCAGAACGGA-TTCCAG are used to define the full length coding sequence of the E. coli DeoD gene and to incorporate Notl sites at both 5' and 3 ' ends of the desired product.
• amplification 94 0 C x 1 minute denaturation, 5O 0 C x 2 minute annealing, and 72 0 C x 3 minute elongation using 1 ng template, 100 microliters of each primer in a 100 microliter reaction mixture containing 2.5 units taq polymerase, 200 micromolar each dNTP, 50 mM KCl, 10 mM Tris Cl (pH 8.3), 1.5 mM MgC12 and 0.01 % gelatin (weight/vol)), a single PCR product of the predicted size is obtained.
• This product is extracted with phenol/ chloroform, precipitated with ethanol, digested with Notl, and gel purified.
• the amplified bacterial PNP sequence is inserted into a eukaryotic expression vector. In order to obtain a vector capable of directing eukaryotic expression of E. coli
• the LacZ gene is excised from pSVB (Clontech, Palo Alto, CA) by digestion with
• the vector backbone was dephosphorylated (calf intestinal alkaline phosphatase, GIBCO BRL, Gaithersburg, MD) and gel purified as above.
• the PNP insert, prepared as above, is then ligated into the Notl ends of the plasmid backbone in order to create a new construct with PNP expression controlled by the SV-40 early promoter. Identity of recombinants and orientation of inserts are confirmed by restriction mapping and by reamplif ⁇ cation of the full length insert from recombinant plasmid using the primers described above. This procedure yields an expression construct SV-PNP.
• HSV vectors exhibit tumor specificity in vivo by selectively targeting the proliferating cells in a growing tumor mass.
• Certain HSV vectors have been used previously in the clinic to examine glioma therapy (for micrometastatic disease) and by regional administration to liver for treatment of metastatic colon cancer Shah, A.C., et al., 2003, J. Neuro-Oncol. 65: 203-226; Markert, J. M., et al., 2000, Gene Therapy 7: 867- 874; and Rampling, R., et al., 2000, Gene Therapy 7: 859-866.
• Sequences encoding PNP and/or APRT are inserted into an HSV targeting plasmid.
• the targeting plasmids are separately co-transfected into rabbit skin cells (RSC) with ClOl viral DNA that is isolated and digested with the restriction enzyme Pad
• the viral genome is reconstituted by homologous recombination and viral plaques selected, propagated, and subjected to two additional plaque purifications on Vero cells.
• Candidate virus clones are confirmed by Southern blot hybridization of restriction enzyme-digested viral DNAs. Detection is performed with alkaline phosphatase, using
• An egr-1 promoter is an example of a promoter included in PNP and APRT HSV constructs for expression of PNP and/or APRT.
• Example 4 Generation of enhanced expression HSV vectors APRT and/or PNP-expressing ⁇ l 34.5- HSVs are constructed using a modification of a technique described by Krisky, D.M., et al., 1997, Gene Therapy 4: 1120-1125.
• C 101 is an HSV vector derived from R3616, described in detail in Whitley, R.J., et al., 1993, J Clin Invest 91(6):2837-43, which contains a CMV-driven transgene cassette introduced within the UL3/UL4 intergenic region. This virus represents a substantial improvement over earlier generation HSV, since 1) it encodes a more active transgene regulatory element, and 2) the UL39 region does not contain a transgene insertion. An intact UL39 region allows improved intratumoral spreading and distribution.
• PNP and APRT HSVs are constructed in which the PNP and/or APRT coding sequence is subcloned from a shuttle plasmid into an HSV targeting plasmid. with sequences homologous to those flanking the UL3/UL4 intergenic region.
• the pCAl 3-wtPNP is used to excise a PNP encoding sequence which is inserted into an HSV targeting plasmid (pCKl 037).
• the resulting targeting plasmid containing PNP, pHNOOl is co-transfected into rabbit skin cells (RSC) with ClOl viral DNA that had previously been isolated and digested with the restriction enzyme Pad to removes a GFP expression cassette from the virus.
• RSC rabbit skin cells
• ClOl viral DNA that had previously been isolated and digested with the restriction enzyme Pad to removes a GFP expression cassette from the virus.
• an APRT encoding sequence is inserted into an HSV targeting plasmid and cotransfected as described.
• the viral genome is reconstituted by homologous recombination and viral plaques are selected, propagated, and subj ected to two additional plaque purifications on Vero cells.
• Candidate virus clones are confirmed by Southern blot hybridization of restriction enzyme-digested viral DNAs (detection performed with alkaline phosphatase; Gene Images AlkPhos Direct DNA labeling system, Amersham-
• E2F1 promoter flanked by Spel and Xhol is generated by PCR, and used to replace the E4 promoter, just upstream of E4. All key regions and PCR inserts are verified by DNA sequencing.
• Various regulatory elements may be included to drive expression of the PNP and/or APRT transgenes in such constructs, illustratively including CMV and E2F1.
• CMV and E2F1 a nucleic acid sequence encoding E. coli PNP or APRT is cloned into the E3 region of pAB27 (Microbix).
• a Clal/BamHI digest is used to excise the CMV promoter and allow substitution of a PCR amplified E2F1 regulatory element.
• the E2F1 promoter is confirmed by DNA sequencing.
• PNP and APRT gene expression cassettes such as CMV driven and E2F1 driven PNP and APRT gene expression cassettes, are cut out of pAB27 by digestion with EcoRI and Hpal and cloned into the corresponding sites of the E3/E4 plasmid described above. A DNA fragment containing these sequences (as well as E2F1 driven E4) is then excised from the E3/E4 plasmid with Srfl and Pad and cloned into the corresponding sites of pAdEasy-1 (Stratagene) to establish the final genomic plasmid.
• CMV driven and E2F1 driven PNP and APRT gene expression cassettes are cut out of pAB27 by digestion with EcoRI and Hpal and cloned into the corresponding sites of the E3/E4 plasmid described above. A DNA fragment containing these sequences (as well as E2F1 driven E4) is then excised from the E3/E4 plasmid with Srfl and Pad and
• APRT overexpression In this example, lentivirus constructs are used to infect the cells. The transduction efficiency achieved at MOI 1-10 is typically sufficient to allow D54 PNP cells co-expressing APRT to be clonally expanded without a selectable marker. Recombinant expression of APRT in specific clones is assayed enzymatically and by RT- PCR. Quantitative or semi-quantitative RT-PCR is performed using a primer set specific for the particular vector that in order to distinguish vector derived mRNA from endogenous APRT message. Such assays may be performed on standard equipment such as an ABI Prism 7500 Sequence Detection System, Assays on Demand, Applied Biosystems. Semi-quantitative RT-PCR analysis and/or enzymatic assays described allow monitoring of APRT overexpression.
• media samples are obtained and the amount of base, F-Ade, in the medium is assessed.
• F-Ade base
• Tumors are infected with both HSV-PNP and HSV-APRT to further improve anti-tumoral response.
• tumors and other tissues will be excised 4 hours after injection of radiolabeled prodrug and the amount of radioactivity determined.
• a survey of tissues e.g. liver, lung, kidney, heart, intestine, marrow, brain, spleen, and gonads
• prodrug in plasma 4 hours after injection of F-araAMP (Parker, W.B., et al., 2002, Cancer Gene
• the homogenate iscentrifuged at 100,000 x g for 60 minutes at 4 0 C and then dialyzed against 1000-fold volume of 100 mM HEPES buffer, pH 7.4 containing 20% glycerol.
• the protein concentration of each sample is determined, and each tissue monitored for PNP activity and APRT as described above.
• Levels of replicating adenoviral vector within tumors and other tissues may be monitored at various time points after prodrug treatment (e.g. 1 , 3 , 5, 7, 14, and 28 days), by harvesting tumors which are then minced/homogenized in a PBS buffer containing
• Example 27 Studies to compare in vivo F-araAMP treatment regimens Treatment-schedule-dependency studies are undertaken early in the course of in vivo evaluation of drugs. It is necessary to identify the optimal treatment schedule in order to design subsequent comparative studies.
• the schedules most often used in schedule-dependency trials of prodrugs such as F-araAMP typically include: a single bolus dose, once daily for five or nine doses, once every 4 days for three or four doses, and some version of every 3 hours for three or eight doses for multiple courses at 4-day intervals.
• a range of dosage levels is used for each schedule to provide dose-response data and to include a toxic dosage level for a benchmark, since it is important to compare various treatment schedules at equitoxic dosages.
• the antitumor activity observed with a given compound may exhibit a striking dependence on the drug treatment schedule employed.
• strong antitumor activity has been observed after relatively short F-araAMP treatment schedules (e.g., QlD x 3 days; or QlD x 3 days, q4h x 3).
• F-araAMP treatment schedules e.g., QlD x 3 days; or QlD x 3 days, q4h x 3.
• modifications of F-araAMP schedules for use with E. coli PNP include 160 mg/kg qldx3-q4hx3; 100 mg/kg q2hx5; qldx3; 167 mg/kg qldx3-q4hx3; 250 mg/kg qldx3; and 25-100 mg/kg q2hx5, qldx3.
• D54 cells seeded inside or outside a cloning ring (removed) are separated by thin barrier (uncrossable) of vacuum grease. All surrounding cells (outside the ring) are D54 parental cells having no E. coli PNP expression.
• cells inside the ring are D54- PNP cells, and in columns B and D, the inside cells are D54 parental cells.
• Columns A and B are treated continuously with 100 micromolar MeP-dR for 6 days. On each day, a row of cells was fixed and stained with crystal violet to monitor cell growth. Because both the PNP prodrug and purine base are freely membrane permeant, gap junctions or cell-to-cell contact are not required for bystander killing mediated by E . coli PNP.
• a more active vectoring system including cloning and purifying recombinant lentivirus, expression of E. coli PNP in D54 tumor cells, isolating and propagating new D54-PNP tumor cell lines, monitoring tumor growth in animals.
• D54 glioma tumors are established from a stable cell line expressing E. coli PNP by a strong, CMV-based promoter in a lentiviral vector.
• Figure 10 illustrates results. Open Circles: Tumors established from 10% PNP expressing/90% non- expressing tumor cells; PNP activity 9600 ⁇ 1300 units on day 14. Open Triangles: 5% expressing, 95% non-expressing tumor cells; PNP activity 5500 ⁇ 1700 units.
• Open Squares Tumors established from 2.5% expressing, 97.5% non-expressing (PNP activity 3600 ⁇ 530 units). Closed symbols depict growth of corresponding (10%, 5%, 2.5%) PNP tumors treated with vehicle (saline control). At least six animals are studied per group. Identically treated animals from each study arm are sacrificed just prior to initiation of MeP-dR therapy (day 14) to measure intratumoral PNP acitivity. Antitumor effects in MeP-dR treatment groups are significantly different from non-treatment groups (p ⁇ 0.05, 2.5% tumors; andp ⁇ .005 for 5% and 10% tumors). These experiments test the influence of substantially higher PNP activities on bystander killing in vivo.
• F-araA is less active than MeP-dR as a substrate for E. coli PNP based upon Vmax/km of the compounds, as calculated from Table 1.
• F-araAMP is capable of mediating anti-tumor effects in vivo.
• tumors established using a first generation system (MuLv-PNP transduced cells, PNP under regulatory control of SV40 promoter) exhibit regressions following MeP-dR (Gadi, V.K., et al., 2003, J. Pharm.
• F-araAMP in these same tumors confers regressions lasting ⁇ 30 days when 100% of cells expressed E. coli PNP, but tumors subsequently escapefrom F-araAMP therapy and progress. Moreover, little or no bystander killing is observed in vivo with F-araAMP following E. coli PNP transduction with MuLv.
• Figure 12 depicts a study in which 100% of cells express E. coli PNP using lentivirus. Complete regression and cures of tumors (12 of 12) are obtained with F- araAMP following a 3 day treatment schedule. Tumors with lower proportions of PNP- expressing cells (10%, 5%, 2.5%), exhibit dose dependence upon both the amount of prodrug added (Figure 13) and the intratumoral PNP activity (Figure 14) compared to controls ( Figure 15). Anti-tumor effects are observed with F-araAMP when as few as 2.5% of tumor cells in vivo express PNP. Adjusting the schedule of F-araAMP may also improve bystander killing in vivo ( Figure 16).
• D54 tumors are a stringent in vivo model for preclinical testing since they are slow growing in mice and resistant to standard cancer agents used against CNS tumors.
• BCNU a clinically approved anti-glioma chemotherapy
• the findings provided indicate the ability to treat otherwise refractory tumors using methods and compositions according to the present invention.
• F-araA is less active as a PNP substrate than MeP-dR, it liberates a more potent agent (F- Ade vs MeP).
• the circulating half life of F-araA is longer than MeP-dR (50 minutes vs. appx. 20 minutes) and peak levels are significantly higher with F-araA than MeP-dR (Parker, W.B., et al., 2002, Cancer Gene Therapy 9, 1-7.).
• FIG 16 shows that multiple F-araAMP schedules exhibit bystander killing. Tumors comprised of 5% PNP expressing cells and 2 different fludarabine schedules are shown. F-araAMP treatment groups are significantly different from non-treatment group (pO.001).
• Ad-PNP mediated cell killing in vitro is shown in Figure 20.
• Confluent HeLa cells are infected with Ad-PNP at various MOIs.
• MeP-dR is added at 40 micrograms/milliliter in one set of cells at 24 hours post infection. At 6 days post infection, all cultures are stained with Crystal Violet to visualize living cells.
• Figure 17 depicts results using an EIa deleted adenoviral vector encoding E. coli PNP as described herein.
• Adenoviral MOFs of 0.1-100 in vitro are sufficient to eliminate populations of cancer cells in combination with MeP-dR by this assay.
• Anti-tumor effects of F-araAMP following delivery of E. coli PNP by Ad-PNP are shown in Figure 18.
• D54 human glioma tumors (appx.250 mg) are injected with an Ad-PNP (2 x 10 9 PFU, open circles) or saline (closed circles).
• PNP activity in tumor extracts taken two days after vector administration is approximately 5000 PNP units.
• Ad-PNP together with F-araAMP confer slowing of tumor growth (closed triangles) (p ⁇ 0.002 compared to virus alone or no treatment controls). No limiting weight loss, animal deaths or other toxicities were noted in this study.
• APRT adenine phosphoribosyltransferase
• APRT adenine phosphoribosyltransferase

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