WO1998025630A1 - Regulation de l'apoptose induite par la transaldolase - Google Patents

Regulation de l'apoptose induite par la transaldolase Download PDF

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WO1998025630A1
WO1998025630A1 PCT/US1997/022770 US9722770W WO9825630A1 WO 1998025630 A1 WO1998025630 A1 WO 1998025630A1 US 9722770 W US9722770 W US 9722770W WO 9825630 A1 WO9825630 A1 WO 9825630A1
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cell
tal
transaldolase
cells
apoptosis
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PCT/US1997/022770
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Andras Perl
Katalin Banki
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The Research Foundation Of State University Of New York
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    • 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.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01002—Transaldolase (2.2.1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention in the fields of medicine and molecular biology relates to the regulation of apoptosis by overexpression or suppressed expression of human transaldolase (TAL) in cells.
  • TAL human transaldolase
  • DNA encoding TAL or DNA antisense to TAL DNA or small molecules which modulate TAL enzymatic activity by influencing the pentose phosphate pathway (PPP) are useful in treating a disease or disorder which is associated with dysregulation of normal apoptotic signalling and processes.
  • Apoptosis is a fundamental form of programmed cell death (PCD) which is indispensable for normal development and maintenance of homeostasis within multicellular organisms (1) .
  • PCD programmed cell death
  • Defects in apoptosis may underlie the etiology of neurodegenerative diseases, cancer, autoimmune diseases, and the acquired immune deficiency syndrome (3) .
  • ROIs reactive oxygen intermediates
  • Many of the chemical and physical stimuli which elicit PCD generate ROIs such as H 2 0 2 and OH " (8) .
  • Low doses of H 2 0 2 induce apoptosis in a variety of cell types, thus establishing oxidative stress as a mediator of apoptosis (7) .
  • the ability of scavengers of ROIs to inhibit apoptosis support this hypothesis (9) . Examples of such scavengers include
  • N-acetylcysteine (NAC) , a precursor of glutathione (GSH) (7,8) and free radical spin traps such as 5 , 5 -dimethyl - 1- pyrroline-1-oxide (DMPO) and 3 , 3, 5, 5-tetramethyl-l- pyrroline-1 -oxide (TMPO) .
  • GAC glutathione
  • TMPO free radical spin traps
  • Bcl-2 the prototype of a novel family of protooncogenes which inhibit apoptosis when induced by any of a number of diverse stimuli, was recently shown to have antioxidant behavior (5,6) .
  • apoptosis and bcl-2 protection were demonstrated in very low oxygen pressure, suggesting that ROIs may not be an absolute requirement for apoptosis (10) .
  • GSH reduced glutathione
  • PPP pentose phosphate pathway
  • the rate limiting enzymes for the two branches are different.
  • the oxidative phase is primarily dependent on glucose-6-phosphate dehydrogenase (G6PD; 12) .
  • G6PD glucose-6-phosphate dehydrogenase
  • Control of the nonoxidative branch, between transaldolase and transketolase, is less well established based on knowledge of the enzyme kinetics. It was suggested that, based on tissue-specific variations in enzymatic activities, TAL may be a rate-limiting enzyme of the nonoxidative branch of the PPP (12,13,14).
  • TAL catalyzes the transfer of a 3 -carbon fragment, corresponding to dihydroxyacetone, to D-glyceraldehyde 3 -phosphate, D-erythrose 4 -phosphate, and a variety of other acceptor aldehydes, including nonphosphorylated trioses and tetroses .
  • Enzymatic activity of TAL is regulated in a tissue-specific (13,14) and developmentally-specific manner (15) . In the brain, TAL is expressed selectively in oligodendrocytes at high levels (16) .
  • myelin sheaths are formed by oligodendrocytes and lesions in the most common demyelinating disease of the CNS, multiple sclerosis (MS) , are characterized by a progressive loss of oligodendrocytes and demyelination.
  • MS multiple sclerosis
  • Oligodendrocytes are extremely sensitive to damage by ROI , such as nitric oxide and tumor necrosis factor- ⁇ .
  • TNF ⁇ TNF ⁇ released by activated macrophages and astrocytes
  • the pentose phosphate pathway fulfills two essential functions in cellular metabolism: (1) the formation of pentose phosphates for synthesis of nucleotides and nucleic acids; and (2) the generation of NADPH for biosynthetic reactions and for maintenance of GSH at a reduced state in which GSH protects sulfhydryl groups and cellular integrity from oxygen radicals.
  • PPP pentose phosphate pathway
  • the present invention identifies for the first time a pivotal role for TAL in regulating the entire PPP and provides a molecular biological and pharmacological basis for modulating TAL expression and activity which is useful in the treatment of a number of diseases characterized by dysfunctional apoptosis.
  • the present inventors discovered that levels of TAL expression can determine susceptibility to apoptosis signals.
  • TAL overexpression of TAL in human T cells was accompanied by a decrease in G6PD and 6-phosphogluconate dehydrogenase (6PGD) activities, a concomitant depletion in NADPH and GSH levels, and increased sensitivity to apoptosis provoked by serum deprivation, H 2 0 2 , nitric oxide (NO) , TNF ⁇ , anti-Fas monoclonal antibody and infection by human immunodeficiency virus (HIV-1) .
  • suppression of TAL activity increased G6PD and 6PGD activities, augmented GSH levels, and inhibited apoptosis.
  • susceptibility to apoptosis is subject to regulation by TAL through control of the balance between the two branches of the PPP and its overall output as measured by NADPH and GSH production.
  • the present invention provides a method to promote apoptosis in a cell comprising providing, either in vi tro or in vivo, to the cell a recombinant TAL DNA molecule in the form of a vector which transforms the cells and which, when expressed, results in overexpression of TAL and stimulation of apoptosis either directly or in response to an apoptotic signal. Also provided is a method to suppress apoptosis in a cell comprising providing, either in vi tro or in vivo, to the cell a recombinant DNA molecule which encodes an RNA molecule that is antisense to all or part of TAL mRNA and which is capable, when expressed, of suppressing TAL expression in the cell. Such suppression of TAL expression results in suppression of apoptosis in response to an apoptotic signal.
  • the invention is directed to a method of stimulating TAL enzymatic activity in a cell by providing to the cell an agent which is a substrate or product of the PPP or which influences the available level of a substrate or product of the PPP such that TAL enzymatic activity is stimulated.
  • Such stimulation results in stimulation or enhancement of apoptosis in the cell either directly or in response to an apoptotic signal .
  • the invention is directed to a method of inhibiting TAL enzymatic activity in a cell by providing to the cell an agent which is a substrate or product of the PPP or which influences the available level of a substrate or product of the PPP such that TAL enzymatic activity is inhibited.
  • Such inhibition results in inhibition or suppression of apoptosis in the cell in response to an apoptotic signal.
  • Such methods are useful in treating a disease or condition characterized by abnormally high levels of, or susceptibility to, apoptosis, or, in contrast, abnormally low levels of, or resistance to, apoptosis.
  • diseases are described below.
  • Figure 1 presents a Western blot analysis of TAL protein levels in cell lines stably transfected with TAL expression vectors oriented in the sense (L26-3/4) and antisense directions (L18-3/1) , in comparison to untransfected Jurkat cells (control) .
  • 40 ⁇ g of protein lysate was loaded in each lane.
  • the TAL protein 38 kDa was detected with rabbit antibody 169 while actin (42 kDa) was visualized with mouse monoclonal antibody C4.
  • TAL/actin ratio in control Jurkat cells was considered as 100%.
  • TAL expression was reduced by 25% in L18-3/1 cells and increased 2.6-fold in L26-3/4 cells;
  • Figure 2 is a set of graphs showing the rate of apoptotic cell death induced by serum withdrawal, 100 ⁇ M
  • FIG 3 shows the time-course of DNA ladder formation in L26-3/4, L18-3/1, and control Jurkat cells during Fas-induced apoptosis.
  • DNA was extracted at the indicated timepoints after treatment with 50 ng/ml anti- Fas antibody and electrophoresed in a 2% agarose gel. Apoptotic cell death, as evidenced by DNA fragmentation into 180-200 bp bands, was accelerated in L26-3/4 cells and suppressed in L18-3/1 cells in comparison to control Jurkat cells; mw, 123 bp molecular weight ladder;
  • Figure 4 presents a flow cytometric analysis of Fas- induced apoptosis in L18-3/1, L26-3/4, and control Jurkat cells.
  • Figure 6 shows the time-course of ROI formation and cell death in Jurkat cells. ROI production was measured in log fluorescence intensity by flow cytometry after labeling with 0.1 ⁇ M DHR for 2 min. % survival was determined by trypan blue exclusion;
  • Figure 7 is a graph showing effect of N- acetylcysteine (NAC) , buthionine sulfoximine (BSO) , and DMPO on GSH levels and TAL and G6PD activities in Jurkat cells;
  • NAC N- acetylcysteine
  • BSO buthionine sulfoximine
  • DMPO DMPO
  • Figure 8 is a graph showing the effect of antioxidants (NAC, NDGA, DEF, Amytal) , prooxidant (BSO) , and spin traps (DMPO, TMPO) on Fas-induced cell death.
  • Jurkat cells were treated with 3 mM NAC, 1 mM BSO, 20 mM DMPO, 10 mM TMPO, 100 ⁇ M desferrioxamine (DEF) , 20 ⁇ M nordihydroguaiaretic acid (NDGA) , and 400 ⁇ M Amytal for 90 min and incubated with 50 ng/ml anti-Fas monoclonal antibody. Cell survival was quantified by trypan blue exclusion. Data represent mean ⁇ SE of three independent experiments;
  • Figure 9 is a graph showing the rate of apoptosis
  • the groups are as follows: Jurkat -tat cells (mock transfected) Jurkat control cells Jurkat L26-3/4 (transfected with TAL DNA)
  • Figure 10 is a graph showing the effect of phosphorylation of TAL by protein kinase C (PKC) .
  • the ordinate shows the enzymatic activity of 50 ng of affinity-purified human recombinant TAL (rTAL-H) measured in the forward (TAL-FOR) and reverse (TAL-REV) reactions.
  • the control group is untreated rTAL-H.
  • rTAL-H was phosphorylated with 25 ng of protein kinase C (Promega, Madison, WI) in the presence of 4 M Tris pH
  • the PKC + INH reaction represents activity of rTAL-H following pretreatment with PKC in the presence of 20 ⁇ M PKC 19-36 inhibitor peptide (GIBCO-BRL) ;
  • Figure 11 is a schematic description of the pentose phosphate pathway and its byproducts showing the enzymes involved in both branches, the sugar phosphate substrates and products, and the generation of NADPH and reduced glutathione .
  • the present invention provides approaches to treating a number of diseases or conditions characterized by altered apoptotic behavior or responsiveness to normal apoptotic signals.
  • Table 1 lists a number of conditions classified in accordance with the apoptosis dysregulation.
  • Subjects having a disease or condition associated with either increased or suppressed apoptosis can be treated using the methods of the present invention.
  • Cells of the subject can be transfected in vi tro or in vivo with a vector comprising (a) TAL-encoding DNA or (b) DNA encoding mRNA which is antisense to TAL mRNA, which, as exemplified below, results (respectively) in either overexpression of TAL and enhanced apoptosis or inhibition of TAL expression and suppressed apoptosis.
  • the above vectors are referred to collectively as TAL DNA construct and more particularly, as either a TAL-encoding DNA construct or a TAL antisense construct .
  • the present invention is used to promote apoptosis by overexpressing TAL or by upregulating TAL activity in a cell for the treatment of diseases and conditions which include those listed in the left column of Table 1 and any other conditions presently known or later discovered which are characterized by abnormally low apoptosis.
  • the preferred targets for overexpression of TAL or upregulation of TAL activity are various tumors and cancer, wherein normal regulation of cell growth is lost in part due to lack of normal apoptosis.
  • Vectors useful for transfecting cells for TAL overexpression are described and exemplified below.
  • a TAL antisense construct is used to deliver DNA which encodes an mRNA molecule or appropriate mRNA fragments which are capable of inhibiting endogenous cellular TAL gene expression leading to the suppression of apoptosis.
  • suppression of apoptosis is achieved by suppression of TAL enzymatic activity in the cell.
  • Such suppression of apoptosis is desirable for the treatment of diseases and conditions which include those listed in the right column of Table 1.
  • Preferred targets of this embodiment include HIV disease, and neurodegenerative and demyelinating diseases such as multiple sclerosis (MS) .
  • MS multiple sclerosis
  • the present inventors have shown that TAL levels are elevated in oligodendrocytes leading to their demise and loss of their yelin production.
  • the invention comprises parenteral administration of a single or multiple doses of the TAL DNA construct in the form of a retroviral vector or naked DNA preparation (see below) to a mammalian subject, preferably a human.
  • an effective amount of the DNA preparation is a function of the nature of the vector, the patient and his clinical status, and can vary from about 0.01 mg/kg body weight to about 1 g/kg body weight. A subject can be given this amount in a single dose or in multiple repeated doses.
  • the TAL DNA construct is used to transfer the TAL gene or antisense sequence to cells of the subject ex vivo followed by administration of the transformed or transfected cells into the subject. Cells expressing the TAL DNA construct are preferably administered at a dose between about 10 s and 10 10 cells on one or several occasions.
  • the dose used in the present invention will in part be dependent upon the health and weight of the recipient, the existence of other concurrent treatment, if any, frequency of treatment, and the nature of the effect desired, for example, eradication of a tumor or treatment of autoimmunity.
  • the TAL DNA construct may be advantageously utilized in combination with other therapeutic agents known to be useful in the treatment of the particular disease or condition being treated.
  • Administration of DNA or a vector or other vehicle containing the DNA or cells, as in the gene therapy embodiments described herein, is performed using any of a number of routes, depending on the location of the cells being targeted.
  • administration may be by any parenteral route, including but not limited to intramuscular, subcutaneous or transdermal, intravenous (including into the portal circulation), intrathecal, intraperitoneal , intragastric and by inhalation or instillation into the lungs.
  • parenteral route including but not limited to intramuscular, subcutaneous or transdermal, intravenous (including into the portal circulation), intrathecal, intraperitoneal , intragastric and by inhalation or instillation into the lungs.
  • Oral administration of certain of the delivery vehicles disclosed herein is also known in the art.
  • a preferred route is by iv injection or infusion.
  • the present invention may be directed to the treatment of disorders associated with increased cell survival.
  • Diseases characterized by the accumulation of cells include cancer, autoimmune diseases, and certain viral illnesses as well as benign hyperplasia and vascular restenosis.
  • Cell accumulation can result from either increased proliferation or the failure of cells to undergo apoptosis in response to appropriate stimuli.
  • bcl2 is a member of a family of genes that can control the apoptotic threshold of a cell (Boise, L.H. et al . , Cell 74 : 597 (1993); E. Y. Lin et al . , J. Immunol . 151 : 1919 (1993); K. M.
  • TAL-encoding DNA construct to overexpress TAL and promote or induce apoptosis, as disclosed herein, provides a novel form of chemotherapeutic intervention against cancer.
  • the role of apoptotic signalling in autoimmune diseases is not clear at the moment, and some controversy exists in the art.
  • Proper regulation of cell death is essential for eliminating potentially autoreactive lymphocytes during development and for removing excess cells after the completion of an immune response. Failure to remove autoimmune cells that arise during development or that develop as a result of somatic mutation during an immune response can result in autoimmune disease.
  • the cell surface receptor Fas is considered critical in regulating cell death in lymphocytes (Watanabe-Fukunaga et al . , Nature 356:314 (1992)).
  • bcl -2 blocks fas-mediated cell death (Itoh, N. et al . , J. Immunol . 151:621-627 (1993)) by inhibiting the action of the cysteine protease interleukin-l -converting enzyme (ICE) .
  • ICE cysteine protease interleukin-l -converting enzyme
  • Transformation of cells with TAL-encoding DNA to promote, or prime the cells for, apoptosis and cell death, as described herein, is therefore useful in arresting the progression of autoimmune diseases such as, for example, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus, just to name a few.
  • the present invention is also useful in the treatment of virus infections.
  • the disruption of cell physiology following viral infection can cause an infected cell to undergo apoptosis (B. Levine et al . , Nature 361 : 139 (1993)).
  • the suicide of an infected cell or its lysis by specific cytotoxic T lymphocytes are both cellular defense mechanisms to prevent viral propagation.
  • T cells can induce cell death by activating the target cell's endogenous cell death program (D. Kagieta et al . , Science 265 : 528 (1994)) .
  • Viruses can circumvent such defenses by disrupting the normal regulation of apoptosis in infected cells.
  • Viruses for which apoptosis-blocking proteins have been identified include adenovirus (Rao, L. et al . , Proc . Na tl . Acad . Sci . USA 83:7742 (1992)), Epstein-Barr virus and African swine fever virus (Neilan, J.G. et al . , J. Virol . 61 ⁇ - 391 (1993)), baculoviruses (Clem, R.J. et al . , Science 254 : 1388 (1991)), and poxviruses (Ray, CA. et al . . Cell 69 : 591 (1992)).
  • DNA constructs of the present invention can be used in an antiviral therapeutic approach, by introduction into cells infected with virus. This is particularly important for latent viral infection or situations in which viral replication does not lead to cell death. Indeed, the prevention of apoptosis by a virus is important for the establishment of viral latency.
  • EBV establishes a latent infection in B lymphocytes and expresses a gene, LMP-I, which specifically upregulates the expression of bcl2, providing a survival advantage to latently infected cells (Henderson et al . , Cell 65 : 1101 (1991)).
  • Chronic Sindbis virus infection also depends on the host cells' expression of jbc!2 (Levine, B.
  • TAL- encoding DNA is introduced into chronically infected cells to overcome the anti -apoptotic action of viral genes, resulting in death of the cells and eradication of the chronic or latent infection.
  • Apoptosis of virus-infected cells has become recognized as an important pathophysiologic mechanism in HIV disease and AIDS (Gougeon, M-L. et al . (1993) Science . 268:1269; Ameisen, J-C. (1992) Immunol . Today. 13 : 388 ) . Furthermore, bystander T cells are at risk for apoptotic death in HIV infected subjects (Finkel, T et al . , (1994) Curr. Opin . Immunol . 6 : 605 ; Banda, N. et al . , (1992) J. Exp . Med . 176 : 1099 ; Newell et al .
  • the present invention can attenuate the apoptosis, primarily of T lymphocytes, caused by HIV infection by blocking expression or enzymatic activity of TAL as described herein.
  • a wide variety of neurological diseases are characterized by the gradual loss of specific sets of neurons (Isacson, O. , Trends Neurosci . 16 : 306 (1993); Heintz, N. , Trends Biochem . Sci . 18 : 151 (1993)).
  • Such disorders include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) , retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration, diseases in which cell death results in specific disorders of movement and central nervous system function. Apoptosis appears to be the mechanism of cell death in such neurodegenerative diseases.
  • Oxidative stress, calcium toxicity, mitochondrial defects, excitatory toxicity, and deficiency of survival factors are proposed pathogenetic determinants (Cow, D.W. , J “ . Neurobiol . 23 : 1261 (1992); Zivetal, I., Neurosci Lett . 170:136 (1994)).
  • Each of these pathways predisposes neurons to apoptosis, either in vi tro or in vivo, whereas overexpression of bcl2 decreases the neurotoxicity of each of these potential inducers ( Zhong, L.T. et al . , Proc. Natl. Acad. Sci U.S.A. 90:4533 (1993)).
  • Neurotrophic growth factors and the extracellular matrix also alter the apoptotic threshold of neural cells (Arenas, E. et al . , Nature 367:368 (1994)). Together, these observations suggest that the threshold for cell death is dynamically regulated such that the apoptotic threshold of a cell is determined by the combined effects of external and internal survival factors. According to the present invention, TAL levels contribute to this emerging model .
  • a form of hereditary ALS results from mutations in the copper-zinc superoxide dismutase gene which renders cells less able to detoxify ROIs.
  • Superoxide-induced death could be specifically inhibited by treatment with survival growth factors or antioxidants (Troy, CM. et al., Proc. Natl. Acad. Sci. U.S.A. 91:6384 (1994)).
  • Retinal degeneration in retinitis pigmentosa results from mutations in any one of three photoreceptor- specific genes: rhodopsin, the ⁇ subunit of cyclic guanosine monophosphate phosphodiesterase, and the peripherin gene.
  • Apoptosis may be initiated in response to the accumulation of mutant proteins or as a result of the altered functional properties of the mutant proteins and results suggest that certain neurotrophic and growth factors can enhance photoreceptor survival .
  • Alzheimer's disease is associated with the progressive accumulation of /3-amyloid peptide in plaques. Mutations in the / ⁇ -amyloid precursor protein are associated with some forms of familial Alzheimer's disease. 3-amyloid peptide induces neurons to undergo apoptosis (Loo, D.T. et al., Proc. Natl. Acad. Sci U.S.A. 90:7951 (1993)), an effect which can be reversed by antioxidants (C. Behl, C. et al., Biochem. Biophys. Res. Commun. 186:944 (1992)).
  • NAIP neuronal apoptosis inhibitory protein
  • IAP baculovirus inhibitor of apoptosis protein
  • the present invention provides methods to inhibit the apoptosis associated with neuronal degeneration in a number of pathogenetic settings by either suppression of the expression of TAL in neurons or by inhibiting neuronal TAL enzymatic activity, as has been described above.
  • the treatments of the present invention may be combined with neuronal growth factors that are know in the art to achieve additive and even synergistic therapeutic effects in neuronal degenerative, as well as in demyelinating diseases and disorders.
  • Plasmids and Vectors The DNA molecules of the present invention may be expressed using any appropriate expression vector as is well-known in the art (Sambrook, J. et al . , Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) .
  • a DNA molecule encoding TAL may be recombined with vector DNA in accordance with conventional techniques, including blunt -ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, ligation with ap- muscularte ligases, or the synthesis of fragments by the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • any of a number of alternate vectors which include the TAL-encoding DNA molecules of the present invention may be selected.
  • Commercially available vectors such as Hook and pCEP4 from Invitrogen may be used.
  • control sequences with tissue specificity for the tissue type of the target cells may be used.
  • promoters with such specific modes of action include the insulin gene promoter for selective expression in the pancreas or the MMTV or lactalbumin promoter for expression in breast tissue.
  • the endogenous translation stop codons may be utilized. If a TAL DNA construct having a C-terminal truncation is used in which the endogenous stop codon is lacking, a stop codon is inserted in the vector just downstream of the cloning site.
  • a selectable marker gene (such as
  • G418-resistance may be added, either on the same plasmid or by contransfection using a second plasmid such as pSV2neo (Southern, P.J. et al . J Mol Appl Genet (1982) 1:327-341) or the plPBl plasmid (Biamonti, G. et al . Nucl Acid Res (1985) 13:5547-5561) .
  • a selection marker useful in vivo is required, for example, the tk gene of HSV (see below) .
  • a promoter is a region of a DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of an operably linked nucleic acid sequence.
  • a promoter sequence is the sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase.
  • Two sequences of a nucleic acid molecule, such as a promoter and a coding sequence, are said to be operably linked when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence.
  • two sequences such as a promoter sequence and a coding sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked coding sequence.
  • two sequences In order to be operably linked it is not necessary that two sequences be immediately adjacent to one another.
  • the promoter sequences of the present invention necessary for expression of the DNA of the invention must be functional in mammalian cells, and may be either eukaryotic or viral promoters. Suitable promoters are inducible, repressible or constitutive.
  • An example of a constitutive promoter is the viral promoter MSV-LTR, which is efficient and active in a variety of cell types, and, in contrast to most other promoters, has the same enhancing activity in arrested and growing cells.
  • Other preferred viral promoters include that present in the CMV-LTR (from cytomegalovirus) (Bashart, M. et al .
  • CdCl 2 heavy metal salts such as CdCl 2 ; the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al . , Nature 290 : 304 - 310 (1981)); and the yeast gal4 gene promoter (Johnston, S.A., et al . , Proc . Natl . Acad . Sci . (USA) 79 : 6911 - 6915 (1982); Silver, P. A., et al . , Proc . Natl . Acad . Sci .
  • the promoter region may further include an octamer region which may also function as a tissue specific enhancer, by interacting with certain proteins found in the specific tissue.
  • the enhancer domain of the DNA construct of the present invention is one which is specific for the target cells to be transfected, or is highly activated by cellular factors of such target cells.
  • vectors plasmid or retrovirus
  • retroviral enhancers e . g. , viral LTR
  • the enhancer is preferably placed upstream from the promoter with which it interacts to stimulate gene expression.
  • the endogenous viral LTR may be rendered enhancer-less and substituted with other desired enhancer sequences which confer tissue specificity or other desirable properties such as transcriptional efficiency on the TAL encoding DNA molecule of the present invention.
  • Such a transactivator is used to stimulate transcription from a promoter sequence, such as the human CMV promoter IE. This is a repressible system in contrast to the estrogen- inducible system described below.
  • a gene controlled by a promoter acting under the influence of the tetracycline- controlled transactivator can be constitutively expressed and turned off by using an effective concentration of tetracycline. Such a system can regulate a gene over about five orders of magnitude.
  • the tetracycline-repressible system functions in vivo in mammals, where tetracycline administration via the diet is used to keep the expression of the inducible gene off. Tetracycline analogs which cross the blood-brain barrier can be used if gene activity is desired in the brain.
  • the TAL DNA is placed under the control of a promoter subject to regulation by a tetracycline-controlled transactivator.
  • a construct in a single vector or preferably two vector form
  • target cells such as tumor cells growing in vivo .
  • tetracycline is withheld so that the TAL is overexpressed.
  • tetracycline or an active congener of tetracycline is administered locally to the cells transfected with the constructs.
  • Effective systemic doses (oral or parenteral) of tetracycline are in the range of about 0.1 mg to 1 g per day.
  • the tetracycline-repressible construct is introduced into selected cells, such as cells of a particular tumor.
  • the transactivator is maintained in the off position using tetracycline until the desirable localization can be demonstrated. At that time, tetracycline is withheld, stimulating expression of TAL leading to more rapid apoptotic death of the transfected cells .
  • inducible promoters well-known in the art can be used to produce analogous inducible systems for expression of the DNA molecules according to the present invention and for the induction of apoptosis in vi tro or in vivo .
  • one means for inducing apoptosis in a controllable manner is to use an TAL DNA construct in combination with inducible or repressible control elements such as an estrogen-inducible system (Brasel ann, S. et al. Proc Natl Acad Sci USA (1993) 90 : 1651 - 1661 ) .
  • the TAL DNA or portion thereof encoding an effective TAL enzyme is controlled by a GAL4-responsive promoter which is transactivated in the presence of 11 - ⁇ estradiol by the GAL-ER-Vpl6 transcription factor, a fusion protein consisting of the DNA-binding domain of GAL4 , the estrogen-binding domain of the estrogen receptor and the transactivation domain of Vpl6 (of
  • Herpes simplex virus For induction of expression of the TAL DNA molecules in an estrogen-inducible system in an animal, local or systemic treatment with estrogen is required.
  • An effective dose of an estrogen is a dose which would trigger the expression of the TAL-encoding DNA to induce apoptosis of cells such as tumor cells.
  • Such doses can be ascertained by one skilled in the art .
  • doses in the range of about 0.05 to 100 mg/kg of an estrogen are used in a single dose or in multiple doses over a period of about one week to about 2 months, or even longer.
  • Forms and preparations of estrogen and their usage in animals, particularly in humans, are well- known in the art (Gil an, A.G. et al .
  • Estrogen analogs which are capable of specifically activating the exogenous transactivator while having fewer biological effects and side effects are preferred.
  • Ionizing radiation has been used to activate the transcription of exogenous genes that encode a cytotoxic protein such as TNF ⁇ (Weichselbaum, R.R. et al . , Int . J. Radiation Oncology Biol . Phys . 24 : 565 - 561 (1992)). This may be accomplished through the use of radiation-responsive elements distal to the transcription start site of such genes. See, for example, Hallahan, D. et al . Proc . Natl . Acad. Sci . USA 88:2152-2160 (1991); Datta, R. et al .
  • the present invention provides methods for the spatial and temporal control of gene therapy using a TAL DNA construct with such a radiation-inducible promoter to activate TAL and thereby induce apoptosis.
  • This method for treating neoplastic disease also takes advantage of the direct anti-tumor effects of the radiation itself, resulting in an additive or synergistic interaction between the cytotoxic action of the TAL overexpression and radiation.
  • For treating tumor metastases it is possible to radiate metastases in one site or in multiple organs such that radiation will preferentially activate TAL overexpression in the irradiated volume.
  • This approach is also applicable to local disease where radiosensitizers can be used in combination with irradiation for direct cytotoxicity and/or activation of transcription of TAL with subsequent apoptotic tumor cell death.
  • the present invention has advantages over the system using TNF described by Hallahan, Weichselbaum and colleagues for sparing surrounding tissue because TAL activation is intracellular . Only cells carrying the construct will be killed whereas TNF ⁇ is activated, diffuses out and acts regionally (and may even reach more distant sites where it could exert undesired toxic effects) .
  • the TAL-encoding DNA is placed in a vector under control of a radiation-inducible promoter.
  • a genetic construct with a VP-16 DNA sequence that encodes a known powerful transactivating protein attached to the DNA coding sequence derived from the DNA binding domain or the Lac repressor is inserted downstream of Cis-acting elements which bind radiation- inducible proteins. These constructs are useful in amplifying radiation-induced signals. This construct would be cotransfected with the plasmid containing multiple DNA binding sites for the Lac repressor protein cloned upstream of genes which when activated alter the phenotypic response of tumors to radiation.
  • TAL DNA or an active polynucleotide fragment thereof is recombined with a replication- deficient adenovirus type 5 (McGrory, et al . Virology 163 : 614 - 611 (1988)) to yield a vector designated Ad.Egr-TAL (similar to the Ad.Egr-TNF vector made by GenVec, Rockville, MD, and described in Hallahan, D.E. et al . , 1995, supra) .
  • Ad.Egr-TAL similar to the Ad.Egr-TNF vector made by GenVec, Rockville, MD, and described in Hallahan, D.E. et al . , 1995, supra
  • This vector employs the CCA (A+T rich) 6 GG elements (known as CArG elements) within the 5'- untranslated region of the early growth response (Egr-1) promoter 425 bp upstream from the transcription start site (Datta et al . , supra) .
  • a control region containing the 6 CArG elements of the promoter/enhancer region of the Egr-1 gene is ligated upstream of the TAL-encoding DNA.
  • These control elements are known to be inducible in several types of human tumor cells.
  • Other DNA sequences that activate transcription after X-irradiation and which may be used in the present method include AP-1 (Hallahan et al . , 1993, supra) and the N/cRB binding sequence (Brach, M. et al . , J. Clin . Invest . 88 : 691 - 695 (1991)) .
  • Tumor cells are injected with or otherwise administered, on one or on multiple occasions, about 2 x 10 8 PFU of AD5.Egr-TAL.
  • the target tissue typically tumor
  • the preferred radiation regimen can be determined readily by the skilled artisan using conventional clinical judgment.
  • the dose and time course are a function of the nature and extent of disease, the particular promoter used and its responsiveness, and the treatment approach ( e . g. , whether the radiation is being relied upon to kill cells directly, to induce apoptosis through TAL activation or both) .
  • 5 Gy X-irradiation are given four times per week for a total of 50 Gy, for example from a Maxitron generator (1.88 Gy/min) .
  • An advantage of the foregoing method is that transcriptional activation of a promoter is controlled by ionizing radiation within a specific body volume and for a chosen period of time. This achieves both spatial and temporal regulation of TAL transcription and overexpression, allowing apoptosis to be induced at a desired time and in a desired volume of cells or tissue. Such regional radiation exposure avoids the possibility of a broader or systemic apoptosis-inducing effect.
  • the methods of the present invention use a poly- or oligonucleotide which comprises an antisense sequence or encodes an antisense RNA which is antisense to an endogenous DNA or mRNA TAL sequence .
  • the present invention uses antisense oligonucleotides and polynucleotides complementary to the gene or genes encoding TAL in a eukaryotic cell, preferably a human cell.
  • Such antisense oligonucleotides should be at least about six nucleotides in length to provide minimal specificity of hybridization, and may be complementary to one strand of DNA or to mRNA encoding TAL (or to a portion thereof) , or to flanking sequences in genomic DNA which are involved in regulating TAL gene expression.
  • the antisense oligonucleotide may be as large as about 100 nucleotides, and may extend in length up to and beyond the full coding sequence for which it is antisense.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • antisense nucleotide may result in specific alteration, primarily inhibition, of TAL gene expression in cells.
  • antisense see: Albers, B., et al . , MOLECULAR BIOLOGY OF THE CELL, 2nd Ed., Garland Publishing, Inc., New York, NY (1989), in particular, pages 195-196.
  • the antisense oligonucleotide may be complementary to any portion of the TAL encoding sequence.
  • the antisense oligonucleotide may be between about 6 and 100 nucleotides, and may be complementary to the initiation ATG codon and an upstream, non-coding translation initiation site of the TAL sequence.
  • antisense nucleotides complementary primarily for non-coding sequence are known to be effective inhibitors of the expression of genes encoding transcription factors (Branch, M.A. , 1993 Molec . Cell . Biol . 13:4284-4290) .
  • Preferred antisense oligonucleotides are complementary to a portion of the mRNA encoding TAL.
  • a preferred antisense oligonucleotide is a 50mer which is antisense to 50 nucleotides in the 5' half of an RNA transcript of a TAL-encoding cDNA, more preferably any stretch of 50 nucleotides in the first 500 nucleotides of the 5' part of the RNA transcript.
  • the antisense oligonucleotide can be antisense to nucleotides 1-50, 2- 51, 3-52, 4-53,5-54, etc., of the RNA transcript.
  • the antisense oligonucleotide can be shorter, for example a 30-mer, and be antisense to any 30 nucleotide stretch of the RNA transcript, preferably in the first 500 5' nucleotides.
  • the minimal amount of homology required by the present invention is that sufficient to result in sufficient complementarity to provide recognition of the specific target RNA and inhibition or reduction of its translation or function while not affecting function of other mRNA molecules and the expression of other genes.
  • the antisense oligonucleotides of the invention comprise sequences complementary to at least a portion of an RNA transcript of TAL, absolute complementarity, although preferred, may not be required.
  • a sequence "complementary to at least a portion of" another sequence, as referred to herein, may have sufficient complementarity to be able to hybridize with that other sequence in vivo, perhaps forming a stable duplex.
  • the ability to hybridize may depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with the TAL target sequence it may contain and still form a stable duplex.
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting temperature of the hybridized complex as discussed above.
  • antisense RNA oligonucleotides generated intracellularly by transcription can be transcribed from exogenously introduced nucleic acid sequences.
  • antisense RNA may be delivered to a cell by transformation or transfection or infection with a vector, such as a plasmid or a virus, into which is incorporated (a) DNA encoding the antisense RNA; and (b) the appropriate regulatory sequences, including a promoter, to express the antisense RNA in a target host cell.
  • a vector such as a plasmid or a virus
  • the exogenous DNA or a portion thereof may be transcribed, producing an antisense RNA of the invention.
  • Vectors can be plasmid, viral, or others known in the art which are used for replication and expression in eukaryotic, preferably mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in eukaryotic, preferably mammalian, cells. Such promoters can be inducible or preferably are constitutive as described above. Such a vector, preferably a plasmid, becomes chromosomally integrated such that it can be transcribed to produce the desired antisense RNA. Such plasmid or viral vectors can be constructed by recombinant DNA technology methods that are standard in the art .
  • An oligonucleotide between about 6 and about 100 bases in length and complementary to the target sequence of TAL may be prepared by chemical synthesis from mononucleotides or shorter oligonucleotides, or produced by recombinant means .
  • An oligonucleotide between about 6 and about 100 bases in length and complementary to the target sequence of TAL may be synthesized chemically from natural mononucleosides or, alternatively, from mononucleosides having substitutions at the non-bridging phosphorous bound oxygens. Alternatively, the oligonucleotide may be produced by recombinant means .
  • a preferred mononucleoside analogue is a methylphosphonate analogue of the naturally occurring mononucleosides. More generally, the mononucleoside analogue is any analogue whose use results in an oligonucleotide which has improved diffusion through cell membranes or increased resistance to nuclease digestion within the body of a subject (Miller, P.S. et al . , Biochemistry 20:1874-1880 (1981)). Such nucleoside analogues are well-known in the art, and their use in the inhibition of gene expression has been disclosed. See, for example, Miller, P.S. et al., supra .
  • the antisense oligonucleotide molecule of the present invention may be a native DNA or RNA molecule or an analogue of DNA or RNA.
  • the present invention is not limited to use of any particular DNA or RNA analogue, provided it is capable of adequate hybridization to the complementary genomic DNA (or mRNA) of TAL, has adequate resistance to nucleases, and adequate bioavailability and cell uptake.
  • DNA or RNA may be made more resistant to in vivo degradation by enzymes such as nucleases, by modifying internucleoside linkages ( e . g. , methylphosphonates or phosphorothioates) or by incorporating modified nucleosides ( e . g. , 2'-0- methylribose or l'- ⁇ -anomers) .
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil , 5- bromouracil, 5-chlorouracil, 5-iodouracil , hypoxanthine , xanthine, 4 -acetylcytosine, 5- (carboxyhydroxylmethyl) uracil , 5-carboxymethylaminomethyl- ⁇ -thiouridine, 5- carboxymethyl-aminomethyl uracil, dihydrouracil , -D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 3 -methyl-cytosine, 5 -methylcytosine, N6- adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, /3-D-mannosylqueosine, 5-methoxy-carbox
  • the oligonucleotide comprises at least one modified sugar moiety selected from the group including, but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.
  • the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphoridothioate, a phosphoramidothioate, a phosphoramidate, a phosphordiimidate, a methylsphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof .
  • the oligonucleotide is an ⁇ -anomeric oligonucleotide which forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual 3-units, the strands run parallel to each other (Gautier et al . , 1987, Nucl . Acids Res . 15:6625-6641) .
  • the oligonucleotide may be conjugated to another molecule, e . g. , a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization- triggered cleavage agent, etc . , all of which are well- known in the art.
  • Oligonucleotides of this invention may be synthesized by standard methods known in the art, e . g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.) .
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al . , 1988 Nucl . Acids Res . 16: 3209
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al . , 1988 Proc . Natl . Acad . Sci . U. S.A . 85:7448-7451) , etc.
  • a DNA molecule encoding TAL such as disclosed by copending commonly assigned U.S. patent application 08/326,119, filed 19 October 1994, which is hereby incorporated by reference in its entirety) may be used for gene transfer to treat diseases or conditions wherein enhanced or suppressed TAL activity is desired, as is described above.
  • this invention is useful for the treatment of a disease or condition in which it is desired to render cells more or less sensitive to apoptotic signals.
  • in vivo and ex vivo methods Two broad categories of gene transfer methods are utilized in the present invention: in vivo and ex vivo methods. In the latter, DNA transfer is performed ex vivo and the transfected cells are introduced into the subject animal.
  • Gene therapy involves introduction of a foreign gene into a cell and ultimately, into a live animal .
  • Several general strategies for gene therapy have been studied and have been reviewed extensively (Yang, N-S., Cri t . Rev. Biotechnol . 12:335-356 (1992); Anderson, W.F., Science 256 : 808 - 813 (1992); Miller, A.S., Nature 357:455-460 (1992); Crystal, R.G., Amer. J. Med . 92 (suppl 6Aj:44S-52S (1992); Zwiebel, J.A. et al . , Ann . N. Y. Acad . Sci .
  • gene therapy would be accomplished by direct transfer of the functionally active TAL DNA into mammalian somatic tissue or organ in vivo, and more preferably, into cells which are to be killed.
  • DNA transfer can be achieved using a number of approaches described below.
  • an optimal gene delivery system should bind the DNA and make it soluble, effectively transfer the DNA into the cell, protect it from nucleases, release the DNA for efficient activity, and be targetable to specific cells.
  • the optimal system may differ according to the particular gene transfer application, e . g. , systemic versus local delivery, target cell type, etc.
  • use of viral vectors is preferable to the use of plasmid DNA.
  • Examples of successful transfer of genes known in the art include: (a) direct injection of plasmid DNA into mouse muscle tissues leading to indefinite expression of marker genes (Wolff, J.A. et al . , Science 247 : 1465 (1990); Acsadi, G. et al . , The New Biologi st 3 : 11 (1991)); (b) retroviral vectors effective for in vivo and in si tu infection of blood vessel tissues; (c) portal vein injection and direct injection of retrovirus preparations into liver to effect gene transfer and expression in vivo (Horzaglou, M. et al . , J. Biol . Chem . 265 : 11285 (1990); Koleko, M.
  • Retroviral Vectors for in vivo gene transfer into brain tissue (Ahmad, F. et al . , eds, Miami Short Reports - Advances in Gene Technology: The Molecular Biology of Human Genetic Disease, Vol 1, Boehringer Mannheim Biochemicals, USA, 1991) . Retroviral Vectors
  • Retroviral -mediated human gene therapy utilizes amphotrophic, replication-deficient retrovirus systems (Te in, H.M. , Human Gene Therapy 1 : 111 (1990); Temin et al . , U.S. Patent 4,980,289; Temin et al . , U.S. Patent 4,650,764; Temin et al . , U.S. Patent No. 5,124,263; Wills, J.W. U.S. Patent 5,175,099; Miller, A.D., U.S. Patent No. 4,861,719; Miller, A.D., Curr. Top . Microbiol . Immunol . 158 : 1 -24 (1989)) .
  • Retrovirus-mediated gene delivery generally requires target cell proliferation for gene transfer (Miller, D.G. et al . , Mol . Cell . Biol . 10 : 4239 (1990)). This condition is met by the preferred target cells for the present invention, i.e., growing tumor cells or activated lymphocytes in the case of autoimmunity.
  • Gene therapy of cystic fibrosis using transfection by plasmids using any of a number of methods and by retroviral vectors has been described by Collins et al . , U.S. Patent 5,240,846
  • DNA encoding TAL or
  • TAL antisense DNA is packaged into retrovirus vectors using one of several known packaging cell lines that produce replication-defective retroviruses (see, for example, Cone, R.D. et al . , Proc . Na tl . Acad . Sci . USA 81:6349-6353 (1984); Mann, R.F. et al . , Cell 33:153-159 (1983); Miller, A.D. et al . , Molec . Cell . Biol . 5:431-437 (1985); Sorge, J. , et al . , Molec . Cell . Biol . 4:1730-1737 (1984); Hock, R.A. et al .
  • the gene therapy approach can be utilized in a site specific manner to deliver a retroviral vector to the tissue or organ of choice.
  • a catheter delivery system can be used (Nabel, E.G. et al . , Science 244:1342 (1989)).
  • Such methods using either a retroviral vector or a liposome vector, are particularly useful to deliver the gene to a blood vessel wall, or into the blood circulation of a tumor.
  • Other Viral Vectors Other virus vectors may also be used, in particular for human gene therapy, including recombinant adenovirus vectors (Horowitz, M.S., In: VIROLOGY, Fields, B.N. et al .
  • HSV Herpes simplex virus
  • adenovirus vectors for human gene therapy include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organism.
  • vaccinia virus Another vector which can express the TAL DNA molecule of the present invention, and is useful in gene therapy, particularly in humans, is vaccinia virus, which can be rendered non-replicating (U.S. Patents 5,225,336; 5,204,243; 5,155,020; 4,769,330). Descriptions of recombinant vaccinia viruses containing heterologous DNA and their uses in immunization and gene therapy are reviewed in: Moss, B., Curr. Opin . Genet . Dev. (1993) 3:86-90; Moss, B.
  • HVJ enables foreign genes to be introduced directly into the cytoplasm by membrane fusion and the nuclear proteins transport the foreign genes rapidly into the nuclei .
  • a reporter gene was introduced into the kidney of intact rats through a cannula in the renal artery.
  • Tomita N et al . . Cancer Detect Prev (1994) 18:485-491 shows the successful introduction and expression of a human insulin gene in the mouse, with presence of human insulin in the mouse plasma and its reduction of plasma glucose levels.
  • the human renin gene was similarly introduced into adult rat liver resulting in significant elevation of blood pressure for 6 days compared with controls (Tomita, N. et al . , Circ Res (1993) 73 :898-905) .
  • Artificial Viral Envelopes were similarly introduced into adult rat liver resulting in significant elevation of blood pressure for 6 days compared with controls (Tomita, N. et al . , Circ Res (1993) 73 :898-905) .
  • AVE artificial viral envelopes
  • a viral membrane such as HIV-1 or RSV
  • AVE artificial viral envelopes
  • the envelope is preferably produced in a two-step dialysis procedure where the naked envelope is formed initially, followed by unidirectional insertion of the viral surface glycoprotein of interest.
  • This process and the physical characteristics of the resulting AVE are described in detail by Chander e ⁇ al . , (supra) .
  • Examples of AVE systems are (a) an AVE containing the HIV-1 surface glycoprotein gpl60 (Chander et al . , supra ; Schreier et al . , 1995, supra) or glycosyl phosphatidylinositol (GPI) -linked gpl20 (Schreier et al .
  • Naked envelopes (without HIV-1 rgpl60 inserted) show background levels of interaction with target cells, transferring material less efficiently and nonspecifically .
  • vesicles are constructed which mimic the natural membranes of enveloped viruses in their ability to bind to and deliver materials to cells bearing corresponding surface receptors.
  • AVEs are used to deliver genes both by intravenous injection and by instillation in the lungs.
  • AVEs are manufactured to mimic RSV, exhibiting the RSV F surface glycoprotein which provides selective entry into epithelial cells.
  • F-AVE are loaded with a plasmid coding for the TAL (or a reporter gene such as CAT not present in mammalian tissue) .
  • Recipient animals preferably humans, have an effective dose of the gene instilled into their lungs via a syringe connected to a thin endotracheal tube or, more preferably by inhalation.
  • animals sacrificed 48 hr after instillation lungs showed significant activity of CAT above background.
  • rats were injected intravenously with AVEs carrying on their surface a specific lung targeting molecule (which has a high binding affinity to the surface of lung endothelial cells) and carrying a payload of a plasmid encoding (heat-resistant) placental alkaline phosphatase (PAP) .
  • Anesthetized rats were injected with 600 ⁇ l L-AVE containing 100 ⁇ g of the gene product via a tail vein.
  • the AVE system described herein is physically and chemically essentially identical to the natural virus yet is entirely artificial, as it is constructed from phospholipids, cholesterol, and recombinant viral surface glycoproteins . Hence, there is no carry-over of viral genetic information and no danger of inadvertent viral infection. Construction of the AVEs in two independent steps allows for bulk production of the plain lipid envelopes which, in a separate second step, can then be marked with the desired viral glycoprotein, also allowing for the preparation of protein cocktail formulations if desired.
  • Bacterial Delivery Recently attenuated Shigella bacteria were described as a DNA delivery system (Sizemore, D.R. et al . , Science 270:299-302 (1995)). This approach exploits the ability of Shigellae to enter epithelial cells and escape the phagocytic vacuole as a method for delivering the TAL DNA construct into the cytoplasm of the target cell.
  • Invasion with as few as one to five bacteria can result in expression of the foreign plasmid DNA delivered by these bacteria.
  • As little as 4-20 x 10 9 ⁇ g DNA by Shigella was shown to be sufficient for expression of a transfected marker.
  • Shigella-mediated delivery of plasmid DNA can be achieved in various cell types which can be infected by Shigella. Plasmid DNA has been successfully delivered to animals in vivo, including keratoconjunctival delivery in guinea pig and intranasal delivery in mice. It is important that the delivery be done using highly attenuated bacteria for reasons of safety in human subjects. Importantly, this approach is not restricted to Shigella. Shigella invasion genes can be transferred to other bacterial genera such as E. coli.
  • a preferred type of mediator of nonviral transfection in vi tro and in vivo is cationic (ammonium derivatized) lipids. These positively charged lipids form complexes with negatively charged DNA, resulting in DNA charged neutralization and compaction. The complexes endocytosed upon association with the cell membrane, and the DNA somehow escapes the endosome, gaining access to the cytoplasm. Cationic lipid:DNA complexes appear highly stable under normal conditions. Studies of the cationic lipid DOTAP suggest the complex dissociates when the inner layer of the cell membrane is destabilized and anionic lipids from the inner layer displace DNA from the cationic lipid. Several cationic lipids are available commercially.
  • cationic lipids are less efficient than viral vectors.
  • a few cationic lipid compounds (Genetic Engineering News, Nov. 15, 1995, pg. 1) are up to two logs more active in their ability to express a reporter gene (CAT) in mouse lung than the compounds used in earlier gene transfer trials for cystic fibrosis.
  • CAT reporter gene
  • DNA is easier to make than virus.
  • the novel cationic lipid:DNA complexes are 500-fold more active than naked DNA. For delivery to lung, any inflammatory responses accompanying the liposome administration are reduced by changing the delivery mode to aerosol administration which distributes the dose more evenly.
  • lipofection One well-known method for effecting efficient DNA transfection is termed lipofection (Feigner, PL et al . , Proc . Natl . Acad . Sci . USA (1987) 84:7413-7417). Cationic liposomes have been successfully employed to express the CFTR protein in rats and to correct the chloride ion transport defect both in transgenic mice and in human patients. In one embodiment, this method utilizes a synthetic cationic lipid, N-[l-(2,3- dioleyloxy)propyl] -N,N,N-trimethylammonium chloride (DOTMA) .
  • DOTMA N-[l-(2,3- dioleyloxy)propyl] -N,N,N-trimethylammonium chloride
  • DOTMA Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA.
  • DOTMA facilitated fusion of the complex with the membrane of cultured cells resulting in both uptake and expression of the DNA.
  • the technique is considered simple, highly reproducible, and effective for both transient and stable expression of transfected DNA.
  • a method employing cationic liposomes useful for direct gene transfer in the therapy of cancer and other diseases is discussed by Farhood, H. et al . , Ann N Y Acad Sci (1994) 716:23-35.
  • Cationic liposomes mediate efficient delivery of DNA and DNA/protein complex to mammalian cells in vi tro and in vivo .
  • Cationic cholesterol derivatives mixed with phosphatidylethanolamine and sonicated to form small unilamellar vesicles complex with DNA and mediate the entry into the cytosol from the endosome compartment .
  • DC-Choi liposomes has been used in a gene therapy clinical trial for melanoma.
  • Such cationic liposomes were used for the delivery of trans-activating protein factors to regulate and control the expression of delivered transgenes in a protein dose-dependent manner.
  • Human tumor cells selected for cis-platin resistance or isolated from patients who have failed cis-platin therapy are highly transfectable with cationic liposomes.
  • the use of cationic liposomes may be combined with Adeno-associated (AAV) -based plasmids to introduce TAL DNA into cancer cells. This method has been used to transfer the IL-2 gene in human prostate cancer (Vieweg, J et al . Cancer Res (1995) 55:2366-2372).
  • a method for transient expression of genes in normal colonic epithelium involves liposomal gene delivery by rectal catheter infusion. This approach has been used to express a reporter gene and the human APC tumor suppressor gene under control of a constitutive promoter in a rodent model (Westbrook CA et al . , Hum Mol Genet (1994) 3:2005-2010). High efficiency transfection was achieved (close to 100% of epithelial cells expressing the introduced gene) . Expression in this system was transient (consistent with the normal turnover time of gut epithelium) . However, repeated treatments maintained expression. Importantly, for the purposes of inducing apoptosis as described herein, such transient overexpression of the TAL gene may be sufficient to achieve the desired cytotoxic effect .
  • TAL DNA is introduced into cells by using targeted liposomes (Nicolau, C et al . , Proc . Natl . Acad. Sci . USA 80 : 1068 (1983); Soriano et al . , supra) such as immunoliposomes, which can incorporate acylated monoclonal antibodies into the lipid bilayer (Wang et al . , supra) .
  • targeted liposomes Nicolau, C et al . , Proc . Natl . Acad. Sci . USA 80 : 1068 (1983); Soriano et al . , supra
  • immunoliposomes which can incorporate acylated monoclonal antibodies into the lipid bilayer
  • Polyclonal antibodies and mAbs specific for various types of tumors, viral antigens or cell surface markers of various normal cell types are well-known in the art.
  • the TAL DNA is specifically introduced into a selected type of target cell by means of an antibody selective for that cell type
  • an antibody specific for a class or subclass of lymphocytes can be used to target the TAL DNA to a particular lymphocyte population in the treatment of autoimmunity.
  • An antibody specific for a tumor associated antigen is used to target the therapeutic composition to cells of a tumor.
  • Proteoliposome delivery vesicles can be prepared by the protein-cochleate method. Self-assembling lipid- based complexes termed cochleate are used for in vivo DNA transfer (Gould-Fogerite, S. et al., 1985, Anal . Biochem . 148 : 15 -25 ; Mannino, R.J. et al . , 1988, Biotechniques 6:682-690; Papahadjopoulos , D. et al., Biochim . Biophys . Acta, 1975, 394:483-491) .
  • Cochleates are prepared by calcium-induced fusion of phosphatidyl serine-cholesterol liposomes (anionic) resulting in an insoluble jellyroll- like structure.
  • the layers of the jellyroll are composed of alternating sheets of negatively charged phospholipid and calcium.
  • Gould-Fogerite, S. et al . , Gene, 1989, 84:429-438 discloses a system in which proteins mediating the entry of enveloped viruses into cells are integrated in the lipid bilayer, and materials are encapsulated at high efficiency within the aqueous interior of these vesicles.
  • Proteoliposome-mediated delivery of proteins and drugs into entire populations of cells can be achieved in culture with this approach.
  • Material can be delivered gradually by Sendai virus glycoprotein-containing proteoliposomes or synchronous delivery can be achieved by exposing cell -bound influenza glycoprotein vesicles briefly to low pH buffer.
  • chimeric proteoliposome gene-transfer vesicles chimerasomes
  • Stable expression of a bovine papilloma virus-based plasmid in tissue-cultured cells at 100,000 times greater efficiency than calcium phosphate precipitation of DNA, has been achieved.
  • Stable gene transfer and expression in mice has been obtained by subcutaneous injection of chimerasomes containing a plasmid.
  • cochleates are solid, multilayered, lyophilizable precipitates containing no internal aqueous space.
  • a cochleate may be considered a fusion intermediate frozen in time.
  • Benefits of this structure include its ability to provide protection from degradation for associated or encochleated molecules, the nontoxic, nonimmunogenic nature of its components, and its stability (it can be lyophilized) .
  • Animal studies show that oral delivery of DNA wrapped in cochleates can result in systemic responses. This was demonstrated using an 11 kb DNA plasmid encoding the env, tat, and rev genes of HIV1 IIIB driven by a CMV promoter. Both oral and intramuscular administration of the DNA cochleates induced antigen-specific T helper cell responses and cytotoxic lymphocyte activity in mice.
  • conjugate includes (a) a molecule recognizing the target tissue; and (b) a DNA binding compound, such as polylysine, to bind to the TAL DNA being transfected.
  • This conjugate is then complexed with plasmid DNA using known methods for transfer.
  • PVP polyvinyl-based polymer
  • PVP is already used in FDA-approved injectable pharmaceutical formulations. The rationale is to enhance expression by protecting the DNA from degradation while retaining the flexibility to promote good dispersion throughout the muscle. Another desired property is interaction with DNA without condensing it into small particles, based on the expectation that condensation decreases the expression level compared to naked DNA.
  • a PVP formulation With a PVP formulation, a 5-to-10-fold increase was observed compared to naked DNA in the level of expression of a -galactosidase reporter gene on direct administration to rat muscle, as well as improved DNA dispersion throughout the muscle.
  • a muscle-specific human growth hormone gene construct was administered to rats using the PVP formulation a significant biological effect over time was observed compared to controls.
  • cationic lipid/colipid delivery systems may be used.
  • gene delivery to hepatocytes a key concern is to protect DNA in the circulation after systemic delivery before it reaches the liver.
  • a glycopeptide delivery system has been used that incorporates a proprietary small condensing peptide (developed as an alternative to polylysine to condense and protect DNA and allow extravasation of the particles through the liver (Genetic Engineering News, Nov. 15, 1995, pg. 1)) .
  • the peptide is galactosylated to target the asialoglycoprotein receptor in order to promote high affinity and specificity of gene delivery to the hepatocytes.
  • the prototype system incorporates an endosomal release agent (lytic peptide) and a hepatocyte-specific promoter. With this approach the efficiency of transfection in vi tro approaches that achieved with adenovirus .
  • Dendrimers lytic peptide
  • Dendrimers a macromolecular architecture, have become recognized as useful vectors for gene transfection
  • Dendrimers are made up of precise three-dimensional branches called dendrons, with a structure that mimics the bifurcation of tree branches.
  • PAMAM polyamidoamine
  • These are spherical polymers (polycationic) built up like layers of an onion (each layer being referred to as a generation) , with an outside surface of primary amines .
  • Dendrimers are nonimmunogenic and appear to protect DNA against nucleases . To enhance transfection ability, an excess of dendrimer to DNA is preferred.
  • small molecules are used to regulate the PPP, and through this regulation to modulate apoptosis.
  • Such molecules may be substrates of TAL or of other enzymes in this pathway.
  • allopurinol, an analogue of hypoxanthine, or its metabolites inhibits purine oxidase enzymes thereby inhibiting breakdown of purines .
  • TAL which is involved in the conversion of ribose-5-phosphate to glucose-6-phosphate which is a substrate for G6PD.
  • the PPP is subject to regulation and modulation by introduction of an agent which influences the TAL-catalyzed reaction.
  • allopurinol or substrates of TAL are the basis for novel methods for modulating the PPP, influencing TAL activity, and, through this, promoting or suppressing apoptosis. See Figure 11, which illustrates the PPP.
  • Anti-Fas monoclonal antibody CH-11 was obtained from MBL (Watertown, MA) .
  • Human recombinant TNF- ⁇ was obtained from Fisher (Pittsburgh, PA) .
  • Anti-human actin monoclonal antibody was from Boehringer-Mannheim (Indianapolis, IN) . 5, 6-carboxy-2 , 7-dichlorofluorescein-diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR) were obtained from Molecular Probes (Eugene, OR) .
  • TAL-H cDNA clone 4/2-4/1 was inserted into the Hpal site of the metallothionein promoter-driven pMAXRHneo-1 vector (19) following removal of the coding sequence of the p40/tax protein of HTLV-I by cleavage with Hpal .
  • Clones containing the TAL-H cDNA in the sense (pL26-3) and antisense orientation (pL18-3) were selected.
  • 20 ⁇ g of plasmid DNA linearized with Kpnl were used to stably transfect Jurkat cells by electroporation as described (19) .
  • Transfected cells were grown in the presence of 750 ⁇ g/ml G418 and cloned by limiting dilution. Levels of transaldolase expression were measured by enzyme assay and Western blot analyses in the absence and presence of 5 ⁇ M CdCl 2 . TAL protein expression was assessed by Western blot analysis and compared to that of actin using an automated scanning densitometer (Bio-Rad Model GS-670) . Transfection of the pMAXRHneo-1 vector alone had no effect on TAL activity. Due to a leakiness of the promoter, cell lines producing increased and suppressed levels of TAL in the absence of CdCl 2 were obtained. While overexpression and suppression were doubled by incubating the cells with 5 ⁇ M CdCl 2 , CdCl 2 was not utilized in the following experiments to preclude possible interference with apoptotic pathways (20) . Induction of apoptosis
  • Cells were cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 ⁇ g/ml gentamicin at 37°C in a humidified atmosphere with 5% C0 2 . 24 h prior to assays, cells were fed with fresh medium and seeded at a density of 2 x 10 5 cells/ml. CD95/Fas/Apo-l-mediated cell death was induced with 50 or 100 ng/ml anti-Fas monoclonal antibody CH-11.
  • TNF-mediated cell death was induced with 20 ng/ml or 100 ng/ml human recombinant TNF-o; as earlier described (21-23) .
  • Apoptotic cell death was also induced by withdrawal of fetal calf serum or treatment with 50 ⁇ M or 100 ⁇ M H 2 0 2 , or 2.5 , 5, and 10 mM sodium nitroprusside (SNP) , used as a source of exogenous NO (4) .
  • SNP sodium nitroprusside
  • Apoptosis was monitored by observing cell shrinkage, nuclear fragmentation, and quantified by trypan blue exclusion (1) .
  • DNA fragmentation during apoptosis was monitored by agarose gel electrophoresis (24) .
  • Apoptosis was also measured by flow cytometry as previously described (25) . Briefly, following induction of apoptosis, cells were washed in PBS and resuspended in PBS with 0.1% Triton X-100 and 50 ⁇ g/ml propidium iodide . The DNA content was analyzed by using a Beckon Dickinson FACStarplus flow-cytometer .
  • TAL Transaldolase
  • the reverse reaction catalyzed by TAL was tested in the presence of 3.2 mM D-fructose 6-phosphate, 0.2 mM erythrose 4 -phosphate, 0.1 mM NADH, 10 ⁇ g ⁇ -glycerophosphate dehydrogenase/triosephosphate isomerase at a 1:6 ratio in 40 mM triethanolamine pH 7.6, 5 mM EDTA at room temperature by continuous absorbance reading at 340 nm for 8 min (18) .
  • the forward reaction catalyzed by TAL was measured at room temperature in the presence of 50 mM TEA pH 7.4, 5mM MgCl 2 , 3 mM ribose 5- phosphate, 0.9 mM xylulose 5-phosphate, 0.5 mM NADP, 0.2 mM thiamine pyrophosphate, 0.2 U/ml transketolase, 0.4 U/ml phosphoglucose isomerase, and 0.3 U/ml G6PD, following a 10 min lag period, by continuous absorbance reading at 340 nm for 8 min (26) .
  • the enzyme assays were conducted in the activity range of 0.001-0.01 U/ml.
  • TAL activity refers to enzyme activity measurements conducted in the reverse reaction. Enzymatic Activities of Transketolase, glucose 6- phosphate dehydrogenase (G6PD) , and 6-phosphogluconate dehydrogenase (6PGD)
  • Transketolase (TK) activity was measured in 50 mM Tris-HCl pH 7.5, 5 mM MgCl 2 , 0.06 mM thiamine pyrophosphate, 0.1 mM NADH, 10 ⁇ g ⁇ -glycerophosphate dehydrogenase/triosephosphate isomerase at a 1:6 ratio, 5 mM ribose 5-phosphate, and 1.25 mM xylulose 5-phosphate (13) .
  • G6PD was measured in the presence of 120 mM Tris pH 8.4, 10 mM MgCl 2 , 2 mM glucose 6-phosphate, 0.9 mM NADP, and 0.1 U/ml 6PGD (27). 6PGD activity was determined in 120 mM Tris pH 7.7, 10 mM MgCl 2 , 0.9 mM NADP, 2 mM 6- phosphogluconate (27) .
  • Glutathione, NADPH, and NADH levels were measured in
  • Total glutathione content was determined by the enzymatic recycling procedure essentially as described by Tietze (28). 10 6 cells were resuspended in 100 ⁇ l of 4.5% 5-sulfosalicylic acid. The acid-precipitated protein was pelleted by centrifugation at 4°C for 10 min at 2000 x g. The total protein content of each sample was determined using the Lowry assay (29) . GSH content of the aliquot assayed was determined in comparison to reference curves generated with known amounts of GSH.
  • Jurkat-tat cells was transfected with HIV-1 DNA clone 4803 (54) by electroporation at 600 ⁇ F/250V/72 ⁇ . Infectious stock of the strain HIV-14803 was harvested from 24 h supematants of freshly re-infected Jurkat-tat cells and infectious titer was determined by an in si tu infectivity (MAGI) assay (Nagy, K. et al . , (1994) J. Virol . 68:757-765) .
  • MAGI si tu infectivity
  • Non-infected control PBL were cultured under identical conditions. Transmission of HIV-1 was monitored by production of gagp24. Recombinant HIV-1 gag24 protein was utilized as a control antigen. As positive control sera, HIV-1 gag p24 specific polyclonal sheep antibody and monoclonal antibodies to p24 were utilized. Viral reagents were obtained from the National Institutes of Health AIDS Research and Reference Program. Western blot analysis
  • Protein lysates were prepared from cell lines and quantified by the Lowry method. 40 ⁇ g of protein lysate in 10 ⁇ l per well was separated by SDS-PAGE and electroblotted to nitrocellulose. Nitrocellulose strips were incubated in 100 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20, and 5% skim milk with TAL-H-specific rabbit Ab 169 (18) or actin-specific murine monoclonal antibody C4 at a 1000-fold dilution at room temperature overnight. For detection of rabbit antibodies, after washing, the strips were incubated with horseradish peroxidase-conjugated goat anti -rabbit IgG (Boehringer Mannheim, Indianapolis, IN) .
  • horseradish peroxidase-conjugated goat anti -rabbit IgG Boehringer Mannheim, Indianapolis, IN
  • the strips were incubated with biotinylated goat anti -mouse serum and, subsequently with horseradish peroxidase-conjugated avidin (Jackson Laboratories, West Grove, PA) . In between the incubations the strips were vigorously washed in 0.1% Tween-20, 100 mM Tris pH 7.5, and 0.9% NaCl. The blots were developed with a substrate comprised of 1 mg/ml 4-chloronaphthol and 0.003% hydrogen peroxide. Flow cytometric analysis of the rates of intracellular oxidation
  • ROIs were estimated fluorometrically using 5 , 6-carboxy-2 , 7- dichlorofluorescein-diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR) as earlier described (31,32).
  • DCFH-DA 7- dichlorofluorescein-diacetate
  • DHR dihydrorhodamine 123
  • TAL expression influences biochemical processes regulating apoptosis
  • cell lines producing increased and suppressed levels of TAL were generated.
  • Jurkat human T cells were stably transfected with eukaryotic expression vectors containing full length transaldolase cDNA in the sense (pL26-3) or antisense orientation (pL18-3) .
  • Western blot analysis revealed several cell lines carrying pL26-3 and pL18-3 which had either increased or decreased levels of TAL expression, respectively.
  • TAL expression was increased by 160 ⁇ 9% (2.6-fold) in L26-3/4 cells ( Figure 1) and 47 ⁇ 8% in L26-3/2D1 cells, whereas, TAL activity was suppressed by 25 ⁇ 4% in L18-3/1 cells ( Figure 1) and 55 ⁇ 6% in L18-3/1D9 cells in comparison to control Jurkat cells.
  • Transfection with the plasmid vector lacking TAL-H cDNA had no effect on enzymatic activities in pXHIC ⁇ and pXH2D2 cells (Table 2) .
  • TAL expression was evaluated on activities of other key PPP enzymes and intracellular levels of NADPH, NADH, and GSH.
  • Overexpression and increased activity of TAL had no significant effect on TK activities in L26-3/4 and L26-3/2D1 cells.
  • Suppression of TAL was associated with a decrease of TK activity in L18-3/1 cells while TK activity remained unchanged in L18-3/1D9 cells. Since the effect of TAL suppression was greater in L18-3/1D9 than in L18-3/1 cells, reduction of TK in L18-3/1 cells may not be related to diminished TAL activity.
  • EXAMPLE III Sensitivity to apoptotic signals is influenced by levels of TAL expression
  • the level of TAL expression influenced the rate of HIV-induced cell death in Jurkat cells infected with HIV- 1. While increased expression of TAL through overexpression of TAL-encoding DNA accelerated cell death, decreased expression of TAL by expression of TAL antisense DNA inhibited cell death ( Figure 9) .
  • Cell death programs induced by Fas or TNF stimulation or by HIV-1 infection caused alterations of PPP enzyme activities leading to depleted intracellular GSH in a pattern reminiscent of the changes produced by TAL overexpression.
  • DHR is nonfluorescent , uncharged, and readily taken up by cells
  • R123 the product of DHR oxidation
  • the rates of increase in fluorescence of cells treated with 100 ⁇ M H 2 0 2 and 50 ng/ml anti-Fas mAb were evaluated. As shown in Figure 5, relative to H 2 0 2 a smaller but consistent increase in ROI was detected in Fas-stimulated cells as compared with untreated cells. In agreement with earlier data (33) , DHR was a significantly more sensitive detector of increases in ROI levels than DCFH. Production of ROI correlated with the rate of cell death (p ⁇ 0.01, Figure 6) .
  • ROI The involvement of ROI in Fas-dependent signaling was suggested by the above observation of (i) an increased sensitivity to Fas-induced death of cells with increased TAL expression and decreased GSH content and (ii) the production of ROI during Fas-mediated apoptosis. An examination of whether changes in GSH levels influence Fas-induced PCD was therefore done. Under the conditions utilized, none of the agents had significant effect on binding of anti-Fas antibody to its receptor (measured using flow cytometry) .
  • Intracellular GSH levels were raised by as much as two-fold using NAC, a precursor of glutathione (34) , or suppressed to less than 15% of baseline by buthionine sulfoximine (BSO) , an inhibitor of ⁇ -glutamyl-cysteine synthetase (34) ( Figure 7) .
  • NAC a precursor of glutathione
  • BSO buthionine sulfoximine
  • G6PD activity was increased by both NAC (p ⁇ 0.01) and BSO (p ⁇ 0.05) after 24 hr incubation.
  • DMPO and TMPO are nitrones which react with ROIs to form more stable nitroxide radical products (36) and have been shown to protect thymocytes against apoptosis (9) . While DMPO or TMPO had no significant effect on GSH levels, they also inhibited Fas-induced apoptosis (p ⁇ 0.01; Figure 6B(8) ) .
  • Increased phosphorylation was accompanied by an up to three- fold stimulation of TAL enzymatic activity in a whole cell lysate of Jurkat cells.
  • enzymatic activity of recombinant TAL was increased 2.5- fold by phosphorylation with protein kinase C (see Figure 10) .
  • the presence of an inhibitor of PKC during this pretreatment fully reversed the effect, indicating that phosphorylation was the basis of the effect.
  • PMA and calcium ionophore are known to be potent inducers of apoptosis ( Immunol . Rev. 1994, 142:301-320), indicating that phosphorylation of TAL-H can serve as a key signaling mechanism of certain forms of PCD.
  • EXAMPLE VII The nucleotide sequence of the human transaldolase mRNA is provided herein as SEQ ID NO:l. This sequence has been assigned Genbank Accession No. L19437. Antisense oligonucleotides are designed based upon this sequence .
  • TAL Overexpression of TAL in Jurkat human T cells resulted in down-regulation of G6PD and 6PGD activities and a decrease of NADPH and GSH levels. NADH levels were also reduced in TAL-overproducing cells which was consistent with the tendency to maintain NADPH at the expense of NADH by transhydrogenases (37) . Alternatively, decreased TAL expression led to upregulation of G6PD and 6PGD activities and increased GSH levels.
  • TAL a pivotal enzyme of the PPP.
  • Overexpression of TAL increased sensitivity, while suppression of TAL decreased sensitivity to six different apoptotic signals, indicating that TAL expression levels profoundly influence susceptibility to PCD.
  • ROIs The involvement of ROIs in each of the apoptosis signaling pathways examined here was suggested by the finding that TAL expression, via regulation of the PPP and of GSH production, modulated susceptibility to apoptosis. Apoptosis triggered by serum withdrawal (39), and NO has been associated with production of ROIs (4) . Because GSH is the most abundant intracellular thiol compound which neutralizes ROI, a direct correlation of GSH levels with resistance to apoptosis induced by either H 2 0 2 , NO, or serum withdrawal was obtained. In contrast, an involvement of ROIs has not been clearly defined in Fas and TNF-induced apoptotic pathways which are signaled through specific cell surface receptors. The Fas/Apo-1 antigen and the TNF ⁇ receptor are members of the
  • TNF/nerve growth factor receptor superfamily contains canonical cysteine-rich extracellular domains; Fas and the type I TNF receptor, mainly responsible for mediating cytolytic activity of TNF, also have a common 70 amino acid intracellular sequence which may play a role in triggering common cytoplasmic death signals.
  • TNF-mediated apoptosis is known to involve oxidative stress based on (a) the induction of ROI in response to TNF (40,41) and (b) the inhibition of TNF-induced killing by free radical scavengers (40-42) .
  • TNF- but not Fas-mediated cell death could be inhibited by ROI -scavenging compounds (42) .
  • No requirement of ROIs in either TNF- or Fas-mediated apoptosis was suggested by Hug et al . (43) .
  • TNF receptor and involvement of common signal transducers suggests that both receptors may mediate cell death via similar mechanism (44) .
  • ZB4 did accelerate HIV-induced apoptosis, consistent with the observation that binding of HIV gpl20 to CD4 (or other interactions between viral proteins and the T cell surface) may initiate a cell death program (2) and sensitize cells to apoptosis induced by a suboptimal or partial apoptotic stimulus such as an inherently non-cytolytic anti-Fas antibody like ZB4.
  • NAC which increases GSH levels
  • DMPO and TMPO two free radical spin traps which form relatively stable nitroxide radical products with ROIs (40)
  • various antioxidants DEF, NDGA, and Amytal
  • the present results support the notion that the PPP is an important biochemical mechanism regulating sensitivity to cell death programs dependent on formation of ROIs. This is consistent with an essential role of the PPP in the generation of NADPH for the synthesis of GSH which, in turn, protects cellular integrity from oxygen radicals. Levels of TAL expression may have a dominant role in regulating the balance between the two branches of PPP and the ultimate output of GSH. It is concluded that TAL, and possibly other PPP enzymes, serves as a critical determinant of tissue and cell type- specific sensitivity to apoptotic signals.
  • MS Follicular lymphomas Multiple sclerosis
  • TAL-H expression vectors Measurement of the activity of TAL in the forward (FOR) and reverse (REV) directions, TK, G ⁇ PD, and 6PGD (mU/mg protein), and of the levels of , NADH (pmol/ ⁇ g protein), NADPH (pmol ⁇ g protein) and GSH (pmot ⁇ g protein) in Jurkat cells stably transfected with TAL-H expression vectors.
  • L26-3/4 and L26-3/2D1 Gens' were transfected with the sense construct.
  • LI 8-3/1 and L18-3/1D9 cells were transfected with the antisense construct
  • pXH2D2 cells were transfected with the expression vector without TAL-H cDNA.
  • % survival was 2 assessed by trypan blue exclusion after 24 h stimulation with 50 ngml anti-Fas monoclonal antibody. Data show mean ⁇ SD r 32 of eight independent experiments. P values indicate significant differences in enzyme activities, NADH, NADPH and GSH m levels in transfected cells as" compared to those of Jurkat cells. *, p ⁇ 0.05; f, p ⁇ 0.01; $, p ⁇ 0.001. ⁇ >
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Abstract

La transaldolase (TAL) joue un rôle important dans la régulation de la sensibilité des cellules à l'apoptose. L'invention porte sur des méthodes régulant à la hausse l'expression du gène de la TAL par exemple en distribuant à une cellule de l'ADN exogène codant pour la TAL, ou des méthodes qui stimulent l'activité enzymatique de la TAL par exemple en provoquant une phosphorylation à l'aide de la protéine kinase (C), et favorisent la mort cellulaire programmée en réponse à des signaux apoptiques. Inversement, l'inhibition de l'expression du gène de la TAL soit en produisant de l'ADN antisens de la TAL, soit en supprimant l'activité enzymatique de la TAL, rend la cellule résistante aux signaux apoptotiques. L'invention concerne des moyens de traitements d'états caractérisés par une apoptose aggravée comme par exemple des maladies neurodégénératives, des maladies démyélinisantes ou dues au VIH, ou des états dans lesquels l'apoptose à été supprimée de manière inopportune, comme par exemple le cancer, certaines infections virales et l'auto-immunité, en régulant à la hausse ou à la baisse l'expression ou l'activité enzymatique de la TAL.
PCT/US1997/022770 1996-12-13 1997-12-12 Regulation de l'apoptose induite par la transaldolase WO1998025630A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620278A1 (fr) * 1993-03-30 1994-10-19 Ono Pharmaceutical Co., Ltd. Transaldolase humaine et DNA codant pour celle-ci

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620278A1 (fr) * 1993-03-30 1994-10-19 Ono Pharmaceutical Co., Ltd. Transaldolase humaine et DNA codant pour celle-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 20 December 1996, Vol. 271, No. 51, BANKI et al., "Glutathione Levels and Sensitivity to Apoptosisare Regulated by Changes in Transaldolase Expression", pages 32994-33001. *

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