JP2003501097A - Inhibition of apoptosis by adenovirus E3 / 6.7K - Google Patents

Abstract

  (57) [Summary] The present invention provides an immune evasion method and an apoptosis evasion method using E3 / 6.7K protein derived from adenovirus.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to inhibition of apoptosis.

BACKGROUND viral infection of the invention is the injury of a cell, and thereby are induced programmed cell death of the host cell. Many viruses, especially persistent DNA viruses, are able to continue viral replication by modifying the apoptotic response of cells.
Apoptosis is associated with members of the TNF receptor superfamily such as Fas (APO-1
Or CD95) and p55 tumor necrosis factor receptor (p55 TNFR) and death domain containing receptors 3, 4 and 5 (DR3, DR4 and DR5 respectively).
The intracellular factors that cause cell death are highly conserved among species and have been targets for viral inhibitors of apoptosis. Proteins belonging to a very diverse class of viruses are believed to have evolved to block the same cellular apoptotic events. This convergent evolution is evidenced by the class of viral inhibitors of apoptosis. For example, adenovirus E
1B 55K (Debbas and White, 1993), SV40 giant T antigen (Levine, 1997; Lill et al.,
1997) and human papillomavirus E6 (Levine, 1997) inhibit p53-mediated cell lysis. The adenovirus E1B 19K (White, 1996), the Epstein-Barr virus BHRF1 (Henderson et al., 1993) and the African swine fever virus LMW5-HL (Neilan et al., 1993) mimic the cell survival factor Bcl-2. Members of the interleukin 1b-converting enzyme (ICE) family of terminal proteolytic enzymes, also known as caspases, are baculovirus p35.
(Clem et al., 1991; Xue and Horvitz, 1995) and crmA, cowpox serpin protein (Zhou et al., 1997; Tewari and Dixit, 1995). Adenovirus E3 / 10.4K and E3 / 14.5K down-regulate surface Fas (Elsing
And Burgert, 1998; Tollefson et al., 1998; Shisler et al., 1996), that the baculovirus apoptosis inhibitor (IAP) family and its mammalian homologues interact with TNF-α receptor-related factors (TRAFs), It blocks the signaling cascade leading to caspase recruitment events (List
on et al., 1996; Duckett et al., 1996; Deveraux et al., 1997). FADD-like interleukin-1
Activation of β-converting enzyme (FLICE) (also known as caspase-8) by Fas results in virus-FLICE-inhibitory proteins (vFLIPs) (Thome et al., 1997) found in various types of herpesvirus genomes. ), And adenovirus E3 /
Blocked by 14.7K (Chen et al., 1998). Adenovirus (Ad) is a very common human pathogen that causes persistent infection of the respiratory or gastrointestinal tract (Fox et al., 1969; Fox et al., 1977). Persistent infections result from the careful evasion of host defenses. Adenovirus genes that cause immune evasion have been mapped to the early 3 (E3) region of the Ad genome (Wold and Gooding, 1989). Adenovirus is suitable as a gene therapy vector due to its persistence, ease of infection and weak pathogenicity. Currently Ad
Gene transfer vectors are the most effective technique available for gene transduction in vivo. The size of transducing DNA that can be accommodated by adenovirus is over 30 kbp, far superior to all other viral systems. In the case of the Ad vector, the genetic makeup of the original vector was designed to delete a region of the adenovirus genome, which is considered non-essential, to contain a large piece of DNA for the transducing gene. The E3 area is one of the first replacement target areas. The sequence of the 6.7K protein encoded by the E3 region (E3 / 6.7K) has no significant homology to any of the other known proteins. This is Group C Ad2
And is highly conserved between Ad5 adenovirus and Group B Ad3, Ad7 and Ad35 adenoviruses (Hawkins et al., 1995). Ad2 E3 / 6.7K protein (Wilso
n-Rawls et al., 1990) has been shown to be an essential membrane protein localized to the endoplasmic reticulum (ER) (Wilson-Rawls and Wold, 1993). This protein exists in two forms, one in the non-glycosylated form with an apparent molecular weight of 8 kDa and the other in the glycosylated form with an apparent molecular weight of 14 kDa. This protein is targeted to the ER but lacks a cleavable signal sequence and has a hydrophobic central region that is thought to function as a signal anchor (Wilson-Rawls et al., 1994). . A major obstacle to the success of Ad vectors as well as all other gene transfer techniques is the unexpectedly strong immune response against cells infected with the modified adenovirus. A strong immune response to the modified Ad vector is due to the circulating cytokine tumor necrosis factor (TNF) alpha (Elkon et al., 1997) and an innate immune response (Worgall et al., 1997).
) Is mediated by. The negative effects of the immune response may be mitigated by using vectors and immunomodulatory proteins capable of surviving the transduced cells against the immune response (Zhang et al., 1998). Immune response avoidance has also been a central obstacle to the establishment of successful transplantation techniques and the treatment of autoimmune and neurodegenerative diseases. Apoptosis of affected organs is often due to neurodegenerative inflammatory disease. Factors that interfere with apoptosis may result in better treatment of these conditions. Expression systems as cell culture reactors are limited only by the ability of cells to grow and produce proteins of commercial or medical interest (Singh and al-Rubeai, 1998; al-Rubeai, 1998). ). As cells proliferate, they reach a density at which protein production ceases, and the producing cells undergo apoptosis in response to factors that are currently poorly characterized (al-Rubeai and Singh, 1998). The inclusion of anti-apoptotic proteins in the composition of cells to avoid the apoptotic response of cell proliferation during culture may potentially improve protein yield (Simpson et al., 1998). SUMMARY OF THE INVENTION It is shown herein that TNF-α induced apoptosis and TNF-α induced arachidonic acid release are significantly reduced in transfected E3 / 6.7K expressing cells. Further, in the present specification, the mechanism of E3 / 6.7K is shown to be associated with cleavage and inactivation of cytosolic phospholipase A2 (cPLA2). This enzyme
It is important for the production of proinflammatory substances and is involved in the release of arachidonic acid. E
3 / 6.7K has no sequence homology with any of the previously described apoptosis inhibitors. Therefore, E3 / 6.7K is a novel class of viral apoptosis inhibitors localized in the endoplasmic reticulum. The present invention provides a method of immune evasion and apoptosis evasion, which uses the E3 / 6.7K protein from adenovirus as a means. The invention also provides vectors containing the adenovirus E3 / 6.7K region for use in gene therapy or for minimizing transplant rejection. The invention also provides a method of improving protein yield from cell culture. The present invention provides a method for inhibiting apoptosis of a cell, which comprises treating the cell, a mammal containing the cell or a tissue containing the cell with an effective amount of E3 / 6.
Treatment with a .7K polypeptide is included. The processing step can include administering a nucleic acid encoding the polypeptide, thereby expressing the polypeptide intracellularly. This administration is by a viral vector containing the nucleic acid, where the vector is an adenovirus, the nucleic acid is other than the adenovirus native E3 derived nucleic acid or is not found in the adenovirus. It is conditional that it is under the transcriptional control of a promoter.

The method of the present invention involves the degenerative (()
For example, for treating a mammalian individual suffering from a neurodegenerative) disease, an immunodeficiency, or an inflammatory disease.

The invention also provides a method of reducing apoptosis in a tissue or cell population of a patient, the method comprising: (a) collecting tissue or cells from the patient; Cells are treated with an effective amount of E3 / 6.7K polypeptide, and (c
And) returning the treated tissue or cells to the patient. The cell population may contain leukocytes or may be composed of leukocytes.

The invention also provides an E3 / 6.7K polypeptide and a suitable carrier for facilitating delivery of the polypeptide to cells, as well as a non-natural adenovirus E3 nucleic acid that can encode the E3 / 6.7K polypeptide. There is provided a pharmaceutical composition containing a nucleic acid comprising.

The present invention also provides a recombinant virus comprising a nucleic acid encoding an E3 / 6.7K polypeptide, wherein when the virus is an adenovirus, the nucleic acid is a native adenovirus. Conditionally, it is other than the E3 nucleic acid, or is under the transcriptional control of a promoter not derived from adenovirus.

The invention also relates to the use of an E3 / 6.7K polypeptide, a nucleic acid encoding said polypeptide or a vector containing said nucleic acid for the treatment of apoptosis, as well as the manufacture of a medicament for treating apoptosis. Of the E3 / 6.7K polypeptide, a nucleic acid encoding the polypeptide or a vector containing the nucleic acid.

The invention also provides an assay for a drug that modulates the anti-apoptotic activity of an E3 / 6.7K polypeptide, the assay being suspected of including the polypeptide and the drug. Included is mixing with the sample and determining if the anti-apoptotic activity is modulated. The mixing can be in cells or extracts of cells that are rescued from apoptosis by the E3 / 6.7K polypeptide expressed in or administered to the cells. The determination may be performed by detecting or measuring TNF-α activity (eg, arachidonic acid release).

Detailed Description As used herein in describing the present invention, E3 / 6.7K protein and E3 / 6.7K
The term 6.7K polypeptide includes the proteins encoded by the nucleic acids shown in Figure 1 or fragments thereof; the Ad2 or Ad5 adenovirus serotype proteins shown in Figure 2 or fragments thereof; or Ad2 or Ad5 shown in Figure 2. Included are proteins that have at least 70% similarity to the proteins as determined by the Basic Blast search using default parameters. Ad5 protein is actually about
It is 7.1K. Modulation of apoptosis, such as inhibition of apoptosis or rescue of cells from apoptosis, can be measured by various methods known in the art. Such methods include assays that directly measure apoptosis, or assays that measure TNF-α activity as described herein. Gene Therapy Method The isolated nucleic acid molecule shown in FIG. 1, the nucleic acid molecule encoding the E3 / 6.7K protein specified herein, or the nucleic acid molecule complementary to the one described above, can be used in mammalian cells to be treated. A transfection vector can be prepared by incorporating the nucleic acid into a vector suitable for introducing into the vector. Vectors suitable for the above purposes include retroviruses and adenoviruses. Techniques for making transfection vectors containing nucleic acid molecules encoding E3 / 6.7K are well known in the art and are described in "Engineering for Human Gene Therapy (Worki
ng Toward Human Gene Therapy) ”“ Recombinant DNA ”, Chapter 28,
Second edition, edited by Watson, JD et al., New York: Scientific American Books, pp. 567-
581 (1992) and references cited therein, generally. It has been shown that similar gene therapy approaches are or are expected to be effective in the treatment of other mammalian diseases as shown below. Ie cystic fibrosis (D
rumm, ML et al., Cell 62: 1227-1233 (1990); Gregory, RJ et al., Nature 347:
358-363 (1990); Rich, DP et al., Nature 347: 358-363 (1990)), Gaucher's disease (Sorge, J. et al., Proc. Natl. Acad. Sci. USA 84: 906-909 (1987); Fink, J
K. et al., Proc. Natl. Acad Sci. USA 87: 2334-2338 (1990)), certain types of hemophilia (Bontempo, FA petal, Blood 69: 1721-1724 (1987); Palmer, T.
D. et al., Blood 73: 438-445 (1989); Axelrod, JH et al., Proc. Nail. Acad. Sc.
i. USA 87: 5173-5177 (1990); Armentano, D. et al., Proc. Natl. Acad. Sci. US.
A 87: 6141-6145 (1990)) and muscular dystrophy (Partridge, TA et al. Nat.
ure 337: 176-179 (1989); Law, PK et al., Lancet 336: 114-115 (1990); Mor.
gan, JE et al., J. Cell Biol. 111: 2437-2449 (1990)), as well as certain cancers,
For example, metastatic melanoma (Rosenberg, SA et al., Science 233: 1318-1321 (1986);
Rosenberg, SA et al., N. Eng. J. Med. 319: 1676-1680 (1988); Rosenberg, S.
A. et al., N. Eng. J. Mcd. 323: 570-578 (1990)). Gene expression in specific tissues can be enhanced by using various promoters. For example, in neural tissue, the neuron-specific enolase promoter (Ad-NS
E), and in lymphocytes the lck promoter may be used. The organ transplant method E3 / 6.7K has potential applications in tissues and organ transplants to reduce their susceptibility to apoptosis. In particular, E3 / 6.7K can be used to genetically modify endothelial cells or other mammalian cells to allow expression of the E3 / 6.7K protein, which protein can transfect TNF- in transfected cells. It specifically inhibits α-induced apoptosis. E3 / 6.7K can also be used for transplanting cells genetically modified to express an inhibitory protein (E3 / 6.7K) or a tissue or organ containing such cells. This is especially the method of transplanting modified xenogeneic or allogeneic cells, tissues or organs; recombinant genes, recombinant proteins and recombinant vectors for obtaining them; and similarly modified cells, tissues Alternatively, it is used for organs and non-human transgenic animals or somatic cell recombinant animals. Suitable methods for inserting foreign cells or DNA into animal tissue include microinjection, manipulation of embryonic stem (ES) cells, electroporation, cell gun, transfection-k, transduction, retroviral infection, etc. To be By inserting the gene into a germ cell (eg, a fertilized egg), a transgenic non-human animal carrying the gene can be produced, which gene is then transmitted to the offspring.

The gene can also result in somatic recombination by inserting it into somatic cells, but the gene from this animal is not transmitted to the offspring.

[0011] In one embodiment, transcription of the gene is under the control of an inducible promoter, thus expression of the recombinant protein may be delayed for an appropriate period of time prior to transplantation.
In other embodiments, the gene is inserted at a specific locus, such as a thrombomodulin or P-selectin locus. Subsequently, when the construct is introduced into embryonic stem (ES) cells, the progeny produced will express the construct in the vascular endothelium. With respect to gene delivery, retroviral vectors, and replication defective retroviral vectors lacking one or more of the gag, pol and env sequences specifically required for retroviral replication, are well known in the art, These can be used to transform endothelial cells. The ability of adenovirus to attach to cells at low ambient temperatures is advantageous in a transplant environment where it can facilitate gene transfer during cryopreservation. Other means of targeting gene delivery include DNA-protein conjugates, liposomes and the like. The cell or cell population may be treated in vivo or in vitro according to the present invention. For example, for in vivo processing, the p65RHD vector may be used in cells, tissues or organs
It can be inserted by direct infection in situ. For example, the blood vessels of organs such as the kidney
Temporarily clamp the blood circulation and perfuse the vessel with a solution containing a transducible vector construct containing the E3 / 6.7K gene for a sufficient time to allow the gene to be inserted into cells of the organ. It can then be unclamped to restore blood flow to the organ and resume its normal function.

[0012] In other embodiments, the modification of the cells can be performed ex vivo. The cell population is
It may be taken from a donor or patient, genetically modified by inserting vector DNA and then transplanted into the patient or syngeneic or allogeneic recipient. For example, an organ may be harvested from a donor, subjected to the perfusion step ex vivo, and the organ reimplanted into the donor or transplanted into another recipient of the same or a different species. Preferably, the region encoding the above protein is placed under the control of a constitutive or inducible promoter. The advantage of utilizing an inducible promoter for transplantation purposes is that the desired high levels of transcription / expression for active genes / proteins can be delayed for an appropriate period of time prior to transplantation. For example, transcription can be triggered as desired in response to a predetermined stimulus, such as the presence of tetracycline in the intracellular milieu. Examples of tetracycline-inducible promoters suitable for use in the present invention are disclosed in Furte et al., PEAS US 91 (1994) 9302-9306. Alternatively, a promoter system in which transcription is initiated by the absence of tetracycline is described in Gossen and Bujard, PEAS URSA 90 (1992) 5547-51. The following terms are explained in this section: "Allogeneic" means allogeneic donors and recipients. "Syngeneic" means that the donor and recipient are genetically identical. "Self" means that the donor and recipient are the same individual. "Xeno" and "xenotransplant" mean the condition in which the donor and recipient of the implant are of different species. Methods for Preparing, Expressing and Administering Peptides The polypeptides of the invention or their derivatives can be administered as pharmaceutical compositions that can be formulated by various methods. For example, such formulations may be solutions or suspensions. However, as is well known, the peptides may also be formulated for therapeutic administration as tablets, pills, capsules, sustained release formulations or powders. The preparation of therapeutic compositions containing polypeptides as active ingredients is well known in the art. Typically, such compositions are in injectable form,
For example, it is prepared as a liquid preparation or suspension. Polypeptides to be used in accordance with the present invention are described, for example, by Bodansky, M., "Principles of Peptide Synthesis" (1993) Springer Verlag, Be.
It can be synthesized using standard techniques such as those described in rlin. Automated peptide synthesizers are commercially available (eg Advanced ChemTech Model 396; Mil
ligen / Biosearch 9600). The peptide was purified by high performance liquid chromatography,
It can be analyzed by mass spectrometry. The one or more modifying groups may be modified by standard methods, for example by modification of amino groups, amino groups, carboxyl groups, hydroxyl groups or other suitable reactive groups of the amino acid side chains of the peptide, or modification of either end of the peptide May be added to peptides (eg Greene, TW and Wuts, PGM "Protective Groups in Organic Synthesis" (1991) John
Wyley & Sons Inc., New York). Polypeptides may also be prepared according to standard recombinant DNA techniques using nucleic acid molecules encoding the peptides described above. The nucleotide sequence encoding the desired peptide can be determined according to the genetic code, and oligonucleotides having this sequence can be labeled with standard DNA synthesis methods (eg, automated DNA
Synthetic method) or using standard molecular biology techniques such as site-directed mutagenesis, polymerase chain reaction, and / or restriction enzyme digestion, It may be synthesized by deriving such DNA from a natural gene or cDNA. The production of recombinant adenovirus proteins is known in the art and also described in the literature described herein. The present invention includes the use of nucleic acids encoding the proteins and polypeptides of interest in the present invention. The nucleic acid of the invention may be inserted into a recombinant vector to facilitate peptide expression in host cells by recombinant DNA technology. Accordingly, the invention also provides a vector comprising the nucleic acid molecule of the invention. The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include circular double-stranded DNA plasmids and viral vectors. Certain vectors, such as those of bacterial origin and episomal mammalian vectors, are capable of autonomous replication in a host cell. Other vectors, such as non-episomal mammalian vectors, are those that can integrate into the host cell's genome upon introduction into the host cell and thereby replicate with the host cell genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked, and are referred to as expression vectors. The nucleotide sequence encoding the polypeptide to be used in the present invention may be operably linked to one or more regulatory sequences selected on the basis of the host cell to be used for expression. This means that the sequence encoding the peptide is linked to regulatory sequences so that the peptide can be expressed. Such regulatory sequences include promoters, enhancers, polyadenylation signals and other expression control elements, such as Goddel's "Gene Expression Techniques: Methods in Enzymology (Gene
Expression Technology: Methods in Enzymology) 185 "(1990) Academic Pre
ss, San Diego, California. Regulatory sequences direct constitutive expression in many types of host cells, or direct expression only in specific tissues or cells. Regulatory elements may direct expression in a regulatable manner, eg only in the presence of inducing agents. Suitable expression vectors for adenovirus polypeptides are known in the art and include those described in the references mentioned herein. Proteins and polypeptides of interest in the present invention may include amino acid sequences not derived from adenovirus, such as fusion proteins. The fusion protein can include a polypeptide of the invention fused to a peptide sequence that facilitates transport of the polypeptide across the cell membrane. The invention also provides derivatives of the proteins and polypeptides of the invention, such as the immunogenicity of the polypeptide or protein,
Derivatives for enhancing biological activity or pharmacokinetic properties are included. Furthermore, the polypeptide of the present invention may be modified by labeling or by coupling with another agent in order to facilitate detection or recovery of the polypeptide of the present invention.
Examples of such labeling include conjugation with enzymes or other detectable labels such as radioactive elements. Examples of modifications that affect pharmacokinetic properties include N- or C-terminal modifications that reduce the ability of the polypeptides of the invention to function as substrates for carboxypeptidases or aminopeptidases (eg, amide groups or D
Modification to contain amino acids), or myristoylation to improve accessibility to the interior of the cell. Examples of suitable parenteral administration include intravenous, subcutaneous and intramuscular routes.
Intravenous administration can be used to provide rapid control of peak plasma concentrations of the drug, which may be required to treat acute episodes of airway hyperreactivity, for example. Encapsulation of drugs in liposomes can help improve drug half-life and targeting to respiratory epithelia. By incorporating a ligand that binds to airway-specific macromolecules outside the liposome, it may be possible to improve the selectivity of liposome targeting to the airways. Alternatively, the drug-degrading microspheres (eg, poly (DL
-Intramuscular or subcutaneous depot injection, with or without (lactide-coglycolide), achieves sustained sustained drug release that may be necessary to suppress the development of airway hyperreactivity. You can. To improve the convenience of the dosage form, it is possible to use intraperitoneally implantable containers and septa, such as the Percuseal system (available from Pharmacia). Improved convenience and patient compliance are also provided by injector pens (eg Novo Pin or Q-pen) or needleless jet injectors (eg Bioject, Mediject or Becton).
Dickinson's). Other precise controlled releases such as zero order sustained release or pulsatile release can also be achieved as required with implantable pumps. Examples include subcutaneously implantable osmotic pumps available from ALZA, such as ALZET osmotic pumps. Nasal delivery may be achieved by incorporating the protein drug into a bioadhesive particle carrier (<200 μm) in combination with a suitable absorption enhancer. Bioadhesive particle carriers include cellulose, polyacrylates or polycarbophils, and absorption enhancers include phospholipids or acylcarnitines. Available systems include those developed by Dan Biosys and Scios Nova. Oral delivery may be achieved by formulating the drug in enteric coated capsules designed to release the drug in the intestine, where digestive protease activity is low. Examples include the OROS-CT / Osmet. TM and PULSINCAP. TM systems (ALZA and Sch
erer Drug Delivery Systems). Other systems utilize azo-crosslinkable polymers that are degraded by colon-specific bacterial azoreductase, or pH-sensitive polyacrylate polymers that are activated by elevated pH in the colon. The system may be used in combination with a wide range of available absorption enhancers. Targeting delivery of high doses of drug to hyperresponsive sites of the respiratory tract can be accomplished directly by pulmonary delivery. The lower respiratory tract epithelium is highly permeable to a wide range of proteins (eg granulocyte colony stimulating factor) with molecular sizes up to 20 kDa. It is also possible to dry atomize the protein in a suitable carrier such as mannitol, sucrose or lactose. Inhale, Dura
, A dry powder inhaler similar in principle to a propellant-loaded dose-adjusted inhaler designed by Fisons, Fisons (Spinhaler), Glaxo (Rotahaler) or Astra (Turbohaler) to deliver to the distal alveolar surface. May be. Solution formulations with or without liposomes can be delivered using an ultrasonic nebulizer. For further discussion on this subject, see the following references: McElvaney et al., J. Clin.
Invest., 90,1296-1301 (1992); and Vogelmeier et al., J. Appl. Physiol.,
69, 1843-1848 (1990). The amount of pharmaceutical composition to be used depends on the recipient and the condition to be treated. The required amount can be determined without undue experimentation by protocols known to those skilled in the art.
Alternatively, the required amount may be calculated based on the determination of the amount of tryptase that should be inhibited to treat the condition. Since it is recognized that the active substances contemplated by the present invention are non-toxic, the treatment preferably involves the administration of an amount of the active ingredient in excess of the optimal required amount. Virus strains and tissue culture Wild-type Ad5 (Ad5wt) can be found in the American Type Culture Collection (Rockville, M
(Aryland, USA) and dl739, E3 / 6.7K defective virus mutant (dl739)
(Brady et al., 1992), courtesy of WS M Wold. The above two adenoviruses of group C have a high degree of similarity, except that the expression of the E3 / 6.7K protein is lost in dl739 as previously reported (Brady et al., 1992). ). Both serotypes of virus were added to minimal essential medium (Gibco BRL Life Technologies Inc., Gaithersburg, Maryland, US) supplemented with 10% fetal calf serum (FCS).
A) were grown in monolayer cultures of A549 cells grown in. Two to five days after Ad5 inoculation, cells were frozen / thawed twice, sonicated for 30 seconds three times, and centrifuged at 500 xg for 5 minutes. The supernatant was collected and its virus titer was propagated on a 6-well plate
It was measured by plaque assay in A549 single cell layer. The titer was 10 ⁸ to 10 ⁹ plaque forming units (pfu) / ml. Control inoculum was prepared from uninfected A549 cells that were treated similarly to the infected cells above.

Tracheal Inoculation and Viral Plaque Assay Two groups of 24 mice were anesthetized with halothane. 60 μ for one group of mice
The other group of mice was infected with 10 ⁷ pfu of Ad5wt in the medium of 1 by the intranasal route,
10 ⁷ pfu of dl739 in 60 μl of medium was infected by the intranasal route. In addition, 6 mice were infected with sterile medium alone. Six animals from each of the two groups were sacrificed by overdosing halothane for 2 hours on days 1, 3 and 7 post infection. Two mock-infected mice were sacrificed 1, 3 and 7 days post infection. The left lung was removed and frozen in liquid nitrogen for use in the viral plaque assay. Right lung is 4%
Paraformaldehyde in PBS, pH 7.4 (0.149M NaCl, 0.012M Na ₂ HPO ₄ , 0.0
It was expanded with 04M KH ₂ PO ₄ ) and embedded in paraffin.

The virus titer virus plaque assay was used to quantify the number of replicating viruses in the mouse lung. Approximately 200 mg of lung was homogenized in 1 ml of sterile Polytron containing MEM on ice. The homogenate was centrifuged at 10,000 g for 2 minutes, the supernatant was taken out and stored at -70 ° C. Titers of lung homogenate supernatants were determined in 10 ^- well MEM / 10% in 6-well plates using 10 ^-1 to 10 ^-6 decimal dilutions in MEM prepared from mouse lung homogenate supernatants from all time groups. Measured by plaque assay in A549 cell monolayer cultures grown in FBS. . Inoculate each well with 500 μl of diluted supernatant and
Adsorption was performed on the single cell layer of A549 cells at 1 ° C. for 1 hour. Agarose overlay after adsorption (0.9% agarose, MEM, 2% FCS and 0.001 Neutral Red, 37 ° C)
I went. Plaques were counted after 10-14 days, normalized to lung volume and (log pfu
/ g lung tissue). Histological scoring 4 μm paraffin-embedded lung tissue sections were mounted on glass slides and stained with hematoxylin and eosin. A separate observer, unaware of the experimental treatment of tissue sections, scored airway mucosa, adventitia and adventitia for inflammation. The histopathological stage was 0 = no inflammation, 1 = mild inflammation, 2 = moderate inflammation, 3 = severe inflammation for each feature. The scores for each feature were summed to give a maximum of 9 total inflammation scores per animal. The average inflammation score is 3 for each animal.
Calculated by dividing by. Mean values and standard deviations were calculated for each experimental group. Statistical analysis Comparisons between the two viruses were made with respect to virus titer, inflammation score and time using bidirectional ANOVA. The significance level was p <0.05. The cDNA of the plasmid construct E3 / 6.7K was obtained by PCR amplifying the region encoding the E3 / 6.7K ORF from the vector carrying the Ad2 E3 region (provided by WSM Wold). The PCR product was Xh from the BPV cDNA expression vector pBCMGSneo (Karasuyama and Melchers, 1988).
It was cloned into the oI site and sequenced to confirm the correct sequence.
5'-ACCACCATGAGCAATTCAAGTAACTC (Forward primer; Fig. 1) 5'-CCTTATCTTGGATGTTGCCCCCAG (Reverse primer; Fig. 1) 24 hours after infection with the above primer and Ad2 and Ad5 to isolate E3 / 6.7K cDNA Template DNA purified from later HEK-293 cells was used. The reaction cocktail included template DNA, 0.5 μM forward and reverse primers, 250
μM of each nucleotide, 5 U of Pfu polymerase (Canadian Lif in 1 × Pfu buffer
e Technologies, 2270 Industrial St., Burlington, Ontario). The reaction conditions were as follows: melting of the double-stranded DNA (95 ° C, 30 seconds), followed by annealing (57 ° C, 30 seconds), followed by 30 seconds of warming with a gradient to 72 ° C, and further 30 seconds. Continuation of extension. In most cases, 30 cycles of the above PCR reaction will produce sufficient DNA for most applications. The newly generated E3 / 6.7K cDNA contained modifications not found in the native E3 nucleic acid (highlighted in bold in Figure 1 and the primer sequences above). Both modifications promote translation initiation at the E3 / 6.7K initiation site and increase protein production in transformed cells. E3 / 6
The natural form of .7K is very inefficient. The forward primer provides an E3 / 6.7K start site with an optimal upstream Kozak consensus sequence. The reverse primer is a natural TGA stop codon (Ochre stop codon (TAA))
Has been modified to replace. The latter modification eliminates the start site of E3 / 19K, which overlaps with the sequence of E3 / 6.7K in the native E3 nucleic acid and thus causes only slight translation of E3 / 6.7K from the native sequence. Become. Generation of a stable U937 cell line expressing E3 / 6.7K U937 human histiocytic lymphoma cells (Sundstrom and Nilsson, 1976) were obtained from the ATCC (CRL 1593) and contained 5% CO ₂ and 100%. Maintained in RPMI 1640, 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin and 100 μg / ml streptomycin under an atmosphere of% humidity. Cells were transfected with the appropriate construct by using DMRIE-C cationic lipid reagent (obtained from Life Technologies) according to the manufacturer's protocol. Transfected cells were maintained in medium containing geneticin G-418 sulfate to a final concentration of 800 μg / ml. Media and supplements were purchased from Life Technologies. Subclones of the transfected cell line were generated by serial dilution and examined by Northern blotting for E3 / 6.7K expression. Expression of E3 / 6.7K mRNA was very similar in all tested clones. All G-418 resistant cells that survived the selection process were pooled and used in in vitro assays to avoid mutations in the clone known to occur in U937 cells. Labeling of transfected cell-derived proteins, U937 cells containing the transfected or said vector a vector carrying the immunoprecipitation and Western blotting E3 / 6.7K, grown in suspension until the logarithmic growth phase It was 10 ⁸ cells were harvested, washed, and intracellular cysteine and methionine were added to a pre-warmed methionine / cysteine-free basal medium (without FCS)
Depleted by incubating at a concentration of × 10 ⁶ cells / ml for 1 hour at 37 ° C. A total of 2 × 10 ⁷ cells were 5 × in pre-warmed methionine / cysteine-free medium containing 0.5 mCi / ml [35S] -cysteine (Amersham) and 0.2 mCi / ml [35S] -methionine (Amersham). Labeling was performed at a concentration of 10 ⁶ cells / ml for 1 hour. After washing the cells, freshly prepared lysis buffer (1% TritonX-100, 1% BSA (bovine serum albumin), 1 mM iodoacetamide, 1 mM PMSF, 2.5 TIU / ml aprotinin, 0.01M Tris pH8.0, 0.14M It was thawed on ice in NaCl). Samples were counted by TCA sedimentation and protein A-Sepharose CL- for each sample at approximately 10 ⁷ cpm.
O / N was demonstrated with 4B and the supernatant was immunoprecipitated with a polyclonal rabbit antiserum raised against the C-terminal part of E3 / 6.7K and protein A-Sepharose. The pellet was denatured in SDS / sample buffer and the Tricine-SDS PAGE gel was a 16.5% T, 3% C preparative gel (Schagger with 10% T, 3% C spacer gel).
And von Jagow, 1987). Alternatively, cell lysates corresponding to 10 ⁵ cells were denatured in SDS-PAGE loading buffer and 10% glycine SDS-PAG was added.
E-gel system was loaded, separated, blotted on Immobilon-P PVDF membrane (Millipore) and probed with cPLA2 rabbit polyclonal antiserum (Cayman Chemical). The signal was detected by chemiluminescence using a horseradish peroxidase-conjugated goat anti-rabbit antiserum using the ECL kit (Biorad). Arachidonic acid release assay Cells were incubated with 10% Hyclone FCS, RPMI 1640, 2mM L-glutamine, 10m over several days.
After growing at low density in MHEPES, they were harvested and washed twice with PBS, 1% BSA. About 5
× 10 ⁶ cells (5 × 10 ⁵ cells / ml) were added to 0.4 μCi / ml [3H] arachidonic acid [5,6,8,9,
Labeling was performed in the same medium as above supplemented with 11,12,14,15-3H (N)] (0.1 mCi / ml stock; New England Nuclear) for 20 hours. Cells were washed twice with RPMI 1640, 0.2% BSA and incubated in wash medium for 1 hour to minimize spontaneous release of [3H] arachidonic acid. Cell numbers were standardized in all cell lines and 400 μl of cell suspension was aliquoted into each well of a 24-well plate containing 100 μl of treatment medium (2 × 10 ⁵ cells / well, Equivalent to 1.4 x 10 ³ counts / well). The assay was prepared to be performed in triplicate and cells were either media alone or media containing 20 ng / ml human rTNF-α (2000 U / ml) (Boehringer, Mannheim) or 10 μg / ml cycloheximide, or 20 ng / ml. Stimulation was done with either medium containing a combination of ml TNF-α and 10 μg / ml cycloheximide. Twenty hours after the treatment, the cells were centrifuged, and 100 μl of the supernatant of 500 μl in total was mixed with 3 ml of scintillation fluid and counted. For each cell line, 3 samples were lysed in lysis buffer and the lysates were used to determine the total counts of [3H] arachidonic acid incorporated. The number of counts of [3H] arachidonic acid released per minute was expressed as the average percentage (%) of total [3H] arachidonic acid incorporated. Annexin V-FACS Apoptosis Assay Annexin V-FITC (PharMingen) was used to measure the binding of Annexin V to the externalized phosphatidylserine. The protocol below is based on the manufacturer's Annexin V-FITC staining protocol. Cells are maintained for 10 days in 10% Hyclone FCS, RPMI 1640, 2 mM L-glutamine, 1
After low density growth in 0 mM HEPES, 5 × 10 ⁶ cells were harvested and washed twice with PBS. The cells resuspended in the above medium can be used alone or 100 ng / ml (10,000 U / ml
) Human rTNF-α or a medium containing 200 μg / ml cycloheximide or 10
Treated for 7 hours with medium containing a combination of 0 ng / ml TNF-α and 200 μg / ml cycloheximide. Cells were mixed with 1x binding buffer (10 mM Hepes /
Resuspended at 1 × 10 ⁶ cells / ml in NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ). 1 x 1
0 ⁵ cells (the suspension of 100 [mu] l) were mixed with 5μl Annexin V-FITC. One sample of cells was used to set baseline fluorescence without staining. Cells were examined on a Beckton Dickson FACS analyzer using a fluorescence activated cell sorter (FACS). Preparation of Ad Vector for Gene Therapy The backbone used for gene therapy is the SV5 backbone (Che
n 1997 PNAS). This backbone chain has been used successfully to transduce the dystrophin gene in vivo. The backbone chain lacks the E1 and E2 regions. The SV5 Ad vector has a replication defect because it does not contain the above-mentioned two regions, and therefore, it is safe for use and elicits a reduction in inflammatory response. The cDNA encoding E3 / 6.7K is under the control of the actin promoter and CMV enhancer, and by using it in addition to SV5, E3 / 6.7K can be integrated as an immunomodulatory protein and is called SV5-6.7. A new vector was created. Preparation of apoptosis-resistant producer cells Chinese hamster oocyte (CHO, a hybridoma resistant to apoptosis)
The production of cells or insect cells followed the same method as the transfection of U937 cells with the following exceptions. That is, at the end of selection in G-418, cells were selected, propagated by cloning and screened for expression of the protein of interest. One of ordinary skill in the art will readily appreciate that other suitable modifications and adaptations to the methods and uses described herein will be apparent and can be made without departing from the scope of the invention or any embodiments thereof. sell. In view of the described aspects of the invention, that aspect can be more clearly understood with reference to the examples described below. The above examples have been described herein merely for purposes of illustration and are not intended to limit the invention. Persistent viral titer increase and low inflammatory response caused by Example E3 / 6.7K
In the presence of reduced E3 / 6.7K, comparing mice infected with the E3 / 6.7K deficient virus (dl739) (Brady et al., 1992) with wild-type virus (Ad5wt),
Persistent viral titers increase during the course of infection. The titer of dl739 (deficient in E3 / 6.7K) was significantly higher than that of Ad5 wild type (Ad5wt) one day after inoculation (p <0.001). As time goes by, the virus is eliminated and dl
The titer of 739 was reduced (p <0.001). In contrast, the Ad5wt titer did not change significantly over the 7 day experimental period. The rapid decrease in dl739 over 7 days
It is thought to be due to a strong host response due to increased inflammation in the absence of 3 / 6.7K. Inflammation in the perivascular area and adventitia of the airways was stronger in animals infected with dl739 than in animals infected with Ad5wt over the 7 day experimental period (
p = 0.025). In addition, a significant increase in inflammation was observed from day 3 to day 7 for both types of virus (p = 0.029).

Reduced TNF-α mediated arachidonic acid release in the presence of E3 / 6.7K E3 / 6.7K may affect the cellular response to inflammatory cytokines. U937 cell line was transfected with E3 / 6.7K cDNA and E3 / 6.7K expression was immunoprecipitated using a polyclonal rabbit antiserum raised against a peptide derived from the C-terminus of E3 / 6.7K. Method and SDS-PAGE electrophoresis. E3 / 6.7
U937 cells (U937-E3 / 6.7K) transfected with K cDNA showed [3H] arachidonic acid release when stimulated with TNF-α, and U937 cells transfected with vector alone (
50% reduction compared to U937neor). TNF-α and cycloheximide (CHX; TNF)
-A3 / 6.7K still increased the release of [3H] arachidonic acid by 60% compared to U937neor, even though the stimulation was increased by the addition of (-a protein synthesis inhibitor that acts in concert with α). Had the ability to reduce. In the presence of E3 / 6.7K, [3
The induced release level of [H] arachidonic acid is reduced.

Reduction of TNF-α-induced apoptosis in the presence of E3 / 6.7K TNF-α-induced apoptosis was assayed by measuring phosphatidylserine externalization with FITC-labeled Annexin V ( Martin et al., 1995). Cells expressing E3 / 6.7K show U93 after stimulation with TNF-α.
The percentage (%) of apoptotic cells was reduced by 55% compared to 7neor. U937-E3 / 6
The .7K cells had a 65% reduction in apoptosis compared to U937neor after augmented stimulation with a combination of TNF-α and CHK. The presence of E3 / 6.7K reduced the apoptotic response in U937 cells upon TNF-α stimulation or combined TNF-α and CHK stimulation.

Cleavage of cPLA2 to the 78 kDa form after TNF-α induction in the presence of E3 / 6. 7K The expression of cPLA2 in U937 cells after induction with TNF-α was assayed. The cPLA2 antiserum recognizes two forms of the enzyme, one of which is larger than about 110 kDa and the second of which is 78 kDa. A significant difference was observed between U937neor cells and U937-E3 / 6.7K in terms of the ratio between the 110kDa and 78kDa forms of cPLA2.
TNF-α is U937neor cells (the major morphology migrates as a 110 kDa protein) and U937-E3 / 6.7K cells (the most major morphology of cPLA2 is 78 kDa) after induction with TNF-α.
It is not considered to change the above ratio in. Antisera were raised against the peptide corresponding to residues 443-462 of the cPLA2 sequence. Therefore, the only fragment detected by immunoblotting after cleavage is isolated from U937 cells.
A 78 kDa fragment corresponding to the 1-522 amino acid sequence of PLA2 (Sharp et al., 1991).

Having described the various aspects of the invention in detail, it will be apparent that changes and modifications to these aspects described herein are within the scope of the claims herein. All publications and patent specifications cited herein are incorporated by reference.

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66-76. Wilson-Rawls, J., Saha, SK, Krajcsi, P., Tollefson, AE, Gooding, L
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[Brief description of drawings]

FIG. 1 shows a natural nucleic acid sequence (SEQ ID NO: 1) capable of encoding an E3 / 6.7K protein corresponding to the wild type (Wt.) Of adenovirus serotype Ad2, and a forward primer (
FP; SEQ ID NO: 3) and reverse primer (RP; SEQ ID NO: 4) and alignment with polymerase chain reaction (PCR) nucleic acid product (SEQ ID NO: 2) expected when amplifying wild type sequence (Wt.) Is a nucleic acid sequence showing The start codon is underlined. Nucleic acids shown in bold in the forward primer (FP) indicate modifications to give the Kozak consensus sequence. The nucleic acids shown in bold in the reverse primer (RP) are stop codons that have been modified to facilitate translation.

FIG. 2 is an amino acid sequence showing an alignment of the amino acid sequences of E3 / 6.7K proteins of Ad2 adenovirus serotype (SEQ ID NO: 4) and Ad5 adenovirus serotype (SEQ ID NO: 5). The Ad 2 E3 / 6.7K amino acid sequence is 61 amino acids long and the Ad 5 E3 / 6.7K amino acid sequence is 63 amino acids long.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] January 8, 2001 (2001.1.8)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 2

[Correction method] Change

[Contents of correction]

FIG. 2 is an amino acid sequence showing an alignment of the amino acid sequences of E3 / 6.7K proteins of Ad2 adenovirus serotype (SEQ ID NO: 5) and Ad5 adenovirus serotype (SEQ ID NO: 6). The Ad 2 E3 / 6.7K amino acid sequence is 61 amino acids long and the Ad 5 E3 / 6.7K amino acid sequence is 63 amino acids long.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 29/00 4C086 A61P 25/28 37/00 4C087 29/00 43/00 105 37/00 C12N 7/00 43/00 105 C12Q 1/02 C12N 7/00 G01N 33/15 Z C12Q 1/02 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, T , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT , RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Moses, Alexandre, Earl. Canada Buoy 6 Tea 1 Z 3 British Columbia, Bankover, University of British Columbia, Health Sciences Mall 2222, Biomedical Research Center (72) Inventor Jeffries, Wilfred, A .. Canada Buoy 6Ti 1Z 3 British Columbia, Vancouver, University of British Columbia, Health Sciences Mole 2222, Biomedical Research Center (72) Inventor Vitalis, Timothy, Zet. Canada Buoy 6 Z 1 1 W 8 British Columbia, Vancouver, Butlerd Street 1081, Saint Pauls Hospital, University of British Columbia, Department of Pathology and Laboratory Medicine. Palmonaly Research Laboratory (72) Inventor Grant, Jason, Robert Canada Buoy 6B 5X 5 British Columbia, Bankover, Camby Street 950, Sweet 902 F Term (reference) 2G045 AA40 BB20 CB01 DA12 DA13 DA14 DA20 DA36 FB03 FB06 FB07 4B024 AA01 AA11 BA80 CA04 DA03 EA04 FA02 GA11 HA11 HA15 HA17 4B063 QA20 QQ08 QQ73 QX07 4B065 AA95X AA95Y AB01 BA02 CA24 CA44 CA46 4C084 AA02 AA06 AA13 BA03 DC50 NA14 ZA152 ZB072 ZB112 ZB212 4C086 AA01 AA03 AA04 EA16 MA01 MA04 NA14 ZA15 ZB07 ZB11 ZB21 4C087 AA01 AA03 BB63 BC83 CA04 CA12 NA14 ZA15 ZB07 ZB11 ZB21

Claims (17)

[Claims] 1. A method of inhibiting apoptosis of a cell, comprising treating the cell, a mammal containing the cell or a tissue containing the cell with an effective amount of an E3 / 6.7K polypeptide. Method. 2. The method of claim 1, wherein the treating step comprises administering to the cell a nucleic acid encoding the polypeptide, thereby expressing the polypeptide intracellularly. 3. The administration is administration of a viral vector containing the above nucleic acid, and when the viral vector is adenovirus, the nucleic acid is other than the natural adenovirus E3 nucleic acid, or The method of claim 2, with the proviso that it is under the transcriptional control of a promoter that is not derived. 4. The method according to claim 1, 2 or 3, wherein the cell is a culture of a eukaryotic cell. 5. The method of claim 1, 2 or 3 wherein the cells are of a mammalian individual suffering from a degenerative, immunodeficient, inflammatory or neurodegenerative disease. 6. A method of reducing apoptosis in a patient's tissue or cell population, comprising: (a) harvesting the tissue or cells from the patient, and (b) using the tissue or cells in an effective amount of E3 / 6.
.7 treatment with the polypeptide and (c) returning the treated tissue or cells to the patient. 7. The method of claim 6, wherein the treatment comprises administering a nucleic acid encoding the polypeptide, thereby expressing the nucleic acid in cells or tissues. 8. A pharmaceutical composition comprising an E3 / 6.7K polypeptide and a suitable carrier for facilitating delivery of the polypeptide to cells. 9. A nucleic acid comprising a non-natural adenovirus E3 nucleic acid capable of encoding an E3 / 6.7K polypeptide. 10. A recombinant virus comprising a nucleic acid encoding an E3 / 6.7K polypeptide, wherein when the virus is an adenovirus, the nucleic acid is other than the native adenovirus E3 nucleic acid, Alternatively, the recombinant virus described above, which is under the transcriptional control of a promoter not derived from adenovirus. 11. The recombinant virus according to claim 10, wherein the nucleic acid is operably linked to a promoter, the virus has a replication defect, and the polynucleotide is The above recombinant virus, which is expressed when the virus infects a eukaryotic cell. 12. Use of an E3 / 6.7K polypeptide, a nucleic acid encoding said polypeptide or a vector containing said nucleic acid for the treatment of apoptosis. 13. E3 for producing a medicament for treating apoptosis.
Use of a /6.7K polypeptide, a nucleic acid encoding said polypeptide or a vector containing said nucleic acid. 14. An assay for a drug that modulates the anti-apoptotic activity of an E3 / 6.7K polypeptide, which comprises mixing the polypeptide with a sample suspected of containing the drug and modulating the anti-apoptotic activity. The above assay, comprising determining whether or not it has been rated. 15. A mixture of E3 / 6 expressed in or administered to cells.
15. The assay of claim 14, which is performed in cells or extracts of cells that are rescued from apoptosis by a .7K polypeptide. 16. The assay according to claim 15, wherein the determination is performed by detecting or measuring TNF-α activity. 17. The assay of claim 16, wherein the activity is characterized by arachidonic acid release from cells.