JP2002512984A - Treatment of endotoxemia - Google Patents

Abstract

(57) SUMMARY The present invention provides therapeutic agents comprising the Lps ^d gene that down-regulate LPS-mediated signaling and thus can be administered to treat or prevent the development of endotoxemia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Cross-Reference to Related Applications This application claims the priority of US Provisional Application No. 60 / 083,210, filed Apr. 27, 1998.

[0002] The invention described in the referenced herein of government authorization, the National Institutes of Health (National Institute of Health)
Partially supported by Approval Nos. 1 ROI AI39159-OlAl and 1 ROI CA70854-01. The federal government has certain rights in the invention.

FIELD OF THE INVENTION The present invention relates to the use of the Lps ^d gene as a therapeutic for treating and preventing endotoxemia.

[0004] BACKGROUND A) LPS lipopolysaccharide endotoxin invention (LPS) is either certain bacteria (some of these are derived from typhoid, dysentery, and cause disease, such as urinary tract infections in the cell walls of) Or macromolecules derived from other bacteria that are commonly present in the intestinal tract of animals and humans and do not normally cause disease. All of these bacteria have a common allogeneic cell wall and are classified as Gram-negative based on their staining characteristics.

[0005] LPS induces the production and release of immunoreactive cytokines and other mediators in the inflammatory response of animals. Cytokines are highly active substances produced by various cells, such as mononuclear phagocytes and lymphocytes, which stimulate other cells and each other. Endothelial cells and granulocytes are also major targets for endotoxin. Intensive research is currently underway to gain a clearer understanding of how endotoxins act on specific receptor molecules and signaling pathways in host animal cells.

[0006] B) The cellular response to endotoxin is under genetic control, as shown by detailed studies in LPS-responsive genetic mice. The genetic basis underlying the host's multiple responses to LPS is based on the discovery of the C3H / HeJ mutant mouse strain in 1968 (Sultzer, Nature 219, 1253-125).
4 (1968)). The strain is poorly responsive to the immunostimulatory and pathophysiological effects of the lipid A component of LPS, and therefore has a fundamental defect in its response to LPS as compared to relative responder animal lines. Conceivable.

[0007] C3H / HeJ defects are special and appear in various forms. For example, C3H / HeJ B cells do not proliferate or differentiate upon exposure to LPS, and their proliferation of thymic lymphocytes induced by Concanavalin A is not enhanced by LPS (Sultzer et al., Nature
New Biology (London) 240,198-200 (1972); Sultzer, Abst. Am. Soc. Microbi
ol. P. 85, M69 (1973); Tanabe et al., Microbiol. Immunol. 23,1097-1104 (1979).
Morrison et al., Adv. Immunol. 28, 294-312 (1979); Sultzer in "Beneficial.
effects of Endotoxin ", Ch 11, Plenum, NY (1983); Sultzer et al., Immunobiolog.
y 187,257-271 (1993); Glode et al., LM and DL Rosenstreich, J. Immunol.
117,2061,2068 (1976); Sultzer, BM Infection and Immunity 13,1579-158
4 (1976)). Macrophage phagocytosis and cytotoxicity are not stimulated by LPS, but stimulated by LPS in responder animal cells Tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF), interleukin-1 Cytokines such as (IL-1) and prostaglandin are not induced in C3H / HeJ macrophages (Morrison et al., Adv. Immunol. 28, 294-312 (1979)). In addition, C3H / HeJ mice are more resistant to lethal endotoxin shock than normal endotoxin-responsive animal strains (Sultzer, Nature 219, 1253-1254 (1968)).

The failure of C3H / HeJ cells to respond to LPS is not due to the absence of helper cells or the presence of suppressor cells, or the inability of LPS to bind to another immune responsive cell due to such events (Sultzer in "Benef
icial effects of Endotoxin ", Ch 11, Plenum, NY (1983). Rather, the proposed explanation is in terms of defective trigger receptors, or signaling in the cells following the first interaction of LPS with the cells. Concentrated on some kind of malfunction (Sultzer
Et al., Immunobiology 187,257-271 (1993)).

[0009] From the results of classical type mating experiments with the C3H / HeJ strain and various responding animal strains, the mitogenic response to LPS is governed by a single locus containing a codominant allele (Morrison et al., Adv. Immunol. 28,294-31
2 (1979); Sultzer in "Beneficial effets of Endotoxin", Ch 11, Plenum, NY
(1983); Sultzer et al., Immunobiology 187, 257-271 (1993); Glode et al., J. Immun.
ol 117,2061-2068 (1976); Sultzer, Infection and Immunity 13,1579-1584 (1
976)). Watson regulates Mup-1 and B cell activation, although the locus (Lps ⁿ ) is on chromosome 4 and linked to the major urine protein locus (Mup-1) Lyb 2; 2; 4; 6 genes were found to be downstream (Watson et al., J.
Immunol. 120,422-429 (1979)). The C3H / HeJ mouse strain contains a mutant Lps ^d allele.

C) Isolation and characterization of the Lps ⁿ gene Sultzer et al., Immunobiology 187,257-271 (1993) describe a cDNA library consisting of six sublibraries derived from spleen cells of C3H / OuJ (responsive) mice. The production of is described. When one sublibrary of 2 x 10 ⁴ independent clones was introduced into spleen cells of C3H / HeJ LPS non-responsive mice, plaque-forming cells reactive with sheep erythrocytes after exposure to LPS in culture. It increased about 4-fold.

[0011] Kang et al., Infect. Immun. 64,4612-4617 (1996) describe further functional screening and fractionation of C3H / OuJ cDNA libraries and lipopolysaccharide-mediated B cell responses in C3H / HeJ spleen B cells. The isolation of a cDNA clone capable of recovery is described. The cDNA of 1166 nucleotides (SEQ ID NO: 1) contains a 648 nucleotide open reading frame encoding a protein of 216 amino acids (SEQ ID NO: 2).

[0012] Sequence analysis, Lps ⁿ genes This is a LPS responsive genes have been shown to encode the Ran / TC4 is important GTP ase for nuclear localization (Raetz, CR H
., Biochemistry of Endotoxins. Ann. Rev. Biochem. 59,129-170 (1990); Wat.
son et al., J. Immunol. 114,1462-1468 (1975); Kabir et al., Infect. Immun. 15,156-
164 (1977)). At the amino acid level, Lps ⁿ is the Ran / TC4 G found in humans and dogs
It is identical to TPase and has 98.6% and 82.8% homology to Ju93 protein in chicken and Spil protein in yeast, respectively (Drivas et al., Mol Cell Biol 1
0,1793-1798 (1990); Dupree et al., Gene 120,325-326 (1992); Trueb et al., FEBS 30
6,181-184 (1992); Matsumoto et al., Cell 66,347-360 (1991)). References (Kang et al.,
The entire disclosures of Drivas et al., Dupree et al., Trueb et al., And Matsumoto et al.) Are hereby incorporated by reference. The DNA level, Lps ⁿ has human TC4 / Ran, dog TC4 / Ran, chicken Ju93 and yeast Spil respectively 89.7% 90.7% homology 85.4% and 68.7% (supra). Therefore, Lps ⁿ molecules, in particular, five domains G1~G5 of the molecule that has been shown to be involved in binding and hydrolysis of guanine nucleotides are highly conserved throughout evolution.

[0013] In summary, Lps ⁿ genes is derived from LPS-responsive mice, such as C3H / HeOuJ, it encodes a functional gene product present in the activated antibody producing B intracellular polyclonal. And the introduction of Lps ⁿ cDNA into C3H / HeJ B cells results in a sufficient restoration of their responsiveness to LPS stimulation and an increase in antibody producing plaque forming cells (PFC) to the level of responding animals. This gene is found not only in B cells, but also in other cell types that respond to LPS, and in cells that produce cytokines and other factors that can be toxic to the host in large amounts. It is believed that it will be.

D) Endotoxemia LPS has a number of pathophysiological effects, one of which is endotoxemia or septic shock caused by large amounts of endotoxin in the blood. It has about 40% mortality. The majority of cases of septic shock are due to Gram-negative bacteria in the blood. However, septic shock syndrome can also be caused by other organisms, including gram-positive bacteria and fungi. Nevertheless, a detailed study of the causes of septic shock has shown that
A major factor in progression has been demonstrated to be the release of LPS from Gram-negative bacteria, followed by a high degree of cell activation by the effects of endotoxin on various cells in the body. As a result, the host is unable to withstand much cellular material, which leads to circulatory failure, shock and death.

[0015] Sepsis is often the cause of death in intensive care units, leaving significant public health problems. When sepsis occurs, key proinflammatory cytokines are involved, including TNF-α, IL-1 and IL-6. The results of clinical trials to suppress TNF using either anti-TNF antibodies or fusion proteins consisting of the extracellular domain of TNF and the Fc portion of IgG were disappointing (Dellinger et al., Chest 111,744-753). (1997)
Grau et al., Nature Medicine 3,1193-1195 (1997)).

[0016] Although potent antibiotics and drugs are available for treating cardiopulmonary failure, improved therapeutic and / or prophylactic methods for inhibiting endotoxemia, particularly in the early stages of sepsis, are available. There is still a great need for treatment.

[0017] SUMMARY OF THE INVENTION The present invention consists of administering Lps ^d gene effective amount to a patient in need of treatment of endotoxemia, provides a method for treating endotoxemia in said patient. The Lps ^d gene differs from the Lps ⁿ gene in that it contains a mutation, which results in a less responsive lipopolysaccharide endotoxin response in cells containing the mutation. In one embodiment of the invention, the Lps ^d gene differs from the corresponding Lps ⁿ gene of the same species by a mutation in the 3 'untranslated region of the Lps ⁿ cDNA. In a preferred embodiment,
The mutation involves a nucleotide substitution at position 870 of the Lps cDNA, provided that the numbering of the positions begins with the translation initiation codon of the DNA. In a preferred embodiment, the Lps ^d gene is a human gene. In another preferred embodiment, the Lp
The ^sd gene is a mouse gene. Since mouse and human Lps encode the same protein, mouse genes can be used for therapy. In a more preferred embodiment, the Lps ^d gene is administered in the form of a viral vector. In a most preferred embodiment, the viral vector is a retroviral vector. In another most preferred embodiment, the viral vector is an adenovirus vector.

The present invention also provides a method for preventing endotoxemia in a patient in need of prevention of endotoxemia, comprising administering an effective amount of the Lps ^d gene to the patient. In a preferred embodiment, the Lps ^d gene is administered in the form of a viral vector. In a most preferred embodiment, the viral vector is a retroviral vector. In another most preferred embodiment, the viral vector is an adenovirus vector.

The present invention further relates to the use of the Lps ^d gene for the manufacture of a medicament for treating or preventing endotoxemia in a patient.

Other aspects and advantages of the present invention are described in the drawings and the following detailed description of the preferred embodiments.

DETAILED DESCRIPTION A) Definitions and General Methodology The following definitions will assist in understanding the scope and practice of the present invention.

“LPS” means lipopolysaccharide endotoxin.

“Lps ⁿ ” and “Lps ^d ” are used to describe allelic types of normal and low responsive lipopolysaccharide endotoxin responsive genes (for LPS responsiveness), respectively, and are related to the source animal species It does not do. Lps ^d includes any gene that encodes an LPS protein, which gene contains a mutation (cells containing this type of mutation produce a poorly responsive LPS response). Lps ⁿ gene is Ran / Tc4 or TC4 / Ra
Also known as n. The term "Lps gene" is intended to include both Lps ⁿ and Lps ^d forms, as well as other natural or laboratory-generated forms of the LPS endotoxin response gene.

“Lps ⁿ protein” and “Lps ^d protein” are used to describe the polypeptide encoded by the Lps ⁿ and Lps ^d genes, respectively. The term "Lps protein" is intended to include both the Lps ⁿ and Lps ^d proteins, as well as other natural or laboratory made forms of the LPS endotoxin responsive gene product.

According to one embodiment of the invention, the Lps ^d gene differs from the corresponding Lps ⁿ gene of the same organism by a mutation in the region encoding the mRNA 3 ′ untranslated region (3 ′ UTR).
This mutation causes a difference in the mRNA secondary structure of the mutated region. In a more preferred embodiment, the mutation is a single point mutation, e.g., C3H / HeJ
It is a mutant TC at position 870 of the mouse Lps cDNA (where the numbering of the positions begins with the translation initiation codon (ATG)).

In general, the nomenclature used below, as well as the laboratory techniques for cell culture, molecular genetics, nucleic acid chemistry and hybridization described below are those that are known and widely used in the art. is there. Recombinant nucleic acid method, polynucleotide synthesis,
Standard techniques are used for cell culture and transgene introduction. Typically,
Enzymatic reactions, oligonucleotide synthesis and purification steps are performed according to the manufacturer's instructions. These techniques and procedures are generally known in the art and are well known to those of skill in the art and various general literature such as Maniatis (Molecular Cloning, Cold Spring H
arbor Laboratories, 1982) and Ausubel (Current Protocols in Molecular B)
iology, Wiley and Sons, 1987), which is incorporated herein by reference.

[0027] B) vectors and gene delivery methods human and mouse Lps ⁿ proteins have the same amino acid sequence, Lps ⁿ (TC4 / Ran mouse and human DNA level) gene showing homology 89.7%. Thus, mutant human Lps ^d gene (cDNA) can be created using site-directed mutagenesis techniques or by combining the human Lps ⁿ gene with a restriction fragment containing a mouse Lps ^d mutation. it can. In one embodiment, the mutation is C3H / HeJ
Lps ⁿ characteristic of mouse It is a TC mutation at position 870 of cDNA. The human Lps ^d gene is L
It can be used for gene therapy to down-regulate the response to PS and therefore treat or prevent the onset of endotoxemia in patients.
Alternatively, since the mouse and human Lps proteins are identical, the mouse Lps ^d gene can be used as a therapeutic sequence for human gene therapy.

[0028] Mutations of Lps ⁿ genes results in hyporesponsiveness of the LPS response can be derived from the known methods of mutagenesis, and screened for LPS responsiveness of the resulting mutant. Lps ⁿ Mutate the cDNA and use a suitable vector, for example, 5 '
And 3 ′ MMLV LTR, splice donor and splice acceptor sites,
N2 retroviral vector containing a neomycin resistance marker gene (Wong et al., Mol
Cell. Biol. 9, 798-808 (1989); Wong et al., Gines Dev. 1, 358-365 (1987)). Using the obtained pN2-Lps ^d plasmid DNA, virus packaging cells are transfected, and a virus supernatant is obtained from the cells. The virus particles are sedimented by known methods. The N2-Lps ^d vector is used to infect macrophages, for example, the Ana I macrophage cell line from normal inbred mice. Transduced cells are assayed for TNFα production after LPS stimulation.

Down-regulation of LPS-mediated signals in transduced cells is measured by a decrease in TNFα production relative to non-transduced macrophage control cells. Mutant cDNAs that cause down-regulation of LPS responsiveness when introduced into macrophages are selected. Representative techniques for retroviral delivery of Lps ^d mutant cDNA to target effector cells are described in the Examples below.

[0030] Preferably, the mutation in the Lps ⁿ gene that results in LPS hyporesponsiveness
It significantly reduces PS responsiveness but does not completely eliminate it.

Preferably, the Lps ^d gene is delivered to the patient in the form of a viral vector. Virus vectors that can be used in the present invention include adenovirus vectors and retrovirus vectors.

[0032] Adenoviruses are eukaryotic DNA viruses that can be modified to deliver nucleic acids efficiently to a variety of cell types. Various serotypes of adenovirus exist, including type 2 and 5 human adenoviruses and adenoviruses of animal origin. Preferably, the replication-defective adenovirus vector of the present invention comprises an ITR,
encapsidation) sequences and nucleic acids of interest. More preferably, at least the E1 region of the adenovirus vector is non-functional. E3 region (WO95 / 0
2697), the E2 region (see WO94 / 28938), the E4 region (see WO94 / 28152, WO94 / 12649 and WO95 / 02697) or other regions, including any of the late genes L1-L5, have also been modified. Good.

[0033] The replication defective recombinant adenovirus of the present invention can be produced by techniques known to those skilled in the art. In particular, it can be produced by homologous recombination of an adenovirus with a plasmid carrying the DNA sequence of interest. Homologous recombination is achieved by co-transfection of the adenovirus and the plasmid into a suitable cell line. The cell line used must be one which can be transformed by the above components and which contains sequences which can complement the defective region of the replication defective adenovirus. An example of a cell line that can be used is the human embryonic cell line 293 (Graham et al., J. Gen. Virol. 36, 59 (1977)) in which the left-hand portion (12%) of the Ad5 adenovirus genome is integrated into the genome. And cell lines capable of complementing E1 and E4 functions as described in WO94 / 26914 and WO95 / 02697. Recombinant adenovirus can be recovered and purified using standard biological techniques well known to those skilled in the art.

[0034] Adenovirus vectors are generally preferred for the treatment of endotoxemia, and generally about 24 hours.
The treatment should be effective within hours. Adenovirus vectors can be produced at high titers (eg, 10 ¹⁰ -10 ¹² infectious units per ml) and transiently express the LPS non-responsive phenotype in a non-tissue specific manner Can be used for

A retrovirus is an integrating virus that infects dividing cells in general. The retroviral genome contains two LTRs, an enveloped sequence, and three coding regions (gag, pol and env). Construction of recombinant retroviral vectors is known to those skilled in the art.

In a recombinant retroviral vector, the gag, pol and env genes are generally completely or partially deleted and replaced with a heterologous nucleic acid sequence of interest. These vectors are M-MuLV, MSV (Moloney murine sarcoma virus), HaSV (
It can be constructed from different types of retroviruses, such as Harvey's sarcoma virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus), and Friend virus.

Generally, to construct a recombinant retrovirus containing a sequence of the present invention, L
A plasmid containing the TR, the encapsulating sequence and the coding sequence is first constructed. This construct is used to transfect a packaging cell line. This cell line is able to provide in trans the retroviral function lacking the plasmid. As a result, generally the packaging cell line is capable of expressing the gag, pol and env genes. Such packaging cell lines have been described in the prior art. In particular, cell line PA317 (U.S. Pat. No. 4,861,719), PsiCRIP cell line (WO90 /
02806), and the GP + env Am-12 cell line (WO89 / 07150). Recombinant retroviral vectors can be purified by standard techniques known to those skilled in the art.

For delivery to cells that do not divide, retroviral vectors derived from lentiviruses such as HIV-1, HIV-2 and SIV can be used. These viruses are
-Pseudotyping can be performed using the surface glycoproteins of MuLV or other viruses such as vesicular stomatitis virus (VSV). Production of high titers of HIV-1 pseudotyped with VSV glycoproteins was performed by Bartz and Vodicka (Methods 12 (4): 337-42 (August 1997)) and production of multi-attenuated lentiviral vectors Zufferey et al. (
Nature Biotechnology 15: 871-75 (September 1997)). Such lentiviral vectors can infect nondividing cells, have a broad host range, and can be concentrated to high titers by ultracentrifugation.

[0039] Chimeric adenovirus / retroviral vector systems can also be used to achieve efficient gene delivery and long term gene expression. Feng et al. (Nature Biotechnology 15: 866-70 (Nature Biotechnology 15: 866-70)) used an adenovirus vector to create transient retroviral producer cells in vivo so that progeny retroviral particles could infect neighboring cells. September 1997)).

For the prevention of endotoxemia in patients at high risk of developing the disease, retroviral vectors are generally preferred. For example, burn patients develop endotoxemia at an almost 100% rate. Since retroviral vectors can be integrated into hematopoietic stem cells, they can provide a repository for LPS-resistant T cells, B cells and macrophages.

The recombinant virus is administered to the patient in an amount sufficient to treat or prevent the onset of endotoxemia. The effective amount may vary depending on the characteristics of the patient, the type and severity of the condition to be treated, the desired duration of treatment, the mode of administration, and other factors. An effective amount can be determined by a physician or other qualified healthcare professional. The recombinant virus of the present invention is generally formulated and administered in a dosage form of about 10 ⁴ to 10 ¹⁴ pfu.

EXAMPLES The practice of the present invention will be described with reference to the following examples. These examples are merely illustrative and do not limit the scope of the invention.

Example 1 Isolation and Characterization of C3H / HeJ Mouse Lps ^d Gene A study by Kang et al. (Infect. Immun. 64, 4612-4617 (1996)) showed that the Lps ⁿ gene had C3H
/ HeJ spleen B cells could restore lipopolysaccharide-mediated B cell responses.

The Lps gene was isolated from the pCD-HeJ cDNA library to show that the Lps gene is defective in the genome of the C3H / HeJ mouse and that this defect explains the low responsiveness to LPS stimulation .

Using RNA from C3H / HeJ spleen B cells, an Okayama-Berg cDNA expression library (O
Kayama, H. and Berg, P., Mol. Cell Biol. 2, 161-170 (1982))
. Apply the pCD-HeJ cDNA library onto eight dishes (150 mm dish).
5.8x10 per piece^FourClones), Lps obtained from cells of C3H / HeOuJ LPS-responsive mice ⁿ This library was probed with a 0.9 kb BamHI-PstI fragment from the cDNA.
(Infect. Immun. 64, 4612-4617 (1996)). 25 positive clones identified
.

As compared with the size of the C3H / HeOuJ (wild-type) Lps ⁿ cDNA, out of the 25 independent clones analyzed, 8 were full-length cDNAs. Next, these independent full-length cDNs
Four of the A clones (Lps ^d- 4, -10, -15 and -17) were sequenced. The sequences of these 4 independent clones were compared to wild-type Lps ⁿ cDNA.

All four clones showed the same point mutation at position 870 of the Lps ⁿ cDNA (residue numbers were counted from ATG). As shown in FIGS. 1A-1D, the T residue was replaced by a C residue. Interestingly, this single point mutation is in the 3 'untranslated region.
According to Northern (RNA) blot analysis, Lps m between C3H / HeJ and C3H / HeOuJ cells
There are no significant differences at the RNA level.

These data indicate that the point mutation present in the mutated HeJ Lps ^d gene
It shows that H / HeJ cells explain the low responsiveness to LPS.

The secondary structure of Lps ⁿ and Lps ^d mRNA was compared using the SQUIGGLES program (Wisconsin) of the GCG sequence analysis package. FIGS.7A (Lps ⁿ ) and 7B (Lps ^d ) show the surprising difference in RNA secondary structure in the region adjacent to the point mutation, and no significant structural differences in the region far from the point mutation. Indicates no difference. A single base substitution present in the 3'UTR of Lps ^d mRNA binds regulatory proteins and imposes post-transcriptional regulation, resulting in translation efficiency, mRNA stability, transport or intracellular localization Suggests the existence of a unique mRNA domain that influences the expression. To further characterize the wild-type and mutant Lps gene products and their role in response to LPS, Lps ⁿ and Lps ^d retroviral vectors were constructed.

Example 2 Construction of Lps ⁿ and Lps ^d Retrovirus Vectors The Lps ⁿ and Lps ^d cDNAs of Example 1 were converted to N2 retrovirus vectors (Wong et al.,
Cell. Biol. 9, 798-808 (1989); Wong et al., Genes Dev. 1, 358-365 (1987)).
At the XhoI site. N2 retroviral vectors are 5 'and 3' MMLV
Includes LTR, splice donor and splice acceptor sites, and neomycin resistance marker gene.

From the XhoI restriction fragment derived from pCD-LPS (Infect. Immun. 64, 4612-4617 (1996)), Lp
The s ⁿ cDNA was isolated and ligated into the XhoI restriction fragment site of the N2 retroviral vector, and pN2-
Lps ⁿ was prepared. Using the same procedure but using the mutated Lps ^d cDNA, p
The N2-Lps ^d vector was created.

To generate retroviral stocks carrying Lps ⁿ and Lps ^d sequences, GP / E virus packaging cells (Markowitz et al., Supra ⁾ by calcium phosphate co-precipitation using pN2-Lps ⁿ or pN2-Lps ^d plasmid DNA. J. Virol. 62, 1120-1124 (
1988)) were transfected separately. Positive transfected cells were selected in media containing 1 mg / ml G418. Individual G418 resistant clones were isolated, expanded and characterized.

To make a concentrated virus supernatant, the virus was collected from the producer cells and used in 600 ml of virus supernatant in 0.4 M NaCl and 8.5% PEG8000 (Sigma) for 1-1.5 hours at 4 ° C. The virus sedimentation was performed with continuous stirring. 7500
The precipitate was collected by centrifugation at rpm for 10 minutes. This pellet is the original volume of 1
% NTE buffer (filter-sterilized 100 mM NaCl, 10 mM Tris-Cl pH 7.4, 1
mM EDTA). Next, this solution was passed through a Sepharose CL-4B column at 4 ° C. Several fractions were collected and pooled. Each pooled fraction is filter sterilized,
As described in the literature (Wong et al., Mol. Cell. Biol. 9, 798-808 (1989)).
Virus titers were tested by G418 resistance using NIH / 3T3 cells.

[0054] N2 parent vector, shown in 3C N2-Lps ⁿ and N2-Lps ^d vector construction from each view 3A.

Example 3 Retrovirus of Lps ⁿ and Lps ^d genes to spleen B cells of C3H / HeJ mouse
Introducing A) red blood cells were removed from the transgenic C3H / HeJ mice spleen B cells by retroviruses, subjected to retroviral infection in presence of 1-100 [mu] g / ml LPS, and then incubated with S1 stromal cell conditioned medium .
This medium is known to stimulate pro-B cell proliferation (Collins et al., J. Immunol.
l. 138, 1082-1087 (1987); Dorshkind et al., J. Immunol. Methods 123, 93-101 (
1989); Narendran et al., Eur. J. Immunol. 22, 1001-1006 (1992)). To maximize the efficiency of retroviral infection, a fresh stock of concentrated virus supernatant was repeatedly fed into the culture and recultured, as shown in the flow chart of FIG. N2 parental vector, N2-Lps ⁿ or N2-Lps viral titer of concentrated stock of ^d vectors were normalized to 2x10 ⁶ G418 resistant colonies per ml.

B) MTT Proliferation Assay At various time points after LPS stimulation and initiation of retroviral infection, cells were harvested and MTT [3
-[4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide
Proliferation assay (Han et al., Proc. Natl. Acad. Sci. 92, 11014-11018 (1995)).

Ten million red blood cell-depleted C3H / HeJ spleen cells were incubated for 24 hours in 10 ml of S17CM (0, 12, 24, 48 hours alignment in S17 cells, 10% fetal bovine serum and 100 mm containing 50 μM 2-RPM containing mercaptoethanol
For 6 hours. After incubation, non-adherent cells were collected and re-infected with 100 ml of the infection mixture in 100 mm plates. The infection mixture contains 3 ml of concentrated virus supernatant (titer 2 × 10 ⁶ virus / ml), 1 ml of S17CM, 10 ml
μg / ml of polybrene, containing 10-100 μg / ml of LPS extracted from hot phenol from Salmonella typhimurium for 16 hours. After 16 hours, the cells were collected and reinfected with a fresh infection mixture for another 16 hours. After infection, 2 ml of S17CM
The harvested cells were stimulated with 10 μg / ml or 100 μg / ml LPS in and incubated in 24-well plates for 0, 12, 24, 48 hours. Next, 100 μl of cells infected with the retrovirus and stimulated with LPS were placed in a 96-well plate. 1
0 μl of 5 mg / ml MTT was added, mixed with the cells, and the plate was placed in an incubator. After 4 hours incubation, 100 μl in isopropanol
Of 0.04N HCl was added to the wells and mixed well. The OD at 570 nm was read using a microplate reader.

At the 32 hour time point (= 0 hour incubation shown in FIG. 2), no significant differences in cell growth were found in any of the cultures (FIG. 4A). 44 hours (= Fig.2
12-hour incubation), cells infected with N2-Lps ⁿ showed a pronounced increase in cell proliferation, but not in cells infected with N2-Lps ^d or N2 (Figure 4B). Up to 4 times the LPS response between N2-Lps ⁿ infected cultures and N2 or N2-Lps ^d infected cultures at 68 h (= 48 h incubation shown in FIG. 2) after the start of the infection culture. Differences were seen. And at 92 hours, the response began to decrease.

The LPS response of the N2-Lps ⁿ infected culture of C3H / HeJ spleen B cells, observed 44 hours after the start of the infection culture, is the LPS response of C3H / HeOuJ responsive spleen B cells 24 hours after LPS stimulation. Equivalent to response (data not shown). This delay (1) even after standing for 48 hours or more, about 50% of the cells only N2-Lps ⁿ vectors that were not transduced by, and (2) the incorporation and transduced expression of genes of the virus Lag time is required. Cell proliferation was clearly pronounced in cultures infected with N2-Lps ⁿ than cultures infected with N2 or N2-Lps ^d. This is because the number of proliferating small lymphocytes and cells undergoing blastogenesis was much higher in the former culture than in the latter culture (data not shown). In addition, differentiation of polyclonal B cells also occurred. This is because, C3H / HeJ transduced with N2-Lps ⁿ vector rather than N2 or N2-Lps ^d vector
Spleen B cells can be stimulated with LPS to produce antibodies similar to those previously described in hemolytic sheep red blood cells (Kang et al., Infect. Immun. 64, 4612-4617 (1996)). Because it was done.

C) RT-PCR Assay A response or lack of response to LPS is indicated by the corresponding wild-type or mutant
To further demonstrate successful retroviral transfer of DNA, RT-PCR assays were performed on cells from all cultures. Using primers 'specific three oligo primers and Lps ⁿ or Lps ^d cDNA' specific 5 to the sequence of the SV40 promoter present in the retroviral vector.

Total RNA was extracted from spleen B cells infected with retrovirus using 4M GTC and phenol / chloroform. CDNA was prepared using AMV-RT. For the PCR reaction,
Two primers, a 5 'primer from the pCD portion of the XhoI fragment (TCTAAAAG
CTGCTGCAGG: SEQ ID NO: 3) and 3 ′ primer derived from Lps cDNA (GTACACGATCTGC
A 907 base pair fragment was generated using TTAGC: SEQ ID NO: 4). FIG. 5 shows the results.

[0062] The predicted 907 bp band was seen with N2-Lps ⁿ and N2-Lps ^d virus cultures transduced, not observed in cultures transduced with N2 virus, on the gene Successful transduction and expression of the transduced gene was confirmed. This primer
Specific to Lps RNA. Because when genomic DNA is used for PCR reaction,
This is because the 970 bp band shown in FIG. 5 was not observed. This is because the 5 'primer was specific for the vector sequence located upstream of the Lps cDNA. Therefore, the introduction and expression of Lps ^d cDNA rather Lps ⁿ cDNA in spleen B cells C3H / HeJ mice, resulted in recovery of significant response to LPS stimulation. Classical breeding experiments with the C3H / HeJ line and other responder cell lines suggest that the mitogenic response of LPS is governed by a single locus consisting of codominant alleles. From the results shown here, it will be apparent that Ran is an Lps responsive gene with a defect in the genome of C3H / HeJ LPS hyporesponsive mice.

Example 4 Retroviral introduction of Lps ^d gene into macrophage cells of normal inbred mice Ana I is a macrophage cell line derived from normal inbred mice, and GG2EE cells are C3
Macrophage cells derived from H / HeJ low responder mice. Ana I cells were prepared using N2
Infected with -Lsp ^d retroviral vectors, after transduced cells LPS stimulation and assayed for TNFα production at various time points.

For retroviral transduction, 1 × 10 ⁶ suspended cells were incubated with 5 ml of retroviral supernatant in the presence of 10 μg / ml polybrene.
The cells were incubated at 37 ° C. under 5% CO ₂ for 4 hours with frequent shaking. The cells are then centrifuged at 1500 rpm for 10 minutes, and D ₁₀ G (10% heat-inactivated FBS
D supplemented with 2 mM L-glutamine, 10 μg / ml gentamicin and 1 mg / ml G418
Washed with MEM), and resuspended in 5 ml of D ₁₀ G. 1.6 ml of methylcellulose was mixed with 0.4 ml of the retroviral transduced cells and 1 mg / ml G418 to prepare a methylcellulose mixture. This mixture was plated on 35 mm plates (about 1.1 ml per plate). Colonies were picked, expanded and pooled after two weeks.

For LPS stimulation, cells (Ana I, GG2EE and retrovirus-transduced Ana I cells) were seeded in 24-well plates (5 × 10 ⁶ cells / well) at 37 ° C.
It was incubated under 5% CO _2. Next, cells were diluted with 0,2 using 100 ng / ml LPS.
Stimulated for 4, 48 or 72 hours. Supernatants were collected and the amount of TNFα was measured using an ELISA assay.

For the TNFα assay, 50 μl of TNFα capture antibody (2 μg / ml) was added to each well of the microplate and the plate was incubated at 4 ° C. overnight. Next, the plates were washed four times with PBST, blocking buffer (200 μl) was added to each well, and the plates were incubated at room temperature for 30 minutes. Subsequently, the plates were washed four times with PBST and samples (100 μl / well) were added into each well. Plates were incubated overnight at 4 ° C., followed by four washes with PBST. For detection of TNFα, biotinylated TNFα antibody (1 μg / ml, 100 μl / well) was added to the wells, the plate was incubated for 1 hour at room temperature and washed with PBST. Streptavidin-HRP conjugate enzyme (1: 1000 dilution, 100 μl / well) was added and the plate was incubated at room temperature for 30 minutes, followed by 8 washes with PBST. ABTS substrate (100 μl / well) was added and the plate was incubated at room temperature for color development. Color was measured by reading at OD 405 nm. FIG. 6 shows the results.

# 1, # 7, and # 9 were independent clones, while P30 was cells derived from a mixture of 30 independent clones. This experiment demonstrates that L
Expression of ps ^d gene, showing that down-regulates LPS-mediated signaling in these cells. Thus, the Lps ^d gene can be used as therapeutic DNA to treat or prevent the occurrence of endotoxemia in a patient.

Example 5 Construction of an Adenovirus Vector for In Vivo Adenovirus Lps Transfer and Virus Production The Lps ^d , Lps ⁿ and GFP genes were cloned into the Ad5 adenovirus vector as described below, and the The E1 region was replaced. The construction of this vector is shown schematically in FIG.

The Lps ⁿ and Lps ^d cDNAs were derived from pCD-Lps ⁿ and pCD-Lps ^d (the latter containing a TC mutation at position 870 of the cDNA and supplied by StemCell Therapeutics).
At 9 and 35, they were excised using BamHI. They were separately inserted into the E1 region of the Ad5 genome and driven by the CMV promoter. pEGFP plasmid (Clontech, L
a Jolla) -derived GFP (green fluorescent protein) gene was excised with BamHI and NotI, inserted into the same site, and similarly driven by the CMV promoter. Recombinant adenovirus lacks E1 and thus is not replication-competent. However, they can replicate in 293 cells containing the E1 gene. For production of recombinant virus, 293 cells were transfected with recombinant Ad5 DNA. Ten days after transfection, cells were scraped and collected from the culture flask. After three cycles of freezing in an ethanol / dry ice bath and rapid thawing at 37 ° C., 1 ml of the virus lysate was used to infect 293 cells in 20 150 mm plates. Three days later, virus was recovered as described above, purified by cesium chloride density gradient centrifugation, and dialyzed three times against PBS. To determine the virus titer, 293 cells were seeded in 6-well plates. Twenty-four hours after plating, they were infected with serial dilutions of the virus stock. Two hours after infection, remove the medium and add 2% FBS to the cells.
And 1% agarose. After 10 days, the number of viral plaques was recorded. The adenovirus vector had a titer of 10 ¹² pfu / ml.

Example 6 In Vivo Adenovirus Lps Transduction In this study, outbred CD1 mice purchased from Charles River were induced to experience septic shock and were treated with Lps ^d cDNA, Lps ⁿ cDNA or GFP cDNA. Was carried out. Outbred CD1 mice were used because the degree of heterogeneity in the experimental results can be observed.

A. The inoculated endotoxins LD ₅₀ and LD ₁₀₀ were determined to be 0.6 mg and 1 mg, respectively, using mice. Thereafter, each mouse was toxin 1mg inner equivalent to LD ₁₀₀ was inoculated by the intraperitoneal route. After 1 hour, Ad5-Lps ^d viruses were administered intravenously liquid Ad5-Lps ⁿ virus or Ad5-GFP virus 10 ¹⁰ pfu containing 0.5ml each mouse.

B. Surviving animals were observed at 6-12 hour intervals. The results are shown in FIG. All of the mice treated with 1 mg LPS died within 36 hours. The Ad5-Lps ^n, or treated mice were similar by Ad5-GFP. Half of the mice treated with Lps ^d died within the same time, while the other half survived (FIG. 9). Heterogeneity of survival after transfection treatment was correlated with the use of outbred CD1 mice. One mouse in this surviving group initially showed cachexia but became healthy after recovery. No mice treated with the Ad5 virus alone died, indicating no toxicity. Thus, endotoxin-sensitive mice can be rescued from septic shock by introduction of the mutant Lps ^d cDNA.

C. To demonstrate the presence of PCR adenovirus gene transfer in transduced mice , by Ad5-Lps ^d , or
The Ad5-Lps ^n, peripheral blood mononuclear cells of mice transduced, for RNA extraction in different times after in vivo gene transfer, and collected in the following description as.

CD1 mice were inoculated through the tail vein with approximately 10 ¹⁰ pfu of infectious recombinant adenovirus particles. After 2, 4, 6, 10 and 24 hours, whole blood was collected in hematocrit tubes. After centrifugation, the leukocyte buffy coat layer was collected and lysed in GTC buffer (Wong et al., J. Virol. 68, 5523-5531, 1994) for total RNA extraction.
The final volume of each RNA sample was 10 μl, all of which were used as described (supra) for reverse transcription using oligo-dT primers. Total RT reaction volume was 20 μl. 2 μl of the 20 μl were used for nested PCR amplification similar to those reported by the inventors (supra). For each reaction, 1.5 units of Taq polymerase (Sigma) were used in a 50 μl reaction volume. For the first round of PCR amplification, samples were first denatured at 95 ° C for 5 minutes, followed by 20 cycles of denaturation (94 ° C,
1 min), annealing (60 ° C., 1 min), and primer extension (72 ° C., 1 min). In the last cycle, the primer extension reaction was extended to 72 ° C. for 10 minutes. For the first round of PCR amplification, the 5 'primer sequence (T7 sense) was 5'-TA
A TAC GAC TCA CTA TAG GGA GA-3 '(SEQ ID NO: 5), which is specific for the T7 promoter sequence. 3 'primer sequence (D2 antisense) is 5'-GAA ATT
CAG AAA GGA AAC AAC TCT GTT CCA-3 ′ (SEQ ID NO: 6)
Antisense sequence specific for cDNA (starting at position 810nt). The expected size of the amplified DNA fragment is 1020 bp. For the second round of nested PCR amplification, 2 μl of the 50 μl reaction volume from the first round PCR amplification was used for the second PCR amplification. The 5 'primer was the pcDNA3 (Invirogen) sense sequence 5'-ACT ATA GGG AGA CCC AAG CT-3' (SEQ ID NO: 7), which bound to the Lps cDNA in the adenovirus vector during construction. Specific to the resulting pcDNA3 vector sequence. 3 'primer (R1 antisense) sequence is 5'-AGC AGT CGT CTG
AGC AAC CT-3 '(SEQ ID NO: 8), which is specific for Lps cDNA (starting at position 606nt). The expected size of the PCR amplification product is 790 bp. Samples were first denatured at 95 ° C. for 5 minutes, followed by 30 cycles of 1 minute at 94 ° C., 1 minute at 60 ° C., and 1 minute at 72 ° C. Subsequently, the primer extension reaction was extended and performed at 72 ° C. for 10 minutes.

D. Results of PCR The RNA of cells that passed only 2 hours after in vivo gene transfer contained a 793 bp band, suggesting the presence of adenovirus mRNA (FIG. 10). ). No such band was observed in the absence of reverse transcription or the absence of cDNA template (FIG. 10). These data suggest that expression of adenovirus genes could occur as early as 2 hours after intravenous vector inoculation.

All publications cited, including those disclosing synthetic methods, preparation methods and analytical methods, are hereby incorporated by reference.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes of the invention and, therefore, may be read from the foregoing specification as indicating the scope of the invention. Rather, reference should be made to the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1A-1D show Lps ^d 2 shows the DNA sequence of an independent clone of the cDNA. The top line is Lps ⁿ isolated from wild-type LPS responder C3H / HeOuJ mice Sequence of cDNA. Lps ^d-
4, -10, -15 and -17 are the sequences of four independently selected plasmid DNAs containing the C3H / HeJ cDNA. Numbering starts at A (position 1) of the translation start site ATG,
G is bold and underlined. The DNA of all four clones contains the same mutation at position 870, with the T residue replaced by a C residue.

FIG. 2 shows a flowchart of retroviral gene transfer into primary spleen B cells of C3H / HeJ mice.

3A is a schematic diagram of the N2 retroviral vector, FIG. 3B is a schematic diagram of the N2-Lps ⁿ retroviral vector, FIG. 3C is a schematic diagram of the N2-Lps ^d retroviral vector.

FIGS. 4A-4D show MTT proliferation assays after initiation of LPS stimulation and retroviral infection.

FIG. 5 shows an RT-PCR assay of spleen B cells infected with retrovirus. MM = molecular weight marker. Similar results were obtained in three or more replicates.

FIG. 6. Macrophage cell line from normal inbred mice (AnaI), macrophage cell line from C3H / HeJ LPS low responder mice (GG2EE), and AnaI cells infected with retrovirus N2-Lps (d) ( # 1, # 7, # 9 and P30) shows stimulation of TNFα expression by LPS.

7A and 7B show Lps ⁿ and Lps ^d , respectively. 2 shows the secondary structure of the 3 ′ untranslated region of mRNA. Position 870 is the position of a single base substitution from T (Lps ⁿ ) to C (Lps ^d ).

FIG. 8 is a schematic diagram showing the construction of an Ad5-Lps adenovirus vector. The black box is the viral ITR (Inverted Terminal Repeat) required for viral DNA replication. "Ψ"
Refers to the packaging array. CMV is the cytomegalovirus promoter. In this construct, the E1 region of Ad5 is Lps ⁿ cDNA (1.1kb), Lps ^d cDNA (1.1kb
) Or green fluorescent protein (GFP) gene (0.8 kb).

FIG. 9 is a diagram plotting endotoxin resistance after adenovirus introduction of Lps ^d , Lps ⁿ or green fluorescent protein (GFP) cDNA into LPS-sensitive mice. 1 mg for each mouse
LD ₁₀₀ endotoxin doses were administered and one hour later, mice were injected intravenously with various adenovirus supernatants containing 10 ¹⁰ infectious virus particles. The animals were then observed over time. Dead (closed circles) and surviving (open circles) animals were recorded.

FIG. 10 shows adenovirus gene expression in peripheral blood mononuclear cells from mice infected with Ad5-Lps ^d or Ad5-Lps ⁿ at various time points. Nested R using specific primers described in Example 6 for total RNA extracted from peripheral blood mononuclear cells
T-PCR amplification was performed. The expected size of the amplified fragment is 793 bp.

──────────────────────────────────────────────────続 き Continuation of front page (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, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, 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, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Wong, Peter, M. ,C. United States 19035 Lafayette Road, Gladwin, PA 1927 F-term (reference) 4B024 AA01 CA04 DA03 EA02 GA11 HA17 4C084 AA13 MA66 NA14 ZA511 4C086 AA01 EA16 NA14 ZA51 4C087 AA01 BC83 CA12 NA14 ZA51

Claims (13)

[Claims] 1. A method for treating endotoxemia in a patient in need thereof, comprising administering an effective amount of the Lps ^d gene to the patient in need of the treatment. 2. The method of claim 1, wherein said Lps ^d gene differs from a corresponding Lps ⁿ gene of the same species by mutation of the 3 'untranslated region of Lps cDNA. 3. The method of claim 2, wherein the mutation comprises a nucleotide substitution at position 870 of the cDNA, where the numbering of the position begins with the translation initiation codon. 4. The method according to claim 1, wherein said Lps ^d gene is administered in the form of a viral vector. 5. The method according to claim 4, wherein said viral vector is a retroviral vector. 6. The method of claim 4, wherein said viral vector is an adenovirus vector. 7. A method for preventing endotoxemia in a patient, which comprises administering an effective amount of the Lps ^d gene to the patient in need of prevention of endotoxemia. 8. The method of claim 7, wherein the Lps ^d gene differs from a corresponding Lps ⁿ gene of the same species by a mutation in the 3 'untranslated region of the Lps cDNA. 9. The method of claim 8, wherein the mutation comprises a nucleotide substitution at position 870 of the cDNA, where the numbering of the position begins with the translation initiation codon. 10. The method according to claim 7, wherein said Lps ^d gene is administered in the form of a viral vector. 11. The virus vector is a retrovirus vector.
The method according to claim 10. 12. The virus vector is an adenovirus vector.
The method according to claim 10. 13. The method of claim 7, wherein said patient is a burn patient.