EP1073471A1 - Method of treating endotoxemia - Google Patents

Abstract

The invention provides a therapeutic agent comprising an Lpsd gene, which downregulates the transduction of LPS-mediated signals and can therefore be administered in order to treat or prevent the occurrence of endotoxemia.

Description

METHOD OF TREATING ENDOTOXEMIA

Cross-Reference to Related Application

This application claims priority from United States provisional application Ser. No. 60/083,210, filed April 27, 1998.

Reference to Government Grant

The invention described herein was supported in part by National Institutes of Health grants 1RO1AI39159-01A1 and 1RO1CA70854-01. The Federal government has certain rights in the invention.

Field of the Invention The invention relates to the use of the Lps^d gene as a therapeutic for the treatment and prevention of endotoxemia.

Background of the Invention

A) LPS

Lipopolysaccharide endotoxin (LPS) is a complex macromolecule that comes from the cell walls of certain bacteria, some of which cause diseases like typhoid fever, dysentery, and urinary tract infections and from other bacteria which are common inhabitants of animal and human intestinal tracts but ordinarily do not cause disease. All of these bacteria have in common the same type of cell wall and are classified as Gram-negative based on their staining properties. 2 -

LPS induces the production and release of immunologically active cytokines and other mediators of the animal's inflammatory response. Cytokines are very active substances produced by various cells such as the mononuclear phagocyte and lymphocytes which stimulate other cells as well as each other. The endothelial cells and granulocytes are also primary targets of endotoxins. Specific receptor molecules and signal transduction pathways in the cells of host animals are being intensively studied at present to gain a clearer understanding of how the endotoxin works.

B) The Genetics of LPS Responsiveness The cellular responses to endotoxin are under genetic control as shown by extensive studies in the mouse. The underlying genetic basis for the multiple response of the host to LPS was initially defined with the discovery of the C3H/HeJ mutant mouse strain in 1968 (Sultzer, Nature 219, 1253-1254 (1968)). The strain is hyporesponsive to the immunostimulatory and pathophysiological effects of the lipid A component of LPS and, therefore, is considered to have a fundamental deficiency in its reactions to LPS as compared to closely related responder strains.

The C3H/HeJ defect is specific and expressed in a variety of ways. For example, C3H/HeJ B cells do not proliferate or differentiate when exposed to LPS, and the proliferation of their thymocytes induced by concanavalin A is not enhanced by LPS (Sultzer et al. , Nature New Biology (London) 240, 198-200 (1972); Sultzer, Abst. Am. Soc. Microbiol. 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. , Immunobiology 187, 257-271 (1993); Glode et al. , L.M. and D.L. Rosenstreich, J. Immunol 117, 2061, 2068 (1976); Sultzer, B.M. Infection and Immunity 13, 1579-1584 (1976)). Macrophage phagocytosis and cytotoxicity are not stimulated by LPS and the cytokines such as Tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF), interleukin-1 - 3 -

(IL-1) and the prostaglandins, which are stimulated by LPS in responder cells, are not induced in C3H/HeJ macrophages (Morrison et al. Adv. Immunol. 28, 294-312 (1979)). Furthermore, the C3H/HeJ mouse is highly resistant to lethal endotoxin shock as compared to normal endotoxin responder 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 for that matter the deficient binding of LPS to otherwise immunocompetent cells (Sultzer in "Beneficial effects of Endotoxin", Ch 11, Plenum, NY (1983). Rather, the explanations offered focus on a deficient trigger receptor or a failure somewhere in signal transduction after the initial interaction of the cells with LPS (Sultzer et al. , Immunobiology 187, 257-271 (1993)).

From the results of classical type breeding experiments with the C3H/HeJ strain and various responder strains, it was found that the mitogenic response to LPS is governed by a single locus composed of co-dominant alleles (Morrison et al. Adv. Immunol. 28, 294-312 (1979); Sultzer in "Beneficial effets of Endotoxin", Ch 11, Plenum, NY (1983); Sultzer et al. , Immunobiology 187, 257-271 (1993); Glode et al. J. Immunol 117, 2061-2068 (1976); Sultzer, Infection and Immunity 13, 1579-1584 (1976)). Watson determined that the locus (Lpsⁿ) was on chromosome 4 and linked to the major urinary protein locus (Mup- 1), but downstream from Mup-1 and the Lyb2;2;4;6 genes which control B cell activation (Watson et al. J. Immunol. 120, 422-429 (1979)). The C3H/HeJ mouse strain contains the mutant Lps^d allele.

C) Isolation and Characterization of Lps" Gene Sultzer et al , Immunobiology 187, 257-271 (1993) describe the preparation of a cDNA library consisting of six sublibraries from C3H/OuJ (responder) mouse spleen cells. The introduction of one sublibrary consisting of 2 x 10⁴ independent clones into C3H/HeJ LPS non-responder spleen cells produced, after exposure to LPS in culture, approximately a four- fold increase in plaque-forming cells reactive to sheep red blood cells.

Kang et al , Infect. Immun. 64, 4612-4617 (1996) describe the further functional screening and fractionation of the C3H/OuJ cDNA library, and the isolation of a cDNA clone which is capable of restoring the lipopolysaccharide-mediated B cell response in C3H/HeJ splenic B cells. The 1166 nucleotide cDNA (SEQ ID NO: l) includes a 648 nucleotide open reading frame encoding a 216 amino acid protein (SEQ ID NO:2).

Sequence analysis showed that this LPS responsive gene, the Lps" gene, encodes Ran/TC4, a GTPase important for nuclear transport (Raetz, C.R.H., Biochemistry of Endotoxins. Ann. Rev. Biochem. 59, 129-170 (1990); Watson et al , J. Immunol 114, 1462-1468 (1975); Kabir et al. , Infect. Immun. 15, 156-164 (1977)). At the amino acid level, Lps" is identical to the Ran/TC4 GTPase found in humans and dogs, and has 98.6% and 82.8% homology to the Ju93 and Spil proteins in chicken and yeast, respectively (Drivas et al. , Mol Cell Biol 10, 1793-1798 (1990); Dupree et al , Gene 120, 325-326 (1992); Trueb et al , FEBS 306, 181-184 (1992); Matsumoto et al , Cell 66, 347-360 (1991)). The entire disclosures of each of Kang et al. , Drivas et al. , Dupree et al , Trueb et α/.and Matsumoto et α/.are incorporated herein by reference. At the DNA level, the homology of Lps" to human TC4/Ran, dog TC4/Ran, chicken Ju93 and yeast Spil 89.7%, 90.7%, 85.4% and 68.7%, respectively. Id. Therefore, the Lps" molecule is highly conserved throughout evolution, particularly the five domains of the molecule, G1-G5, which have been shown to be involved in the binding and hydrolysis of guanine nucleotides. In summary, the Lps" gene encodes a functional gene product which is present in polyclonally activated antibody producing B cells from LPS- responder mice such as the C3H/HeOuJ, and introduction of the Lps" cDNA into C3H/HeJ B cells is sufficient to restore their responsiveness to LPS stimulation, resulting in the increase of antibody producing plaque-forming cells (PFCs) to a responder level. It is believed that this gene will be found not only in B cells, - 5

but also in other cell types which respond to LPS and in cells as well which produce cytokines and other factors that in large amounts can be deleterious to the host.

D) Endotoxemia

There are many pathophysiological effects of LPS, one of which is endotoxemia or septic shock which results from large amounts of endotoxin in the blood and has an approx. 40% fatality rate. The majority of the cases of septic shock are a consequence of Gram-negative bacteria in the blood. The septic shock syndrome can, however, be induced by other organisms including Gram-positive bacteria and fungi. Nevertheless, extensive investigations into the causes of septic shock have established that a key factor in the development of septic shock is the release of LPS from Gram-negative bacteria and the subsequent effects of the endotoxin on various cells in the body which become highly activated. As a result, the host is overwhelmed with many cell substances, and this can lead to circulatory failure, shock, and death.

Sepsis is a frequent cause of death in intensive care units and it remains a major public health problem. When sepsis occurs, major proinflammatory cytokines are involved including TNF-alpha, IL-1 and IL-6. Clinical trial results of inhibiting TNF using either anti-TNF antibodies or fusion proteins composed of the extracellular domain of the TNF with the Fc portion of IgG have been disappointing (Dellinger et al. , Chest 111, 744-753 (1997); Grau et al , Nature Medicine 3, 1193-1195 (1997)).

Although powerful antibiotics and drugs to treat cardiopulmonary failure are available, there remains a great need for improved therapeutic and/or prophylactic treatment regimens for the control of endotoxemia, especially during the early stages of sepsis. 6 -

Summarv of the Invention

The present invention provides a method of treating endotoxemia in a patient in need of such treatment comprising administering an effective amount of an Lps^d gene. The Lps" gene differs from the Lps" gene in that it contains a mutation which results in a hyporesponsive lipopolysaccaride endotoxin response in cells containing such a mutation. In one embodiment of the invention, the Lps^d gene differs from the corresponding Lps" gene of the same species by a mutation in the 3' untranslated region of the Lps" cDNA. In a preferred embodiment, the mutation comprises a nucleotide substitution at a position 870 of the Lps DNA, wherein the position numbering commences 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 Lps^d gene is a murine gene. As the murine and human Lps encode identical proteins, the murine gene may be used for therapy. In a more preferred embodiment the Lps^d gene is administered in the form of a viral vector. In one most preferred embodiment the viral vector is a retroviral vector. In another most preferred embodiment the viral vector is an adenoviral vector.

The invention also provides a method of preventing endotoxemia in a patient in need of such treatment comprising administering an effective amount of an Lps^d gene. In a preferred embodiment, the Lps^d gene is administered in the form of a viral vector. In one most preferred embodiment the viral vector is a retroviral vector. In another most preferred embodiment the viral vector is an adenoviral vector.

The invention also constitutes the use of an Lps^d gene for the preparation of a medicament to treat or prevent endotoxemia in a patient.

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

Figures 1A-1D show the DNA sequence of independent clones of an Lps^d cDNA. The top line is the sequence of Lps" cDNA, isolated from wildtype LPS responder C3H/HeOuJ mice. Lps^d -4, -10, -15 and -17 are sequences of four independently selected plasmid DNAs containing C3H/HeJ cDNA. The numbering starts at A (position 1) of the translation start site, ATG, which is indicated by bold type and underlining. The DNA of all four clones contained the same mutation at position 870, where a T residue was substituted with C residue. Figure 2 shows a flow chart of the retroviral gene transfer into primary splenic B cells of C3H/HeJ mice.

Figure 3 A is a diagram of the N2 retrovirus vectors, Figure 3B is a diagram of the N2-Lps" retrovirus vectors, and Figure 3C is a diagram of the N2-Lps^d retrovirus vectors. Figures 4A-4D show MTT proliferation assays after initiation of

LPS stimulation and retroviral infection.

Figure 5 shows RT-PCR assays of retrovirus infected splenic B cells. MM = Molecular Weight Marker. Similar results were obtained in three or more repeated experiments. Figure 6 shows the stimulation of TNFα expression by LPS in macrophage cell lines derived from normal inbred mice (Ana I), from C3H/HeJ LPS hyporesponder mice (GG2EE), and in Ana I cells infected with the retrovirus N2-Lps(d) (#1, #7, #9, and P30).

Figures 7A and 7BB respectively show the secondary structure of the 3 '-untranslated regions of Lps" and Lps^d mRNA. Position 870 is the site of a single base substitution from T (Lpsⁿ ) to C (Lps^d).

Figure 8 is a schematic of the construction of Ad5-Lps adenovirus vectors. The black boxes are viral ITR (Invert Terminal Repeat), necessary for viral DNA replication. "Ψ" refers to the packaging sequence. CMV is cytomegalovirus promoter. In the constructs, the El region of Ad5 was replaced with Lps" (l. lkb) cDNA, Lps" cDNA (1.1 kb) or the green fluorescent protein (GFP) gene (0.8 kb).

Figure 9 is a plot of endotoxin-resistance after adenoviral transfer of an Lps^d, Lps" or green fluorescent protein (GFP) cDNA into LPS sensitive mice. Each mouse received an LD₁₀₀ endotoxin dose of 1 mg and, one hour later, various adenoviral supernatants carrying 10¹⁰ infectious virus particles were injected into the mice intravenously. The animals were then observed over time. Dead (close circles) and live animals (open circles) were recorded as shown.

Figure 10 shows the expression of adenoviral genes in peripheral blood mononuclear cells from mice infected with Ad5-Lps^d or Ad5-Lps" at various time points. Nested RT-PCR amplification was performed using specific primers as described in Example 6 on total RNA extracted from peripheral blood mononuclear cells. The expected size of the amplified fragment is 793bp.

Detailed Description of the Invention A) Definitions and General Methodology

The following definitions are intended as an aid to understanding the scope and practice of the present invention.

"LPS" means lipopolysaccharide endotoxin.

"Lpsⁿ" and "Lps^d" are used to describe the normal (with respect to LP S responsiveness) and hyporesponsive lipopolysaccharide endotoxin response gene allelic forms respectively, without regard to the species of origin. Lps^d includes any gene which encodes an LPS protein, but which gene contains a mutation which results in a hyporesponsive LPS response in cells containing such a mutation. The Lps" gene is also known as Ran/Tc4 or TC4/Ran. The term "Lps gene" includes the Lps" and Lps^d forms, as well as other forms, both naturally occurring and created in the laboratory, of the LPS endotoxin response gene.

"Lps" protein" and "Lps^d protein" are used to describe the polypeptides that are encoded by the Lps" and Lps^d genes respectively. The term "Lps protein" includes the Lps" and Lps^d proteins, as well as other forms, both naturally occurring and created in the laboratory, of the LPS endotoxin response gene product.

According to one embodiment of the invention, the Lps^d gene differs from the corresponding Lps" gene of the same organism by a mutation in the region encoding the mRNA 3' - untranslated region (3' UTR). The mutation gives rise to a difference in mRNA secondary structure in the area of the mutation. In a more preferred embodiment, the mutation is a single point mutation, such as the mutation T-C in the Lps cDNA at position 870 (position numbering starting from the translation initiation codon (ATG)) of C3H HeJ mice.

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation. Generally enzymatic reactions, oligonucleotide syntheses, and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are known to the skilled artisan, including Maniatis (Molecular Cloning, Cold Spring Harbor Laboratories, 1982), and Ausubel (Current Protocols in Molecular Biology, Wiley and sons, 1987), which are incorporated herein by reference.

B) Vectors and Methods of Gene Delivery

The human and murine Lps" proteins have an identical amino acid sequence, and at the DNA level the murine and human Lps" (TC4/Ran) genes show 89.7% homology. A mutant human Lps^d gene (cDNA) can therefore be created by using the technique of site-directed mutagenesis or, in the alternative, by combining the human Lps" gene with a restriction fragment which contains the murine Lps^d mutation. In one embodiment, the mutation is the T-C mutation at position 870 10

of the Lps" cDNA characteristic of C3H/HeJ mice. The human Lps^d gene can be used as a gene therapeutic to down regulate the response to LPS and therefore to treat or prevent the occurrence of endotoxemia in a patient. Alternatively, since the murine and human Lps proteins are identical, the murine Lps^d gene may be utilized as the therapeutic sequence for human gene therapy.

Mutations in the Lps" gene which give rise to a hyporesponsive LPS response may be induced by known mutagenesis techniques, coupled with screening of the resulting mutant claims for LPS responsiveness. Lps" cDNA is mutated and cloned into an appropriate vector, e.g., the N2 retroviral vector (Wong et ai, Mol. Cell. Biol. 9, 798-808 (1989); Wong et al, Genes Dev. 1, 358-365 (1987) which contains 5' and 3' MMLV LTRs, splice donor and splice acceptor sites, and a neomycin resistance marker gene. The resulting pN2-Lps plasmid DNA is used to transfect virus packaging cells, from which a viral supernatant is obtained. Virus particles are precipitated by known techniques. The N2-Lps^x vector is used to infect macrophages, e.g. the Ana I macrophage cell line derived from normal inbred mice. The transduced cells are assayed for the TNFα production following LPS stimulation.

Down regulation of LPS-mediated signals in the transduced cells is determined by a decrease in TNFα production over montransduced macrophase control cells. Mutant cDNAs which upon introduction into macrophages induce down-regulation of LPS responsiveness are selected. Representative techniques for retroviral delivery of Lps^x mutant cDNA into target effector cells are described in the examples below.

Preferable, the mutation in the Lps" gene which gives rise to LPS hyporesponsive ness will significantly decease, but not completely abrogate LPS responsiveness in the host cell.

The Lps^d gene is preferably delivered to the patient in the form of a viral vector. Viral vectors that may be used in the present invention include adenoviral vectors and retroviral vectors. 11

Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver nucleic acid to a variety of cell types. Various serotypes of adenovirus exist, including type 2 and type 5 human adenoviruses and adenoviruses of animal origin. Preferably, the replication defective adenoviral vectors according to the invention comprise the ITRs, an encapsidation sequence, and the nucleic acid of interest. Still more preferably, at least the El region of the adenoviral vector is nonfunctional. Other regions may also be modified, including the E3 region (see WO95/02697), the E2 region (see WO94/28938), the E4 region (see W094/28152, WO94/12649, and WO 95/02697), or in any of the late genes L1-L5 The replication defective recombinant adenoviruses according to the invention can be prepared by techniques known to a person skilled in the art. In particular they can be prepared by homologous recombination between an adenovirus and a plasmid which carries the DNA sequence of interest. Homologous recombination is effected following cotransfection of the adenovirus and plasmid into an appropriate cell line. The cell line employed should be transformable by said components and contain sequences which are able to complement the defective regions in the replication defective adenovirus. Examples of cell lines which may be used are the human embryonic cell line 293 (Graham et al, J. Gen. Virol. 36, 59 (1977)) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described in WO94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified using standard biological techniques which are well known to those having ordinary skill in the art. Adenoviral vectors are generally preferred for the treatment of endotoxemia, where effective treatment must generally occur within about 24 hours. Adenoviral vectors can be produced at high titers (e.g. 10¹⁰-10¹² infectious units per ml), and can be used to transiently express the LPS nonresponsive phenotype in a non-tissue-specific manner. 12

Retroviruses are integrating viruses which generally infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant retroviral vectors is known to those of skill in the art. In recombinant retroviral vectors, the gag, pol, and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as M-MuLV, MSV (murine Moloney sarcoma virus), HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus) and Friend virus.

In general, in order to construct recombinant retroviruses containing a sequence according to the invention, a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art. In particular the cell line PA317 (US 4,861,719), the PsiCRIP cell line (WO90/02806), and the GP+envAm-12 cell line ( WO89/07150) may be mentioned. Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Retroviral vectors derived from lentiviruses such as HIV-1 , HIV-2, and SIV can be used for delivery to nondividing cells. These viruses can be pseudotyped with the surface glycoproteins of other viruses, such as M-MuLV or vesicular stomatitis virus (VSV). The production of high titer HIV-1 pseudotyped with VSV glycoprotein has been disclosed by Bartz and Vodicka (Methods 12(4):337-42 (August 1997)), and multiply attenuated lentiviral vectors have been disclosed by 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. 13

Chimeric adenoviral/retroviral vector systems can also be used to achieve efficient gene delivery and long term gene expression. A chimeric viral system in which adenoviral vectors are used to produce transient retroviral producer cells in vivo, such that progeny retroviral particles infect neighboring cells has been described by Feng et al. (Nature Biotechnology 15:866-70 (September 1997)).

Retroviral vectors are generally preferred for the prevention of endotoxemia in patients who are at a high risk of developing this condition. As an example, burn patients develop endotoxemia at close to a 100% rate. Because retroviral vectors can integrate into hematopoietic stem cells, they can give rise to a reservoir of LPS resistant T cells, B cells, and macrophages.

Recombinant viruses are administered to a patient in an amount sufficient to treat or prevent the occurrence of endotoxemia. Effective amounts vary depending on the characteristics of the patient, the type and severity of the condition being treated, the desired duration of treatment, the method of administration, and other parameters. Effective amounts may be determined by the physician or by another qualified medical professional. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 10⁴ and 10¹⁴ pfu.

Examples

The practice of the invention is illustrated by the following examples. These examples are illustrative only, and do not limit the scope of the invention.

Example 1

Isolation and Characterization of the C3H/HeJ Mouse Lps^d Gene The studies of Kang et al. (Infect. Immun. 64, 4612-4617 (1996)) showed that the Lps" gene is capable of restoring the lipopolysaccharide-mediated B cell response in C3H/HeJ splenic B cells. 14

To demonstrate that the Lps gene is defective in the genome of C3H/HeJ mice, and that this defect accounts for the hyporesponsiveness to LPS stimulation, the Lps gene was isolated from a pCD-HeJ cDNA library.

An Okayama-Berg cDNA expression library (Okayama, H., and Berg, P., Mol Cell Biol 2, 161-170 (1982)) was constructed using RNA from C3H/HeJ splenic B cells. The pCD-HeJ cDNA library was spread over 8 dishes (of 5.8 xlO⁴ clones per 150mm dish), and the library was probed with the 0.9kb BamHl-Pstl fragment of Lps" cDNA obtained from cells of C3H/HeOuJ LPS responder mice (Infect. Immun. 64, 4612-4617 (1996)). Twenty five positive clones were identified.

Among the twenty five independent clones analyzed, eight of them were full length cDNAs, as determined by comparison with the size of the C3H HeOuJ (wild type) Lps" cDNA. Sequencing was then performed on four of these independent full length cDNA clones (Lps^d -4, -10, -15 and -17). The sequence of these four independent clones was compared with that of the wildtype Lps" cDNA.

All four clones showed the same point mutation at position 870 of the Lps" cDNA (residue numbering starts from ATG), where a T residue was substituted with a C residue, as shown in Figures 1 A- ID. Interestingly, this single point mutation occurs in the 3 '-untranslated region. By Northern (RNA) blot analysis, there is no significant difference between the Lps mRNA levels in C3H/HeJ and C3H/HeOuJ cells.

These data indicate that the point mutation present in the mutated HeJ Lps^d gene accounts for the hyporesponsiveness of C3H/HeJ cells to LPS. The secondary structures of the Lps" and Lps^d mRNA were compared using the SQUIGGLES program of the GCG Sequence analysis Package (Wisconsin). Figures 7A (Lps") and 7B (Lps^d ) show a striking difference in RNA secondary structure in areas proximal to the point mutation, and no significant structural difference in areas distal to the point-mutation. The single base substitution present in Lps^d mRNA at the 3 ' UTR suggests the presence of a unique 15 -

mRNA domain to which regulatory protein(s) may bind and impose a post- transcriptional regulation, affecting either the translational efficiency, mRNA stability, transport or intracellular localization. Lps" and Lps^d retroviral vectors were constructed in order to further characterize the wildtype and mutant Lps gene products, and their role in the response to LPS.

Example 2

Construction of Lps" and Lps^d Retroviral Vectors

The Lps" and Lps^d cDNAs from Example 1 were cloned into the

Xho 1 site of the N2 retroviral vector (Wong et al, Mol. Cell. Biol. 9, 798-808 (1989); Wong et al, Genes Dev. 1, 358-365 (1987)). The N2 retroviral vector comprises 5' and 3' MMLV LTRs, splice donor and splice acceptor sites, and a neomycin resistance marker gene.

The Lps" cDNA was isolated from the Xho I restriction fragment from pCD-LPS (Infect. Immun. 64, 4612-4617 (1996)) and ligated into the Xho I restriction fragment site of N2 retrovirus vector to produce pN2-Lps". The same procedure was used to generate the pN2-Lps^d vector, except that the mutated LPS^d cDNA was used.

To generate retroviral stocks carrying the Lps" and Lps^d sequence, GP/E virus packaging cells (Markowitz et α/. , J. Virol. 62, 1120-1124 (1988)) were separately transfected with pN2-Lps" or pN2-Lps^d plasmid DNA by calcium phosphate DNA co-precipitation. Positive transfectants were selected in medium containing lmg/ml G418. Individual G418 resistant clones were isolated, expanded and characterized.

To produce a concentrated viral supernatant, virus was harvested from the producer cells and six hundred milliliters of viral supernatant were used for virus precipitation in 0.4MNaCl and 8.5% PEG 8000 (Sigma) for 1 to 1.5 hours at 4°C with continuous stirring. The precipitate was collected by centrifugation at 7,500 rpm for ten minutes. The pellet was dissolved in NTE buffer ( 1 OOmM NaCl, lOmM Tris-Cl pH7.4, ImM EDTA, filter sterilized) in 1% of the original volume. 16

This solution was then loaded onto a Sepharose CL-4B column at 4°C. Several fractions were collected and pooled. Each pooled fraction was filter-sterilized and tested for viral titer by virtue of G418 resistance using NIH/3T3 cells, as described (Wong et al., Mol. Cell. Biol. 9, 798-808 (1989).

The construction of the N2 parental vector, N2-Lpsⁿ and N2-Lps^d vectors is shown in Figures 3A-3C, respectively.

Example 3

Retroviral Transfer of Lps" and Lps^d Genes Into Splenic B Cells from C3H/HeJ Mice

A) Retroviral Gene Transfer

Splenic B cells from C3H/HeJ mice were depleted of red blood cells, subjected to retroviral infection in the presence of 1 -100 μg/ml LPS, and then cultured in S 17 stromal cell conditioned medium, which is known to stimulate pre- B cell growth (Collins etal.,J. Immunol. 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, cultures were refed with new stocks of concentrated viral supernatants repeatedly, as described in the flow chart shown in Figure 2. Viral titers from concentrated stocks of either the N2 parental vector, N2-Lps" or N2-Lps^d vectors were normalized to 2 x 10⁶ G418^r colonies per ml.

B) MTT Proliferation Assays

At various time point after initiation of LPS stimulation and retroviral infection, the cells were harvested and subject to the MTT [3-94,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide] proliferation assay (Han et al, Proc.Natl.Acad.Scl 92, 11014-11018 (1995)).

Ten million C3H/HeJ Splenic cells, depleted of Red blood cells, were placed in a 100mm cell culture plates containing 10ml of S17CM (RPMI containing 10% fetal calf serum and 50 μM 2-mercaptoethanol incubated in S17 17

cells for 24 hours, at 0, 12, 24 48 hours of alignments) for 6 hours. After incubation, non-adherent cells were collected and re-infected in a 100 mm plate with 10ml of infection mixture. The infection mixture contained 3 ml of the concentrated viral supernatant with the titer of 2x10⁶ virus/ml, 1 ml of S 17CM, 10 μg/ml polybrene, and 10-100μg/ml of hot phenol extracted LPS from Salmonella typhimurium) for 16 hours. Sixteen hours later, the cells were collected and re- infected with fresh infection mixture for another 16 hours. After infection, collected cells were stimulated with 10 μg/ml or 100 μg/ml of LPS in 2 ml of S 17CM and incubated in a 24-well plate for 0, 12, 24, 48 hours. Subsequently, 100 μl of the retroviral infected and LPS stimulated cells were placed in a 96 well plate. 10 μl of the 5 mg/ml MTT was added and mixed with cells then the plate was placed in an incubator. After 4 hours of incubation, 100 μl of 0.04N HCl in isopropanol was added to wells and mixed very thoroughly. O.D. at 570nm was read in a microplate reader. At 32 hours (= 0 hour incubation shown in Figure 2), no significant difference in cell proliferation was found among all cultures (Figure 4A). At 44 hours (= 12 hours incubation shown in Figure 2), N2-Lps" infected cells, but not N2-Lps^d cells nor N2-infected cells, showed a clear increase in cell proliferation (Figure 4B). At 68 hours after initiation of infection culture (= 48 hours incubation shown in Figure 2), there was a maximal 4-fold difference in the LPS response between the N2-Lpsⁿ-infected culture and the N2- or N2-Lps^d -infected culture. This response subsequently started to decline at 92 hours.

The LPS response in the N2-Lps" infected culture of C3H/HeJ splenic B cells observed at 44 hours is equivalent to that of C3H/HeOuJ responder splenic B cells 24 hours after LPS stimulation (data not shown). The delay is attributable to (1) only about 50% of the cells were transduced with the N2-Lps" vector after more than 48 hours, and (2) a lag time is necessary for viral integration and expression of the transduced gene. Cell proliferation was obviously more prominent in the N2-Lps" - infected culture than the N2- or N2-Lps^d - infected cultures, as the number of both proliferating small lymphocytes and cells - 18 -

undergoing blastogenesis was much higher in the former than the latter cultures (data not shown). Further, polyclonal B cell differentiation also took place, as splenic B cells from C3H/HeJ transduced with the N2-Lps", but not with N2 nor N2-Lps^d vectors, could be stimulated with LPS to produce antibody in lysing sheep red blood cells similar to what has been described previously (Kang et al , Infect. Immun. 64, 4612-4617 (1996)).

C) RT-PCR Assays

To further show that the response, or a lack of response, to LPS is due to the successful retroviral transfer of the corresponding wildtype or mutant Ran cDNA, RT-PCR assays were performed on cells from all cultures. A 5' oligo primer specific for a sequence of SV40 promoter that is present within the retrovirus vector, and a 3' primer specific for the Lps" or Lps^d cDNA were used.

Total RNA was extracted from retrovirus infected splenic B cells using 4M GTC and phenol/chloroform. AMV-RT was used to generate cDNA. Two primers were used for PCR reaction: a 5 ' primer (TCTAAAAGCTGCTGCAGG, SEQ ID NO:3) from the pCD portion of the Xho I fragment, and a 3' primer (GTACACGATCTGCTTAGC, SEQ ID NO:4) from the Lps cDNA, to generate a 907 base pair fragment. The results are shown in Figure. 5. The predicted 907 bp band was seen in cultures transduced with N2-

Lps" and with N2-Lps^d, but not with N2 virus, confirming the successful transfer and expression of the transduced genes. This primer is specific for Lps RNA because when the genomic DNA was used for PCR reaction, the 907bp band shown in Figure 5 could not be observed. This was because the 5' primer was specific for the vector sequence upstream of the Lps cDNA. Thus the introduction and expression of Lps" cDNA, but not Lps^d cDNA, in splenic B cells from C3H/HeJ mice resulted in restoration of a significant response to LPS stimulation. Classical breeding experiments with the C3H/HeJ strain and other responder strains suggest that the mitogenic response of LPS is governed by a single locus composed of 19

codominant alleles. From the results shown here, it would appear that Ran is the Lps responsive gene, which is defective in the genome of C3H/HeJ LPS hyporesponsive mice.

Example 4 Retroviral Transfer of the Lps^d Gene Into Macrophage Cells from

Normal Inbred Mice

Ana I is a macrophage cell line derived from normal inbred mice, and GG2EE cells are macrophage cells derived from C3H/HeJ hyporesponder mice. Ana 1 cells were infected with the N2-Lps(d) retroviral vector from Example 2, and the transduced cells were assayed for TNFα production at various times after LPS stimulation.

For the retroviral transduction, 1X10⁶ cells in suspension were incubated with 5 ml retroviral supernatant in the presence of 10 μg/ml polybrene. The cells were incubated at 37°C, 5% C0₂, for four hours with frequent shaking. The cells were then centrifuged at 1500 rpm for ten minutes, washed with D,₀G (DMEM supplemented with 10% heat-inactivated FBS, 2mM L-glutamine, 10 μg/ml gentamycin and 1 mg/ml G418) and resuspended in 5 ml D,₀G. A methylcellulose mixture was prepared by combining 1.6 ml methylcellulose with 0.4 ml retrovirus transduced cells, and 1 mg/ml G418, and the mixture (approx. 1.1 ml per plate) was plated in 35 mm dishes. Colonies were picked, expanded, and pooled after two weeks.

For the LPS stimulation, cells (Ana I, GG2EE, and retrovirus transduced Ana I cells) were seeded in 24 well plates (5X10⁵ cells/well) and incubated at 37 °C, 5% CO₂. The cells were stimulated with 100 ng/ml LPS for 0, 24, 48, or 72 hours. The supernatants were collected, and the amount of TNF A was measured using an ELISA assay.

For the TNFα assays, 50 μl of TNFα capture antibody (2 μg/ml) was added to each well of a microplate, and the plate was incubated at 4°C overnight. The plate was then washed four times with PBST, blocking buffer (200 μl) was added to each well, and the plate was incubated at room temperature for 30 min. - 20

The plate was then washed four times with PBST, and samples (100 μl per well) were added to each well. The plate was incubated overnight at 4°C, then washed four times with PBST. For the TNFα detection, biotinylated TNFα antibody (1 μg/ml, 100 μl/well) was added to the wells and the plate was incubated at room temperature for 1 hr, the washed with PBST. Streptavidin-HRP conjugated enzyme (1 :1000 dilution, 100 μl/well) was added and the plate was incubated at room temperature for 30 min., then washed eight times with PBST. ABTS substrate was added (100 μl per well) and the plate was incubated at room temperature for color development. The color was measured by reading OD 405 nm. The results are shown in Figure 6.

#1, #7, and #9 were independent clones, whereas P30 were cells derived from a mixture of 30 independent clones. This experiment shows that expression of the Lps^d gene in Ana I responder cells down regulates the transduction of LPS-mediated signals in these cells. The Lps^d gene can therefore be used as a therapeutic DNA to treat or prevent the occurrence of endotoxemia in patients.

Example 5

Construction of Adenovirus Vectors and Virus Production for in Vivo Adenoviral Lps Transfer Lps^d, Lps" and GFP genes were cloned into and replaced the El region of the Ad5 adenoviral vector as follows. The construction of the vectors is schematically shown in Figure 8.

Lps" and Lps^d cDNAs were excised with BamHl at sites 3249 and 35 from pCD-Lps" and pCD-Lps^d (the latter, containing the T-C mutation at cDNA position 870, was provided by StemCell Therapeutics). They were separately inserted in the El region of the Ad5 genome and driven by CMV promoter. The GFP (green fluorescence protein) gene from the pEGFP plasmid (Clontech, La Jolla) was excised with BamHl and Notl and inserted at the same site and driven by CMV promoter as well. The recombinant adenoviruses are replication defective 21

since they are El deleted. However, they can replicate in 293 cells containing the El gene. For the production of recombinant viruses, 293 cells were transfected with recombinant Ad5 DNA. Ten days post-transfection, the cells were collected by scraping them off culture flasks. After three cycles of freezing in an ethanol/ dry ice bath and rapid thawing at 37°C, one ml of viral lysate was used to infect 293 cells in twenty 150mm plates. Three days later, viruses were harvested as described above and purified by cesium chloride banding and dialyzed against PBS three times. To determine virus titer, 293 cells were seeded in 6 well plates. Twenty-four hours after plating, they were infected with serial dilutions of the virus stocks. Two hours after infection, the medium was removed and cells were overlaid with media containing 2% FBS and 1% agarose. Ten days later, the number of virus plaques was recorded. The adenovirus vectors have a titer of 10¹² pfu/ml.

Example 6 In Vivo Adenoviral Lps Transfer

In this study, out-bred CDl mice purchased from Charles River were induced to undergo septic shock and were treated with vectors carrying the Lps^d, Lps" or GFP cDNA. Out-bred CDl mice were used so that the degree of heterogeneity in the experimental outcome could be observed.

A. Inoculation

LD₅₀ and LD₁₀₀ of endotoxin were determined to be 0.6 mg and 1 mg, respectively, using the mice. After that, each mouse was inoculated intraperitoneally with 1 mg endotoxin, equivalent to LD₁₀₀. One hour later, 0.5 ml of Ad5-Lps^d, Ad5-Lps" or Ad5-GFP virus containing 10¹⁰ pfu was administered into each mouse intravenously. - 22

B. Survival

The animals were observed at 6-12 hour intervals. The results are set forth in Figure 9. All mice treated with 1 mg LPS died within 36 hours, as did mice treated with Ad5-Lps" or with Ad5-GFP. Half of the mice treated with Lps^d died within the same period of time, but the other half survived (Figure 9). Heterogeneity of survival after gene transfer treatment correlated with use of out- bred CDl mice. One mouse within this group that survived manifested cachexia initially but became healthy after recovery. None of the mice treated with Ad5 virus alone died, indicating absence of toxicity. Thus, transfer of the mutant Lps^d cDNA can rescue endo toxin-sensitive mice from septic shock.

C. PCR of Transduce Mice

To verify the presence of adenoviral gene transfer, peripheral blood mononuclear cells of mice transduced with Ad5-Lps^d or Ad5-Lps" at different times after in vivo gene transfer were collected for RNA extraction, as described as follows.

CDl mice were inoculated intravenously through the tail vein with approximately 10¹⁰pfu infectious recombinant adenovirus particles. At 2, 4, 6, 10 and 24 hours later, whole blood was collected into hematocrit tubes. After centrifugation, the leukocyte buffy coat layer was harvested and lysed in GTC buffer (Wong et al. , J. Virol. 68, 5523-5531 , 1994) for total RNA extraction. Final volume of each RNA sample was lOμl; all of which were used for reverse transcription using oligo-dT primer as described (Id.). The total RT reaction volume was 20μl. Two out of the 20μl were used for nested PCR amplification similar to those in our previous report (Id.). For each reaction, 1.5 unit of Taq polymerase (Sigma) was used in a 50μl reaction volume. For the first round of PCR amplification, the samples were first denatured at 95 °C for 5 minutes, then subjected to 20cycles of denaturation (94°C for 1 min.), annealing (60°C for 1 min.) and primer extension (72 °C for 1 min.). At the last cycle, there was an - 23

extended primer extension at 72 °C for 10 minutes. For the first round of PCR amplification, the 5' primer sequence (T7 sense) was 5'- TAA TAC GAC TCA CTA TAG GGA GA-3' (SEQ ID NO:5), which is specific for T7 promoter sequence, the 3' primer sequence (D2 antisense) was 5'- GAA ATT CAG AAA GGA AAC AAC TCT GTT CCA-3' (SEQ ID NO:6), which is an antisense sequence specific for Lps cDNA (starting at position 810 nt). The expected size of the amplified DNA fragment was 1020bp. For the second round of the nested PCR amplification, 2μl out of 50μl total reaction volume from the first round PCR amplification was used for the second PCR reaction. The 5' primer was pcDNA3 (Invitrogen) sense sequence 5'-ACT ATA GGG AGA CCC AAG CT-3 ' (SEQ ID NO:7), which is specific for pcDNA3 the vector sequence attached to the Lps cDNA in the adenovirus vectors during construction. The 3' primer (Rl antisense) sequence was 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 amplified product would be 790bp. The samples were first denatured at 95 °C for 5 minutes, then subjected to 30 cycles of 94°C for 1 minute, 60°C for 1 minute, and then 72 °C for 1 minute, followed by an extended primer extension reaction of 72 °C for 10 minutes.

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

All of the references cited, including those which disclose synthetic, preparative and analytical procedures, are incorporated herein by reference.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, - 24

reference should be made to the appended claims, rather than to the foregoing specification, as indication the scope of the invention.

Claims

25CLAIMS

1. A method of treating endotoxemia in a patient in need of such treatment comprising administering an effective amount of an Lps^d gene.

2. The method according to claim 1 wherein the Lps^d gene differs from the corresponding Lps" gene of the same species by a mutation in the 3' untranslated region of the Lps cDNA.

3. The method according to claim 2 wherein the mutation comprises a nucleotide substitution at position 870 of said cDNA, wherein the position numbering commences with the translation initiation codon.

4. The method of claim 1 wherein the Lps^d gene is administered in the form of a viral vector.

5. The method of claim 4 wherein the viral vector is a retroviral vector.

6. The method of claim 4 wherein the viral vector is an adenoviral vector.

7. A method of preventing endotoxemia in a patient in need of such treatment comprising administering an effective amount of an Lps^d gene.

8. The method according to claim 7 wherein the Lps^d gene differs from the corresponding Lps" gene of the same species by a mutation in the 3' untranslated region of the Lps cDNA. - 26

9. The method according to claim 8 wherein the mutation comprises a nucleotide substitution at position 870 of said cDNA, wherein the position numbering commences with the translation initiation codon.

10. The method of claim 7 wherein the Lps^d gene is administered in the form of a viral vector.

11. The method of claim 10 wherein the viral vector is a retroviral vector.

12. The method of claim 10 wherein the viral vector is an adenoviral vector.

13. The method of claim 7 wherein the patient is a burn patient.