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|Publication number
|Publication type
|Application number
|Publication date
|Oct 11, 2011
|Filing date
|Dec 1, 2004
|Priority date
|Dec 1, 2003
|Fee status
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|Publication number
|Sunil Chada
|Original Assignee
|Introgen Therapeutics
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|BiBTeX, EndNote, RefMan
|Patent Citations (74), Non-Patent Citations (296), Referenced by (2), Classifications (15), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application claims the benefit of the filing date of U.S. provisional patent application Ser. No. 60/526,031, filed Dec. 1, 2003, the entire contents of which are hereby incorporated by reference.
1. Field of the Invention
The present invention relates generally to the fields of molecular biology, infectious diseases, and pharmacology. More particularly, it concerns methods of suppressing or preventing an infection of a subject or a cell by a pathogen using an MDA-7 polypeptide or a nucleic acid encoding an MDA-7 polypeptide.
2. Description of Related Art
Infections by pathogens are a major cause of morbidity and mortality in the U.S., and throughout the world. Successful vaccination programs against smallpox and polio, between 1950 and 1970, led to a general view by public health authorities, particularly in the West, that the war against infectious diseases was effectively over and some countries scaled-back health measures. However, the emergence of HIV and multi-resistant organisms has shown that constant vigilance is needed where pathogens are concerned.
In addition, in light of the events of Sep. 11, 2001, many anti-terrorist experts and government officials believe that the prospect of a bioterrorism attack against the United States using pathogens is highly likely. For example, terrorists may have the ability of gaining access to pathogens that are associated with substantial morbidity and mortality. Large scale release of a highly infectious pathogenic agent could infect the inhabitants of targeted cities and other areas. The attacks could result in long- and short-term health consequences and possibly death. Thus, in addition to the continuing need for the possibility of mass treatment of pathogens, there is an urgent need to develop strategies to prevent the morbidity and mortality associated with a bioterrorist attack wherein the public is exposed to deadly pathogens.
Viruses pose the threat of inflicting serious morbidity and mortality on the population. As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular RNA. These mechanisms serve to redirect the translational apparatus to favor viral transcripts, and they often come at the expense of the host cell.
One such mechanism whereby viruses ensure translational efficiency involves the cellular kinase known as interferon-induced, double-stranded (ds) RNA-activated serine/threonine protein kinase (PKR). PKR is involved in the regulated of apoptosis, cell-proliferation, signal transduction, and differentiation of cells. PKR is believed to localize in the cytoplasm and nucleolus of the cell, and associates with ribosomes. Overexpression or activation of PKR in HeLa, COS-1, U937, and NIH-3T3 cells has been shown to lead to apoptosis. In addition, mouse embryo fibroblasts from PKR knock-out mice are resistant to apoptotic cell death in response to dsRNA, tumor necrosis factor, and lipopolysaccharide. RNaseL and PKR mediate the IFN-induced antiviral response of the host, which is required to limit viral protein synthesis and pathogenesis. As part of the innate immune response to viral infection, RNaseL and PKR are activated by dsRNAs produced as intermediates in viral replication.
PKR also mediates the antiviral actions of interferon, in part by phosphorylating the alpha subunit of eukaryotic initiation factor 2 (eIF-2α), resulting in acute inhibition of mRNA translation and a concomitant block in viral replication (Merrick and Hershey, 1996; Meurs et al., 1990). In addition, PKR facilitates IFN-induced transcriptional programs by participating in the activation of nuclear factor kappa B and INF-regulatory factor 1 (Kumar et al., 1997).
Many viruses inhibit PKR activity. One such example is Hepatitis C virus (HCV), a member of the Flaviviridae, which mediates persistent infection within a majority of infected individuals. Many strains of HCV are resistant to alpha interferon (IFN) therapy. It has recently been shown that HCV represses PKR function through the actions of the viral NS5A protein, which binds and inhibits PKR in vivo (Gale et al., 1998, Gale et al., 1999). Other examples of viruses that inhibit PKR activity include adenovirus, EBV, poxvirus, influenza virus, reovirus, HIV, polio, HSV, and SV40 (Gale et al., 2000). Inhibition of PKR results in inhibition of apoptosis, with resulting continued translation of viral proteins.
Another cellular regulatory mechanism involved in protecting against viral infection involves the PRK-like endoplasmic-reticulum (ER)-resident kinase (PERK). PERK is a type I transmembrane protein that attenuates protein synthesis during endoplasmic reticulum (ER) stress by phosphorylating serine 51 on eIF2α. The HCV envelope protein, E2, can serve as a pseudosubstrate inhibitor of PERK and a potential viral regulator of the ER stress response (Pavio et al., 2003).
Another mediator of ER stress is the Unfolded Protein Response (UPR) (Gething and Sambrook, 1992). The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes and is responsible for maintaining cellular homeostasis. Transient stresses such as glucose deprivation or perturbation of calcium or redox homeostasis can result in transient inhibition of protein translation and growth arrest. Prolonged UPR activation leads to activation of death-related signaling pathways and ultimately, to apoptotic death (Kaufman, 2002; Ron, 2002). The UPR is used by many endoplasmic reticulum-tropic viruses, such as HCV, flaviviruses, HHV6, rubella, LCMV, HIV, and Hepatitis B virus (HBV), to facilitate their life cycle and pathogenesis. Note that many viruses, especially positive strand viruses, modify host intracellular membranes to create a suitable compartment for viral replication—this requirement for virus-induced intracellular membrane production results in activation of UPR.
It is clear that pathogens other than viruses can activate the PKR and UPR defense systems. For example, PKR is activated in response to Salmonella infection (Shtrichman et al., 2002). Shtrichman et al. (2002) describes tissue-selectivity of interferon-stimulated gene expression in mice infected with Dam (+) versus Dam (−) Salmonella enterica serovar Typhimurium strains. In addition, the UPR serves as a defense system in plants (Oh et al., 2003). Oh et al. (2003) have shown conservation between animals and plants of the cis-activating element involved in the UPR.
The intracellular mediators, IRE1 and PERK are activated by ER stress, which can be induced by high-level glycoprotein expression. All enveloped viruses produce excess glycoproteins that could elicit PERK and IRE1 activation to meet the need for increased folding and secretory capacity. Thus, UPR activation is a common feature of viral replication.
Therefore, there is the need for novel agents that can be applied in the prevention and treatment of infections of subjects by viruses and other pathogens. Such novel methods may target not only immunostimulation, but may also be directed at interfering with viral replication through activation of PKR and the UPR response. These agents can be applied in ameliorating the effects of bioterrorism attacks wherein the public is exposed to pathogens.
Melanoma differentiation associated gene-7 (mda7) is a tumor suppressor gene. Numerous studies have documented the growth suppression effected by elevated expression of MDA-7 protein in various types of cancer cells (reviewed in Fisher et al., 2003).
The inventors have discovered that MDA-7 functions in a manner that involves certain pathways in cancer cells that are involved in pathogen infection and pathogen replication. The inventors propose to exploit these properties of MDA-7 by applying MDA-7 to the suppression and prevention of infections by viruses and other pathogens.
In addition, the inventors have discovered that Ad-mda7 transduction of lung cancer cells results in activation of the UPR. It has been discovered that MDA-7 expression in the ER elicited potent cell death in tumor cells and thus it appears necessary for MDA-7 to enter the secretory pathway to be effective in inducing apoptosis. In view of the fact that the UPR is exploited by viruses, these findings further support the inventors' discovery that MDA-7 can be applied in the prevention and treatment of infections of subjects by pathogens.
Certain embodiments of the present invention are generally concerned with methods of suppressing or preventing an infection of a subject by a pathogen, that involve administering to the subject a composition that includes: (a) a therapeutically effective amount of an MDA-7 polypeptide or a nucleic acid encoding the MDA-7 polypeptide; and (b) a pharmaceutically acceptable preparation suitable for delivery to said subject, wherein the MDA-7 suppresses or prevents the infection.
A pathogen is defined herein to include any living microorganism that can cause disease or dysfunction in a cell or subject. The methods of the present invention contemplate any type of pathogen. For example, the pathogen may be a bacterium, virus, fungus, protozoan, or helminth. One of ordinary skill in the art would be familiar with the variety of pathogens that are known to exist.
Any bacterium is contemplated for inclusion as a pathogen. For example, the pathogen may be a gram negative bacterium, a gram positive bacterium, or a bacterium for which gram staining is not applicable.
In certain embodiments of the present invention, the bacterium is a Pneumococcus species, Streptococcus species, Enterococcus species, Staphylococcus species, Hemophilus species, Pseudomonas species, Brucella species, Bordetella pertussis, Neisseria species, Moraxella catarrhalis, Corynebacterium species, Listeria monocytogenes, Nocardia asteroides, Bacteroides species, Leptospira species, Klebsiella pneumoniae, Escherichia coli, Proteus species, Serratia species, Acinetobacter species, Yersinia pestis, Francisella tularensis, Enterobacter species, Helicobacter species, Legionella species, Shigella species, Mycobacterium species, Bacillus species, or Yersinia species.
The species of Streptococcus may, for example, be S. pyogenes, S. agalactiae, S. equi, S. canis, S. bovis, S. equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis, S. mutans, viridans streptococci, or peptostreptococci. The species of Enterococcus may be Enterococcus faecalis or Enterococcus faecium. The species of Staphylococcus may be, for example, Staphylococcus epidermidis or Staphylococcus aureus. The species of Pseudomonas may be Pseudomonas aeruginosa, Pseudomonas pseudomallei, or Pseudomonas mallei. The species of Brucella may be Brucella melitensis, Brucella suis, or Brucella abortus. The species of Neisseria may be may be Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Corynebacterium pseudodiphtheriticum, Corynebacterium urealyticum, Corynebacterium hemolyticum, or Corynebacterium equi. The species of Mycobacterium may be M. tuberculosis, M. bovis, M. avium complex, M. marinum, M. fortuitum, or M. kansaii. The species of Bacillus may be B. cereus, B. thuringiensis, or B. anthracis. The species of Yersinia may be Y. pestis, Y. enterocolitica, or Y. pseudotuberculosis.
Any type of virus is encompassed within the definition of pathogen. For example, in some embodiments, the virus is an endoplasmic reticulum-tropic virus. Examples of endoplasmic reticulum-tropic viruses include Hepatitis C virus, a flavivirus species, HHV6, rubella, LCMV, HIV, and Hepatitis B virus. In other embodiments, for example, the virus is influenza A, influenza B, influenza C, parainfluenza, paramyxoviruses, Newcastle disease virus, respiratory syncytial virus, measles, mumps, adenoviruses, adenoassociated viruses, parvoviruses, Epstein-Barr virus, rhinoviruses, coxsackieviruses, echoviruses, reoviruses, rhabdoviruses, coronavirus, polioviruses, herpes simplex virus, cytomegaloviruses, papillomaviruses, virus B, varicella-zoster, poxviruses, rabies, picomaviruses, rotavirus, or Kaposi associated herpesvirus. The definition of pathogen also encompasses fungi. Any fungus is contemplated for inclusion in the definition of pathogen, as long as it meets the definition of being a pathogen. For example, in some embodiments, the fungus is a Absidia species, Actinomadura madurae, Actinomyces species, Allescheria boydii, Alternaria species, Anthopsis deltoidea, Apophysomyces elegans, Arnium leoporinum, Aspergillus species, Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris species, Blastomyces dermatitidis, Candida species, Cephalosporium species, Chaetoconidium species, Chaetomium species, Cladosporium species, Coccidioides immitis, Conidiobolus species, Corynebacterium tenuis, Cryptococcus species, Cunninghamella bertholletiae, Curvularia species, Dactylaria species, Epidermophyton species, Epidermophyton floccosum, Exserophilum species, Exophiala species, Fonsecaea species, Fusarium species, Geotrichum species, Helminthosporium species, Histoplasma species, Lecythophora species, Madurella species, Malassezia furfur, Microsporum species, Mucor species, Mycocentrospora acerina, Nocardia species, Paracoccidioides brasiliensis, Penicillium species, Phaeosclera dematioides, Phaeoannellomyces species, Phialemonium obovatum, Phialophora species, Phoma species, Piedraia hortai, Pneumocystis carinii, Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizopus species, Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Torulopsosis species, Trichophyton species, Trichosporon species, Ulocladium chartarum, Wangiella dermatitidis, Xylohypha species, Zygomyetes species, Thermomucor indicae-seudaticae, or a Radiomyces species.
Pathogens may also include protozoans or helminths, such as Cryptosporidium, Entamoeba, Plasmodium, Giardia, Leishmania, Trypanasoma, Trichomonas, Naegleria, Isospora belli, Toxoplasma gondii, Trichomonas vaginalis, Wunchereria, Ascaris, Schistosoma species, Cyclospora species, or Chlamydia species.
The methods of the present invention contemplate any type of subject. For example, the subject may be a mammal. In certain embodiments, the mammal is a human.
Any method of administration of the composition is completed by the present invention. Methods of administration are discussed further in the specification below. In some embodiments, administering involves providing to the subject an expression cassette that includes a promoter, active in the subject, operably linked to a polynucleotide encoding an MDA-7 polypeptide.
In some embodiments, the expression cassette is carried in a viral vector. For example, the viral vector may be an adenoviral vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, or a poxviral vector. In certain particular embodiments, the viral vector is an adenoviral vector.
In other embodiments, the expression cassette is carried in a nonviral vector. For example, the nonviral vector may include a lipid, such as a liposome.
Any MDA-7 polypeptide or nucleic acid encoding an MDA-7 polypeptide is contemplated for inclusion in the methods of the present invention. MDA-7 polypeptides and polynucleotides encoding an MDA-7 polypeptide is discussed further in the specification below. In certain embodiments, the MDA-7 polypeptide includes amino acids 49 to 206 of SEQ ID NO: 2. In certain other embodiments, the MDA-7 polypeptide includes a sequence of SEQ ID NO: 2.
Any pharmaceutically acceptable preparation suitable for delivery to the subject is contemplated for inclusion in the present invention. One of ordinary skill in the art would be familiar with the many types of preparations that can be formulated. Examples are discussed further in the specification below. For example, in some embodiments, the composition is formulated for oral administration, topical administration, intralesional injection, or intravenous administration.
In other embodiments of the present invention, the method involves the additional administration of at least one additional agent to prevent or suppress the infection in the subject. The administration of any one or more additional agent to prevent or suppress the infection is contemplated by the present invention. Examples of such agents are discussed further in the specification below.
Certain other embodiments of the present invention are generally concerned with methods of suppressing or preventing a viral infection of a cell, that involve obtaining an MDA-7 polypeptide or a nucleic acid encoding the MDA-7 polypeptide; and contacting the cell with the MDA-7 polypeptide or the nucleic acid encoding the MDA-7 polypeptide, wherein the MDA-7 suppresses or prevents infection of the cell.
The viral infection may be caused by any type of virus. For example, the viral infection may be caused by an oncogenic virus or an endoplasmic reticulum-tropic virus. For example, the endoplasmic reticulum-tropic virus may be Hepatitis C virus, a flavivirus species, HHV6, rubella, LCMV, HIV, or Hepatitis B virus.
Other examples of viruses which may cause the viral infection include influenza A, influenza B, influenza C, parainfluenza, paramyxoviruses, Newcastle disease virus, respiratory syncytial virus, measles, mumps, adenoviruses, adenoassociated viruses, parvoviruses, Epstein-Barr virus, rhinoviruses, coxsackieviruses, echoviruses, reoviruses, rhabdoviruses, coronavirus, polioviruses, herpes simplex virus, cytomegaloviruses, papillomaviruses, virus B, varicella-zoster, poxviruses, rabies, picornaviruses, rotavirus, or Kaposi associated herpes virus.
Any cell is contemplated for inclusion in the methods of the present invention. For example, in certain embodiments the cell is a mammalian cell, such as a human cell. In other embodiments, the cell is a cancer cell.
As with the methods discussed above, the MDA-7 polypeptide or nucleic acid encoding the MDA-7 polypeptide may include an expression cassette comprising a promoter, active in the cell, operably linked to a polynucleotide encoding an MDA-7 polypeptide. The parameters pertaining to expression cassettes discussed above also apply to these methods. For example, the expression cassette may be carried in a viral vector. Viral vectors are discussed further in the specification below.
The viral vector may, for example, be an adenoviral vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, or a poxviral vector. In other embodiments, the expression cassette is carried in a non-viral vector. The nonviral vector may include, for example, a lipid.
MDA-7 polypeptides and polynucleotides encoding MDA-7 polypeptides have been previously discussed, and are discussed further in the specification below. For example, the MDA-7 polypeptide may include a sequence of SEQ ID NO: 2.
In other embodiments, the methods of the present invention may further include administering at least one additional agent to prevent or suppress the viral infection in the cell. One of ordinary skill in the art would be familiar with the numerous agents that are available to prevent or suppress viral infection in a cell.
As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Melanoma differentiation associated gene-7 (mda-7) is a tumor suppressor gene. Numerous studies have documented the growth suppression effected by elevated expression of MDA-7 protein in various types of cancer cells (reviewed in Fisher et al., 2003). MDA-7 over-expression in normal cells, however, lacks the cytotoxic effects seen in cancer cells (Mhashilkar et al., 2001; Lebedeva et al., 2003).
Although primarily studied in the context of its tumor suppressor activity, MDA-7 has other activities. For instance, it has potent anti-angiogenic activity. In particular, the anti-angiogenic activity of MDA-7 is 50-fold more potent than endostatin. It has also been shown to have substantial pro-apoptotic activity.
MDA-7 has also been shown to exhibit immunostimulatory activity against cancer cells. Its immunosuppressive function includes potent inhibition of proinflammatory cytokine synthesis, including the inhibition of synthesis of IFN-γ, TNF-α, and IL-6 (De Waal Malefyt et al., 1991). MDA-7 has also been found to induce secretion of high levels of IL-6, TNF-α, and IFN-γ from human PBMC as measured by ELISA (Caudell et al., 2002).
Based upon its chromosomal localization in the IL-10 family cluster and its limited amino-acid identity to IL-10, mda-7 has now been designated as IL-24, and recent data indicate that this molecule functions as a pro-Th1 cytokine in human PBMC (Caudell et al., 2002). Further studies have demonstrated expression of mda-7 mRNA and MDA-7 protein in immune cells (Huang et al., 2001; Caudell et al., 2002). Thus mda-7 is an unusual pro-Th1 cytokine with pro-apoptotic activity.
Ad-mda 7 has been shown to induce and activate PKR in cancer cells, which leads to phosphorylation of the eIF-2α and the induction of apoptosis (Pataer et al., 2002). Treatment with 2-aminopurine (2-AP), a serine/threonine kinase inhibitor, inhibits PKR activation, eIF-2α phosphorylation, and apoptosis induction by Ad-mda7. Additionally, PKR null but not wild-type fibroblasts are resistant to Ad-mda7-induced apoptosis. Mouse embryo fibroblasts from PKR knockout mice are resistant to Ad-mda7-induced apoptosis (Pataer et al., 2002)]. In addition, immunofluorescence studies demonstrate that MDA-7 colocalizes with endogenous PKR protein, further indicating that MDA-7 is a substrate for PKR. These results suggest that the activation of PKR and its downstream targets may be a critical pathway for Ad-mda7-mediated apoptosis in cancer cells.
The inventors have discovered that MDA-7 functions in a manner that involves certain pathways in cancer cells that are involved in pathogen infection and replication. However, MDA-7 has never been contemplated for use as a therapeutic agent against infections by viruses and other pathogens. The inventors propose to exploit these properties of MDA-7 by applying MDA-7 to the suppression and prevention of infections by viruses and other pathogens.
The inventors seek to exploit the fact that MDA-7 functions in a manner that involves certain pathways in cancer cells that are involved in pathogen infection and pathogen replication by applying MDA-7 as a therapeutic and preventive agent against pathogen infection. For example, compositions that include MDA-7 can be applied in methods pertaining to the treatment and prevention of viral infection. Furthermore, MDA-7 may be applied as a therapeutic and preventive agent in bioterrorism attacks wherein the public is exposed to pathogens.
The methods of the present invention involve administering to a subject or contacting a cell with an MDA-7 polypeptide or a nucleic acid encoding an MDA-7 polypeptide. MDA-7 is a putative tumor suppressor that has been shown to suppress the growth of cancer cells that are p53-wild-type, p53-null and p53-mutant.
mda-7 is a tumor suppressor gene which was identified using a subtractive hybridization approach from human melanoma cells induced to differentiate with IFN-beta and mezerein (Jiang et al., 1995). mda-7 mRNA was expressed in normal melanocytes and early stages of melanoma, but was lost during melanoma progression (Jiang et al., 1995). Transfection of mda-7 expression plasmid into various tumor cell lines resulted in suppression of growth (Jiang et al., 1996). Based on these observations it was suggested that mda-7 is a novel tumor suppressor gene whose expression must be inhibited for tumor progression.
The tumor suppressor activities of mda-7 gene transfer have now been well established. Numerous studies have documented the growth suppression effected by elevated expression of MDA-7 protein in cancer cells from lung, breast, melanoma, prostate, mesothelioma, glioma etc (reviewed in Fisher et al., 2003). MDA-7 overexpression in normal cells however, lacks the cytotoxic effects seen in cancer cells (Mhashilkar et al., 2001; Lebedeva et al., 2003). It has recently demonstrated that Ad-mda7 causes growth suppression, cell cycle block, and apoptosis in non-small cell lung cancer cells in vitro and in vivo using xenograft models (Saeki et al., 2000; Saeki et al., 2002; Pataer et al., 2002).
The mda-7 gene is located on chromosome 1 at 1q32, and is contained within a cytokine cluster that encodes IL-10; IL-19 and IL-20 (Mhashilkar et al., 2001; Blumberg et al., 2001; Huang et al., 2001). Based upon its chromosomal localization in the IL-10 family cluster and its limited amino-acid identity to IL-10, mda-7 has now been designated as IL-24, and recent data indicate that this molecule functions as a pro-Th1 cytokine in human PBMC (Caudell et al., 2002). Further studies have demonstrated expression of mda-7 mRNA and MDA-7 protein in immune cells (Huang et al., 2001; Caudell et al., 2002). Thus mda-7 is an unusual pro-Th1 cytokine with pro-apoptotic activity.
Based upon its chromosomal localization in the IL-10 family cluster and its limited amino-acid identity to IL-10, mda-7 has now been designated as IL-24, and recent data indicate that this molecule functions as a pro-Th1 cytokine in human PBMC (Caudell et al., 2002). Further studies have demonstrated expression of mda-7 mRNA and MDA-7 protein in immune cells (Huang et al., 2001; Caudell et al., 2002). Thus mda-7 is an unusual pro-Th1 cytokine with pro-apoptotic activity.
A number of studies have evaluated molecules involved in mediating the apoptotic response of tumor cells to Ad-mda7. These studies have used cell lines from different tumor types and have shown regulation of a variety of diverse cellular mediators. For example, analysis of molecules involved in the effector phase of the apoptotic pathway has shown that MDA-7 can increase expression of p53, BAK, BAX, activate caspases 3, 8 and 9 and increase mitochondrial cytochrome c release in lung and breast cell lines (Mhashilkar et al., 2001; Saeki et al., 2000; Saeki et al., 2002; Pataer et al., 2002; Pataer et al., 2003). Analyses of signaling pathways have revealed Ad-mda 7 regulation of iNOS and MAPK in melanoma (Ekmekcioglu et al., 2003, Sarkar et al., 2002), and jun kinase, PI3K and PKR in lung and breast tumor cells (Pataer et al., 2002; Kawabe et al., 2002; Mhashilkar et al., 2003). Thus, different signaling pathways may be responsible for induction of apoptosis in different tumor types. This concept was recently validated by demonstrating that MDA-7 mediated killing of breast cancer cells was blocked by inhibitors of MAPK or MEKK pathways, whereas blocking these pathways had minimal effects against lung tumor cells (Mhashilkar et al., 2003).
Recent studies have demonstrated that Ad-mda7 transduction causes high levels of MDA-7 protein to be released from transduced cells (Mhashilkar et al., 2001).
MDA-7 mRNA has been identified in human PBMC (Ekmekcioglu et al., 2001), and no cytokine function of human MDA-7 protein was reported. MDA-7 has been designated as IL-24 based on the gene and protein sequence characteristics (NCBI database accession XM—001405). The murine MDA-7 protein homolog FISP (IL-4-Induced Secreted Protein) was reported as a Th2 specific cytokine (Schaefer et al., 2001). Transcription of FISP is induced by TCR and IL-4 receptor engagement and subsequent PKC and STAT6 activation as demonstrated by knockout studies. Expression of FISP was characterized but no function has been attributed yet to this putative cytokine. The rat MDA-7 homolog C49a (Mob-5) is 78% homologous to the mda-7 gene and has been linked to wound healing (Soo et al., 1999; Zhang et al., 2000). Mob-5 was also shown to be a secreted protein and a putative cell surface receptor was identified on ras transformed cells (Zhang et al., 2000). Therefore, homologues of the mda-7 gene and the secreted MDA-7 protein are expressed and secreted in various species. However, no data has emerged to show MDA-7 has cytokine activity. Such activity has ramifications for the treatment of a wide variety of diseases and infections by enhancing immunogenicity of an antigen.
The mda-7 cDNA (SEQ ID NO: 1) encodes a novel, evolutionarily conserved protein of 206 amino acids (SEQ ID NO: 2) with a predicted size of 23.8 kDa. The deduced amino acid sequence contains a hydrophobic stretch from about amino acid 26 to 45, which has characteristics of a signal sequence. The protein sequence shows no significant homology to known proteins with the exception of a 42 amino acid stretch that is 54% identical to interleukin 10 (IL-10). Structural analysis has determined that MDA-7 (IL-BKW or IL-20) displays the structural characteristics of the cytokine family (WO 98/28425, specifically incorporated herein by reference). The structural characteristics and limited identity across a small stretch of amino acids implies an extracellular function for MDA-7. The expression of MDA-7 is inversely correlated with melanoma progression as demonstrated by increased mRNA levels in normal melanocytes as compared to primary and metastatic melanomas as well as decreased MDA-7 expression in early vertical growth phase melanoma cells selected for enhanced tumor formation in nude mice. Additional information and data regarding MDA-7 can be found in U.S. patent application Ser. Nos. 09/615,154 and 10/017,472, which are herein incorporated by reference in their entirety.
Additional studies have shown that elevated expression of MDA-7 suppressed cancer cell growth in vitro and selectively induced apoptosis in human breast cancer cells as well as inhibiting tumor growth in nude mice (Jiang et al., 1996; Su et al., 1998). Jiang et al. (1996) report findings that mda-7 is a potent growth suppressing gene in cancer cells of diverse origins including breast, central nervous system, cervix, colon, prostate, and connective tissue. A colony inhibition assay was used to demonstrate that elevated expression of MDA-7 enhanced growth inhibition in human cervical carcinoma (HeLa), human breast carcinoma (MCF-7 and T47D), colon carcinoma (LS174T and SW480), nasopharyngeal carcinoma (HONE-1), prostate carcinoma (DU-145), melanoma (HO-1 and C8161), glioblastome multiforme (GBM-18 and T98G), and osteosarcoma (Saos-2). Mda-7 overexpression in normal cells (HMECs, HBL-100, and CREF-Trans6) showed limited growth inhibition indicating that mda-7 transgene effects are not manifest in normal cells. Taken together, the data indicates that growth inhibition by elevated expression of MDA-7 is more effective in vitro in cancer cells than in normal cells.
Su et al. (1998) reported investigations into the mechanism by which MDA-7 suppressed cancer cell growth. The studies reported that ectopic expression of MDA-7 in breast cancer cell lines MCF-7 and T47D induced apoptosis as detected by cell cycle analysis and TUNEL assay without an effect on the normal HBL-100 cells. Western blot analysis of cell lysates from cells infected with adenovirus mda-7 ("Ad-mda-7") showed an upregulation of the apoptosis stimulating protein BAX. Ad-mda7 infection elevated levels of BAX protein only in MCF-7 and T47D cells and not normal HBL-100 or HMEC cells. These data lead the investigators to evaluate the effect of ex vivo Ad-mda7 transduction on xenograft tumor formation of MCF-7 tumor cells. Ex vivo transduction resulted in the inhibition of tumor formation and progression in the tumor xenograft model.
In certain embodiments of the present invention, the mda-7 is provided as a nucleic acid expressing the MDA-7 polypeptide. In specific embodiments, the nucleic acid is a viral vector, wherein the viral vector dose is or is at least 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015 or higher pfu or viral particles. In certain embodiments, the viral vector is an adenoviral vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, a polyoma viral vector, an alphaviral vector, a rhabdoviral vector, or a herpesviral vector. Most preferably, the viral vector is an adenoviral vector. In other specific embodiments, the nucleic acid is a non-viral vector.
In certain embodiments, the nucleic acid expressing the polypeptide is operably linked to a promoter. Non-limiting examples of promoters suitable for the present invention include a CMV IE, dectin-1, dectin-2, human CD11c, F4/80, SM22 or MHC class II promoter, however, any other promoter that is useful to drive expression of the mda-7 gene or the immunogen of the present invention, such as those set forth herein, is believed to be applicable to the practice of the present invention.
1. Nucleic Acids, Vectors and Regulatory Signals
The present invention concerns methods that involve compositions that include polynucleotides or nucleic acid molecules relating to the mda-7 gene and its gene product MDA-7. These polynucleotides or nucleic acid molecules are isolatable and purifiable from mammalian cells. It is contemplated that an isolated and purified MDA-7 nucleic acid molecule, either the secreted or full-length version, that is a nucleic acid molecule related to the mda-7 gene product, may take the form of RNA or DNA. Similarly, the nucleic acid molecule related to the immunogenic molecule may take the form of RNA or DNA. As used herein, the term "RNA transcript" refers to an RNA molecule that is the product of transcription from a DNA nucleic acid molecule. Such a transcript may encode for one or more polypeptides.
As used in this application, the term "polynucleotide" refers to a nucleic acid molecule, RNA or DNA, that has been isolated free of total genomic nucleic acid. Therefore, a "polynucleotide encoding MDA-7" refers to a nucleic acid segment that contains MDA-7 coding sequences, yet is isolated away from, or purified and free of, total genomic DNA and proteins. When the present application refers to the function or activity of a MDA-7-encoding polynucleotide or nucleic acid, it is meant that the polynucleotide encodes a molecule that has the ability to enhance an immune response. Further, a "polynucleotide encoding an immunogen" refers to a nucleic acid segment that contains an immunogenic coding sequences, yet is isolated away from, or purified and free of, total genomic DNA and proteins. When the present application refers to the function or activity of an immunogen encoding an immunogen, it is meant that the polynucleotide encodes an immunogenic molecule that has the ability to induce an immune response in the body of a human.
The term "cDNA" is intended to refer to DNA prepared using RNA as a template. The advantage of using a cDNA, as opposed to genomic DNA or an RNA transcript is stability and the ability to manipulate the sequence using recombinant DNA technology (see Sambrook, 2001; Ausubel, 1996). There may be times when the full or partial genomic sequence is some. Alternatively, cDNAs may be advantageous because it represents coding regions of a polypeptide and eliminates introns and other regulatory regions.
It also is contemplated that a given MDA-7-encoding nucleic acid or mda-7 gene from a given cell may be represented by natural variants or strains that have slightly different nucleic acid sequences but, nonetheless, encode a MDA-7 polypeptide; a human MDA-7 polypeptide is a specific embodiment. Consequently, the present invention also encompasses derivatives of MDA-7 with minimal amino acid changes, but that possess the same activity.
The term "gene" is used for simplicity to refer to a functional protein, polypeptide, or peptide-encoding unit. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. The nucleic acid molecule encoding MDA-7 or another therapeutic polypeptide such as the immunogen may comprise a contiguous nucleic acid sequence that is 5 or more nucleotides nucleotides, nucleosides, or base pairs in length. For example, in certain embodiments, the nucleic acid molecule is 5, 6, 7, 8, 9, 10, 20, 40, 100, 150, 200, 400, 600, 800, 1200, 3000, 6000, 9000, 12000 or more nucleotides, nucleosides, or base pairs in length. Such sequences may be identical or complementary to SEQ ID NO: 1 (MDA-7 encoding sequence).
"Isolated substantially away from other coding sequences" means that the gene of interest forms part of the coding region of the nucleic acid segment, and that the segment does not contain large portions of naturally-occurring coding nucleic acid, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the nucleic acid segment as originally isolated, and does not exclude genes or coding regions later added to the segment by human manipulation.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a MDA-7 protein, polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO: 2, corresponding to the MDA-7 designated "human MDA-7" or "MDA-7 polypeptide."
The term "a sequence essentially as set forth in SEQ ID NO: 2" means that the sequence substantially corresponds to a portion of SEQ ID NO: 2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO: 2.
The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences that have about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, about 70% to about 80%, and more preferably about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO: 2 will be sequences that are "essentially as set forth in SEQ ID NO: 2" provided the biological activity of the protein is maintained. In particular embodiments, the biological activity of a MDA-7 protein, polypeptide or peptide, or a biologically functional equivalent, comprises enhancing an immune response. Further, in particular embodiments, the biological activity of an immunogen, an immunogenic molecule that is a protein, polypeptide or peptide, or a biologically functional equivalent, comprises immunogenicity, which refers to the molecule's ability to induce an immune response in the body of a human. In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO: 1. The term "essentially as set forth in SEQ ID NO: 1" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO: 1 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO: 1. Again, DNA segments that encode proteins, polypeptide or peptides exhibiting MDA-7 activity will be most some.
In particular embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating DNA sequences that encode MDA-7 polypeptides or peptides that include within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to MDA-7 polypeptides. In other embodiments, the invention relates to an isolated nucleic acid segment and recombinant vectors incorporating DNA sequences that encode an immunogen, protein, polypeptide or peptides that include within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to the immunogen.
Vectors of the present invention are designed, primarily, to transform cells with a therapeutic mda-7 gene under the control of regulated eukaryotic promoters (i.e., inducible, repressible, tissue specific). Also, the vectors may contain a selectable marker if, for no other reason, to facilitate their manipulation in vitro. However, selectable markers may play an important role in producing recombinant cells.
TABLES 1 and 2, below, list a variety of regulatory signals for use according to the present invention.
Inducible Elements
Phorbol Ester (TPA)
Palmiter et al., 1982; Haslinger
Heavy metals
and Karin, 1985; Searle et al.,
1985; Stuart et al., 1985;
Imagawa et al., 1987; Karin et
al., 1987; Angel et al., 1987b;
McNeall et al., 1989
MMTV (mouse
Huang et al., 1981; Lee et al.,
1981; Majors and Varmus, 1983;
Yamamoto et al., 1983; Lee et
al., 1984; Ponta et al., 1985;
Si.e.,i et al., 1986
Tavernier et al., 1983
Imperiale and Nevins, 1984
Phorbol Ester (TPA)
Angel et al., 1987a
Phorbol Ester (TPA)
Angel et al., 1987b
Phorbol Ester (TFA)
Angel et al., 1987b
Murine MX
Interferon, Newcastle
Hug et al., 1988
Disease Virus
GRP78 Gene
Resendez et al., 1988
Kunz et al., 1989
Rittling et al., 1989
MHC Class I
Blanar et al., 1989
Gene H-2κb
Ela, SV40 Large T
Taylor et al., 1989; Taylor and
Kingston, 1990a,b
Phorbol Ester-TPA
Mordacq and Linzer, 1989
Hensel et al., 1989
Thyroid Hormone
Chatterjee et al., 1989
α Gene
Other Promoter/Enhancer Elements
Immunoglobulin Heavy Chain
Banerji et al., 1983; Gillies et al., 1983;
Grosschedl and Baltimore, 1985; Atchinson and
Perry, 1986, 1987; Imler et al., 1987; Neuberger
et al., 1988; Kiledjian et al., 1988;
Immunoglobulin Light Chain
Queen and Baltimore, 1983; Picard and
Schaffner, 1985
T-Cell Receptor
Luria et al., 1987, Winoto and Baltimore, 1989;
Redondo et al., 1990
HLA DQ α and DQ β
Sullivan and Peterlin, 1987
Goodbourn et al., 1986; Fujita et al., 1987;
Goodbourn and Maniatis, 1985
Greene et al., 1989
Interleukin-2 Receptor
Greene et al., 1989; Lin et al., 1990
MHC Class II 5
Koch et al., 1989
MHC Class II HLA-DRα
Sherman et al., 1989
Kawamoto et al., 1988; Ng et al., 1989
Muscle Creatine Kinase
Jaynes et al., 1988; Horlick and Benfield, 1989;
Johnson et al., 1989a
Prealbumin (Transthyretin)
Costa et al., 1988
Elastase I
Omitz et al., 1987
Karin et al., 1987; Culotta and Hamer, 1989
Pinkert et al., 1987; Angel et al., 1987
Albumin Gene
Pinkert et al., 1987; Tronche et al., 1989, 1990
Godbout et al., 1988; Campere and Tilghman,
Bodine and Ley, 1987; Perez-Stable and
Constantini, 1990
Trudel and Constantini, 1987
Cohen et al., 1987
Triesman, 1985; Deschamps et al., 1985
Edlund et al., 1985
Neural Cell Adhesion Molecule
Hirsch et al., 1990
Latimer et al., 1990
H2B (TH2B) Histone
Hwang et al., 1990
Mouse or Type I Collagen
Rippe et al., 1989
Glucose-Regulated Proteins
Chang et al., 1989
(GRP94 and GRP78)
Rat Growth Hormone
Larsen et al., 1986
Human Serum Amyloid A (SAA)
Edbrooke et al., 1989
Troponin I (TN I)
Yutzey et al., 1989
Platelet-Derived Growth Factor
Pech et al., 1989
Duchenne Muscular Dystrophy
Klamut et al., 1990
Banerji et al., 1981; Moreau et al., 1981; Sleigh
and Lockett, 1985; Firak and Subramanian,
1986; Herr and Clarke, 1986; Imbra and Karin,
1986; Kadesch and Berg, 1986; Wang and
Calame, 1986; Ondek et al., 1987; Kuhl et al.,
1987 Schaffner et al., 1988
Swartzendruber and Lehman, 1975; Vasseur et
al., 1980; Katinka et al., 1980, 1981; Tyndell et
al., 1981; Dandolo et al., 1983; Hen et al., 1986;
Si.e.,i et al., 1988; Campbell and Villarreal,
Kriegler and Botchan, 1983; Kriegler et al.,
1984a,b; Bosze et al., 1986; Miksicek et al.,
1986; Celander and Haseltine, 1987; Thiesen et
al., 1988; Celander et al., 1988; Chol et al.,
1996; Reisman and Rotter, 1989
Papilloma Virus
Campo et al., 1983; Lusky et al., 1983;
Spandidos and Wilkie, 1983; Spalholz et al.,
1985; Lusky and Botchan, 1986; Cripe et al.,
1987; Gloss et al., 1987; Hirochika et al., 1987,
Stephens and Hentschel, 1987
Hepatitis B Virus
Bulla and Siddiqui, 1988; Jameel and Siddiqui,
1986; Shaul and Ben-Levy, 1987; Spandau and
Lee, 1988
Human Immunodeficiency Virus
Muesing et al., 1987; Hauber and Cullan, 1988;
Jakobovits et al., 1988; Feng and Holland, 1988;
Takebe et al., 1988; Berkhout et al., 1989;
Laspia et al., 1989; Sharp and Marciniak, 1989;
Braddock et al., 1989
Weber et al., 1984; Boshart et al., 1985;
Foecking and Hofstetter, 1986
Gibbon Ape Leukemia Virus
Holbrook et al., 1987; Quinn et al., 1989
The promoters and enhancers that control the transcription of protein encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex patterns of transcriptional regulation.
The term "promoter" will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator proteins.
At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between elements is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Aside from this operational distinction, enhancers and promoters are very similar entities.
Promoters and enhancers have the same general function of activating transcription in the cell. They are often overlapping and contiguous, often seeming to have a very similar modular organization. Taken together, these considerations suggest that enhancers and promoters are homologous entities and that the transcriptional activator proteins bound to these sequences may interact with the cellular transcriptional machinery in fundamentally the same way.
In some embodiments, the promoter for use in the present invention is the cytomegalovirus (CMV) promoter. This promoter is commercially available from Invitrogen in the vector pcDNAIII, which is some for use in the present invention. Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Below are a list of additional viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the present invention. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or a heterologous protein of interest.
Another signal that may prove useful is a polyadenylation signal. Such signals may be obtained from the human growth hormone (hGH) gene, the bovine growth hormone (BGH) gene, or SV40.
The use of internal ribosome binding sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5-methylated cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
In any event, it will be understood that promoters are DNA elements which when positioned functionally upstream of a gene leads to the expression of that gene. Most transgene constructs of the present invention are functionally positioned downstream of a promoter element.
2. Proteins, Peptides and Polypeptides
a. Biologically Functional Equivalents
The present invention is directed to enhancing an immune response by providing an effective amount of a MDA-7 polypeptide. In certain embodiments, the MDA-7 polypeptide is directly provided. In specific embodiments, the MDA-7 polypeptide is provided before therapy. In specific embodiments, the MDA-7 polypeptide is administered at the same time as administration of an immunogenic molecule, such as an antigen, for purposes of immune therapy. In other specific embodiments, the MDA-7 polypeptide is provided after therapy, and in some instances, after providing an immunogenic molecule for purposes of treating, diagnosing or prognosing induction of an immune response.
Additional embodiments of the invention encompass the use of a purified protein composition comprising MDA-7 protein, truncated versions of MDA-7, and peptides derived from MDA-7 amino acid sequence administered to cells or subjects for the inhibition of angiogenesis. Truncated molecules of MDA-7 include, for example, molecules beginning approximately at MDA-7 amino acid residues 46-49 and further N-terminal truncations. Specifically contemplated are molecules start at residue 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, and 182, and terminate at residue 206. In additional embodiments, residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46 are included with other contiguous residues of MDA-7, as shown in SEQ ID NO: 2.
As will be understood by those of skill in the art, modification and changes may be made in the structure of a MDA-7 polypeptide or peptide, an immunogenic molecule, or an immunogenic product and still produce a molecule having like or otherwise desirable characteristics. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on molecules such as Tat and RNA polymerase II. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of HIV polypeptides or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.
In terms of functional equivalents, the skilled artisan also understands it is also well understood by the skilled artisan that inherent in the definition of a biologically-functional equivalent protein or peptide, is the concept of a limit to the number of changes that may be made within a defined portion of a molecule that still result in a molecule with an acceptable level of equivalent biological activity. Biologically-functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. In particular, where small peptides are concerned, less amino acids may be changed. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.
It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in the active site of an enzyme, or in the RNA polymerase II binding region, such residues may not generally be exchanged. This is the case in the present invention, where residues shown to be necessary for inducing an immune response should not generally be changed, which is contemplated for both the MDA-7 polypeptide and the immunogen product.
Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape, and type of the amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all a similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape. Therefore, based upon these considerations, the following subsets are defined herein as biologically functional equivalents: arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine.
To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within �2 is preferred, some, those which are within �1 are particularly preferred, some, and those within �0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0�1); glutamate (+3.0�1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5�1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within �2 is preferred, those which are within �1 are particularly preferred, and those within �0.5 are even more particularly preferred.
While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons may encode the same amino acid.
b. Synthetic Peptides
The compositions of the invention may include a peptide modified to render it biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Pat. No. 5,028,592, incorporated herein by reference, protected peptides often exhibit increased pharmacological activity.
Compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids, or a mixture thereof. The use of D-amino acids may confer additional resistance to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.
c. In Vitro Protein Production
Following transduction with a viral vector according to some embodiments of the present invention, primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well known to those of ordinary skill in the art.
One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production and/or presentation of proteins. The gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions. The gene for virtually any polypeptide may be employed in this manner. The generation of recombinant expression vectors, and the elements included therein, are discussed above. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
Another embodiment of the present invention uses autologous B lymphocyte cell lines, which are transfected with a viral vector that expresses an immunogen product, and more specifically, an protein having immunogenic activity. Other examples of mammalian host cell lines include Vero and HeLa cells, other B- and T-cell lines, such as CEM, 721.221, H9, Jurkat, Raji, etc., as well as cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
A number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection: for dhfr, which confers resistance to; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418; and hygro, which confers resistance to hygromycin.
Animal cells can be propagated in vitro in two modes: as non-anchorage-dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).
Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent cells.
The observed upregulation of the apoptosis-related BAX gene in p53 null cells indicates that MDA-7 is capable of using p53-independent mechanisms to induce the destruction of cancer cells. It has been observed that adenoviral-mediated overexpression of MDA-7 led to the rapid induction and activation of PKR with subsequent phosphorylation of eIF-2α, other PKR target substrates and apoptosis induction. Specific inhibition of PKR by 2-aminopurine (2-AP) in lung cancer cells abrogates Ad-mda7 induced PKR activation, PKR substrate target phosphorylation and apoptosis induction. As evidenced by PKR null fibroblasts, Ad-mda7 apoptosis is dependent on a functional PKR pathway. These characteristics indicate that MDA-7 has broad therapeutic, prognostic and diagnostic potential as an inducer of PKR and, consequently, an enhancer of an induced immune response.
PKR exerts antiviral and anticellular functions, and is involved in regulating a number of physiologic processes that include cell growth and differentiation (U.S. Pat. No. 6,326,466; Feng et al., 1992; Petryshyn et al., 1988; Petryshyn et al., 1984; Judware et al., 1991), tumor suppression (Koromilas et al., 1992; Meurs et al., 1993), and modulation of signal transduction pathways (Leonardo et al., 1989; Kumar et al., 1994; Maran et al., 1994).
Upregulation of PKR leads to the induction of apoptosis in various cancer cell lines. Furthermore, in myelodysplasias, critical tumorigenic deletions of the IRF-1 gene on chromosome 5q (Beretta et al., 1996) appear associated with decreased PKR levels and immunohistochemical analyses of lung and colorectal cancers demonstrate an association with PKR expression and prolonged survival (Haines et al., 1992). PKR appears to mediate anti-tumorigenic activity through the activation of multiple transduction pathways culminating in growth inhibition and apoptosis induction. Activation of these pathways occurs after the latent, inactive homodimeric form is induced by activating signals to undergo conformational changes leading to auto-phosphorylation and activation (Vattem et al., 2001). Once activated, PKR is able to phosphorylate various substrate targets, which are important in growth control and apoptosis induction (Saelens et al., 2001; Sudhakar et al., 2000). Stimulation of the immune system has been linked to apoptosis (Albert et al., 1998; Restifo et al., 2001). Further, artificial induction of apoptosis has been demonstrated to enhance the immunogenicity of a vaccine due to the stimulatory effect of dendritic cells that became activated by transfection of the apoptotic cells (Chattergoon et al., 2000).
The present invention pertains to methods of suppressing or preventing an infection of a subject by a pathogen. The invention also pertains to methods of suppressing or preventing a viral infection of a cell. Subjects who are infected by a pathogen may or may not be symptomatic.
Concerning viruses, any virus is contemplated for inclusion in the methods of treatment of the present invention. One of ordinary skill in the art would be familiar with the many different types of viral pathogens that are known. The following viruses are mentioned by way of example: influenza A, B and C, parainfluenza, paramyxoviruses, Newcastle disease virus, respiratory syncytial virus, measles, mumps, parvoviruses, Epstein-Barr virus, rhinoviruses, coxsackieviruses, echoviruses, reoviruses, rhabdoviruses, lymphocytic choriomeningitis, coronavirus, polioviruses, herpes simplex, human immunodeficiency viruses, cytomegaloviruses, papillomaviruses, virus B, varicella-zoster, poxviruses, rubella, rabies, picomaviruses, rotavirus and Kaposi associated herpes virus.
In certain embodiments of the present invention, the virus is an endoplasmic reticulum-tropic virus. This is a class of viruses that utilize the endoplasmic reticulum (ER) for production of critical viral proteins. Any endoplasmic reticulum tropic virus is contemplated by the present invention. For example, the virus may be Hepatitis C virus, a flavivirus species, HHV6, rubella, LCMV, HIV, or Hepatitis B virus.
In certain embodiments, the pathogen is a bacterium. Any bacterium is contemplated for suppression and prevention by the methods of the present invention. For example, the bacterium may be a gram negative bacterium, a gram positive bacterium, or a bacterium for which gram staining is not applicable. Examples of such bacteria include, but are not limited to, the 83 or more distinct serotypes of pneumococci, streptococci such as S. pyogenes, S. agalactiae, S. equi, S. canis, S. bovis, S. equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis, S. mutans, other viridans streptococci, peptostreptococci, other related species of streptococci, enterococci such as Enterococcus faecalis, Enterococcus faecium, staphylococci, such as Staphylococcus epidermidis, Staphylococcus aureus, Hemophilus influenzae, Pseudomonas species such as Pseudomonas aeruginosa, Pseudomonas pseudomallei, Pseudomonas mallei, brucellas such as Brucella melitensis, Brucella suis, Brucella abortus, Bordetella pertussis, Borellia species, such as Borellia burgedorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Corynebacterium pseudodiphtheriticum, Corynebacterium urealyticum, Corynebacterium hemolyticum, Corynebacterium equi, etc. Listeria monocytogenes, Nocordia asteroides, Bacteroides species, Actinomycetes species, Treponema pallidum, Leptospirosa species, Haemophilus species, Helicobacter species, including Helicobacter pylori, Treponema species and related organisms. The invention may also be useful against gram-negative bacteria such as Klebsiella pneumoniae, Escherichia coli, Proteus, Serratia species, Acinetobacter, Yersinia pestis, Francisella tularensis, Enterobacter species, Bacteriodes and Legionella species, Shigella species, Mycobacterium species (e.g., Mycobacterium tuberculosis, Mycobacterium bovis or other mycobacteria infections), Mycobacterium avium complex (MAC), Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium kansaii, Yersinia infections (e.g., Yersinia pestis, Yersinia enterocolitica or Yersinia pseudotuberculosis) and the like. In addition, the invention in contemplated to be of use in controlling protozoan, helminth or other macroscopic infections by organisms such as Cryptosporidium, Entamoeba, Plasmodiium, Giardia, Leishmania, Trypanasoma, Trichomonas, Naegleria, Isospora belli, Toxoplasma gondii, Trichomonas vaginalis, Wunchereria, Ascaris, Schistosoma species, Cyclospora species, for example, and for Chlamydia trachomatis and other Chlamydia infections such as Chlamydia psittaci, or Chlamydia pneumoniae, for example. Of course it is understood that the invention may be used on any pathogen against which an effective antibody can be made.
Fungal and other mycotic pathogens (some of which are described in Beneke, 1979; Smith, 1989; and Scrip's Antifungal Report, 1992) are also contemplated as a pathogen addressed by the methods of the present invention. Fungal disease contemplated in the context of the invention includes, but is not limited to, Aspergillosis, Black piedra, Candidiasis, Chromomycosis, Cryptococcosis, Onychomycosis, or Otitis extema (otomycosis), Phaeohyphomycosis, Phycomycosis, Pityriasis versicolor, ringworm, Tinea barbae, Tinea capitis, Tinea corporis, Tinea cruris, Tinea favosa, Tinea imbricata, Tinea manuum, Tinea nigra (palmaris), Tinea pedis, Tinea unguium, Torulopsosis, Trichomycosis axillaris, White piedra, and their synonyms, to severe systemic or opportunistic infections, such as, but not limited to, Actinomycosis, Aspergillosis, Candidiasis, Chromomycosis, Coccidioidomycosis, Cryptococcosis, Entomophthoramycosis, Geotrichosis, Histoplasmosis, Mucormycosis, Mycetoma, Nocardiosis, North American Blastomycosis, Paracoccidioidomycosis, Phaeohyphomycosis, Phycomycosis, Pneumocystic pneumonia, Pythiosis, Sporotrichosis, and Torulopsosis, and their synonyms, some of which may be fatal. Known fungal and mycotic pathogens include, but are not limited to, Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria boydii, Alternaria spp., Anthopsis deltoidea, Apophysomyces elegans, Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Candida spp., Cephalosporium spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis, Conidiobolus spp., Corynebacterium tenuis, Cryptococcus spp., Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp., Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Madurella spp., Malassezia furfur, Microsporum spp., Mucor spp., Mycocentrospora acerina, Nocardia spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phoma spp., Piedraia hortai, Pneumocystis carinii, Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizopus spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Wangiella dermatitidis, Xylohypha spp., Zygomyetes spp. and their synonyms. Other fungi that have pathogenic potential include, but are not limited to, Thermomucor indicae-seudaticae, Radiomyces spp., and other species of known pathogenic genera.
In addition, the invention may prove useful in controlling protozoan or macroscopic infections by organisms such as Cryptosporidium, Isospora belli, Toxoplasma gondii, Trichomonas vaginalis, Cyclospora species, for example, and for Chlamydia trachomatis and other Chlamydia infections such as Chlamydia psittaci, or Chlamydia pneumoniae, for example.
Certain embodiments of the invention pertain to methods utilizing compositions that include an expression cassette. In particular, in certain embodiments, administering the composition to the subject may involve providing to the subject an expression cassette comprising a promoter, active in the subject, operably linked to a polynucleotide encoding an MDA-7 polypeptide. In certain embodiments that pertain to suppressing or preventing a viral infection of a cell, the MDA-7 polypeptide or nucleic acid encoding the MDA-7 polypeptide may comprise an expression cassette that includes a promoter, active in the cell, operably linked to a polynucleotide encoding an MDA-7 polypeptide.
Throughout this application, the term "expression cassette" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein or polypeptide, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a mRNA into a polypeptide.
In order for the expression cassette to effect expression of a polypeptide, the polynucleotide encoding the polynucleotide will be under the transcriptional control of a promoter. A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrase "operatively linked" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. One of skill in the art would understand how to use a promoter or enhancer to promote expression of an MDA-7 polypeptide.
In certain embodiments of the invention, the delivery of an expression cassette in a cell may be identified in vitro or in vivo by including a marker in the expression vector. The marker would result in an identifiable change to the transfected cell permitting easy identification of expression. The selectable marker employed is not believed to be important, so long as it is capable of being expressed along with the polynucleotide of the expression cassette. Examples of selectable markers are well known to one of skill in the art.
A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.
In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). One of skill in the art would be familiar with use of IRES in expression cassettes.
Expression cassettes can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. See Carbonelli et al. (1999); Levenson et al. (1998); Cocea (1997). "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. One of skill in the art would understand how to use these signals to effect proper polyadenylation of the transcript.
In certain embodiments of the present invention, the expression cassette comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis and, in some cases, integrate into the host cell chromosomes, have made them attractive candidates for gene transfer in to mammalian cells. However, because it has been demonstrated that direct uptake of naked DNA, as well as receptor-mediated uptake of DNA complexes, expression vectors need not be viral but, instead, may be any plasmid, cosmid or phage construct that is capable of supporting expression of encoded genes in mammalian cells, such as pUC or Bluescript™ plasmid series. One of ordinary skill in the art would be familiar with use of viruses as tools to promote expression of the polypeptide.
In certain embodiments of the invention, a treated cell may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
Certain embodiments of the present invention pertain to methods wherein the expression cassette that includes a promoter, active in the subject or cell, is operably linked to a polynucleotide encoding an MDA-7 polypeptide, and wherein the expression cassette is carried in a viral vector. In other embodiments, the expression cassette is carried in a nonviral vector.
1. Viral Transformation
One method for delivery of the expression cassette involves the use of an viral expression vector. Any viral expression vector is contemplated for application in the present invention.
a. Adenoviral Infection
In certain embodiments of the present invention, the viral vector is an adenoviral vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a recombinant gene construct that has been cloned therein.
The vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5-tripartite leader (TPL) sequence which makes them some mRNA's for translation.
In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.
Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the some helper cell line is 293.
Racher et al. (1995) have disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
The adenovirus vector may be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the some starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region. Thus, it will be most convenient to introduce the transforming construct at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109-1013 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Animal studies have suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).
b. Retroviral Infection
The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).
Concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, packaging cell lines are available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).
c. AAV Infection
Certain embodiments of the present invention involve use of a viral expression vector wherein the vector is an adeno-associated virus (AAV) vector. AAV is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988), which means it is applicable for use with the present invention. Details concerning the generation and use of rAAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated herein by reference.
Studies demonstrating the use of AAV in gene delivery include LaFace et al. (1988); Zhou et al. (1993); Flotte et al. (1993); and Walsh et al. (1994). Recombinant AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al., 1994; Lebkowski et al., 1988; Samulski et al., 1989; Shelling and Smith, 1994; Yoder et al., 1994; Zhou et al., 1994; Hermonat and Muzyczka, 1984; Tratschin et al., 1985; McLaughlin et al., 1988) and genes involved in human diseases (Flotte et al., 1992; Ohi et al., 1990; Walsh et al., 1994; Wei et al., 1994). Recently, an AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis.
AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992). In the absence of coinfection with helper virus, the wild-type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al., 1990; Samulski et al., 1991). rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and Smith, 1994). When a cell carrying an AAV provirus is superinfected with a helper virus, the AAV genome is "rescued" from the chromosome or from a recombinant plasmid, and a normal productive infection is established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990; Muzyczka, 1992).
Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; each incorporated herein by reference) and an expression plasmid containing the wild-type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference). The cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function. rAAV virus stocks made in such fashion are contaminated with adenovirus which must be physically separated from the rAAV particles (for example, by cesium chloride density centrifugation). Alternatively, adenovirus vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the adenovirus helper genes could be used (Yang et al., 1994; Clark et al., 1995). Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al., 1995).
d. Other Viral Vectors
Other viral vectors may be employed as constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
A molecularly cloned strain of Venezuelan equine encephalitis (VEE) virus has been genetically refined as a replication competent vaccine vector for the expression of heterologous viral proteins (Davis et al., 1996). Studies have demonstrated that VEE infection stimulates potent CTL responses and has been suggested that VEE may be an extremely useful vector for immunizations (Caley et al., 1997). It is contemplated in the present invention, that VEE virus may be useful in targeting dendritic cells.
With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. Chang et al. (1991) recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991).
In still further embodiments of the present invention, the nucleic acid encoding a MDA-7 to be delivered is housed within an infective virus that has been engineered to express a specific binding ligand. Alternatively, the nucleic acid encoding the MDA-7 polypeptide to be delivered is housed within an infective virus that has been engineered to express an immunogen. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via asialoglycoprotein receptors.
For example, to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).
2. Non-Viral Delivery
In addition to viral delivery of the nucleic acid encoding a MDA-7 protein, the following are additional methods of recombinant gene delivery to a given host cell and are thus considered in the present invention.
a. Lipid Mediated Transformation
In a further embodiment of the invention, the gene construct may be entrapped in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is a gene construct complexed with Lipofectamine (Gibco BRL).
Lipid-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated the feasibility of lipid-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
Lipid based non-viral formulations provide an alternative to adenoviral gene therapies. Although many cell culture studies have documented lipid based non-viral gene transfer, systemic gene delivery via lipid based formulations has been limited. A major limitation of non-viral lipid based gene delivery is the toxicity of the cationic lipids that comprise the non-viral delivery vehicle. The in vivo toxicity of liposomes partially explains the discrepancy between in vitro and in vivo gene transfer results. Another factor contributing to this contradictory data is the difference in lipid vehicle stability in the presence and absence of serum proteins. The interaction between lipid vehicles and serum proteins has a dramatic impact on the stability characteristics of lipid vehicles (Yang and Huang, 1997). Cationic lipids attract and bind negatively charged serum proteins. Lipid vehicles associated with serum proteins are either dissolved or taken up by macrophages leading to their removal from circulation. Current in vivo lipid delivery methods use subcutaneous, intradermal, intratumoral, or intracranial injection to avoid the toxicity and stability problems associated with cationic lipids in the circulation. The interaction of lipid vehicles and plasma proteins is responsible for the disparity between the efficiency of in vitro (Felgner et al., 1987) and in vivo gene transfer (Zhu et al., 1993; Philip et al., 1993; Solodin et al., 1995; Liu et al., 1995; Thierry et al., 1995; Tsukamoto et al., 1995; Aksentijevich et al., 1996).
Recent advances in lipid formulations have improved the efficiency of gene transfer in vivo (Smyth-Templeton et al., 1997; WO 98/07408). A novel lipid formulation composed of an equimolar ratio of 1,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP) and cholesterol significantly enhances systemic in vivo gene transfer, approximately 150-fold. The DOTAP:cholesterol lipid formulation is said to form a unique structure termed a "sandwich liposome". This formulation is reported to "sandwich" DNA between an invaginated bi-layer or 'vase' structure. Beneficial characteristics of these lipid structures include a positive colloidal stabilization by cholesterol, two dimensional DNA packing and increased serum stability.
The production of lipid formulations often is accomplished by sonication or serial extrusion of liposomal mixtures after (I) reverse phase evaporation (II) dehydration-rehydration (III) detergent dialysis and (IV) thin film hydration. Once manufactured, lipid structures can be used to encapsulate compounds that are toxic (chemotherapeutics) or labile (nucleic acids) when in circulation. Lipid encapsulation has resulted in a lower toxicity and a longer serum half-life for such compounds (Gabizon et al., 1990). Numerous disease treatments are using lipid based gene transfer strategies to enhance conventional or establish novel therapies, in particular immune therapies.
In certain embodiments of the invention, the lipid vehicle may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of lipid-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the lipid vehicle may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the lipid vehicle may be complexed or employed in conjunction with both HVJ and HMG-1.
1. Therapeutic Amounts
One of ordinary skill in the art would be familiar with administration of a therapeutic amount of a composition to a subject. Whether a certain amount of a composition is therapeutic depends on the goal of the treating physician. For example, in some embodiments of the present invention, the goal is prevention of pathogen infection. In other embodiments, the goal is suppression of an already infected subject or cell. One of ordinary skill in the art would be familiar with clinical endpoints for measuring response in a subject. An endpoint may be, for example, improvement in clinical symptoms of a previously infected subject, or absence of active symptoms of infection where the goal is prevention.
One of ordinary skill in the art would be familiar with dosing regimens which can be applied in the context of the present invention. For example, the composition may be administered continuously over a defined period of time, such as over 1 hour, at periodic intervals over the course of a day. A course of therapy may last for a period of time as determined by one of ordinary skill in the art.
The treatments may include various "unit doses." Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the present invention may conveniently be described in terms of plaque forming units (pfu) or viral particles for a viral construct. Unit doses range from 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013 pfu or viral particles (vp) and higher.
Protein may be administered to a patient in doses of or of at least about 0.01 ng/ml or any amount greater than 0.01 mg/ml.
2. Injectable Compositions and Formulations
Any method of administration of the therapeutic compositions of the present invention is contemplated. One of ordinary skill in the art would be familiar with the wide range of methods of delivery of a therapeutic composition to a subject. For example, the composition may be administered parenterally, intravenously, intradermally, intramuscularly, or even intraperitoneally as described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363, which are specifically incorporated herein by reference in their entirety.
Injection of nucleic acid constructs may be delivered by syringe or any other method used for injection of a solution, as long as the expression construct can pass through the particular gauge of needle required for injection. A novel needless injection system has recently been described (U.S. Pat. No. 5,846,233) having a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery. A syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Pat. No. 5,846,225).
Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
3. Combination Treatments
Certain embodiments of the present invention involve administering to the subject or contacting the cell with at least one additional agent to prevent or suppress the infection of the subject or cell, respectively.
In some embodiments, the MDA-7 therapy is used in conjunction with another antiviral agent. One of ordinary skill in the art would be familiar with the wide range of antiviral agents that are available.
In other embodiments, the MDA-7 therapy is used in conjunction with an agent that prevents or suppresses infection by acting as an immunogenic agent.
The MDA-7 therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
Various combinations may be employed, for example gene therapy is "A" and the immunogenic molecule given as part of an immune therapy regime, such as an antigen, is "B":
Administration of the therapeutic expression constructs of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
In yet another embodiment, the MDA-7 therapy is provided as part of a therapeutic regimen that involves the administration of one or more other forms of gene therapy. Delivery of a vector encoding mda-7 in conjunction with a second vector encoding another agent that can prevent or suppress infection by a pathogen will have a combined inducing effect on target tissues. Alternatively, a single vector encoding both genes may be used.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
1. Cell Lines and Cell Culture
All the tumor cell lines utilized were obtained from American Type Culture Collection (ATCC, Rockville, Md.). The cell lines evaluated were: lung (H1299, A549), prostate (PC3), MCF-7 (breast) and 293 HEK cells. The cells were cultured according to supplier's recommendation. Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics, (Walkersville, Md.). The cells were free of mycoplasma and were used in the log phase of growth. Cells were harvested with 0.125% Trypsin-1.3 mM EDTA (GIBCO-BRL, Life Technologies, Grand Island, N.Y.). Brefeldin A and Tunicamycin were purchased from Sigma Chemicals (St. Louis, Mo.).
2. Plasmids
pSHOOTER expression plasmids (Invitrogen, Carlsbad, Calif.) direct subcloned proteins to the cytoplasm, nucleus, or endoplasmic reticulum (ER). Each vector construct (containing an engineered C-terminal myc tag) was used to subclone mda-7 cDNA. The vector directing proteins to the cytoplasm contains a standard expression vector backbone, while the vectors directing proteins to the nucleus and ER contain signal sequences appropriate to those compartments. The nuclear targeting vector has an N-terminal nuclear targeting signal, while the ER targeting vector has an N-terminal ER signal peptide sequence and a C-terminal ER retention sequence. mda-7 was subcloned into these vectors using PCR to delete both the stop codon and the region encoding the first 48 amino acids, constituting the secretion signal, from full length mda-7 cDNA. PCR was also used to provide restriction sites compatible with the Invitrogen targeting vectors, and in frame with the C-terminal myc tag contained in the vectors. The primers sequences used for cloning are as follows: forward PCR primer (with SalI site): ttttttGTCGACatggcccagggccaagaattcc (SEQ ID NO: 3); Reverse PCR primer (with NotI site): ttttttGCGGCCGCgagcttgtagaatttctgc (SEQ ID NO: 4). All plasmids were sequenced to confirm orientation and in-frame sequence.
3. Recombinant Adenovirus Production and Gene Transfer
Construction and purification of replication-deficient human type 5 Adenovirus (Ad5) carrying the mda-7 gene was described previously (Mhashilkar et al., 2001). Cell lines were infected with Ad-mda7 or Ad-luc with indicated MOIs. Cells were plated at 105-106 cells/well in a 6-well plate format for protein expression, trypan-blue exclusion assay or apoptosis assays. For transfection with pShooter vectors, 1 μg of plasmid DNA was used to transfect cells using lipofectamine protocol as described (Mhashilkar et al., 2003).
4. Microarray Analysis
MICROMAX Human cDNA System I—Direct kit (NEN Life Science Products, Inc.) containing 2400 human cDNAs were used. mRNA was isolated from H1299 cells treated with Ad-mda7 or Ad-luc (1000 vp/cell for 24 hr) and analyzed according to manufacturer's instructions.
5. Western Blot Analyses
Cell lysates (105-106 cells) were suspended in 500 μl of Laemmli buffer with 5% 2-mercapto-ethanol (2ME) or supernatants (1:1 mixing with Laemmli buffer+2ME), were analyzed by SDS gel electrophoresis and western blot analysis. Expression of various proteins was determined by using the following primary antibodies: rabbit or mouse anti-human MDA-7 (Introgen Therapeutics, Inc), goat anti-human BiP/GRP-78, rabbit anti-human GADD34, goat anti-human PP2A, goat anti-human PERK, rabbit anti-human XBP-1 (Santa Cruz Biotechnologies, Santa Cruz, Calif.), rabbit anti-human caspase 7 and caspase-9 (Cell Signaling Technology, Beverly, Mass.). Antibodies against beta-actin, and alpha-tubulin were purchased from Sigma. Following incubation with horseradish peroxidase-labeled secondary antibody (Amersham Biosciences, Piscataway, N.J.) the proteins were visualized on enhanced chemiluminescence film (Hyperfilm, Amersham Biosciences, Piscataway, N.J.) by application of Amersham's enhanced chemiluminescence western blotting detection system. All westerns were performed 2-4 times.
6. Production of Recombinant MDA-7 Protein
Secreted MDA-7 protein was purified from the supernatants of MDA-7 baculovirus-infected cells. Briefly, Hi-Five cells (Invitrogen) were infected with MDA-7 baculovirus at an MOI of 5, and supernatants harvested after 72 hours of growth. For purification, polyclonal rabbit anti-MDA-7 antibody was cross-linked to immobilized protein G using the Seize-X Kit (Endogen), and incubated with supernatant at 4� C. for 4 hours. After washing, bound protein was eluted according to manufacturer's instructions, neutralized, and dialyzed against PBS. MDA-7 protein-containing fractions were verified by Western blot and quantified by ELISA. Bacterial MDA-7 was produced by cloning the human mda-7 cDNA (residues 55-206) into the p202 expression vector (kindly provided by Dr. Jim Sacchetini, TAMU). This plasmid expresses a His(6)/Maltose Binding Protein (MBP)/MDA-7 fusion protein after induction with IPTG. Recombinant His(6)-MBP-MDA-7 protein was partially purified with Ni-NTA Agarose resin (Qiagen). The protein was eluted from the column and MDA-7 was released from the His(6)-MBP carrier protein by overnight cleavage with TEV protease. The MDA-7 protein was purified to homogeneity by anion exchange chromatography.
7. Immunofluorescence Assays
Cells were seeded into multi-well chamber slides and transfected with plasmid DNA or transduced with Ad-vectors. Untreated cells served as controls in these experiments. Forty-eight hours after transfection, cells were washed in PBS, fixed with cold ethanol:acetic acid (95:5 vol/vol), and stained for MDA-7 using mouse anti-human MDA-7 antibody and Texas Red-conjugated anti-mouse secondary antibody (Vector Laboratories, Burlingame, Calif.). Slides were mounted using anti-fade mounting reagent (Vector Laboratories) and analyzed using a Nikon Fluorescent microscope. Photographs were taken using a Nikon Digital camera DXM1200 System. All assays were performed at least 3 times.
8. Cell Viability Assays
Cell viability was determined by trypan blue exclusion assay or Live/Dead assay. Cells were trypsinized and a small aliquot was suspended 1:1 volume with 0.1% trypan blue. Total cell numbers and cell viability counts were assessed using a hemocytometer under light microscopy. For Live/Dead assay (Molecular Probes, Eugene Oreg.) cells were plated in chamber slides (Nunc) and then treated with pShooter-plasmid vectors or with Ad− vectors. Two-four days later the cells were treated with the probes, Calcein AM and Ethidium homodimer (both at 5 uM final conc. in PBS), for 30 minutes and then live (green) and dead (red) cells were counted via fluorescence microscopy. All assays were performed at least three times.
9. FACS Analyses and Annexin V Assay
Cells were analyzed for apoptosis using the ApoAlert Annexin V-FITC kit (CLONTECH). Briefly, vector-transduced cells (105-106 cells total) were washed extensively in PBS and then incubated with Annexin V-FITC reagent for 30 minutes on ice. Cells were then washed with PBS and processed for FACS analysis and fluorescence microscopy as above. Assays were performed 2-3 times.
10. Statistical Analysis
The statistical significance of the experimental results was calculated using Student's t-test. The level of significance was set at p<0.05.
1. Ad-mda7 Induces Apoptosis and Secretion of MDA-7 Protein
The mda-7 coding region possesses an unusually long leader sequence (48 amino-acids). Analysis of the predicted primary amino-acid sequence indicates that the MDA-7 protein contains a prototypic signal sequence, which is likely responsible for directing secretion of the protein (
H1299 NSCLC tumor cells were transduced with Ad-mda7 or Ad-luc and analyzed for cytotoxicity. H1299 cells were killed in a temporal- and dose-dependent manner by Ad-mda7 (
High levels of MDA-7 (>100 ng/ml) are secreted from tumor cells (
2. Ad-mda7 Activates Stress Proteins Associated with UPR (Unfolded Protein Response)
Previous studies have demonstrated that Ad-mda7 transduction of tumor cells results in activation of molecules involved in apoptosis and death signaling pathways (p53, BAX, BAK, TRAIL, c-jun, JNK, caspases, p38, PI3K and beta-catenin) (Mhashilkar et al., 2001; Saeke et al., 2000; Saeki et al., 2002; Pataer et al., 2002; Sarkar et al., 2002; Kawabe et al., 2002; Mhashilkar et al., 2003). Microarray analyses performed on Ad-mda7 treated H1299 cells indicated activation of a number of additional stress-related genes, especially those involved in regulation of protein folding and secretion (TABLE 3).
TABLE 3 demonstrates microarray analysis of Ad-mda7 gene regulation in H1299 cells. Cells were treated with Ad-mda7 or Ad-luc (1000 vp/cell for 24 hr) and mRNA isolated. The samples were used to evaluate mRNA changes in transduced cells by performing microarray analyses using the MICROMAX (NEN) arrays. Analyses of datasets showed regulation of stress and protein folding pathways (n.d.: no data). Ad-mda7 induced protein changes in H1299 cells. Cells were treated with Ad-luc or Ad-mda7 and protein changes in MDA-7 expressing cells relative to control cells were evaluated using western analyses. All assays were performed at least twice.
MDA-7 Regulates Stress Genes involved in Protein-Folding
Hsp 70-5
BiP/grp 78
↑ 12.8
↑ 5
Hsp 90
ATP-regulated molecular chaperone
↓ 2.0
↑ <1.5
tra 1
ER heat shock protein (grp 94/gp 96)
↑ 8.4
Heat shock protein
↑ 7.7
Hsp 60
↑ 1.4
Hsp 70-1
↑ 1.4
↑ <1.5
protein kinase
overexpression leads to tumor
↓ 2.7
inhibitor p58
formation in mice
Protein phosphatase 2A; ser/thr p'tase
↑ 2.8
↑ 2.5
Strong regulation, at a transcriptional level of PP2A, hsp 70-5 (BiP), HSJ1 and tra 1, was noted. Therefore, protein expression of various stress-related molecules (BiP, GADD34, PP2A) was evaluated after Ad-mda7 transduction of H1299 cells (
3. MDA-7 is Heavily Glycosylated
The higher molecular weight of the secreted MDA-7 protein, as shown in
4. Secreted MDA-7 is Not Required for Cell Death in NSCLC Cells
The cytotoxicity induced by Ad-mda7 in the presence of these glycosylation and secretion inhibitors was next evaluated. TUN and BFA levels were titrated until inhibition of secretion of MDA-7 protein was detected. As shown in TABLE 3 and
In BFA treated cells, the 19 kD unglycosylated protein is observed, in addition to a higher molecular weight smear indicative of partially mature glycosylated products which are blocked in transport from the ER. Note that no maturation to the 40 kD secreted product is evident. Additional lower molecular weight species are observed; these are likely degradation products. Blocking secretion of MDA-7 using glycosylation inhibitors such as TUN or BFA did not abrogate cell killing by Ad-mda7 (TABLE 3,
To address whether apoptosis induction is due to intracellular MDA-7 or is simply a non-specific effect due to protein accumulation in the ER, we performed similar experiments using an adenoviral vector encoding endostatin as a control glycosylated secreted protein. Neither BFA nor tunicamycin significantly modulated cell killing due to Ad-luc or Ad-endostatin (
Intracellular MDA-7 Mediates Apoptosis in H1299 cells
Annexin V
MDA-7 Protein
Ad-mda7 + Tun.
H1299 cells were treated with 1000 vp/cell Ad-mda7 and the indicated concentrations of tunicamycin. Cells were harvested 24 hr later and analyzed for intracellular and secreted MDA-7 protein by western blot and apoptosis via Annexin V assay. Control cultures of cells treated with only tunicamycin were also assessed for apoptosis (Tun. Toxicity).
These results suggest that it is the intracellular and not the secreted form of MDA-7 that is primarily responsible for eliciting cell death in H1299 cells. This hypothesis was tested by adding secreted MDA-7 protein to na�ve lung tumor cells and monitoring cell death. Initial studies adding supernatant from Ad-mda7 transduced cells did indicate cell death; however careful analysis demonstrated that the cell death was caused by residual Ad-mda7 vector in the culture supernatant. When culture supernatants were treated to minimize Ad-mda 7 contamination, negligible cell death was induced by MDA-7 containing supernatants. To eliminate any confounding aspect of Ad vector toxicity, this study was repeated using MDA-7 containing supernatant from stably expressing 293-mda7 cells applied to tumor cell cultures (
5. Subcellular Targeting of MDA-7
The question of whether MDA-7 was being released into the cytosol or nucleus during supraphysiologic expression in Ad-mda7 infected cells and whether this was responsible for inducing death was next addressed. To investigate the effects of subcellular localization of MDA-7 protein on cell viability, we constructed expression vectors designed to target MDA-7 expression to different subcellular compartments. In constructing these re-targeting vectors, the secretion signal sequence in the mda-7 cDNA was first deleted and an ATG inserted to generate an initiator codon. As shown in
The vectors were transiently transfected into H1299 cells, and subsequent targeted MDA-7 protein expression analyzed by immunocytochemistry. As shown in
Addition of the myc tag did not adversely affect MDA-7 protein stability as a control full length mda-7 expression plasmid (without a myc tag) expressed comparable steady-state levels of MDA-7 protein. The myc tag did not appear to interfere with protein processing or secretion as the full length myc-tagged protein showed 2 intracellular MDA-7 bands similar to the full length MDA-7 protein expressed by full length plasmid or Ad-mda7 (
6. Subcellular Localization of MDA-7 Determines Cytotoxicity
Hoechst staining of nuclei was used as a screen to evaluate cytotoxic effects of re-targeted MDA-7 expression. Nuclear or cytoplasmic targeted MDA-7 expression had no effect on nuclear morphology. Cells containing FL and ER-localized MDA-7 protein, however, had disrupted nuclear morphology indicative of apoptosis. To determine the anti-tumor effects of targeted MDA-7 protein expression, cell viability, apoptosis induction and colony formation activity in a panel of tumor cell lines was evaluated.
Cell viability was assessed using a fluorescence-based assay, which uses calcein staining to identify viable cells (green). When cells die, they become membrane permeant and take up EtBr dye and stain red. Transient transfection of plasmid constructs into H1299 lung cancer cells demonstrated high level expression of targeted MDA-7 proteins (
The targeted mda-7 expression constructs incorporate a neomycin resistance marker and were stably transfected into various tumor cell lines and assessed for colony formation (
Materials and Methods
1. Clinical Protocol.
An open-label, Phase I, dose-escalation study was conducted to evaluate safety and determine the MTD of the adenovirus-mda-7 construct, INGN 241, when administered intratumorally to advanced cancer patients. The pharmacokinetics of INGN 241 DNA, MDA-7 protein, and the humoral immune response to INGN 241 were evaluated in order to better understand their effects on both safety and efficacy.
Eligible patients with defined entry criteria were enrolled into one of 8 treatment cohorts (TABLE 5) and the dominant, symptom-causing tumor was identified and treated as the indicator tumor.
Patient Cohorts and Treatment Profile
Diagnosis of Evaluable
no (no.
of pts)
No. of Viral Particles
(no. tested)
1 (1)
2 � 1010
+24 hrs
BrCa (1)
2 (1)
2 � 1011
+24 hrs
CoCa (1)
3 (3)
2 � 1012
+24 hrs
SCCHN (1), Mel (1), LCL (1)
4 (3)
2 � 1012
+48 hrs
BrCa (1), CoCa (1), ChCa (1)
5 (2)
2 � 1012
+96 hrs
BrCa (1), Mel (1)
6 (1)
2 � 1012, divided dose
+48 hrs
Mel (1)
7 (5)
2 � 1012
Day 30
TCC (1), Mel (1), CoCa (1),
8 (5)
2 � 1012;
Day 30
Mel (3), SCCHN (2)
twice weekly � 3
1BrCa, breast carcinoma; CoCa, colon carcinoma; SCCHN, squamous cell carcinoma of the head and neck, Mel, melanoma; LCL, large cell lymphoma; ChCa, cholangiocarcinoma; TCC, transitional cell carcinoma.
The first two cohorts (1 patient each) received 2�1010 virus particles (vp) or 2�1011 vp (cohort 2). Excisional biopsies were obtained at 24 hours post treatment. Cohorts 3, 4 and 5 (3 patients per cohort) received 2�1012 vp, and had excisional biopsies at 24 hours (cohort 3), 48 hours (cohort 4), and 96 hours (cohort 5), respectively. Each resected lesion was serially sectioned and analyzed to determine the radius of diffusion of injection solution, distribution and concentration of the viral agent, transgene protein expression, and the resulting biologic outcome. One patient was enrolled into cohort 6 and receive 2�1012 vp in divided doses administered to different sections of the indicator tumor and excisional biopsies were obtained at 48 hours.
To assess longer term effects of MDA-7 expression, 5 patients with unresectable disease were entered into cohort 7, who receive 2�1012 vp and underwent incisional or core biopsies at pretreatment and at 30 days post treatment. Cohort 8 included 5 patients who received single injections of 2�1012 vp twice a week for 3 weeks (total of 6 injections per cycle). All patients were analyzed throughout the trial for development of toxicity that may be related to either the agent or the injection. The clinical protocol was approved by the US Oncology Institutional Review Board (IRB). All human subjected-related protocols for laboratory analyses that were performed at Baylor University Medical Center were reviewed and approved by the IRB for Human Protection at Baylor University Medical Center.
INGN 241 comprises a replication defective Ad5 backbone with E1 and partial E3 deletions. An expression cassette comprising the CMV IE promoter, the wild type mda-7 transgene ORF and the SV40 polyadenylation sequence were cloned into the E1 region of the construct. Vector was double-plaque-purified and correct sequence confirmed as described (Mhashilkar et al., 2001). Clinical material was prepared under cGMPs and complies with guidances for testing for freedom from adventitious agents. INGN 241 was provided as a frozen vial suspension (3.0 m, 1�1012 vp/ml) in a neutral buffer containing saline and 10% glycerol. The vials were thawed to ambient temperature and mixed with a tracking dye (Isosulfan blue) immediately prior to injection.
3. Immunohistochemical Analysis
A previously described automated immunoperoxidase staining technique (Nemunaitis, 1998) was used to characterize MDA-7 protein expression. Briefly, serial sections of formalin-fixed, paraffin-embedded tissue block were deparaffinized in xylene and graded alcohols, then incubated with 1% H2O2 with appropriate washings. Antigen retrieval was carried out with 0.01 M citrate buffer in the microwave, followed by incubations in H2O2 in methanol, 0.05% Triton-X 100 washed in phosphate buffered saline (PBS), and normal goat serum. MDA-7 expression was determined with the avidin-biotin-complexed immunoperoxidase reaction (DAB detection kit, Ventana Medical System) following initial incubation with affinity-purified rabbit anti-human MDA-7 antibody (Introgen Therapeutics), using the Ventana 320ES System (Ventana Medical Systems, Tucson, Ariz.). The reaction was compared with a negative control antibody stained slide and graded in a blinded fashion as negative (−), weak ("+", ≦25% positive cells), moderate ("++", 26-50% positive cells), or strong ("+++", >50% positive cells). Ad-mda 7 transduced cells were used as a positive control.
Determinations of beta-catenin and iNOS expression were carried out in the same manner, following treatment with the relevant primary antibody (mouse anti-human β-catenin antibody C19220, 2.5 ug/ml, BD Biosciences; mouse anti-human iNOS monoclonal antibody N32020, 5 ug/ml; BD Pharmingen).
4. Quantification of Apoptosis by TUNEL
A TUNEL method (DeadEnd Colorimetric Apoptosis Detection Systems, Promega) was used to detect DNA fragmentation in situ. Briefly, tissue sections were deparaffinized in xylene/graded alcohol, and fixed with in 4% paraformaldehyde in PBS before and after proteinase K treatment (20 μg/ml, 15 min, 23 C). An in situ Tdt (terminal deoxynucleotidyl transferase) reaction was carried out with biotinylated nucleotides according to manufacturer's protocols. Apoptotic cells were identified by light microscopy as cells with definitive brown staining in the nucleus that were rounded or shrunken.
5. Serum Cytokine Analysis
ELISA assays (R&D Quantikine kits, Minneapolis, Minn.) were used to quantify patient serum cytokine levels at defined time points before and after treatment as described previously (Nemunaitis '01). Briefly, patient peripheral blood was collected by venipuncture. Serum samples were extracted after clotting and stored at −80� C. Serial serum samples from the same patient were analyzed simultaneously, using cytokine-specific immunoassay reagents according to manufacturer's protocols. The colorimetric reaction was quantified as a function of OD absorbance at 540 nm (SpectraMax 340, Molecular Devices, Sunnyvale, Calif.). Cytokine concentration was calculated according to a reference standard curve and OD values of known, graded concentrations of the recombinant cytokine. The minimal detectable concentration, defined by OD readings at ≧3-fold higher than background, is as follows: IFNγ: <3 pg/ml; TNFα: <4 pg/ml; IL-13β: <1 pg/ml; IL-10: <2 pg/ml; IL-2: <7 pg/ml; IL-6: <0.7 pg./ml; GM-CSF: <3 pg/ml. The % increase in cytokine level at any time point post-treatment was determined through comparison with the baseline level in serum harvested before INGN 241 injection. Based on inter- and intra-sample variations, increases in cytokine level of ≧50% over baseline were considered significant.
6. Flow Cytometric Immunophenotype Analysis
Peripheral blood immunophenotype analysis was carried by a two color immunofluorescence reaction and flow cytometric analysis as described previously (Zhang, 2000). Briefly, patient or normal healthy donor peripheral blood was collected by venipuncture. 100 μl of whole blood was treated with 20 μl of the following reactant mixtures in order to determine the frequency distribution of T, B and NK cell subsets: CD45-FITC/CD14-PE, CD3-FITC/CD19-PE, CD4-FITC/CD8-PE, CD13-FITC, CD20-FITC, CD56-FITC (all from BD Biosciences). The reactants were fixed with 1% paraformaldehyde before flow cytometric analysis (Becton Dickinson FACScan) with the CELLQUEST software (Becton Dickinson).
7. Anti-Adenovirus Antibodies
Serum samples were evaluated for anti-Adenovirus type 5 antibodies using an indirect immunofluorescence assay to detect humoral immune responses against vector as previously reported (Nemunaitis et al., 2000). Post-treatment samples were compared to based one pre-treatment anti-Ad antibody titers (defined as 1).
8. Detection of Viral DNA and RNA
Tumor samples were evaluated for the presence of INGN 241 DNA, mRNA expression from INGN 241, and the expression of a panel of cytokine genes using real-time PCR, TaqMan chemistry, and the ABI Prism's 7700 Sequence Detection System. A real-time PCR method was developed to target the junction of the vector CMV promoter with the 5′ end of mda-7 cDNA. Each specimen's DNA was analyzed in duplicate reactions containing 100 ng total DNA. A third reaction was spiked with 100 copies of the target to check for inhibitors of the PCR. INGN 241 derived RNA was measured using a two-step RT-PCR method. First, cDNA was generated from total tumor RNA in a reverse transcription reaction primed with random hexamers. The cDNA was then amplified using the INGN 241 qPCR assay. The number of copies of INGN 241 transcripts per microgram of total RNA was calculated using a standard curve of INGN 241 in vitro runoff transcripts. The expression of tumor mRNAs for human interleukin 6, interleukin 10, and interferon-gamma was determined using Applied Biosystems assay reagents. Expression of each cytokine was analyzed relative to the expression of human GAPDH.
9. INGN 241 qPCR assay primers and probe sequences
1. Transgene Expression Following Intratumoral Injection with INGN 241
Patients were stratified into 8 cohorts according to viral dose (2�1010 to 2�1012), time for post-treatment biopsy (24 h to 30 days), and treatment mode (single, divided dose or multiple injections) as described above (see TABLE 5). To better understand the safety and efficacy of INGN 241 treatment, cell and molecular analyses were performed to characterize transgene expression and pharmacokinetics, biologic activity, and immune response.
For patients entered into cohorts 1-6, the vector treated tumor was resected at 24-96 hours post-injection. Serial sections were evaluated for vector penetration and biologic outcome. All pretreatment biopsies were MDA-7 negative by IHC. All INGN 241 injected lesions showed high levels of MDA-7 immunoreactivity in the center (injection site). Signal intensity was reduced at the periphery, although 6 of 8 lesions still showed MDA-7 immunoreactivity (
Cohorts 3-5 received the same intratumoral viral dose (2�1012 vp) whereas the injected tumor was biopsied at 24, 48, and 96 hrs respectively. This limited analysis with 2-3 patients per time point nonetheless illustrated the sustained expression of the MDA-7 protein (
2. Tumor Apoptotic Activity Correlated with MDA-7 Penetration
Pronounced apoptotic activity was observed in INGN 241-injected tumors in areas of MDA-7 expression, when the injected lesions were resected after 24-96 hours Apoptotic induction was highest in the high dose group (2�1012 vp) and could still be detected at the tumor periphery in 4 of 6 evaluable patients (
To correlate apoptotic induction with MDA-7 expression, TUNEL reactions in serial sections from individual injected tumors were contrasted with their pattern of MDA-7 immuno-histochemical reactivity. A graded pattern of MDA-7 expression was consistently observed, with up to 90% of MDA-7-positive tumor cells at the site of injection. Peripheral sections generally displayed proportionately decreased MDA-7 expression (0-25%). The frequency of apoptosis displayed a similarly graded pattern which correlated with MDA-7 expression (
3. INGN 241 Pharmacokinetics
Further evaluations of intratumoral INGN-241 pharmacokinetics were carried out by quantifying vector DNA recovered from serial sections of injected tumors, and the presence of viral DNA in plasma of patients over time. The total number of vector DNA copies in the tumor was compared with total vector injected to yield the % vector recovered from tumor as a function of time. 11.7% of total viral DNA remained in the injected tumor lesion after 24 hours, which was reduced to 1.7% and 0.03% of input load at days 2 and 4. Circulating INGN 241 vector was evaluated in patient serum using quantitative DNA PCR. INGN 241 vector DNA was transiently detected in plasma in both a dose and time dependent manner. INGN 241 was detectable in plasma within 30 minutes after intratumoral injection in all patients analyzed. In cohort 1 and 2 patients, plasma vector was undetectable by 24 hr whereas higher doses of vector were not cleared until 24-72 hr post-injection. In all patients evaluated, circulating vector was no longer detectable after 72 hours. The maximal amount of injected vector detected in the circulation constituted approximately 3% of the intratumorally injected dose. The level of circulating viral DNA in patient plasma represented 0.0025% (5.1�107 vp/ml in an estimated total plasma volume of 3 liters) of input load, based on DNA decay analysis.
4. Molecular Outcome of Intratumoral INGN-241 Treatment
Immunohistochemical evaluations were carried out to quantify β-catenin and iNOS expression in INGN 241-treated lesions. Four patients in cohorts 1-5 were diagnosed with metastatic breast cancer at the time of treatment. Their untreated tumors displayed a uniform, diffuse nuclear/cytoplasmic pattern of β-catenin expression. Three cases underwent a distinct redistribution of this protein to the plasma membrane post-treatment. While β-catenin redistribution was not evident in other non-breast tumors examined, the majority of these cases (6 of 8 tested) exhibited a significantly decreased level of beta-catenin expression following intratumoral INGN 241 (TABLE 6).
β-Catenin Expression in INGN-241-Treated Patient Specimens
Effect of Treatment on on
β-Catenin Expression
Redistribution to
7/AdrCor Ca
8/Chol Ca
10/JRG; Mel
12/BVR; Mel
13/MHH; BrCa
No. positive/no. evaluable
1Patient 4 was not examined due to lack of specimen. Patient 5 was diagnosed with large cell lymphoma which did not express β-catenin.
By comparison, it is unclear whether INGN-241 treatment altered iNOS expression substantially in an array of tumor types. However, 2 of 2 melanoma cases from cohorts 1-6 exhibited decreased iNOS expression post-treatment. iNOS has been reported as a putative prognostic marker in melanoma and recent studies have shown that Ad-mda7 and secreted MDA-7 protein can down-regulate iNOS expression in melanoma tumor cells via regulation of IRF-1/IRF-2 signaling. Altogether, 4 of 9 cases of various solid tumor types showed a decrease of iNOS following INGN-241 treatment.
5. Immune Activation by INGN-241
To identify systemic immune activation events following intratumoral INGN 241, serum cytokines were quantified at various times after INGN 241 injection. The majority of patients had significantly elevated levels of serum IL-6 (18 of 21 patients), IL-10 (19/21) and TNFα (12/21) following INGN 241 injection. Some of the patients demonstrated increased levels of IFNγ (8/21), GM-CSF (4/21) and IL-2 (2/21) that were ≧50% higher than baseline level within 8 days post-injection. Post-treatment levels of IL-6, IL-10 and IFNγ constituted up to 20-fold higher than pretreatment level. The initial peak responses of IL-6 and TNFα responses occurred at 6 hours post-treatment for most patients, whereas maximal INFγ and IL-10 responses occurred at day 2 post-injection (
Further analysis was carried out to characterize cytokine response of the 4 patients who responded clinically to INGN-241 (pts 83, 84, 87, 88). Responding patients exhibited significantly higher increases in TNFα (0-22,617%, n=4, vs. 0-161% in non-responders, n=17; p=0.015, df=19) and IL-6 (0-16,636%, n=4; vs. 0-25,85%, n=17; p=0.028, df=19) at 6 hours post-injection, while no significant difference was evident with respect to IL-10 (p=0.3) or IFNgamma (p=0.2) expression in the patient subsets.
For patients who received 2�1011 or 2�1012 vps, 10 of 21 exhibited an increase in the post-treatment frequency of CD3+ CD8+ peripheral blood T cells. Despite the interpatient variation of the frequency distribution of CD8+ T cells before treatment, there was a significant elevation in % CD8-positive T cells (P<0.03, df=40) at day 15 post-injection (from mean pretreatment level of 31�2.4% to 38�2.6% at day 15) (
14 patients in cohorts 1-7 were tested for the presence of anti-adenoviral antibodies. All had elevated antibody titers post-treatment that increased exponentially with time, with a median titer that was 64-fold higher than pre-treatment level at day 15 post-injection.
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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