EP2747552A1

EP2747552A1 - Inducible mouse models of lung injury and lung diseases

Info

Publication number: EP2747552A1
Application number: EP12826382.9A
Authority: EP
Inventors: Vrushank Girishbhai DAVE
Original assignee: University of South Florida
Current assignee: University of South Florida
Priority date: 2011-08-23
Filing date: 2012-08-23
Publication date: 2014-07-02
Also published as: WO2013028871A1

Abstract

The present invention provides inducible transgenic mouse models of lung cancer and lung remodeling diseases. In one embodiment, the present invention provides pre-clinical mouse models in which lung cancer and a lung disease of interest can be induced "at will" at any time points by the presence and withdrawal of doxycycline. The transgenic mouse model of the present invention recapitulates molecular, cellular and pathological characteristics of human lung cancer and lung remodeling diseases including emphysema, COPD, and pulmonary fibrosis.

Description

DESCRIPTION

INDUCIBLE MOUSE MODELS OF LUNG INJURY AND LUNG DISEASES CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application Serial No. 61 /526,546, filed August 23, 201 1 , which is herein incorporated by reference in its entirety.

BACKGROUND

Lung cancer causes over 1 million deaths worldwide and over 160,000 deaths in the

United States each year. The long-term survival rate from lung cancer is less than 2%. The low survival rate from lung cancer is primarily due to the fact that lung tumors can develop "acquired resistance" to systemic and targeted therapies and re-grow after surgical resection. Unfortunately, the extremely heterogeneous nature of malignant lung tumors and genetic diversity amongst patients makes understanding of the mechanism of oncogenesis difficult. Development of therapy for lung cancer is challenging.

Lung remodeling pathologies include chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), and are characterized by profound abnormalities in inflammatory and fibrotic pathways. Molecular basis of aberrant lung remodeling pathologies remains unclear. While animal models of lung fibrosis have been developed by chemical injury or viral infection to the lungs, these models are not informative of the molecular basis of lung fibrosis because the sequential molecular events that cause fibrosis are difficult to discern.

There is a need for molecularly defined animal models of lung fibrosis that allow for determination of molecular events in the initiation of fibrotic pathways. There is also a need for a pre-clinical animal models of lung cancer. The improved animal models of lung cancer and lung diseases would provide mechanistic understanding of molecular targets, useful for development of treatment regimens for these diseases. BRIEF SUMMARY

The present invention provides inducible transgenic mouse models of lung cancer and lung remodeling diseases. In one embodiment, the present invention provides pre-clinical mouse models in which lung cancer or lung remodeling pathology can be induced at any time points by the presence and withdrawal of doxycycline. The transgenic mouse model of the present invention recapitulates molecular, cellular, and pathological characteristics of human lung cancer and lung remodeling pathology, and thus is useful for elucidation of the precise mechanism of pathogenesis as well as for development of therapeutic agents. In one embodiment, the present invention provides an inducible transgenic mouse that is a model of lung cancer, wherein the genome of the transgenic mouse comprises:

a nucleic acid molecule encoding a reverse tRA (rtTA), operably linked to a first lung-specific promoter (e.g., the surfactant protein C promoter (SPCp), Clara cell secretory protein (CCSP) promoter);

a nucleic acid molecule encoding a Cre recombinase protein, operably linked to a second promoter (e.g., the CMV promoter) under the control of a TetO operator;

a loxP-flanked nucleic acid molecule encoding a tumor suppressor protein operably linked to a third promoter; and

a nucleic acid molecule encoding an oncoprotein operably linked to a fourth promoter and under the control of a lox-stop-lox (LSL) sequence;

wherein the presence of doxycycline activates the binding of the rtTA protein to the TetO operator;

wherein the binding of the Cre recombinase to the loxP sites results in the excision of the nucleic acid molecule encoding the tumor suppressor protein; and

wherein lung cancer is induced after doxycycline is administered to the transgenic mouse.

In another embodiment, the present invention provides an inducible transgenic mouse of lung remodeling diseases, wherein the genome of the transgenic mouse comprises:

a nucleic acid molecule encoding a reverse tRA (rtTA), operably linked to a first lung-specific promoter;

a nucleic acid molecule encoding a Cre recombinase protein, operably linked to a second promoter under the control of a TetO operator; and

a loxP-flanked nucleic acid molecule encoding a von Hippel-Lindau (VHL) tumor suppressor protein operably linked to a third promoter;

wherein the presence of doxycycline activates the binding of the rtTA protein to the

TetO operator; wherein the binding of the Cre recombinase to the loxP sites results in the excision of the nucleic acid molecule encoding a von Hippel-Lindau (VHL) tumor suppressor protein; and

wherein after doxycycline is administered to the transgenic mouse, a lung remodeling disease is induced.

In one embodiment, the present invention provides cells, tissues, organs of the inducible transgenic mouse of the present invention.

Also provided are uses of the inducible transgenic mouse model of the present invention, as well as cells, tissues, organs thereof, for elucidation of disease mechanism and for development of therapeutic regimes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows embodiments of the inducible transgenic mouse model of lung cancer.

Figure 2 shows an inducible mouse model of lung hyperplasia with inactivation of

Pten in the respiratory epithelium. (A) Deletion of PTEN is achieved in mice bearing loxP- flanked exon V of Pten (Pterl'^ox/flox). Pten^llox/f!ox mice are mated to SP-C-rtTA^'/tg mice and the TetO-Cre'^g/~ mice. Administration of doxycycline to dams deleted exon V of Pten in the respiratory epithelium of the embryos, termed PtenD/D mice. (B) Genotype analysis using PGR on genomic DNA using primers flanking loxP sites in exon V identifies Pteri^{vt t}, and Pteri^loxh genotypes. M, DNA molecular weight marker. (C) Hematoxylin/eosin staining of lung sections from PterP/⁰ mice demonstrated normal branching morphogenesis and postnatal lung formation at 6 weeks of age.

Figure 3 shows bronchiolar hyperplasia of the inducible transgenic mouse of lung hyperplasia. Selective deletion of PTEN gene in the lung using the SPC-rtTA/Tet-O-Cre system caused epithelial hyperplasia within 6 weeks. As compared to control littermates, the bronchial epithelium in Pten⁰/¹ mice is hypercellular (A, B). Papillae consisting of fibrovascular cores are lined by a hypercellular epithelium protruded into the bronchiolar lumens of Pten mice. In contrast, bronchioles in control littermates lack papillae and are lined by a pseudostratified or single layer epithelium (C, D). Papillae contained fibrovascular cores consisting of stroma with its associated collagen as highlighted by trichrome stain (E, F). n > 4 mice per genotype. Lung images shown are from 6-weekold mice, but are also representative of lungs from mice at 5 months of age. Figure 4 shows an embodiment of the inducible transgenic mouse model of lung cancer (A). (B) shows administration of doxycycline induces the loss of PTEN expression and hyperactivation of KRas^G12D in the inducible transgenic mouse lung cancer of the present invention. (C) shows that the loss of PTEN expression and hyperactivation of KRas^G12D result in lung cancer with aggressive adenocarcinomas.

Figure 5 shows an embodiment of the inducible transgenic mouse model of lung cancer. The administration of doxycycline induces the loss of PTEN expression and hyperactivation of KRas ¹²⁰ in the inducible transgenic mouse lung cancer of the present invention, and results in lung cancer with aggressive adenocarcinomas.

Figure 6(A, B) shows an embodiment of inducible transgenic mouse model of lung remodeling disease of emphysema, COPD, and pulmonary fibrosis. (C) shows that the administration of doxycycline induces the loss of VHL expression in the inducible transgenic mouse lung remodeling disease of the present invention. The severity of emphysema / COPD phenotype after 2 weeks, 4 weeks, and 8 weeks of doxycycline administration is shown.

Figure 7 shows that spontaneous pulmonary fibrosis is induced after doxycycline administration without experimental injury to the lungs. This model can serve as a clinical animal model for idiopathic pulmonary fibrosis (IDF).

Figure 8 shows that after doxycycline administration, VHL expression is inactivated in the inducible mouse model of the present invention. Mice with VHL deficiency in lungs are prone to the development of pulmonary fibrosis after exposure to toxin or suffering from inj ury, when compared to normal lungs without VHL deficiency.

DETAILED DESCRIPTION

The present invention provides inducible transgenic mouse models of lung cancer and lung remodeling diseases. In one embodiment, the present invention provides pre-clinical mouse models with inherent genetic modifications that allows for induction of lung cancer and lung remodeling diseases "at will" in the adult mouse by the presence and withdrawal of doxycycline. The transgenic mouse model of the present invention recapitulates molecular, cellular and pathological characteristics of human lung cancer and other lung diseases.

In one embodiment, the inducible mouse model of lung cancer is developed by breeding LKBl-loxp mouse (Source: NCI) into CCSP-rtTA/OtetCRE/ /LSL-RAS-G 12D mouse (Fig. 6). In one embodiment, the present invention provides mouse models of lung cancer and lung remodeling diseases, including emphysema/ chronic obstructive pulmonary disease (COPD), respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and idiopathic pulmonary fibrosis (IPF). One advantage of mouse models of the present invention is that the mice are normal and do not develop lung diseases until doxycycline is administered (for example, via oral administration of doxycycline through food or water). Thus, unlike animal models with congenital genetic lesions, the lung disease of interest can be induced at any given time point of interest in the life of a mouse. For example, genes of interest can be modified in embryonic and late fetal/perinatal period to produce mouse model of BPD and RDS, or at adult stage to produce mouse models of emphysema/COPD, pulmonary fibrosis, pulmonary inflammation and injury.

Inducible Transgenic Mouse Model of Lung Injury and Disease

In one embodiment, the present invention provides an inducible transgenic mouse that is a model of lung cancer, wherein the genome of the transgenic mouse comprises:

a nucleic acid molecule encoding a Cre recombinase protein, operably linked to a second promoter under the control of a TetO operator;

a nucleic acid molecule encoding an oncoprotein operably linked to a fourth promoter and under the control of a lox-stop-Iox (LSL) sequence;

wherein the presence of doxycycline activates the binding of the rtTA protein to the TetO operator; and

wherein the binding of the Cre recombinase to the loxP sites results in the excision of the nucleic acid molecule encoding the tumor suppressor protein.

In one embodiment, after doxycycline is administered to the inducible transgenic mouse of lung cancer of the present invention, lung cancer is induced.

In another embodiment, the present invention provides an inducible transgenic mouse that is a model of lung remodeling diseases, wherein the genome of the transgenic mouse comprises:

a nucleic acid molecule encoding a reverse tRA (rtTA), operably linked to a first lung-specific promoter; a nucleic acid molecule encoding a Cre recombinase protein, operably linked to a second promoter under the control of a TetO operator; and

wherein the presence of doxycycline derivative activates the binding of the rtTA protein to the TetO operator; and

wherein the binding of the Cre recombinase to the loxP sites results in the excision of the nucleic acid molecule encoding a von Hippel-Lindau (VHL) tumor suppressor protein.

In one embodiment, after doxycycline is administered to the inducible transgenic mouse of lung remodeling disease of the present invention, emphysema, COPD, or idiopathic pulmonary fibrosis is induced.

Lung immaturity and resultant surfactant deficiency cause respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD), which are common disorders contributing to morbidity and mortality in preterm infants. In one embodiment, the present invention provides a mouse model of RDS/BPD that recapitulates human condition.

In one embodiment, the present invention provides an inducible mouse model of emphysema and COPD wherein the expression of VHL gene can be inactivated by administration of doxycycline. When doxycycline is administered at different time intervals early during development, the severity of emphysema disease can be regulated, thereby controlling the disease conditions in patients. Figure 7B shows the inactivation of VHL expression at different time points in the mouse model produces emphysema / COPD phenotype with different degrees of severity.

In one embodiment, the present invention provides an inducible mouse model of spontaneous pulmonary fibrosis; this mouse model recapitulates molecular and cellular features of human idiopathic pulmonary fibrosis (IPF). Lung fibrosis develops slowly and in a spontaneous manner in humans, and the etiology of IPF remains unknown. Currently, there is no animal model available for spontaneous pulmonary fibrosis. Also, there lacks effective treatment for IPF, and the survival time is usually 2-3 years. As shown in Figures 8 and 9, the inducible mouse model of the present invention is useful for elucidation of the precise mechanisms of pulmonary fibrosis and for development of targeted drug therapies in interstitial lung diseases/pulmonary fibrosis. Lung-specific promoters useful according to the present invention include, but are not limited to, surfactant protein C promoter (SPCp) and Clara cell secretory protein (CCSP) promoter.

Tetracycline (Tet)-controlled transcriptional activation is a method of inducible expression where transcription is reversibly controlled by the presence or absence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). Gene expression is activated as a result of binding of the Tet-off or Tet-on protein to tetracycline response elements (TREs) located within an inducible promoter. Both the Tet-on and Tet-off proteins activate gene expression. The Tet-Off protein activates gene expression in the absence of a tetracycline derivative - doxycycline (Dox), whereas the Tet-on protein activates gene expression in the presence of Dox.

In the Tet-off system, the tetracycline transactivator (tTA) protein, which is created by fusing the TetR (tetracycline repressor) protein (obtainable from Escherichia coli bacteria) with the VP16 protein (obtainable from the Herpes Simplex Virus), binds on DNA at a TetO operator. Once bound the TetO operator activates the promoter coupled to the TetO operator, thereby activating the transcription of the nearby gene. Tetracycline derivatives bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of target genes.

In the Tet-On system, when the tTA protein is bound by doxycycline, the doxycycline-bound tTA is capable of binding the TetO operator. Thus the introduction of doxycyline to the system initiates the transcription of the genetic product. The Tet-on system is sometimes preferred for the faster responsiveness.

The reverse tTA (rtTA) is a complementary genetic module for rapid gene activation by addition of Dox (Tet-on). See Kistner et ah, Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice, Proc. Natl. Acad. Sci. U.S.A. Vol. 93, pp. 10933- 10938 (1996), which is hereby incorporated by reference in its entirety.

The Tet-on advanced transactivator (also known as rtTA2^s-M2) is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-on. In addition, its expression is considered to be more stable in eukaryotic cells due to being human codon optimized and utilizing three minimal transcriptional activation domains. Tet-on 3G (also known as rtTA-V l O) is similar to Tet-on Advanced, and is human codon optimized and composed of three minimal VP16 activation domains. The Tet-on 3G is sensitive to 100-fold less Dox than the original Tet-on. In one embodiment of a tetracycline-responsive regulatory expression element, a tetracycline-controlled reverse transactivator (rtTA) comprises a tetR (e.g., from Escherichia coli Tn l O); a mammalian transcription factor VP 16 transactivating domain serving as an effector; and a tissue-specific promoter controlling the rtTA effector transcription. In the presence of doxycycline, the rtTA binds to a (TeTO)7 operator (a seven tandemly repeated TetO sequence) placed upstream of a CMV promoter that drives expression of a transgene. As a result, transgene expression can be switched on or off by administration and withdrawal of doxycycline.

The Cre/LoxP recombination system is a site-specific recombinase technology useful for performing site-specific deletions, insertions, translocations, and inversions in the DNA of cells or transgenic animals. The Cre recombinase protein (encoded by the locus originally named as "causes recombination") consists of four subunits and two domains, the larger carboxyl (C-terminal) domain, and smaller amino (N-terminal) domain. The lox P (locus of X-over PI ) is a site on the Bacteriophage PI and consists of 34 bp. The results of Cre- recombinase-mediated recombination depend on the location and orientation of the loxP sites, which can be located cis or trans. In case of cis-localization, the orientation of the loxP sites can be the same or opposite. In case of trans-localization, the DMA strands involved can be linear or circular. The results of Cre recombinase-mediated recombination can be excision (when the loxP sites are in the same orientation) or inversion (when the loxP sites are in the opposite orientation) of an intervening sequence in case of cis loxP sites, or insertion of one DNA into another or translocation between two molecules (chromosomes) in case of trans loxP sites. Cre-Lox recombination system is known in the art, see, for example, Andras Nagy, Cre recombinase: the universal reagent for genome tailoring, Genesis 26:99-109 (2000), which is hereby incorporated by reference in its entirety.

The Lox-Stop-Lox (LSI,) cassette prevents expression of the transgene in the absence of Cre-mediated recombination. In the presence of Cre recombinase, the LoxP sites recombine, and the stop cassette is deleted. The Lox-Stop-Lox (LSL) cassette is known in the art. See, Allen Institute for Brain Science, Mouse Brain Connectivity Alias, Technical White Paper: Transgenic Characterization Overview (2012).

In one embodiment, the nucleic acid molecule encoding a tumor suppressor protein is flanked by cis-loxP sites that are in the same orientation; accordingly, binding of the Cre recombinase to the loxP sites results in the excision of the flanked nucleic acid molecule encoding the tumor suppressor protein. The term "constitutive promoter," as used herein, refers to its ordinary meaning that is an unregulated promoter that allows for continual transcription of its associated gene. Constitutive promoters useful according to the present invention include, but are not limited to, cytomegalovirus (CMV) promoter, CMV-chicken beta actin promoter, ubiquitin promoter, JeT promoter, SV40 promoter, elongation Factor 1 alpha (EF1 -alpha) promoter, RSV promoter, and Mo-MLV-LTR promoter.

The term "tumor suppressor gene," as used herein, refers to its ordinary meaning that is a gene that protects a cell from one step on the path to cancer, and if repressed or silenced, leads deregulated cell division and/or overexpression of a proto-oncogene or oncogene. Tumor supressor gene products repress genes that are essential for the continuing of the cell cycle. Effectively, if these genes are expressed, the cell cycle will not continue, effectively inhibiting cell division. Tumor suppressor gene products couple the cell cycle to DNA damage. Thus, these gene products activate cell cycle checkpoints and DNA repair mechanisms that stall or prevent cell division. If the damage cannot be repaired, the cell initiates apoptosis, or programmed cell death. Some tumor supressor gene products (tumor suppressor proteins) are involved in cell adhesion, and thus, prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis. These proteins are also known as metastasis suppressors.

In certain embodiments, the genome of the transgenic mouse comprises a loxP- flanked nucleic acid molecule encoding a tumor suppressor protein including, but not limited to, phosphatase and tensin homolog deleted on chromosome 1 0 (PTEN), p53, von Hippel- Lindau (VHL) tumor suppressor protein, LKB l tumor suppressor kinase, the retinoblastoma (RB) protein, tuberous sclerosis protein 1 (TSC1), the pi 6 tumor suppressor protein (also known as cyclin-dependent kinase inhibitor 2A (CDKN2A), p l 6Ink4A, multiple tumor suppressor 1 (MTS-1 )), epidermal growth factor receptor-L858R mutant (EGFR-L858R), and CAG-LSL-EGFR-WT.

The term "oncogene" as used herein, refers to its ordinary meaning that is a gene, one or more forms of which can cause oncogenesis or tumor formation. Exemplary oncogenes include, but are not limited to, growth factors, transciption factors, regulatory proteins, e.g., GTPases and receptors, and cell cycle proteins. The term "proto-oncogene," as used herein, refers to its ordinary meaning that is a gene modified, directly or indirectly, that cause oncogenesis and tumor formation. Examples of oncogenes include, but are not limited to, activated or mutated versions of RAS, MYC, SRC, FOS, JUN, MYB, ABL, BCL2, HOX11, HOX11L2, TAL1/SCL, LMOl, LM02, EGFR, MYCN, MDM2, CDK4, GLI1, IGF2, EGFR, FLT3-ITD, TP53, PAX3, PAX7, BCR/ABL, HER2 NEU, FLT3R, FLT3-ITD, TAN1, PTC, B-RAF, E2A- PBX1 , and NPM-ALK, as well as fusion of members of the PAX and FKHR gene families, WNT, MYC, ERK EGFR, FGFR3, CDH5, KIT, RET, and TRK. Other exemplary oncogenes are well known in the art and several such examples are described in, for example, The Genetic Basis of Human Cancer (Vogelstein, B. and Kinzler, K. W. eds. McGraw-Hill, New York, N.Y., 1998).

In one embodiment, the transgenic mouse model has a genome that comprises a nucleic acid molecule encoding the KRAS-G12-D mutant protein.

The present invention encompasses the use of oncogenes, proto-oncogenes, tumor suppressor genes, or nucleic acid molecules encoding oncoproteins, proto-oncoproteins, tumor suppressor proteins that can be obtained from publically known sequences obtaintable from, for example, in the GenBank database.

In one embodiment, the nucleic acid molecules useful in the genetic constructs of the invention are DNA.

In one embodiment, the present invention provides cells (e.g., lung epithelium cells), tissues, and organs of the inducible transgenic mouse of the present invention.

Expression Constructs

The present invention also embodies expression constructs, vectors, as well as host cells useful for producing transgenic mouse models of the present invention.

As used herein, the term "expression construct" refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence.

Expression constructs of the invention will also generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed.

Thus, a person of ordinary skill in the art can select regulatory elements for use in, for example, bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a peptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.

As used herein, the term "operably linked" refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner. In general, operably linked components are in contiguous relation. Sequence(s) operably-linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably-linked to a promoter when the promoter is capable of directing transcription of that coding sequence.

A "coding sequence" or "coding region" is a polynucleotide sequence that is transcribed into mRNA and/or translated into a polypeptide. For example, a coding sequence may encode a polypeptide of interest. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3 '-terminus.

The term "promoter," as used herein, refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule. A promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.

In addition to a promoter, one or more enhancer sequences may be included such as, but not limited to, cytomegalovirus (CMV) early enhancer element and an SV40 enhancer element. Additional included sequences are an intron sequence such as the beta globin intron or a generic intron, a transcription termination sequence, and an mRNA polyadenylation (pA) sequence such as, but not limited to, SV40-pA, beta-globin-pA, the human growth hormone (hGH) pA and SCF-pA. The term "polyA" or "p(A)" or "pA" refers to nucleic acid sequences that signal for transcription termination and mRNA polyadenylation. The polyA sequence is characterized by the hexanucleotide motif AAUAAA. Commonly used polyadenylation signals are the SV40 pA, the human growth hormone (hGH) pA, the beta-actin pA, and beta-globin pA. The sequences can range in length from 32 to 450 bp. Multiple pA signals may be used.

The term "vector'^* is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information (e.g., a polynucleotide of the invention) to a host cell. The terms "expression vector" and "transcription vector" are used interchangeably to refer to a vector that is suitable for use in a host cell (e.g., a subject's cell) and contains nucleic acid sequences that direct and/or control the expression of exogenous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present. Vectors useful according to the present invention include plasmids, viruses, BACs, YACs, and the like. Particular viral vectors illustratively include those derived from adenovirus, adeno-associated virus and lentivirus.

As used herein, the term "isolated" molecule (e.g. , isolated nucleic acid molecule) refers to molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

The term "recombinant" is used to indicate a nucleic acid construct in which two or more nucleic acids are linked and which are not found linked in nature.

The term "nucleic acid" as used herein refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide.

The term "nucleotide sequence" is used to refer to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.

The term "expressed" refers to transcription of a nucleic acid sequence to produce a corresponding mRNA and/or translation of the mRNA to produce the corresponding protein.

Expression constructs can be generated recombinantly or synthetically or by DNA synthesis using well-known methodology.

The term "regulatory element" as used herein refers to a nucleotide sequence which controls some aspect of the expression of an operably linked nucleic acid sequence.

Exemplary regulatory elements illustratively include an enhancer, an internal ribosome entry site (IRES), an intron; an origin of replication, a polyadenylation signal (pA), a promoter, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid sequence. Those of ordinary skill in the art are capable of selecting and using these and other regulatory elements in an expression construct with no more than routine experimentation.

Optionally, a reporter gene is included in the transgene construct. The term "reporter gene" as used herein refers to gene that is easily detectable when expressed, for example via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, and the like. Exemplary reporter genes include but are not limited to green fluorescent protein (GFP; see Mistili and Spector, Nature Biotechnology 15:961 -964 (1997), eGFP, YFP, eYFP, CFP, eCFP, BFP, eBFP, MmGFP, a modified GFP, dsRed (red fluorescent protein, RFP), luciferase and beta-galactosidase (lacZ). The production of recombinant nucleic acids, vectors, transformed host cells, proteins and protein fragments by genetic engineering is well known.

If desired, the vector may optionally contain flanking nucleic sequences that direct site-specific homologous recombination. The use of flanking DNA sequence to permit homologous recombination into a desired genetic locus is known in the art. At present it is preferred that up to several kilobases or more of flanking DNA corresponding to the chromosomal insertion site be present in the vector on both sides of the encoding sequence (or any other sequence of this invention to be inserted into a chromosomal location by homologous recombination) to assure precise replacement of chromosomal sequences with the exogenous DNA. See e.g. Deng et al, 1993, Mol. Cell. Biol 13(4):2134-40; Deng et al, 1992, Mol Cell Biol 12(8):3365-71 ; and Thomas et al, 1992, Mol Cell Biol 12(7):2919-23. It should also be noted that the cell of this invention may contain multiple copies of the gene of interest.

Transformed host cells are cells which have been transformed or transfected with vectors containing nucleic acid constructs of the invention and may or may not transcribe or translate the operative ly associated nucleic acid of interest.

Methods of Making Transgenic Non-Human Animals

Any of various methods can be used to introduce a transgene into a non-human animal to produce a transgenic animal. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection, viral infection and transformation of embryonic stem cells and iPS cells. Methods for generating transgenic animals that can be used include, but are not limited to, those described in J. P. Sundberg and T. Ichiki, Eds., Genetically Engineered Mice Handbook, CRC Press; 2006; M. H. Hofker and I. van Deursen, Eds., Transgenic Mouse Methods and Protocols, Humana Press, 2002; A. L. Joyner, Gene Targeting: A Practical Approach, Oxford University Press, 2000; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; 2002, ISBN-10: 0879695919; K. Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol. Biol. 2002; 185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 978047015180; Meyer et al. PNAS USA, vol. 107 (34), 15022-15026.

Methods for making inducible mouse model of lung hyperplasia can be found at Dave et ah, Conditional deletion of Pten causes bronchilar hyperplasia, Am J. Respir. Cell. Mol. Biol. Vol. 38 pp337-345 (2008), which is herein incorporated by reference in its entirety.

Applications of the Inducible Transgenic Mouse Model of Lung Injury or Diseases

The mouse models of lung cancer and lung remodeling diseases can be used to elucidate the disease mechanisms and to develop and evaluate molecularly targeted therapies for lung diseases.

In one embodiment, the inducible mouse model of lung cancer can develop aggressive tumors within weeks after induction (e.g., the presence of tetracycline or tetracycline derivatives such as doxycycline); in contrast, it takes 6- 12 months for tumor develop in existing mouse models of lung cancer. Thus, the mouse model of the present invention can be used to test a battery of drugs in a very short period of time, useful for in vivo semi-high throughput screening.

In another embodiment, the mouse model of the present invention can be used to derive and enrich cell lines that have a large population of cancer like-stem cells, useful as therapeutic targets for development of stem-cell based cancer therapy.

In one embodiment, the present invention provides a method of screening therapeutic agents for treating a lung disease of interest. In one embodiment, the method comprises: providing an inducible mouse of the present invention; administering doxycycline to the inducible transgenic mouse; administering a candidate therapeutic agent to the mouse; and determining the effect of the candidate therapeutic agent on the lung disease of interest. The candidate therapeutic agent may be administered by any suitable technique, including but not limited to, oral administration, pulmonary administration, aerosol administration, parenteral administration (subcutaneous injection, intramuscular injection, intraveneous injection, etc.), transdernal administration, etc. In one embodiment, the present invention can be used to screen for therapeutic agents for lung cancer, emphysema/ chronic obstructive pulmonary disease (COPD), respiratory distress syndrome (RDS), and pulmonary fibrosis (including idiopathic pulmonary fibrosis (1PF)).

EXAMPLE

Following are examples that illustrate procedures and embodiments for practicing the invention. The examples should not be construed as limiting. EXAMPLE 1 - INDUCIBLE MOUSE MODEL OF LUNG CANCER

Figures 1 and 4 show embodiments of the inducible transgenic mouse model of lung cancer of the present invention. In one embodiment as shown in Fig. 1A, the inducible transgenic mouse model of lung cancer has a genome comprising a human-3.7-SPC-rtTA DNA promoter element; a TetO-CMV-Cre DNA promoter element; an expression construct comprising at least one tumor suppressor gene (TSG) (e.g., PTEN, LKB 1), wherein the TSG is flanked by loxP DNA elements; and an expression construct comprising at least one oncogene (e.g., KRas), wherein the promoter and the coding region of the oncogene is separated by a loxP-flanked transcription termination sequence (e.g., LSL). In another embodiment as shown in Fig. I B, the inducible transgenic mouse model of lung cancer has a genome comprising a rat-2.3-CCSP-rtTA DNA promoter element; a TetO-CMV-Cre DNA promoter element; an expression construct comprising at least one tumor suppressor gene (TSG) (e.g., PTEN, LKB 1), wherein the TSG is flanked by loxP DNA elements; and an expression construct comprising at least one oncogene (e.g., KRas), wherein the promoter and the coding region of the oncogene is separated by a loxP-flanked transcription termination sequence (e.g., LSL).

As shown in Figure 4, in the presence of doxycycline, rtTA binds to the TeTO/CMV promoter, thereby activating the expression of Cre recombinase. The Cre recombinase catalyzes the recombination of DNA between the loxP sites, thereby resulting in the excision of the DNA elements (e.g., PTEN, LSL) flanked by the loxP sites. Doxycycline causes the inactivation of PTEN and hyperactivation of KRas, resulting in the development of lung cancer (Fig. 4C). In an alternative embodiment of the inducible transgenic mouse model as shown in Figure 5, doxycycline causes the inactivation of LKB l and the hyperactivation of KRas, thereby resulting in the development of lung cancer. EXAMPLE 2 - INDUCIBLE MOUSE MODEL OF LUNG HYPERPLASIA

Figure 2 shows an embodiment of the inducible transgenic mouse model of lung hyperplasia. In one embodiment as shown in Fig. 2A, the inducible transgenic mouse model of lung hyperplasia has a genome comprising a human-3.7-SPC-rtTA DNA promoter element; a TetO-CMV-Cre DNA promoter element; and an expression construct comprising at least one tumor suppressor gene (TSG) (e.g., PTEN), wherein the TSG is flanked by loxP DNA elements.

Briefly, triple transgenic mice in which PTEN is conditionally inactivated in the fetal lung epithelium are obtained using the SPC-rtTA/TetO-Cre system. Mice bearing loxP- flanked exon V of Pten are produced and maintained as homozygotes in a mixed FVBN/129S4/SvJae6 background. To achieve lung epithelial specific deletion of the PTEN gene, these mice are first mated to SP-C-rtTA2^{~ tg} mice and TetO-Cre^tg/- m ice expressing Cre recombinase. Further back-crossings give triple-transgenic mice harboring SP-C-rtTA^' ^;'^sPetO-Cretg ^sCnbf^ox/flox alleles. Doxycycline in food (25 mg/g; Harlan Teklad, Madison, WI) is administered to dams from embryonic day 0.5 to day 14.5, producing experimental animals: triple transgenic mice SPC-rtTA/TetO-Cre'^Pten^ ¹, herein called and control transgenic littermates: SPC-rtTA'⁸'- or TetO- Cre*'

The mice are housed in humidity- and temperature-controlled rooms on a 12-hour light/12-hour dark cycle with food and water ad libitum. There is no serologic or histologic evidence of either pulmonary pathogens or infections in sentinel mouse colonies. Gestation is dated 0.5 by vaginal plug. The mice are sacraficed by injection of anesthetic to obtain lung tissue between 4 and 6 weeks for biochemical and histochemical analyses.

The results show that the administration of doxycycline to the mouse model of hyperplasia causes deletion of the loxP-flanked PTEN gene, leading to inactivation of PTEN expression. The inactivation of PTEN causes hyperplasia / metaplasia in the mouse lung (Fig. 3). EXAMPLE 3 - INDUCIBLE MOUSE MODEL OF EMPHYSEMA, COPD, AND

PULMONARY FIBROSIS

Figure 6 shows an embodiment of the inducible transgenic mouse model of emphysema, COPD, and/or pulmonary fibrosis. In one embodiment, inducible transgenic mouse model of lung cancer has a genome comprising a 2.3-r-CCSP-rtTA DNA promoter element, a (TetO)7-CMV-Cre DNA promoter element, and an expression construct comprising at least a nucleic acid encoding Vhl operably linked to a promoter, wherein the nucleic acid molecule is flanked by loxP DNA elements.

As shown in Fig. 7, the treatment of doxycycline causes the inactivation of VHL expression in the mouse lung, thereby inducing spontaneous pulmonary fibrosis in the transgenic mouse model without other experimental injury; this can be used as a clinical model for idiopathic pulmonary fibrosis (IPF). As shown in Fig. 8, the administration of chemical toxins (e.g., bleomycin) to the lung increases the severity of pulmonary fibrosis in the transgenic mouse model of the present invention.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

REFERENCES

Allen Institute for Brain Science, Mouse Brain Connectivity Altas, Technical White Paper: Transgenic Characterization Overview (2012). Dave et al., Conditional deletion of Pten causes bronchilar hyperplasia, Am J. Respir. Cell. Mol. Biol. Vol. 38 pp337-345 (2008), which is herein incorporated by reference in its entirety.

Kistner et ah, Doxycycline-raediated quantitative and tissue-specific control of gene expression in transgenic mice, Proc. Natl. Acad. Sci. U.S.A. Vol. 93, pp. 10933-10938 (1996).

Nagy, Cre recombinase: the universal reagent for genome tailoring, Genesis 26:99-109 (2000).

Claims

CLAIMS I claim:

1 . A cell of an inducible transgenic mouse that is a model of lung cancer, wherein the genome of the cell of the transgenic mouse comprises:

2. An inducible transgenic mouse that is a model of lung cancer, comprising a cell according to claim 1.

3. The inducible transgenic mouse according to claim 2, wherein the first lung-specific promoter is selected from a surfactant protein C promoter (SPCp) and a Clara cell secretory protein (CCSP) promoter.

4. The inducible transgenic mouse according to claim 2, wherein the second promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, CMV-chicken beta actin promoter, ubiquitin promoter, JeT promoter, SV40 promoter, elongation Factor 1 alpha (EF 1 -alpha) promoter, RSV promoter, and Mo-MLV-LTR promoter.

5. The inducible transgenic mouse according to claim 2, wherein the second promoter is a cytomegalovirus (CMV) promoter.

6. The inducible transgenic mouse according to claim 2, wherein the nucleic acid encoding the Cre recombinase protein is operably linked to a (TetO)7/CMV promoter element.

7. The inducible transgenic mouse according to claim 2, wherein the tumor suppressor protein is selected from the group consisting of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), p53, von Hippel-Lindau (VHL) tumor suppressor protein, LKB 1 tumor suppressor kinase, the retinoblastoma (RB) protein, tuberous sclerosis protein 1 (TSC 1), the pi 6 tumor suppressor protein (also known as cyclin-dependent kinase inhibitor 2A (CDKN2A), pl 6Ink4A, multiple tumor suppressor 1 (MTS- 1)), epidermal growth factor receptor-L858R mutant (EGFR-L858R), and CAG-LSL-EGFR-WT.

8. The inducible transgenic mouse according to claim 7, wherein the tumor suppressor protein is selected from phosphatase and tensin homolog deleted on chromosome 10 (PTEN), p53, von Hippel-Lindau (VHL) tumor suppressor protein, and LKB 1 tumor suppressor kinase.

9. The inducible transgenic mouse according to claim 2, wherein the oncoprotein is selected from a mutated or activated version of a protein selected from the group consisting of RAS, MYC, SRC, FOS, J UN, MYB, ABL, BCL2, HOX1 1 , HOX1 1 L2, TAL1/SCL, LMOl , LM02, EGFR, MYCN, MDM2, CDK4, GLI1 , IGF2, EGFR, FLT3-ITD, TP53, PAX3, PAX7, BCR/ABL, HER2/NEU, FLT3R, FLT3-ITD, TA 1 , PTC, B-RAF, E2A-PBX1 , and NPM-ALK, as well as fusion of members of the PAX and FKHR gene families, WNT, MYC, ERK EGFR, FGFR3, CDH5, KIT, RET and TRK.

1 0. The inducible transgenic mouse according to claim 2, wherein the oncoprotein is the KRAS-G 12-D mutant protein.

1 1. The cell according to claim 1, which is a lung epithelial cell or a lung tumor cell.

12. A cell of an inducible transgenic mouse that is a model of a lung remodeling disease selected from emphysema, COPD, or idiopathic pulmonary fibrosis, wherein the genome of the cell of the transgenic mouse comprises:

wherein the binding of the Cre recombinase to the loxP sites results in the excision of the nucleic acid molecule encoding a von Hippel-Lindau (VHL) tumor suppressor protein; and

wherein after administration of doxycycline to the transgenic mouse, a lung remodeling disease selected from emphysema, COPD, or idiopathic pulmonary fibrosis is induced.

13. An inducible transgenic mouse comprising a cell according to claim 12.

14. The inducible transgenic mouse according to claim 13, wherein the first lung-specific promoter is selected from a surfactant protein C promoter (SPCp) and a Clara cell secretory protein (CCSP) promoter.

15. The inducible transgenic mouse according to claim 13, wherein the second promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, CMV-chicken beta actin promoter, ubiquitin promoter, JeT promoter, SV40 promoter, elongation Factor 1 alpha (EF1 -alpha) promoter, RSV promoter, and Mo-MLV-LTR promoter.

16. The inducible transgenic mouse according to claim 13, wherein the second promoter is a cytomegalovirus (CMV) promoter.

17. The inducible transgenic mouse according to claim 13, wherein the nucleic acid molecule encoding the Cre recombinase protein is operably linked to a (TetO)7/CMV promoter element.

1 8. The cell according to claim 12, which is a lung epithelial cell.

19. A method for screening a therapeutic agent for treatment of lung cancer, wherein the method comprises:

providing an inducible transgenic mouse of claim 2;

administering doxycycline to the inducible transgenic mouse;

administering a candidate therapeutic agent to the transgenic mouse; and

determining whether the candidate therapeutic agent provides treatment of lung cancer in the transgenic mouse.

20. A method according to claim 19, wherein doxycycline is administered to the transgenic mouse.

21 . A method for screening a therapeutic agent for treatment of a lung remodeling disease selected from emphysema, COPD, and idiopathic pulmonary fibrosis, wherein the method comprises:

providing an inducible transgenic mouse of claim 13;

administering doxycycline to the inducible transgenic mouse;

administering a candidate therapeutic agent to the transgenic mouse; and

determining whether the candidate therapeutic agent provides treatment of a lung remodeling disease selected from emphysema, COPD, and idiopathic pulmonary fibrosis in the transgenic mouse.

22. A method according to claim 21 , wherein doxycycline is administered to the transgenic mouse.