Patent Application
20060052327
The invention relates to the use of double-strand inhibitory RNA (iRNA) to silence genes. In particular, the invention provides methods to use iRNA to silence genes in a cell specific manner.
RNA interference (RNAi) is a post-transcriptional process triggered by the introduction of double-stranded RNA, which leads to gene silencing in a sequence-specific manner (1). Specific gene silencing may be achieved in a variety of cell systems using chemically synthesized or in vitro transcribed small interfering RNA (siRNA) (2) as well as PCR or DNA vector-based short hairpin RNA (shRNA) (3-6). A few promoters have been reported to drive shRNA expression in cells, including RNA polymerase III-based promoters, U6 and H1, and RNA polymerase II promoter, CMV. However, the use of these promoters to drive shRNA expression in animals would silence a gene in all types of cells and thus produce undesirable effects in non-target cells. The lack of cell specificity limits the use of this technique in vivo. Cell-specific targeting of siRNA is an important issue to consider in RNAi therapeutics (7). If one wants to silence a gene in a particular type of cell without affecting its expression in other types of surrounding cells, a cell-specific promoter has to be used. So far, there are no successful reports in the use of a cell-specific promoter to drive shRNA expression in cells. Most recently, the use of tissue-specific recombination to produce tissue-specific knock-down has been reported (8).
The prior art has thus-far failed to provide methods that employ gene specific silencing that is cell-specific to treat conditions associated with the expression of a specific gene.
The present invention provides methods that utilize the technique of RNA interference to bring about specific gene silencing in order to treat conditions associated with the expression of specific genes. However, in contrast to the prior art, the present methods do so in a cell-specific manner, i.e. the silencing of the targeted gene of interest occurs only (or at least primarily) in a single, targeted cell type. Thus, an undesirable feature of the prior art (silencing a targeted gene in all types of cells, which may produce undesirable effects in non-target cells) is eliminated. This is accomplished by creating constructs in which transcription of the silencing (interfering) RNA is driven by a promoter that is active only in the type of cell that is targeted (a cell specific promoter). Introduction of such a construct into a targeted cell will result in transcription of the RNA because the promoter will be activated inside the targeted cell. However, the same construct, when introduced into a different type of cell, will not be active, and the RNA will not be transcribed. Thus, the silencing RNA is produced only in cells in which the promoter that drives its transcription is active.
In particular, a strategy for specifically silencing genes in alveolar epithelial type II cells of mammalian lungs is described herein. Such a strategy allows the silencing of one or more genes in this type of cell in order to treat disease conditions that results from or are exacerbated by expression of the targeted genes. In addition, the ability to do so allows direct elucidation of the function of a targeted gene in targeted cells, thereby facilitating the development of more refined disease models. As is described in detail in the Examples section, advenoviral vectors containing various shRNA under the control of SP-C promoter were constructed. Using these vectors, specific silencing of target genes in type II cells, but not in other lung cells, was demonstrated both in vitro and in vivo. These results provide proof of principal of the inventive concept, and it could be expected that one of ordinary skill in the art could use the invention to target almost any particular cell or tissue type in vivo or in vitro, and to silence a gene in only that cell type using RNA silencing.
The invention provides a method for the treatment of conditions caused or exacerbated by expression of a specific gene by decreasing translation of mRNA encoded by the specific gene in a specific type of cell. The method includes the step of providing to the specific type of cell a vector comprising i) expressible DNA encoding RNA capable of forming a dsRNA structure, in which a nucleotide sequence of a portion of the dsRNA structure is identical to a nucleotide sequence of a portion of the mRNA; and ii) a promoter sequence operationally linked to the expressible DNA. The promoter sequence is active only in the specific type of cell (i.e. targeted operation is achieved by having the dsRNA formed in specific types of cells. In one embodiment, the specific type of cell is a lung cell, and in particular may be an alveolar Type II cell. However, cancer cells or other cells of interest can be subjected to targeted gene silencing according to this invention. In one embodiment, the promoter is SP-C. In one embodiment, the dsRNA structure is shRNA. In one embodiment, the vector is an adenoviral vector.
The invention further provides a vector which comprises: i) expressible DNA encoding RNA capable of forming a dsRNA structure, in which a nucleotide sequence of a portion of the dsRNA structure is identical to a nucleotide sequence of a portion of an mRNA of interest; and ii) a promoter sequence operationally linked to the expressible DNA, wherein the promoter sequence is active only in a specific type of cell. In one embodiment, the specific type of cell is a lung cell, and in particular may be an alveolar Type II cell. In one embodiment, the promoter is SP-C. In one embodiment, the dsRNA structure is shRNA. In one embodiment, the vector is an adenoviral vector.
The invention further provides a method of reducing expression of a specific gene by decreasing translation of mRNA encoded by the specific gene in lung cells or other cells in vivo in a patient in need thereof. The method comprises the step of administering to the lung cells a vector comprising i) expressible DNA encoding RNA capable of forming a dsRNA structure, in which a nucleotide sequence of a portion of the dsRNA structure is identical to a nucleotide sequence of a portion of the mRNA; and ii) a promoter sequence operationally linked to the expressible DNA, wherein the promoter sequence is active only in the lung cells. In one embodiment, the lung cell is an alveolar Type II cell. In one embodiment, the promoter is SP-C. In one embodiment, the dsRNA structure is shRNA. In one embodiment, the vector is an adenoviral vector.
The present invention provides methods of gene silencing by RNA interference that are cell-specific. That is, the invention can eliminate or reduce the expression of a specific gene in a specific type of cell or tissue or interest, in vitro and in vivo. The method involves creating constructs that encode the interfering (silencing) RNA in which a promoter that is active in only one type of cell mediates transcription of the RNA. Thus, when the construct is administered to an individual, even though the construct may enter many different types of cells in the individual, the RNA will be produced only in the one type of cell in which the promoter is active.
The methods of the present invention involve the silencing of a specific gene (a “gene of interest” or “targeted gene” or “selected gene”). By “silencing” a gene, we mean that expression of the gene product is reduced or eliminated, in comparison to a corresponding control gene that is not being silenced. Those of skill in the art are familiar with the concept of comparing results obtained with control vs. experimental results. Without being bound by theory, it is believed that RNAi is characterized by specific mRNA degradation after the introduction of homologous double stranded RNA (dsRNA) into cells. The dsRNA is recognized and processed into small interfering RNAs (siRNAs) of 19-25 nucleotides in length by an endonuclease enzyme dimer termed Dicer (RNase III family). These siRNAs, in turn, target homologous RNA for degradation by recruiting the protein complex, RNA-induced silencing complex (RISC). The complex recognizes and cleaves the corresponding mRNA (Dykxhoom D M, Novina C D and Sharp P A, Nature Review, 4: 457-467, 2003; Mittal V, Nature Reviews, 5: 355-365, 2004).
In general, “reduced or eliminated” refers to a reduction or elimination of detectable amounts of the gene product by an amount in the range of at least about 10% to about 100%, or preferably of at least about 25% to 100%, or more preferably about 50% to about 100%, and most preferably from about 75% to about 100%. If desired, a reduction or elimination may be determined by any of several methods that are well known to those of skill in the art, and may vary from case to case, depending on the gene that is being silenced. For example, such a reduction or elimination of the expression of the gene may be determined by quantification of the gene product (e.g. by determining the quantity of a protein, polypeptide or peptide that is made) or quantification of an activity of the gene product (e.g. an activity such as enzymatic activity, signaling or transport activity, activity as a structural component of the cell, activity to change cell behaviors, activity to kill bacteria or viruses, activity to induce gene expression, etc.), or by observation and quantification of a phenotypic characteristic of the targeted cell in comparison to a control cell (e.g. lack of ability to proliferate, differentiate, or undergo apoptosis, etc). Any suitable means to determine whether or not a targeted gene has been silenced may be used. Further, the result of silencing of the gene in a cell may be highly variable, e.g. the cell may die, or become quiescent; the metabolism of the cell may be altered; the cell may lose the ability to metastasize; etc. The specific effect of silencing the gene is not a key feature of the invention, so long as the effect results in a desired outcome (e.g. ameliorating an undesired condition, or bringing about a desired condition, in the cell).
The constructs utilized in the practice of the invention include at least one cell-specific promoter that is operationally linked to nucleotides (usually DNA) encoding an RNA molecule. By “operationally linked” we mean that, in the vector, the promoter is associated with the nucleotides encoding the RNA in a manner that allows the promoter to drive transcription (i.e. expression) of the RNA from the nucleotides. Transcription of RNA from, e.g. a DNA template is well-understood. Those of skill in the art will recognize that many such cell-specific promoters are known, and additional cell-specific promoters are continually being discovered. All such cell-specific promoters are encompassed by the present invention.
The promoters that are employed in the invention are cell-specific. Those of skill in the art will recognize that some tissues are made up of a single type of cell, or some types of cells are expressed only in a particular tissue, and thus, the promoter may be referred to as a “tissue-specific” promoter. In addition, some promoters may be specific for more than one, but not all, cells. These promoters may also be used in the practice of the invention, so long as it is desired to silence a gene in all cells in which the promoter is active. Examples of cell (or tissue)-specific promoters and the cells for which they are specific include but are not limited to
The RNA molecule that is encoded by the construct of the present invention ultimately forms a double-strand RNA molecule within the cell in which it is transcribed. In general, one strand of the double-strand RNA structure will be in the range of from about 10 to about 30 ribonucleotides in length, and preferably from about 19 to about 25 ribonucleotides in length. Those of skill in the art will recognize that several viable strategies exist for forming such double-strand RNA. For example, a single RNA molecule that includes two regions that are homologus to each other and that will thus hybridize may be utilized. In this case, a hairpin loop will be formed. Alternatively, two separate RNA segments that are homologus to each other and that will thus hybridize may be formed. Other alternatives include microRNA-based hairpin RNA, etc. In one embodiment of the invention, only one gene is silenced in a particular, targeted cell type. However, this need not be the case. For example, provision of multiple constructs with the same cell-specific promoter but which encode different silencing RNAs may be used within the practice of the invention. This is illustrated in
In one embodiment of the invention, the promoter that is used is a constitutive promoter. However, in another embodiment, the promoter that is utilized is an inducible promoter. In this case, the formation of the silencing dsRNA in a targeted cell is not only cell specific, but expression of the RNA is activated or induced by a signal from the environment. Those of skill in the art will recognize that many suitable inducible promoters exist that could be used in the practice of the invention, examples of which include but are not limited to: (1) tetracycline-inducible system: The shRNA expression is under the control of the modified U6, H1, or 7SK promoter, in which the tetracycline operator (TetO) sequence is added. The tetracycline repressor (tTR) or tTR-KRAB expression is under the control of cell-specific promoter, such as SP-C promoter. In the absence of an inducer, the tTR or t-TR-KRAB binds to TetO and inhibits the expression of shRNA. The addition the inducer, doxycycline (DOX) removes the tTR or tTR-KRAB from the TetO and thus induces the transcription of shRNA in a cell-dependent manner since tTR or tTR-KRAB is only expressed in a specific cell type. (2) IPTG-inducible system. This is similar to (1) above except that TetO and tTR are replaced with lac operator and lac repressor, respectively. The inducer in this case is isopropyl-thio-beta-D-galactopyranoside (IPTG). (3) CER inducible system: a neomycin cassette (neo) is inserted into the U6 or H1 promoter that drives shRNA expression. The insertion disrupts the promoter activity and thus no transcription of shRNA occurs. However, the cell-specific expression of Cre recombinase under the control of a cell-specific promoter restores the promoter activity and thus the expression of shRNA in a specific cell type. The inducer in this case is tamoxifen. (4) Ecdysone-inducible system. The inducible ecdysone-responsive element/Hsmin (ERE/Hsmin) is added to U6 promoter that controls the expression of shRNA. The expression of two proteins, VgEcR and RXR are driven by cell-specific promoters. In the presence of the inducer, MurA, VgEcR and RXR form a dimer and bind to ERS/Hsmin to initiate the transcription of shRNA in a specific cell type. It will be understood that a construct can have more than one constitutive promoter, as well as combinations of constitutive and inducible promoters.
The methods of the invention involve creating constructs (e.g. vectors) that contain at least one cell-specific promoter that is operationally connected to DNA that encodes RNA for silencing a specific gene. In addition, the constructs are suitable for administration to individuals that are to be treated by the methods. In a preferred embodiment of the present invention, the construct is an adenoviral vector for delivery as disclosed herein. However, those of skill in the art will recognize that many other systems for delivering a nucleic acid to cells already exist or are currently under development, and would be suitable for use in the practice of the present invention. For example, other vectors (both viral and non-viral) may be utilized (e.g. plasmids, viral particles, baculovirus, phage, phagemids, cosmids, phosmids, bacterial artificial chromosomes, viral DNA, P1-based artificial chromosomes, yeast plasmids, and yeast artificial chromosomes, and the like. Some forms of viral vectors may be especially useful (e.g. viral vectors such as retrovirus, lentivirus, adenovirus or adenovirus-associated vectors). Alternatively, the construct may be delivered via liposomes or liposome-type delivery systems, or via attenuated bacterial delivery systems, by binding (either covalently or non-covalently) to another molecule which enhances delivery, by direct injection of the construct, or by catheterization, and the like. Further, other procedures which enhance the delivery of nucleic acids into cells may be utilized in conjunction with the practice of the present invention, e.g. various means of altering cell membrane permeability (e.g. ultrasound, exposure to chemicals or membrane permeability altering substances, and the like). Any appropriate means of delivery of the construct may be utilized in the practice of the present invention.
The present invention also provides a therapeutic composition comprising an effective dose of construct as described herein. The construct may conveniently be provided in the form of formulations suitable for administration to mammals. In addition, a suitable administration format may be determined by a medical practitioner for each patient individually. Suitable pharmaceutically acceptable carriers (e.g. aqueous, oil-based, etc.) and their formulation are described in standard formulations treatises, e.g., Remington's Pharmaceuticals Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A. “Parental Formulations of Proteins and Peptides: Stability and Stabilizers”, Journals of Parental Sciences and Technology, Technical Report No. 10, Supp. 42:2S (1988). Constructs of the present invention should preferably be formulated in solution at neutral pH, for example, about pH 6.5 to about pH 8.5, more preferably from about pH 7 to 8, with an excipient to bring the solution to about isotonicity, for example, 4.5% mannitol or 0.9% sodium chloride, pH buffered with art-known buffer solutions, such as sodium phosphate, that are generally regarded as safe, together with an accepted preservative such as metacresol 0.1% to 0.75%, more preferably from 0.15% to 0.4% metacresol. The desired isotonicity may be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions. If desired, solutions of the above compositions may also be prepared to enhance shelf life and stability. The therapeutically useful compositions for use in the practice of the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be mixed to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water and/or a buffer to control pH or an additional solute to control tonicity.
In a preferred form for use by a physician, the compositions will be provided in dosage form containing an amount of a construct that will be effective in one or multiple doses to induce RNA silencing. As will be recognized by those in the field, an effective amount of therapeutic agent will vary with many factors including the age and weight of the patient, the patient's physical condition, the type of condition being treated, and other factors.
The effective dose of the constructs of this invention will typically be in the range of about 107 to about 1012 pfu (plaque forming units).
The delivery of the constructs may be in general local or systemic, and may be accomplished by a variety of methods, including but not limited to injection, positive pressure, continuous flow infusion, oral or intravenous administration, inhalation, and the like. Any suitable delivery means may be utilized in the practice of the present invention. Further, the constructs may be delivered in conjunction with other therapies.
The methods of the invention can be used to treat conditions that are caused at least in part by the expression of a particular gene. In general, conditions that are treated by the methods of the invention are those in which the phenotypic expression of the targeted gene would generally be considered unfavorable or untoward for the individual in whom the gene is expressed. For example, the expression of the gene may lead to or contribute to the development of symptoms of a disease, or may predispose an individual in whom the gene is expressed to the development of such symptoms. Such conditions include but are not limited to:
Those of skill in the art will recognize that the RNAi technology of the present invention can be used to treat any condition for which it is desired to reduce or eliminate the expression of a particular gene or genes in a particular type of cell or cells. (See, for example, Uprichard S L, FEBS Letters, in press (2005), available on-line, Leung R K M, Whittaker, P A, Pharmacology & Therapeutics, 107: 222-239 (2005); Shankar P, Manjunath N, and Lieberman, J. JAMA, 293: 1367-1373, 2005)
However, the genes that are silenced (i.e. those whose expression is eliminated or reduced) need not be directly related to recognized “diseases” as such. For example, in some cases it may be desirable to silence genes related metabolism (e.g. to bring about weight loss, to lower cholesterol, etc.).
In one embodiment of the invention, the genes that are targeted for silencing are located in lung cells, i.e. the targeted or selected cells are lung cells. The lung is one of the major targets for gene therapy. Alveolar epithelium is composed of morphologically and functionally distinct type I and type II cells. A number of genes that are expressed in a highly cell-selective manner in the respiratory epithelium have been isolated and characterized. These include the genes encoding surfactant protein (SP)-A, B, and C, and the Clara cell secretory protein. Among them, SP-C is exclusively expressed in alveolar type II cells of the distal airways. The human SP-C promoter has been successfully used to express a transgene in a cell-specific manner (9). This, in a preferred embodiment of the invention, the cells that are targeted are alveolar type II lung cells, the gene that is targeted for silencing is a gene that is expressed in an alveolar type II cells lung cells, and the promoter that drives expression of the silencing RNA is one that is active only in alveolar type II lung cells, such as the SP-C promoter.
The present invention also has useful applications as a laboratory tool. The ability to selectively silence a single gene, or specific combinations of genes, within a particular cell type allows the elucidation of the function of a specific gene (or specific combination of genes) in the cell type. The ability to do so provides a useful tool for understanding the role of specific genes in cellular metabolism, in susceptibility to disease, disease progression, or other possible functions of the gene.
Example 1. RNA interference (RNAi) is sequence-specific post-transcriptional gene silencing. Although it is widely used in the loss-of-function studies, none of current RNAi technologies can achieve cell-specific gene silencing. Here, we report a cell-specific RNAi system using an alveolar epithelial type II cell—specific promoter: surfactant protein C (SP-C) promoter. We show that the SP-C-driven small hairpin RNAs specifically depress the expression of exogenous reporter (enhanced green fluorescent protein) and endogenous genes (lamin A/C and annexin A2) in alveolar type II cells, but not other lung cells using in vitro cell and organ culture as well as in vivo. The present study provides an efficient strategy in silencing a gene in one type of cell without interfering with other cell systems and may have a significant impact on RNAi therapy.
Materials and Methods
Design and construction of shRNA and viral vectors: A DNA fragment containing 3.7-kb of human SP-C promoter, rtTA coding sequence and 0.45-kb SV40 poly A was PCR amplified with Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and the primer pair, 5′-CACCTCGAGATCGATGAAGACTGCTGCTCTCTACCACGTT-3′ (SEQ ID NO: 1) and 5′-TTCGAACGCGTAATTCGAGCTCGGTACCCGGGGATCAGACATGA-3′ (SEQ ID NO: 2) from pSP-C-rtTA vector (9). pENTR/SP-C-rtTA vector was obtained by directionally cloning the purified PCR product into pENTR/D-Topo vector (Invitrogen, Carlsbad, Calif.). Later, pENTR/SP-C-rtTA vector was digested by Sal I and EcoR I restriction enzymes to remove the rtTA fragment between SP-C promoter and poly A sequences. The annealed shRNAs with Sal I-EcoR I overhangs were then cloned into SP-C vector through Sal I-EcoR I sites to obtain a new vector, pENTR/SP-C-shRNA-pA with a poly A terminal sequence. We replaced the poly A sequence with a minimal poly A (mA) at the EcoR I and BamH I restriction sites using two annealed oligonucleotides:
This minimal poly A sequence has been successfully used for CMV-driven shRNA (3). The shRNAs targeted to enhanced green fluorescent protein (EGFP), lamin A/C (Lamin), annexin A2 (AII), and an unrelated siRNA negative control (Con) contain a sense strand siRNA 19- or 21-nucleotide sequence, followed by a short spacer (5′-TTCAAGAGA-3′), an antisense strand, and two thymidines. Four sets of oligonucleotides with Sal I and EcoR I overhangs were synthesized:
All the shRNA sequences were subcloned into the pENTR vector with SP-C promoter and the minimal poly A sequences between the Sal I-EcoR I sites. The final clones were verified by DNA sequencing. The CMV-driven EGFP expression cassette was PCR-amplified using pEGFP-N1 (Clontech, Palo Alto, Calif.) as a template, and cloned into pENTR/D-Topo vector. All the inserts in pENTR vector were switched into the adenoviral vector, pAd/PL-DEST, through the Gateway technique (Invitrogen, Carlsbad, Calif.). The resulting adenoviral plasmids (Ad/SP-C-shEGFP, Ad/SPC-shLamin, Ad/SP-C-shAII, and Ad/CMV-EGFP) were linearized by Pac I and purified with GENECLEAN Turbo kits (Qbiogene, Carlsbad, Calif.). Using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) as a transfection reagent, Pac I-linearized adenoviral plasmids were transfected into 293A cells for the generation of adenovirus. The adenoviruses were concentrated and purified by a cesium chloride density gradient ultracentrifugation (10). Infectious units and particle titers were determined by plaque assay and OD260 (11).
Cell culture and adenoviral infection: Alveolar type II cells were isolated from rat lungs as previously described (12). A mixed lung cell preparation was obtained by using elastase digestion (4.5 units/ml) as previously described (13). The resulting cell mixture consisted of alveolar type I cells, type II cells, Clara cells, ciliated airway epithelial cells, fibroblasts, macrophages, and lymphocytes. To preserve type II cell phenotype, type II cells and mixed lung cells were cultured on an air-liquid culture system as previously described (14). The cells (3×106) were plated on a 30 mm filter insert (Millipore, Bedford, Mass.) coated with rat-tail collagen and Matrigel (4:1, vol/vol, Collaborative Biomedical Products, Bedford, Mass.). One ml of DMEM containing 5% rat serum, 10 ng/ml keratinocyte growth factor, and 10 nM dexamethasone were added to each side of the insert. The plates were placed on a rocking rotator inside an incubator with 5% CO2. After a 20-h culture, 0.3 ml of the same medium containing adenoviruses (400 mutiplicities of infection or MOI) was added to the apical surface, and 1.5 ml of the medium was placed on the outside of the insert. A 1:8 ratio was used when Ad/CMV-EGFP and Ad/SP-C-shRNA adenovirus were added simultaneously. The medium was changed on alternate days. On the 6th day, the type II cells were directly examined for the EGFP fluorescence, and the mixed cells collected by disrupting the collagen gel with a pipette and filtering through 160-μm pore-size filter. Cells were cytospinned to coverslips for immunocytochemistry
In vitro lung organ culture: The lungs were excised from three-day-old rat pups and cut transversely into 5-mm slices using a sterile blade. The lung slices were cultured on a 30 mm filter insert. Serum free, hormone-free BGJb medium (0.3 ml) containing 0.2 mg/ml ascorbic acid, 0.5 units/ml penicillin, and 0.5 g/ml streptomycin was added on the inside of insert and 1.5 ml outside of insert (15). In this design, the lung slices were situated just above the median and remained suffused with medium by capillary action through the membrane. The adenovirus (6×109 particles) was added on the inside of the insert at the time of plating. After culturing for 6 days, the organ culture was fixed with 4% (w/v) formaldehyde, embedded in paraffin, sectioned, and immunostained with anti-annexin II and anti-SP-C antibodies.
Adenoviral delivery into the rat lungs: Adult male Sprague-Dawley rats (200-250 g) were used for in vivo studies. Oklahoma State University Animal Use and Care Committee approved the animal procedures. Endotracheal intubation and administration of the virus was done as described earlier (16). In brief, the animals were anesthetized with an intraperitoneal injection of Ketamine and Xylazine. The epiglottis and trachea of the animal were visualized using a modified intubation wedge. The animals were then orally intubated using a sterile 18-guage intravenous catheter. Immediately prior to the administration of the adenovirus, the animals were forced to exhale by circular compression of the thoracic cavity and then 200-400 μl of the adenovirus (5×1011 particles) in phosphate buffered saline containing 50% Survanta (Abbot laboratories, Columbus, Ohio), 1 mg/ml of protamine sulfate, and 250 μg/ml hydrocortisone (17;18) was administered. The virus was incubated for 10 min with protamine sulfate before being administrated into the animal. Animals were sacrificed on the 5th day. The lungs were instilled with 4% formaldehyde (w/v) in phosphate buffered saline (pH 7.4), embedded in paraffin, and processed for immunostaining.
Northern Blotting: Total RNA was isolated with TRI reagents (Molecular Research Center, Inc., Cincinnati, Ohio) from the cultured type II cells. RNA (20 μg/lane) were electrophoretically separated on a 15% polyacrylamine-7 M urea gel and transferred by electroblotting onto to Hybond N+ membrane (Amersham Pharmacia Biotech). The sense oligonucleotides (100 pmoles) of shEGFP or shCon were end-labeled with polynucleotide kinase and [32P]-ATP (150 μCi), purified through a G-25 MicroSpin Column (Amersham Pharmacia Biotech), heated for 5 min to 65° C., and then used for hybridization at 37° C. overnight. Membrane was washed for 2 times of 5 min interval at room temperature in 2×SSC plus 0.1% SDS, 3 times for 10 min in 0.1×SSC plus 0.1% SDS, and exposed on BioMax MS films (Kodak).
Reverse Transcription/Polymerase Chain Reaction: After being treated with DNase, 1 μg of total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (200 U) in presence of 100 ng 18-mer oligo(dT) and 5 ng EGFP reverse primer. One μl of cDNA were used to amplify the EGFP fragment with 1× PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 200 μM of primers, and Taq DNA polymerase. For normalization of RNA loading, the housekeeping gene, β-actin, was also amplified from each sample. The primer sequences are as follows: EGFP, forward, 5′-TGCCACCTACGGCAAGCTGA-3′ (SEQ ID NO: 13) (111-130), reverse, 5′-TCGATGTTGTGGCGGATCTT-3′ (SEQ ID NO: 14) (499-518); β-actin, forward, 5′-GGCATTGTAACCAACTGGGACGATATG-3′ (SEQ ID NO: 15) (220-246), reverse, 5′-TTCATGGATGCCACAGGATTCC-3′ (SEQ ID NO: 16) (807-828). PCR amplification was performed using the following conditions: 1 cycle of 95° C. for 3 min, 25 cycles of 94° C. for 30 sec, 56° C. for 1 min, and 72° C. for 1 min, followed by a final elongation step of 72° C. for 7 min. After amplification, 10 μl aliquots of PCR products from each condition were separated on a 1.5% agarose gel. Signals were quantified by densitometric analysis using Bio-Rad Quantity One 4.0.3 software.
Western blotting: Cells were lyzed at 4° C. for 1 h in the lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1% Triton X-100, 10 mM EGTA, 2 mM EDTA, 20 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride). Ten to twenty μg of the proteins were resolved on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and electrophorically transferred onto a nitrocellulose membrane. The membranes were blocked with Tris-buffered saline plus 0.1% Tween 20 (TTBS) containing 5% non-fat milk for 1 h, incubated with the appropriate primary antibodies (anti-β-actin, 1:4000 dilution; anti-GFP, 1:1000 dilution) in TTBS containing 1% BSA for 2 h, followed by incubation with secondary antibodies (horseradish peroxidase-conjugated IgG, 1:5,000 dilution) for 1 h. Finally, the proteins were visualized by enhanced chemiluminescence reagents. Signals were quantified by densitometric analysis using Bio-Rad Quantity One 4.0.3 software.
Immunostaining: Immunohistochemistry and immunocytochemistry were performed as described previously (19). The primary and secondary antibodies were: polyclonal goat anti-SP-C (1:50), monoclonal anti-lamin A/C (1:50), and polyclonal rabbit anti-annexin II (1:50) antibodies (all from Santa Cruz Biotechnology, Santa Cruz, Calif.) and Alexa 488 or Alexa 546 -conjugated anti-goat (1:200), Alexa-488-conjugated anti-mouse (1:200), Cy 3-conjugated anti-mouse (1:200), and Alexa 546-conjugated anti-rabbit (1:200) IgG (Molecular Probes, Eugene, Oreg.).
Results and Discussion
To determine whether the SP-C promoter can be used to produce siRNA and silence gene expression in a cell-specific fashion, we constructed an adenoviral vector, in which shRNA in the form of a small hairpin structure, was placed under the control of the 3.7-kb human SP-C promoter, and followed by a minimal poly A (
Northern blotting analysis showed that Ad/SPC-shEGFP or Ad/SPC-shCon infected type II cells expressed a ˜57 base pair RNA specific to shEGFP or shCon sequences (
About 40 different cell types exist in the lungs. To further demonstrate the specificity of SP-C-driven shRNA, we prepared a lung cell mixture from the elastase-digested rat lungs (13) and used it for gene silencing. The cell preparation contains alveolar type I and type II cells, Clara cells, ciliated airway epithelial cells, fibroblasts, macrophages, and lymphocytes. The mixed cells were cultured on an air-liquid cell culture system as described above and infected with Ad/CMV-EGFP in the presence of Ad/SP-C-shEGFP or Ad/SP-C-shCon. EGFP fluorescence was monitored with a fluorescence microscope, and type II cells were identified by immunostaining using anti-SP-C antibodies. SP-C expression was taken into consideration to ascertain the gene silencing occurred only in type II cells and not in other cells. In the Ad/SP-C-shEGFP-treated group, EGFP signals was markedly decreased in a majority of type II cells (arrows,
The ability of shRNAs controlled by SP-C promoter to work on endogenous genes was tested. Two endogenous genes with diverse functions and different cellular locations were selected, lamin A/C and annexin A2. Lamin A/C is a nuclear membrane protein involved in the organization of nuclear architecture (20). Annexin A2 is a cytosolic Ca2+-dependent phospholipid-binding protein and plays an important role in the membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells (21). The siRNA sequence, targeted to the coding region of 607 to 625 of rat lamin A/C gene, was chosen based on the previous reports on human lamin A/C gene, (2;4). In this region, there is one base difference between rat and human sequences, which we switched to the rat sequence (C611→T611 ). A mixed lung cell culture was infected with Ad/SP-C-shLamin or Ad/SP-C-shCon. After a 4-day culture, the cells were double-labeled with anti-lamin A/C and anti-SP-C antibodies to determine the protein expression level of lamin A/C and to identify alveolar type II cells, respectively. As shown in
Dissection of lung cells from rat lung tissues and the cell isolation procedure may alter cell function and activate gene expression. Therefore, an in vitro model of neonatal rat lung organ culture was used to further test the specific silencing of an endogenous gene, annexin A2. This organ culture system maintains the cellular architecture and thus intracellular contacts and communications (15). We have previously screened six in vitro transcribed siRNA sequences targeted to different regions of the rat annexin A2 gene and found that the siRNA sequence targeted to 129-147 nucleotide was the most effective in silencing annexin A2 protein expression in type II cells (22). This annexin A2 siRNA sequence was used in the present study to construct an adenoviral vector, Ad/SP-C-shAII. Double-labeling with anti-annexin A2 and SP-C antibodies was used to determine annexin A2 protein expression level (red) and to identify type II cells (green), respectively. The infection of the neonatal lung organ culture with Ad/SP-C-shAII generated green-positive (type II cells) and red-negative (annexin A2 expression level) cells (
Finally, to examine whether SP-C-driven shRNA works in vivo, we directly delivered the adenoviral vector, Ad/SP-C-shAII, into rat lungs and determined annexin A2 expression in type II cells. The virus was delivered to rat lungs intrabronchially. Surfactant and protamine were included to enhance the efficiency of adenovirus-mediated siRNA expression, and corticosteroids were used to inhibit inflammation and minimize virus-related toxicity (17;18). Rats were sacrificed and the lungs infused with 4% (w/v) formaldehyde 5 days after delivery. The tissue section was double-stained with anti-annexin A2 and anti-SP-C antibodies. In the Ad/SP-C-shAII-treated group, we found that annexin A2 was silenced in type II cells (green-positive and red-negative cells) (
The most commonly used plasmids for expressing siRNAs in cells contain RNA polymerase III-based promoters such as U6 and H1 or RNA polymerase II promoters such as CMV (3-6). Although those promoters have a wide applicability in cell systems in vitro, their lack of cell specificity limits their usage in vivo. We have demonstrated that SP-C-driven shRNA expression effectively and specifically silences exogenous and endogenous gene expression in type II lung cells using in vitro cell and organ culture as well as in vivo. The current studies, therefore, establish proof-of-principle for using a cell-specific promoter to depress gene expression in a particular type of cell. This provides an efficient strategy in targeting and silencing a specific gene in vivo in one type of cell without interfering with other cell systems. For example, in order to target an oncogene in cancer cells, but not normal cells, a telomerase reverse transcriptase (TERT) promoter (23) would be used to drive shRNA expression in cancer cells. Considerable interest has developed in the potential for RNAi therapy. As a therapeutic agent, there is a great need for delivering siRNA to and thus silencing a gene in a particular type of target cell. This strategy may have a significant impact on RNAi therapy.
3. Xia, H., Mao, Q., Paulson, H. L. and Davidson, B. L. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol, 20, 1006-1010.
This application claims benefit of U.S. provisional patent application U.S. 60/607,671, filed Sep. 7, 2004, the complete contents of which are hereby incorporated by reference.
The Government of the United States of America may have certain rights in this invention pursuant to Grant No. 5 R01 HL52146-09 and Grant No. 1 R01 HL071628-01A1 awarded by the National Institute of Health.
| Number | Date | Country | |
|---|---|---|---|
| 60607671 | Sep 2004 | US |
