Patent Application
20170191057
The present disclosure relates to the field of oncology, biomarkers, biology and drug discovery. Particularly, the disclosure relates to compositions useful for studying the cell cycle, RNA-editing enzymes, monitoring of disease progression, and pharmacological screening. The disclosure also includes methods for expanding stem cells. The disclosure also relates to compositions and methods for inhibiting the action of double-stranded RNA-specific adenosine deaminases, or ADAR enzymes. The disclosure includes methods and compositions for treating, ameliorating or preventing diseases and conditions, such as cancer, including cancers associated with stem cells such as, without limitation, a myeloproliferative neoplasm like chronic myeloid leukemia (CML) or acute myeloid leukemia (AML).
RNA editing is a post-transcriptional processing mechanism that results in an RNA sequence that is different from that encoded by the genomic DNA and thereby diversifies the gene product and function. The type of RNA editing that is most prevalent in higher eukaryotes converts adenosine residues into inosine (A-to-I editing) in double-stranded RNA (dsRNA) through the action of double-stranded RNA-specific adenosine deaminases (Bass B L, Weintraub H, Cell, 1988; 55(6):1089-98). RNA-specific adenosine deaminases include ADAR1 (also known as ADAR), ADAR2 (ADARB1), and ADAR3 (ADARB2). ADAR1 and ADAR2 are active in embryonic cell types, and ADAR3 may play a nonenzymatic regulatory role in RNA-editing activity (Osenberg S, et al., GPLoS One, 2010, 5(6):e11173; Chen C X et al., RNA, 2000, 6(5):755-767.
The ADAR1 enzyme destabilizes double-stranded RNA through conversion of adenosine to inosine. The ADAR1 enzyme modifies cellular and viral RNAs, including coding and noncoding RNAs. ADAR1 targets double-stranded RNA hairpin-containing loop structures, such as microRNAs (miRNAs) by operating through base-pairing with complementary sequences within an mRNA molecule leading to mRNA degradation and gene silencing. ADAR activity is suggested in various tumor types (Galeano F. et al., Semin Cell Dev Biol, 2012 23(3):244-250; Cenci et al., J Biol Chem, 2008, 283(11):7251-7260).
Traditional treatments for myeloproliferative neoplasm are a great financial burden on patients. Moreover, they are not efficient at eradicating cancer stem cells (CSC), such as leukemia cancer stem cells (LCS). Studies suggest that LCS promote therapeutic resistance, relapse and disease progression, the leading causes of leukemia mortality, as a result of enhanced survival and self-renewal combined with a propensity to become dormant in supportive microenvironments. Therapies capable of breaking LSC quiescence while sparing normal hematopoietic stem cell (HSC) function have remained elusive. New drugs that target cancer stem cells are urgently needed for patient care as well as research tools that aid in the studies of the role cancer stem cells play in cancer. The disclosure herein provides drugs and research tools useful in prognostication, pharmacological screening, treating and studying cancer.
Disclosed herein are methods of expanding cells, such as stem cells and hematopoietic cells, comprising increasing ADAR1 activity in such cells, such as by overexpression of ADAR1 on a plasmid. In some aspects of this embodiment, the cells are cord blood cells (CB), such as cord blood cells that are CD34+. Included in this embodiment are the cell or cells that overexpress ADARI.
Another embodiment disclosed herein are methods for transplanting hematopoietic-reconstituting cells into a subject in need thereof, the method comprising administering to the subject CD34+ cells having enhanced expression of ADAR1. In an aspect of this embodiment, the subject has a disorder treatable by hematopoietic stem cell transplantation, such as a hematopoietic deficiency or malignancy.
In a related embodiment are methods for reconstituting hematopoietic cells into subjects in need thereof comprising: a) collecting cord blood cells from a donor or donors; b) transducing into the cells from step (a) a vector that overexpresses ADARI; c) expanding the transduced cells for about 3-5 days; d) collecting the cells from step (c); and e) transplanting the expanded cells from step (D) into the subject in need of treatment. In some aspects of this method the cord blood cells are CD34+. In some embodiments of this method the subject is being treated for leukemia, lymphoma or other blood-related diseases. In some aspects of this method the subjects blood supply has been damaged by chemotherapy, radiation, or toxic agent and/or the patient needs a bone marrow transplant. In some aspects of this method the cells to be expanded are transduced with a vector that overexpresses ADAR1 such as a lentiviral vector. In some embodiments of the method successful expansion is measured by an elevation in CD45+ as compared to the CD45+ levels in cord blood cells without overexpression of ADAR1 or a normal control. In some embodiments the expanded cells are CD34+ CD38− and Lin− human stem cells.
Also disclosed herein are vectors that overexpress ADAR1 and vectors that express a mutant version of ADAR1 protein wherein the mutation results in an ADAR1 protein that retains dsDNA and dsRNA binding capacity but has reduced ability to convert A-to-I activity. In aspects of this embodiment, the mutant ADAR1 protein has a mutation in the active RNA-editing sites, such as a point mutation, for example, a point mutation at nt5293 which results in an A to C mutation resulting in glutamic acid to alanine change. In some aspects the vector is a human-specifc lentivaral vector. Also included in this embodiment are cells containing the disclosed ADAR1 vectors. In some embodiments the cells are K562 cells.
Another embodiment disclosed herein is a reporter vector for measuring A-to-I editing comprising dual-luciferase, or enhanced green fluorescence protein (EGFP) or enhanced yellow fluorescence protein (EYFP) and a stop codon (TAG) in a hairpin structure which is part of the promoter sequence, wherein the stop codon is removed due to RNA editing resulting in a reporter gene signal being generated as a readout of RNA-editing level. In some aspects this vector contains an opposite oriented Alu-sequence to detect RNA editing in non-coding regions. Included in this embodiment are cells containing the vectors, such as stem cells and cancer stem cells. In an aspect of this embodiment are methods for measuring and tracing the A-to-I RNA-editing changes in a cancer stem cell comprising introducing the vectors into cancer stem cells and correlating changes in A-to-I RNA editing after exposure to agents. In some aspects the cancer stem cells are in in vitro stromal co-culture system or in vivo xenograft mouse models.
In another embodiment are vectors comprising wild-type GLI2 cloned into pcDH-EF1-T2A-cop and ligated in frame with the FLAG epitope. In a related embodiment are vectors comprising mutant GLI2 wherein the transcription activation domain has been deleted, In some embodiments the mutant GLI2 is cloned into pcDH-EF1-T2A in frame with the FLAG epitope. Included in these embodiments are cells containing the GLI2 wild-type and mutant GLI2 vectors. The cells can be stem cells, such as cancer stem cells.
Also disclosed herein are vectors comprising a bi-cistronic fluorescent ubiquitination-based cell-cycle indicator (FUCCI) reporter in a vector that is suitable for use in mammalian cell populations and which are useful for studying the cell cycle. In some embodiments the vector contains a bi-cistronic FUCCI reporter that was generated by cloning mCherryhCdt1 and Venus-hGeminin into vector pcDH-EF1-T2A. Included in this embodiment are cells that contain the bi-cistronic FUCCI reporter vectors. The cells can be stem cells, such as cancer stem cells.
In another embodiment are methods for treating, ameliorating or preventing diseases and conditions associated with the down regulation of one or more microRNA identified in
As used herein, the term “subject” refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey); other mammals such as rodents (mice, rats), cattle, pigs, horses, sheep, goats, cats, dogs; and/or birds, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of an, agent, composition, compound or drug, then the subject/patient has been the object of treatment, observation, and/or administration of the composition, compound or drug.
In an embodiment disclosed herein are methods for monitoring if a subject with cancer or an inflammatory disease should be treated or enrolled in a clinical trial comprising: (a) isolating blood cells from the subject during or after treatment; (b) processing the isolated stems cells to detect and/or quantitate one or more microRNA identified in
In another embodiment disclosed herein are primers comprising, consisting of, or consisting essentially of the primers identified in
Disclosed herein are methods for screening for agonists and inhibitors of RNA editing comprising: (a) contacting a CD34+ cell which overexpresses ADAR1 with a test compound; and (b) determining whether the test compound acts as an agonist or inhibitor. In some aspects the determination is measured using the fluorescent A-to-I editing reporter described in
An embodiment disclosed here is a method of treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation and/or self-renewal (or self-renewal capacity) of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, comprising, administering 8-azaadenosine or an 8-azaadenosine derivative. In some aspects the 8-azaadenosine derivative is a compound as shown in
In still other embodiments are methods for detecting leukemic progression into blast phase comprising the steps of: (a) collecting a blood sample from a subject with leukemia; (b) isolating mononuclear cells from the blood sample; (c) isolating CD34+ cells; (d) isolating RNA from the CD34+ cells; (e) generating cDNA from the isolated RNA; (f) performing quantitative PCR using one or more primer sets for the genes listed in Table 1—MDM2, APOBEC3D, GLI1, AZIN1, SRPN, GSK3B, PTPN14, SF3B3, ABI1, LYST; and (g) quantifying the data to determine the relative A-to-G(I) editing ratios in one or more of MDM2, APOBEC3D, GLI1, AZIN1, SRPN, GSK3B, PTPN14, SF3B3, ABI1, LYST and MDM4, wherein if the ratio of edited is higher than a normal standard or a previous determination from the same patient obtained in chronic phase such increased editing activity indicates the patient may be in blast phase or entering blast phase and should be treated or entered into a clinical trial. In some embodiments of this method, the one or more primer sets are represented by SEQ ID Nos. 1-4 for MDM2 position 69237534; SEQ ID NOS. 5-8 for APOBEC3D position 39415872; SEQ ID NOS:9-12 for APOBEC3D position 39415911; SEQ ID NOS:13-16 for SRP9 position 225976198; SEQ ID NOS: 17-20 for Gli1 position 57864624; SEQ ID NOS:21-24 for Gli1 position 57864911(negative control); SEQ ID NOS:25-28 for GSK3B position 119545199; SEQ ID NOS:29-32 for AZIN position 103841636; SEQ ID NOS:33-36 for PTPN14 position 214529774; SEQ ID NOS:37-40 for SF3B3 position 70610885; SEQ ID NOS:41-44 for ABI1 position 27049636; SEQ ID NOS:45-48 for LYST position 235990569; and SEQ ID NOS:49-52 for MDM4 position 204521159. In some embodiments of this method, the cDNA preparation utilizes gene transcription with reverse transcription and gene-specific primers and/or random hexamer primers only and/or a supermix containing both random hexamer and oligo-dT primers.
Disclosed herein are methods for testing whether an agent is effective for reducing the editing activity of ADAR1 comprising: (a) adding the agent to cells that have ADAR1 activity; (b) isolating RNA from the cells of step (a); (c) generating cDNA from the isolated RNA; (d) performing quantitative PCR using one or more primer sets for the genes listed in Table 1—MDM2, APOBEC3D, GLI1, AZIN1, SRPN, GSK3B, PTPN14, SF3B3, ABI1, LYST; and (e) quantifying the data to determine the relative A-to-G(I) editing ratios in one or more of MDM2, APOBEC3D, GLI1, AZIN1, SRPN, GSK3B, PTPN14, SF3B3, ABI1, LYST and MDM4, wherein if the ratio of edited/wild-type is lower than cells not treated with the tested agent indicates that the agent lowers the editing activity of ADAR1 and may be useful for treating conditions associated with increased levels of ADAR1 activity, such as leukemia and inflammatory diseases. In some embodiments of this method the one or more primer sets are represented by SEQ ID Nos. 1-4 for MDM2 position 69237534; SEQ ID NOS. 5-8 for APOBEC3D position 39415872; SEQ ID NOS:9-12 for APOBEC3D position 39415911; SEQ ID NOS:13-16 for SRP9 position 225976198; SEQ ID NOS: 17-20 for Gli1 position 57864624; SEQ ID NOS:21-24 for Gli1 position 57864911(negative control); SEQ ID NOS:25-28 for GSK3B position 119545199; SEQ ID NOS:29-32 for AZIN position 103841636; SEQ ID NOS:33-36 for PTPN14 position 214529774; SEQ ID NOS:37-40 for SF3B3 position 70610885; SEQ ID NOS:41-44 for ABI1 position 27049636; SEQ ID NOS:45-48 for LYST position 235990569; and SEQ ID NOS:49-52 for MDM4 position 204521159. In some aspects of this embodiment, the cells that have ADAR1 activity are cells containing a wild-type ADAR1 expression vector. In other aspects of the methods the cells are K562 cell. In some embodiments, the K562 cells are stably-transduced with lentiviral-ADAR1. In other aspects of this method the cells treated with an agent are CD34+ cells obtained from primary CML patients; or cells obtained from an immunocompromised non-human animal transplanted with primary blast crisis cells, such as, without limitation, a mouse.
In another embodiment are methods for detecting leukemic progression into blast phase comprising the steps of: (a) collecting a blood sample from a subject with leukemia; (b) isolating mononuclear cells from the blood sample; (c) isolating CD34+ cells; (d) isolating RNA from the CD34+ cells; (e) converting the RNA from step (d) into cDNA; (f) evaluating miRNA expression using MiScript qPCR array; and (g) determining if one or more of mir26a-5p, mir26b-5p, mir155-5P, mir21-5P, mir125a-5P, mir23b-3P, let7c, let7e, mir150-5p, or let7d are downregulated as compared to a normal control or a previous sample from the subject while in chronic phase, wherein if one or more of mir26a-5p, mir26b-5p, mir155-5P, mir21-5P, mir125a-5P, mir23b-3P, let7c, let7e, mir150-5p, or let7d are downregulated indicates that the patient is in or entering blast phase and should be treated or enrolled in a clinical trial.
Also disclosed herein are methods for testing whether an agent is effective for reducing the editing activity of ADAR1 comprising: (a) adding the agent to cells that have ADAR1 activity; (b) isolating RNA from the cells of step (a); (c) generating cDNA from the isolated RNA; (d) evaluating miRNA expression using MiScript qPCR array; (e) determining if one or more of mir26a-5p, mir26b-5p, mir155-5P, mir21-5P, mir125a-5P, mir23b-3P, let7c, let7e, mir150-5p, or let7d are upregulated as compared to cells not treated with the tested agent, wherein if one or more of mir26a-5p, mir26b-5p, mir155-5P, mir21-5P, mir125a-5P, mir23b-3P, let7c, let7e, mir150-5p, or let7d are upregulated this may indicate that the tested agent inhibits ADAR1 activity and may be useful to treat conditions associated with high ADAR1 editing activity, such as leukemia or inflammatory diseases. In some embodiments of this method, the cells that have ADAR1 activity are cells containing a wild-type ADAR1 expression vector. In other embodiments of this method, the cells are K562 cells. In other embodiments of this method the cells are K562 cells stably-transduced with lentiviral-ADAR1.
As used herein, the terms “compositions,” “drug,” “agent,” “compound,” and “therapeutic agent” are used interchangeably, and may include, without limitation, small molecule compounds, biologics (e.g., antibodies, proteins, protein fragments, fusion proteins, glycoproteins, etc.), nucleic acid agents (e.g., antisense, RNAi/siRNA, shRNA, and microRNA molecules, etc.), vaccines, etc., which may be used for therapeutic and/or preventive treatment of a disease (e.g., malignancy).
“Therapeutically effective amount,” or “therapeutic effect,” as used herein, refers to a minimal amount or concentration of an agent, composition, compound and/or drug that, when administered alone or in combination, is sufficient to provide a therapeutic benefit in the treatment of the condition, or to delay or minimize one or more symptoms associated with the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent. The therapeutic amount need not result in a complete cure of the condition; partial inhibition or reduction of the malignancy being treated is encompassed by this term.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes except to the extent they are inconsistent with the disclosures herein.
Disclosed herein are compositions and methods useful for studying the cell cycle, RNA-editing enzymes, monitoring of disease progression, and pharmacological screening based on RNA editing detection. Also disclosed herein are research tools and methods useful to study stem cells and diseases associated with stem cells, including cancer stem cells. Specifically these tools involve ADAR1 constructs, GLI2 reporters, and cell-cycle fluorescent indicators.
Data disclosed herein indicates that ADAR1 accelerates G0 to G1 phase transition in normal hematopoietic stem cells (cord blood CD34+ population), coupled with increased cell size and elevated expression of Ki67. The expanded population maintains stemness without any significant increase in differentiation. ADAR1 shows a preference of localization to the cell nucleus, suggesting the A-to-I editing events happen in the nucleus. qRT-PCR microarray of cell-cycle genes indicates that p21 expression level is reduced by >70% when ADAR1 is overexpressed. Therefore, ADAR1 is a useful tool for in vitro expansion of normal hematopoietic stem cells (
Moreover, shRNA knockdown of ADAR1 in CML BC sample shows a reduction of engraftment in bone marrow and spleen, and an enrichment of G0 population in the remaining cells. A decrease of self-renewal capacity as demonstrated by serial engraftment suggests the residual LSC failed to propagate (
Also disclosed herein are vectors such as lentiviral vectors that overexpress a mutant version of ADAR1 protein. The point mutation locates in the active RNA-editing sites, and when mutated, the ADAR1 protein will retain dsDNA and dsRNA binding capacity, but lacks the ability to convert A-to-I. These constructs are useful for understanding the functionality of ADAR1 (
In another embodiment, disclosed herein is a human-specific quantitative fluorescence dsRNA lentiviral reporter that can accurately measure aberrant A-to-I editing activity using luciferase activity or EGFP/YGFP as readout (see reference, Gommans et al, 2010). This reporter was cloned into a pCDH lentiviral plasmids (
Such lentiviral A-to-I editing reporters can be used to measure and trace the A-to-I RNA editing changes in LCS and other CSC with in vitro stromal co-culture system and in vivo xenograft mouse models, and correlate these changes with exposure to agents such as toxins and other chemicals, including effects on therapeutic outcome.
We have also observed an expansion cord blood cells ex vivo. This could serve as a new blood supply for patients who have had theirs destroyed by chemotherapy or radiation to treat leukemia, lymphoma and other blood-related diseases (
In one embodiment, the procedure can start with collection of cord blood cells from a donor or donors which is then transduced with lentivirus that overexpresses ADARI RNA editing enzyme. After about 3-5 days, the cells are collected and transplanted back into patients who received therapy to enable a healthy blood system to reestablish quickly. In some aspects the transplanted cells are CD34+. Successful expansion can be determined, without limitation, by measuring the elevation in CD45+ (
The data in
Currently, there are few methods to visualize cell cycle progression in LSC. Human LSC are characterized as quiescent self-renewing cells that drive leukemic transformation of myeloproliferative neoplasms and myelodysplastic syndromes. Additionally, current methods require sequential transduction and clonal selection. These methods and conditions are not feasible to study primary patient samples and LSC, which are generally limited in sample size. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI (fluorescent ubiquitination-based cell-cycle indicator) probes. The development of this new reporter offers a new and more effective method to track cell cycle progression in limited mammalian cell populations, including cell cycle changes in CSC. These constructs can identify changes in the cell cycle leading to new strategies to combat chemoresistance, and for screening new candidate therapeutics.
Bi-cistronic FUCCI reporter was generated by subcloning mCherry-hCdt1 into BspE1/SalI digested pcDH-EF1-T2A-copGFP and ligated in frame. Venus-hGeminin was subcloned into XbaI/BamHI digested pcDH-EF1-T2A-mCherry-hCdt1 and ligating in frame (
GLI2 Wild Type and mutant GLI2 Vectors: Wild type and mutant versions of GLI2 were generated by either subcloning full length human GLI2 or transcriptionally inactive GLI2 into XbaI/NotI digested pcDH-EF1-T2A-copGFP and ligated in frame with the FLAG epitope. Mutant GLI2 was generated by deletion of transcriptional activation domain of GLI2 denoted as pcDH-EF1-ΔTAD GLI2-T2A-copGFP (
While miRNA alterations have been associated with CML in the context of bulk tumor, no published studies have focused on CML LSC population that behaves remarkably different from bulk tumor and is responsible for disease progression and relapse. Disclosed herein is the finding that in Chronic Myeloid Leukemia (CML) progression is driven by ADAR1-dependent regulation of microRNA. Without wishing to be bound by any particular theory, it is postulated that in blast crisis, when ADAR1 p150 is upregulated, ADAR1 affects microRNA regulation by editing of pri-microRNA and pre-microRNA (
Also, disclosed herein is finding that blast crisis CD34+ cells show downregulation of 13 microRNA compared to CD34+ Chronic Phase. This can be related to overexpression of ADAR1 in Blast Crisis. ADAR1, by editing microRNA precursors negatively affect complete maturation of microRNA mediated by RNAse Drosha and DICER, thus leading to degradation of the edited product and a consequent downregulation of the edited microRNA (
Another embodiment disclosed herein is the finding that Lenti-viral overexpression of ADAR1 in chronic phase primary samples leads to a statistically significant downregulation of 10 microRNA, thus, showing a microRNA expression profile more similar to that seen in blast crisis (
Lenti-viral overexpression of ADAR1 in CD34+ Cord blood cells leads to the significant downregulation of 16 microRNA (
In Blast Crisis, where ADAR1 is upregulated and induces malignant progenitor reprogramming, 10 out the 13 microRNA that are downregulated in comparison with chronic phase, are in common with the downregulated microRNA in ADAR1 overexpressing Cord blood (
Since in both ADAR1 transduced cord blood and blast crisis, we observed a downregulation of the family of reprogramming microRNA let7, we overexpressed let7a in cord blood and evaluated the expression of the target LIN28, as well as its effect on self-renewal. Let7a overexpression leads to a statistical significant decrease of the efficiency of colony formation. This reduction is driven by the reduction in GM colonies. Moreover, overexpression of let7a decreases self-renewal as shown by the reduction of number of replating colonies. Further, Lin28 is statistically downregulated after let7-a overexpression. ADAR1, by downregulating let7 family increases self-renewal (
Whole transcriptome RNA sequencing (RNA-Seq) revealed increased adenosine to inosine (A-to-I) RNA editing during CML progression concentrated within primate specific Alu-containing transcripts. However, detection of RNA editing by RNA-Seq in rare cell populations can be technically challenging, costly and requires PCR validation by direct sequencing which cannot reliably detect low levels of RNA editing, or cloning which is labor-intensive and low-throughput. RNA Editing Site-Specific qPCR (RESSq-PCR) provides a rapid, clinically amenable method to detect aberrant primate-specific RNA editing at CSC-associated loci.
To develop a model system for development of the RESSq-PCR diagnostic assay, we established an in vitro model of stable ADAR1 expression in leukemia cells, wherein the BCR-ABL+ human leukemia cell line K562 was stably transduced with lentiviral human ADAR1-IRES-GFP (K562-ADAR1) or vector ORF (K562-ORF). Positively-transduced cells were identified on the basis of high GFP expression, FACS-purified to establish stable cell lines, and expanded under routine culture conditions. High levels of ADAR1 lentivirus and total human ADAR1 mRNA in K562-ADAR1 cells were confirmed by qRT-PCR using lentiviral-specific and human-specific primers, respectively (
We previously identified 274 differentially edited sites in CML blast crisis versus chronic phase, and selected top candidates from this dataset to develop site-specific primers and validate RNA editing sites as potential biomarkers of disease progression in primary patient samples. Candidate sites for validation and qRT-PCR assay development were selected from our previous RNA-sequencing data on the basis of greatest average change in RNA editing, with a focus on selecting sites within functionally relevant target genes involved in stem cell survival and self-renewal pathways, along with 3 known RNA editing sites within stem cell regulatory genes and cancer-associated pathways (
In order to implement rapid, inexpensive and clinically-amenable detection of RNA-edited transcripts that predict leukemic progression, we utilized a novel RNA editing site-specific quantitative RT-PCR (RESS-qPCR) primer design strategy. Since RNA-edited transcripts are predicted to differ at only one nucleotide position, developing highly specific primers for traditional qRT-PCR assays requires sensitive and selective primer design strategies. We have previously developed qRT-PCR primers that specifically recognize a gene product with one point mutation (JAK2 V617F11), and here we employed a similar strategy in designing RESSq-PCR primers (
In
All primer sets generated single bands in cDNA by gel analysis. In independent experiments, RESSq-PCR accurately detected robust RNA editing in K562-ADAR1 cells (
As proof-of-concept that small molecule inhibitors could block RNA editing activity, CD34+ cells from a CML blast crisis patient were treated with vehicle or 1 μM 8-azaadenosine (Veliz et al., JACS 2003) for 24 hours and co-cultured on humanized bone marrow stromal cells (SL/M2) for 5 days. Direct sequencing of two CSC-specific RNA editing biomarkers in APOBEC3D showed a reduction in edit/wild-type sequence ratios following 24 hrs of 8-azaadenosine treatment (
We have also demonstrated that let7 regulation by BCR-ABL and ADAR editing of miRs is niche dependent (
In alternative embodiments, nucleic acids of the invention are made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
The nucleic acids used to practice this invention, whether RNA, iRNA, shRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
Alternatively, nucleic acids used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066.
Techniques for the manipulation of nucleic acids used to practice this invention, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P I artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; P 1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages or plasmids.
Nucleic acids or nucleic acid sequences used to practice this invention can be an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. Compounds use to practice this invention include “nucleic acids” or “nucleic acid sequences” including oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA, shRNA) of genomic or synthetic origin which may be single-stranded or double-stranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practice this invention include nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. Compounds use to practice this invention include nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156. Compounds use to practice this invention include “oligonucleotides” including a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that may be chemically synthesized. Compounds use to practice this invention include synthetic oligonucleotides having no 5′ phosphate, and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
In alternative aspects, compounds used to practice this invention include genes or any segment of DNA or RNA involved in producing a polypeptide chain; it can include regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening sequences (introns) between individual coding segments (exons). “Operably linked” can refer to a functional relationship between two or more nucleic acid (e.g., DNA or RNA) segments. In alternative aspects, it can refer to the functional relationship of transcriptional regulatory sequence to a transcribed sequence. For example, a promoter can be operably linked to a coding sequence, such as a nucleic acid used to practice this invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. In alternative aspects, promoter transcriptional regulatory sequences can be operably linked to a transcribed sequence where they can be physically contiguous to the transcribed sequence, i.e., they can be cis-acting. In alternative aspects, transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
In alternative aspects, the invention comprises use of “expression cassettes” comprising a nucleotide sequence used to practice this invention, which can be capable of affecting expression of the nucleic acid, e.g., a structural gene or a transcript (e.g., encoding a DRP or antibody) in a host compatible with such sequences. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, in one aspect, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers.
In alternative aspects, expression cassettes used to practice this invention also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a “vector” used to practice this invention can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice this invention can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors used to practice this invention can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). In alternative aspects, vectors used to practice this invention can include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice this invention can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.
In alternative aspects, “promoters” used to practice this invention include all sequences capable of driving transcription of a coding sequence in a cell, e.g., a mammalian cell such as a brain cell. Thus, promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter used to practice this invention can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
“Constitutive” promoters used to practice this invention can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation. “Inducible” or “regulatable” promoters used to practice this invention can direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions.
Cloning and transduction of pCDH vectors is described in the user manual for pCDH cDNA Cloning and Expression Lentivectors from System Biosystems, and which is incorporated herein by reference.
Methods related to ADAR1 as it relates to cancer stem cells, including full transcriptome RNA sequencing, assays and the like are disclosed in WO 2013/036867 and Jiang et al., Proc. Natl Acad Sci USA 2013, 110(3):1041-1046, which are incorporated herein by reference.
RNA Editing Site-Specific qPCR is described in Ye S, Dhillon S, Ke X, Collins A R & Day I N (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29, E88, and Chen et al., Anal Biochem, 2008, 375(1):46-52. Other methods for determining RNA editing sites are disclosed in, for example, Chateigner-Bouting and Small, Nucl Acids Res, 2007, 35(17), e114 and Paz et al., Genome Res., 2007, 17(11):1586-1595.
The invention provides kits comprising compositions and/or instructions for practicing methods of the invention. As such, kits, cells, vectors, primers, and the like can also be provided. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
A large collection of leukemia patient samples and normal age-matched control bone marrow samples were obtained from consenting patients. Peripheral blood or bone marrow samples were processed by Ficoll density centrifugation and viable cells stored in liquid nitrogen. Normal peripheral blood mononuclear cells (MNC) were obtained from AllCells (Alameda, Calif.). Mononuclear cells from normal or CML patients were then further purified by magnetic bead separation of CD34+ cells (MACS; Miltenyi, Bergisch Gladbach, Germany) for subsequent FACS-purification of hematopoietic progenitor cells (CD34+CD38+Lin−) that represent the LSC fraction in BC CML (Jamieson et al.). Datasets from previous RNA-seq analyses of purified CML LSC are available through the NIH Sequence Read Archive (SRA), accession ID SRP028528.
For primary patient-derived LSC purification, CD34-selected cells were stained with fluorescent antibodies against human CD34, CD38, lineage markers (cocktail, all antibodies from BD Biosciences, San Diego, Calif.) and propidium iodide as previously described (Jiang et al., Jamieson et al, and Abrahamsson et al.). Following staining, cells were analyzed and sorted using a FACS Aria II, and hematopoietic progenitor (CD34+CD38+Lin−) populations were isolated. Freshly-sorted cells were collected in lysis buffer (Qiagen, Germantown, Md.) for RNA extraction followed by RNA-seq or qRT-PCR analyses as previously described (Jiang et al.).
For PCR and targeted Sanger sequencing analysis, 1-2 μL of first-strand cDNA templates were prepared for PCR in 25-50 μL reaction volumes using the high-fidelity KOD Hot Start DNA Polymerase kit according to the manufacturer's instructions (EMD Millipore, Temecula, Calif.). “Outer” primers (Additional file 2: Table S2) used for sequencing produce predicted amplicons of approximately 150-250 nucleotides in length, and flank each editing site with approximately 50-100 bp on either side of the editing site to facilitate successful sequencing analysis. PCR cycling conditions were as follows: 95° C. for 2 minutes, followed by 35 cycles of 95° C. for 20 seconds, 62° C. for 10 seconds and 70° C. for 10 seconds, with a final extension step of 70° C. for 30 seconds. Production of amplicons of the predicted size was verified for each outer primer set by DNA gel electrophoresis using 10-20 μL of the completed reaction mixture separated on 2% agarose gels containing ethidium bromide and visualized under UV light. Then, 15 μL of each reaction was processed within 24 hrs for PCR purification and sequencing was performed on ABI 3730xl DNA Sequencers (Eton Bioscience, San Diego, Calif.). Sanger sequencing was carried out using the reverse outer primer used for PCR amplification, so edited loci are identified in the reverse complementary sequence as T/C nucleotides, except in cases where the gene products are transcribed from the reverse strand. Sequence chromatograms were analyzed using 4Peaks (by A. Griekspoor and Tom Groothuis) and peak heights calculated using ImageJ (NIH). For RNA editing analysis of sequencing chromatograms, ratios of edited/WT peaks were calculated using the raw peak amplitude of each sequence trace.
K562 cells (ATCC, Manassas, Va.) were maintained in complete medium containing DMEM (Life Technologies, Carlsbad, Calif.), 10% fetal bovine serum (FBS), 1% Glutamax (Life Technologies), and 1% penicillin-streptomycin (Life Technologies). Parental cell lines and stably-transduced lines were authenticated as K562 by routine qRT-PCR analysis of BCR-ABL transcript levels (Jiang et al.). Mouse bone marrow stromal cell lines (SL and M2) expressing human interleukin-3 (IL-3), stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF), which support erythroid and myeloid cell expansion and differentiation, were maintained under standard culture conditions, as previously described (Hogge et al.). Briefly, SL cells were grown in complete medium containing DMEM, 10% FBS, 1% Glutamax, and 1% penicillin-streptomycin, while M2 cells were grown in complete medium containing RPMI, 10% FBS, 1% Glutamax, and 1% penicillin-streptomycin (all from Life Technologies). Every four passages, cells were selected by addition of G418 and hygromycin to the culture media for one passage (3-4 days), to maintain human cytokine expression (Hogge et al.). All cell lines were maintained in T-25 or T-75 culture flasks and were passaged at dilutions of 1:5-1:10 every 2-4 days. Low passage aliquots of cells were thawed every two months to ensure consistency of experiments.
We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP (Jiang et al.). For production of the catalytically-inactive ADAR1 mutated (ADAR1m) lentivirus, site-directed mutagenesis was carried out using the QuikChange II Site-Directed Mutagenesis Kit (Agilent) according to manufacturer's instruction. Mutagenic primers were designed to produce a nucleotide substitution of A5293C, which generates an E912A amino acid change and abolishes RNA editase activity (Lai et al.). Primers contained the desired mutation and anneal to the same sequence on opposite strands of the plasmid [(FW 5′-GTCAATGACTGCCATGCAGCAATAATCTCCCGG-3′ (SEQ ID NO:53), REV 5′-CCGGGAGATTATTGCTGCATGGCAGTCATTGAC-3′ (SEQ ID NO:54)]. XLI super competent cells were transformed with amplification products, after digestion with DpnI. Colonies were screened to identify mutated clones by DNA sequencing (Sanger sequencing, Eton Bioscience). Lentiviruses including control vectors (ORF) were produced according to established methods (Goff et al.) with some batches of lentivirus being produced by the GT3 Viral Vector Core Facility (UCSD). We have previously validated lentivirus transduction efficiency in normal cord blood, 293T cells and K562 cells, with an increase of approximately five-fold overexpression of ADAR1 transcripts confirmed by qRT-PCR analysis (Jiang et al).
Transduction of Human Cell Lines and Primary Cells with Lentiviral-ADAR1:
For preparation of stably-transduced K562 cell lines, 50,000 wild-type (wt) K562 cells were plated into 96-well U-bottom plates in complete culture medium and transduced with lentiviral vectors expressing GFP (ORF), ADAR1-GFP, or ADAR1m-GFP at multiplicities of infection (MOI) from 50-200. After transduction, cultures were expanded for at least 5 passages and then processed for FACS purification of GFP-positive cells to establish pure stably-transduced lines. Stable expression of lentivirus-enforced ADAR1 conferring increased transcript levels of human ADAR1 in K562-ADAR1 cells was confirmed at every 5 passages by qRT-PCR.
For transduction of human normal HSC and CML progenitors, 50,000 CD34-selected (MACS, Miltenyi, Auburn, Calif.) cells were plated in 96-well U-bottom plates in StemPro media (Life Technologies) supplemented with human cytokines (IL-6 10 ng/mL, FLT3 50 ng/mL, SCF 50 ng/mL, and Tpo 10 ng/mL) as previously described Jiang et. al and Abrahamsson et al. Twenty-four hours later, cells were transduced with lentiviral vectors (ADAR1 or ORF control, MOI=50-100) for up to five days. For co-culture experiments, CD34-selected CP CML cells were transferred three days after transduction (MOI=75) to monolayers of mouse bone marrow stromal cell cultures containing a 1:1 mixture of irradiated SL and M2 cells (50,000 total stromal cells per well in 24-well plates) (Goff et al.). Primary transduced cells were maintained in co-culture for 5-days in Myelocult (Stem Cell Technologies, Vancouver, Canada) and then the total culture was harvested in lysis buffer for RNA extraction and qRT-PCR and RESSq-PCR analyses.
For purification of stably-transduced K562 cell lines, K562 cells transduced with lentiviral-ADAR1 or ORF controls (MOI=50-200) were collected (minimum 1×106 cells), washed in HBSS containing 2% FBS (staining media), and sorted using a FACS Aria II for high GFP signal to purify the highly-transduced cell population. Purified cells were collected in complete media and maintained under routine culture conditions for K562 cells. The lentiviral-ORF and ADAR1 vectors include a blasticidin-resistance gene, but we observed no significant change in ADAR1 expression in our stably-transduced cell lines following selection with blasticidin, and therefore no subsequent selection method was used after FACS purification.
Cell lines, lentivirus-transduced primary hematopoietic cells, or FACS-purified primary cells were harvested in lysis buffer (Qiagen). RNA was purified using RNeasy extraction kits with a DNase (Qiagen) incubation step to digest any trace genomic DNA present. For RNA extraction from cell line lysates, 1-2×106 cells were extracted using RNeasy mini columns, and for primary cells, 5-10×104 cells were lysed and extracted using RNeasy micro columns. Genomic DNA was purified from equal numbers of cells lysed separately using the QIAamp DNA Blood Mini Kit (Qiagen) including an RNase A incubation step to digest any RNA present (Qiagen). RNA was stored at −80° C. and gDNA stored at −20° C. Immediately prior to reverse transcription of RNA samples, nucleic acid concentrations were quantified on a NanoDrop 2000 spectrophotometer (Thermo Scientific), and purity was considered acceptable if A260/A280 values were >1.8. For standard qRT-PCR analysis of relative mRNA expression levels, DNA was synthesized using 50 ng-1 μg of template RNA in 20 μL reaction volumes using the First-Strand SuperScript III Reverse Transcriptase Supermix (Life Technologies) followed by incubation with RNase H according to the manufacturer's protocol and as described previously (Abrahamsson et al.). All cDNA products were stored at −20° C.
Because RNA editing events often occur in pre-processed RNA species, for cDNA preparation, we tested three different conditions, including (1) reverse transcription with gene-specific primers, (2) random hexamer primers only, or (3) a supermix containing both random hexamers and oligo-dT primers. Using cDNA prepared with all three methods was suitable for detection of intronic regions in cDNA prepared from DNase-digested RNA extracts, and detected increased RNA editing in K562-ADAR1 cells. We therefore proceeded with the standard supermix reverse transcription method for RESSq-PCR, as this would provide the most versatility for use of valuable human tissue samples and would allow analysis of total mRNA expression of other genes in the same samples.
Primers were synthesized by ValueGene (San Diego, Calif.) and diluted to 10 μM working dilutions in DNase/RNase-free water. qRT-PCR was performed in duplicate using cDNA (1 μL reverse transcription product per reaction) on an iCycler (Bio-Rad, Hercules, Calif.) using SYBR GreenER Super Mix (Life Technologies) in 25-μL volume reactions containing 0.2 μM of each forward and reverse primer. Cycling conditions were as follows: 50° C. for 2 minutes, then 95° C. for 8 minutes and 30 seconds, followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 60 seconds. Melting curve analysis was performed on each plate according to the manufacturer's instructions. For standard qRT-PCR, HPRT mRNA transcript levels were used to normalize Ct values obtained for each gene, and relative expression levels were calculated using the 2−ddCt method. To ensure validity of results, only Ct values <35 were used in gene expression analyses. All primer sets were tested in a no-template control (NTC) reaction containing only water instead of cDNA, and all gave Ct values >35 in NTC reactions. Production of a single amplicon of the expected size was verified for each primer set by DNA gel electrophoresis on 2% agarose gels containing ethidium bromide. For all cell line experiments, assays were repeated at least three times using separate RNA extracts and cDNA preparations.
In order to implement a rapid, cost-effective and clinically amenable method to detect a CSC-specific RNA editing fingerprint of cancer progression, we devised an RNA editing site-specific primer design strategy that is compatible with SYBR green qRT-PCR protocols (RESSq-PCR). Since RNA-edited transcripts are predicted to differ from wild-type (WT) sequences at only one nucleotide position, detection of RNA editing by qRT-PCR requires highly sensitive and selective primer design strategies. We have previously developed qRT-PCR primers that specifically recognize a gene product with a single point mutation (JAK2 V617F (Geron et al.), and here we employed a similar approach in designing RESSq-PCR primers. Allele-specific PCR strategies, based on positioning the 3′ base of a PCR primer to match one variant allele, have been used for the detection of SNPs and mutations in human genomic DNA or cDNA Ye et al., however are not routinely used in quantitative detection of RNA single nucleotide modifications.
The RESSq-PCR assay primer design was applied to specific cancer and stem cell-associated loci (
qRT-PCR data were measured as a continuous outcome and each group was assessed for distribution. For normally distributed data, the Student's t-test was applied to compare differences in RNA expression and editing ratios calculated by Sanger sequencing and RESSq-PCR, and values are expressed as means±SEM. Experiments were performed in triplicate on blind-coded samples, and all statistical analyses were performed using GraphPad Prism (San Diego, Calif.).
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/14686 | 2/5/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61936314 | Feb 2014 | US |
