Patent Application: US-201515117038-A

Abstract:
compositions and methods for expanding cd34 + cells , performing research related to cancer stem cells , rna - editing enzymes and for monitoring , diagnosing and treating , ameliorating and preventing diseases such as cancers or inflammatory diseases .

Description:
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 & gt ; 70 % when adar1 is overexpressed . therefore , adar1 is a useful tool for in vitro expansion of normal hematopoietic stem cells ( fig1 ). the data in fig1 was obtained by transducing cord blood cd34 + cells with adar1 or orf lentivirus . after 5 - days , the cells were collected for facs cell cycle analysis . 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 ( fig2 ). the data in fig2 was generated as follows : cd34 + cells from cml bc patient were transduced with lentivirus expressing shadar1 or shcontrol backbone for 3 - days . the cells were then transplanted into immunocompromised mice . after 20 weeks , the mice were sacrificed and the bone marrow ( bm ) and spleen ( sp ) were collected . cd34 + cells from bone marrow were serial transplanted to examine the self - renewal capacity . facs analysis indicated both the primary and serial engrafted samples have an increased g0 population . 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 ( fig3 ) 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 ( fig3 ). the reporter contains a stop codon ( tag ) in a hairpin structure as part of the promoter sequence . when rna editing occurs , the stop codon is removed ( tgg ) and luciferase or fluorescent signal will serve as a readout of rna editing level . in an aspect of this embodiment , an opposite orientated alu - sequence is introduced into the hairpin structure to detect rna editing in non - coding regions . the reporter &# 39 ; s efficiency was confirmed in k562 leukemia cell line that stably overexpresses adar1 ( approximate ˜ 10 fold by western blot ). the results indicate a 5 - fold increase in luciferase signal compared to k562 cells transduced with backbone lentivirus ( fig3 b ). in an aspect of this embodiment , the dsrna reporter hairpin is introduced into a lentiviral backbone using luciferase activity or egfp / eyfp as readout so that efficient transduction can be produced in quiescent myelodysplastic syndrome ( mds ) and aml lscs . 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 ( fig4 ). it has been difficult to successfully expand normal blood stem cells in vitro since the cells are likely to differentiate into mature blood cells . our finding of adar1 indicates it is able to expand hematopoietic stem cells by 10 - 20 fold without significant increase in differentiation . it provides a safe and efficient method to produce large quantity of blood stem cell for bone marrow transplant and other medical needs , such for subjects who have had their blood cells destroyed by chemotherapy or radiation to treat , without limitation , 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 + ( fig4 ). this method enables quick transplantation of donor blood cells immediately after patients receive chemotherapy or radiation . the increase in number of healthy donor blood also helps to provide a faster and better recovery . since the culture condition is very simple and the turn - over - time is short , this method provides a more efficient alternative to existing technologies . the data in fig4 was obtained as follows : facs - purified cd34 + cd38 − lin − hsc ( n = 3 ) were transduced with adar1 or orf backbone . the pictures were taken 3 - days post transduction ( fig4 a ), when the cells were collected for qrt - pcr analysis . genes involved in cell cycle arrest ( cdkn1a and cdkn2a ) were both down - regulated by adar1 ( fig4 b ). cord blood cd34 + cells were transduced with adar1 or orf and transplanted into immunodeficient mice . after 12 weeks , bone marrow were collected and analyzed by facs ( fig4 c ). 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 ( fig6 ). 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 ( fig5 ). fig5 b shows the fold expression of gli2 in gli2 transduced skno - 1 cells using the gli2 vector . fig5 c show western blots of cells transduced with the gli2 vector . 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 ( fig7 ). this discovery is unique since it allows one to predict leukemia progression by identifying edited mirna generated by malignant - specific a - to - i rna editase adar1 . this will also allow one to use edited mirna signature , instead of a general mirna profile as disease biomarkers . moreover , the identification of disease progression markers can help identify and develop new targeted therapies . in an aspect of this embodiment , this discovery allows for the development of a detection platform of edited microrna in lsc population from primary patients and to validate their role as diagnostic and prognostic biomarkers with primary leukemia patient at different disease stages and following different treatments . 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 ( fig8 ). the data in fig8 was generated as follows : primary cml patient samples and normal blood were cd34 selected for rna extraction . cdna was prepared in a reverse - transcription reaction using miscript rtii kit ( qiagen ) and used as a template to profile the expression of the 84 most abundantly expressed and best characterized mirnas by using miscript mirna pcr array ( qiagen ), which contains mirna specific miscript primer assays . qrt - pcr was performed with sybr green kit ( qiagen ). qrt - pcr for the validation of array results was performed by using mirna specific primer assays and sybr green kit ( qiagen ). normalization was performed by using miscript primer control ( rnu6 - 2 ). 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 ( fig9 ). the data generated in fig9 was obtained as follows : cp cd34 + cells were transduced with lentiviral vectors overexpressing human adar1 or gfp - expressing lentiviral backbone . cells were collected for rna extraction and mirna expression was evaluated by miscript qpcr array , and differentially expressed mirnas was validated by qrt - pcr with specific primers . lenti - viral overexpression of adar1 in cd34 + cord blood cells leads to the significant downregulation of 16 microrna ( fig1 ). the data shown in fig1 was generated as follows cp cd34 + cells were transduced with lentiviral vectors overexpressing human adar1 or gfp - expressing lentiviral backbone . cells were collected for rna extraction and mirna expression was evaluated by miscript qpcr array , and differentially expressed mirnas was validated by qrt - pcr with specific primers . 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 ( fig1 ). 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 ( fig1 ). the data shown in fig1 was obtained as follows : cd34 + cord blood n = 3 cells were transduced with lentivirus overexpressing let - 7a or gfp expressing lentiviral backbone . after 48 hours hematopoietic progenitor assays was performed by plating cells into methocult medium . after 2 weeks , total colony number fig1 a and change in individual colony types fig . b between mirna - overexpressing and the control conditions was analyzed . individual colonies were replated into fresh methocult medium and replating efficiency after 2 weeks of culture was evaluated as a measure of self - renewal capacity ( n = 2 ) fig1 c . 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 ( fig1 ). cell types with high levels of endogenous adar1 expression ( undifferentiated human embryonic stem cells ) were used as a positive control for comparison to enforced adar1 overexpression in k562 cells ( fig1 ). for the data shown in fig1 a , k562 cells were transduced with lentiviruses expressing human adar1 - gfp ( lenti - adar1 ) or backbone - gfp ( lenti - orf ) control . cells were facs - purified for gfp - positive cells and expanded in culture . 106 cells were harvested and rna was extracted and reverse transcribed using a cdna supermix containing random hexamers and oligodt . adar1 lentivirus and total adar1 levels were measured by qpcr . in fig1 b relative levels of adar1 isoforms ( p150 , p110 ), adar2 and rna editing target gene mdm2 were evaluated in wild - type ( wt ) k562 , undifferentiated hesc ( hues16 undiff ), 293 cells and mouse bone marrow stromal cells ( sl / m2 ). 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 ( fig1 ). 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 ( fig1 and fig1 ). for each adar target site , two sets of primers containing non - genomic sequences were designed : one pair detecting the wild - type transcript ( an “ a ” base ), and one pair detecting the edited transcript ( a “ g ” base representing inosine substitution ) ( fig1 and table 1 ). an additional mismatch was incorporated upstream of the 3 ′ primer end to enhance allelic discrimination . in fig1 , cdna and genomic dna ( gdna ) from k562 - adar1 cells were analyzed by ressq - pcr with primers specific for wild - type ( wt , a ), edit ( g ) or positive ( outer flanking ) gene sequences at rna editing sites in mdm2 , apobec3d , gli1 , gsk3b , and azin1 site - specific primers distinguished g ( i ) bases at rna editing sites in cdna and as predicted gave no signal in gdna ( fig1 ). 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 ( fig1 ). relative a - to - i rna editing ratios were increased by 2 to 3 fold in adar1 - expressing cells compared to vector controls ( orf ) at sites in mdm2 , apobec3d , gli1 and azin1 transcripts ( fig1 ). increased a - to - i changes in rna editing sites were confirmed by targeted sequencing ( fig1 , 20 ). 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 ( fig2 , 26 , 27 ). these data show that ressq - pcr for these and other rna editing biomarkers can form the basis of screening methods to test activity of rna editing inhibitor compounds derived from 8 - azaadenosine and related small molecules ( fig2 and ep0066918 ) to identify novel agonists and inhibitors of rna editing in high - throughput ressq - pcr and rna editing reporter screens . thus , ressq - pcr and rna editing inhibitors is an allele - specific diagnostic assay that can be used for detection of primate - specific rna editing events in cancer stem cells . we have also demonstrated that let7 regulation by bcr - abl and adar editing of mirs is niche dependent ( fig2 - 23 ) and that jak2 overexpression regulated let7 family members ( fig2 ). the data shown in fig2 was obtained by transducing cd34 + cb n = 3 with lenti - bcr - abl and expression of let7 family was evaluated by rt - qpcr . lentiviral over - expression of bcr - abl in cb n = 3 do not affect expression of members of let7 family . 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 & amp ; 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 &# 39 ; 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 & amp ; 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 &# 39 ; 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 &# 39 ; 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 ). 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 × 10 6 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 × 10 6 cells were extracted using rneasy mini columns , and for primary cells , 5 - 10 × 10 4 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 & gt ; 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 &# 39 ; 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 &# 39 ; 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 & lt ; 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 & gt ; 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 ( fig1 ). efficiency of all primer sets ( fig1 and table was tested using serial dilutions of k562 - adar1 cdna . primer sets were tested experimentally for human specificity and were considered to be human - specific if they returned ct values & gt ; 35 in cdna prepared from mouse bone marrow stromal cell controls . editing site - specific primers for some loci ( fig1 ) either failed to discriminate between cdna and gdna , or k562 - adar1 cells did not display increased editing by sanger sequencing , and therefore were not continued for assay development . ressq - pcr was performed in duplicate using cdna ( 1 - 5 μl reverse transcription product per reaction ) or gdna ( 10 - 200 ng input gdna ) on an icycler ( bio - rad ) 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 the same as for standard qrt - pcr . relative rna editing rates ( relative edit / wt rna ) were calculated using the following calculation : 2 −( ct edit - ct wt ) . qrt - pcr data were measured as a continuous outcome and each group was assessed for distribution . for normally distributed data , the student &# 39 ; 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 .). jiang q , crews l a , barrett c l , chun h j , court a c , isquith j m , zipeto m a , goff d j , minden m , sadarangani a , et al : adar1 promotes malignant progenitor reprogramming in chronic myeloid leukemia . proc natl acad sci usa 2013 , 110 : 1041 - 1046 . jamieson c h , ailles l e , dylla s j , muijtjens m , jones c , zehnder j l , gotlib j , li k , manz m g , keating a , et al : granulocyte - macrophage progenitors as candidate leukemic stem cells in blast - crisis cml . n engl j med 2004 , 351 : 657 - 667 . abrahamsson a e , geron i , gotlib j , dao k h , barroga c f , newton i g , giles f j , durocher j , creusot r s , karimi m , et al : glycogen synthase kinase 3beta missplicing contributes to leukemia stem cell generation . proc natl acad sci usa 2009 , 106 : 3925 - 3929 . hogge d e , lansdorp p m , reid d , gerhard b , eaves c j : enhanced detection , maintenance , and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor , interleukin - 3 , and granulocyte colonystimulating factor . blood 1996 , 88 : 3765 - 3773 . gommans w m , mccane j , nacarelli g s , maas s . a mammalian reporter system for fast and quantitative detection of intracellular a - to - i rna editing levels . analytical biochemistry . 2010 ; 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( 2008 ) visualizing spatiotemporal dynamics of multicellular cell - cycle progression . cell . february 8 ; 132 ( 3 ): 487 - 98 . pmid : 18267078 veliz e a , easterwood l m & amp ; beal p a ( 2003 ) substrate analogues for an rna - editing adenosine deaminase : mechanistic investigation and inhibitor design . j am chem soc 125 , 10867 - 10876 . ye s , dhillon s , ke x , collins a r & amp ; day i n ( 2001 ) an efficient procedure for genotyping single nucleotide polymorphisms . nucleic acids res 29 , e88 . 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 .