Patent Application: US-41033306-A

Abstract:
the present invention provides molecular methods for efficiently transforming the genome of common disease - transmitting parasites , such as plasmodium falciparum . the transformation efficiencies are improved up to 100 times over those conventionally known . the methods provide high saturation of the target parasite genome , of 50 % or greater , and target non - specifically ttaa - rich sites in the parasite genome . the invention also discloses a model that may be used to functionally annotate the genome of the plasmodium falciparum , thus permitting the design and screening of compounds that may be useful in the control and inhibiting of diseases caused and transmitted by these parasites , including malaria . highly efficient and multi - site integrating transposons , particularly piggybac transposons , which provide for random and multi - site integration into parasite genomes in the presence of a helper plasmid , are also presented .

Description:
it is advantageous to define several terms before describing the invention . it should be appreciated that the following definitions are used throughout this application . where the definition of terms departs from the commonly used meaning of the term , applicant intends to utilize the definitions provided below , unless specifically indicated . for the purposes of the present invention , the term “ spacer ” refers to sequences , for example from 3 base pairs ( bp ) to about 31 base pairs ( bp ) or more in length , separating the 5 ′ and 3 ′ ( respectively ) terminal repeat and internal repeat sequences of the piggybac transposon . for the purposes of the present invention , the term “ vector ” refers to any plasmid containing piggybac ends that is capable of moving foreign sequences into the genomes of a target organism or cell . for the purposes of the present invention , the term “ plasmid ” refers to any self - replicating extrachromosomal circular dna molecule capable of maintaining itself in bacteria . for the purposes of the present invention , the term “ transgenic organism ” refers to an organism that has been altered by the addition of foreign or introduced dna sequences ( i . e ., not naturally occurring or native dna sequence and / or inserted at a new ( non - native site ) chromosomal location ) in to its genome . for the purposes of the present invention , the term “ genetic construct ” refers to any artificially assembled combination of nucleic acid , including dna and / or rna , sequences . for the purposes of the present invention , the term “ helper construct ” refers to any plasmid construction that generates the piggybac transposase gene product upon transfection of cells or injection of embryos . for purposes of the present invention , the term “ cell ” referres to any eukaryotic or prokaryotic cell capable of being genetically manipulated from its native , wild type genetic content . the methods of the present invention provide for more highly efficient and predictable techniques for manipulating and using the lepidopteron transposon derived piggybac for the transformation and study of disease transmitting parasites , as well as the diseases that are manifest by these parasites . the following non - limiting examples are illustrative of the present invention , and should not be construed to constitute any limitation of the invention as it is described in the claims appended hereto . the present example demonstrates the utility of the present invention for use as a highly efficient method for transforming disease transmitting parasites , such as p . falciparum , using the piggybac constructs and a helper plasmid as defined herein . these methods provide a p . falciparum transformation technique that is at least 100 times more efficient than those previously available . a minimal piggybac transposon vector , pxl - bacii - dhfr , was created by cloning the human dihydrofolate reductase ( hdhfr ) coding sequence under the control of plasmodium 5 ′ and 3 ′ regulatory elements of calmodulin and histidine rich protein - 2 , respectively , in the plasmid vector , pxl - bacii . 24 this drug resistance cassette was flanked by the 3 ′ inverted terminal repeat ( itr1 ) and the 5 ′ inverted terminal repeat ( itr2 ) of the piggybac element ( fig1 a ). the itrs are oriented such that , upon transposition , they will carry the drug - resistance cassette into the plasmodium genome without any of the plasmid backbone . a helper plasmid , phth , was created by cloning the piggybac transposase coding sequence under the control of heat shock protein 86 ( hsp 86 ) regulatory elements to mobilize the piggybac element in the erythrocytic stages of p . falciparum ( fig1 b ). intended only for transient transfection , this helper plasmid contained no selectable marker . mature blood - stage p . falciparum nf54 parasites were purified by isolation on a magnetic column ( miltenyi biotec ). the paramagnetic hemozoin ( heme polymer ) present in the food vacuole of the parasites allows the separation of parasitized erythrocytes from the uninfected erythrocytes . 25 the purified parasitized erythrocytes were then cultured in rbcs loaded with plasmids pxl - bacii - dhfr and phth 26 ( fig1 c ). purification through the magnetic column ensured invasion of only dna - loaded erythrocytes , whereupon parasites spontaneously acquired plasmids from the erythrocytes . 26 after 1 - 4 generations of parasite growth in dna - loaded erythrocytes , wr99210 was used to select parasites expressing hdhfr . drug - resistant parasites were obtained from eight different transfected cultures , and southern blot hybridizations were performed using an hdhfr probe . novel hybridization bands were detected in each parasite population , in addition to the episomal band , indicating multiple unique integrations of the piggybac element into the p . falciparum genome ( fig2 a ). the average transformation efficiency of piggybac was estimated from eight independently transfected parasite populations to be 6 . 4 - 12 . 6 × 10 − 4 ( table 1 ). this transformation efficiency is approximately 100 times more than what has previously been reported for plasmodium . 10 there was no evidence for piggybac insertions in the absence of the helper plasmid . the present example demonstrates the utility of the present invention for providing stable genetically modified malarial parasites , plasmodium falciparum , that was achieved using the piggybac construct defined herein in the presence of a helper plasmid . the transformed p . falciparum were stable for at least 20 generations in the absence of a helper plasmid . the present example also demonstrates the utility of the method for providing multiple random insertions into the p . falciparum genome using the piggybac constructs in the presence of a helper plasmid . in order to test the stability of piggybac integrations in the genome , parasites from populations “ 1 ” and “ 2 ” were cloned by a limiting dilution method . 27 southern blot hybridizations with an hdhfr probe identified clones with integrations into the genome . clones a1 , b8 , b12 , c12 and f4 derived from population “ 1 ” appeared to have the same integration , “ a ”, while clones b4 and g5 that were derived from population “ 2 ” had two different integrations , “ b ” and “ c ” ( fig2 b ). these clones were maintained in culture for 20 + generations in the absence of the helper plasmid . the integrated piggybac cassette was stable in all the clones as seen by southern blot hybridizations . hence , there was not any endogenous transposase activity . multiple site transformation of p . falciparum genome using piggybac construct method the present example demonstrates the utility of the present invention for providing multiple , random insertions into a p . falciparum genome . to identify the sites of integration in the transformed populations , inverse pcr analyses were performed at the itr2 of piggybac 28 . the inverse pcr products were then cloned into the pgem t - easy vector ( promega ) and sequenced . from the multiple integrations obtained in the transformed populations , nine ( 9 ) different insertion sites were isolated and identified . these insertions represented the predominant population in each transfection study , and therefore were identified with ease ( fig2 a ). the nine ( 9 ) identified insertions were dispersed in different chromosomes throughout the genome of the parasite ( fig3 ). sequence analysis of these nine insertions confirmed a consensus ttaa - site specific integration of the piggybac element into the parasite genome , as expected for authentic transposition ( fig4 ). integration of the itr1 of the piggybac element was confirmed in separate pcr reactions using locus - specific primers and a primer in the itr1 of piggybac . sequence analysis confirmed the ttaa duplication at the itr1 end of the insertion for all integrations , except for integrations “ b ”, “ g ”, and “ h ” due to the at - rich repeat regions in those sequences . instead , the complete integration of the dhfr cassette was confirmed in these populations by southern hybridization rflp analysis . all of the identified piggybac integrations occurred outside of the predicted orfs , 29 - 30 except for integration “ b ” which had the insertion approximately 100 bp downstream of the start codon of a hypothetical asparagine - rich protein ( pfd0200c ), thereby disrupting the putative orf of this gene . five insertions , “ c ”, “ f ”, “ g ”, “ h ”, and “ i ” were in the 5 ′ region of the closest orfs . insertions “ c ”, “ i ” and “ j ” were located approximately 1000 bp 5 ′ to the nearby orf . insertions “ f ” and “ g ” were approximately 300 bp upstream , respectively . insertions “ a ”, “ d ” and “ e ” were 100 bp , 150 bp and 465 bp downstream of the closest orfs , respectively . further analysis will characterize the effects of these insertions on gene expression in these transgenic parasite - lines . based on the distribution of these piggybac integration sites in the non - coding regions , it is not clear whether this apparent bias is significant . given the higher at richness of the non - coding regions ( 86 %) of p . falciparum verses its coding regions ( 74 . 2 %), such an apparent bias may reflect a greater probability of a ttaa target for piggybac insertion occurring in the noncoding regions . also , p . falciparum has a low gene density with long intergenic regions , which could increase the chances of insertions occurring in the intergenic regions . the present example demonstrates the high distribution of ttaa sites in the p . falciparum genome , and the amenability of these sites for manipulating the p . falciparum genome using these sites as targets for genetic integration using the piggybac transformation system and helper plasmid . the pattern of this distribution of ttaa target sites in the p . falciparum genome is also identified in the present example . the present example also demonstrates the utility of the piggybac - transformation system as a useful tool in large - scale genetic screening protocols . 183 , 422 ( 59 . 5 %) of ttaa sites were found in the non - coding regions of the p . falciparum genome , and 124 , 733 ( 40 . 5 %) of ttaa sequences were found in the est sequences of p . falciparum . from this , approximately five targets in each p . falciparum gene were identified . 29 , 30 the identification of these multiple ttaa sites provided a mechanism for transforming p . falciparum at an extremely high efficiency . using an hdhfr - tagged piggybac transposon , a transgenic p . falciparum population was generated by bonafide transpositional integration into the genome , in the presence of a transposase - expressing helper plasmid . insertions were obtained randomly throughout the p . falciparum genome at high transformation efficiency , and the genomic insertion sites were rapidly identified by using an inverse pcr technique . parasites with single transposon insertions were cloned out from mixed populations and the integrated transposons in these transformed parasite lines were stable for many generations , thus confirming their utility for phenotypic analyses . piggybac - mediated transformation protocols were adapted for conditions compatible for large - scale genetic screening , further corroborating the tremendous utility of this technique . the practicality of such a useful application was demonstrated by the transfected parasite populations 3 - 6 ( fig2 a ), which were transformed in a 96 - well microtiter plate . multiple integrations occurred in these small parasite populations , thereby achieving very high transformation efficiencies . this demonstration of the ability to transform p . falciparum with relative ease and high efficiency , by using only a few thousand parasites in a small culture volume , confirmed the suitability of piggybac to be used for large - scale genetic screens . the piggybac transposition system is demonstrated to be an important new genetic tool for manipulation of the p . falciparum genome . this is the first report of high efficiency transposition in this deadly human pathogen . with this efficient integration system , many genetic strategies that have eluded plasmodium research will now be feasible . this methodology , being used in the blood stages , is unable to modify genes that are absolutely essential for the blood - stage development of the parasite . to overcome this , piggybac mobilization can be carried out in the other life cycle stages of the parasite by using a helper plasmid designed for sexual stage - specific expression with another selectable marker . the ability of the piggybac transposable system for use in large - scale mutagenesis of p . falciparum , will provide new insight into the complex genetic structure of the malaria parasite and greatly accelerate efforts to develop novel intervention strategies . the present example describes an efficient helper plasmid that may be used in the practice of the herein described transformation methods using the piggybac transposon construct . in some initial studies , the pfhsp86 promoter was used to drive transposase expression in p . falciparum . this promoter was chosen because it is known to be effective for transgene expression in transfected p . falciparum . this helper plasmid has been re - engineered to boost transposase expression ( fig5 ) using a ‘ head - to - head ’ arrangement of the calmodulin 5 ′ utr with other promoters to significantly enhance transgene expression . this design may be used to boost transposase expression from the helper plasmid , reinforcing the principle that mobilization of piggybac can be regulated temporally and quantitatively by altering the non - coding regulatory sequences flanking the piggybac orf . as an example , the strong calmodulin promoter is inserted in place of the hsp86 promoter of phth and p . falciparum dhfr promoter is inserted upstream of the calmodulin 5 ′ utr in an inverse or head - to - head orientation . this arrangement of promoters can generate substantially higher reporter gene expression levels than plasmid constructs with only a single promoter . this design of the helper plasmid may be further modified by addition of a selectable marker bsd ( phth - bsd ) or neo ( phth - neo ) in order to create a helper line of parasite that constitutively expresses transposase , carrying the helper plasmid as a stable episome . mobilization of piggybac in other development stages may be achieved by addition of a stage - specific promoter in place of hsp86 in the phth as well as a drug resistance cassette . the present example is presented to demonstrate the utility of the present invention for providing a transformation system for a disease - transmitting parasite , such as plasmodium falciparum , that provides a population of transformed p . falciparum having a highly saturated transformed genome . in p . falciparum , the total number of target ttaa insertion sites is 328 , 861 with 159 , 841 in the cds . although the number of ttaa sites per gene varies considerably , the average number of ttaa sites per gene is & gt ; 20 . the pattern of piggybac insertions within p . falciparum genes occur primarily in the 5 ′ utr and just after the 5 ′ start site ( fig6 ). it is expected that 10 , 000 mutations will represent about 50 % saturation of the p . falciparum genome . higher saturation levels will become progressively less efficient as redundant mutations occur and as multiple ttaa in the same locus are hit . the tendency to target the 5 ′ regions of genes facilitates targeting designs for functional annotation of malarial genes , using promoter trap experiments ( fig7 ) or n - terminal exon trapping of insertional tagging . high saturation mutagenesis may be used to demonstrate genes essential for parasite development in humans , genes vital for parasite survival , etc . the present example is presented to demonstrate the utility of the present invention for use as a technique to annotate a genome of a parasite , such as the malarial parasite , p . falciparum , using the herein described piggybac constructs and helper plasmid technique . a simple promoter trap strategy was used that relied solely on an indigenous promoter upstream of the transposon insertion for expression ( fig8 , 9 and 10 ). the drug selectable marker had a promoter - less drug selection cassette . this design was sufficient to isolate parasites transformed with this transposable element , establishing our ability to identify genes of interest through non specific targeting strategies that rely on a phenotype selection . the present example is provided to demonstrate the utility of the invention for providing an optimized piggybac transformation vector that includes substituted codons . these substituted piggybac constructs have enhanced transformation efficiency potential for transforming the p . falciparum genome . piggybac transposase has a single open reading frame ( orf ) of 1785 nucleotides , and our analysis found that 50 of its 594 codons are rare codons for p . falciparum . rare codons were defined as occurring in ≦ 10 % of the all p . falciparum orfs . 29 table 2 identifies the amino acids having rare p . falciparum codons present in the piggybac transposase and the more common codon that will be used to replace the native piggybac nucleotide . in all cases , it is the third base that is replaced . codon usage in the rodent malaria parasites is similar . codon optimization provides optimal expression in the organisms of interest . the present example presents the native sequence of the open reading frame ( orf ) of the piggybac transposon . particular identified “ rare codons ” within this sequence , rare relative to p . falciparum naturally occurring codons , are identified ( see table 2 ) and replaced so as to provide the modified piggybac construct having improved efficiency as described in example 8 . in pxl - baciii - hdhfr , the drug selection cassette has been re - engineered to have the hrp3 promoter ( fig5 ). the calmodulin promoter of pxl - bacii - hdhfr will be replaced with the hrp3 promoter to create pxl - baciii - hdhfr . the calmodulin 5 ′ utr used has bidirectional promoter activity , which will drive transcription of the genes adjacent to the piggybac insertion . such expression of the adjacent gene will create a phenotype for the altered expression of the gene . the present example is provided to demonstrate the utility of the invention for providing an asymmetric piggybac transformation vector that includes an asymmetric arrangement of inverted repeat ( itr ) elements . these asymmetric piggybac constructs have the potential for permanently inactivating p . falciparum genes in a manner suitable for creation of an attenuated parasite vaccine . an asymmetric arrangement of the inverted repeats necessary for piggybac insertion and excision from genomic dna flank a drug selection cassette or other transgene . piggybac transposase does not operate by scanning , but identifies the itr termini directly , so remobilization is unbiased in terms of the itr used , and an equal number of mobilizations will occur with itrs in tandem . this strategy relies on a second mobilization , leaving an orphan arm of the original transposon , which will disrupt the targeted gene , to inactivate expression or generate a direct protein fusion . all documents , patents , journal articles and other materials cited in the present application are hereby incorporated by reference . although the present invention has been fully described in conjunction with several embodiments thereof with reference to the accompanying drawings , it is to be understood that various changes and modifications may be apparent to those skilled in the art . such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims , unless they depart therefrom . the following references are hereby specifically incorporated by reference herein in their entirety . 1 . van dijk , m . r ., waters , a . p . & amp ; janse , c . j . stable transfection of malaria parasite blood stages . science 268 , 1358 - 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