Patent Application: US-99263806-A

Abstract:
it was the object of the present invention to provide rna with increased stability and translation efficiency and means for obtaining such rna . it should be possible to obtain increased grades of expression by using said rna in gene therapy approaches .

Description:
according to the invention , standard methods may be used for preparing recombinant nucleic acids , culturing cells and introducing nucleic acids , in particular rna , into cells , in particular electroporation and lipofection . enzymatic reactions are carried out according to the manufacturers &# 39 ; instructions or in a manner known per se . according to the invention , a nucleic acid molecule or a nucleic acid sequence refers to a nucleic acid which is preferably deoxyribonucleic acid ( dna ) or ribonucleic acid ( rna ). according to the invention , nucleic acids comprise genomic dna , cdna , mrna , recombinantly prepared and chemically synthesized molecules . according to the invention , a nucleic acid may be in the form of a single - stranded or double - stranded and linear or covalently closed circular molecule . “ mrna ” means “ messenger rna ” and refers to a “ transcript ” which is produced using dna as template and which itself codes for a peptide or protein . an mrna typically comprises a 5 ′- untranslated region , a protein - encoding region and a 3 ′- untranslated region . mrna has a limited half time both in cells and in vitro . according to the invention , mrna may be prepared from a dna template by in vitro transcription . it may be modified by further stabilizing modifications and capping , in addition to the modifications according to the invention . the term “ nucleic acid ” furthermore also comprises a chemical derivatization of a nucleic acid on a nucleotide base , on the sugar or on the phosphate , and nucleic acids containing non - natural nucleotides and nucleotide analogs . according to the invention , a “ nucleic acid sequence which is derived from a nucleic acid sequence ” refers to a nucleic acid containing , in comparison with the nucleic acid from which it is derived , single or multiple nucleotide substitutions , deletions and / or additions and which is preferably complementary to the nucleic acid from which it is derived , i . e . there is a certain degree of homology between said nucleic acids and the nucleotide sequences of said nucleic acids correspond in a significant direct or complementary manner . according to the invention , a nucleic acid derived from a nucleic acid has a functional property of the nucleic acid from which it is derived . such functional properties include in particular the ability to increase , in a functional linkage to a nucleic acid which can be transcribed into rna ( transcribable nucleic acid sequence ), the stability and / or translation efficiency of rna produced from this nucleic acid in the complete rna molecule . according to the invention , “ functional linkage ” or “ functionally linked ” relates to a connection within a functional relationship . a nucleic acid is “ functionally linked ” if it is functionally related to another nucleic acid sequence . for example , a promoter is functionally linked to a coding sequence if it influences transcription of said coding sequence . functionally linked nucleic acids are typically adjacent to one another , where appropriate separated by further nucleic acid sequences , and , in particular embodiments , are transcribed by rna polymerase to give a single rna molecule ( common transcript ). the nucleic acids described according to the invention are preferably isolated . the term “ isolated nucleic acid ” means according to the invention that the nucleic acid has been ( i ) amplified in vitro , for example by polymerase chain reaction ( pcr ), ( ii ) recombinantly produced by cloning , ( iii ) purified , for example by cleavage and gel - electrophoretic fractionation , or ( iv ) synthesized , for example by chemical synthesis . an isolated nucleic acid is a nucleic acid available to manipulation by recombinant dna techniques . a nucleic acid is “ complementary ” to another nucleic acid if the two sequences can hybridize with one another and form a stable duplex , said hybridization being carried out preferably under conditions which allow specific hybridization between polynucleotides ( stringent conditions ). stringent conditions are described , for example , in molecular cloning : a laboratory manual , j . sambrook et al ., eds ., 2nd edition , cold spring harbor laboratory press , cold spring harbor , n . y . 1989 or current protocols in molecular biology , f . m . ausubel et al ., eds ., john wiley & amp ; sons , inc ., new york , and refer , for example , to a hybridization at 65 ° c . in hybridization buffer ( 3 . 5 × ssc , 0 . 02 % ficoll , 0 . 02 % polyvinylpyrrolidone , 0 . 02 % bovine serum albumin , 2 . 5 mm nah 2 po 4 ( ph7 ), 0 . 5 % sds , 2 mm edta ). ssc is 0 . 15 m sodium chloride / 0 . 15 m sodium citrate , ph 7 . after hybridization , the membrane to which the dna has been transferred , is washed , for example , in 2 × ssc at room temperature and then in 0 . 1 - 0 . 5 × ssc / 0 . 1 × sds at temperatures up to 68 ° c . according to the invention , complementary nucleic acids have nucleotides which are at least 60 %, at least 70 %, at least 80 %, at least 90 %, and preferably at least 95 %, at least 98 % or at least 99 %, identical . the term “% identical ” is intended to refer to a percentage of nucleotides which are identical in an optimal alignment between two sequences to be compared , with said percentage being purely statistical , and the differences between the two sequences may be randomly distributed over the entire length of the sequence and the sequence to be compared may comprise additions or deletions in comparison with the reference sequence , in order to obtain optimal alignment between two sequences . comparisons of two sequences are usually carried out by comparing said sequences , after optimal alignment , with respect to a segment or “ window of comparison ”, in order to identify local regions of corresponding sequences . the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by smith and waterman , 1981 , ads app . math . 2 , 482 , with the aid of the local homology algorithm by neddleman and wunsch , 1970 , j . mol . biol . 48 , 443 , and with the aid of the similarity search algorithm by pearson and lipman , 1988 , proc . natl . acad . sci . usa 85 , 2444 or with the aid of computer programs using said algorithms ( gap , bestfit , fasta , blast p , blast n and tfasta in wisconsin genetics software package , genetics computer group , 575 science drive , madison , wis .). percentage identity is obtained by determining the number of identical positions in which the sequences to be compared correspond , dividing this number by the number of positions compared and multiplying this result by 100 . for example , the blast program “ blast 2 sequences ” which is available on the website http :// www . ncbi . nlm . nih . gov / blast / b12seq / wblast2 . cgi may be used . “ 3 ′ end of a nucleic acid ” refers according to the invention to that end which has a free hydroxy group . in a diagrammatic representation of double - stranded nucleic acids , in particular dna , the 3 ′ end is always on the right - hand side . “ 5 ′ end of a nucleic acid ” refers according to the invention to that end which has a free phosphate group . in a diagrammatic representation of double - strand nucleic acids , in particular dna , the 5 ′ end is always on the left - hand side . in particular embodiments , a nucleic acid is functionally linked according to the invention to expression control sequences which may be homologous or heterologous with respect to the nucleic acid . a transcribable nucleic acid , in particular a nucleic acid coding for a peptide or protein , and an expression control sequence are “ functionally ” linked to one another , if they are covalently linked to one another in such a way that transcription or expression of the transcribable and in particular coding nucleic acid is under the control or under the influence of the expression control sequence . if the nucleic acid is to be translated into a functional peptide or protein , induction of an expression control sequence functionally linked to the coding sequence results in transcription of said coding sequence , without causing a frame shift in the coding sequence or the coding sequence being unable to be translated into the desired peptide or protein . the term “ expression control sequence ” comprises according to the invention promoters , ribosome - binding sequences and other control elements which control transcription of a gene or translation of the derived rna . in particular embodiments of the invention , the expression control sequences can be regulated . the precise structure of expression control sequences may vary depending on the species or cell type but usually includes 5 ′- untranscribed and 5 ′- and 3 ′- untranslated sequences involved in initiating transcription and translation , respectively , such as tata box , capping sequence , caat sequence and the like . more specifically , 5 ′- untranscribed expression control sequences include a promoter region which encompasses a promoter sequence for transcription control of the functionally linked gene . expression control sequences may also include enhancer sequences or upstream activator sequences . the nucleic acids specified herein , in particular transcribable and coding nucleic acids , may be combined with any expression control sequences , in particular promoters , which may be homologous or heterologous to said nucleic acids , with the term “ homologous ” referring to the fact that a nucleic acid is also functionally linked naturally to the expression control sequence , and the term “ heterologous ” referring to the fact that a nucleic acid is not naturally functionally linked to the expression control sequence . the term “ promoter ” or “ promoter region ” refers to a dna sequence upstream ( 5 ′) of the coding sequence of a gene , which controls expression of said coding sequence by providing a recognition and binding site for rna polymerase . the promoter region may include further recognition or binding sites for further factors involved in regulating transcription of said gene . a promoter may control transcription of a prokaryotic or eukaryotic gene . a promoter may be “ inducible ” and initiate transcription in response to an inducer , or may be “ constitutive ” if transcription is not controlled by an inducer . an inducible promoter is expressed only to a very small extent or not at all , if an inducer is absent . in the presence of the inducer , the gene is “ switched on ” or the level of transcription is increased . this is usually mediated by binding of a specific transcription factor . examples of promoters preferred according to the invention are promoters for sp6 , t3 or t7 polymerase . according to the invention , the term “ expression ” is used in its most general meaning and comprises production of rna or of rna and protein . it also comprises partial expression of nucleic acids . furthermore , expression may be transient or stable . with respect to rna , the term “ expression ” or “ translation ” refers in particular to production of peptides or proteins . the term “ nucleic acids which can be transcribed to give a common transcript ” means that said nucleic acids are functionally linked to one another in such a way that , where appropriate after linearization such as restriction enzyme cleavage of the nucleic acid molecule comprising said nucleic acids , in particular of a closed circular nucleic acid molecule , transcription under the control of a promoter results in an rna molecule comprising the transcripts of said nucleic acids covalently bound to one another , where appropriate separated by sequences located inbetween . according to the invention , the term “ transcription ” comprises “ in vitro transcription ”, wherein the term “ in vitro transcription ” relates to a method in which rna , in particular mrna , is synthesized in vitro in a cell - free manner , i . e . preferably by using appropriately prepared cell extracts . the preparation of transcripts preferably makes use of cloning vectors which are generally referred to as transcription vectors and which are included according to the invention under the term “ vector ”. the term “ nucleic acid sequence transcribed from a nucleic acid sequence ” refers to rna , where appropriate as part of a complete rna molecule , which is a transcription product of the latter nucleic acid sequence . the term “ nucleic acid sequence which is active in order to increase the translation efficiency and / or stability of a nucleic acid sequence ” means that the first nucleic acid is capable of modifying , in a common transcript with the second nucleic acid , the translation efficiency and / or stability of said second nucleic acid in such a way that said translation efficiency and / or stability is increased in comparison with the translation efficiency and / or stability of said second nucleic acid without said first nucleic acid . in this context , the term “ translation efficiency ” relates to the amount of translation product provided by an rna molecule within a particular period of time and the term “ stability ” relates to the half life of an rna molecule . the 3 ′- untranslated region relates to a region which is located at the 3 ′ end of a gene , downstream of the termination codon of a protein - encoding region , and which is transcribed but is not translated into an amino acid sequence . according to the invention , a first polynucleotide region is considered to be located downstream of a second polynucleotide region , if the 5 ′ end of said first polynucleotide region is the part of said first polynucleotide region closest to the 3 ′ end of said second polynucleotide region . the 3 ′- untranslated region typically extends from the termination codon for a translation product to the poly ( a ) sequence which is usually attached after the transcription process . the 3 ′- untranslated regions of mammalian mrna typically have a homology region known as the aauaaa hexanucleotide sequence . this sequence is presumably the poly ( a ) attachment signal and is frequently located from 10 to 30 bases upstream of the poly ( a ) attachment site . 3 ′- untranslated regions may contain one or more inverted repeats which can fold to give stem - loop structures which act as barriers for exoribonucleases or interact with proteins known to increase rna stability ( e . g . rna - binding proteins ). 5 ′- and / or 3 ′- untranslated regions may , according to the invention , be functionally linked to a transcribable and in particular coding nucleic acid , so as for these regions to be associated with the nucleic acid in such a way that the stability and / or translation efficiency of the rna transcribed from said transcribable nucleic acid are increased . the 3 ′- untranslated regions of immunoglobulin mrnas are relatively short ( fewer than about 300 nucleotides ), while the 3 ′- untranslated regions of other genes are relatively long . for example , the 3 ′- untranslated region of tpa is about 800 nucleotides in length , that of factor viii is about 1800 nucleotides in length and that of erythropoietin is about 560 nucleotides in length . it can be determined according to the invention , whether a 3 ′- untranslated region or a nucleic acid sequence derived therefrom increases the stability and / or translation efficiency of rna , by incorporating the 3 ′- untranslated region or the nucleic acid sequence derived therefrom into the 3 ′- untranslated region of a gene and measuring whether said incorporation increases the amount of protein synthesized . the above applies accordingly to the case in which according to the invention a nucleic acid comprises two or more 3 ′- untranslated regions which are preferably coupled sequentially with or without a linker inbetween , preferably in a “ head - to - tail relationship ” ( i . e . the 3 ′- untranslated regions have the same orientation , preferably the orientation naturally occurring in a nucleic acid ). according to the invention , the term “ gene ” refers to a particular nucleic acid sequence which is responsible for producing one or more cellular products and / or for achieving one or more intercellular or intracellular functions . more specifically , said term relates to a dna section which comprises a nucleic acid coding for a specific protein or a functional or structural rna molecule . the terms “ polyadenyl cassette ” or “ poly ( a ) sequence ” refer to a sequence of adenyl residues which is typically located at the 3 ′ end of an rna molecule . the invention provides for such a sequence to be attached during rna transcription by way of a dna template on the basis of repeated thymidyl residues in the strand complementary to the coding strand , whereas said sequence is normally not encoded in the dna but is attached to the free 3 ′ end of the rna by a template - independent rna polymerase after transcription in the nucleus . according to the invention , a poly ( a ) sequence of this kind is understood as meaning a nucleotide sequence of at least 20 , preferably at least 40 , preferably at least 80 , preferably at least 100 and preferably up to 500 , preferably up to 400 , preferably up to 300 , preferably up to 200 , and in particular up to 150 , consecutive a nucleotides , and in particular about 120 consecutive a nucleotides , wherein the term “ a nucleotides ” refers to adenyl residues . in a preferred embodiment , a nucleic acid molecule according to the invention is a vector . the term “ vector ” is used here in its most general meaning and comprises any intermediate vehicles for a nucleic acid which , for example , enable said nucleic acid to be introduced into prokaryotic and / or eukaryotic host cells and , where appropriate , to be integrated into a genome . such vectors are preferably replicated and / or expressed in the cell . vectors comprise plasmids , phagemids or virus genomes . the term “ plasmid ”, as used herein , generally relates to a construct of extrachromosomal genetic material , usually a circular dna duplex , which can replicate independently of chromosomal dna . according to the invention , the term “ host cell ” refers to any cell which can be transformed or transfected with an exogenous nucleic acid . the term “ host cell ” comprises , according to the invention , prokaryotic ( e . g . e . coli ) or eukaryotic cells ( e . g . yeast cells and insect cells ). particular preference is given to mammalian cells such as cells from humans , mice , hamsters , pigs , goats , primates . the cells may be derived from a multiplicity of tissue types and comprise primary cells and cell lines . specific examples include keratinocytes , peripheral blood leukocytes , bone marrow stem cells and embryonic stem cells . in other embodiments , the host cell is an antigen - presenting cell , in particular a dendritic cell , a monocyte or a macrophage . a nucleic acid may be present in the host cell in a single or in several copies and , in one embodiment is expressed in the host cell . according to the invention , a peptide or protein encoded by a nucleic acid may be a peptide or protein which is located in the cytoplasma , in the nucleus , in the membrane , in organelles or is secreted . they include structural proteins , regulatory proteins , hormones , neurotransmitters , growth - regulating factors , differentiation factors , gene expression - regulating factors , dna - associated proteins , enzymes , serum proteins , receptors , medicaments , immunomodulators , oncogenes , toxins , tumor antigens or antigens . said peptides or proteins may have a naturally occurring sequence or a mutated sequence in order to enhance , inhibit , regulate or eliminate their biological activity . the term “ peptide ” refers to substances which comprise two or more , preferably 3 or more , preferably 4 or more , preferably 6 or more , preferably 8 or more , preferably 10 or more , preferably 13 or more , preferably 16 or more , preferably 20 or more , and up to preferably 50 , preferably 100 or preferably 150 , consecutive amino acids linked to one another via peptide bonds . the term “ protein ” refers to large peptides , preferably peptides having at least 151 amino acids , but the terms “ peptide ” and “ protein ” are used herein usually as synonyms . the terms “ peptide ” and “ protein ” comprise according to the invention substances which contain not only amino acid components but also non - amino acid components such as sugars and phosphate structures , and also comprise substances containing bonds such as ester , thioether or disulfide bonds . the invention provides for nucleic acids , in particular rna , to be administered to a patient . in one embodiment , nucleic acids are administered by ex vivo methods , i . e . by removing cells from a patient , genetically modifying said cells and reintroducing the modified cells into the patient . transfection and transduction methods are known to the skilled worker . the invention also provides for nucleic acids to be administered in vivo . according to the invention , the term “ transfection ” refers to introducing one or more nucleic acids into an organism or into a host cell . various methods may be employed in order to introduce according to the invention nucleic acids into cells in vitro or in vivo . such methods include transfection of nucleic acid - capo 4 precipitates , transfection of nucleic acids associated with deae , transfection or infection with viruses carrying the nucleic acids of interest , liposome - mediated transfection , and the like . in particular embodiments , preference is given to directing the nucleic acid to particular cells . in such embodiments , a carrier used for administering a nucleic acid to a cell ( e . g . a retrovirus or a liposome ) may have a bound targeting molecule . for example , a molecule such as an antibody specific to a surface membrane protein on the targeted cell , or a ligand for a receptor on the target cell may be incorporated into or bound to the nucleic acid carrier . if administration of a nucleic acid by liposomes is desired , proteins binding to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation in order to enable targeting and / or absorption . such proteins include capsid proteins or fragments thereof which are specific to a particular cell type , antibodies to proteins that are internalized , proteins targeting an intracellular site , and the like . “ reporter ” relates to a molecule , typically a peptide or protein , which is encoded by a reporter gene and measured in a reporter assay . conventional systems usually employ an enzymatic reporter and measure the activity of said reporter . the term “ multiple cloning site ” refers to a nucleic acid region containing restriction enzyme sites , any one of which may be used for cleavage of , for example , a vector and insertion of a nucleic acid . according to the invention , two elements such as nucleotides or amino acids are consecutive , if they are directly adjacent to one another , without any interruption . for example , a sequence of x consecutive nucleotides n refers to the sequence ( n ) x . “ restriction endonuclease ” or “ restriction enzyme ” refers to a class of enzymes that cleave phosphodiester bonds in both strands of a dna molecule within specific base sequences . they recognize specific binding sites , referred to as recognition sequences , on a double - stranded dna , molecule . the sites at which said phosphodiester bonds in the dna are cleaved by said enzymes are referred to as cleavage sites . in the case of type iis enzymes , the cleavage site is located at a defined distance from the dna binding site . according to the invention , the term “ restriction endonuclease ” comprises , for example , the enzymes sapi , ecii , bpii , aari , aloi , baei , bbvci , ppii and psri , bsrd1 , btsi , eari , bmri , bsai , bsmbi , faui , bbsi , bcivi , bfuai , bspmi , bseri , ecii , btgzi , bpuei , bsgi , mmei , cspci , baei , bsami , mva1269i , pcti , bse3di , bsemi , bst6i , eam1104i , ksp632i , bfii , bso31i , bsptni , eco31i , esp3i , bfui , acc36i , aari , eco57i , eco57mi , gsui , aloi , hin4i , ppii , and psri . “ half life ” refers to the time required for eliminating half of the activity , amount or number of molecules . the present invention is described in detail by the following figures and examples which should be construed by way of illustration only and not by way of limitation . on the basis of the description and the examples , further embodiments are accessible to the skilled worker and are likewise within the scope of the invention . in order to study the effects of the rna modifications according to the invention on the level and duration of expression , a number of ivt vectors were prepared which served as template for in vitro transcription ( fig3 ). the reporter genes for egfp and d2egfp , two molecules with different half lives ( hl ), were inserted into the vectors , thereby enabling the influence of the rna modifications according to the invention to be analyzed . fluorescence decreases with an average hl of 17 . 3 h for egfp and 2 h for d2egfp . these constructs were used for preparing in vitro - transcribed egfp rna and d2egfp rna , respectively . transfection of cells with the in vitro - transcribed rna modified according to the invention and effect on rna translation and stability in vitro - transcribed egfp rna and d2egfp rna were used for transfecting k562 cells ( human , leukemia ) by means of electroporation . the transfection efficiency was & gt ; 90 % in k562 cells . this was followed by assaying the action of the rna modifications described in human dendritic cells ( dcs ) which are the most important modulators of the immune system . this approach is immunologically relevant because rna - transfected dcs can be considered for vaccination . immature dcs are located in the skin and in peripheral organs . here they are in an immature state which is characterized by well - studied surface markers and which is functionally distinguished by high endocytotic activity . an immunogenic stimulus such as , for example , an infection with pathogens , triggers a dc maturation process . at the same time , said stimulus initiates dc migration into the regional lymph nodes , where said dcs are the most effective inducers of t cell and b cell immune responses . the mature state of said dcs is also characterized by expression of surface markers and cytokines studied in detail and by a characteristic dc morphology . there are established cell culture systems for differentiating immature human dcs from blood monocytes . these may be caused to mature by various stimuli . the transfection efficiency in primary dendritic cells was 70 - 80 %. the dcs were stained with anti - cd80 , anti - cd83 , anti - cd86 and anti - hla - dr antibodies which recognize specific dc maturation markers , and analyzed by flow cytometry ( fig4 ). the level and duration of expression were determined with the aid of facs - kalibur by way of determining the egfp fluorescence intensity . the amount of rna in the cells was determined with the aid of a quantitative rt - pcr . a . effect of an open - ended poly ( a ) sequence on rna translation and stability both the tumor cell line k562 and immature dcs ( idc ) were shown to translate rna having an open - ended poly ( a ) sequence more efficiently and over a longer period of time than rna having a masked - end poly ( a ) sequence ( fig5 a ). the translation efficiency for an unmasked - end poly ( a ) sequence in immature dcs is increased by a factor of 1 . 5 , with poly ( a ) sequences of equal length . moreover , said modification results in higher rna stability ( fig5 b ). a 4 to 5 - fold amount of rna can be detected in immature dcs which had been transfected with rna having an unmasked poly ( a ) sequence 48 h after electroporation . b . effect of the poly ( a ) sequence length on rna translation and stability the analysis of rna having poly ( a ) sequences of 16 bp , 42 bp , 51 bp , 67 bp , 120 bp , 200 bp , 300 bp and 600 bp in length revealed that extension of said poly ( a ) sequence up to 120 a nucleotides increases transcript stability and translation and that an extension going beyond that has no positive effect . this effect is observed both in k562 cells and in immature dcs ( idc ) ( fig6 a and 6 b ). extending the poly ( a ) sequence from 51 to 120 a nucleotides produces a 1 . 5 to 2 - fold increase in translation efficiency . this effect is also reflected in rna stability ( fig6 c ). c . effect of the occurrence of a 3 ′- untranslated region on rna translation and stability a time course with k562 cells and immature dcs confirmed that introducing a 3 ′- untranslated region ( utr ) of human beta - globin results in increasing expression of an rna transcript . in addition , it was demonstrated that a double 3 ′- untranslated region ( utr ) of human beta - globin results in an enhanced level of expression after 24 h , which markedly exceeds the combined effect of two individual utrs ( fig7 ). d . effect of a combination of the above - described modifications on rna translation and stability according to the invention , a combination of the above - described modifications in an rna transcript was shown to increase the translation efficiency of egfp in immature and also in mature dcs by a factor of greater than five ( fig8 ). presentation of a peptide expressed via in vitro - transcribed rna with increased stability and translation efficiency by mhc molecules according to the invention , the use of rna constructs modified according to the invention was shown to increase peptide - mhc presentation on the cell surface . to this end , the nucleic acid sequence coding for egfp in the ivt vectors described was replaced with a nucleic acid sequence coding for the ova257 - 264 epitope ( siinfekl ), and the constructs were compared with one another . the target cells used for transfection were el4 cells ( murine , t cell lymphoma ). in order to quantify siinfekl peptides presented by mhc molecules , the cells were stained with an anti - h2 - k b - ova257 - 264 antibody at various time points after electroporation , and the fluorescence intensity of a secondary antibody was determined with the aid of facs - kalibur ( fig9 ). furthermore , the siinfekl peptide was cloned into the vector which reflected all optimizations ( pst1 - sec - siinfekl - 2bgutr - a120 - sap1 ) and into a vector with standard features ( pst1 - sec - siinfekl - a67 - spe1 ). ivt rna derived from both vectors was electroporated into el4 cells and bmdcs . ova - peptide / k b complexes were found on the cell surface in substantially greater numbers and were maintained over a longer period of time after electroporation of the rna modified according to the invention , sec - siinfekl - 2 - bgutr - a120 ( fig1 ). effect of a transfection of cells with in vitro - transcribed rna coding for a peptide to be presented on the expansion of antigen - specific t cells in order to evaluate the effect on stimulatory capacity , ot - i - tcr was employed which had been used intensively in the c57bl / j6 ( b6 ) background in order to detect mhc class i presentation of the siinfekl peptide . ot - i cd8 + t cells which are transgenic with regard to the t cell receptor ( tcr ) and which recognize the k b - specific peptide siinfekl from chicken ova ( ova 257 - 264 ), was kindly provided by h . schild ( institute of immunology , university of mainz , germany ). on day 0 , animals underwent adoptive transfer with ot - i - cd8 + t cells . to this end , splenocytes were prepared from tcr tg ot - i mice and introduced into the tail vein of c57bl / j6 recipient mice . the cell number was adjusted to 1 × 10 5 tcr tg cd8 + t cells . on the next day , 1 × 10 6 bmdcs of c57bl / j6 mice which had been electroporated with 50 pmol of siinfekl - encoding rna construct variants and had been allowed to mature by means of poly ( i : c ) for 16 hours were administered ip to mice . on day 4 , ot - i - cd8 + t cells were measured in peripheral blood with the aid of the tetramer technology . to this end , retroorbital blood samples were taken and stained with anti - cd8 ( caltag laboratories , burlingame , usa ) and siinfekl tetramer ( h - 2 kb / siinfekl 257 - 264 ; beckman coulter , fullerton , usa ). in vivo expansion of antigen - specific tcr - transgenic cd8 + t cells was found to be substantially improved when using sec - siinfekl - 2bgutr - a120 rna for antigen supply in comparison with sec - siinfekl - a67 - acuag rna ( fig1 a ). in order to evaluate , whether stabilized ivt rna constructs for antigen supply also improve antigen - specific stimulation of human t cells , hcmv - pp65 , the immunodominant antigen of human cytomegalovirus which is often used for validating autologous stimulation of polyepitopic t cell reactions , was employed . cd4 + and cd8 + t cells which had been purified from hcmv - seropositive healthy donors by positive magnetic cell sorting by means of antibody - coated microbeads ( miltenyi biotec , bergisch - gladbach , germany ) were cocultured with 2 × 10 5 autologous dcs which had been electroporated with the corresponding ivt rna variants coding for pp65 . an expansion of t cells , measured on day 7 in an ifn - γ - elispot using autologous dcs which had been pulsed with a pool of overlapping peptides covering the entire pp65 protein sequence , or with a control protein , demonstrated the superiority of sec - pp65 - 2bgutr - a120 , with the effects with regard to expansion of cd4 + t cells being the most pronounced ( fig1 b ). bargmann , c . i ., hung , m . c ., and weinberg , r . a . 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