Christopher D Herring

Christophe Chevillard

Sean L Johnston

Peter J Wettstein

Roy Riblet
email: riblet@cerfnet.com

Vector-Hexamer PCR Isolation of All Insert Ends from a YAC Contig of the Mouse Igh Locus

We have developed a simple PCR strategy, termed vector-hexamer PCR, that is unique in its ability to easily recover every insert end from large insert clones in YAC and BAC vectors. We used this method to amplify and isolate all insert ends from a YAC contig covering the mouse Igh locus. Seventy-seven ends were amplified and sequenced from 36 YAC clones from four libraries in the pYAC4 vector. Unexpectedly, 40% of the insert ends of these YACs were LINE1 repeats. Nonrepetitive ends were suitable for use as probes on Southern blots of digested YACs to identify overlaps and construct a contig. The same strategy was used successfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBAC11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investigation.

One of the primary goals of genome research is the production of genetic and physical maps of whole genomes. Such maps are a necessary resource for the identification of all functional genes and the localization of specific genes associated with disease. The main tools to build physical maps are large genomic insert libraries in yeast and bacterial artificial chromosome vectors (YACs and BACs, respectively) [START_REF] Burke | Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors[END_REF][START_REF] Shizuya | Cloning and stable maintenance of 300 kilobase-pair fragments of human DNA in Esherichia coli[END_REF]. Such libraries consist of multiple genome equivalents in tens of thousands of clones.

To begin the construction of the physical map of a genomic region YAC and BAC libraries are screened to identify clones carrying markers mapped to the region. This can be accomplished by either hybridization on densely gridded filters or PCR on hierarchical or multidimensional pools [START_REF] Green | Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction[END_REF]. Next, both ends of each clone are isolated for use as hybridization probes and sequenced to design new PCR markers. These probes and PCR markers are used to assess the overlap of the clones and to ''walk'' if needed, that is, to rescreen the libraries to isolate additional clones that overlap the end and extend the developing contig. The walking strategy is effective but time consuming, making the choice of end isolation strategy critical. The method should be simple, rapid, and completely efficient, successful at every end.

Early methods to isolate insert ends involved subcloning into phage or plasmid vectors and screening subclone libraries with YAC vector arms to identify the junction fragments. Simpler, faster PCR-based methods are more generally used, including IRS PCR, inverse PCR, and vectorette PCR. Inter repeat sequence (IRS)-PCR takes advantage of the frequent occurrence of repeat elements in the genome (Alu for human and B1 for mouse). PCR amplification is performed between Alu (or B1) consensus sequences and the YAC vector sequence [START_REF] Nelson | Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources[END_REF][START_REF] Breukel | Vector-Alu PCR: A rapid step in mapping cosmids and YACs[END_REF]). However, it fails in many cases because it relies on the presence of a repeat element within a short distance from the end. Another problem is the generation of extraneous non-end products from amplification between repeats. Inverse PCR relies on restriction enzyme cleavage at sites within the vector and the genomic insert [START_REF] Triglia | A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences[END_REF][START_REF] Silverman | Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3[END_REF][START_REF] Silverman | Isolating vector-insert junctions from yeast artificial chromosomes[END_REF]. The fragments produced are then self-ligated and the end is amplified using divergent vector primers. Although conceptually simple, this strategy is limited to the use of a small number of restriction enzymes with appropriate YAC arm sites, and frequently it failed in our hands. Vectorette PCR involves the ligation of a ''bubble'' linker to digested total YAC DNA and subsequent recovery of the insert end by amplification between this linker primer and nested YAC arm primers [START_REF] Riley | A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones[END_REF]. This is a very effective strategy but somewhat complex in operation.

We have developed a method for the isolation of insert ends from YACs that uses PCR exclusively, involves a minimum number of manipulations, and does not require adapters, linkers, or ligations. On the basis of restriction-site PCR [START_REF] Sarkar | Restriction-site PCR: A direct method of unknown sequence retrieval adjacent to a known locus by using universal primers[END_REF][START_REF] Johnston | A novel method for sequencing members of multi-gene families[END_REF], and drawing on degenerate oligonucleotide primed (DOP)-PCR [START_REF] Wesley | Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer[END_REF][START_REF] Telenius | Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer[END_REF], this generalized strategy, called vector-hexamer PCR, uses hexamer primers designed by adding a 6-bp sequence to the 3Ј end of an arbitrary ''anchor'' sequence. Three rounds of PCR are performed using a nested series of vector arm primers in conjunction with one hexamer primer. It is similar to the DOPvector PCR [START_REF] Wesley | Rapid sequential walking from termini of cosmid, P1 and YAC inserts[END_REF][START_REF] Wu | DOP-vector PCR: A method for rapid isolation and sequencing of insert termini from PAC clones[END_REF] and thermal assymetric interlaced (TAIL)-PCR [START_REF] Liu | Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[END_REF] methods described for cosmid, P1, YAC, and BAC end isolation but is more general in that it incorporates a variety of alternative primers and is less complex than either method. It is completely effective, recovering every end that we have attempted.

In this paper we demonstrate the use of vectorhexamer PCR for the isolation of insert ends from BACs and two varieties of YAC vector clones from the mouse immunoglobulin heavy chain (Igh) locus located near the telomere of chromosome 12. Whereas the human IGH locus has been cloned recently in YACs and characterized [START_REF] Cook | A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q[END_REF], knowledge of the organization of the mouse locus is still based mainly on genetic and deletion mapping [START_REF] Tutter | Structure, map position, and evolution of two newly diverged mouse Ig VH gene families[END_REF][START_REF] Mainville | Deletional mapping of fifteen mouse Vh gene families reveals a common organization for three Igh haplotypes[END_REF]. It is comprised of >100 gene segments spread over more than a centiMorgan of recombination distance. To investigate this complex locus we identified a series of YACs containing all Igh gene elements. To assemble a contig the insert ends were isolated from all 36 YAC clones by vector-hexamer PCR to characterize YAC overlap and to detect chimeric clones. A description of this simple, rapid, and efficient method is presented here.

RESULTS AND DISCUSSION

The vector-hexamer technique to recover clone insert ends uses a nested series of vector arm primers in conjunction with a hexamer primer that anneals to multiple sites randomly located in the clone insert (Fig. 1a). The primers are shown in Table 1. The hexamer primers correspond to a variety of restriction enzyme recognition sites and frequently occurring sequences; in principle, the hexamer can be any useful sequence. For these short sequences to act as primers, five cycles of low annealing temperature (35°C) PCR are used to begin the amplification (Fig. 1b). In addition to the hexamer sequence, the hexamer primers contain an anchor sequence at the 5Ј end; for most of them this is the T7 promoter sequence. The incorporation of the anchor sequence into the products from the first five cycles of PCR allows the annealing temperature of subsequent cycles of PCR to be increased to 60°C (Fig. 1c-e).

In the pYAC4 vector the cloning site is in the SUP4 nonsense suppressor tRNA gene (Fig. 1a). Because there are related tRNA genes in the yeast host, primers that are designed in the area immediately adjacent to the cloning site amplify host sequences along with the YAC insert end. In early experiments, when the initial primer was L-II or R-II, yeast background bands were isolated and sequenced in 5 of 21 PCR products. When L-I and R-I primers, located outside of the SUP4 gene (Fig. 1a) were used for the first round of PCR, yeast products were incorrectly recovered in 6 of 87 cases. Additional measures to minimize and identify amplification of yeast background include amplifying and analyzing many YACs at once with the same hexamer primer to identify common yeast fragments and using an ''empty YAC'' negative control (AB1380 transformed with linearized pYAC4).

First round PCR products (Figs. 1c and2a) typically show either a pattern of yeast bands present in every YAC or nothing at all. Second-round, nested PCR products (Figs. 1d and2b) typically consist of faint YAC-specific end products among persistent yeast bands. These products can be isolated and sequenced, but not reliably. Third round, further nested, amplification (Figs. 1e and2c) is necessary to generate enough insert end product to sequence routinely. These PCR products consist of several YAC-specific bands between ∼100 and 2000 bp, often with one or two very intense bands (estimated 200-2000 ng). As seen in Figure 2, c and d, yeast and vector bands are often present in the third-round product but are identified by their presence in mul-tiple lanes and the control. The importance of multiple hexamer primers is illustrated in Figure 2d. The YAC RAD52-y99A9 yields a difficultly large, possibly yeast product with the BanI primer (left), apparently only yeast-derived products with the PstI primer (center), but a useful 200-bp product with the SduI primer (right).

Vector-hexamer PCR was used to amplify and isolate 77 ends from 36 Igh region YACs using 9 different hexamer primers. More than two ends were isolated from some clones because five of them contained more than one YAC. Table 2 shows the size and intensity of the best product from each vector-hexamer amplification that was performed. Also listed are the GenBank accession numbers for each of the 77 insert end sequences. Useful, medium to strong intensity end products were readily obtained, usually with several hexamer primers. In two instances five, and even six, restriction site hexamer primers failed, but in these cases use of the two DOP primers succeeded.

Apparently two DOP primers were the most uniformly successful in comparing the different hexamer primers; however, these were incorporated in the primer panel after the technique had been refined and have been less extensively tested. Highly successful primers include DdeI+G, PstI, and SduI. The efficiency of the PvuII primer differed greatly between the left and the right arm in this study.

We were able to examine the annealing specificity of hexamer primers in cases in which two separate end products of different lengths were sequenced from the same YAC end. As seen in Table 3, almost every example shows at least one mismatch from the hexamer sequence, usually in the 5Ј half. This result was reproducible-in cases in which two different length end products were generated from the same vector-hexamer reaction, the smaller fragment could be regenerated by reamplifying the gelpurified larger fragment, even without low annealing temperature cycles.

Sequence Analysis

The PCR insert end products were sequenced with the third or fourth nested vector arm primer. Sometimes the hexamer primer was used as well to sequence the reverse strand. For many ends several products made with different hexamer primers were sequenced. All end sequences were submitted to the BLAST e-mail server [START_REF] Altschul | Basic local alignment search tool[END_REF]). Of the 77 ends, 12 were similar to known sequences from the Igh locus, usually sequences flanking coding regions. Known genes other than Igh or repeat elements were identified by five YAC ends, but they The later cycles of the first round of PCR are performed at a higher annealing temperature so that the entire hexamer primer stably anneals only to products from the initial cycles. (d,e) The second and third rounds of PCR reamplify the vector/insert junction products from the first PCR with nested vector primers to increase the specificity and yield of the amplification. The fourth nested vector primer allows reduction of vector arm contribution to the PCR product for use as a hybridization probe.

VECTOR-HEXAMER PCR ISOLATION OF YAC ENDS

GENOME RESEARCH 675

appear to be the result of YAC chimerism or other artifact as they hybridized only to the YACs from which they were isolated, or they matched sequences from Escherichia coli.

Repetitive sequences were identified in 33 of 77 ends (43%) by BLAST analysis. Twenty-nine ends belonged to the LINE1 or L1 family of high copy number repetitive sequences, and the others included a B1 repeat, an intracisternal A-particle (IAP) env gene, a CT microsatellite repeat, and a flanking sequence of a presumed novel transposon [START_REF] Bultman | Molecular analysis of reverse mutations from nonagouti (a) to black-and-tan (at) and white-bellied agouti (aw) reveals alternative forms of agouti transcripts[END_REF]. The incidence of L1 elements at YAC ends is markedly higher than the 5% incidence observed in a YAC contig on chromosome 11 [START_REF] Nehls | YAC/P1 contigs defining the of 56 microsatellite markers and several genes across a 3.4 cM interval on mouse Chromosome 11[END_REF] but more similar to the 25% seen in the Igk locus [START_REF] Schupp | A yeast artificial chromosome contig spanning the mouse immunoglobulin kappa light chain locus[END_REF]) and the even higher incidence in the MHC locus [START_REF] Jones | MHC class I gene organization in >1.5-Mb YAC contigs from the H2-M region[END_REF]. Clearly the frequencies of L1 elements at different genomic sites varies considerably. This is difficult to assess properly, as they are repeats and therefore, most were not mapped in our study or in the other studies. These YACs are potentially chimeric, and accurate comparison is not possible.

Hybridization Results

Fifty-six vector-hexamer PCR products were used as hybridization probes on YAC or other mapping blots. Six were unsuitable because of repetitive sequence content, but this did not always make an end product un- Ambiguous nucleotides are denoted with standard IUPAC/IUB codes. References for previously described primers are (a) primers 1089 and 1091 in [START_REF] Riley | A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones[END_REF]), (b) [START_REF] Johnston | A novel method for sequencing members of multi-gene families[END_REF], (c) [START_REF] Telenius | Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer[END_REF]), (d) [START_REF] Weier | Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes[END_REF], (e) R. Maatman; Jackson Laboratory, (f) [START_REF] Wang | Construction and characterization of a human chromosome 2-specific BAC library[END_REF]). (g) The pRML1 (L-I and L-II) and pRML2 (R-I and R-II) nesting primers were designed using primer 0.5 [START_REF] Lincoln | PRIMER: A computer program for automatically selecting PCR primers[END_REF]). (h) The pRML1 L-III and pRML2 R-III primers are the T3 and T7 promoter sequences flanking the insert in the assembled YAC [START_REF] Spencer | Targeted recombination-based cloning and manipulation of large DNA segments in yeast[END_REF]. suitable. Nine ends that contained repeats were successfully used as probes, in most cases when the similarity to the repeat was low. Useful probes were generated when similarity was high if nonrepetitive sequence was detected beyond a repeat element and could be isolated. For example, in one case a new PCR primer was designed to amplify the unique region, and in another the PCR product was digested at a restriction site near the end of the repeat and gel purified.

Of the 50 useful YAC insert end probes thatwere generated, 37 hybridized to other Igh YACs, whereas 13 probes hybridized only to their source YACs. In the latter cases, the overlap of other YACs ruled out incomplete coverage and indicated that these ends are not present in the Igh locus. Two of these probes were from clones containing more than one YAC; the remaining 11 ends from 36 YACs indicate chimerism.

Extension of Vector-Hexamer PCR to Other Vectors

The new WI/MIT-820 YAC library is intended to be the basis for development of a complete physical map of the mouse genome [START_REF] Haldi | A comprehensive large-insert yeast artificial chromosome library for physical mapping of the mouse genome[END_REF]. This library is constructed in a newer vector system, pRML1/2, and required design of new nested vector primers, shown in Table 1. In an initial test they were used in conjunction with only the two DOP hexamer primers to attempt recovery of the ends of 14 Igh region YACs from this library. Twenty-two ends were amplified and sequenced (data not shown), and we expect that the few remaining ends can be recovered using other hexamer primers. Consistent with our findings in Igh clones from the other YAC libraries 8 of the 22 ends (36%) were repeat sequences, and 6 of the repeats were L1 sequences.

We have also used vectorhexamer PCR to isolate and sequence the ends of BAC clones in the pBeloBAC11 vector (primers in Table 1). All 28 ends were readily obtained from the 14 BAC clones studied (data not shown). It is an advantage that vector-hexamer PCR can be used to amplify

METHODS YAC and BAC Libraries

YAC and BAC clones were obtained from the following libraries. The Princeton University C57BL/6J mouse YAC library [START_REF] Burke | A mouse genomic library of yeast artificial chromosome clones[END_REF][START_REF] Rossi | Genomic analysis using yeast artificial chromosome library with mouse DNA inserts[END_REF]) can be screened by PCR using DNA pools obtainable from S.M. Tilghman (Howard Hughes Medical Institute, Princeton University, NJ). The ICRF C3H mouse YAC library [START_REF] Larin | Yeast artificial chromosome libraries containing large inserts from mouse and human DNA[END_REF]) is accessed through H. Lehrach at the Resource Center/Primary Database (RZPD) of the German Human Genome Project. See the World Wide Web information server of the RZPD at http://www.rzpd.de. The first Whitehead Institute (WI-I) C57BL/6J mouse YAC library [START_REF] Kusumi | Construction of a large-insert yeast artificial chromosome library of the mouse genome[END_REF]) can be screened by PCR using pools available from Research Genetics (Huntsville, AL). The second Whitehead (WI/MIT-820) C57BL/6J mouse YAC library [START_REF] Haldi | A comprehensive large-insert yeast artificial chromosome library for physical mapping of the mouse genome[END_REF]) can be screened by PCR using pools obtained from Research Genetics (see http://wwwgenome.wi.mit.edu). The Saint Mary's Hospital Medical School RAD52 C57BL/10 mouse YAC library [START_REF] Chartier | Construction of a mouse yeast artificial chromosome library in a recombination-deficient strain of yeast[END_REF]) is available for PCR screening using DNA pools obtained from Dr. S.D.M. Brown at the Mouse Genome Center, Harwell, UK (see http://www.mgc.har.mrc.ac.uk). The 129/Sv mouse BAC library was prepared by Dr. Bruce Birren at the Whitehead Institute Genome Center and can be screened by PCR or high-density filter hybridization; DNA pools and filters are available from Research Genetics (Huntsville, AL).

Vector-Hexamer PCR

This is a heminested strategy and is diagrammed in Figure 1; the primers used are listed in Table 1. First-round reactions consisted of 2 µl ן01 PCR buffer [500 mM KCl, 100 mM Tris (pH 8.0), 15 mM MgCl 2 , 1 mg/ ml Knox gelatin], 0.4 µl dNTP solution (2.5 mM each dNTP), 0.08 µl Taq polymerase (5 U/µl), 1 µl (40 ng) of the outermost left arm or right arm nested vector primer (L-I or R-I; Table 1), 1 µl (40 ng) hexamer primer, 1 µl YAC DNA (25 ng/µl, as prepared in [START_REF] Ausubel | Current protocols in molecular biology[END_REF], and water to a total volume of 20 µl. The PCR program was five cycles (94°C for 30 sec, 35°C for 60 sec, 72°C for 3 min), immediately followed by 25 cycles (94°C for 30 sec, 60°C for 30 sec, 72°C for 3 min) in an Ericomp Twinblock Easycycler (San Diego, CA). Multiple YACs and a negative control YAC lacking insert (see below) were amplified at the same time to allow identification of yeast background products. For the second round of nested PCR the same conditions as the first round PCR were used, except that the template was 1 µl of first-round PCR product and the vector primer was the second nested vector arm primer, L-II or R-II. The PCR program was 30 cycles [94°C for 30 sec, 50°C (pYAC4 left arm) or 60°C (pYAC4 right arm) for 30 sec, 72°C for 3 min]. For the third round 1 µl of secondround product was used as template in a 40-µl total volume reaction with the third nested vector primers. Volumes of all reagents (except template) were doubled. The PCR program was 30 cycles (94°C for 30 sec, 55°C for 30 sec, 72°C for 3 min). A negative control was a YAC clone identical to the experimental YACs but lacking insert. This control was created by transforming yeast host AB1380 in lithium acetate with linearized pYAC4 (both available from ATCC, nos. 20843 and 67379, respectively), as described in [START_REF] Ausubel | Current protocols in molecular biology[END_REF]. Sequences from short YAC end PCR products in which the hexamer primer was discernible at the end were aligned to sequences from longer PCR products generated from the same YAC end with the same or a different hexamer primer. The sequence of the hexamer primer incorporated into the short product was compared to the corresponding sequence in the longer product (priming site). Mismatched nucleotides in the priming site are underlined.

Table 2. (Continued from facing page.) The 36 Igh YAC clones in this study are listed vertically in the top half for the left-arm ends and in the bottom half for the right-arm ends. Hexamer primers are listed across the top. Results of each attempt to recover a YAC insert end with vector-hexamer PCR are indicated. Reactions that did not produce any visible YAC end product are denoted X; blank cells are combinations of YAC end and hexamer primer that were not tried. For each successful reaction the size (in bp) and intensity are given for the most readily sequenceable product (those of the greatest intensity and closest to 400 bp). The yield of the PCR is indicated as intense product (∼0.5-2.0 µg) (q); medium intensity ( ); and faint product (᭺). Sequencing was usually successful with medium-and high-intensity fragments but only rarely with faint products. The size listed is the third-round product, which includes ∼75 bp of vector sequence. Where an end product was successfully sequenced the GenBank accession no. is given to the right of the intensity indicator.

Where GenBank entries are the compilation of several sequences obtained with different hexamer primers the same accession no. appears several times in a row. In certain cases, the sequence from the vector primer did not overlap the reverse sequence from the hexamer primer and multiple accession numbers are listed for one YAC end amplification.

The pYAC4 plasmid was isolated from bacterial culture with a standard alkaline lysis procedure and linearized with BamHI before being used to transform the yeast. BAC insert ends were recovered in a similar manner using as initial template either ∼100 ng of BAC DNA purified on Qiagen columns according to the manufacturer's instructions or 1-2 µl of a crude ''boil prep'' made by picking a bacterial colony into 20 µl of water, boiling for 5 min, and centrifuging particulate matter. For the SP6 end different primers were used for amplification and sequencing as shown in Table 1. For the T7 end amplification and sequencing were done with the same T7 promoter primer. Multiple BACs and a negative control (pBeloBAC11 without insert) were amplified at the same time to allow identification of bacterial background products. The BAC PCR program was 35 cycles [94°C for 30 sec, 60°C (Sp6 amplification primer) or 55°C (T7 amplification primer) for 30 sec, 72°C for 1 minute].

Gel Analysis

Thirty-five microliters of third round PCR products was electrophoresed on 10 ן 8 ן 0.15-cm vertical 10% 37.5:1 polyacrylamide (AMRESCO, Solon, OH) gels. Gels were stained with ethidium bromide. Bands ranging in size from 120 bp to ∼1500 bp (optimally 300-500 bp) were isolated if they were reasonably bright and did not appear in other YAC lanes or the negative control using the same hexamer primer. Gel segments measuring ∼2 ן 5 ן 1.5 mm were excised with a scalpel and placed in individual yellow 200-µl pipette tips. These tips were put into 0.65-ml microcentrifuge tubes with the pipette tips protruding through holes that had been punched in the bottom of the tubes with an 18-gauge needle. The 0.65-ml tubes were placed into 2-ml microcentrifuge tubes and spun for 1 min in a microcentrifuge at 12,000g. The gel fragments were extruded through the yellow tips into the bottom of the 2.0-ml tubes as a fine paste. The tip and 0.65-ml tubes were discarded and 30 µl of water were added. They were protected from light and agitated overnight to elute the DNA.

Sequencing

Insert ends were sequenced with the ABI Prism dye terminator cycle sequencing kit and ABI373 automated sequencer (Perkin Elmer, Foster City, CA). The gel fragment homogenates were centrifuged briefly and up to 11 µl of eluate were removed from the top and used as template with 1 µl of L-III or R-III primer (20 ng/µl). Use of this primer resulted in the presence of the vector/insert junction (EcoRI site) in the sequence confirming that the correct product was obtained. Temperature cycling was 25 cycles (96°C for 30 sec, 50°C for 15 sec, 60°C for 4 min) in an Ericomp Twinblock Easycycler. The excess terminators were removed by phenol:chloroform extraction and ethanol precipitation or by ethanol precipitation alone. The same methodologies were used for BAC insert end sequencing with the SP6 and T7 sequencing primers in Table 1.

Figure 1

 1 Figure 1 Schematic representation of vectorhexamer PCR. (a) The YAC vector/insert junction (right or left arm) before amplification. The YAC vector is shown in red (left) with nested vector priming sites in green. The first nested priming site is located outside the SUP4 gene. The mouse genomic insert is shown in blue (right) with two particular hexamer sequences shown in red. (b) The initial cycles of amplification at low annealing temperature. The vector primer (in green) anneals to its complement in the YAC arm, from which a new strand is synthesized (broken line). The hexamer primer is shown annealing to a target site in the mouse insert, its hexamer portion shown in red and its 20-bp anchor sequence in orange. The low annealing temperature (35°C) allows pairing and priming by the 3Ј hexamer alone (shown in expanded view). (c)The later cycles of the first round of PCR are performed at a higher annealing temperature so that the entire hexamer primer stably anneals only to products from the initial cycles. (d,e) The second and third rounds of PCR reamplify the vector/insert junction products from the first PCR with nested vector primers to increase the specificity and yield of the amplification. The fourth nested vector primer allows reduction of vector arm contribution to the PCR product for use as a hybridization probe.

Figure 2

 2 Figure 2 Vector-hexamer PCR products. (a) Products of the initial PCR using the outermost YAC vector right arm primer R-I with the PvuII primer. Only nonspecific products are visible. (b) Products of the second round of PCR using the first nested right arm primer R-II with the PvuII primer. Both nonspecific and a few specific products are visible. (c) Products of the third round using the second nested right arm primer R-III with the PvuII primer. Bright YAC-specific products are apparent in many lanes. (d) Third-round PCR products from several different YACs made with the left arm L-III primer in conjunction with three different hexamer primers. The center gel demonstrates a background band amplified from the yeast host near the position of the largest size marker band (X174 digested with HincII) in all four YACs and the control YAC.

Figure 2 (

 2 See facing page for legend.) VECTOR-HEXAMER PCR ISOLATION OF YAC ENDS GENOME RESEARCH 677 (See facing page for footnote.)

Table 1 . Primers

 1

	Annealing
	temp.	Ref.

Table 2 . YAC Insert End Recovery with Vector-Hexamer PCR BAC

 2 ends directly from bacterial colonies, and usually with just one round of PCR, simplifying the procedure and eliminating the expense of BAC DNA purifications.

Table 3 . Hexamer Priming Fidelity

 3

	Accession	Priming		Primer
	no.	site	Primer	sequence

ACKNOWLEDGMENTS

We are grateful for the generous assistance and access to YAC libraries and screening pools provided by D. Koos, G. Guan, and S. Tilghman; F. Chartier, G. Argyropoulos, and S.D.M. Brown; and E. Brundage. This work was supported by National Institutes of Health grant AI23548.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked ''advertisement'' in accordance with 18 USC section 1734 solely to indicate this fact.


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3

In vitro transcribed (IVT) messenger RNA (mRNA) constitutes a versatile platform to deliver antigenic information to the immune system. Systemic administration of mRNA packaged into Lipid based mRNA nanocomplexes (LR) has emerged as a particularly powerful approach to yield potent antitumor T-cell responses. [START_REF] Broos | Particle-Mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-Specific T-Cell Responses Despite the Induction of Type I Interferon[END_REF][START_REF] Kranz | Systemic RNA Delivery to Dendritic Cells Exploits Antiviral Defence for Cancer Immunotherapy[END_REF][START_REF] Pektor | Toll Like Receptor Mediated Immune Stimulation can be Visualized in vivo by [18F]FDG-PET[END_REF] Inevitably, systemic administration of mRNA encapsulated into nanoparticles comes at the expense of an increased risk of adverse events. Toxicity to systemic administration of mRNA nanoparticles is multifaced but closely linked to the secretion of inflammatory cytokines. [START_REF] Zhang | Implications of Pharmacokinetic Behavior of Lipoplex for its Inflammatory Toxicity[END_REF] These inflammatory responses are associated with liver damage and hematological toxicities and should hence be minimized to increase safety. [START_REF] Xue | Nanotoxicity: a Key Obstacle to Clinical Translation of siRNA-Based Nanomedicine[END_REF][START_REF] Zatsepin | Lipid Nanoparticles for Targeted siRNA Delivery -Going From Bench to Bedside[END_REF] To avoid these inflammatory responses, the RNA field has designed several mRNA modifications that strongly reduce RNA sensing by innate sensors. [START_REF] Kormann | Expression of Therapeutic Proteins after Delivery of Chemically Modified mRNA in Mice[END_REF][START_REF] Kariko | Incorporation of Pseudouridine into mRNA Yields Superior Nonimmunogenic Vector with Increased Translational Capacity and Biological Stability[END_REF][START_REF] Andries | N(1)-Methylpseudouridine-Incorporated mRNA Outperforms Pseudouridine-Incorporated mRNA by Providing Enhanced Protein Expression and Reduced Immunogenicity in Mammalian Cell Lines and Mice[END_REF] Although avoidance of innate activation is vital when using mRNA in the context of protein replacement therapy, a certain level of innate immune activation needs to be maintained to evoke T-cell immunity in the context of mRNA vaccination. Identification of an mRNA nanoparticle format that combines excellent immunogenicity with sufficient (inflammatory) safety thereby represents a major challenge to enable safe application of systemic mRNA vaccines.

LR elicit potent cytokine responses reminiscent of systemic viral infections upon systemic administration that can be a cause of adverse effects, ranging from mild flulike symptoms to liver toxicities and auto-immune pathologies. [START_REF] Crow | Type I Interferon in the Pathogenesis of Lupus[END_REF][START_REF] Rizza | Recent Advances on the Immunomodulatory Effects of IFN-Alpha: Implications for Cancer Immunotherapy and Autoimmunity[END_REF][START_REF] Pott | Type I and III Interferon in the Gut: Tight Balance between Host Protection and Immunopathology[END_REF] Hybrid Lipidshell Polymer core mRNA nanoparticles (LPR) might represent valuable alternatives to LR as they combine improved colloidal stability with reduced cytotoxicity. [START_REF] Rezaee | Progress in the Development of Lipopolyplexes as Efficient Non-Viral Gene Delivery Systems[END_REF][START_REF] Goncalves | Lipopolyplexes Comprising Imidazole/Imidazolium Lipophosphoramidate, Histidinylated Polyethyleneimine and siRNA as Efficient Formulation for siRNA Transfection[END_REF] Moreover, the physicochemical properties of LPR are likely to result in a differential interaction of the delivered mRNA with innate RNA sensors, which in turn might alter the immunogenicity and safety profile of LPR relative to LR. In this study, we thereby addressed the T-cell responses and inflammatory responses to an advanced LPR platform comprising a lipid shell endowed with Mannose Receptor targeting moieties. [START_REF] Perche | Enhancement of Dendritic Cells Transfection In Vivo and of Vaccination Against B16F10 Melanoma with Mannosylated Histidylated Lipopolyplexes Loaded with Tumor Antigen Messenger RNA[END_REF][START_REF] Barbeau | Synthesis of a Trimannosylated-Equipped Archaeal Diether Lipid for the Development of Novel Glycoliposomes[END_REF] LPR exhibited excellent hemocompatibility and largely restricted mRNA expression to splenic antigen presenting cells upon systemic administration. Immunization with LPR instigated potent T-cell immunity and showed superior effectiveness in controlling tumor growth compared to 4 intravenous immunization with antigen mRNA electroporated dendritic cells (DCs) and LR. Early innate responses to LPR were characterized by a type I IFN signature in the spleen. Nonetheless, conversely to LR, LPR did not depend on these type I IFN responses to generate cytolytic effectors. This striking behavior of LPR enabled the generation of a less pro-inflammatory yet equally potent systemic LPR vaccine by usage of N1-methylpseudo-uridine nucleoside modified mRNA (see Graphical Table of Contents).

RESULTS AND DISCUSSION:

Synthesis and characterization of Tri-Mannosylated LPR LPR nanoparticles were produced by a well-established two-step approach, comprising first the complexation of mRNA to a cationic polypeptide PEG-HpK at mRNA/polymer at weight ratio of 1/3 and then a subsequent mixing of the generated mRNA polyplexes with liposomes at mRNA/lipid weight ratio of 1/2. [START_REF] Wang | Systemic Delivery of Modified mRNA Encoding Herpes Simplex Virus 1 Thymidine Kinase for Targeted Cancer Gene Therapy[END_REF] The previously described polyethylene glycol (PEG)ylated derivative of histidylated polylysine was used to complex the mRNA into polyplexes. Liposomes were derived from those reported by Perche et al. yet the mono-mannose bearing lipid was replaced by a tri-mannose bearing diether lipid as this was demonstrated to further increase the selectivity of Mannose Receptor targeting in vitro. [START_REF] Perche | Enhancement of Dendritic Cells Transfection In Vivo and of Vaccination Against B16F10 Melanoma with Mannosylated Histidylated Lipopolyplexes Loaded with Tumor Antigen Messenger RNA[END_REF][START_REF] Barbeau | Synthesis of a Trimannosylated-Equipped Archaeal Diether Lipid for the Development of Novel Glycoliposomes[END_REF] The LPR generation and characteristics are shown in Figure 1A-B. The complexation of mRNA was confirmed by the absence of mRNA migration in an agarose gel electrophoretic mobility shift assay (Figure 1C).

Moreover, mRNA was stable as evidenced by the absence of degradation when LPR was mixed with Fetal Clone I serum in contrast to 'naked' mRNA (Figure 1C). To assess mRNA integrity upon LPR incorporation, we extracted the mRNA from LPR using TRizol extraction agent and performed capillary gel electrophoresis (Agilent). As can be appreciated from Figure 1D, mRNA integrity was not affected upon LPR generation. The morphology of LPR was assessed by Transmission electron microscopy (TEM). Liposomes exhibited a spherical shape with a laminar lipid bilayer structure (Figure 1E). Tri-mannosylated LPR displayed the core morphology of mRNA/PEG-HpK polyplexes, which appeared surrounded by a laminar lipid bilayer structure of liposomes (Figure 1F-G). How polyplexes become encapsulated into 5 liposomes is not yet fully understood. The charge of mRNA polyplex is close to neutrality due to the presence of PEG on the polymer, this prevents strong repulsion between cationic liposomes and polyplexes favoring their interaction and encapsulation of polyplexes into the liposomes. The interaction may be also favored by (i) local high concentration of PEG favoring lipid mixing and/or bilayer destabilization and (ii) interaction between imidazole groups of the polyplex and those of the polar heads of lipids. Altogether, those interactions could favor polyplexes encapsulation.

The association of mRNA, lipids and polymer was addressed by flow cytometry through usage of fluorescein-labelled liposomes, Cy3-labelled mRNA and Cy5-labelled polymer.

The results indicated that the liposomes, the polymer and the mRNA were associated in a same particle. Based on the side and forward scatters of polyplexes, liposomes and LPR, no free polyplexes were detected in the LPR solution indicating that all polyplexes were encapsulated inside liposomes (Figure S1).

Intravenous LPR administration targets and activates splenic antigen presenting cells

The spleen constitutes the lymphoid organ where T-cell immunity against blood-borne antigens is initiated and thereby represents the major target of systemic mRNA vaccines. The functional bio-distribution of mRNA was assessed through incorporation of Firefly Luciferase (Fluc) encoding mRNA into LPR and full body bioluminescence imaging (BLI). A rapid and sustained Fluc expression was observed in the spleen, whereas no significant expression was detectable in other body parts. These data were confirmed by organ isolation, which showed an exclusive splenic BLI signal (Figure 2A-B). Of note, LPR were previously reported to be also delivered in other organs and notably in the liver. [START_REF] Perche | Enhancement of Dendritic Cells Transfection In Vivo and of Vaccination Against B16F10 Melanoma with Mannosylated Histidylated Lipopolyplexes Loaded with Tumor Antigen Messenger RNA[END_REF] Nevertheless, no luciferase expression was observed in the liver. Splenic expression was strongly diminished in transgenic CD11c-diphtheria toxin (DT) receptor mice treated with DT prior to Fluc mRNA LPR administration (Figure 2C), suggesting mRNA expression predominantly occurs in DCs. To further delineate the location of the mRNA uptake we incorporated Cy5-labeled mRNA into LPR. Sections obtained from spleens dissected four hours post injection were stained for CD3 and B220 to respectively visualize T cells and B cells present in the white pulp. Large 6 numbers of Cy5-labeled LPRs accumulated in the area surrounding the white pulp, which corresponds to the marginal zone sinuses of the spleen (Figure S2). To characterize more specifically the cell types that express LPR delivered mRNA, we injected ROSA26-loxP-Stop-loxP RFP transgenic mice with LPR that contain CRE Recombinase mRNA. [START_REF] Luche | Faithful Activation of an Extra-Bright Red Fluorescent Protein in "Knock-In" Cre-Reporter Mice Ideally Suited for Lineage Tracing Studies[END_REF] In these mice, cells expressing CRE will remove the floxed stop codon enabling Red Fluorescent Protein expression to unfold. Spleen sections stained for CD11c and MOMA-1 revealed RFP expression in CD11c DCs, yet also in MOMA-1 macrophages (Figure 2D). When LR were modified with tri-mannose, a BLI signal was also detected in the spleen, but the BLI signal in LPR injected mice was higher than in LR injected mice (Figure S3). The higher BLI signal suggested either a better transfection efficiency of DC with LPR than with LR or a better delivery of LPR in the spleen notably in splenic DCs.

DC maturation constitutes an essential prerequisite for efficient T-cell priming and

effector/memory T-cell differentiation. We thereby determined the maturation status of CD8a DCs and of CD11b DCs -the two major conventional DC populations present in the spleen. [START_REF] Mildner | Development and Function of Dendritic Cell Subsets[END_REF] An overview of the gating strategy applied to identify splenic DC subsets is given in Figure S4. In response to systemic LPR administration, CD8a DCs -the crosspresenting DC subset generally considered vital for initiation of CD8 T-cell immunityexhibited a pronounced upregulation of MHC class II, CD86 and CD40. [START_REF] Li | Cross-Dressed CD8Alpha+/CD103+ Dendritic Cells Prime CD8+ T Cells Following Vaccination[END_REF] CD86 and CD40 were also upregulated on the CD11b DC subset, albeit to a lesser extent (Figure 2E). Taken together, these data demonstrate that systemic administration of LPR not only targets mRNA expression to the relevant antigen presenting cells of the spleen, but also properly activates them to subsequently prime T cells.

Systemic administration of LPR instigates superior T-cell immunity compared to LR

Next, we determined the capacity of systemic LPR vaccination to stimulate CD8 T-cell immune responses against the model antigen ovalbumin (OVA) and against the Human Papillomavirus 16 (HPV16) oncoprotein E7. Cytolytic T-cell responses were quantified after single LPR administration using a well-established in vivo killing assay. [START_REF] Durward | Antigen Specific Killing Assay Using CFSE Labeled Target Cells[END_REF] LPR immunization induced strong target cell lysis against both antigens. Addition of TriMix 7 mRNA -a mixture of mRNAs encoding the immune-stimulatory proteins CD40L, CD70 and caTLR4 -to the antigen encoding mRNA further enforced the evoked cytolytic Tcell responses (Figure 3A). [START_REF] Bonehill | Enhancing the T-Cell Stimulatory Capacity of Human Dendritic Cells by Co-Electroporation with CD40L, CD70 and Constitutively Active TLR4 Encoding mRNA[END_REF][START_REF] Van Lint | Optimized Dendritic Cell-Based Immunotherapy for Melanoma: the TriMix-Formula[END_REF] These responses increased in a dose dependent fashion, evidenced by the increased percentages of OVA-specific CD8 T cells and by the elevated numbers of IFN-γ secreting OVA-specific T cells at the higher dose (Figure S5A-B). Repeated immunizations with OVA/TriMix mRNA LPR profoundly expanded the circulating antigen-specific T-cell pool, resulting in an impressive percentage of OVA-specific CD8 T cells (51% +/-10%) 19 days after the initiation of immunization (Figure 3B). These strong OVA-specific T-cell responses were sustained over a long period of time and slowly decreased, as over 20% of all peripheral CD8 T cells remained antigen-specific one month after the third immunization. Subsequent boosting resulted in a rapid recall response, indicative of memory conversion (Figure 3B). After the final boost, a high fraction of splenic antigen-specific CD8 T cells produced IFN-γ (38%+/-4%) and a significant amount of antigen-specific T cells co-produced IFN-γ and TNF-α (6%+/-1%), indicative of a polyfunctional effector phenotype, which has been associated with improved T cell functionallity (Figure 3C). [START_REF] Han | Polyfunctional Responses by Human T Cells Result from Sequential Release of Cytokines[END_REF] Compared to LR with the same lipid composition, mice bearing TC-1 tumor cells injected with LPR encoding E7 demonstrated the advantage for the addition of the PEG-HpK on the Tri-mannosylated liposomes (Figure S6). The results were in line with those reported by Mockey et al. showing that LR was not an efficient formulation to induce a specific immune response upon intravenous injection. [START_REF] Mockey | mRNA-Based Cancer Vaccine: Prevention of B16 Melanoma Progression and Metastasis by Systemic Injection of MART1 mRNA Histidylated Lipopolyplexes[END_REF] In addition, the advantage of the Tri-mannosylated liposomes over non-mannosylated liposomes is also shown, and in line with results previously reported. [START_REF] Perche | Enhancement of Dendritic Cells Transfection In Vivo and of Vaccination Against B16F10 Melanoma with Mannosylated Histidylated Lipopolyplexes Loaded with Tumor Antigen Messenger RNA[END_REF] Thus, even with Tri-Man targeting DCs, the efficiency of LR was still lower than with LPR.

Systemic LPR administration elicits profound antitumor immunity

The therapeutic benefit of systemic LPR immunization was assessed in the aggressive TC-1 tumor model, which expresses the HPV16 oncoprotein E7. Therapeutic vaccination consisted of three IV immunizations with E7/TriMix mRNA LPR. Antitumor efficacy was benchmarked against immunization with LR (Figure 4A-B) and ex vivo generated DCs electroporated with E7/TriMix mRNA (Figure S7A-C). TriMix/antigen 8 mRNA electroporated DCs were selected as benchmark as this approach was demonstrated to be sufficiently powerful to yield clinical benefit in melanoma patients in a phase II study. [START_REF] Wilgenhof | Phase II Study of Autologous Monocyte-Derived mRNA Electroporated Dendritic Cells (TriMixDC-MEL) Plus Ipilimumab in Patients With Pretreated Advanced Melanoma[END_REF] Systemic LPR treatment dramatically improved the median survival time of TC-1 inoculated mice and was even superior in controlling tumor growth in comparison to treatment with LR and electroporated DCs, respectively. As a consequence, these data highlight the capacity of LPR to yield effective antitumor immunity.

LPR induced type I IFN are dispensable for cytolytic T-cell differentiation mRNA complexed to lipid carriers (LR) instigates vigorous type I interferon (IFN)

responses upon in vivo administration. Alike the LR described previously, LPR evoked transiently elevated IFN-α titers in blood (Figure 5A). [START_REF] Broos | Particle-Mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-Specific T-Cell Responses Despite the Induction of Type I Interferon[END_REF][START_REF] Kranz | Systemic RNA Delivery to Dendritic Cells Exploits Antiviral Defence for Cancer Immunotherapy[END_REF] To pinpoint the anatomical location of type I IFN induction a transgenic IFN-β reporter mouse strain was used. [START_REF] Lienenklaus | Novel Reporter Mouse Reveals Constitutive and Inflammatory Expression of IFN-Beta In Vivo[END_REF] In this mice strain, a Luciferase encoding gene sequence was placed under control of the IFN-β promoter. Full body imaging revealed a strong bioluminescence signal confined to the spleen of LPR injected mice, whereas no IFN-β promoter activation was observed in non-lymphoid organs typically associated with nanoparticle accumulation such as liver and lungs (Figure 5B). This selective type I IFN induction can be considered highly beneficial, as the presence of type I IFN in lymphoid tissues is likely to maximize T cell immunity, whereas absence of type I IFN induction in vital organs such as lungs and liver should minimize adverse effects.

NanoString transcriptome analysis further confirmed the existence of a strong antivirallike type I IFN signature in the spleen (Figure 5C), with upregulation of mRNAs encoding IFN-β, IFN-α isoforms and downstream Interferon Stimulated Genes (ISGs).

Transcripts encoding the intracellular RNA sensors RIG-I (ddx58) and MDA-5 (Ifih1) were upregulated alongside transcripts that encode the 2'-5'-Oligoadenylate Synthetases (OAS) 2 and 3. OAS2 and OAS3 are activated by double stranded (ds) RNA and induce RNA cleavage by activation of RNAseL. [START_REF] Donovan | Structural Basis for Cytosolic Double-Stranded RNA Surveillance by Human Oligoadenylate Synthetase 1[END_REF] Transcripts for endosomal TLRs recognizing viral RNAs (TLR3, TLR7) were increased, whereas transcript levels for TLR recognizing bacterial ligands (TLR4 and TLR5) remained unaffected or were even slightly downregulated. In addition to these typical antiviral mediators, spleens of We next analyzed the impact of N1mψ nucleoside modified mRNA on the biodistribution and intensity of mRNA expression upon systemic LPR administration. LPR containing N1mψ nucleoside modified Fluc mRNA retained the spleen-centered expression pattern of unmodified mRNA (Figure S9A) and induced elevated levels of Fluc expression compared to LPR containing unmodified mRNA (Figure S9B).

N1mψ modified mRNA does not hamper antitumor T-cell immunity to LPR

To address how the combination of elevated antigen expression levels with reduced inflammatory responses upon use of N1mψ nucleoside modified mRNA impacts the magnitude of the LPR instigated T-cell responses, we quantified the percentages of circulating antigen-specific T cells and their IFN-γ secretion upon restimulation. N1mψ nucleoside modified mRNA did not interfere with the initial priming and expansion of antigen-specific T cells. Instead, after the third immunization, mice immunized with N1mψ nucleoside modified mRNA LPR even displayed elevated percentages of antigen-specific CD8 T cells (Figure 7A-C). The fraction of OVA specific CD8 T cells that rapidly responded with IFN-γ secretion upon in vitro peptide restimulation was however reduced from 60% (unmodified mRNA) to approximately 40% in the modified mRNA group (Figure 7D). These data support a model in which usage of modified mRNA in LPR context supports elevated T cell proliferation -by enhancing antigen expression and presentation -yet results in relatively lower percentages of T cells displaying immediate effector function, which most likely is a consequence of reduced inflammatory cytokines titers that impose effector function. Combined, these data fit into the hypothesis that high TCR stimulation combined with low to moderate levels of cytokines promote central memory T cell responses, whereas TCR stimulation in the context of high inflammation rather promotes effector/effector memory differentiation.

The functionality of the elicited T cells was assessed by comparison of antigen-specific cytolytic activity after immunization with of OVA/TriMix unmodified mRNA LPR or with OVA/TriMix N1mψ nucleoside modified mRNA LPR (Figure 7E-F). Cytolytic effector Tcell responses were not dampened by N1mψ nucleoside modified mRNA. In line with our observations in Ifnar -/-mice, this striking feature was intrinsic to LPR, as LR (identical lipid composition yet lacking the polymeric LPR core) largely lost their capacity to elicit cytolytic effectors upon incorporation of N1mψ nucleoside modified mRNA (Figure 7E). To assess whether these features are specific to the tri-mannosylated LPR system applied in this study or represent a common property of LPR, we designed an LPR system with a lipid shell composed of RNAiMAX. [START_REF] Broos | Particle-Mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-Specific T-Cell Responses Despite the Induction of Type I Interferon[END_REF] RNAiMAX based LPR and LR behaved identical to the tri-mannosylated LPR/LR system: equal cytolytic activity when N1mψ nucleoside modified mRNA is applied in an LPR context, whilst a reduction in cytolytic activity upon N1mψ nucleoside modified mRNA delivery in an LR context (Figure 7F). The better quality of the immune response including the amount of antigenspecific CD8 T cells (Figure 7G) induced with LPR as compared to LR might be due to supramolecular organization of mRNA with the polymer and liposomes in LPR and of 12 mRNA with liposomes in LR, which likely makes the difference. In the LPR formulation, the mRNA is condensed with the polymer and then encapsulated in a liposome. In contrast, mRNA is sandwiched between lipid layers in LR. The targeting to DCs of LR and LPR by mannose moieties via the mannose receptor would not be different.

However, the presence of the mRNA-condensing polymer would modify the release and/or intracellular trafficking of mRNA and therefore the sensing by pattern recognition receptors as those involved in IFN type I activation. We showed that similar effect but in a lower extent was obtained with RNAiMAX-based LR and LPR. Therefore, the mRNA complexation with the cationic polymer would be rather responsible than the nature of liposomes.

The antitumor capacities of systemic immunization with N1mψ nucleoside modified mRNA LPR and unmodified mRNA LPR were compared in the subcutaneous TC-1 (Figure 7H-J) and B16-OVA tumor models (Figure 7J). The growth curve shows the lack of growth control for LR with N1mψ nucleoside modified mRNA, as opposed to the LPR treated mice where no significant differences were observed between the two treatment groups. Similar observations were obtained in the highly aggressive subcutaneous B16-OVA model, with both treatment modalities being equally potent in stalling tumor growth (Figure 7J). Taken together, these data demonstrate LPR can be combined with N1mψ nucleoside modified mRNA to improve inflammatory safety upon systemic administration without hampering the functionality and antitumor efficacy of the evoked T cell response.

Incorporation of N1mψ nucleoside modified mRNA reduces LPR evoked type I IFN on human PBMCs

Finally, we aimed to assess the translational potential of using N1mψ modified nucleoside mRNA LPR by assessing hemocompatibility, transfection efficiency and cytokine responses on human peripheral blood mononuclear cells (PBMCs). The hemocompatibility of LPR was addressed on human blood according to the guidelines of the US Nanotechnology Characterization Laboratory (NCL). LPR were evaluated at mRNA plasma-concentrations corresponding to intravenous injection of respectively 1250 µg (223 ng/ml), 250 µg (44.6 ng/ml) or 50 µg of mRNA (8.9 ng/ml). At none of the Page 12 of 40 assayed doses, LPR induced significant complement activation -as determined by Western Blot quantification of C3a cleavage (Figure 8A-B). Hemolysis was determined through measurement of hemoglobin release on Li-heparin anticoagulated blood from two different healthy donors. None of the LPR incubated samples showed significant hemolysis according the NCL criteria (Figure 8C). Finally, platelet aggregation was quantified on pooled platelet rich plasma (PRP) obtained from four healthy donors. At the evaluated doses, LPR did not evoke significant platelet aggregation (Figure 8D).

To assess whether LPR can transfect human antigen presenting cells, we incubated monocyte derived DCs from healthy donors with LPR containing eGFP mRNA. As can be appreciated from Figures S10A-B, LPR were capable of mediating DC transfection without severe impact on cell viability. Finally, we addressed to which extent the reduction in inflammatory cytokines observed in mice upon usage of N1mψ modified mRNA could be extended to human PBMCs. Alike our findings in mice, incorporation of N1mψ nucleoside modified mRNA strongly reduced IFN-α and IFN-β titers produced by human PBMCs upon incubation with LPR, suggesting that our data can be translated to the human setting (Figure 8E-F).

CONCLUSIONS:

In conclusion, we have demonstrated that systemic immunization with LPR comprising The HPV16 E7-derived peptide RAHYNIVTF and the OVA-derived peptide SIINFEKL were purchased from Eurogentec (Belgium). mRNA production. mRNA encoding caTLR4, mouse CD70, mouse CD40L, Fluc, eGFP, HPV16 E7/DCLamp, tNGFR and OVA was used throughout the study. The mRNA was transcribed from the following previously described plasmids pEtheRNA-v2-TLR4, pEtheRNA-v2-moCD70, pEtheRNA-v2-moCD40L, pEtheRNA-v2-Fluc, pEtherna-v2-eGFP, pEtheRNA-v2-sig-E7/16-DCLco, pGEM-tNGFR and pGEM-li80tOVA, respectively. [START_REF] Broos | Particle-Mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-Specific T-Cell Responses Despite the Induction of Type I Interferon[END_REF][START_REF] Van Lint | Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy[END_REF][START_REF] Bialkowski | Intralymphatic mRNA Vaccine Induces CD8 T-Cell Responses that Inhibit the Growth of Mucosally Located Tumours[END_REF] The mRNA transcription and quality control were performed as previously described. [START_REF] Van Lint | Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy[END_REF]

Lipids, Liposomes and polymer. The trimannosyl diether lipid (TriMan-Lip), O,Odioleyl-N-[3N-(N-methylimidazolium iodide) propylene] phosphoramidate (Lip1) and

O,O-dioleyl-N-histamine phosphoramidate (Lip2) were synthesized as described. [START_REF] Barbeau | Synthesis of a Trimannosylated-Equipped Archaeal Diether Lipid for the Development of Novel Glycoliposomes[END_REF][START_REF] Mevel | Synthesis and Transfection Activity of New Cationic Phosphoramidate Lipids: High Efficiency of an Imidazolium Derivative[END_REF] TriManlip100 liposomes were prepared at 5.4 mM by mixing in ethanol Lip1, Lip2 and TriMan-Lip in the percentage of 47.5%, 47.5% and 5%, respectively. Solution was then evaporated until formation of a film. The film was hydrated for 12 h at 4°C in 1 mL of 10 mM RNase free HEPES buffer, pH 7.4, vortexed and then the suspension was sonicated for 15 min at 37 kHz using a Bioblock ultrasonic bath (Bioblock Scientific, Illkirch, France). Liposomes were dialyzed (Dialysis Tubing Cellulose membrane; MWCO: 12.4 kDa; size: 33 x 21 mm, Sigma) at 4°C for 6 hours and then overnight against 500 mL 10 mM RNase free HEPES buffer, pH 7.4. The lipid concentration was determined with Nile Red. The amount of mannosylated lipid per liposome was determined using the colorimetric resorcinol/sulfuric assay. [START_REF] Monsigny | Colorimetric Determination of Neutral Sugars by a Resorcinol Sulfuric Acid Micromethod[END_REF] PEGylated and histidinylated polylysine (PEG-HpK; average Mw of 75.4 kDa) was poly-L-lysine of degree of polymerization substituted at 45% with histidine residues and one mPEG molecule of 5 kDa prepared as described. [START_REF] Mockey | mRNA-Based Cancer Vaccine: Prevention of B16 Melanoma Progression and Metastasis by Systemic Injection of MART1 mRNA Histidylated Lipopolyplexes[END_REF][START_REF] Midoux | Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes[END_REF] LPR preparation. LPRs were prepared as previously described. [START_REF] Perche | Enhancement of Dendritic Cells Transfection In Vivo and of Vaccination Against B16F10 Melanoma with Mannosylated Histidylated Lipopolyplexes Loaded with Tumor Antigen Messenger RNA[END_REF] In brief, mRNA was first complexed with PEG-HpK by vortex at an mRNA/PEG-HpK weight ratio of 1/3. The resulting polyplexes were incubated with TriManlip100 liposomes or RNAiMAX liposomes at an mRNA/liposome ratio of 1/2. LR were formed by mixing mRNA with TriManlip100 liposomes or RNAiMAX liposomes at an mRNA/liposome ratio of 1/2.

Electrophoretic mobility shift assay and particle characterization. The size and zetapotential of LPR was measuredusing SZ-100 Analyser (Horiba Scientific, les Ulis, France), the electrophoretic mobility shift assay were performed as described earlier. 1

Transmission electron Microscopy (TEM).

The sample solutions were deposited on carbon grid (for 10 min) and then rinsed three times with distilled water. The samples were then stained with uranyl acetate for 30 sec. Finally, the grids were washed twice with distilled water prior to air-drying. The samples were observed under TEM (CM20 Philips, FEI company, Oregon, USA) equipped with LaB6 filament and operating at 160 kV.

Immunization of mice.

Mice were immunized at the indicated dose and mRNA composition intravenously formed as LPR in a final volume of 250 µl 10 mM Hepes and 5 % glucose (pH 7.4).

In vivo bioluminescence imaging. In vivo bioluminescence imaging was conducted on the Photoimager Optima (Biospacelab, France) using the Photo Acquisition software 16 Version 3.4 (Biospacelab, France) and the analysis M3 Vision Software 1.0.7.1178 (Biospacelab, France) as previously described. [START_REF] Bialkowski | Adjuvant-Enhanced mRNA Vaccines[END_REF] In vivo killing assay. The assay was performed as previously described. NanoString analysis. Total RNA was isolated from the spleen of mice using the SV Total RNA Isolation System (Promega, Madison, USA). The Samples were then analyzed with the PanCancer Immune Profiling Panel according to the manufacturer's instructions and run on the nCounter SPRINT Profiler. The data were analyzed using the nSolver software.

Platelet aggregometry. Platelet aggregations were performed according to procedure ITA-2.2 described by the NCL, with minor modifications. In brief, blood was withdrawn from healthy volunteers and anticoagulated with 3.2% w/v trisodium citrate. Whole blood was centrifuged for 8 minutes at 200 x g or for 10 minutes at 2500 x g to prepare platelet-rich plasma (PRP) or platelet-poor plasma (PPP), respectively. PRP and PPP Hemolysis assay. Hemolytic properties of nanoparticles were assessed according to procedure ITA-1 described by the NCL. In short, blood anticoagulated with lithium heparin was collected from healthy volunteers and assessed for the presence of < 1 mg/mL free plasma hemoglobin using a calibration curve of human hemoglobin (Sigma-Aldrich, Steinheim, Germany) dissolved in Drabkin's Reagent (Sigma-Aldrich), which was prepared according to the manufacturer's instructions. Whole blood was diluted to a total hemoglobin concentration of 10 mg/mL using PBS, and mixed with 8 volumes of nanoparticles in PBS in triplicates. PBS and 1% of Triton X-100 served as negative and positive controls for hemolysis, respectively. Mixtures were incubated for 3 hours at 37°C, and mixed every 30 minutes by inverting. Cells were removed by centrifuging at 800 x g for 15 minutes, and supernatants were transferred to clear 96-well plates.

Sample inhibition/enhancement controls were prepared by spiking positive control supernatant with nanoparticles. All samples and controls were mixed with equal volumes of Drabkin's reagent and measured using a SpectraMax M2e microplate reader (Molecular Devices, UK) at 540 nm. Calibration curves of human hemoglobin in Drabkin's reagent were used to calculate the concentration of free hemoglobin in the supernatants. Hemolysis was expressed as the percentage of free hemoglobin compared to total blood hemoglobin.

Flow cytometry. Spleen DC maturation was assessed using CD11c-efluor610, CD11b-APC-efluor780, MHCII-efluor450, CD40-PE, CD86-APC, CD8-PE-Cy7 (all eBioscience).

Quantification of OVA-specific and E7-specific CD8 T cells was performed using APClabelled dextramers (Immudex) according to the manufacturer's instructions. CD3-BV421, CD8-PerCP-Cy5.5, IFN-γ-APC and TNF-α-PE-Cy7 (all BD Biosciences) were used for intracellular cytokine staining using the BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. The data were collected using the LSRFortessa or FACSCanto (BD) and the analysis of the data was performed using the FACSDiva (BD) and Flow Jo software.

9 LPR

 9 Figure S8A-B). Bioluminescence imaging of IFN-β reporter mice confirmed the reduced IFN-β promoter activation upon injection of N1mψ nucleoside modified mRNA LPR (Figure 6C).

  histidinylated polylysine and Tri-Mannosylated and imidazoylated liposomes elicits strong cytolytic T-cell responses that confer high antitumor efficacy. In contrast to LR made with same liposomes, cytolytic T-cell responses to LPR immunization did not require type I IFN responses. Incorporation of N1mψ modified mRNA increased mRNA expression levels and reduced inflammatory responses, without hampering T-cell mediated antitumor efficacy. LPR displayed excellent hemocompatibility on human blood and incorporation of N1mψ modified mRNA into LPR dramatically reduced type I IFN secretion upon incubation with human PBMCs. Taken together, by combining excellent immunogenicity with improved inflammatory safety, those LPR constitute an interesting alternative to the current developed LR. Page 13 of 40 MATERIALS AND METHODS: Mice. 6-12 week old female C57BL/6 mice were purchased from Charles River (France). Ifnar /-were kindly provided by C. Libert (Ghent University, Belgium). CD11c-DTR mice and ROSA26-loxP-Stop-loxP RFP transgenic mice were kindly provided by B. Lambrecht (Ghent University, Belgium). IFN-β reporter mice were provided by S. Lienenklaus (Hannover Medical School). All mice were housed in individually ventilated cages and handled according to the regulations of the Animal Ethics and Animal Care Committee of the Vrije Universiteit Brussel. Cell line and reagents. The TC-1 cell line was obtained from T.C. Wu (Johns Hopkins Medical Institution, Baltimore, Maryland, USA). The expression of the viral proteins HPV16-E6 and HPV16-E7 was confirmed by RT-PCR. The HEK 293T cells and the melanoma B16-OVA cells were obtained and cultured as recommended by the American Type Culture Collection.

17 from

 17 three donors was pooled. Subsequently, 450 µL of pooled PRP was added to glass cuvettes with a stirring bar set at 1200 rpm in a Model 700 Chrono-log aggregometer (Chrono-log corporation, Havertown, USA) and maintained at 37°C. After 2 minutes, when a stable baseline was obtained, 50 µL of nanoparticle solutions in PBS were added in duplicate and light transmission was measured for 7 minutes. 450 µL of pooled PPP mixed with 50 µL PBS served as a background reference. AGGRO/LINK software (Chrono-log corporation) was used to calculate the area-under-the-curve (AUC) ranging from 2 until 9 minutes for each sample, which was expressed as a percentage of the AUC of positive control samples (1 µg/mL Collagen Reagens HORM Suspension, Takeda, Austria).Complement activation assay.Qualitative analysis of complement activation was performed according to procedure ITA-5.1 of the NCL, with minor modifications. In brief, pooled PPP was prepared from blood (anticoagulated with 3.2% w/v trisodium citrate) from two healthy donors as described above. Pooled PPP was mixed with an equal volume of 0.2 µm filtered veronal buffer (10 mM barbital, 145 mM sodium chloride, 0.5 mM magnesium chloride, 0.15 mM calcium chloride, pH 7.2) and aliquoted in 20 µL aliquots for each sample to be tested in duplicate. Ten microliters of nanoparticle solution was added to the mixture and briefly vortexed. PBS was used as negative control, and 0.37 mg/mL of Cobra Venom Factor (CVF, Quidel Corporation, Kornwestheim, Germany) was used as a positive control. Samples were incubated for 30 minutes at 37°C. Proteins were electrotransferred to Immobilon-FL polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 50% v/v Odyssey blocking buffer (LI-COR Biosciences, Leusden, The Netherlands) in Tris-buffered saline (TBS), followed by incubation with goat anti-C3 polyclonal antibody (Protos Immunoresearch, Burlingame, USA), 1:2000 diluted in 50% v/v Odyssey blocking buffer in TBS with 0.1% v/v Tween20 (TBS-T). Blots were washed and probed with IRDye 800CW donkey anti-goat antibody (LI-COR Biosciences), 1:7500 diluted in 50% v/v Odyssey blocking buffer in TBS-T and visualized using an Odyssey Infrared Imager system (LI-COR Biosciences) at 800 nm. Band intensities of C3 cleavage products (~40 kDa) were measured with Odyssey application software (version 3.0.16, LI-COR), 18 normalized for lane background and expressed as a ratio compared with negative control samples.

Confocal imaging.Figure 1 .

 1 photon laser. Images were analyzed with Imaris software.

Figure 2 .

 2 Figure 2. Systemic LPR administration targets and activates splenic DCs. (A-B) Bioluminescence imaging (BLI) was performed 24 hours after the intravenous administration of 20 µg Fluc mRNA as LPR (n = 9). The whole body BLI is depicted in (A) and the organs isolated after 24 hours are shown in (B). BLI of isolated organs 1) spleen 2) lung 3) liver 4) inguinal lymph nodes 5) intestines 6) kidney 7) heart 8) stomach. (C) Intensity of bioluminescence in spleens of CD11c-DTR mice after injection of Fluc mRNA LPR (20 µg mRNA). -DT = without Diphtheria Toxin; + DT = with Diphtheria Toxin. Data are a representative of two independent experiments (n = 4). * P < 0.05 (Unpaired t-test). (D) Immunohistochemistry images of spleen sections after injection of ROSA26-loxP-Stop-loxP RFP transgenic mice with Cre mRNA LPR (n = 4) Spleens were stained for MOMA-1 and for CD11c to visualize metallophilic marginal zone macrophages and DCs. Triangles in R1 indicate RFP expression in MOMA-1+ cells whereas arrows in R2 indicate RFP expression in CD11c + cells (E) Flow cytometric analysis of the expression of MHCII, CD86 and CD40 on splenic CD8a DCs and CD11b DCs after IV injection of PBS (untreated) or Fluc mRNA LPR. Data are

Figure 3 .

 3 Figure 3. Systemic LPR administration instigates potent T-cell immunity. (A) Cytolytic T-cell response as measured by the % target cell lysis after single immunization with LPR. Mice were immunized with OVA mRNA (10 µg) or E7 mRNA (10 µg) supplemented with either irrelevant control mRNA (15 µg) or with TriMix mRNA (5 µg/component). Data are shown as means +/-SD and are a representative of two independent experiments (n = 4). **P < 0.01; * p < 0.05 (Two-way ANOVA followed by Bonferroni's multiple comparisons test). (B) Schematic overview of the immunization and sampling schedule applied. Mice were immunized at days 0, 7, 14 and 60 with OVA/TriMix (10 µg/15 µg) mRNA LPR. Blood samples were collected for quantification of the percentages of OVA-specific CD8 T cells at days 0, 5, 12, 19, 35, 55 and 65. Spleens were collected at day 65 for quantification of IFN-γ and TNF-α production by OVA-specific CD8 T cells. Flow cytometry analysis of the percentages of circulating OVA-specific CD8 T cells. (C) Flow cytometry analysis of cytokine production by OVAspecific T cells with (red) and without ex vivo peptide (blue) re-stimulation. Percentages of IFN-γ+, TNF-α+ and IFN-γ+ TNF-α+ OVA-specific CD8 T cells. Results are shown as means +/-SD (n =6). **** P < 0.0001; ns = non-significant (Two-way ANOVA followed by Bonferroni's multiple comparisons test).

Figure 4 .

 4 Figure 4.Systemic LPR administration elicits profound antitumor immunity. (A) TC-1 inoculation and treatment schedule. Mice were immunized with E7/TriMix (10 µg/15 µg) mRNA LR or LPR. (B) Spaghetti plots showing tumor growth rates of individual mice.

Figure 5 .

 5 Figure 5. LPR induced type I IFN are dispensable for cytolytic T-cell differentiation. (A) Serum IFN-α titers as measured by ELISA at the indicated time intervals after systemic administration of LPR (25 µg Fluc mRNA). Data are shown as means +/-SD (n = 4). *** P < 0.001; ** p < 0.01; ns = non-significant (One-way ANOVA analysis followed by Bonferroni's multiple comparisons test). (B) In vivo BLI images showing the anatomical distribution of IFN-β activation as reflected by luciferase expression in the IFN-β reporter mice. Mice were injected with 25 µg OVA mRNA (n=3).

Page 29 of 40 ( C )

 40C NanoString transcriptome profiling of spleens three hours after systemic LPR administration. (D-F) Cytolytic T-cell responses as measured by the % target cell lysis in wild type mice and Ifnar -/-mice after single immunization with LPR containing respectively OVA/TriMix mRNA (D), OVA/ctrl mRNA (E) or with LR containing OVA/TriMix mRNA (F). Data are shown as means +/-SD (n= 4-8). Data shown are a representative of three independent experiments. *** P < 0.001; ns = non-significant (unpaired t-test).

Figure 6 .

 6 Figure 6. N1mψ modified mRNA reduces inflammatory responses to systemic LPR immunization. (A-B) Quantification of inflammatory cytokine responses in blood of LPR injected mice relative to untreated mice at two hours and six hours' post injection. (A) Heat map representation after injection of unmodified Fluc mRNA LPR (25 µg mRNA) and N1mψ nucleoside modified Fluc mRNA LPR (25 µg mRNA). (B) Graphs showing the differential induction of IFN-α, CXCL-10, IL-12 and IL-6 to unmodified mRNA LPR and N1mψ mRNA LPR. Data are shown as log 2 fold change in titers when compared to untreated mice. Data are shown as means +/-SD (n = 4). **** P < 0.0001; *** p < 0.001; ** p < 0.01; ns = non-significant (Two-way ANOVA followed by Bonferroni's multiple comparisons test. (C) In vivo BLI images showing similar anatomical distribution and reduced intensity of IFN-β activation in N1mψ mRNA LPR as reflected by luciferase expression in the IFN-β reporter mice. Mice were injected with 25 µg OVA mRNA (n=3). (D) IFN-α and IFN-β titers in supernatants of human PBMCs incubated with the indicated doses of unmodified Fluc mRNA LPR and N1mψ Fluc mRNA LPR.

Figure 7 .

 7 Figure 7. N1mψ nucleoside modified mRNA does not hamper antitumor T-cell immunity to LPR. (A-C) Flow cytometry analysis of antigen-specific T cells upon immunization with respectively unmodified mRNA or N1mψ mRNA LPR. (A) Schematic overview of immunization and sampling schedule (B) Representative flow cytometry plots depicting the percentages of OVA-specific T cells after the third immunization with unmodified mRNA or N1mψ mRNA LPR. (C) Percentages of OVA-specific CD8 T cells (left panel) and E7-specific CD8 T cells (right panel) after the second and third immunization with unmodified mRNA or N1mψ mRNA LPR. (n=8). (D) The graph

Figure 8 .

 8 Figure 8. Characterization of LPR hemocompatibility on human blood. (A) Analysis of complement activation by Western Blot. Isolated platelet poor plasma of healthy donors was incubated with LPR at the indicated mRNA concentrations. Cobra Venom factor (CVF) was used as a positive control. SDS PAGE gels were stained with a polyclonal antibody for C3a to quantify C3a cleavage. Cleavage product band intensity was quantified and compared with PBS control. (B) Graph showing the relative increase in cleaved C3a/uncleaved C3a ratio compared to PBS. (C) Quantification of the percentage hemolysis upon incubation of Li-heparin anticoagulated blood obtained from two different healthy donors with LPR at the indicated mRNA concentrations. (D) Quantification of platelet aggregation. Platelet aggregation was assessed on pooled platelet rich plasma from three healthy donors using a Chrono-LOG aggregometer. Collagen was used as positive control. (E) IFN-α and (F) IFN-β titers in supernatans of human PBMCs incubated with the indicated doses of unmodified Fluc mRNA LPR and N1mψ Fluc mRNA LPR.

Figure 2 .Figure 3 .

 23 Figure 2. Systemic LPR administration targets and activates splenic DCs. 182x181mm (300 x 300 DPI)

Figure 4 .

 4 Figure 4.Systemic LPR administration elicits profound antitumor immunity 66x35mm (300 x 300 DPI)

Figure 5 .

 5 Figure 5. LPR induced type I IFN are dispensable for cytolytic T-cell differentiation 243x289mm (300 x 300 DPI)

Figure 6 .Figure 7 .

 67 Figure 6. N1mψ modified mRNA reduces inflammatory responses to systemic LPR immunization 172x146mm (300 x 300 DPI)

  [START_REF] Broos | Particle-Mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-Specific T-Cell Responses Despite the Induction of Type I Interferon[END_REF]

	Cytokine secretion by peripheral blood mononuclear cells (PBMCs). PBMCs were
	isolated from whole blood and cultured according to procedure ITA-10 described by the
	Nanoparticle Characterization Laboratory (NCL) of the National Cancer Institute
	(http://ncl.cancer.gov). In brief, blood was withdrawn from healthy volunteers and
	anticoagulated with lithium heparin. Withdrawal of blood samples was approved by the
	University Medical Center Utrecht (UMCU) Ethics Committee. Cells were resuspended
	in culture medium (RPMI 1640; ThermoFisher Scientific) containing 10% fetal bovine
	serum (FBS) and 100 U/mL penicillin and streptomycin) at a final concentration of 1.3 x
	10

6 cells/mL. 800 µL of cell suspension was seeded in 24-well plates and mixed with 200 µL of nanoparticle solution in duplicate in PBS. Cells were cultured at 37°C and 5% CO 2 for 24 hours, after which supernatants were collected. Cytokine release profiles were determined using an in-house developed and validated (ISO9001 certified) multiplex immunoassay (Laboratory of Translational Immunology, UMCU) based on Luminex technology (xMAP, Luminex, Austin, USA). Data was acquired with a Bio-Rad FlexMAP3D system (Bio-Rad laboratories, Hercules, USA) with xPONENT software version 4.2 (Luminex).

ACKNOWLEDGEMENTS

We thank P. Roman, E. Vaeremans, E. Heirman, E. De Smet, J. Corthals, J. Puttemans, C. Mahmoud and F. Geldholf for their technical assistance. We thank C. Goyvaerts for the breeding of transgenic mice and M. Keyaerts to provide the bioluminescence device. We thank S. Akhter, A. Sauldubois and C. Andreazza from the "Centre de Microscopie Electronique" of University of Orléans, France for assistance in TEM data acquisition. This work was supported by grants from the Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy, the National Cancer Plan of the Federal Ministry of Health, the Stichting tegen Kanker, the Vlaamse Liga tegen Kanker, an Integrated Project and an EU FP7-funded Network of Excellence, an IWT-TBM program, the FWO-Vlaanderen, Fonds Germaine Eisendrath-Dubois and the Scientific Fund Willy Gepts of the University Hospital Brussels. KVdJ, LB, PTJ and KB are supported by the IWT. The production of mRNA was supported by eTheRNA immunotherapies, a spin-off company of the Vrije Universiteit Brussel, founded by K.

Thielemans.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: kris.thielemans@vub.ac.be *E-mail: kbreckpo@vub.ac.be *E-mail: chantal.pichon@cnrs.fr ORCID ID Kevin Van der Jeught: 0000-0002-4626-2343